tretinoin and Breast-Neoplasms

The knowledge of the structure, function, and abundance of specific proteins related to the EMT process is essential for developing effective diagnostic approaches to cancer with the perspective of diagnosis and therapy of malignancies. The success of all-

Topics: Animals; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Metastasis; Neoplasm Proteins; Receptors, Retinoic Acid; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; 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Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; 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Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; 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YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Although integrity of the p53 signaling pathway in a given tumor was expected to be a critical determinant of response to therapies, most clinical studies failed to link p53 status and treatment outcome. Here, we present two opposite situations: one in which p53 is an essential effector of cure by targeted leukemia therapies and another one in advanced breast cancers in which p53 inactivation is required for the clinical efficacy of dose-dense chemotherapy. If p53 promotes or blocks therapy response, therapies must be tailored on its status in individual tumors.

Topics: Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Breast Neoplasms; Female; Humans; Leukemia, Promyelocytic, Acute; Molecular Targeted Therapy; Oncogene Proteins, Fusion; Oxides; Treatment Outcome; Tretinoin; Tumor Suppressor Protein p53

All-trans retinoic acid (ATRA) is the most important active metabolite of vitamin A controlling segmentation in the developing organism and the homeostasis of various tissues in the adult. ATRA as well as natural and synthetic derivatives, collectively known as retinoids, are also promising agents in the treatment and chemoprevention of different types of neoplasia including breast cancer. The major aim of the present article is to review the basic knowledge acquired on the anti-tumor activity of classic retinoids, like ATRA, in mammary tumors, focusing on the underlying cellular and molecular mechanisms and the determinants of retinoid sensitivity/resistance. In the first part, an analysis of the large number of pre-clinical studies available is provided, stressing the point that this has resulted in a limited number of clinical trials. This is followed by an overview of the knowledge acquired on the role played by the retinoid nuclear receptors in the anti-tumor responses triggered by retinoids. The body of the article emphasizes the potential of ATRA and derivatives in modulating and in being influenced by some of the most relevant cellular pathways involved in the growth and progression of breast cancer. We review the studies centering on the cross-talk between retinoids and some of the growth-factor pathways which control the homeostasis of the mammary tumor cell. In addition, we consider the cross-talk with relevant intra-cellular second messenger pathways. The information provided lays the foundation for the development of rational and retinoid-based therapeutic strategies to be used for the management of breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; ErbB Receptors; Female; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PubMed; Receptor Cross-Talk; Receptors, Notch; Retinoids; Transforming Growth Factor beta; Tretinoin; Wnt Proteins

To be maximally effective, therapy of cancer must be directed against both the resting stem cells and the proliferating cells of the cancer. The cell populations of both normal and cancer tissues consist of resting stem cells, proliferating transit-amplifying cells, terminally differentiating cells and dying (apoptotic) cells. The difference between normal tissue renewal and growth of cancers is that some of the transit-amplifying cells in the cancer population do not mature into terminally differentiating cells, but instead continue to proliferate and do not die (maturation arrest). Because of this the number of cancer cells increase, whereas the cell population of normal tissues remains a relatively constant. Conventional radiation treatment and chemotherapy kill the actively proliferating transit- amplifying cells of the cancer. Differentiation therapy, using specific targeted inhibitors of activation, effectively induces differentiation of the proliferating transit-amplifying cancer cells. However, even if the proliferating cancer cells are completely inhibited or eliminated, the cancer stem cells may restore the transit-amplifying population, so that clinical remission is usually temporary. The hypothesis presented in this paper is that successful cancer therapy must be directed against both the resting stem cells and the proliferating cells of the cancer. This may be possible if specific stem cell signals are inhibited using gene therapy, while at the same time attacking proliferating cells by conventional radiation treatment or chemotherapy. With advances in approaches using specific inhibitory RNA, such combination therapy may now be possible, but critical problems in delivering the inhibitory effect specifically to the cancer stem cells have yet to be worked out.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Lineage; Cell Proliferation; Embryonal Carcinoma Stem Cells; Female; Genetic Therapy; Humans; Leukemia; Models, Biological; Neoplasms; Neoplastic Stem Cells; RNA Interference; Stem Cells; Teratocarcinoma; Tretinoin

Breast cancer still remains a major problem in its incidence, morbidity and mortality; therefore, more effective strategies for its prevention are urgently needed. Retinoids, natural and synthetic derivatives of vitamin A, possess antiproliferative and proapoptotic properties, making them a promising class of chemopreventive agents against breast cancer. The efficacy of all-trans retinoic acid, 9-cis-retinoic acid, LGD1069 (Targretin, bexarotene), and N-(4-hydroxyphenyl)retinamide (fenretinide) as breast cancer chemopreventive agents is being studied. A better understanding of the molecular mechanisms of action of these agents should lead to improvements in their clinical application. In this review, we discuss the mechanisms by which retinoids exert their antiproliferative and apoptotic effects in breast cancer cells.

Topics: Alitretinoin; Anticarcinogenic Agents; Apoptosis; Bexarotene; Breast Neoplasms; Cell Cycle; Cell Division; Female; Fenretinide; Humans; Retinoids; Tetrahydronaphthalenes; Tretinoin

Chemoprevention of cancer is reviewed from the viewpoints of action mechanisms and methodology of clinical trials in order to introduce promising agents discovered by in vitro and/or in vivo studies to applications in humans. The clinical trial procedure essentially follows the phase study which has been employed for chemotherapeutic drugs. Chemoprevention of bladder cancer, prostate cancer, gastric cancer, hepatocellular carcinoma, breast cancer, head and neck cancer, colorectal cancer and lung cancer is reviewed, mainly focusing on clinical trials. Previous clinical trials have shown the effectiveness of the following: polyprenoic acid (acyclic retinoid) for hepatocellular carcinoma; tamoxifen for breast cancer; retinoic acids for head and neck tumor; and aspirin, a COX-2 inhibitor, for colorectal cancer. Despite the advantageous effects of some of these agents, their toxic effects must also be of concern at the same time. For example, in a chemoprevention trial of lung cancer, beta-carotene was unexpectedly found to increase the risk of lung cancer among high-risk groups. It is also noted that large-scale clinical trials demand large research grants, which may not be affordable in Japan. Chemoprevention is still an emerging field of oncology where researchers in both basic and clinical sciences face great challenges.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; beta Carotene; Breast Neoplasms; Clinical Trials as Topic; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Colorectal Neoplasms; Female; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Neoplasms; Prostatic Neoplasms; Tamoxifen; Tretinoin; Urinary Bladder Neoplasms

The sodium iodide symporter (NIS) is an intrinsic plasma membrane protein that mediates the active transport of iodide in the thyroid gland and a number of extrathyroidal tissues, in particular lactating mammary gland. Because of its crucial role in the ability of thyroid follicular cells to trap iodide, cloning of NIS opened an exciting and extensive new field of thyroid-related research. Cloning and molecular characterization of NIS allowed investigation of its expression and regulation in thyroidal and nonthyroidal tissues, and its potential pathophysiological and therapeutic implications in benign and malignant thyroid disease. In addition to its key function in thyroid physiology, NIS-mediated iodide accumulation allows diagnostic thyroid scintigraphy as well as effective therapeutic application of radioiodine in benign and malignant thyroid disease. Characterization and application of NIS as a novel therapeutic gene and the presence of high native NIS expression in the majority of breast cancers further suggest a promising role of NIS in diagnosis and therapy of cancer outside the thyroid gland.

Topics: Biological Transport, Active; Breast; Breast Neoplasms; Carcinoma, Papillary; Carcinoma, Papillary, Follicular; Female; Genetic Therapy; Humans; Iodine; Lactation; Male; Mutation; Prostatic Neoplasms; Symporters; Thyroid Diseases; Thyroid Gland; Thyroid Neoplasms; Thyroiditis, Autoimmune; Tretinoin

Retinoids have been reported to inhibit the growth of several breast cancer cell lines in culture and to reduce breast tumor growth in animal models. Furthermore, retinoic acid (RA) can augment the action of other breast cancer cell growth inhibitors both in vitro and in vivo. Clinically, interest has increased in the potential use of retinoids for the prevention and treatment of human breast cancer. The regulation of cell growth and differentiation of normal, premalignant, and malignant cells by retinoids is mediated by the RA receptors (RARs) and retinoid X receptor. One of the target genes of retinoid receptors is RARbeta2. A growing body of evidence supports the hypotheses that the RARbeta2 gene is a tumor suppressor gene and the chemopreventive effects of retinoids are due to induction of RARbeta2. RARbeta2 expression is reduced in many malignant tumors including breast carcinoma. This paper will briefly discuss basic aspects of retinoids and retinoid acid receptor. In particular, we review what is now known for inactivation mechanism of RARbeta2 and its role in tumor cell growth inhibition.

Topics: Breast Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A 69-year-old woman developed microgranular acute promyelocytic leukemia (APL-M3) 10 months after receiving adjuvant cyclophosphamide, doxorubicin, and paclitaxel for breast cancer. Replicate bone marrow aspirate karyotypes contained a translocation between the long arms of chromosomes 15 and 17, but not at breakpoints typical for APL. Fluorescence in situ hybridization paints and RARalpha/PML cosmid probes verified that the breakpoints on chromosomes 15 and 17 were proximal to both the PML and RARalpha genes; t(15;17)(q13;12). Although the patient received induction chemotherapy and a several month trial of all-trans retinoic acid (ATRA), there was no clinical improvement or hematological remission. We suspect that this patient developed postchemotherapy secondary APL with an atypical t(15;17), which rendered her leukemic cells unresponsive to ATRA therapy.

Topics: Aged; Bone Marrow; Breast Neoplasms; Chromosome Breakage; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Drug Resistance, Neoplasm; Female; Humans; In Situ Hybridization, Fluorescence; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Translocation, Genetic; Tretinoin

A growing body of evidence supports the hypothesis that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene which induces apoptosis and that the chemopreventive and therapeutic effects of retinoids are due to induction of RAR-beta2. During breast cancer progression, RAR-beta2 is reduced or even lost. It is known from studies of other tumor-suppressor genes that methylation of the 5'-region is the cause of loss of expression. Several groups demonstrated that this is also true for the RAR-beta2 in breast cancer by treating breast cancer cell lines with a demethylating agent and examining expression of the RAR-beta2 gene in response to a challenge with retinoic acid. Studies using sodium bisulfite genomic sequencing as well as methylation specific PCR showed that a number of breast cancer cell lines as well as breast cancer tissue showed signs of methylation. The RAR-beta2 gene was unmethylated in non-neoplastic breast tissue as well as in other normal tissues. A combination of retinoic acid with demethylating agents as well as with histone deacetylase inhibitors acts synergistically to inhibit growth. This review presents data that suggest that treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality. Both the time of commencement of chemoprevention and the choice of substances that are able either to prevent de novo methylation or to reverse methylation-caused gene silencing may be important considerations.

Topics: Base Sequence; Breast Neoplasms; Chemoprevention; DNA Methylation; Down-Regulation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The erbB2 gene, which encodes a transmembrane growth factor receptor, is overexpressed in approximately 30% of breast cancers. Overexpressing this gene makes breast cancers resistant to certain chemotherapeutic agents. In this article, we review what is known about ErbB2-mediated chemoresistance and the controversies surrounding it. We also examine the antiapoptotic function of erbB2 as one of the molecular mechanisms of ErbB2-mediated Taxol resistance and describe several emerging strategies for overcoming intrinsic ErbB2-mediated chemoresistance. Finally, we discuss future avenues for studies of chemosensitivity in ErbB2-overexpressing breast cancers that may lead to the development of effective biology-based treatment strategies.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Drug Resistance; Female; Gene Expression; Genes, erbB-2; Humans; Paclitaxel; Trastuzumab; Tretinoin

Protein kinase C (PKC) isoforms are serine/threonine kinases involved in signal transduction pathways that govern a wide range of physiological processes including differentiation, proliferation, gene expression, brain function, membrane transport and the organization of cytoskeletal and extracellular matrix proteins. PKC isoforms are often overexpressed in disease states such as cancer. In this review, PKC in a variety of cancers is discussed along with some specific cell biological mechanisms by which PKC exerts its function(s). The PKC family consists of several isoforms comprising three groups: classical, novel and atypical. Although PKC has been investigated for around 2 decades, only recently has the specific function of each isoform started to be elucidated and the isoforms evaluated for use as targets of drug action. Phorbol esters such as the tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol (DAG) activate classical and novel PKC isoforms. Naturally occurring retinoids, antisense oligonucleotides against specific PKC isoforms and specific PKC inhibitors can block this activation. Beta carotene and retinoid derivatives act as anticarcinogenic agents and can antagonize some of the biological actions of phorbol esters and oxidants. Another important area of investigation is the use of antisense oligonucleotides to inhibit specific PKC isoforms. These compounds have proven effective in reducing specific types of cancer in rodents and humans and are currently used in clinical trials. This review examines PKC isoforms as a target of drug action with special emphasis on their use in cancer therapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Drug Delivery Systems; Enzyme Inhibitors; Gastrointestinal Neoplasms; Humans; Isoenzymes; Lung Neoplasms; Neoplasm Proteins; Neoplasms; Oligonucleotides, Antisense; Protein Kinase C; Retinoids; Staurosporine; Tretinoin

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Breast Neoplasms; Cell Division; Drug Synergism; Female; Humans; Mammary Neoplasms, Experimental; Melatonin; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Retinoids, the natural and synthetic vitamin A derivatives, are known to inhibit the proliferation of lung cancer and breast cancer cells and the growth of carcinogen-induced bronchogenic squamous cell carcinoma and mammary tumors, and have been used as chemoprevention agents against both types of cancer. However, clinical trials of retinoids in patients with advanced lung cancer and breast cancer have not been successful. In studying how retinoid sensitivity is lost in cancer cells, we have found that lack of the retinoic acid receptor beta (RAR beta) gene expression and its abnormal regulation by retinoic acid (RA) are common features in human lung cancer and breast cancer cells. The absence and abnormal RA regulation of RAR beta correlates with the loss of anti-proliferation effect of RA in hormone-independent breast cancer cells, and is due to different abnormalities found in cancer cells. Furthermore, expression of RAR beta gene in hormone-independent breast cancer cells restores their RA sensitivity. These data demonstrate that RAR beta can mediate the growth inhibitory effect of RA and suggest that the lack of RAR beta may contribute to retinoid resistance in certain cancer cells.

Topics: Breast Neoplasms; Cell Differentiation; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Topics: Adenocarcinoma; alpha Catenin; Animals; beta Catenin; Breast Neoplasms; Cadherins; Cell Polarity; Cytoskeletal Proteins; Cytoskeleton; Desmoplakins; Dogs; Epithelium; Fetal Proteins; Flavones; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Macromolecular Substances; Multigene Family; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Protein Conformation; Receptor, IGF Type 1; Structure-Activity Relationship; Tamoxifen; Trans-Activators; Tretinoin; Tumor Cells, Cultured

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; HL-60 Cells; Humans; Neoplasms; Tretinoin

A number of lines of evidence suggest that IGFs are important mitogens in human breast cancer: (1) IGFs are the most potent growth factor in human breast cancer cells; (2) estrogen stimulates expression of IGF-II and the type 1 IGF receptor; and (3) stromal cells express IGFs, which may act in a paracrine manner. Numerous studies have demonstrated that IGFBPs modulate the mitogenic effects of IGFs in the local environment. In particular, we have recently demonstrated that IGFBP-3 inhibits the growth of Hs578T and MDA-MB-231 human breast cancer cells in an IGF-independent manner. Further studies revealed the existence of cell surface-associated IGFBP-3 receptors. Receptor binding and the subsequent antiproliferative action of IGFBP-3 was inhibited by IGFs, owing to the formation of an IGF-IGFBP-3 complex that prevents the binding of IGFBP-3 to its receptors. In addition, exogeneously added soluble heparin or heparan sulfate inhibited the binding of IGFBP-3 to the cell surface in a dose-dependent manner. However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically remove, IGFBP-3 binding was only minimally affected. These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting receptor binding of IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of IGFBP-3. Additionally, the role of IGFBP-3 in the antiproliferative effects of transforming growth factor (TGF)-beta and retinoic acid (RA) is supported by our observations that: (1) inhibition of IGFBP-3 gene expression using an IGFNBP-3 antisense oligodeoxynucleotide not only blocks TGF-beta and RA simulation of IGFBP-3 production by up to 90%m but also inhibits their antiproliferative effects by 40-60%; and (2) treatment with IGF-II and IGF-II analogs diminish TGF-beta effects by blocking TGF-beta induced binding of IGFBP-3 to the cell surface. Taken together, our results support the hypothesis that IGFBP-3 is an important antiproliferative factor in human breast cancer, acting in an IGF-independent manner in addition to its ability to modulate the binding of IGF peptides to IGF receptors.

Topics: Breast Neoplasms; Cell Division; Endopeptidases; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Models, Biological; Oligonucleotides, Antisense; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

We propose that the secreted protein pleiotrophin (PTN) is a major factor in the malignant progression of breast cancer. This hypothesis is based on the growth-stimulatory effects of PTN on cells in vitro and in vivo and on its high levels of expression in 60% of tumor samples from breast cancer patients. The stimulation of proliferation and tube formation of endothelial cells by PTN suggests that it can serve as an angiogenesis factor during tumor growth. We hypothesize that PTN has the potential to support growth of breast cancer at its primary site and to enhance the ability of tumor cells to metastasize. Furthermore, we suggest that specific endocrine signals interact to regulate the expression of PTN in vitro and in vivo. Finally, we propose that understanding the functions of PTN and its hormonal regulation can lead to the development of novel therapeutic strategies for breast cancer.

Topics: Breast Neoplasms; Carrier Proteins; Cell Division; Cytokines; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tretinoin; Tumor Cells, Cultured

In this review I summarize the experimental data in favor of the notion that control of epidermal growth factor (EGF) receptor (R) and/or c-erbB-2 protooncogene expression by specific autocrine growth factors and certain classical endocrine hormones serves as a transducer of extracellular signals that ultimately lead to growth responses in breast carcinoma cells. I summarize some new results on the role of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and TGF beta in the control of EGF-R protooncogene expression in human breast carcinoma cells. Furthermore, the data embracing the hypothesis that the growth actions of hormone receptors that are homologous to the v-erbA oncogene (estrogens, progesterone, thyroid hormones, retinoic acid, and vitamin D) are mediated, in part, by modulating EGF-R and/or c-erbB-2 protooncogene transcription are reviewed. Finally, I develop the theme that cooperation of certain c-erb-A-related, c-erbB-2 and/or EGF-R gene products contribute to the uncontrolled growth of human mammary carcinoma cells. From the evidence reviewed, one can infer that elucidation of the molecular control of EGF-R/c-erbB-2 gene expression by c-erbA-related gene products may lead to both a better understanding of breast carcinogenesis and a new therapeutic approach directed at controlling the transcriptional responses of EGF-R/c-erbB-2 genes.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cholecalciferol; Epidermal Growth Factor; ErbB Receptors; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Hydrocortisone; Progesterone; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tretinoin; Triiodothyronine

Topics: Adjuvants, Immunologic; Animals; Breast Neoplasms; Carcinogens; Cricetinae; Diterpenes; Female; Humans; Keratoacanthoma; Lung Neoplasms; Male; Mice; Neoplasms; Prostatic Neoplasms; Rats; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

Topics: Anthraquinones; Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Breast Neoplasms; Cisplatin; Dose-Response Relationship, Drug; Female; Genetic Techniques; Hodgkin Disease; Hormones; Humans; Immunotherapy; Interferons; Male; Mechlorethamine; Methotrexate; Mitoxantrone; Neoplasm Metastasis; Neoplasms; Neoplasms, Germ Cell and Embryonal; Nitrosourea Compounds; Osteosarcoma; Prednisone; Procarbazine; Prognosis; Receptors, Cell Surface; Tretinoin; Vincristine

Topics: Breast Neoplasms; Etretinate; Humans; Isomerism; Isotretinoin; Skin Diseases; Skin Neoplasms; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; 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Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

Topics: Aldehyde Oxidoreductases; Animals; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Signal Transduction; Tretinoin

We investigated a combination therapy with weekly paclitaxel and all trans-retinoic acid (ATRA) for tolerability, response to treatment, time to progression and survival in previously treated patients with metastatic or recurrent breast cancer. Our rationale was based on preclinical studies demonstrating potentiation of the cytotoxic effects of taxanes and induction of differentiation by ATRA.. Seventeen patients with previously treated metastatic or recurrent breast cancer were enrolled to a regimen of all-trans retinoic acid (Vesanoid, tretinoin, Hoffman-La Roche, Inc.) 45 mg/m(2) PO daily for 4 days starting 2 days before a 1 h treatment with paclitaxel (Taxol, Bristol-Myers Squibb, Plainsboro, NJ) 80 mg/m(2) IV administered weekly for 3 weeks, repeated in 28 day cycles until disease progression or until no longer tolerated. Patients were evaluated for toxicity, response, time to progression and survival. Patients were primarily African American and Latino, representative of the population served by our Cancer Center.. The regimen was relatively well tolerated. There were nine grade 3 and one grade 4 toxic events. We administered 162 treatment cycles with a mean of 7.5 per patient (range 1-22, median 5). Three patients had a partial response (17.6%) and ten patients had stable disease (58.8%), with an overall clinical benefit of 76.4%. Median time to progression was 6.0 months (range 1-21, mean 7.7 months). Fourteen evaluable patients had a median survival of 16 months (range 1-68 months, mean 25.2 months).. The data suggest this is a well tolerated regimen with modest response rates but with time to progression and survival rates similar to those reported for paclitaxel alone and relatively high rates of stable disease in this sample of patients.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Black or African American; Breast Neoplasms; Disease Progression; Female; Hispanic or Latino; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Paclitaxel; Pilot Projects; Survival Rate; Treatment Outcome; Tretinoin

Failure to eradicate all cancer stem cells, lymphocytopenia, and high levels of vascular endothelial growth factor (VEGF) may explain the limited efficacy of high dose-chemotherapy (HDCT) with peripheral progenitor cell transplantation (PBPCT) in high-risk early breast cancer with more than 10 axillary nodes (HRBC).. With the aim of increasing patient's lymphocyte count and reducing VEGF, wich could translate into an improved immune function and a better clinical outcome, patients with HRBC, received HDCT, PBPCT and immunotherapy with interleukin-2 (IL-2) and 13-cis retinoic acid (RA).. A total of 30 HRBC patients were entered into the study. Grade 4 hematological toxicity was universal, while major adverse effects of IL-2 were fever, rash and autoimmune reactions. After a median follow-up of 61 months, immune function improved with a statistically significant increase of lymphocyte count and a decrease in VEGF levels. This translated into an unexpected 5-year relapse-free and overall survival rates of 76% and 85%, respectively.. These data show that IL-2 and RA administration after HDCT and PBPCT is feasible and, as well as giving a statistically significant improvement in lymphocyte count and a decrease of VEGF, also seems to improve the expected clinical outcome.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Ductal, Breast; CD4-CD8 Ratio; Combined Modality Therapy; Cyclophosphamide; Epirubicin; Female; Fluorouracil; Hematopoietic Stem Cell Transplantation; Humans; Interleukin-2; Killer Cells, Natural; Methotrexate; Middle Aged; Neoplasm Staging; Prospective Studies; Treatment Outcome; Tretinoin

Maintenance chemotherapy provides only a modest survival advantage in metastatic breast cancer (MBC). We have previously shown that a maintenance immunotherapy (MI) regimen based on low-dose interleukin-2 (IL-2) and 13-cis retinoic acid (RA) improved the lymphocyte and natural killer cell (NK) counts, and CD4+/CD8+ ratio in patients with a clinical benefit from chemotherapy. With the aim of improving progression-free survival (PFS), 100 consecutive MBC patients with a clinical benefit from chemotherapy were treated with an MI. Patients with MBC were eligible if they had no evidence of progression after 6-8 courses of epirubicin-paclitaxel induction chemotherapy. Treatment consisted of low-dose IL-2 and oral RA given until progression. The primary endpoint was progression-free survival (PFS); secondary endpoints were toxicity, overall survival (OS), and changes in immunological parameters. From 04/1997 to 04/2002, 100 patients with MBC were enrolled. After a median follow-up of 49 months, median PFS and OS were 37.1 and 57.5 months, respectively. No WHO grade 3 or 4 toxicity was observed; grade 2 cutaneous toxicity and autoimmune reactions occurred in 19 and 16% of patients, respectively. A sustained improvement in lymphocytes, NKs, and in the CD4+/CD8+ ratio was observed, with respect to baseline values. In conclusion, MI with IL-2 and RA in MBC patients who do not progress after 6-8 courses of chemotherapy is well-tolerated, improves lymphocyte, NK, CD4+/CD8+ ratio, and appears to delay disease recurrence. A randomized trial is warranted.

Topics: Adult; Aged; Breast Neoplasms; Disease-Free Survival; Female; Humans; Immunotherapy; Interleukin-2; Kaplan-Meier Estimate; Killer Cells, Natural; Lymphocyte Count; Lymphocytes; Middle Aged; Tretinoin

To evaluate whether all-trans-retinoic acid (ATRA) is able to modulate the hemostatic system in patients with solid tumors, we studied patients with locally advanced breast cancer who were enrolled in a Phase Ib study of ATRA +/- Tamoxifen (Tam). In this study, two groups of 15 patients/each were treated for 21 days before operation with ATRA at three doses (15, 45, or 75 mg/m(2)/day on alternate days) given alone (group 1) or in combination with Tam (group 2). One additional group received Tam alone. Plasma samples were evaluated for hypercoagulation markers (FVIIa, F1+2, TAT, D-dimer), fibrinolysis proteins (t-PA, PAI-1), and coagulation inhibitors (protein C, AT). At baseline, cancer patients had FVIIa, F1+2, TAT, and PAI-1 significantly greater than control subjects. During treatment, in the patients given ATRA alone, hypercoagulation markers appeared unmodified. Instead, subjects given Tam alone had a significant elevation of FVIIa, F1+2, and TAT versus baseline. However, in the ATRA + Tam groups, hypercoagulation markers were decreased compared with Tam alone. These results suggest that in selected conditions, pre-operative ATRA may modulate the hypercoagulable state of breast cancer patients.

Topics: Aged; Antineoplastic Agents; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Breast Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Middle Aged; Prospective Studies; Tamoxifen; Thrombophilia; Tissue Plasminogen Activator; Tretinoin

To determine the overall and dose-limiting toxicities (DLTs) of alitretinoin (9-cis-retinoic acid) in combination with tamoxifen and the pharmacokinetics of alitretinoin alone and when combined with tamoxifen in patients with metastatic breast cancer. The effect of tamoxifen and alitretinoin on MIB-1, a marker of proliferation, in unaffected breast tissue was explored.. Eligible patients had metastatic breast cancer. Previous tamoxifen therapy was allowed. Planned dose levels for alitretinoin ranged from 50 to 140 mg/m2/d with 20 mg/d tamoxifen in all patients after 4 weeks of alitretinoin as a single agent. Plasma concentrations of alitretinoin and retinol were measured at baseline and after 1, 2, and 3 months. Breast core biopsies were obtained at baseline and after 2 months of therapy.. Twelve patients with metastatic breast cancer received a total of 86 cycles of therapy. At 90 mg/m2/d, three of five patients experienced a DLT: grade 3 headache, grade 3 hypercalcemia, and grade 3 noncardiogenic pulmonary edema. At 70 mg/m2/d, one of six patients experienced a DLT (headache), and this level was considered the maximal tolerated dose in this study. Three toxicities occurred that had not been reported previously with alitretinoin: an asymptomatic delay in dark adaptation, a marked decrease in high-density lipoprotein cholesterol, and the occurrence of enthesopathy. Two of the nine assessable patients had a durable clinical response: one partial response and stable disease for 18 months and one complete response in continuous remission for 48+ months. Both responding patients were estrogen receptor-positive and had had previous tamoxifen therapy. There was a high degree of interpatient variability of plasma alitretinoin concentrations, although a significant decline in alitretinoin plasma levels over time was observed. MIB-1 scores declined in four of the eight paired breast specimens obtained.. The combination of tamoxifen and alitretinoin is well tolerated and has antitumor activity in metastatic breast cancer. The recommended phase II dose is 70 mg/m2/d with 20 mg/d tamoxifen.

Topics: Adult; Aged; Alitretinoin; Antigens, Nuclear; Antineoplastic Agents; Area Under Curve; Biomarkers; Breast Neoplasms; Cholesterol; Chromatography, High Pressure Liquid; Female; Humans; Ki-67 Antigen; Middle Aged; Nuclear Proteins; Tamoxifen; Tretinoin

High insulin-like growth factor-I (IGF-I) levels are associated with an increased risk of breast cancer in premenopausal women. Because the synthetic retinoid fenretinide showed a beneficial effect on second breast cancers in premenopausal women in a Phase III trial, we studied its long-term effects on IGF-I levels. We measured, at yearly intervals for up to 5 years, the circulating levels of IGF-I, IGF binding protein (BP)-3, and their molar ratio in 60 subjects < or = 50 years of age and 60 subjects > 50 years of age allocated either to fenretinide or no treatment. In women < or = 50 years of age, measurements of IGF-II, IGFBP-1, and IGFBP-2 were also performed. The associations between biomarkers and drug or metabolite plasma concentrations were also investigated. All biomarkers were relatively stable over 5 years in the control group. Compared with controls and after adjustment for baseline, treatment with fenretinide for 1 year induced the following changes: IGF-I, -13% [95% confidence interval (CI), -25 to 1%] in women < or = 50 years of age and -3% (95% CI, -16 to 13%) in women > 50 years of age; IGFBP-3, -4% (95% CI, -12 to 6%) in both age groups; IGF-I:IGFBP-3 molar ratio, -11% (95% CI, -22 to 1%) in women < or = 50 years of age and 1% (95% CI, -11 to 16%) in women > 50 years of age. These effects were apparently maintained for up to 5 years, although fewer samples were available as time progressed. No change in other IGF components was observed. Drug and metabolite concentrations were negatively correlated with IGF-I and IGF-I:IGFBP-3 molar ratio in women < or = 50 years of age. Fenretinide induces a moderate decline of IGF-I levels in women < or = 50 years of age. The association between IGF-I change and the reduction of second breast cancers in premenopausal women warrants further study.

Topics: Adult; Aged; Analysis of Variance; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Drug Administration Schedule; Female; Fenretinide; Follow-Up Studies; Humans; Insulin-Like Growth Factor I; Long-Term Care; Middle Aged; Neoplasm Staging; Reference Values; Treatment Outcome; Tretinoin

There are strong data showing that increased breast cancer risk is associated with increased mammographic density. Tamoxifen has been shown to decrease the risk of invasive breast cancer and decrease breast density. We sought to demonstrate and calculate the extent of change in mammographic density in women who have taken tamoxifen for up to 2 years. We evaluated mammograms from 28 high-risk women who were taking tamoxifen. Four different methods of evaluation were used: (a) two qualitative methods (Wolfe criteria and the American College of Radiology Breast Imaging and Reporting Data System criteria); (b) one semiquantitative method (mammograms were assigned one of five semiquantitative scores by visual inspection); and (c) one quantitative method (computer-aided calculation of fibroglandular area from digitized mammograms). The Wolfe criteria showed a 0.03 category decrease per year (P = 0.50). The American College of Radiology Breast Imaging and Reporting Data System criteria showed a 0.1 category decrease per year (P = 0.12). Semiquantitative criteria showed a 0.2 category decrease per year (P = 0.039). Digitized scores showed a 4.3% decrease per year (P = 0.0007). In conclusion, tamoxifen causes a decrease in mammographic density with use, an effect that is better quantitated with semiquantitative criteria or digitized images. Density change might become useful as a surrogate end point for the effect of tamoxifen and other chemopreventive measures, although our data do not predict an individual's degree of risk reduction.

Topics: Adult; Age Factors; Aged; Anticarcinogenic Agents; Antineoplastic Agents; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Feasibility Studies; Female; Humans; Mammography; Middle Aged; Pilot Projects; Postmenopause; Radiographic Image Enhancement; Reproducibility of Results; Selective Estrogen Receptor Modulators; Tamoxifen; Tretinoin

In addition to suppressing breast cancer cell growth, retinoids potentiate growth inhibition in human breast cancer when tested in vitro and in vivo with tamoxifen and/or interferon. The purpose of this study was to ascertain the biologic effects of all-trans-retinoic acid (ATRA) administered alone and with tamoxifen +/- interferon and to identify the relationship between ATRA plasma concentrations and optimal biological dose (the lowest dose that produces a biological response). Three consecutive groups of 15 patients with locally advanced operable breast cancer were treated, in accordance with good clinical practice (GCP) requirements, with ATRA at 3 dose levels alone or with tamoxifen +/- alpha-interferon 2a at flat doses. After 3 weeks, the tumors were surgically removed. Biological parameters measured at the beginning (in biopsy tissue) and end (in surgical tissue) of the study were compared. The optimal biological dose for ATRA was 15 mg/m2/day. Treatments influenced tumor grade but not cell cycle kinetics (G0-G1 phase) or proliferation (Ki67 levels). ATRA induced progesterone receptors independent of dose level and co-administered drugs, but did not induce estrogen receptors when administered alone. Retinoic acid receptor (RAR)-alpha was not affected by treatment and RAR-alpha was moderately influenced whereas RAR-beta (concomitantly with transforming growth factor-beta) was induced in 33% of patients by ATRA alone. ATRA pharmacokinetics were dose- and time-dependent. Neither the ATRA + tamoxifen nor the ATRA + tamoxifen + interferon combinations potentiated the ATRA-induced biological changes. Future studies evaluating the role of RAR-beta as a biological marker of retinoid activity are warranted.

Topics: Aged; Aneuploidy; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Bone Marrow Diseases; Breast Neoplasms; Carcinoma; Drug Administration Schedule; Drug Interactions; Female; Follow-Up Studies; Headache; Humans; Hypercholesterolemia; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Mastectomy; Middle Aged; Neoplasm Proteins; Receptors, Retinoic Acid; Receptors, Steroid; Recombinant Proteins; Safety; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome; Tretinoin

Surrogate end point biomarkers for risk assessment and efficacy of potential chemopreventive agents are needed to improve the efficiency and reduce the cost of chemoprevention trials. It is imperative to develop the best clinical breast model for translational surrogate end point biomarker studies, especially with respect to accrual feasibility. We have initiated a prospective study to develop biomarkers for tamoxifen and N-[4-hydroxyphenyl] retinamide by administering either a placebo or both drugs for 2-4 weeks to women with ductal carcinoma in situ or early invasive cancers in the interval between the initial diagnostic core biopsy and definitive surgery. The principle end point is pretreatment versus posttreatment tumor levels of Ki-67; a number of other exploratory markers will also be examined. The planned target sample size is 100 patients. Between February 1997 and February 2000, 4514 women who had either an abnormal mammogram or a diagnosed breast cancer were screened for the study. Of these 4514 screened patients, 52 (1%) were registered on the study. Major factors of nonparticipation in the remaining 4462 women were as follows: (a) no evidence of malignancy (2081 patients; 46%); (b) ineligible per protocol criteria (575 patients; 13%); (c) preoperative chemotherapy/tamoxifen (520 patients; 11%); (d) surgery scheduling conflict (360 patients; 8%); (e) outside needle biopsy (221 patients; 5%); (f) no residual disease after excisional biopsy (345 patients; 8%); and (g) second opinion only (123 patients; 3%). Other nonparticipation factors included fine needle aspiration only, refusal, tumor size > 2 cm, and estrogen replacement therapy (35 patients each; 2% each). The protocol was amended in midstudy to allow outside needle biopsy, tumor > 2 cm, and estrogen replacement therapy. Accrual to biomarker (nontherapeutic) protocols with delay in definitive cancer surgery is challenging but feasible. Although some accrual problems remain, we have nonetheless succeeded in recruiting 50% of our target sample size in a 3-year period.

Topics: Adult; Aged; Anticarcinogenic Agents; Antineoplastic Agents; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Middle Aged; Patient Selection; Prospective Studies; Research Design; Risk Assessment; Sensitivity and Specificity; Tamoxifen; Tretinoin

Because tamoxifen and all-trans-retinoic acid (ATRA) have additive antitumor effects in preclinical systems, we performed a Phase I/II clinical trial of this combination in patients with advanced breast cancer. Patients with potentially hormone-responsive advanced breast cancer were enrolled. All received 20 mg of tamoxifen by mouth daily. Consecutive cohorts of 3-6 patients were treated on odd-numbered weeks with ATRA at doses of 70, 110, 150, 190, or 230 mg/m2/day. Twenty-six patients were entered in this trial; 25 were evaluable. A dose of 230 mg/m2 ATRA produced unacceptable headache and dermatological toxicity, but doses < or = 190 mg/m2 were tolerable. Two of 7 patients with measurable disease responded. Seven of 18 patients with evaluable, nonmeasurable disease achieved disease stability for more than 6 months. Plasma AUCs on day 1 of successive weeks of treatment were stable over time. A nonsignificant decrease in serum insulin-like growth factor I levels was noted during treatment, but this trend was similar to that observed in three "control" patients treated with tamoxifen alone. When given with daily tamoxifen, the maximum tolerated dose of ATRA that could be given on alternate weeks was 190 mg/m2/day. This schedule of ATRA resulted in repeated periods of exposure to potentially therapeutic concentrations of ATRA. Declines in the serum insulin-like growth factor I concentrations observed in patients treated with tamoxifen and ATRA were similar to those observed in patients treated with tamoxifen alone. Objective responses were observed, some in patients who had previously progressed while receiving tamoxifen, suggesting that further studies would be of interest.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Breast Neoplasms; Drug Administration Schedule; Female; Humans; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Tamoxifen; Tretinoin

The purpose of this trial was to evaluate tumor cytoreduction by all-trans retinoic acid (ATRA) in patients with metastatic breast cancer and to characterize the initial pharmacokinetics of this agent.. The study was a single institution, phase II study. The treatment regimen consisted of ATRA administered orally at a dose of 50 mg/m2 three times a day for 14 consecutive day of a 21-day cycle. Cycles were repeated until disease progression, unacceptable toxicity or patient withdrawal. Plasma samples were obtained following the first dose of ATRA for pharmacokinetic analysis.. A total of 17 patients with metastatic breast cancer were enrolled in the study, and 14 completed at least one cycle of therapy and were evaluable for response. One patient achieved a partial response in soft tissue of 4 months duration. Three patients had stable disease for 4, 2, and 2 months duration. The remainder had progressive disease. ATRA was reasonably well tolerated. Pharmacokinetic analysis revealed a high degree of interpatient variability in systemic exposure following the initial dose of ATRA.. We conclude, that in the dose and schedule tested, ATRA does not have significant activity in patients with hormone-refractory, metastatic breast cancer. Future studies should focus on more intensive investigation of those individuals with very high or low ATRA initial systemic exposure in the hope of expanding our understanding of ATRA's clinical pharmacology, ultimately leading to improved efficacy.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Area Under Curve; Breast Neoplasms; Female; Humans; Middle Aged; Treatment Outcome; Tretinoin

N-(4-hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid which reduces the incidence of experimental tumours in animals and has been chosen for its weak toxicity to be tested as a chemopreventive agent in humans. The mechanism of antineoplastic action is still unknown but a possible immunoenhancing effect may be postulated. We investigated the NK activity of PBMC from a group of women treated with 4-HPR as a part of a large scale randomised phase III trial on chemoprevention of contralateral disease in mastectomised women. After 180 days of treatment the NK activity was augmented 1.73 times as compared to that of patients given a placebo. The NK activity of PBMC from 4-HPR treated women is maximised, being higher than the basal and even the rIL-2 or alfa-rIFN stimulated activity of controls. For this reason in the majority of cases it cannot be further augmented by incubation with either rIL-2 or alfa-rIFN in vitro. The increased NK activity of 4-HPR treated women is not due to an enhanced production of endogenous IL-2, because PBMC cultures from patients treated with 4-HPR or placebo, incubated in vitro with a panel of different stimulators (recall antigens, PHA, allogeneic and xenogeneic cells) produce similar amounts of IL-2. The functional activity, but not the number of NK cells is increased in 4-HPR treated women. The mechanism by which 4-HPR stimulates NK activity is not a function of direct action on NK cells. Indeed incubation of PBMC from blood donors with 4-HPR or its major metabolite N-(4-methoxyphenyl) retinamide (4-MPR) does not modify their natural cytotoxicity.

Topics: Adult; Aged; Breast Neoplasms; Cytokines; Cytotoxicity, Immunologic; Female; Fenretinide; Humans; Immunophenotyping; Interleukin-2; Killer Cells, Natural; Leukocytes, Mononuclear; Middle Aged; Tretinoin; Tumor Cells, Cultured

Monitoring of fenretinide (4HPR) levels, kinetics, and effects on retinal was performed in patients who participated in a phase I trial and who continued to be treated for 5 years as phase III trial patients. Accumulation of 4HPR in the breast was also assessed.. Plasma concentrations of 4HPR, of its main metabolite N-(4-methoxyphenyl)retinamide (4MPR), and of retinol were assayed by high-performance liquid chromatography (HPLC) in breast cancer patients treated orally with 4HPR 200 mg/d for 5 years with a 3-day drug interruption at the end of each month.. 4HPR, at 200 mg/d, resulted in average 4HPR plasma levels of approximately 1 mumol/L, which remained steady and caused steady retinol level reduction; 4MPR levels, similar to those of 4HPR, slightly but significantly increased during the first 35 months, but at 5 years they were similar to those at 5 months. During daily treatment, baseline retinol concentrations were reduced by 71%; after a 3-day drug interruption, all patients recovered and the mean reduction was 38%. After discontinuation of 5-year treatment, 4HPR and 4MPR half-lives (t1/2 beta) were 27 and 54 hours, respectively, similar to those reported after 28 daily treatments. After 6 and 12 months, the concentrations of 4HPR were at the limit of detectability (0.01 mumol/L), whereas those of 4MPR were five times higher. Baseline retinol concentrations were already recovered after 1 month. Accumulation of this retinoid in the breast was evidenced by concentrations of 4HPR and 4MPR in nipple discharge and in breast biopsies that were 10 and 20 times higher, respectively, than those found in plasma.. 4HPR, at 200 mg/d for 5 years, resulted in constant drug plasma levels and constant retinol level reduction. After treatment interruption, 4HPR plasma concentrations decreased at the limit of detectability at 6 months and baseline retinol plasma concentrations were recovered after 1 month.

Topics: Adult; Aged; Analysis of Variance; Breast Neoplasms; Female; Fenretinide; Humans; Male; Middle Aged; Tretinoin; Vitamin A

Fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR] is an effective agent for the inhibition of N-nitroso-N-methylurea-induced breast cancer in rats. This compound has been studied extensively and proved to be safer and less teratogenic than many other retinoids. A major characteristic of 4-HPR is its ability to concentrate in the granular and fat tissue of the breast instead of in the liver. Between January and June 1986, we carried out a phase I study on 101 patients divided into four randomized groups receiving placebo and 100, 200, and 300 mg/day of 4-HPR. Patients received the drug for 6 months without any major toxic effect. This finding was confirmed by another 6-month study in which patients received a common dose of 200 mg/day. In March 1987, a phase III study was started to evaluate the effectiveness of 4-HPR in preventing contralateral primary tumors in women who had already been treated for breast cancer. If 4-HPR succeeds in preventing second primaries in breast cancer patients, it may be useful for a wider group of subjects at high risk for breast cancer. This randomized study was designed with two arms: an intervention group versus a group receiving no treatment. Patients in the intervention group will be treated with 200 mg/day 4-HPR for 5 years. Patients in the control group will not be treated. A further 2 years of follow-up is planned for both groups. Currently, 2450 patients have been recruited. We expect a total accrual of 3500 patients by the end of 1992.

Topics: Adult; Aged; Breast Neoplasms; Drug Evaluation; Female; Fenretinide; Follow-Up Studies; Humans; Middle Aged; Tretinoin

A group of 53 patients initially participating in a phase I trial with the synthetic retinoid fenretinide was assessed for the long-term tolerability of this compound. The patients were evaluated after 42 months of drug intake at a dose of 200 mg/day, including a 3-day drug interruption at the end of each month, by the following examinations: a dermatological visit; an ophthalmological evaluation including an ophthalmological questionnaire and an electroretinogram (ERG); a study on blood chemistry and plasma retinol levels; a study on bone densities and on skeletal X-rays; and finally a psychological evaluation including various tests for anxiety, depression and overall mood. The results show that prolonged administration of fenretinide is well tolerated. No acute nor severe toxicity was observed and thus this compound can be considered a good candidate for chemoprevention trials in a variety of patient populations.

Topics: Affective Symptoms; Bone Density; Breast Neoplasms; Drug Eruptions; Drug Evaluation; Electroretinography; Female; Fenretinide; Follow-Up Studies; Humans; Tretinoin; Vision, Ocular; Vitamin A

Retinoids, the natural and synthetic analogs of vitamin A, are growth-inhibiting and differentiation-inducing agents and show clinical promise as chemopreventive and antineoplastic agents. Fenretinide, a new synthetic retinoid, has antitumor activity in certain in vitro and in vivo model systems and was relatively nontoxic in phase I trials. Based on these data, we designed a phase II study of Fenretinide involving 31 patients with advanced breast cancer [15] and melanoma [16], two cancers shown to be responsive to this agent in preclinical models. Fenretinide was inactive in patients with advanced disease. Toxicity was mild, and reversible. Mucocutaneous side effects occurred in 16 (52%) patients. Nyctalopia developed in three patients one of whom developed decreased B-wave amplitude of the scotopic electroretinogram. The minimal toxicity and significant activity in preclinical studies make this an attractive agent for future breast cancer chemoprevention studies.

Topics: Adult; Aged; Breast Neoplasms; Drug Evaluation; Female; Fenretinide; Humans; Male; Melanoma; Middle Aged; Tretinoin

Fenretinide, N-(4-hydroxyphenyl)retinamide (HPR), is a synthetic retinoid which has been proven effective in inducing cell differentiation and in inhibiting carcinogen induced mammary tumors in rodents. Because of its efficacy and low toxicity in animals, HPR has been proposed for chemopreventive evaluation in humans. Thus, a randomized trial has been conducted to select a dose which can be administered over a lengthy period of time and with acceptable toxicity. The retinoid was administered orally to patients already operated on for breast cancer in daily doses of 100, 200 and 300 mg for 6 months and subsequently at 200 mg for another 6 months. No acute toxicity was found. Dermatological toxicity was minimal and no liver function abnormalities were observed. Nausea and headaches were infrequent and always mild. Menstrual irregularities were recorded with similar frequency in the treatment and placebo groups and appeared to be more age related than drug dependent. After 6 months of treatment one of 25 patients taking 300 mg HPR daily experienced impaired night vision, confirmed by the electroretinogram, and resolved by interruption of treatment. Because the 300 mg daily dose is possibly associated with impaired dark adaptation, the recommended dose for chemoprevention trials of HPR is 200 mg per day.

Topics: Adult; Aged; Breast Neoplasms; Drug Eruptions; Drug Evaluation; Drug Tolerance; Female; Fenretinide; Humans; Middle Aged; Pruritus; Random Allocation; Tretinoin

Fenretinide (HPR) is a synthetic retinoid which has been shown to cause a reduction in the incidence of carcinogen-induced epithelial tumors in experimental animals, and it has been chosen to be tested as a chemopreventive agent in humans. A study on plasma concentrations of HPR, of its metabolite N-(4-methoxyphenyl)retinamide (MPR), and on its effects on endogenous retinol was performed in groups of 14 to 18 breast cancer patients who received p.o. daily doses of placebo or 100, 200, and 300 mg of HPR for 6 mo and subsequently 200 mg for an additional 6 mo. After the first 5 mo of treatment, there was a linear relationship between doses of HPR administered and HPR, MPR, and retinol levels. HPR and MPR levels increased with the increase in dose, whereas retinol levels decreased, and the reduction was statistically significant compared with the placebo group after all the doses tested. Plasma retinol binding proteins (RBP) decreased proportionally to retinol (r = 0.96). The effect of HPR on retinol and RBP occurred early, since retinol and RBP levels had already been decreased, compared with the initial levels, by 38% and 26%, respectively, 24 h after a 200-mg HPR dose. After 12 mo of treatment, in patients treated with 200 mg daily, the dose chosen for a chemopreventive trial, HPR and retinol levels were similar to those found at 5 mo, suggesting no drug accumulation and no further retinol reduction, whereas MPR levels were higher. Following interruption of treatment, as HPR decreased, retinol increased with a linear relationship between log levels (r = 0.78); after about 50 days, HPR was present in trace amounts, and retinol levels were in the range of those of the placebo group. These data show that HPR treatment lowers retinol and RBP plasma concentrations. This effect is related to HPR levels and is reversible on cessation of HPR administration.

Topics: Antineoplastic Agents; Breast Neoplasms; Drug Administration Schedule; Drug Evaluation; Female; Fenretinide; Follow-Up Studies; Humans; Tretinoin; Vitamin A

One hundred and one patients participating in a phase I study with the synthetic retinoid 4-HPR (4-hydroxyphenyl retinamide) were evaluated. The study was set up by Veronesi et al. during 1986 at the National Cancer Institute of Milan. The patients were randomized into 4 groups of therapy: 25 in the placebo group, 25 in the group receiving a daily dose of 100 mg of HPR, 26 in the group receiving 200 mg/day of HPR, and 25 in the group receiving 300 mg/day of HPR. All patients were previously treated at our Institute for breast cancer. None had received adjuvant therapy, chemotherapy or hormone therapy. After 4-5 months from the beginning of treatment, all patients received a series of tests to evaluate anxiety, depression and sexual life. Moreover, during one the follow-up checkups after 4-5 months, the patients filled-out a self-scoring mood questionnaire. The results did not show any particular differences between the groups, although we found that the administered drug and experimental setting do not interfere with the psychologic state of the participating patients.

Topics: Adaptation, Psychological; Adult; Affect; Anxiety; Breast Neoplasms; Depression; Drug Evaluation; Female; Fenretinide; Humans; Middle Aged; Random Allocation; Sexual Behavior; Tretinoin

Topics: Breast Neoplasms; Clinical Trials as Topic; Female; Fenretinide; Hormones; Humans; Tretinoin

Breast cancer stem cells (BCSCs) are the culprit of triple-negative breast cancer invasiveness and are heterogeneous. It is recognized that the combination of chemotherapy and differentiation therapy for killing BCSCs and non-BCSCs simultaneously is a reliable strategy. In this study, an oil-in-water nanoemulsion was prepared by high-pressure homogenization with coencapsulation of all-trans retinoic acid (ATRA) and doxorubicin (DOX). The preparation process was simple, and the production was easy to scale up. The particle size of the nanoemulsion was 127.2 ± 2.0 nm. Cellular toxicity assay showed that the composite index of the ATRA and DOX was less than 1 and exhibited a fine combined effect.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Doxorubicin; Female; Humans; NIMA-Interacting Peptidylprolyl Isomerase; Tretinoin; Triple Negative Breast Neoplasms

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERβ1 and ERβ2 (isoforms of ERβ), the second ER isotype. At present, the impact of ERβ isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERβ1 or ERβ2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERβ1 and MCF7-ERβ2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERβ1 cells and cancer-promoting effects in MCF7-ERβ2 cells. Our data are favorable to ERβ1 being a marker of responsiveness and ERβ2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Resistance, Neoplasm; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptor Modulators; Female; Fulvestrant; Humans; Neoplasm Recurrence, Local; Protein Isoforms; Tamoxifen; Tretinoin

This paper expands the current state of knowledge on impact of retinoids on redox status of cytochrome c in cancers. Little is known how the expression of cytochromes may influence the development of cancers. We studied the effect of the redox status of the central iron ion in heme of cytochrome c. We determined the redox status of the iron ion in cytochrome c in mitochondria, cytoplasm, lipid droplets, and endoplasmic reticulum of the human breast cancer cells by Raman imaging. We incubated human breast adenocarcinoma cells (SK-BR-3) with retinoic acid, retinol and retinyl ester (palmitate) at concentration of 50 μM for 24 h. We recorded the Raman spectra and images of human breast cancer in vitro SK-BR-3 cells receiving redox stimuli by retinoic acid, retinol and retinyl ester (palmitate). The paper provides evidence that retinoic acid and retinol are pivotally important for mitochondrial energy homeostasis by controlling the redox status of cytochrome c in the electron transport chain controlling oxidative phosphorylation and apoptosis. We discussed the role of retinoids in metabolism and signaling of cancer cells. The paper provides experimental support for theoretical hypothesis how retinoic acid/retinol catalyse resonance energy transfer reactions and controls the activation/inactivation cycle of protein kinase PKCδ.

Topics: Breast Neoplasms; Cytochromes c; Endoplasmic Reticulum; Female; Humans; Iron; Oxidation-Reduction; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

Breast cancer is one of the most prevalent and deadly cancers among women worldwide. Recently, natural compounds have been widely used for the treatment of breast cancer. Present study evaluated antiproliferative and anti-metastasis activities of two natural compounds of dandelion and all-trans-retinoic acid (ATRA) in human MCF-7 and MDA-MB231 breast cancer cells. We also evaluated the expression of MMP-2, MMP-9, IL-1β, p53, NM23 and KAI1 genes. Data showed a clear additive cytotoxic effect in concentrations of 40 μM ATRA with 1.5 and 4 mg/ml of dandelion extract in MCF-7 and MDA-MB231 cells, respectively. In both cell lines, compared with the untreated cells, the expression levels of MMP-9 and IL-1β were significantly decreased while p53 and KAI1 expression levels were increased. Besides, MMP-2 and NM23 had different expressions in the two studied cell lines. In conclusion, dandelion/ATRA co-treatment, in addition to having strong cytotoxic effects, has putative effects on the expression of anti-metastatic genes in both breast cancer cells.

Topics: Breast Neoplasms; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Plant Extracts; Taraxacum; Tretinoin; Tumor Suppressor Protein p53

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduce

Topics: Breast Neoplasms; Cell Proliferation; Collagen; Epithelial Cells; Female; Gene Expression; Humans; Retinoids; Retinol-Binding Proteins, Cellular; Tretinoin; Tumor Microenvironment; Vitamin A

The combination therapy with different treatment modalities has been widely applied in the clinical applications of cancer treatment. However, it stills a considerable challenge to achieve co-delivery of different drugs because of distinct drug encapsulation mechanisms, low drug loading, and high excipient-related toxicity. Cancer stem cells (CSCs) are closely related to tumor metastasis and recurrence due to high chemoresistance. Herein, we report a stimuli-responsive and tumor-targeted small-molecule self-assembled nanodrug for the combination therapy against CSCs and normal cancer cells. The hydrophobic differentiation-inducing agent (all-trans retinoic acid, ATRA) and hydrophilic anticancer drug (irinotecan, IRI) constitute this amphiphilic nanodrug, which could self-assemble into stable nanoparticles and encapsulate the photothermal agent IR825. Upon cellular uptake, this nanodrug display good release profiles in response to acid and esterase microenvironments by ester linkage. The released drugs not only increase chemotherapy sensitivity by the differentiation of CSCs into non-CSCs, but also exhibit superior cytotoxicity in cancer cells. In addition, IR825 within this nanodrug enables in vivo fluorescence/photoacoustic (PA) imaging allowing for tracking drug distribution. Moreover, the DSPE-PEG-RGD-functionalized nanodrug displayed high tumor accumulation and good biocompatibility, enabling efficient inhibition of tumor growth and tumor metastasis in tumor-bearing mice.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Combined Modality Therapy; Female; Humans; Mice; Nanoparticles; Neoplastic Stem Cells; Tretinoin; Tumor Microenvironment

Developing chemotherapy with nanoparticle-based prodrugs provides promising strategies for improving the safety and delivery of anti-cancer drugs therapeutics and effective cancer treatment. Herein, we developed a pH-sensitive prodrug delivery system (All-Trans-Retinoic Acid (ATRA) grafted poly (β-amino esters) (PBAE) copolymers, ATRA-g-PBAE) for delivery of ATRA with some physicochemical and biological properties. The in vitro release of ATRA-g-PBAE prodrug nanoparticles (PNPs) was sustained-release and pH-sensitive. The cytotoxicity and uptake of different preparations in vitro were evaluated on MCF-7 cells at pH 7.4 and 5.5. The carrier PBAE had no cytotoxicity, and ATRA-g-PBAE PNPs could significantly inhibit cell growth at pH 5.5. MCF-7 cells treated with Cy5.5 grafted PBAE (Cy5.5-PBAE) showed stronger fluorescence signals at pH 5.5. Meanwhile, ATRA-g-PBAE PNPs entered the cell via a clathrin-mediated endocytic pathway. Subsequently, PBAE protonation facilitated the escape of PNPs from the lysosome and released the drug. ATRA-g-PBAE seems promising as a novel pH-sensitive prodrug to overcome the limitations of ATRA for breast cancer therapy.

Topics: Breast Neoplasms; Female; Humans; Hydrogen-Ion Concentration; Nanoparticles; Prodrugs; Tretinoin

All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.

Topics: Animals; Breast Neoplasms; Caspase 3; Female; Focal Adhesion Protein-Tyrosine Kinases; HeLa Cells; Humans; Mice; Retinoids; Tretinoin; Vitamin A

Topics: Anti-Bacterial Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; MCF-7 Cells; Quinolines; Receptor, Bradykinin B2

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Chitosan; Disease Models, Animal; Doxorubicin; Drug Liberation; Drug Synergism; Female; Folic Acid; Gene Knockdown Techniques; Humans; Mice; Micelles; Nanoparticle Drug Delivery System; Neoplastic Stem Cells; NIMA-Interacting Peptidylprolyl Isomerase; Tissue Distribution; Tretinoin

Tumour heterogeneity remains a major challenge in cancer therapy owing to the different susceptibility of cells to chemotherapy within a solid tumour. Cancer stem-like cells (CSCs), which reside in hypoxic tumour regions, are characterized by high tumourigenicity and chemoresistance and are often responsible for tumour progression and recurrence. Here we report a nanotherapeutic strategy to kill CSCs in tumours using nanoparticles that are co-loaded with the differentiation-inducing agent, all-trans retinoic acid, and the chemotherapeutic drug, camptothecin. All-trans retinoic acid is released under hypoxic conditions, leading to CSC differentiation in the hypoxic niche. In differentiating CSC, the reactive oxygen species levels increase, which then causes the release of camptothecin and subsequent cell death. This dual strategy enables controlled drug release in CSCs and reduces stemness-related drug resistance, enhancing the chemotherapeutic response. In breast tumour mouse models, treatment with the nanoparticles suppresses tumour growth and prevents post-surgical tumour relapse and metastasis.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Camptothecin; Cell Differentiation; Drug Delivery Systems; Drug Liberation; Drug Resistance, Neoplasm; Female; Humans; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Neoplastic Stem Cells; Rats, Wistar; Tissue Distribution; Tretinoin; Xenograft Model Antitumor Assays

Retinoic acid-induced 2 (RAI2) has been shown to be a putative suppressor of the early hematogenous dissemination of tumor cells to the bone marrow in breast cancer. Here, we investigated the associations of RAI2 mRNA and protein expression with clinicopathological factors and prognosis in breast cancer patients with long-term follow-up.. Invasive breast cancer tissues (n = 604) were analyzed for RAI2 mRNA expression. We examined the associations of clinicopathological factors with the expression levels of RAI2 mRNA in these samples. We also analyzed RAI2 protein expression by immunohistochemistry in invasive breast cancer tissues (n = 422).. We identified significant positive associations between low expression of RAI2 mRNA and shorter disease-free survival (DFS), breast-cancer-specific survival (BCSS), and overall survival (OS) in breast cancer patients. We also identified significant positive associations between negative for RAI2 protein expression and shorter DFS, BCSS, and OS in breast cancer patients. Low RAI2 mRNA and negative for RAI2 protein expression were positively associated with larger tumor size, higher tumor grade, and ERα-negativity. Multivariate analyses indicated that not only RAI2 mRNA but also RAI2 protein expression were independent risk factors for both DFS and BCSS in breast cancer patients. The median follow-up periods were 10.3 and 9.3 years for the RAI2 mRNA and protein expression analyses, respectively.. Our findings suggest that RAI2 has a role in the metastasis of breast cancer, and that RAI2 expression could be a promising candidate biomarker of prognosis in breast cancer patients.

Topics: Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Female; Humans; Intercellular Signaling Peptides and Proteins; Prognosis; Tretinoin

Retinoic acid (RA) binding proteins, CRABP1 and CRABP2, are molecular chaperones that mediate intracellular activity of RA, the key promoter of cell differentiation with tumor suppressor activity. One of the main functions of CRABP2 is delivery and transfer of RA to the nuclear receptors RAR/RXR, which leads to activation of the transcription of a wide range of retinoid-responsive genes. The functions of CRABP1 are less studied but are apparently associated with sequestration of RA in cytoplasm and limitation of its transcriptional activity, suggesting involvement of this protein in the development of RA resistance. The mechanisms regulating activity of CRABP1 are also poorly understood. Comparison of the CRABP1 level in tumor cell lines of various origins, performed for the first time here, showed absence of the CRABP1 protein in the cell lines of tumors considered to be RA-resistant, and pronounced production of this protein in the RA-sensitive cells. However, analysis carried out with a panel of breast cancer cell lines with different levels of RA-sensitivity showed that there was no correlation between the production of CRABP1 protein and the sensitivity of the cells to RA. At the same time, we found strong correlation between the expression of CRABP1 and CRABP2 proteins in all studied cell types, regardless of their origin and RA-sensitivity/resistance. Moreover, suppression of the CRABP1 level in both RA-sensitive and RA-resistant cells was shown in the cells with cells with knockdown of CRABP2 gene. The revealed CRABP2-dependent regulation of CRABP1 production is a new mechanism of the intracellular retinoic signaling system.

Topics: A549 Cells; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplasms; Receptors, Retinoic Acid; Signal Transduction; Tretinoin

Delivery of combination chemotherapeutic agents to the tumor via nanovesicles has the potential for superior tumor suppression and reduced toxicity. Herein, we prepare a block copolymer (mPH-RA) composed of methoxy-poly(ethylene glycol) (mPEG), b-poly(N-(2 hydroxypropyl) methacrylamide) (pHPMA), and all-trans retinoic acid (ATRA) by conjugating ATRA to the pre-formed copolymer, mPEG-b-pHPMA(mP-b-pH). Doxorubicin-loaded micelles, Dox@mP-b-pH, and Dox@mPH-RA were characterized by determining particle size, zeta potential, % DL, EE, Dox release, hemolysis study, and by DSC. The Dox@mPH-RA micelles (mPH-RA: Dox ratios of 10:0.5-2) displayed nano-size (36-45 nm), EE. 26-74%, and DL. 2.9-5.6%. Dox@mPH-RA micelles displayed the highest penetrability and cytotoxicity than free Dox and Dox@mP-b-pH micelles in breast cancer cell lines. Dox@mPH-RA exhibited the highest induction of apoptosis (94.1 ± 3%) than Dox (52.1 ± 4.5%), and Dox@mP-b-pH (81.7 ± 3%), and arrested cells in the highest population in G2 and S phase. Dox@mPH-RA increased the t

Topics: Acrylamides; Breast Neoplasms; Doxorubicin; Drug Carriers; Drug Therapy, Combination; Female; Humans; Hydrogen-Ion Concentration; Micelles; Polyethylene Glycols; Tretinoin

Liposomal drug delivery has become an established technology platform to deliver dual drugs to produce synergistic effects and reduce the adverse effects of traditional chemotherapy. Gambogic acid (GA) and retinoic acid (RA) are both effective anticancer components, but their low water-solubility (gambogic acid < 0.0050 mg/mL, retinoic acid 0.0048 < mg/mL) makes it difficult to load both drugs into the liposomes actively using the conventional method. We have successfully used solvent-assisted active loading technology (SALT) to load the insoluble drugs into the internal water phase via water-miscible organic solvent. Gambogic acid and retinoic acid co-encapsulated liposomes (weight ratio of GA to RA = 1:2, GRL) exhibited the strongest synergistic effect; combination index (CI) was 0.614 in 4T1 cells. Our studies demonstrated that GRL had uniform droplet size of about 130 nm, high stability, and controlled release behavior. GRL outperformed gambogic acid and retinoic acid solution (GRS) in pharmacokinetic profiles for a longer half-life and increased AUC. Comparing to GRS, GL, and RL, GRL showed increased cytotoxicity and apoptosis in 4T1 cells and showed the strongest anti-tumor ability in the in vivo anti-tumor efficacy. Overall, the SALT was a promising method to active loading poorly soluble drugs into liposomes, and the results showed GRL possessed a great potential for use in synergistic anticancer therapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Capsules; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Combinations; Drug Compounding; Drug Liberation; Female; Liposomes; Mice; Particle Size; Solvents; Tretinoin; Xanthones; Xenograft Model Antitumor Assays

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Epithelial-Mesenchymal Transition; Female; Humans; Organotin Compounds; Proteomics; Retinoid X Receptors; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Tretinoin; Trialkyltin Compounds; Vimentin

Pt-ATRA, a carboplatin analogue containing an all-trans retinoic acid (ATRA) derivative ligand, was synthesized via a click reaction. Upon cellular internalization, Pt-ATRA exhibits a dual function, releasing an active Pt(ii) moiety to induce cell apoptosis and ATRA to inhibit tumor metastasis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carboplatin; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Ligands; Mice; Molecular Structure; Neoplastic Stem Cells; Structure-Activity Relationship; Tretinoin

Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment.. PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR.. Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARβ expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression.. Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARβ receptor is required to ensure the effect on this process.

Topics: Animals; Breast; Breast Neoplasms; Cell Differentiation; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; MCF-7 Cells; Mice; Protein Kinase C-alpha; Receptors, Retinoic Acid; Retinoids; Signal Transduction; Tretinoin; Vitamin A

All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied.. We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and -resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data.. ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells.. The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.

Topics: Breast Neoplasms; Cardiolipins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Exome Sequencing; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lipidomics; Mass Spectrometry; Mitochondria; Oxidative Phosphorylation; Single-Cell Analysis; Tretinoin

Trialkyltins and triaryltins function as nuclear retinoid X receptors (RXR) agonists due to their affinity to the ligand-binding domain of RXR subtypes and function as transcriptional activators. We present the data on combined effects of all-trans retinoic acid (ATRA), retinoic acid receptor (RAR) ligand and tributyltin chloride or triphenyltin chloride (RXR ligands) on protein pattern in MDA-MB-231 cells. Proteomic strategies based on bottom-up method were applied in this study. The total cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. By employing PDQuest™ software, we identified more than 30 proteins differently affected by the above compounds. For further studies, we selected specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or to cellular processes as apoptosis, regulation of gene transcription or epithelial-mesenchymal transition (annexin 5, nucleoside diphosphate kinase B and vimentin). We have found that treatment of MDA-MB-231 cells with triorganotins reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin and nucleoside diphosphate kinase B. These results demonstrate that RXR/RAR heterodimer may act under this experimental design as permissive heterodimer allowing activation of RXR by triorganotins.

Topics: Breast Neoplasms; Humans; MCF-7 Cells; Organotin Compounds; Proteomics; Tandem Mass Spectrometry; Tretinoin

Retinoids are derived from vitamin A through a multi-step process. Within a target cell, retinoids regulate gene expression by activating the retinoid acid receptors (RAR) and retinoid x receptors (RXR), which are ligand-dependent transcription factors. Besides its therapeutic use in dermatological disorders, all-trans retinoic acid (ATRA) is successfully utilized to treat acute promyelocytic leukemia (APL) patients. The use of ATRA in APL patients is the first example of clinically useful differentiation therapy. Therapeutic strategies aiming at cancer cell differentiation have great potential for solid tumors, including breast cancer. The few clinical studies conducted with ATRA in breast cancer are rather disappointing. However, these studies did not take into account the heterogeneity of the disease and were conducted on unselected cohorts of patients.We recently showed that ATRA treatment of breast cancer cells induces autophagy, a highly conserved process aiming at degrading and recycling superfluous or harmful cellular components. In addition, autophagy inhibition significantly increases the therapeutic activity of ATRA. This finding is of fundamental importance, since autophagy has a dual role in cancer. Whereas autophagy may be a protective mechanism during the initial phases of cancer development, it may support cancer cell survival in already established tumors. Furthermore, autophagy can lower or enhance therapeutic efficiency, depending on the tumor type and the anticancer agent considered. Therefore, it is important to investigate the role of autophagy in the context of specific tumors and therapeutic approaches. Accurate autophagy studies are challenging given the dynamic nature of the process and the difficulty of measuring the rate of autophagosome degradation (autophagic flux). In this chapter, we provide protocols for a careful assessment of the autophagic flux in ATRA treated 2D and 3D breast cancer cultures.

Topics: Antineoplastic Agents; Autophagy; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Separation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Tretinoin

The NM23 gene is overexpressed in many hematological malignancies and its overexpression predicts poor treatment outcomes. NM23 overexpression is thought to suppress myeloid differentiation of leukemia cells, but the molecular mechanism is unknown. In breast cancer cells, the lysophosphatidic acid (LPA) receptor EDG2/lpa1 was downregulated by NM23-H1 overexpression, and this reciprocal expression pattern was associated with suppressed or induced cell motility/metastasis. Here, we examined the relationship between EDG2 and NM23 expression during myeloid differentiation of leukemia cells. NM23 expression decreased and EDG2 expression increased during all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60, NB4, and THP-1 leukemia cells. Moreover, this inverse correlation was more evident when myeloid differentiation was enhanced by ellagic acid, an inhibitor of NM23 activity. In contrast, there was no inverse correlation between EDG2 and NM23 expression during erythroid differentiation of HEL and K562 cells. ATRA plus LPA enhanced the motility of leukemia cells as well as breast cancer cells in an EDG2-dependent manner. These results suggest a common molecular mechanism between myeloid differentiation of leukemia cells and migration of breast cancer cells depending on NM23 and EDG2 expression levels.

Topics: Breast Neoplasms; Cell Differentiation; Down-Regulation; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; K562 Cells; Leukemia; Lysophospholipids; MCF-7 Cells; Myeloid Cells; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Receptors, Lysophosphatidic Acid; Tretinoin; U937 Cells; Up-Regulation

To build up a combined therapy strategy to address limitations of the enhanced permeability and retention (EPR) effect and improve the efficiency of tumor therapy.. A pH-sensitive nanocomplex for co-delivery of doxorubicin (DOX) and all-trans retinoic acid (ATRA) was developed based on nanodiamonds (DOX/ATRA-NDs) to enhance intracellular retention of drugs. Meanwhile, ultrasound was employed to enhance tumor vascular penetration of DOX-ATRA-NDs.. The distribution of DOX/ATRA-NDs in the tumor tissues increased threefold when ultrasound was applied at 1 MHz and 0.6 W/cm. DOX-ATRA-NDs demonstrate great potential in clinical applications.

Topics: Animals; Breast Neoplasms; Doxorubicin; Drug Carriers; Female; Hep G2 Cells; Humans; Liver Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Nanodiamonds; Tretinoin; Ultrasonic Waves

One of the major problems in breast cancer treatment is pharmacoresistance. Therefore, exploration of treatment alternatives is of clinical relevance. The present work focused on tumor cell-inhibiting effects of a combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and all trans retinoic acid (ATRA) in breast cancer cells.. Breast cancer cell lines (BT-20, BT-474, MDA-MB-231, MDA-MB-436, MDA-MB-453, MCF-7, SKBR3, T47D, ZR-75-1) and the mammary epithelial cell line MCF-10A were treated with TRAIL and ATRA alone and in combination. Cell viability was assessed via 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay, the potential of cell colony formation via clonogenic assay, cell death induction via cell-cycle analysis by fluorescence-activated cell sorting (FACS), terminal deoxynucleotidyltransferase-mediated UTP nick end labeling (TUNEL) assay and Cell death detection ELISA. TRAIL and ATRA evoked synergistic inhibition of breast cancer cell viability based on cytostatic and cytotoxic mechanisms. This correlated with augmented fragmentation of nuclear DNA, up-regulation of TRAIL receptor, down-regulation of cyclin D1 and enhancement of caspase activity. MCF-10A cells were merely slightly susceptible to TRAIL and ATRA.. The cytostatic and cytotoxic effects of the combination of TRAIL and ATRA are tumor cell-selective.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; DNA Fragmentation; Drug Resistance, Neoplasm; Drug Synergism; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Neoplasm Proteins; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tretinoin; Tumor Stem Cell Assay

Deciphering the molecular networks that discriminate organ-confined breast cancer from metastatic breast cancer may lead to the identification of critical biomarkers for breast cancer invasion and aggressiveness. Here metabolomics, a global study of metabolites, has been applied to explore the metabolic alterations that characterize breast cancer progression. We profiled a total of 693 metabolites across 87 serum samples related to breast cancer (46 clinically localized and 41 metastatic breast cancer) and 49 normal samples. These unbiased metabolomic profiles were able to distinguish normal individuals, clinically localized and metastatic breast cancer patients. 9-cis-Retinoic acid, an isomer of all-trans retinoic acid, was identified as a differential metabolite that significantly decreased during breast cancer progression to metastasis, and its levels were also reduced in urine samples from biopsy-positive breast cancer patients relative to biopsy-negative individuals and in invasive breast cancer cells relative to benign MCF-10A cells. The addition of exogenous 9-cis-retinoic acid to MDA-MB-231 cells and knockdown of aldehyde dehydrogenase 1 family member A1, a regulatory enzyme for 9-cis-retinoic acid, remarkably impaired cell invasion and migration, presumably through preventing the key regulator cofilin from activation and inhibiting MMP2 and MMP9 expression. Taken together, our study showed the potential inhibitory role for 9-cis-retinoic acid in breast cancer progression by attenuating cell invasion and migration.

Topics: Adult; Aged; Alitretinoin; Breast Neoplasms; Disease Progression; Female; Humans; Metabolomics; Middle Aged; Tretinoin

N-(4-hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid, less toxic than the parent all-trans retinoic acid (RA). Unlike RA, 4-HPR induces apoptosis in tumor cells. Because 4-HPR can hydrolyze to liberate RA, a potent human teratogen, the unhydrolyzable ketone analog of 4-HPR, 4-hydroxybenzylretinone (4-HBR) has been prepared and has been found to cause apoptosis in tumor cells and shrink carcinogen-induced rat mammary tumors as 4-HPR does. Herein, we examined the mechanism whereby 4-HPR and 4-HBR induce apoptosis and death in breast cancer cells.. Gene expression profiling was conducted in MCF-7 cells over a 1.5- to 6-h time course and changes were validated by quantitative polymerase chain reaction (qPCR). Growth arrest and DNA damage-inducible protein 153 (GADD153 or C/EBP homologous protein, CHOP) was knocked down and the effect on 4-HPR-induced cell death and gene expression was assessed. 4-HPR synergy with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL or Apo2 ligand) was also examined.. Drug treatment induced increased expression of endoplasmic reticulum (ER) stress-related and pro-apoptotic genes. Gene expression changes were verified by qPCR in three invasive ductal breast carcinoma cell lines (MCF-7, T-47D, MDA-MB-231). GADD153 showed the largest increase in the microarray experiment; however, knockdown of GADD153 did not abrogate apoptosis and death. Genes related to the extrinsic pathway of apoptosis including a receptor for TRAIL, death receptor 5 (DR5), were up-regulated by drug treatment. A dose of 4-HPR that alone is ineffective in killing TRAIL-resistant MCF-7 cells, synergized with recombinant TRAIL to induce breast cancer cell death.. 4-HPR and analogs might be useful in sensitizing tumor cells to death receptor agonists.

Topics: Apoptosis; Breast Neoplasms; Cell Death; Cell Division; Cell Line, Tumor; Endoplasmic Reticulum Stress; Female; Fenretinide; Humans; MCF-7 Cells; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Tretinoin; Up-Regulation

Retinoic acid (RA) and unsaturated fatty acids (UFA) are proposed as nutritional anticancer agents. Nonetheless, the activity of their combination on human breast cancer needs further study. Our aim was to evaluate this activity on the MCF-7 and ZR-75-1 cell lines treated with 1 µM RA and 50 µM of γ-linoleic (GLA, ω-6), eicosapentaenoic (EPA, ω-3), oleic (OA, ω-9), or eicosatrienoic (ETA, ω-9) acids. The following cellular responses were compared by ANOVA and Fisher test (P < 0.05): fatty acids, E-cadherin, actin (differentiation), conjugated dienes, γ-glutamyltranspeptidase activity (stress), and viability, which were correlated by partial least squares regression. Although both cell lines responded differentially, RA modified unsaturated fatty acids, increased differentiation, reduced γ-glutamyltranspeptidase, and viability. RA differentiating activity on ZR-75-1 was morphologically enhanced by UFA. Stress induction with γ-glutamyltranspeptidase decrease and conjugated dienes was promoted by ETA in MCF-7, and EPA and OA in ZR-75-1. RA-related reduced viability was potentiated by EPA and OA in both lines. GLA was less active. Therefore, unsaturated fatty acids (ω-3/ω-9) potentiated the multitarget retinoic acid activity against these human breast cancer cells.

Topics: Antigens, CD; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Survival; Fatty Acids, Unsaturated; Female; gamma-Glutamyltransferase; Humans; Tretinoin

The presence of hypoxic areas is common in all breast lesions but no data clearly correlate low oxygenation with the acquisition of malignant features by non-invasive cells, particularly by cells from ductal carcinoma in situ (DCIS), the most frequently diagnosed tumor in women.. By using a DCIS-derived cell line, we evaluated the effects of low oxygen availability on malignant features of non-invasive breast tumor cells and the possible role of all-trans retinoic acid (ATRA), a well-known anti-leukemic drug, in counteracting the effects of hypoxia. The involvement of the β2 isoform of PI-PLC (PLC-β2), an ATRA target in myeloid leukemia cells, was also investigated by specific modulation of the protein expression.. We demonstrated that moderate hypoxia is sufficient to induce, in DCIS-derived cells, motility, epithelial-to-mesenchymal transition (EMT) and expression of the stem cell marker CD133, indicative of their increased malignant potential. Administration of ATRA supports the epithelial-like phenotype of DCIS-derived cells cultured under hypoxia and keeps down the number of CD133 positive cells, abrogating almost completely the effects of poor oxygenation. We also found that the mechanisms triggered by ATRA in non-invasive breast tumor cells cultured under hypoxia is in part mediated by PLC-β2, responsible to counteract the effects of low oxygen availability on CD133 levels.. Overall, we assigned to hypoxia a role in increasing the malignant potential of DCIS-derived cells and we identified in ATRA, currently used in treatment of acute promyelocytic leukemia (APL), an agonist potentially useful in preventing malignant progression of non-invasive breast lesions showing hypoxic areas.

Topics: Antineoplastic Agents; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression; Humans; Hypoxia; Immunohistochemistry; Neoplasm Grading; Neoplasm Staging; Oxygen; Phospholipase C beta; Tretinoin

Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models.. Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD).. In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD.. In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Breast Neoplasms; Cell Line, Tumor; Female; Heterografts; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MCF-7 Cells; Mice; Neoplastic Stem Cells; Neovascularization, Pathologic; Retinal Dehydrogenase; RNA, Messenger; Signal Transduction; Transfection; Tretinoin; Vascular Endothelial Growth Factor A

Previous studies have showed the anticancer effect of the all-trans retinoic acid (ATRA) in many tumors including breast cancer; however, the underlying molecular mechanism is still poorly understood. This study experimentally revealed that ATRA treatment inhibited MCF-7 cell proliferation and promoted its apoptosis, along with an enhanced expression of docking protein 1 (DOK1). ATRA's effects on cell proliferation and apoptosis were prevented by DOK1 knockdown. In addition, the genetic silence of DOK1 can inhibit PPARγ expression and its activity. Moreover, inactivation of PPARγ by its specific inhibitor GW9662 reversed the impacts of ATRA on cell proliferation and apoptosis. Taken together, these results indicate that ATRA-enhanced expression of DOK1 activates PPARγ leading to inhibition of cell proliferation and enhancement of cell apoptosis in MCF-7 cell.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Proliferation; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Phosphoproteins; PPAR gamma; RNA-Binding Proteins; Signal Transduction; Treatment Outcome; Tretinoin

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Interferon Regulatory Factor-3; Interferon Type I; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor, Member 10c; RNA, Double-Stranded; TNF-Related Apoptosis-Inducing Ligand; Toll-Like Receptor 3; Tretinoin

Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5- cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the -1.1 kb essential PRE, and also to a proximal region near -130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the -1.1 kb PRE and the -130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-5; Neoplastic Stem Cells; Progesterone; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Progesterone; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Signal Transduction; Tretinoin

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Ligands; Proteomics; Retinoid X Receptors; Tretinoin; Up-Regulation

Retinoids, derivatives of vitamin A, are key physiological molecules with regulatory effects on cell differentiation, proliferation and apoptosis. As a result, they are of interest for cancer therapy. Specifically, models of breast cancer have varied responses to manipulations of retinoid signaling. This study characterizes the transcriptional response of MDA-MB-231 and MDA-MB-468 breast cancer cells to retinaldehyde dehydrogenase 1A3 (ALDH1A3) and all-trans retinoic acid (atRA). We demonstrate limited overlap between ALDH1A3-induced gene expression and atRA-induced gene expression in both cell lines, suggesting that the function of ALDH1A3 in breast cancer progression extends beyond its role as a retinaldehyde dehydrogenase. Our data reveals divergent transcriptional responses to atRA, which are largely independent of genomic retinoic acid response elements (RAREs) and consistent with the opposing responses of MDA-MB-231 and MDA-MB-468 to in vivo atRA treatment. We identify transcription factors associated with each gene set. Manipulation of the IRF1 transcription factor demonstrates that it is the level of atRA-inducible and epigenetically regulated transcription factors that determine expression of target genes (e.g. CTSS, cathepsin S). This study provides a paradigm for complex responses of breast cancer models to atRA treatment, and illustrates the need to characterize RARE-independent responses to atRA in a variety of models.

Topics: Aldehyde Oxidoreductases; Antineoplastic Agents; Base Sequence; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Interferon Regulatory Factor-1; Nucleotide Motifs; Position-Specific Scoring Matrices; Receptors, Retinoic Acid; Response Elements; Transcription, Genetic; Transcriptome; Tretinoin

The study aimed to investigate the effects of combination treatment of curcumin and β-interferon (IFN-β)/retinoic acid (RA) on breast cancer cells, including cell viability, apoptosis and migration, and to determine the mechanisms related to GRIM-19 through STAT3-dependent and STAT3-independent pathways.. The following groups were used for the in vitro experiment: control siRNA, GRIM-19 siRNA, IFN-β/RA and IFN-β/RA + curcumin. Cell viability is by the MTT method, cell apoptosis by flow cytometry and cell migration by wound healing experiment; GRIM-19, STAT3, survivin, Bcl-2, GADD153 and COX-2 expression was measured by Western blot. In vivo experiment, MCF-7 cells were subcutaneously injected into nude mice.. GRIM-19 siRNA promoted MCF-7 cell proliferation and migration; inhibited cell apoptosis; and promoted the expression of STAT3, survivin, Bcl-2 and MMP-9. IFN-β/RA inhibited cell proliferation and migration; promoted cell apoptosis; up-regulated GRIM-19; and inhibited the expression of STAT3, survivin, Bcl-2 and MMP-9. Combination treatment of curcumin and IFN-β/RA had a stronger effect than that of the IFN-β/RA group. In addition, curcumin and IFN-β/RA combination inhibited the expression of COX-2 and up-regulated GADD153.. Curcumin synergistically increases the effects of IFN-β/RA on breast cancer cells. The mechanism may be related to the up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Curcumin; Drug Synergism; Female; Humans; Interferon-beta; MCF-7 Cells; Mice; Mice, Nude; NADH, NADPH Oxidoreductases; Signal Transduction; STAT3 Transcription Factor; Tretinoin; Up-Regulation; Xenograft Model Antitumor Assays

All-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals.. RNA-sequencing data for 10 080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis.. We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes.. In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Topics: Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Glioma; Humans; Leukemia, Promyelocytic, Acute; Machine Learning; Melanoma; Models, Theoretical; Sequence Analysis, RNA; Tretinoin; Uveal Neoplasms

The key molecular mechanism governing the cancer cell state (stem cell-like state vs differentiation state) to control the cancer stem cell (CSC) pool remains elusive. This study provides the first evidence showing that all-trans retinoic acid (ATRA) induces the interaction and chromatin recruitment of a novel RARβ-TET2 complex to epigenetically activate a specific cohort of gene targets, including MiR-200c. TET2-activated miR-200c further targets and suppresses PKCζ, a cell polarity protein that has a pivotal role in directing asymmetric division of mammalian stem cells to sustain the stem cell pool. Our data reveal that pharmacological concentration of ATRA effectively downregulates PKCζ through activation of miR-200c, leading to a decrease of the stem cell-like populations from non-tumorigenic mammary epithelial cells and non-aggressive breast cancer cells. However, aggressive breast cancer cells that manifest TET2-miR-200c dysregulation sustain a CSC pool highly resistant to ATRA, where inhibition of PKCζ directs the resistant CSCs to the luminal cell-like state and sensitization to tamoxifen, resulting in abrogation of mammary tumor growth and progression. Together, these findings elucidate a novel RARβ-TET2-miR-200c-PKCζ signaling pathway that directs cancer cell state changes and also provide previously unidentified therapeutic implications for PKCζ inhibitors in diminishment of breast CSCs to eradicate breast cancer.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Dioxygenases; DNA-Binding Proteins; Female; Humans; Mice; Mice, Nude; Protein Kinase C; Proto-Oncogene Proteins; Signal Transduction; Tretinoin; Xenograft Model Antitumor Assays

Functional studies indicate that essential cellular processes are controlled by Vitamin A derivatives. Among these the retinoic acid isoforms, all-trans- and 9-cis (9cRA), regulate the expression of various genes in both physiological and pathological conditions. Using several in vitro experimental models such as pancreatic β-cells, pre-adipocytes and breast cancer cells with different phenotypes, we demonstrated the capability of 9cRA to modulate myotrophin (Mtpn) and miR-375 expressions. The 9cRA effect in pancreatic β-cells line INS-1 832/13 point out a decreased expression of Mptn at both mRNA and protein levels associated to a concomitant increase of miR-375. We also studied the effect of this molecule on 3T3-L1 pre-adipocytes cells demonstrating a down-regulation of Mtpn and a dramatic increase of miR-375. Moreover, in the in vitro breast cancer model such as MDA-MB-231 and MCF-7 cells, 9cRA showed different effect on both Mtpn and miR-375 expression. In INS-1 832/13, 3T3-L1 pre-adipocytes and MCF-7 but not in MDA-MB-231, the effect of 9cRA on Mptn gene expression and its miR was under the control of RARs and RXRs receptors, as revealed by the exposure of these cell line to LE540 or HX603 receptor antagonists. In our findings 9cRA emerges has a hormone with a regulatory action on miR-375 that in most cases interfere with Mtpn expression.

Topics: Alitretinoin; Benzazepines; Benzoates; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Secreting Cells; Intercellular Signaling Peptides and Proteins; MCF-7 Cells; MicroRNAs; RNA, Messenger; Tretinoin; Vitamin A

All-trans retinoic acid (ATRA) has been shown to enhance the expression of connexin 43 (Cx43) and the bystander effect (BSE) in suicide gene therapy. These in turn improve effects of suicide gene therapies for several tumor types. However, whether ATRA can improve BSE remains unclear in suicide gene therapy for breast cancer. In the present study, MCF-7, human breast cancer cells were treated with ATRA in combination with a VEGFP-TK/CD gene suicide system developed by our group. We found that this combination enhances the efficiency of cell killing and apoptosis of breast cancer by strengthening the BSE in vitro. ATRA also promotes gap junction intercellular communication (GJIC) in MCF-7 cells by upregulation of the connexin 43 mRNA and protein in MCF-7 cells. These results indicate that enhancement of GJIC by ATRA in suicide gene system might serve as an attractive and cost-effective strategy of therapy for breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Bystander Effect; Connexin 43; Female; Gene Expression Regulation, Neoplastic; Genes, Transgenic, Suicide; Genetic Therapy; Humans; MCF-7 Cells; Tretinoin

Radiotherapy is of critical importance in the treatment of breast cancer. However, not all patients derive therapeutic benefit and some breast cancers are resistant to the treatment, and are thus evidenced with prospective distant metastatic spread and local recurrence. In this study, we investigated the potential therapeutic effects of all-trans retinoic acid (ATRA) on radiation-resistant breast cancer cells and the associated invasiveness.. The MCF7/C6 cells with gained radiation resistance after a long term treatment with fractionated ionizing radiation were derived from human breast cancer MCF7 cell line, and are enriched with cells expressing putative breast cancer stem cell biomarker CD44(+)/CD24(-/low)/ALDH(+). The enhanced invasiveness and the acquired resistances to chemotherapeutic treatments of MCF7/C6 cells were measured, and potential effects of all-trans retinoic acid (ATRA) on the induction of differentiation, invasion and migration, and on the sensitivities to chemotherapies in MCF7/C6 cells were investigated.. MCF7/C6 cells are with enrichment of cancer stem-cell like cells with positive staining of CD44(+)/CD24(-/low), OCT3/4 and NANOG. MCF7/C6 cells showed an increased tumoregensis potential and enhanced aggressiveness of invasion and migration. Treatment with ATRA induces the differentiation in MCF7/C6 cells, resulting in reduced invasiveness and migration, and increased sensitivity to Epirubincin treatment.. Our study suggests a potential clinic impact for ATRA as a chemotherapeutic agent for treatment of therapy-resistant breast cancer especially for the metastatic lesions. The study also provides a rationale for ATRA as a sensitizer of Epirubincin, a first-line treatment option for breast cancer patients.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Combined Modality Therapy; Female; Humans; Hyaluronan Receptors; Neoplasm Invasiveness; Neoplastic Stem Cells; Radiation Tolerance; Tretinoin

Breast cancer is the most common malignancy in women, with metastases being the cause of death in 98%. In previous works we have demonstrated that retinoic acid (RA), the main retinoic acid receptor (RAR) ligand, is involved in the metastatic process by inhibiting migration through a reduced expression of the specific migration-related proteins Moesin, c-Src, and FAK. At present, our hypothesis is that RA also acts for short periods in a non-genomic action to cooperate with motility reduction and morphology of breast cancer cells. Here we identify that the administration of 10(-6) M RA (10-20 min) induces the activation of the migration-related proteins Moesin, FAK, and Paxillin in T-47D breast cancer cells. The phosphorylation exerted by the selective agonists for RARα and RARβ, on Moesin, FAK, and Paxillin was comparable to the activation exerted by RA. The RARγ agonist only led to a weak activation, suggesting the involvement of RARα and RARβ in this pathway. We then treated the cells with different inhibitors that are involved in cell signaling to regulate the mechanisms of cell motility. RA failed to activate Moesin, FAK, and Paxillin in cells treated with Src inhibitor (PP2) and PI3K inhibitor (WM), suggesting the participation of Src-PI3K in this pathway. Treatment with 10(-6) M RA for 20 min significantly decreased cell adhesion. However, when cells were treated with 10(-6) M RA and FAK inhibitor, the RA did not significantly inhibit adhesion, suggesting a role of FAK in the adhesion inhibited by RA. By immunofluorescence and immunoblotting analysis we demonstrated that RA induced nuclear FAK translocation leading to a reduced cellular adhesion. These findings provide new information on the actions of RA for short periods. RA participates in cell adhesion and subsequent migration, modulating the relocation and activation of proteins involved in cell migration.

Topics: Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Nucleus; Female; Focal Adhesion Protein-Tyrosine Kinases; HSP27 Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Microfilament Proteins; Paxillin; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Transport; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; src-Family Kinases; Tretinoin; Tumor Suppressor Protein p53

Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression.

Topics: Breast Neoplasms; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Phosphoproteins; Phosphorylation; Proteome; Signal Transduction; Transcriptome; Tretinoin

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; Epithelial Cells; Female; Humans; Morphogenesis; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Retinoic Acid Receptor alpha; Tretinoin

Co-delivery of all-trans-retinoic acid and paclitaxel using albumin-bound nanoparticles demonstrated a significantly improved anti-metastatic effect to breast cancer both in vitro and in vivo. Notably, the co-delivery nanoparticles exhibited more pronounced therapeutic effects than the combination of two free drugs or two HSA loaded single drugs.

Topics: Albumin-Bound Paclitaxel; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Humans; Mice; Models, Molecular; Nanoparticles; Neoplasm Metastasis; Protein Conformation; Tretinoin

It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all-trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA-induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone-independent breast cancer treatment.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Nucleolus; Female; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Humans; Mice; Mice, Inbred BALB C; Protein Isoforms; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase Inhibitors; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Combination treatment through simultaneous delivery of two or more drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employ a combination therapy for eliminating both the bulk tumor cells and the rare cancer stem cells (CSCs) that have a high self-renewal capacity and play a critical role in cancer treatment failure. All-trans-retinoic acid (ATRA), a powerful differentiation agent of cancer stem cells and the clinically widely used chemotherapy agent doxorubicin (DOX) are simultaneously encapsulated in the same nanoparticle by a single emulsion method. It is demonstrated that ATRA and DOX simultaneous delivery-based therapy can efficiently deliver the drugs to both non-CSCs and CSCs to differentiate and kill the cancer cells. Differentiation of CSCs into non-CSCs can reduce their self-renewal capacity and increase their sensitivity to chemotherapy; with the combined therapy, a significantly improved anti-cancer effect is demonstrated. Administration of this combinational drug delivery system can markedly augment the enrichment of drugs both in tumor tissues and cancer stem cells, prodigiously enhancing the suppression of tumor growth while reduce the incidence of CSC in a synergistic manner.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Delivery Systems; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Injections, Intravenous; Mice, Inbred NOD; Mice, SCID; Nanoparticles; Neoplasms; Neoplastic Stem Cells; Particle Size; Proliferating Cell Nuclear Antigen; Static Electricity; Tretinoin

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.

Topics: Adaptor Proteins, Vesicular Transport; Alitretinoin; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Movement; Cofilin 1; Cytoskeleton; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Female; HSP27 Heat-Shock Proteins; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Proteomics; snRNP Core Proteins; Tretinoin

All-trans retinoic acid (ATRA) is one of the most successful examples of differentiation agents and histone deacetylase inhibitors, such as tributyrin (TB), are known for their antitumor activity and potentiating action of drugs, such as ATRA. Nanostructured lipid carriers (NLC) represent a promising alternative to the encapsulation of lipophilic drugs such as ATRA. This study aims to develop, characterize and evaluate the cytotoxicity of ATRA-TB-loaded NLC for cancer treatment.. The influence of in situ formation of an ion pairing between ATRA and a lipophilic amine (benethamine) on the characteristics of NLC (size, zeta potential, encapsulation efficiency) was evaluated. TB, a butyric acid donor, was used as a component of the lipid matrix. In vitro activity on cell viability and distribution of cell cycle phases were evaluated for MCF-7, MDA-MB-231, HL-60 and Jurkat cell lines.. The presence of the amine significantly increased the encapsulation efficiency of ATRA in NLC. Inhibition of cell viability by TB-ATRA-loaded NLC was more pronounced than the free drug. Analysis of the distribution of cell cycle phases also showed increased activity for TB-ATRA-loaded NLC, with the clear effect of cell cycle arrest in G0/G1 phase transition. The presence of TB played an important role in the activity of the formulation.. Taken together, these findings suggest that TB-ATRA-loaded NLC represents a promising alternative to intravenous administration of ATRA in cancer treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Drug Carriers; Drug Combinations; Female; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Jurkat Cells; Leukemia; Lipids; MCF-7 Cells; Nanostructures; Tretinoin; Triglycerides

A new linear-dendritic copolymer composed of poly(ethylene glycol) (PEG) and all-trans-retinoic acid (ATRA) was synthesized as the anticancer drug delivery platform (PEG-G3-RA8). It can self-assemble into core-shell micelles with a low critical micelle concentration (CMC) at 3.48 mg/L. Paclitaxel (PTX) was encapsulated into PEG-G3-RA8 to form PEG-G3-RA8/PTX micelles for breast cancer treatment. The optimized formulation had high drug loading efficacy (20% w/w of drug copolymer ratio), nanosized diameter (27.6 nm), and narrow distribution (PDI = 0.103). Compared with Taxol, PEG-G3-RA8/PTX remained highly stable in the serum-containing cell medium and exhibited 4-fold higher cellular uptake. Besides, near-infrared fluorescence (NIR) optical imaging results indicated that fluorescent probe loaded micelle had a preferential accumulation in breast tumors. Pharmacokinetics and biodistribution studies (10 mg/kg) showed the area under the plasma concentration-time curve (AUC0-∞) and mean residence time (MRT0-∞) for PEG-G3-RA8/PTX and Taxol were 12.006 ± 0.605 mg/L h, 2.264 ± 0.041 h and 15.966 ± 1.614 mg/L h, 1.726 ± 0.097 h, respectively. The tumor accumulation of PEG-G3-RA8/PTX group was 1.89-fold higher than that of Taxol group 24 h postinjection. With the advantages like efficient cellular uptake and preferential tumor accumulation, PEG-G3-RA8/PTX showed superior therapeutic efficacy on MCF-7 tumor bearing mice compared to Taxol.

Topics: Animals; Breast Neoplasms; Dendrimers; Drug Delivery Systems; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Paclitaxel; Polyethylene Glycols; Rats; Tretinoin

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Catalysis; Catalytic Domain; Cell Line, Tumor; Dose-Response Relationship, Drug; Female; Fibroblasts; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Leukemia, Promyelocytic, Acute; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Transplantation; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphates; Phosphorylation; Proline; Tretinoin; Triple Negative Breast Neoplasms

Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all-trans retinoic acid (ATRA) sensitivity. Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER(+) carcinomas are ATRA sensitive, while triple-negative (Basal) and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER(+) and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; Gene Expression Profiling; Gene Silencing; Humans; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transplantation, Heterologous; Tretinoin

MicroRNAs (miRNAs) are short non-coding RNA molecules that play a critical role in mRNA cleavage and translational repression, and are known to be altered in many diseases including breast cancer. MicroRNA-10a (miR-10a) has been shown to be deregulated in various cancer types. The aim of this study was to investigate miR-10a expression in breast cancer and to further delineate the role of retinoids and thyroxine in regulation of miR-10a.. Following informed patient consent and ethical approval, tissue samples were obtained during surgery. miR-10a was quantified in malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) tissues by RQ-PCR. Gene expression of Retinoic Acid Receptor beta (RARβ) and Thyroid Hormone receptor alpha (THRα) was also quantified in the same patient samples (n = 168). The in vitro effects of all-trans Retinoic acid (ATRA) and L-Thyroxine (T4) both individually and in combination, on miR-10a expression was investigated in breast cancer cell lines, T47D and SK-BR-3.. The level of miR-10a expression was significantly decreased in tissues harvested from breast cancer patients (Mean (SEM) 2.1(0.07)) Log10 Relative Quantity (RQ)) compared to both normal (3.0(0.16) Log10 RQ, p < 0.001) and benign tissues (2.6(0.17) Log10 RQ, p < 0.05). The levels of both RARβ and THRα gene expression were also found to be decreased in breast cancer patients compared to controls (p < 0.001). A significant positive correlation was determined between miR-10a and RARβ (r = 0.31, p < 0.001) and also with THRα (r = 0.32, p < 0.001). In vitro stimulation assays revealed miR-10a expression was increased in both T47D and SK-BR-3 cells following addition of ATRA (2 fold (0.7)). While T4 alone did not stimulate miR-10a expression, the combination of T4 and ATRA was found to have a positive synergistic effect.. The data presented supports a potential tumour suppressor role for miR-10a in breast cancer, and highlights retinoic acid as a positive regulator of the microRNA.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Female; Fibroadenoma; Gene Expression; Humans; MicroRNAs; Middle Aged; Receptors, Retinoic Acid; Thyroid Hormone Receptors alpha; Tretinoin; Young Adult

Owing to the pharmacological potential of ATRA (all trans-retinoic acid), a series of retinamides and a 1-(retinoyl)-1,3-dicyclohexylurea compound were prepared by reacting ATRA with long chain alkyl or alkenyl fatty amines by using a 4-demethylaminopyridine (DMAP)-catalyzed N,N¢-dicyclohexylcarbodiimide (DCC) coupling. The successful synthesis of the target compounds was demonstrated using a range of spectroscopic techniques. The cytotoxicity of the compounds was measured along with their ability to induce cell cycle arrest and apoptosis in human cancer cell lines MCF-7 (breast cancer) and HepG2 (liver cancer) and normal human cell line HEK293 (embryonic kidney). The results of cytotoxicity and flow cytometry data showed that the compounds had a moderate to strong effect against MCF-7 and HepG2 cells and were less toxic to HEK293 cells. N-oleyl-retinamide was found to be the most potent anticancer agent and was more effective against MCF-7 cells than HepG2 cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line; Cell Line, Tumor; Female; HEK293 Cells; Hep G2 Cells; Humans; Liver Neoplasms; MCF-7 Cells; Tretinoin; Urea

SKBR3-cells, characterized by ERBB2/RARA co-amplification, represent a subgroup of HER2+ breast-cancers sensitive to all-trans retinoic acid (ATRA) and Lapatinib. In this model, the two agents alone or in combination modulate the expression of 174 microRNAs (miRs). These miRs and predicted target-transcripts are organized in four interconnected modules (Module-1 to -4). Module-1 and Module-3 consist of ATRA/Lapatinib up-regulated and potentially anti-oncogenic miRs, while Module-2 contains ATRA/Lapatinib down-regulated and potentially pro-oncogenic miRs. Consistent with this, the expression levels of Module-1/-3 and Module-2 miRs are higher and lower, respectively, in normal mammary tissues relative to ductal-carcinoma-in-situ, invasive-ductal-carcinoma and metastases. This indicates associations between tumor-progression and the expression profiles of Module-1 to -3 miRs. Similar associations are observed with tumor proliferation-scores, staging, size and overall-survival using TCGA (The Cancer Genome Atlas) data. Forced expression of Module-1 miRs, (miR-29a-3p; miR-874-3p) inhibit SKBR3-cell growth and Module-3 miRs (miR-575; miR-1225-5p) reduce growth and motility. Module-2 miRs (miR-125a; miR-193; miR-210) increase SKBR3 cell growth, survival and motility. Some of these effects are of general significance, being replicated in other breast cancer cell lines representing the heterogeneity of this disease. Finally, our study demonstrates that HIPK2-kinase and the PLCXD1-phospholipase-C are novel targets of miR-193a-5p/miR-210-3p and miR-575/miR-1225-5p, respectively.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Carrier Proteins; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Female; Humans; Lapatinib; MicroRNAs; Phosphoinositide Phospholipase C; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Quinazolines; Receptor, ErbB-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

Topics: Breast Neoplasms; Catalytic Domain; Drug Discovery; Female; Half-Life; Humans; Leukemia, Promyelocytic, Acute; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Signal Transduction; Tretinoin

All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Receptor, Notch1; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tretinoin

Breast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model.. The bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo.. We found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA.. Our results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial Cells; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice, Inbred BALB C; Microscopy, Fluorescence; Models, Biological; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Burden

The retinoic acid (RA)-metabolizing enzyme CYP26A1 has been shown to efficiently enhance the oncogenic potential of breast cancer, suggesting a potential oncogenic function. We previously demonstrated that CYP26A1 confers unique cell survival properties by modulating the expression of a variety of genes and identified a number of genes that drive the cells into the oncogenic state. Accumulating evidence suggested that fascin is overexpressed in various types of cancer, primarily leading to increased cell motility. Therefore, in the present study, we examined fascin, an actin-bundling protein, using immunohistochemical and SA-β-gal staining as well as TUNEL and colony forming assays. The results of the present study showed that the expression levels of fascin increased significantly in response to CYP26A1 overexpression and, conversely, treatment with all-trans RA downregulated the expression of fascin. In addition, primary breast carcinoma samples, particularly hormone receptor-negative carcinomas and CYP26A1-overexpressing cancers, expressed elevated levels of fascin. Notably, fascin contributed to the ability of breast carcinoma cells to escape premature senescence and exhibit enhanced cell apoptotic resistance, promoting anchorage-independent growth properties. Fascin also promoted cell motility and the invasiveness of CYP26A1-expressing breast carcinoma cells. These data suggest that fascin expression is modulated by the intracellular RA status regulated by the expression of CYP26A1 and plays a significant role in the malignant behavior of CYP26A1-expressing breast carcinoma cells. CYP26A1 exerts oncogenic functions during breast carcinogenesis. Therefore, CYP26A1-mediated oncogenic characteristics may be partially responsible for the elevated expression of fascin.

Topics: Breast Neoplasms; Carrier Proteins; Cell Survival; Cell Transformation, Neoplastic; Cytochrome P-450 Enzyme System; Female; Humans; MCF-7 Cells; Microfilament Proteins; Retinoic Acid 4-Hydroxylase; Tretinoin

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Benzamides; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Histone Deacetylase Inhibitors; Humans; Letrozole; Mice, Nude; Nitriles; Pyridines; Receptor, ErbB-2; Tretinoin; Triazoles; Xenograft Model Antitumor Assays

Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer.. CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis.. Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.. CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Intracellular Space; Models, Biological; Neoplasm Grading; Prognosis; Protein Transport; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Triple Negative Breast Neoplasms

Interactions between cancer cells and microenvironment are emerging issue in tumor progression. Aldehyde dehydrogenase 1 (ALDH1) is a recognized cancer stem cell marker but little is known about its role in intratumoral stroma. Therefore, we focused on ALDH1 expression in tumor-associated stroma of breast carcinomas (BrCa). Stromal and tumoral ALDH1 expression was evaluated immunohistochemically in BrCa and their lymph node metastases (LNMs), and related to clinico-pathological characteristics, patients' outcome, presence of CD68, HLADR, retinoic acid (RA) in stroma, and selected proteins in tumor cells. ALDH1(+) stromal cells were detected in 53% of 374 BrCa and 61% of 102 LNMs. ALDH1(+) stroma in primary tumor correlated to longer disease-free (p = 0.030), metastasis-free (p = 0.024), and overall survival (p = 0.043) having an independent prognostic impact on DFS (multivariate analysis, p = 0.047). It was associated with concomitant presence of HLA-DR(+) stromal cells and RA in tumor cells (both p < 0.001), and inversely associated with vimentin expression in tumor cells (p = 0.036). ALDH1(+) stroma in LNMs correlated inversely to presence of disseminated tumor cells in patients' bone marrow (p = 0.014) and was independent prognosticator of shorter DFS and MFS (multivariate analysis, p = 0.004 and p = 0.002, respectively). In conclusion, ALDH1 expression in tumor-associated stromal cells indicates reduced BrCa progression, possibly via RA secretion.

Topics: Aged; Aldehyde Dehydrogenase 1 Family; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Breast Neoplasms; Carcinoma; Cohort Studies; Disease Progression; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Germany; HLA-DR Antigens; Humans; Immunohistochemistry; Isoenzymes; Kaplan-Meier Estimate; Lymphatic Metastasis; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplastic Stem Cells; Poland; Retinal Dehydrogenase; Stromal Cells; Tissue Array Analysis; Tretinoin

The transcription factor Kruppel-like factor 2 (KLF2) displays anticarcinogenic activities but the mechanism that underlies this activity is unknown. We show here that KLF2 is markedly downregulated in human breast cancers and that its expression positively correlates with breast cancer patient survival. We show further that KLF2 suppresses tumor development by controlling the transcriptional activity of the vitamin A metabolite retinoic acid (RA). RA regulates gene transcription by activating two types of nuclear receptors: RA receptors (RARs), which inhibit tumor development, and peroxisome proliferator-activated receptor β/δ (PPARβ/δ), which promotes tumorigenesis. The partitioning of RA between these receptors is regulated by two carrier proteins: cellular retinoic acid-binding protein 2 (CRABP2), which delivers RA to RARs, and fatty acid-binding protein 5 (FABP5), which shuttles ligands to PPARβ/δ. We show that KLF2 induces the expression of CRABP2 and RARγ and inhibits the expression FABP5 and PPARβ/δ thereby shifting RA signaling from the pro-carcinogenic FABP5/PPARβ/δ to the growth-suppressing CRABP2/RAR path. The data thus reveal that KLF2 suppresses tumor growth by controlling the transcriptional activities of RA.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Fatty Acid-Binding Proteins; Female; Heterografts; Humans; Kruppel-Like Transcription Factors; MCF-7 Cells; Mice; Mice, Nude; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Signal Transduction; Transcription, Genetic; Tretinoin

We have previously reported that the major isoform of Flt1/VEGFR-1 expressed in MDA-MB-231 breast cancer cells was a truncated intracellular isoform transcribed from intron 21 (i21 Flt1). This isoform upregulated the active form of Src and increased breast cancer cell invasiveness. Since expression of the transmembrane and soluble Flt1 isoforms of HUVEC is activated by Notch signaling, we wondered whether the expression of the intracellular isoform i21 Flt1 was also dependent on Notch activation. We report here that the expression of i21 Flt1 in HUVEC and MDA-MB-231 cells is downregulated by the γ-secretase inhibitor DAPT. In addition, treatment of MDA-MB-231 cells with siRNA specific for Notch-1 and Notch-3 downregulates the expression of i21 Flt1. In agreement with these findings, HUVEC and MDA-MB-231 breast cancer cells, cultured on dishes coated with recombinant human Dll4 extracellular domain, express higher levels of i21 Flt1. In cancer cells, Flt1 is a target of the micro RNA family miR-200. In MDA-MB-231 breast cancer cells, the truncated intracellular isoform i21 Flt1 is also negatively regulated by miR-200c. Retinoic acid interferes i21 Flt1 expression by downregulating Notch-3 and upregulating miR-200 expression. Treatment of MDA-MB-231 breast cancer cells with both a γ-secretase inhibitor and retinoic acid suppresses the expression of i21 Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Breast Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Dipeptides; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Signaling Peptides and Proteins; MicroRNAs; Protein Isoforms; Receptor, Notch1; Receptor, Notch3; Receptors, Notch; Tretinoin; Up-Regulation; Vascular Endothelial Growth Factor Receptor-1

Myeloid-derived suppressor cells (MDSCs) are emerging as potential promoters of metastatic tumor growth, and there is interest in targeting immature MDSCs by inducing their differentiation into more mature myeloid cells. We used all-trans retinoic acid (ATRA) to differentiate MDSCs in mice bearing metastatic 4T1 or 4TO7 murine mammary tumors, and assessed the immune-suppressive mechanisms and potencies of different myeloid cell subpopulations. Metastatic mammary tumors induced the accumulation of distinct populations of immature CD11b(+)Gr1(+)F4/80(-)Ly6C(mid)Ly6G(+) MDSCs ("Gr1(+) cells") and mature CD11b(+)Gr1(-)F4/80(+) cells ("F4/80(+) cells") in metastatic target organs. ATRA triggered the differentiation of Gr1(+) cells into F4/80(+) cells in the lungs and, unexpectedly, enhanced pulmonary metastatic tumor growth. We found that F4/80(+)Ly6C(-)Ly6G(-) mature macrophages (Ms) were up to 30-fold more potent immune suppressors than Gr1(+) cells on a per-cell basis, which we postulate may contribute to the increased metastatic growth observed with ATRA treatment. F4/80(+) cells and Gr1(+) cells used different reactive oxygen species (ROS)-mediated mechanisms of immunosuppression ex vivo, with F4/80(+) cells producing higher levels of ROS, which is consistent with their superior immunosuppressive abilities. These data highlight the potent immunosuppressive functions of Ms, reveal that Ms can suppress T cell responses via ROS production, and suggest that ROS inhibitors may be useful in promoting antitumor immune responses. Our findings also caution against using ATRA to modulate myeloid cell differentiation and function to treat breast cancer metastases in the lung, and support the development of therapeutic strategies to enhance antitumor immunity by targeting myeloid cells as a collective group.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Disease Models, Animal; Female; Immunophenotyping; Lung Neoplasms; Macrophages; Mice; Mice, Transgenic; Myeloid Cells; Neoplasm Metastasis; Phenotype; Reactive Oxygen Species; Receptors, Cell Surface; T-Lymphocytes; Tretinoin

The RNA-binding protein Quaking (QKI) is known to be essential for embryonic development and postnatal myelination. Forkhead box O1 (FOXO1) is a critical tumor suppressor for cell proliferation control. Dysregulation of FOXO1 expression has been observed in a variety of cancers. In the present study, we demonstrated that QKI decreased FOXO1 mRNA expression at the post-transcriptional level. QKI was able to bind the 3'UTR of FOXO1 mRNA directly and decreased its mRNA stability. To determine whether QKI-mediated post-transcriptional repression of FOXO1 indeed plays a role in cancer cells, we first detected both QKI and FOXO1 expression in four breast cancer cell lines. FOXO1 expression was extremely low in these cell lines, whereas QKI expression was relative high. Knockdown of QKI significantly restored FOXO1 expression. ATRA, an inducer of apoptosis or differentiation, dramatically enhanced FOXO1 expression while it repressed QKI expression. Importantly, the ATRA-induced increase in FOXO1 expression was dependent on QKI-mediated post-transcriptional regulation. Consistently, 5-FU, a widely used chemotherapeutic agent, increased FOXO1 expression via inhibition of QKI. In summary, our study provides initial evidence demonstrating that QKI-mediated repression of FOXO1 may be one of the factors contributing to the oncogenesis and progression of breast carcinoma, which suggests that targeting QKI may serve as a novel strategy to sensitize breast cancers to chemotherapy.

Topics: Antimetabolites, Antineoplastic; Breast Neoplasms; Drug Screening Assays, Antitumor; Female; Fluorouracil; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; MCF-7 Cells; RNA Stability; RNA-Binding Proteins; Transcription, Genetic; Tretinoin

Breast cancer is the most common type of cancer in women in many areas and is increasing found in developing countries, where the majority of cases are diagnosed in late stages. Retinoic acids, through their associated nuclear receptors, exert intoxicating effects on cell growth, differentiation and apoptosis, and hold significant promise in relation to cancer therapy and chemoprevention. To enhance our understanding of the molecular mechanisms associated with retinoic acids in the breast cancer cell line MCF-7 in a time-dependent manner, we conducted a proteomic analysis of MCF-7 cells using the 2-DE couple with high-throughput mass spectrometry and bioinformatics tools. In the 2-DE patterns of MCF-7 cells treated with retinoic acid in a time-dependent manner, 35 protein spots were found to be differentially expressed. These were 17 increased, 4 decreased, and 14 unevenly expressed protein spots, all of which were analyzed using LTQ-FTICR mass spectrometry. Furthermore, five candidate proteins, up-regulated, were validated by western blotting. These were nucleoredoxin, latexin, aminomethyltransferase, translationally controlled one tumor protein, and rab GDP dissociation inhibitor β. These observations represent novel findings leading to new insight into the exact mechanism behind the effect of retinoic acids in MCF-7 cells while also identifying possible therapeutic targets for breast cancer diagnosis and novel drug development paths for the treatment of this disease.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cell Survival; Computational Biology; Female; Humans; MCF-7 Cells; Proteome; Proteomics; Reproducibility of Results; Tretinoin

Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor β (RARβ) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARβ protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARβ gene expression that was greatest after 72 hrs with a concentration 1 μM. High concentrations of RA increased the expression of RARβ causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARβ-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARβ gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c-Src and FAK expressions. RARβ is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.

Topics: Actin Cytoskeleton; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; CSK Tyrosine-Protein Kinase; Female; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Humans; Immunoenzyme Techniques; Microfilament Proteins; Receptors, Retinoic Acid; Retinoids; RNA, Small Interfering; src-Family Kinases; Tretinoin; Tumor Cells, Cultured

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Autophagy; Biological Availability; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Synergism; Endoplasmic Reticulum Stress; Female; Humans; Imidazoles; Intracellular Space; Rats; Rats, Sprague-Dawley; Thapsigargin; Tretinoin; Up-Regulation; Xenograft Model Antitumor Assays

All-trans-retinoic acid (RA) is a promising agent for breast cancer treatment, but it induces several adverse effects and the few clinical trials performed up to now in breast cancer patients have provided disappointing results. The combination of RA and antiestrogenic compounds, such as tamoxifen, synergistically decreases the proliferation of breast cancer cells and an interplay between retinoid and estrogen signaling has begun to be unraveled, turning these combinations into an appealing strategy for breast cancer treatment. This review focus on the current knowledge regarding the interplay between retinoid and estrogen signaling in breast cancer and the combinations of RA with antiestrogens, aiming their future utilization in cancer therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Estrogen Receptor Modulators; Estrogens; Female; Humans; Male; Receptor Cross-Talk; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoids; Signal Transduction; Tretinoin

To explore the effect and probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethyl- phenyl ester (ATPR) on apoptosis of MDA-MB-231 breast cancer cells.. MTT assays were performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all- trans retinoic acid (ATRA) and ATPR. Morphologic changes were observed by microscopy. The apoptosis rates and cell cycling of MDA-MB-231 cells treated with ATRA or ATPR were assessed using flow cytometry analysis. Expression of retinoic acid receptor and phosphorylation of ERK, JNK, p38 proteins were detected by Western blotting.. Treatment of the cells with the addition of 15 μmol/L ATPR for 48 h clearly demonstrated reduced cell numbers and deformed cells, whereas no changes in the number and morphology were observed after treatment with ATRA. The apoptosis rate was 33.2% after breast cancer MDA-MB-231 cells were treated by ATPR (15 μmol/L) whereas ATRA (15 μmol/L) had no apoptotic effect. ATPR inhibited the phosphorylation of ERK, JNK, and p38 while ATRA had no significant effect. ATPR inhibited the expression of BiP and increased the expression of Chop at the protein level compared with control groups, ATRA and ATPR both decreased the protein expression of RXR α, ATPR reduced the protein expression of RARβ and RXRβ while ATRA did not decrease RARβ or RXRβ.. ATPR could induce apoptosis of breast cancer MDA-MB-231 cells, possible mechanisms being binding to RARβ/RXRβ heterodimers, then activation of ER stress involving the MAPK pathway.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Female; Flow Cytometry; Humans; Phosphorylation; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The Na(+)/I(-) symporter (NIS (SLC5A5)) is a transmembrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. NIS-mediated iodide uptake in thyroid cells is the basis for targeted radionuclide imaging and treatment of differentiated thyroid carcinomas and their metastases. Furthermore, NIS is expressed in many human breast tumors but not in normal non-lactating breast tissue, suggesting that NIS-mediated radionuclide uptake may also allow the imaging and targeted therapy of breast cancer. However, functional cell surface NIS expression is often low in breast cancer, making it important to uncover signaling pathways that modulate NIS expression at multiple levels, from gene transcription to posttranslational processing and cell surface trafficking. In this study, we investigated NIS regulation in breast cancer by MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling, an important cell signaling pathway involved in oncogenic transformation. We found that MEK inhibition decreased NIS protein levels in all-trans retinoic acid/hydrocortisone-treated MCF-7 cells as well as human breast cancer cells expressing exogenous NIS. The decrease in NIS protein levels by MEK inhibition was not accompanied by a decrease in NIS mRNA or a decrease in NIS mRNA export from the nucleus to the cytoplasm. NIS protein degradation upon MEK inhibition was prevented by lysosome inhibitors but not by proteasome inhibitors. Interestingly, NIS protein level was correlated with MEK/ERK activation in human breast tumors from a tissue microarray. Taken together, MEK activation appears to play an important role in maintaining NIS protein stability in human breast cancers.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Humans; Hydrocortisone; Iodides; Lysosomes; Mitogen-Activated Protein Kinase Kinases; Nitriles; Protein Kinase Inhibitors; Proteolysis; RNA, Messenger; Symporters; Tretinoin

We characterized the cellular properties of cancer stem-like cells (CSLCs) isolated from immortalized MDA-MB453 human breast cancer cells in culture. We showed that although the expression of Octamer-binding transcription factor-4 (OCT4) correlates to stemness in these CSLCs, OCT4 knockdown does not induce their differentiation. Our results suggest that the differentiation program in MDA-MB453 CSLCs is blocked at a step upstream of the transcription of the OCT4 promoter, allowing CSLCs to maintain their population through asymmetric cell division during many repeated passages. Comparative expression analysis indicates that only a subset of genes and signaling pathways known to be associated with survival and maintenance of CSCs are selectively expressed in CSLCs, as compared with non-CSLCs fractionated from the same parental MDA-MB453 cells. These results suggest that selective expression of a limited number of genes may be sufficient for establishment and maintenance of CSLCs with high tumorigenicity.

Topics: Autophagy; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Endoplasmic Reticulum Stress; Female; Gene Expression Profiling; Histones; Humans; Neoplastic Stem Cells; Octamer Transcription Factor-3; Tretinoin; Ubiquitination

More than 70% of breast cancers in women require estrogens for cell proliferation and survival. 17β-estradiol (E2) effect on mammary target cells is almost exclusively mediated by its binding to the estrogen receptor-α (ERα) that joins chromatin where it assembles active transcription complexes. The proliferative and pro-survival action of estrogens is antagonized in most cases by retinoic acid (RA), even though the cognate retinoic acid receptor-α (RARα) cooperates with ERα on promoters of estrogen-responsive genes. We have examined at the molecular level the crosstalk between these nuclear receptors from the point of view of their control of cell growth and show here that RA reverts estrogen-stimulated transcription of the pivotal anti-apoptotic bcl-2 gene by preventing demethylation of dimethyl lysine 9 in histone H3 (HeK9me2). As we previously reported, this is obtained by means of E2-triggered activation of the lysine-specific demethylase 1 (LSD1), an enzyme that manages chromatin plasticity in order to allow specific movements of chromosomal regions within the nucleus. We find that E2 fuels LSD1 by inducing migration of the catalytic subunit of protein kinase A (PKA) into the nucleus, where it targets estrogen-responsive loci. RA rescues LSD1-dependent disappearance of H3K9me2 at bcl-2 regulatory regions upon the prevention of PKA assembly to the same sites.

Topics: Breast Neoplasms; Catalytic Domain; Chromatin; Colforsin; Cyclic AMP-Dependent Protein Kinases; Estrogens; Female; Flavonoids; Histone Demethylases; Histones; Humans; Isoquinolines; MAP Kinase Signaling System; MCF-7 Cells; Methylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sulfonamides; Transcription, Genetic; Tretinoin

To explore the effect and its probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethyl-phenyl ester (ATPR) on the migration of human breast cancer MDA-MB-231 cells.. MTT assay was performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all-trans retinoic acid (ATRA) and ATPR. The effect of ATPR and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, an inhibitor of p38, on the migration of MDA-MB-231 cells were analyzed by wound healing assay. The expression of MLCK and phosphorylation of myosin light chain (MLC), ERK, JNK, p38 proteins were detected by western blot. After the cells were treated by ATRA and ATPR, the proliferation and migration of breast cancer MDA-MB-231 cells were inhibited significantly. The IC 50 of ATRA and ATPR is 34.08 μmol/l and 18.06 μmol/l respectively. The relative migration rate of MDA-MB-231 cells treated with ATPR reached 50% at 48 h while the ATRA group is over 90%. The relative migration rate of ML-7 group and SB group had significant decrease compared with control group. The expression level of MLCK and phosphorylation of MLC of breast cancer cells was reduced when the cells were treated by ATPR with 48 h, the phosphorylation of ERK, JNK and p38 in breast cancer also reduced when cells were treated by ATPR with 2 h. In addition, ML-7 (50 μmol/l) could inhibit the phosphorylation of p38 and SB (50 μmol/l) could inhibit the expression of MLCK and phosphorylation of MLC.. ATPR had a better inhibition on the proliferation and the migration of breast cancer MDA-MB-231 cells than ATRA, and its probable mechanism was associated with the down regulation of expression of MLCK and phosphorylation of MLC protein involving p38-MAPK pathway.

Topics: Antineoplastic Agents; Azepines; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inhibitory Concentration 50; Myosin-Light-Chain Kinase; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Retinoids; Tretinoin

To detect the inhibitory effect of all-trans retinoic acid(ATRA) on breast cancer stem cells (CSCs).. The inhibitory effect of ATRA on MCF-7 and SK-BR-3 cell lines was analyzed using a Cell Counting Kit-8 (CCK-8). The proportion of CD44(+)CD24(-) tumor cells of the two cell lines were measured before and after the ATRA treatment, and the role of ATRA in the regulation of CSC self-renewing ability was evaluated with a tumor sphere assay. The tumor spheres were grown in an adherent culture to evaluate the ATRA-induced differentiation of breast cancer stem cells.. ATRA effectively inhibited the unsorted cells and stem cells, but the CSCs were more sensitive to ATRA. At a concentration of 10(-6) mol/L, the inhibitory rate of MCF-7 unsorted cells and stem cells were (8.66 ± 1.06)% and (21.09 ± 3.25)%, respectively (P = 0.004). For SK-BR-3 cells, the rates were (39.19 ± 1.47)% and (51.22 ± 2.80)%, respectively (P = 0.005). The self-renewing ability of the CSCs was impaired by ATRA at a concentration of 10(-6) mol/L. The rate of MCF-7 and SK-BR-3 stem cells to form tumor sphere was 5.2% (5/96) and 13.5% (13/96), respectively. For the control group, it was 86.5% (83/96) and 93.8% (90/96), respectively (P < 0.001). ATRA also promoted the CD44(+)CD24(-) subpopulation to differentiate. SK-BR-3 stem cells were grown in an adherent culture. After using ATRA, the proportion of CD44(+)CD24(-) cells was (48.1 ± 2.5)% and that of the control group was (86.6 ± 2.5)% (P < 0.001).. ATRA effectively inhibits breast NCSCs and CSCs, but CSCs are more sensitive to ATRA. ATRA impairs the self-renewing ability of CSCs and promotes CSCs to differentiate.

Topics: Antineoplastic Agents; Breast Neoplasms; CD24 Antigen; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Humans; Hyaluronan Receptors; Neoplastic Stem Cells; Tretinoin

Toll-like receptor 3 (TLR3) is a key effector of the innate immune system against viruses. Activation of TLR3 exerts an antitumoral effect through a mechanism of action still poorly understood. Here we show that TLR3 activation by polyinosinic:polycytidylic acid induces up-regulation of microRNA-29b, -29c, -148b, and -152 in tumor-derived cell lines and primary tumors. In turn, these microRNAs induce reexpression of epigenetically silenced genes by targeting DNA methyltransferases. In DU145 and TRAMP-C1 prostate and MDA-MB-231 breast cancer cells, we demonstrated that polyinosinic:polycytidylic acid-mediated activation of TLR3 induces microRNAs targeting DNA methyltransferases, leading to demethylation and reexpression of the oncosuppressor retinoic acid receptor beta (RARβ). As a result, cancer cells become sensitive to retinoic acid and undergo apoptosis both in vitro and in vivo. This study provides evidence of an antitumoral mechanism of action upon TLR3 activation and the biological rationale for a combined TLR3 agonist/retinoic acid treatment of prostate and breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Male; Mice; Mice, Nude; MicroRNAs; Neoplasms; Poly I-C; Prostatic Neoplasms; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; Toll-Like Receptor 3; Tretinoin; Xenograft Model Antitumor Assays

Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers.. Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines.. Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARA-amplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1.. The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting.

Topics: Antibodies, Monoclonal, Humanized; Breast Neoplasms; Carcinoma, Ductal, Breast; Drug Resistance, Neoplasm; Female; Gene Amplification; Humans; Leukemia, Promyelocytic, Acute; Middle Aged; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Trastuzumab; Tretinoin; Tumor Cells, Cultured

Recently developed genomics-based tools are allowing repositioning of Food and Drug Administration (FDA)-approved drugs as cancer treatments, which were employed to identify drugs that target cancer stem cells (CSCs) of breast cancer. Gene expression datasets of CSCs from six studies were subjected to connectivity map to identify drugs that may ameliorate gene expression patterns unique to CSCs. All-trans retinoic acid (ATRA) was negatively connected with gene expression in CSCs. ATRA reduced mammosphere-forming ability of a subset of breast cancer cells, which correlated with induction of apoptosis, reduced expression of SOX2 but elevated expression of its antagonist CDX2. SOX2/CDX2 ratio had prognostic relevance in CSC-enriched breast cancers. K-ras mutant breast cancer cell line enriched for CSCs was resistant to ATRA, which was reversed by MAP kinase inhibitors. Thus, ATRA alone or in combination can be tested for efficacy using SOX2, CDX2, and K-ras mutation/MAPK activation status as biomarkers of response.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Drug Approval; Gene Expression Profiling; Humans; Neoplastic Stem Cells; Reproducibility of Results; Sensitivity and Specificity; Treatment Outcome; Tretinoin; United States; United States Food and Drug Administration

Retinoids, acting through their cognate nuclear receptors, are crucial transcriptional regulators of many cellular processes such as differentiation, development, apoptosis, carbohydrate and lipid metabolism, homeostasis, etc. The aim of this study was the exploration of molecular mechanisms in relation to therapy of human breast cancer. One of the efficient strategies is identification of biomarkers as important tools in early cancer diagnosis and advisable treatment. Retinoids have been regarded as important therapeutic agents for many types of cancers, including human breast cancer. The effects of all-trans retinoic acid and 9-cis retinoic acid or their combination on proteomic pattern in human MCF-7 breast cancer line were investigated. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and identified by matrix assisted laser desorption ionization technique with time of flight mass analyzer (MALDI-TOF/TOF). Our data offer novel information on the proteomic pattern of proteins evaluated after treatment of MCF-7 cells with retinoic acid isomers.

Topics: Adenocarcinoma; Alitretinoin; Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Female; HSP90 Heat-Shock Proteins; Humans; Isomerism; MCF-7 Cells; Molecular Sequence Data; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin

Amphiphilic copolymer of folate-conjugated dextran/retinoic acid (FA/DEX-RA) was self-assembled into micelles by direct dissolution method. Magnetic iron oxide nanoparticles (MNPs) coated with oleic acid (OA) were prepared by hydrothermal method and encapsulated within the micelles. Doxorubicin HCl was loaded in the magnetic micelles. The characteristics of the magnetic micelles were determined by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM). The crystalline state of OA-coated MNPs and their heat capacity were analyzed by X-ray diffraction (XRD) and differential scanning calorimetry (DSC) methods, respectively. The iron content of magnetic micelles was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Bovine serum albumin (BSA) was used to test the protein binding of magnetic micelles. The cytotoxicity of doxorubicin loaded magnetic micelles was studied on MCF-7 and MDA-MB-468 cells using MTT assay and their quantitative cellular uptake by fluorimetry method. TEM results showed the MNPs in the hydrophobic core of the micelles. TGA results confirmed the presence of OA and FA/DEX-RA copolymer on the surface of MNPs and micelles, respectively. The magnetic micelles showed no significant protein bonding and reduced the IC₅₀ of the drug to about 10 times lower than the free drug.

Topics: Breast Neoplasms; Dextrans; Doxorubicin; Drug Delivery Systems; Female; Ferric Compounds; Folic Acid; Humans; Magnetite Nanoparticles; MCF-7 Cells; Micelles; Tretinoin

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

Topics: Azacitidine; Binding Sites; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Embryonal; Cell Movement; Decitabine; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Embryonal Carcinoma Stem Cells; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Reporter; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Luciferases; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Nuclear Receptor Subfamily 6, Group A, Member 1; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; RNA Interference; RNA, Messenger; Time Factors; Tissue Array Analysis; Transcription, Genetic; Transfection; Tretinoin; Valproic Acid

Due to its ability to regulate the growth, differentiation and apoptosis of cancer cells, retinoic acid (RA) is considered a signaling molecule with promising therapeutic potential in oncology. In this study, we show that RA is able to induce the intrinsic ability of breast cancer cells to recognize double-stranded RNA (dsRNA) through the upregulation of Toll-like receptor 3 (TLR3) expression. RA, co-administered with the dsRNA mimicker polyinosinic-polycytidylic acid (poly(I:C)), synergizes to mount a specific response program able to sense dsRNA through the concurrent upregulation of TLR3, the dsRNA helicases melanoma differentiation-associated antigen-5 (MDA-5) and RA-inducible gene-1 (RIG-1), and the dsRNA-activated protein kinase (PKR) expression, leading breast cancer cells to specifically express downstream transcriptional targets of dsRNA sensors, such as interferon-β (IFNβ), interleukin-8 (IL-8), chemokine (C-C motif) ligand 5 (CCL5), and C-X-C motif Chemokine 10 (CXCL10). A TLR3-dependent apoptotic program is also induced by RA and poly(I:C) co-treatment that correlates with the induction of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and contributes to block breast cancer cell proliferation. The mechanisms of apoptosis induced by RA/poly(I:C) in breast cancer cells involve type I IFN autocrine signaling, caspase-8 and caspase-3 activation, as well as TRAIL signaling. Our results reveal important links among RA, TLR3 and TRAIL and highlight the combined use of RA and poly(I:C) as a potential effective tumor therapy by improving the apoptotic response of cancer cells with low sensitivity to the action of synthetic dsRNA.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; DEAD-box RNA Helicases; Drug Synergism; eIF-2 Kinase; Female; Humans; Interferon Type I; Interferon-beta; Interferon-Induced Helicase, IFIH1; Membrane Proteins; Nerve Tissue Proteins; Poly I-C; Receptors, Cell Surface; Recombinant Proteins; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Toll-Like Receptor 3; Transcriptional Activation; Tretinoin; Up-Regulation

Aberrations in DNA methylation patterns have been reported to be involved in driving changes in the expression of numerous genes during carcinogenesis and have become promising targets for chemopreventive action of natural compounds. In the present study, we investigated the effects of all-trans retinoic acid (ATRA), vitamin D₃ and resveratrol alone and in combination with adenosine analogues, 2-chloro-2'-deoxyadenosine (2CdA) and 9-β-d-arabinosyl-2-fluoroadenine (F-ara-A), on the methylation and expression of phosphatase and tensin homologue (PTEN) tumour suppressor gene in MCF-7 and MDA-MB-231 breast cancer cells. The present results showed that in non-invasive MCF-7 cells, ATRA, vitamin D₃ and resveratrol possess high efficacy in the reduction of PTEN promoter methylation. It was associated with PTEN induction as well as DNA methyltransferase down-regulation and p21 up-regulation after treatments with vitamin D₃ and resveratrol, suggesting a complex regulation of the DNA methylation machinery. Vitamin D₃ and resveratrol improved the inhibitory effects of 2CdA and F-ara-A on PTEN methylation in MCF-7 cells; however, only the combined action of vitamin D₃ and 2CdA boosted the induction of PTEN expression, suggesting a cooperation of these compounds in additional processes driving changes in PTEN expression. In contrast, in highly invasive MDA-MB-231 cells, only vitamin D₃ reduced PTEN methylation and induced its expression without notable effects in combined treatments. The present results suggest that natural compounds can find application in epigenetic anticancer therapy aimed at inhibition of promoter methylation of tumour suppressor genes and induction of their expression at early stages of carcinogenesis.

Topics: Adenocarcinoma; Adenosine; Antimetabolites, Antineoplastic; Antineoplastic Agents, Phytogenic; Antioxidants; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor p21; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Neoplasm Proteins; Promoter Regions, Genetic; PTEN Phosphohydrolase; Resveratrol; RNA, Messenger; Stilbenes; Tretinoin; Vitamins

The majority of breast cancer deaths are because of ineffective treatment of metastatic disease. We previously identified a subpopulation of cells in human breast cancer cell lines that demonstrate high activity of aldehyde dehydrogenase (ALDH) and high expression of CD44. These ALDH(hi)CD44(+) cells displayed enhanced metastatic behavior in vitro and in vivo relative to ALDH(low)CD44(-) cells. The goal of this study was to test the hypothesis that ALDH(hi)CD44(+) breast cancer cells are more resistant to standard cancer therapy, and that inhibiting ALDH activity through all-trans retinoic acid (ATRA) or the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) sensitizes these cells to treatment. ALDH(hi)CD44(+) and ALDH(low)CD44(-) populations were isolated from MDA-MB-231 and MDA-MB-468 cells lines and exposed to chemotherapy (doxorubicin/paclitaxel) or radiotherapy ± ATRA or DEAB. Cell populations were assessed for differences in survival, colony formation, and protein expression related to therapy resistance and differentiation. Significantly more ALDH(hi)CD44(+) cells survived chemotherapy/radiotherapy relative to ALDH(low)CD44(-) cells (P < 0.001). Glutathione-S-transferase pi, p-glycoprotein, and/or CHK1 were overexpressed in ALDH(hi)CD44(+) populations compared with ALDH(low)CD44(-) populations (P < 0.05). Pre-treatment of cell populations with DEAB or ATRA had no effect on ALDH(low)CD44(-) cells, but resulted in significant initial sensitization of ALDH(hi)CD44(+) cells to chemotherapy/radiotherapy. However, only DEAB had a long-term effect, resulting in reduced colony formation (P < 0.01). ATRA also significantly increased expression of CK8/18/19 in MDA-MB-468 ALDH(hi)CD44(+) cells compared with control (P < 0.05). Our novel findings indicate that ALDH(hi)CD44(+) breast cancer cells contribute to both chemotherapy and radiation resistance and suggest a much broader role for ALDH in treatment response than previously reported.

Topics: Aldehyde Dehydrogenase; Antineoplastic Agents; Benzaldehydes; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Female; Humans; Hyaluronan Receptors; Neoplastic Stem Cells; Radiation Tolerance; Tretinoin

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoid-based protocols. Here, we demonstrate that 23-32% of the human ERBB2(+) breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARα. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARα agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib+ATRA. Induction of the retinoid-dependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFβ pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARα.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Synergism; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Amplification; Humans; Lapatinib; Phosphorylation; Quinazolines; Receptor, ErbB-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Smad3 Protein; Transcription, Genetic; Transcriptome; Transforming Growth Factor beta; Tretinoin

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Iodine; Isoenzymes; MAP Kinase Signaling System; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; rac1 GTP-Binding Protein; Rats; Symporters; Thyroid Gland; Tretinoin

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Humans; Inflammation; Interleukin-6; Neoplastic Stem Cells; NF-kappa B; Phenanthrenes; Pioglitazone; PPAR gamma; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Thiazolidinediones; Tretinoin

VN/12-1 is a novel retinoic acid metabolism blocking agent discovered in our laboratory. The purpose of the study was to elucidate the molecular mechanism of anticancer activity of VN/12-1 in breast cancer cell lines and in tumor xenografts. We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR-3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and in combination with autophagy inhibitor chloroquine in an SKBR-3 mouse xenograft model. Short exposure of low dose (<10 μmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G(1)-S phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1 enhanced anticancer activity induced by VN/12-1 in SKBR-3 cells by triggering apoptosis. Importantly, VN/12-1 (5 mg/kg twice weekly) and the combination of VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (P < 0.001 vs. control) and 96.2% (P < 0.001 vs. control), respectively. Our novel findings suggest that VN/12-1 may be useful as a single agent or in combination with autophagy inhibitors for treating human breast cancers. Our data provides a strong rationale for clinical evaluation of VN/12-1 as single agent or in combination with autophagy inhibitors.

Topics: Animals; Apoptosis; Autophagy; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Drug Synergism; Female; Humans; Imidazoles; Mice; Mice, SCID; Tretinoin; Xenograft Model Antitumor Assays

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.

Topics: Alanine; Animals; Antineoplastic Agents; Aspartic Acid; Breast Neoplasms; Cattle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Class Ia Phosphatidylinositol 3-Kinase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Female; Humans; Mutation; Neoplasm Invasiveness; Phosphorylation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Serine; Signal Transduction; src Homology Domains; Time Factors; Transfection; Tretinoin

Melatonin has been shown to inhibit breast cancer cell growth in numerous studies. However, our understanding of the therapeutic effects of this hormone is still marginal and there is little information concerning its combination with other antitumor agents to achieve additional potential benefits. All-trans retinoic acids or somatostatin have been used in combination with melatonin in several pre-clinical and clinical trials, but they have never been combined altogether as an anti-breast cancer treatment. In the present study, we investigated whether the association of melatonin, all-trans retinoic acid and somatostatin leads to an enhanced anticancer activity in MCF-7 breast cancer cells. In such conditions, MCF-7 cells were investigated for cell growth/viability and proliferation, as well as for the expression of cyclin A, and components of the Notch and EGFR pathways, by Western blotting and confocal immunofluorescence. Electrophysiological, morphological, and biochemical analysis were also performed to reveal signs of cell damage and death. We found that melatonin in combination with all-trans retinoic acid and somatostatin potentiated the effects of melatonin alone on MCF-7 cell viability and growth inhibition; this phenomenon was associated with altered conductance through Ca²⁺ and voltage-activated K⁺ (BK) channels, and with substantial impairments of Notch-1 and epidermal growth factor (EGF)-mediated signaling. The combined treatment also caused a marked reduction in mitochondrial membrane potential and intracellular ATP production as well as induction of necrotic cell death. Taken together our results indicate that co-administration of melatonin with all-trans retinoic acid and somatostatin may be of significant therapeutic benefit in breast cancer.

Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Large-Conductance Calcium-Activated Potassium Channels; Melatonin; Membrane Potential, Mitochondrial; Somatostatin; Tretinoin

The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.. FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.. Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; COUP Transcription Factor II; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Grading; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, Retinoic Acid; Repetitive Sequences, Nucleic Acid; RNA-Binding Proteins; Tissue Array Analysis; Transcriptional Activation; Tretinoin

Breast cancers expressing hormone receptors for estrogen (ER) and progesterone (PR) represent ~70% of all cases and are treated with both ER-targeted and chemotherapies, with near 40% becoming resistant. We have previously described that in some ER(+) tumors, the resistant cells express cytokeratin 5 (CK5), a putative marker of breast stem and progenitor cells. CK5(+) cells have lost expression of ER and PR, express the tumor-initiating cell surface marker CD44, and are relatively quiescent. In addition, progestins, which increase breast cancer incidence, expand the CK5(+) subpopulation in ER(+)PR(+) breast cancer cell lines. We have developed models to induce and quantitate CK5(+)ER(-)PR(-) cells, using CK5 promoter-driven luciferase (Fluc) or green fluorescent protein (GFP) reporters stably transduced into T47D breast cancer cells (CK5Pro-GFP or CK5Pro-Luc). We validated the CK5Pro-GFP-T47D model for high-content screening in 96-well microplates and performed a pilot screen using a focused library of 280 compounds from the National Institutes of Health clinical collection. Four hits were obtained that significantly abrogated the progestin-induced CK5(+) cell population, three of which were members of the retinoid family. Hence, this approach will be useful in discovering small molecules that could potentially be developed as combination therapies, preventing the acquisition of a drug-resistant subpopulation.

Topics: Acitretin; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Discovery; Drug Resistance, Neoplasm; Female; Genes, Reporter; Green Fluorescent Proteins; Humans; Hyaluronan Receptors; Isotretinoin; Keratin-5; Luciferases; Miconazole; Neoplastic Stem Cells; Progesterone; Progestins; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Progesterone; Retinoids; Small Molecule Libraries; Tretinoin

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3.

Topics: Alitretinoin; Amino Acid Motifs; Amino Acid Sequence; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chlorocebus aethiops; COS Cells; Embryo, Nonmammalian; Embryonic Development; Female; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Humans; Ligands; Microinjections; Models, Biological; Molecular Sequence Data; Proliferating Cell Nuclear Antigen; Protein Binding; Protein Stability; Replication Protein C; Retinoid X Receptor alpha; Retinoids; Sea Urchins; Tretinoin

In addition to estrogen receptor modulators, retinoic acid and other retinoids are promising agents to prevent breast cancer. Retinoic acid and estrogen exert antagonistic regulations on the transcription of coding genes and we evaluated here whether these two compounds have similar effects on microRNAs. Using an integrative approach based on several bioinformatics resources together with experimental validations, we indeed found that retinoic acid positively regulates miR-210 and miR-23a/24-2 expressions and is counteracted by estrogen. Conversely, estrogen increased miR-17/92 and miR-424/450b expressions and is inhibited by retinoic acid. In silico functional enrichment further revealed that this combination of transcriptional/post-transcriptional regulations fully impacts on the molecular effects of estrogen and retinoic acid. Besides, we unveiled a novel effect of retinoic acid on aerobic glycolysis. We specifically showed that it increases extracellular lactate production, an effect counteracted by the miR-210 and the miR-23a/24-2, which simultaneously target lactate dehydrogenase A and B mRNAs. Together our results provide a new framework to better understand the estrogen/retinoic acid antagonism in breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogens; Female; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactic Acid; MicroRNAs; Transcriptome; Tretinoin

To explore the effects of all trans retinoic acid (ATRA) on the cell proliferation and expression alterations of beta-protein 1 (BP1) in human breast cancer cells lines of MDA-MB-468 and MCF-7.. The proliferation changes were detected by thiazolyl blue tetrazolium bromide (MTT) after the treatment of ATRA. At the dose of 10(-5) mol/L ATRA, the expression of BP1 was measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry.. After the treatment of ATRA, the proliferation of cells and the expression of BP1 decreased. Optical density ratio (ODR) of each group decreased from 0.85 ± 0.01, 0.71 ± 0.01 to 0.75 ± 0.02, 0.72 ± 0.06 at 24 h, 0.55 ± 0.01, 0.52 ± 0.05 at 48 h and 0.34 ± 0.02, 0.48 ± 0.03 at 72 h. Significant differences existed among different time groups (P < 0.01). The mean optical density (MOD) of each group decreased from 0.509 ± 0.081, 0.826 ± 0.015 to 0.509 ± 0.081, 0.826 ± 0.015 at 24 h, 0.270 ± 0.022, 0.641 ± 0.041 at 48 h and 0.145 ± 0.019 and 0.206 ± 0.179 at 72 h. Significant differences existed among different time groups (P < 0.01).. ATRA can inhibit the proliferation and the expression of BP1 in breast cancer cells. And BP1 gene may become a therapeutic target for the ATRA-mediated inhibited growth of breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Homeodomain Proteins; Humans; Transcription Factors; Tretinoin

The sodium iodide symporter (NIS) mediates the active iodide uptake in the thyroid gland as well as lactating breast tissue. Recently, we reported significant stimulation of all-trans retinoic acid (atRA)-induced NIS expression in the estrogen-receptor positive human breast cancer cell line MCF-7 by dexamethasone (Dex) in vitro and in vivo, which might offer the potential to image and treat breast cancer with radioiodine. In this study, based on its known interaction with the pregnane-X-receptor (PXR) forming a heterodimer with the retinoid-X-receptor (RXR), we examined the effect of carbamazepine (CBZ), a potent activator of PXR, on atRA-induced NIS expression and therapeutic efficacy of (131)I in MCF-7 cells. For this purpose, functional NIS expression in MCF-7 cells was examined by iodide uptake assay, quantitative real-time PCR as well as Western blot analysis, followed by investigation of (131)I cytotoxicity in vitro after incubation with CBZ (4, 25, 100 μM) in the presence of atRA (1 μM) with or without Dex (100 nM). Incubation with CBZ stimulated atRA-induced iodide accumulation up to twofold in a concentration-dependent manner, while atRA/Dex-stimulated iodide uptake was further stimulated up to 1.5-fold by additional CBZ treatment based on significantly increased NIS mRNA and protein levels. This stimulatory effect of CBZ was shown to be dependent on the PI3K-Akt pathway without involvement of mTOR. In contrast, treatment with CBZ alone had no effect on functional NIS expression. Moreover, selective cytotoxicity of (131)I was significantly increased from approximately 20% in MCF-7 cells treated with atRA alone to 50% after treatment with CBZ in the presence of atRA, which was further enhanced to 90% after combined treatment with atRA/Dex/CBZ. In conclusion, CBZ represents another potent stimulator of atRA-induced functional NIS expression resulting in an enhanced selective killing effect of (131)I in MCF-7 breast cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Carbamazepine; Cell Line, Tumor; Cell Survival; Dexamethasone; Female; Humans; Iodine Radioisotopes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; Retinoid X Receptors; Symporters; TOR Serine-Threonine Kinases; Tretinoin; Tumor Stem Cell Assay

Aromatase converts androgens into estrogens and is thought to supply a local source of estrogen that facilitates the growth of hormone-responsive tumor cells. Inhibition of aromatase is therefore an important chemopreventive strategy. We investigated the effect of retinol and selected retinoids on the activity and expression of aromatase in two human carcinoma cell lines in vitro. Retinol (ROH) and all-trans retinoic acid (ATRA) significantly inhibited aromatase activity in a concentration-dependent manner in microsomes isolated from JEG-3 human placental carcinoma cells, whereas 9-cis and 13-cis retinoic acid had significant inhibitory activity only at the highest concentrations tested. Similar results were observed in an assay of cellular aromatase activity in MCF-7 human breast cancer cells. Enzyme kinetic studies by double-reciprocal plot demonstrated that ROH inhibited microsomal aromatase activity in a mixed manner. In addition, ROH suppressed both the basal and cAMP-induced expression of aromatase mRNA in MCF-7 cells and inhibited transcription controlled by a cAMP-responsive element. These results suggest that aromatase activity and expression are a molecular target of ROH and chemopreventive retinoids, an activity that may underlie, in part, their inhibitory effects on hormone-dependent cancer.

Topics: Aromatase; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; RNA, Messenger; Tretinoin; Vitamin A; Vitamins

Crocetin is a carotenoid dicarboxylic acid which, in nature, is esterified with glucose or gentiobiose units forming the crocins, abundant components of saffron (a spice with many reputed medicinal uses). We have previously reported that saffron, crocins and crocetin inhibit breast cancer cell proliferation. In order to further study the effect of crocetin on breast cancer cells, we used the highly invasive MDA-MB-231 cells and measured the viability with the WST-1 assay and the invasiveness through a reconstituted basement membrane. After 24 h incubation, crocetin significantly inhibited not only proliferation but also invasion at 1 and 10 µM. Cancer invasiveness and metastasis are associated with the expression of matrix metalloproteinases (MMPs). In order to study the molecular changes of MMP expression that might accompany the observed crocetin effects, gene expression of MMPs was studied by RT-PCR, whereas protein expression and gelatinolytic activity were determined with Western blotting and zymography, respectively. The gene and protein expression of pro-MT1-MMP and pro-MT2-MMP were greatly attenuated by both crocetin and all- TRANS-retinoic acid (ATRA, used as control). Incubation with 10 µM crocetin for 24 h in serum-free conditions reduced pro-MMP-9 activity and pro-MMP-2/MMP-2 protein levels. When cultured in media with sera 2 and 5 %, crocetin at 10 μΜ also reduced gelatinase activity. The above findings show that crocetin, the main metabolite of crocins, inhibits MDA-MB-231 cell invasiveness via downregulation of MMP expression.

Topics: Anticarcinogenic Agents; Antioxidants; Breast Neoplasms; Carotenoids; Cell Line, Tumor; Cell Proliferation; Crocus; Down-Regulation; Female; Flowers; Gelatinases; Humans; Matrix Metalloproteinases; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Vitamin A

The relapse of cancer is mostly due to the proliferation of cancer stem cells which could not be eliminated by a standard chemotherapy. A new kind of all-trans retinoic acid stealth liposomes was developed for preventing the relapse of breast cancer and for treating the cancer in combination with a cytotoxic agent, vinorelbine stealth liposomes. In vitro studies were performed on the human breast cancer MCF-7 and MDA-MB-231 cells. In vivo evaluations were performed on the newly established relapse model with breast cancer stem cells. Results showed that the particle size of all-trans retinoic acid stealth liposomes was approximately 80nm, and the encapsulation efficiency was >90%. Breast cancer stem cells were identified with the CD44(+)/CD24(-) phenotype and characterized with properties: resistant to cytotoxic agent, stronger capability of proliferation, and stronger capability of differentiation. Inhibitory effect of all-trans retinoic acid stealth liposomes was more potent in cancer stem cells than in cancer cells. The mechanisms were defined to be two aspects: arresting breast cancer stem cells at the G(0)/G(1) phase in mitosis, and inducing the differentiation of breast cancer stem cells. The cancer relapse model was successfully established by xenografting breast cancer stem cells into NOD/SCID mice, and the formation and growth of the xenografted tumors were significantly inhibited by all-trans retinoic acid stealth liposomes. The combination therapy of all-trans retinoic acid stealth liposomes with vinorelbine stealth liposomes produced the strongest inhibitory effect to the relapse tumor model. It could be concluded that all-trans retinoic acid stealth liposomes could be used for preventing the relapse of breast cancer by differentiating cancer stem cells and arresting the cell-cycle, and for treating breast cancer as a co-therapy, thus providing a novel strategy for treating breast cancer and preventing relapse derived from breast cancer stem cells.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Female; Humans; Liposomes; Mice; Mice, SCID; Neoplastic Stem Cells; Recurrence; Tretinoin; Vinblastine; Vinorelbine

Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα(+)) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα(+) cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα(+) cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5'-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; MicroRNAs; Receptors, Estrogen; Tissue Plasminogen Activator; Transcriptional Activation; Tretinoin

Na(+)/I(-) symporter (NIS)-mediated iodide uptake into thyroid follicular cells serves as the basis of radioiodine therapy for thyroid cancer. NIS protein is also expressed in the majority of breast tumors, raising potential for radionuclide therapy of breast cancer. KT5823, a staurosporine-related protein kinase inhibitor, has been shown to increase thyroid-stimulating hormone-induced NIS expression, and thus iodide uptake, in thyroid cells. In this study, we found that KT5823 does not increase but decreases iodide uptake within 0.5 h of treatment in trans-retinoic acid and hydrocortisone-treated MCF-7 breast cancer cells. Moreover, KT5823 accumulates hypoglycosylated NIS, and this effect is much more evident in breast cancer cells than thyroid cells. The hypoglycosylated NIS is core glycosylated, has not been processed through the Golgi apparatus, but is capable of trafficking to the cell surface. KT5823 impedes complex NIS glycosylation at a regulatory point similar to brefeldin A along the N-linked glycosylation pathway, rather than targeting a specific N-glycosylated site of NIS. KT5823-mediated effects on NIS activity and glycosylation are also observed in other breast cancer cells as well as human embryonic kidney cells expressing exogenous NIS. Taken together, KT5823 will serve as a valuable pharmacological reagent to uncover mechanisms underlying differential NIS regulation between thyroid and breast cancer cells at multiple levels.

Topics: Animals; Breast Neoplasms; Brefeldin A; Carbazoles; Cell Line; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Hydrocortisone; Iodides; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Rats; Symporters; Thyroid Gland; Thyrotropin; Tretinoin

The presence, relevance and regulation of the Sodium Iodide Symporter (NIS) in human mammary tissue remains poorly understood. This study aimed to quantify relative expression of NIS and putative regulators in human breast tissue, with relationships observed further investigated in vitro.. Human breast tissue specimens (malignant n = 75, normal n = 15, fibroadenoma n = 10) were analysed by RQ-PCR targeting NIS, receptors for retinoic acid (RARα, RARβ), oestrogen (ERα), thyroid hormones (THRα, THRβ), and also phosphoinositide-3-kinase (PI3K). Breast cancer cells were treated with Retinoic acid (ATRA), Estradiol and Thyroxine individually and in combination followed by analysis of changes in NIS expression.. The lowest levels of NIS were detected in normal tissue (Mean(SEM) 0.70(0.12) Log(10) Relative Quantity (RQ)) with significantly higher levels observed in fibroadenoma (1.69(0.21) Log(10)RQ, p<0.005) and malignant breast tissue (1.18(0.07) Log(10)RQ, p<0.05). Significant positive correlations were observed between human NIS and ERα (r = 0.22, p<0.05) and RARα (r = 0.29, p<0.005), with the strongest relationship observed between NIS and RARβ (r = 0.38, p<0.0001). An inverse relationship between NIS and PI3K expression was also observed (r =  0.21, p<0.05). In vitro, ATRA, Estradiol and Thyroxine individually stimulated significant increases in NIS expression (range 6-16 fold), while ATRA and Thyroxine combined caused the greatest increase (range 16-26 fold).. Although NIS expression is significantly higher in malignant compared to normal breast tissue, the highest level was detected in fibroadenoma. The data presented supports a role for retinoic acid and estradiol in mammary NIS regulation in vivo, and also highlights potential thyroidal regulation of mammary NIS mediated by thyroid hormones.

Topics: Breast Neoplasms; Estradiol; Female; Fibroadenoma; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; Symporters; Thyroid Hormones; Thyroxine; Tretinoin

The sodium iodide symporter (NIS) mediates active iodide uptake in lactating breast tissue, and when its levels are enhanced by all-trans retinoic acid (atRA), NIS has been proposed as a target for the imaging and radiotherapy of breast cancer. Importantly, the estrogen receptor α (ERα) is an important regulator of atRA induced NIS gene expression in breast cancer cells. In this study, we investigated the effect of an ER agonist (17β-estradiol, E(2)) or antagonist [trans-hydroxytamoxifen (TOT) or raloxifene (RAL)] treatment on the regulation of NIS gene expression and iodide uptake in an ERα-positive breast cancer (MCF-7) model.. NIS functional activity was measured in vitro by (125)I uptake assay after incubation with E(2) (from 10(-15) to 10(-5) M), TOT (from 5×10(-8) to 5×10(-6) M), or RAL (from 5×10(-8) to 5×10(-6) M) in the presence or absence of atRA (10(-7) M). Under the same conditions, NIS mRNA expression was examined by reverse transcriptase polymerase chain reaction. Athymic mice with MCF-7 xenograft tumors were treated with atRA alone or atRA together with E(2) to evaluate the change of (125)I uptake in tumor tissues in vivo.. In the iodide uptake study in cells, E(2), TOT, or RAL treatment alone did not stimulate (125)I uptake. However, when iodide uptake was stimulated by atRA, cotreatment with E(2), TOT or RAL decreased (125)I uptake in a concentration-dependent manner. The hormone effects on NIS mRNA expression levels in MCF-7 cells were similar. The results of the in vivo biodistribution study showed that (125)I uptake was reduced 50% in tumor tissues of mice treated with atRA/E(2) as compared to tumors treated only with atRA.. Our results suggest that combination treatment of atRA and ER ligands could limit the functional activity of the NIS gene induced by atRA, thereby compromising its use as a target for diagnosis or radiotherapy in breast cancer.

Topics: Animals; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Interactions; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Ligands; Mice; Mice, Inbred BALB C; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; Symporters; Tamoxifen; Tretinoin; Xenograft Model Antitumor Assays

Recent studies using animal models suggest that expression of FABP5 drives the stimulation of cell growth observed in estrogen receptor (ER)-negative breast cancer cells on exposure to retinoic acid (RA). The purpose of this study was to investigate the clinicopathological significance of FABP5 in breast cancer and to evaluate FABP5 as a prognostic marker and a possible novel therapeutic target in breast cancer. Gene expression microarray analysis revealed a significant correlation between elevated FABP5 RNA levels and ER/progesterone receptor (PR)-negative status, high tumor grade, and poor prognosis. Tissue microarray analysis demonstrated similar correlations with cytoplasmic FABP5 protein. Based on multivariate proportional regression analysis, cytoplasmic FABP5 is a significant and independent prognostic marker of overall survival and recurrence-free survival in breast cancer. The effects of FABP5 on tumor growth appear to be mediated primarily through cytoplasmic FABP, because no correlation was found between nuclear FABP5 and ER/PR-negative status, recurrence, and survival. FABP5 knockdown in breast cancer cell lines demonstrates a correlation between FABP5 levels and growth response to RA. We propose a model whereby growth-promoting FABP5 competes with growth-inhibiting CRABP2 for RA, with retention of RA in the cytoplasm by FABP5 preventing the inhibition of tumor growth.

Topics: Aged; Antibody Specificity; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytoplasm; Fatty Acid-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Models, Biological; Multivariate Analysis; Prognosis; Proportional Hazards Models; Receptors, Estrogen; Receptors, Progesterone; Receptors, Retinoic Acid; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis; Tretinoin

The native fluorescence spectra of retinoic acid (RA)-treated and untreated human breast cancerous cells excited with the selective wavelengths of 300 nm and 340 nm were measured and analyzed using a blind source separation method namely Nonnegative Matrix Factorization (NMF). The results show that the fluorophores of human malignant breast cells change their compositions when they are treated with RA. The reduced contribution from tryptophan, NADH and flavin to the fluorescence of the treated breast cancerous cells was observed in comparison with that of the untreated cells. The results indicate that the decrease of adenosine triphosphate (ATP) in the RA-treated cells. The possible clinical applications of this native fluorescence study are discussed.

Topics: Algorithms; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Flavins; Humans; NAD; Reference Standards; Sensitivity and Specificity; Spectrometry, Fluorescence; Tretinoin; Tryptophan

Icb-1 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. To further elucidate the function of the icb-1 gene in differentiation of breast and endometrial cancer cells, we knocked down its expression by means of shRNA transfection. Knockdown of icb-1 inhibited the vitamin D3-induced up-regulation of E-cadherin expression in both MCF-7 and HEC-1B cells. Induction of E-cadherin expression by all-trans retinoic acid (ATRA) was also blocked in both cell lines expressing icb-1 siRNA. Examination of icb-1 and E-cadherin expression in 66 breast cancer tissue samples revealed a significant positive correlation between the two genes. In MCF-7 cells, silencing of the icb-1 gene inhibited the ATRA- and the vitamin D3-induced up-regulation of lactoferrin and estrogen receptor β expression. The data of our knockdown study suggest that icb-1 may act as a mediator of differentiation signals in breast cancer cells induced by ATRA or vitamin D3. These findings together with the observed co-expression of icb-1 with E-cadherin in breast cancer samples support an important role of the icb-1 gene in cancer cell differentiation.

Topics: Breast Neoplasms; Cadherins; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genital Neoplasms, Female; Humans; Intracellular Signaling Peptides and Proteins; Lactoferrin; Neoplasm Proteins; RNA, Small Interfering; Tretinoin

All trans-retinoic acid (ATRA) induces apoptosis and/or differentiation in solid tumors, including breast cancer, and has become a therapeutic tool in this disease. In human promyelocytic leukemia ATRA reduces the expression of cellular procoagulant activities (PCA), i.e. tissue factor (TF) and cancer procoagulant (CP). There are no studies on the effects of ATRA on the PCA of solid tumors, i.e. breast cancer cells. We analyzed different human breast cancer cell lines in order to: 1. characterize the expression of TF and CP; 2. evaluate whether these activities are affected by ATRA; and 3. verify whether a reduction in tumor cell procoagulants may occur in association to apoptosis and growth inhibition induced by ATRA. Two estrogen receptor positive (ER-positive; i.e. MCF7 and ZR75.1) and one estrogen receptor negative (ER-negative; i.e. MDA.MB.231) cell lines were included into the study. The results show that ATRA affected TF in a dose-dependent fashion only in ER-positive cell lines. In particular, at 1 uM ATRA, TF significantly (p < 0.05) decreased by 57%, 44% in MCF7, ZR75.1 cells, respectively. Differently the results show that ATRA dose-dependently affected CP expression in all three cell lines. Specifically, at 1 uM ATRA, CP significantly decreased by 44%, 50% and 25% in MCF7, ZR75.1, and MDA.MB.231. Only in ER-positive cell lines, there was a dose-dependent inhibition of cell growth that became statistically significant at 1 uM ATRA, which was associated to a slight but significant increase in the percentage of apoptotic cells. In conclusion, this study demonstrates for the first time that ATRA downregulates the expression of TF and CP in breast cancer cells. Due to the pivotal role of coagulation activation in tumor progression, the capacity of ATRA to affect also tumor procoagulants, in parallel to cell apoptosis, open new perspectives in tumor therapy.

Topics: Antineoplastic Agents; Apoptosis; Blood Coagulation; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Receptors, Estrogen; RNA, Messenger; Thromboplastin; Time Factors; Tretinoin

The combined treatment with nanomolar doses of the PPARγ ligand Rosiglitazone (BRL) and the RXR ligand 9-cis‑retinoic acid (9RA) induces a p53-dependent apoptosis in MCF7, SKBR3 and T47D human breast cancer cells. Since MCF7 cells express a wild-type p53 protein, while SKBR3 and T47D cells harbor endogenous mutant p53, we elucidated the mechanism through which PPARγ and RXR ligands triggered apoptotic processes independently of p53 transcriptional activity. We showed an upregulation of Bid expression enhancing the association between Bid/p53 in both cytosol and mitochondria after the ligand treatment. Particularly in the mitochondria, the complex involves the truncated Bid that plays a key role in the apoptotic process induced by BRL and 9RA, since the disruption of mitochondrial membrane potential, the induction of PARP cleavage and the percentage of TUNEL-positive cells were reversed after knocking down Bid. Moreover, PPARγ and RXR ligands were able to reduce mitochondrial GST activity, which was no longer noticeable silencing Bid expression, suggesting the potential of Bid in the regulation of mitochondrial intracellular reactive oxygen species scavenger activity. Our data, providing new insight into the role of p53/Bid complex at the mitochondria in promoting breast cancer cell apoptosis upon low doses of PPARγ and RXR ligands, address Bid as a potential target in the novel therapeutical strategies for breast cancer.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Ligands; Mitochondria; PPAR gamma; Retinoid X Receptors; RNA Interference; RNA, Small Interfering; Rosiglitazone; Thiazolidinediones; Tretinoin; Tumor Suppressor Protein p53; Up-Regulation

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17β-estradiol (E₂) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E₂-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E₂-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells.

Topics: Breast Neoplasms; Cell Proliferation; Electrophoretic Mobility Shift Assay; Estradiol; Estrogen Receptor alpha; Female; Genes, erbB-1; HeLa Cells; Humans; Ligands; Promoter Regions, Genetic; Protein Binding; Receptor Cross-Talk; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Sp1 Transcription Factor; Transcriptional Activation; Transfection; Tretinoin

RET, a gene causatively mutated in Hirschsprung disease and cancer, has recently been implicated in breast cancer estrogen (E2) independence and tamoxifen resistance. RET displays both E2 and retinoic acid (RA)-dependent transcriptional modulation in E2-responsive breast cancers. However, the regulatory elements through which the steroid hormone transcriptional regulation of RET is mediated are poorly defined. Recent genome-wide chromatin immunoprecipitation-based studies have identified 10 putative E2 receptor-alpha (ESR1) and RA receptor alpha-binding sites at the RET locus, of which we demonstrate only two (RET -49.8 and RET +32.8) display significant E2 regulatory response when assayed independently in MCF-7 breast cancer cells. We demonstrate that endogenous RET expression and RET -49.8 regulatory activity are cooperatively regulated by E2 and RA in breast cancer cells. We identify key sequences that are required for RET -49.8 and RET +32.8 E2 responsiveness, including motifs known to be bound by ESR1, FOXA1 and TFAP2C. We also report that both RET -49.8 regulatory activity and endogenous RET expression are completely dependent on ESR1 for their (E2)-induction and that ESR1 is sufficient to mediate the E2-induced enhancer activity of RET -49.8 and RET +32.8. Finally, using zebrafish transgenesis, we also demonstrate that RET -49.8 directs reporter expression in the central nervous system and peripheral nervous system consistent with the endogenous ret expression. Taken collectively, these data suggest that RET transcription in breast cancer cells is modulated by E2 via ESR1 acting on multiple elements collectively.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Enhancer Elements, Genetic; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Protein Binding; Proto-Oncogene Proteins c-ret; Response Elements; Tretinoin; Zebrafish

We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoprecipitation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; SOX9 Transcription Factor; Transcription Factor HES-1; Transfection; Tretinoin

Agents capable of increasing radioiodine concentration by stimulating the sodium/iodide symporter (NIS) expression have been extensively investigated for the treatment of certain well-differentiated breast cancers.. In this study, we analyzed the regulation of the NIS and lactoperoxidase (LPO) gene expression in 4 different human breast cancer cell lines, representative of different histotypes of breast cancer.. MCF-7, T-47D, MDA-MB231, and HCC-1937 (the latter carrying the BRCA-1 mutation) were exposed to different stimulators and the levels of NIS and LPO mRNA measured by a quantitative RT-PCR.. All-trans-Retinoic Acid (RA), Dexamethasone (DEX), Trichostatin A (TSA), and Sodium Butyrate (NaB) induced the expression of NIS mRNA in MCF-7 and T-47D cell lines, whereas HCC-1937 and MBA-MB231 were slightly responsive only to the histone-deacetylase inhibitors TSA and NaB. Minor stimulatory effects were detected on LPO mRNA in MCF-7 and T-47D treated with TSA and NaB or RA only in MCF-7, while no effect was detectable in the other two cell lines.. These data indicate that retinoic acid, alone or in combination with DEX, as well as HDAC-inhibitors are very promising agents for a radioiodine- based therapy in a large spectrum of breast cancers, including neoplasms from both basal and ductal cells, especially for the well-differentiated estrogen-dependent tumors. Other molecules or other drug combinations should be tested to extend the same strategy to the less differentiated and more aggressive tumor cells, including those carrying the BRCA mutation.

Topics: Breast Neoplasms; Butyrates; Cell Line, Tumor; Dexamethasone; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lactoperoxidase; RNA, Messenger; Symporters; Tretinoin

It is known that all-trans retinoic acid (RA) is a useful therapeutic anticancer agent in breast cancer that acts by inducing apoptosis and growth inhibition. Insulin-like growth factor-I (IGF-I) is also known to be a growth hormone that plays an important role in cell proliferation and apoptosis. We examined the relationships between RA-induced protein kinase C (PKC)-delta, the secretion and synthesis of IGF-I, and oxidative stress. RA at 10(-8)M and 10(-7)M increased PKC-delta phosphorylation (the ratio of phosphorylated to total PKC-delta) (p<0.05) and decreased the secretion and synthesis of IGF-I (p<0.05) compared to control, with the effects peaking for treatment with 10(-7)M RA for 72h. The silencing of PKC-delta prevented the RA-induced inhibition of the secretion and synthesis of IGF-I and cell viability (p<0.05). Application of 10(-7)M RA for 72h increased the level of thiobarbituric-acid-reactive substances and the expression of inducible nitric oxide synthase relative to control (p<0.05). These increases were blocked by suppressing PKC-delta and by pretreatment with the antioxidants glutathione and diphenyleneiodonium (p<0.05). These antioxidants also reversed the RA-induced inhibition of the secretion and synthesis of IGF-I and cell viability to control levels (p<0.05). The effects of suppressing IGF-I demonstrate that IGF-I plays a critical role in the RA-induced inhibition of the cell viability. These results indicate that the anticancer effects of RA are mediated by inhibition of the secretion and synthesis of IGF-I, and involve a PKC-delta-dependent mechanism, and they provide evidence of an interaction between PKC-delta and reactive oxygen species.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Female; Gene Expression Regulation; Humans; Insulin-Like Growth Factor I; Oxidative Stress; Phosphorylation; Protein Kinase C-delta; Reactive Oxygen Species; Time Factors; Tretinoin; Up-Regulation

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis in animal models and elevated risk for a number of human cancers. Here, we found that CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, is highly expressed in 42% (27/65) of primary breast cancers. We also showed that enhanced expression of CYP26A1 suppresses cellular responses to anoikis and consequently promotes anchorage-independent growth. This transformed phenotype was sufficient to markedly increase tumorigenic and metastatic potential. Suppression of CYP26A1 significantly reversed the CYP26A1-mediated oncogenic characteristics, suggesting a direct link between intracellular RA status and tumorigenicity. Our observations provide strong evidence for oncogenic and cell survival properties of CYP26A1 in carcinogenesis, and suggest mechanisms whereby VAD might promote cancer development.

Topics: Animals; Anoikis; Breast Neoplasms; Carcinogenicity Tests; Carcinogens; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cytochrome P-450 Enzyme System; Disease Models, Animal; Drug Interactions; Gene Expression Profiling; Humans; Mice; Mice, Knockout; Neoplasms; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured; Vitamin A; Vitamin A Deficiency

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)2D3 called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D3-MARRS expression modulates 1,25(OH)2D3 activity in breast cancer cells. Relative levels of 1,25D3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D(3)-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)2D3 ( IC(50) 56+/-24 nM) compared to controls (319+/-181 nM; P<0.05). Reduction in 1,25D3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)2D3. Knockdown of 1,25D3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)2D3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D3-MARRS expression or activity as anticancer agents.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Isoflavones; Paclitaxel; Receptors, Calcitriol; RNA, Catalytic; Tretinoin; Vitamin D

Retinoids, through their cognate nuclear receptors, exert potent effects on cell growth, differentiation and apoptosis, and have significant promise for cancer therapy and chemoprevention. These ligands can determine the ultimate fate of target cells by stimulating or repressing gene expression directly, or indirectly through crosstalking with other signal transducers.. Using different breast cancer cell models, we show here that depending on the cellular context retinoids can signal either towards cell death or cell survival. Indeed, retinoids can induce the expression of pro-apoptotic (i.e. TRAIL, TNF-Related Apoptosis-Inducing Ligand, Apo2L/TNFSF10) and anti-apoptotic (i.e. cIAP2, inhibitor of apoptosis protein-2) genes. Promoter mapping, gel retardation and chromatin immunoprecipitation assays revealed that retinoids induce the expression of this gene mainly through crosstalk with NF-kappaB. Supporting this crosstalk, the activation of NF-kappaB by retinoids in T47D cells antagonizes the apoptosis triggered by the chemotherapeutic drugs etoposide, camptothecin or doxorubicin. Notably apoptosis induced by death ligands (i.e. TRAIL or antiFAS) is not antagonized by retinoids. That knockdown of cIAP2 expression by small interfering RNA does not alter the inhibition of etoposide-induced apoptosis by retinoids in T47D cells reveals that stimulation of cIAP2 expression is not the cause of their anti-apoptotic action. However, ectopic overexpression of a NF-kappaB repressor increases apoptosis by retinoids moderately and abrogates almost completely the retinoid-dependent inhibition of etoposide-induced apoptosis. Our data exclude cIAP2 and suggest that retinoids target other regulator(s) of the NF-kappaB signaling pathway to induce resistance to etoposide on certain breast cancer cells.. This study shows an important role for the NF-kappaB pathway in retinoic acid signaling and retinoic acid-mediated resistance to cancer therapy-mediated apoptosis in breast cancer cells, independently of cIAP2. Our data support the use of NF-kappaB pathway activation as a marker for screening that will help to develop novel retinoids, or retinoid-based combination therapies with improved efficacy.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cytoprotection; Etoposide; Female; Humans; I-kappa B Proteins; Inhibitor of Apoptosis Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Promoter Regions, Genetic; Protein Binding; Receptors, Retinoic Acid; Signal Transduction; Transcription Factor RelA; Transcription, Genetic; Tretinoin; Ubiquitin-Protein Ligases

Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I(-)) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA-protein interaction assays revealed direct association between retinoic acid receptor-alpha (RARalpha) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Conserved Sequence; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Genomics; Humans; Introns; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA Polymerase II; Symporters; Transcription, Genetic; Tretinoin

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFbeta signaling pathway were induced by RA, and specific inhibition of the TGFbeta type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFbeta pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA.

Topics: Antigens, CD; Antineoplastic Agents; Breast Neoplasms; Cadherins; Cell Differentiation; Cell Fusion; Cell Line, Tumor; COUP Transcription Factor II; Endothelial Cells; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Receptors, Transforming Growth Factor beta; Tretinoin

DNA methylation is considered as a potential cause of aberrations in regulation of gene expression during carcinogenesis. Therefore, changes in DNA methylation patterns may be targets for chemoprevention. In the present study, we investigated effects of all-trans retinoic acid (ATRA), vitamin D(3), and resveratrol alone and in combination with adenosine analogues: 2-chloro-2'-deoxyadenosine (2CdA) and 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A), on methylation and expression of retinoic acid receptor beta 2 (RAR beta 2) in MCF-7 and MDA-MB-231 breast cancer cell lines. Alterations in methylation and expression levels after treatment of cells with the tested compounds were evaluated by methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. RAR beta 2 promoter in the tested fragment was partially methylated in MCF-7 cells and non-methylated in MDA-MB-231 cells. In MCF-7 cells, all compounds, except for resveratrol, inhibited promoter methylation and increased expression of RAR beta 2. All natural compounds improved the action of 2CdA and F-ara-A on RAR beta 2 methylation and/or expression. Combination of ATRA or vitamin D(3) with 2CdA was the most effective. In MDA-MB-231 cells, only 2CdA, F-ara-A, and ATRA induced RAR beta 2 expression without any notable effects in combined treatment. Our results demonstrate that both natural compounds and adenosine analogues are able to reduce promoter methylation and/or induce expression of RAR beta 2 in non-invasive MCF-7 cells. Furthermore, the natural compounds improve effects of adenosine analogues, however only at early non-invasive stages of carcinogenesis.

Topics: Adenosine; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cholecalciferol; Cladribine; DNA Methylation; Drug Screening Assays, Antitumor; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Resveratrol; Stilbenes; Transcriptional Activation; Tretinoin; Vidarabine

HER2 and estrogen receptor (ER) are important in breast cancer and are therapeutic targets of trastuzumab (Herceptin) and tamoxifen, respectively. Retinoids inhibit breast cancer growth, and modulate signaling by HER2 and ER. We hypothesized that treatment with retinoids and simultaneous targeting of HER2 and/or ER may have enhanced anti-tumor effects.. The effects of retinoids combined with trastuzumab or tamoxifen were examined in two human breast cancer cell lines in culture, BT474 and SKBR3. Assays of proliferation, apoptosis, differentiation, cell cycle distribution, and receptor signaling were performed.. In HER2-overexpressing/ER-positive BT474 cells, combining all-trans retinoic acid (atRA) with tamoxifen or trastuzumab synergistically inhibited cell growth, and altered cell differentiation and cell cycle. Only atRA/trastuzumab-containing combinations induced apoptosis. BT474 and HER2-overexpressing/ER-negative SKBR3 cells were treated with a panel of retinoids (atRA, 9-cis-retinoic acid, 13-cis-retinoic acid, or N-(4-hydroxyphenyl) retinamide (fenretinide) (4-HPR)) combined with trastuzumab. In BT474 cells, none of the single agents except 4-HPR induced apoptosis, but again combinations of each retinoid with trastuzumab did induce apoptosis. In contrast, the single retinoid agents did cause apoptosis in SKBR3 cells; this was only modestly enhanced by addition of trastuzumab. The retinoid drug combinations altered signaling by HER2 and ER. Retinoids were inactive in trastuzumab-resistant BT474 cells.. Combining retinoids with trastuzumab maximally inhibits cell growth and induces apoptosis in trastuzumab-sensitive cells. Treatment with such combinations may have benefit for breast cancer patients.

Topics: Alitretinoin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Estrogen Antagonists; Female; Humans; Isotretinoin; Receptor, ErbB-2; Receptors, Estrogen; Retinoids; Tamoxifen; Trastuzumab; Tretinoin

The seeming impairment of retinoid metabolism in human breast tumor cells has been attributed to the lower expression of cellular retinol binding proteins (CRBPs), of alcohol/retinol dehydrogenases, or aldehyde/retinaldehyde dehydrogenases. In a previous study we indicated that xanthine dehydrogenase (XDH) is able to oxidize actively both all-trans-retinol (t-ROL) bound to the CRBP (holo-CRBP) and all-trans-retinaldehyde (t-RAL) to all-trans-retinoic acid (t-RA) in human mammary epithelial cells (HMEC). Since both XDH and CRBP are required for the biosynthesis of t-RA, we have inspected their bioavailability in both estrogen-responsive and nonresponsive human mammary epithelial cancer cells. The XDH activity, as assessed in the crude and purified extracts of both MCF7 and MDA-MB 231 cells by measuring the substrate t-RAL (that unlike t-ROL does not need CRBP), was 6 to 10 times lower than that previously encountered in normal HMEC. In addition, CRBP expression was absent in either cell line. Based on this preliminary evidence, we propose here that the low levels of XDH activity and the associated absence of CRBP in both MCF7 and MDA-MB 231 human breast cancer cells might be responsible for the retinoic acid deficiency observed in these cell model systems. This defect may be the crux of the impairment to stem cell differentiation and, hence, may be primarily implicated in human mammary carcinogenesis.

Topics: Breast Neoplasms; Cell Line; Cell Line, Tumor; Chromatography, High Pressure Liquid; Humans; Mammary Glands, Human; Radiometry; Retinol-Binding Proteins, Cellular; Tretinoin; Xanthine Dehydrogenase

Iodide uptake in the thyroid and breast is mediated by the sodium/iodide symporter (NIS). NIS activation is used for radioiodide imaging and therapeutic ablation of thyroid carcinoma. NIS is expressed in >70% of breast cancers but at a level insufficient for radioiodine treatment. All-trans retinoic acid (tRA) induces NIS gene expression and functional iodide uptake in human breast cancer cell lines and mouse breast cancer models. tRA usually regulates gene expression by direct interaction of RA receptor (RAR) with a target gene, but it can also act through nongenomic pathways. We report a direct influence of tRA treatment on the phosphoinositide 3-kinase (PI3K) signal transduction pathway that mediates tRA-induced NIS expression in MCF-7 breast cancer cells. MCF-7 cells express all three RAR isoforms, alpha, beta, and gamma, and RXRalpha. We previously identified RARbeta and RXRalpha as important for NIS induction by tRA. Treatment with LY294002, the PI3K inhibitor, or p85alpha knockdown with siRNA abolished tRA-induced NIS expression. Immunoprecipitation experiments and glutathione S-transferase pull-down assay showed a direct interaction between RARbeta2, RXRalpha, and p85alpha. RA also induced rapid activation of Akt in MCF-7 cells. Treatment with an Akt inhibitor or Akt knockdown with siRNA reduced NIS expression. These findings indicate that RA induction of NIS in MCF-7 cells is mediated by rapid activation of the PI3K pathway and involves direct interaction with RAR and retinoid X receptor. Defining these mechanisms should lead to methods to further enhance NIS expression, as well as retinoid targets that influence growth and differentiation of breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Enzyme Activation; Humans; Iodides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Retinoic Acid; Retinoid X Receptor alpha; Symporters; Tretinoin

Retinoic acid (RA) triggers antiproliferative effects in tumor cells, and therefore RA and its synthetic analogs have great potential as anticarcinogenic agents. Retinoic acid receptors (RARs) mediate RA effects by directly regulating gene expression. To define the genetic network regulated by RARs in breast cancer, we identified RAR genomic targets using chromatin immunoprecipitation and expression analysis. We found that RAR binding throughout the genome is highly coincident with estrogen receptor alpha (ERalpha) binding, resulting in a widespread crosstalk of RA and estrogen signaling to antagonistically regulate breast cancer-associated genes. ERalpha- and RAR-binding sites appear to be coevolved on a large scale throughout the human genome, often resulting in competitive binding activity at nearby or overlapping cis-regulatory elements. The highly coordinated intersection between these two critical nuclear hormone receptor signaling pathways provides a global mechanism for balancing gene expression output via local regulatory interactions dispersed throughout the genome.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor alpha; Estrogens; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Signal Transduction; Tretinoin

The antimalarial drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have potential applications in cancer treatment. The growth of MCF-7 and MDA-MB-231 human breast cancer cells in vitro was inhibited by CQ and HCQ and these cells were more sensitive than nontumorigenic MCF-10A breast epithelial cells. Furthermore, all-trans retinoic acid (ATRA) augmented the anticancer effects of CQ and HCQ as evidenced by significant reductions in Ki67-positive cancer cells and clonogenicity compared with cells treated with CQ or HCQ in the absence of ATRA. As an earlier study suggested that CQ, HCQ, and ATRA are breast cancer cell differentiation agents, these agents were screened in cell-free histone deacetylase (HDAC) and histone acetyltransferase (HAT) assays. ATRA, but not CQ or HCQ, inhibited HDAC activity in HeLa nuclear extracts. Growth inhibitory concentrations of HCQ and ATRA stimulated purified p300/CBP-associated factor, where CBP is the cAMP-response element binding protein, HAT activity. To investigate whether growth inhibitory concentrations of these agents influenced protein acetylation in cells, gel-purified histone H3 and histone H4 were analyzed using mass spectrometry. HCQ alone and HCQ+ATRA treatments altered the acetylation status in the N-terminal lysines of histones H3 and H4 compared with dimethyl sulfoxide (DMSO) controls. The results indicated that HCQ and ATRA regulate protein acetylation events in MCF-7 breast cancer cells, and identify a potential mechanism for their effects on breast cancer cell growth and differentiation.

Topics: Acetylation; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cellular Senescence; Chloroquine; Enzyme Inhibitors; Female; HeLa Cells; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Hydroxychloroquine; Ki-67 Antigen; Mass Spectrometry; Tretinoin; Tumor Stem Cell Assay

Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Breast; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial Cells; Female; Humans; Ligands; NF-kappa B; PPAR gamma; Retinoid X Receptors; Rosiglitazone; Thiazolidinediones; Tretinoin; Tumor Suppressor Protein p53

Mechanisms by which the inhibitory effect of retinoic acid on tumor growth is attenuated as tumors progress to more advanced stages are unclear.. This study utilizes a novel cell culture system of human breast epithelial cells (HBEC). Immortal (M13SV1), weakly tumorigenic (M13SV1-R2), and highly tumorigenic (M13SV1-R2N1) transformed Type I HBEC were derived sequentially from the same parental Type I HBEC (stem cells) developed from reduction mammoplasty of healthy women. Effects of all-trans retinoic acid (AT-RA) on the growth, protein expression of RAR-alpha, beta and gamma, and RARE transcriptional activation were determined.. AT-RA reduces proliferation rates of immortal and weakly tumorigenic cells, but not highly tumorigenic cells. This loss of response of highly tumorigenic cells to AT-RA is associated with overexpression of p185(c-erbB2/neu). It is not associated with decreased RAR-alpha, beta or gamma expression, or activation by AT-RA; RAR-alpha, beta and gamma are expressed and AT-RA increases RARE transcriptional activity in all cell lines tested in this study.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Luciferases; Receptor, ErbB-2; Receptors, Retinoic Acid; Regulatory Elements, Transcriptional; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Transcriptional Activation; Tretinoin

The cancer stem cell (CSC) hypothesis implicates the development of new therapeutic approaches to target the CSC population. Characterization of the pathways that regulate CSCs activity will facilitate the development of targeted therapies. We recently reported that the enzymatic activity of ALDH1, as measured by the ALDELFUOR assay, can be utilized to isolate normal and malignant breast stem cells in both primary tumors and cell lines. In this study, utilizing a tumorsphere assay, we have demonstrated the role of retinoid signaling in the regulation of breast CSCs self-renewal and differentiation. Utilizing the gene set enrichment analysis (GSEA) algorithm we identified gene sets and pathways associated with retinoid signaling. These pathways regulate breast CSCs biology and their inhibition may provide novel therapeutic approaches to target breast CSCs.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Breast Neoplasms; Cell Differentiation; Female; Humans; Isoenzymes; Neoplastic Stem Cells; Retinal Dehydrogenase; Retinoids; Signal Transduction; Tretinoin

Multiple mechanisms regulate cancer-associated telomerase activity at the level of human telomerase reverse transcriptase (hTERT) transcription which may serve as novel targets for anticancer approaches.. The effects of prolonged all-trans retinoic acid (ATRA) exposure on hTERT regulation in estrogen receptor-negative SK-BR-3 breast cancer cells were examined.. ATRA had a profound effect on the morphology and proliferation rate of the SK-BR-3 cells. ATRA also hindered the ability of these cancer cells to grow independently, rendering them more like normal somatic cells. The effect of ATRA on the decrease of telomerase activity was found to be associated with a rapid decrease in histone H3-lysine 9 acetylation (H3-K9-Ac) of the hTERT promoter. Extended-exposure to ATRA in these cells also caused the initiation of a putative compensatory mechanism, counteracting the induced surge in apoptosis.. A rapid decrease of H3-K9 acetylation at the hTERT promoter could be an important mechanism by which ATRA shuts down telomerase activity and mediates its antitumor effects in estrogen receptor-negative breast cancer cells.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Adhesion; Chromatin Immunoprecipitation; DNA Methylation; Female; Histones; Humans; Promoter Regions, Genetic; Receptors, Estrogen; Telomerase; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Novel mutual prodrugs (MPs) of ATRA (all- trans-retinoic acid) and HDIs (histone deacetylase inhibitors) ( 10, 13, 17- 19) connected via glycine acyloxyalkyl carbamate linker (AC linker) or through a benzyl ester linker (1,6-elimination linker) were rationally designed and synthesized. Most of our novel MPs were potent inhibitors of growth of several hormone-insensitive/drug resistant breast cancer cell lines and the hormone-insensitive PC-3 prostate cancer cell line. The novel MPs exhibited differential antiproliferative potencies in both MDA-MB-231 and PC-3 cell lines. Whereas 19 (VNLG/124) [4-(butanoyloxymethyl)phenyl(2 E,4 E,6 E,8 E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI 50 of 10 nM was the most potent MP versus the MDA-MB-231 cells, 13 (VNLG/66) [{ N-[ N-{2-[4-{[3-pyridylmethoxy)carbonyamino]methyl}phenyl) carbonylamino]phenyl} carbamoylcarbamoyloxy}methyl(2 E,4 E,6 E,8 E)-3,7-dimethyl-9-(2,6,6-trimethyl cyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI 50 = 40 nM was the most potent versus the PC-3 cells. MP 19 exhibited the most benefit because its GI 50 of 10 nM versus MDA-MB-231 cells was remarkably 1085-fold lower than that of parent ATRA and over 100000-fold lower than butyric acid (BA).

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbamates; Cell Line, Tumor; Cell Proliferation; Drug Design; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Male; Mice; Mice, SCID; Molecular Structure; Prodrugs; Prostatic Neoplasms; Structure-Activity Relationship; Tretinoin

Many aspects of development, tumor growth and metastasis depend upon the provision of an adequate vasculature. This can be a result of regulated angiogenesis, recruitment of circulating endothelial progenitors and/or vascular trans-differentiation. The present study demonstrates that treatment of SKBR-3 breast cancer cells with retinoic acid (RA), an important regulator of embryogenesis, cancer and other diseases, stimulates the formation of networks in Matrigel. RA-treatment of SKBR-3 cells co-cultured with human umbilical vein endothelial cells resulted in the formation of mixed structures. RA induces expression of many endothelial genes including vascular endothelial (VE) cadherin. VE-cadherin was also induced by RA in a number of other breast cancer cells. We show that RA-induced VE-cadherin is responsible for the RA-induced morphological changes. RA rapidly induced the expression of Sox-9 and ER81, which in turn form a complex on the VE-cadherin promoter and are required to mediate the transcriptional regulation of VE-cadherin by RA. These data indicate that RA may promote the expression of endothelial genes resulting in endothelial-like differentiation, or provide a mechanism whereby circulating endothelial progenitor cells could be incorporated into a growing organ or tumor.

Topics: Antigens, CD; Breast Neoplasms; Cadherins; Cell Differentiation; Cell Line, Tumor; Cell Transdifferentiation; Collagen; DNA-Binding Proteins; Drug Combinations; Gene Expression Regulation, Neoplastic; High Mobility Group Proteins; Humans; Laminin; Models, Biological; Neovascularization, Pathologic; Promoter Regions, Genetic; Proteoglycans; Signal Transduction; SOX9 Transcription Factor; Transcription Factors; Tretinoin; Umbilical Veins

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.

Topics: Adenocarcinoma; Aldehydes; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Cells, Cultured; Gene Expression Regulation; Glutamate-Cysteine Ligase; Humans; Kidney; Lipid Peroxidation; Liver Neoplasms; Male; MAP Kinase Kinase 1; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-E2-Related Factor 2; Response Elements; Retinoid X Receptor alpha; Retinoid X Receptor beta; RNA, Small Interfering; Tretinoin

The sodium iodide symporter (NIS) mediates iodide uptake in the thyroid gland as well as in lactating breast, and is also expressed in the majority of breast cancers. Recently, we have reported stimulation of all-trans retinoic acid (atRA)-induced NIS expression in the human breast cancer cell line MCF-7 by dexamethasone (Dex), resulting in an enhanced therapeutic effect of (131)I in vitro.. In the current study we examined the efficacy of Dex stimulation of atRA-induced NIS expression in vivo in MCF-7 xenotransplants in nude mice.. After systemic treatment with atRA alone or in combination with Dex, iodide accumulation in the tumors was assessed by gamma camera imaging and gamma counter analysis. In addition, NIS expression was examined on RNA and protein level by RT-PCR and immunohistochemistry, respectively.. Using gamma camera imaging after intraperitoneal injection of 18.5 MBq (123)I, no iodide accumulation was detected in tumors of untreated mice or mice treated with atRA only. After combined treatment with atRA/Dex significant (123)I accumulation was detected in MCF-7 xenografts, which, by ex vivo gamma counting revealed a 3.3-fold increase in iodide accumulation as compared to control tumors. Surprisingly, in a subset of mice treated with atRA or atRA/Dex iodide accumulation was also detected in the normal mammary glands. In a normal human mammary epithelial cell line HB-2, however, no functional NIS expression was induced after treatment with atRA and/or Dex in vitro. Further, NIS mRNA and protein expression was detected in atRA/Dex treated MCF-7 tumors by RT-PCR and immunohistochemistry, respectively.. Treatment with Dex in the presence of atRA is able to induce significant amounts of iodide accumulation in breast cancer xenotransplants in vivo due to stimulation of functional NIS protein expression, which opens exciting perspectives for a possible diagnostic and therapeutic role of radioiodine in the treatment of breast cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Dexamethasone; Humans; Immunohistochemistry; Iodides; Mice; Mice, Nude; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Symporters; Tretinoin; Xenograft Model Antitumor Assays

All-trans retinoic acid (tRA) induces differentiation in MCF-7 breast cancer cells, stimulates sodium/iodide symporter (NIS) gene expression, and inhibits cell proliferation. Radioiodine administration after systemic tRA treatment has been proposed as an approach to image and treat some differentiated breast cancer.. The objective of this work was to study the relative role of genomic and nongenomic pathways in tRA stimulation of NIS expression in MCF-7 cells.. We inspected the human NIS gene locus for retinoic acid-responsive elements and tested them for function. The effects of signal transduction pathway inhibitors were also tested in tRA-treated MCF-7 cells and TSH-stimulated FRTL-5 rat thyroid cells, followed by iodide uptake assay, quantitative RT-PCR of NIS, and cell cycle phase analysis.. Multiple retinoic acid response elements around the NIS locus were identified by sequence inspection, but none of them was a functional tRA-induced element in MCF-7 cells. Inhibitors of the IGF-I receptor, Janus kinase, and phosphatidylinositol 3-kinase (PI3K), significantly reduced NIS mRNA expression and iodide uptake in tRA-stimulated MCF-7 cells but not FRTL-5 cells. An inhibitor of p38 MAPK significantly reduced iodide uptake in both tRA-stimulated MCF-7 cells and TSH-stimulated FRTL-5 cells. IGF-I and PI3K inhibitors did not significantly reduce the basal NIS mRNA expression in MCF-7 cells. Despite the chronic inhibitory effects on cell proliferation, tRA did not reduce the S-phase distribution of MCF-7 cells during the period of NIS induction.. The IGF-I receptor/PI3K pathway mediates tRA-stimulated NIS expression in MCF-7 but not FRTL-5 thyroid cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclic AMP-Dependent Protein Kinases; Female; Humans; Insulin-Like Growth Factor I; MAP Kinase Signaling System; Morpholines; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Signal Transduction; Symporters; Tretinoin; Tyrphostins

Stanniocalcin 1 (STC1) and STC2 are secreted, homodimeric glycoproteins that share 30% amino acid sequence identity. Breast tumour gene profiling studies have demonstrated significantly upregulated STC2 expression in hormone-responsive positive breast tumours; therefore, the purpose of this study was to investigate STC2 hormonal regulation and function in breast cancer cells. Here we report that STC2 is expressed in a number of human breast cancer cell lines, regardless of their oestrogen (E(2)) and progesterone (P4) receptor status, and its expression is readily detectable in human and mouse mammary gland tumours. Besides E(2), retinoic acid (RA) and P4 play an important role in the regulation of STC2 expression, not only in MCF-7 but also in other breast cancer and non-breast cell lines. The expression of the related hormone, STC1, is not affected by the above hormones in breast and endometrial cancer cell lines implying a fundamental difference in regulation in cancer cell lines. The induction of STC2 expression by E(2) and RA occurs at the transcriptional level but through intermediary transcription factors. The STC2 proximal promoter region is not responsible for hormonal induction, but exhibits a high basal transcriptional activity. Constitutive STC2 expression in human breast cancer cell lines resulted in significant impairment of cell growth, migration and cell viability after serum withdrawal. In conclusion, STC2 is a downstream target of E(2), P4 and RA signalling pathways. In hormone receptor negative cell lines it can function in a paracrine/autocrine fashion to reduce cell proliferation.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Estradiol; Female; Gene Expression Regulation; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Mammary Neoplasms, Experimental; Mice; Signal Transduction; Tretinoin

Novel aminophenol analogues were synthesized based on the structure of fenretinide (N-(4-hydroxyphenyl)retinamide, 5), which is a potent anticancer agent. Our findings showed that the anticancer activities of 5 were due to the side chain attached to the aminophenol moiety. A p-octylaminophenol (p-OAP) provided the most potent anticancer activity among p-alkylaminophenols examined. In this study, we investigated anticancer activities against various cancer cell lines by the new aminophenols, p-dodecylaminophenol (1), p-decylaminophenol (2), N-(4-hydroxyphenyl)dodecananamide (3), and N-(4-hydroxyphenyl)decananamide (4), which exhibits a side chain as long as 5. Cell growth of breast cancer (MCF-7, MCF-7/Adr(R)), prostate cancer (DU-145), and leukemia (HL60) cells was suppressed by 1 and 2 in a fashion dependent on the length of the alkyl chain attached to the aminophenol. In contrast, 3 and 4 were extremely weak. Compound 5 was less potent than 1. Cell growth of liver cancer (HepG2) was not markedly affected by these compounds. In addition, apoptosis of HL60 cells was induced by 1 and 2 in a chain length-dependent manner, but not by 3 and 4. Incorporation of compounds into HL60 cells was in the order 1>2=3>4. These results indicated that anticancer activities for 1 and 2 are correlated with their incorporation into cancer cells and their capability to induce apoptosis, but not for 3 and 4. Compound 1, a potent anticancer agent with potency strikingly greater than 5, may potentially be useful in clinic.

Topics: Aminophenols; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; DNA Fragmentation; Electrophoresis, Agar Gel; Female; Fenretinide; HL-60 Cells; Humans; Male; Prostatic Neoplasms; Structure-Activity Relationship

All-trans-retinoic acid and the tumor suppressor promyelocytic leukemia protein (PML) are potent regulators of the growth of cancer cells. This study investigates the individual and combined effects of PML, when overexpressed by the recombinant PML adenovirus, and all-trans-retinoic acid on the proliferation of human estrogen-receptor negative SKBR-3 and estrogen-receptor positive MCF-7 breast cancer cell lines. All-trans-retinoic acid caused a significant degree of cell death in SKBR-3 cells and MCF-7 cells, and PML elicited a similar incidence of or slightly more cell death in MCF-7 cells. Dual-treated cells displayed significantly less cell death than did single-treated cells in the same cell line. We concluded that PML and all-trans-retinoic acid cause cell death by different pathways: PML activates ERK1/2, p38 MAPK, and p21; arrests the cell cycle; and later causes cell death; and all-trans-retinoic acid activates proteasome function, caspase cleavage, and apoptosis. The combined use of all-trans-retinoic acid and PML gene therapy may not be the best treatment for patients with cancer, because the ubiquitinylation of PML and its subsequent proteasome-dependent degradation by retinoic acids occur before overexpressed PML exhibits tumor-suppressive activity.

Topics: Blotting, Western; Breast Neoplasms; Cell Death; Cell Line, Tumor; Down-Regulation; Fluorescent Antibody Technique; Humans; Neoplasm Proteins; Nuclear Proteins; Promyelocytic Leukemia Protein; Proteasome Endopeptidase Complex; Transcription Factors; Tretinoin; Tumor Suppressor Proteins; Ubiquitin

The anti-estrogen tamoxifen and vitamin A-related compound, all-trans retinoic acid (RA), in combination act synergistically to inhibit the growth of MCF-7 human breast cancer cells. In the present study, we applied two-dimensional gel electrophoresis based proteomic approach to globally analyze this synergistic effect of RA and tamoxifen. Proteomic study revealed that multiple clusters of proteins were involved in RA and tamoxifen-induced apoptosis in MCF-7 breast cancer cells, including post-transcriptional and splicing factors, proteins related to cellular proliferation or differentiation, and proteins related to energy production and internal degradation systems. The negative growth factor-transforming growth factor beta (TGFbeta) was secreted by RA and/or tamoxifen treatment and was studies as a potential mediator of the synergistic effects of RA and tamoxifen in apoptosis. By comparing protein alterations in treatments of RA and tamoxifen alone or in combination to those of TGFbeta treatment, or co-treatment with TGFbeta inhibitor SB 431542, proteomic results showed that a number of proteins were involved in TGFbeta signaling pathway. These results provide valuable insights into the mechanisms of RA and tamoxifen-induced TGFbeta signaling pathway in breast cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Estrogen Antagonists; Humans; Neoplasm Proteins; Protein Biosynthesis; Proteomics; Tamoxifen; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2.

Topics: Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; G1 Phase; Genes, cdc; Genes, Tumor Suppressor; Humans; Immediate-Early Proteins; Receptors, Retinoic Acid; Signal Transduction; Transcriptional Activation; Tretinoin; Tumor Suppressor Proteins; Up-Regulation

Retinoic acids and their derivatives potentiate anti-cancer effects in breast cancer cells. The aberrant expression of telomerase is critical to the continued proliferation of most cancer cells. Thus, telomerase is an attractive target for chemoprevention and treatment of breast cancer. 9cUAB30 is a novel synthetic retinoid X receptor-selective retinoic acid (RA) that effectively reduces the tumorigenic phenotype in mouse breast carcinoma with lower toxic effects than natural retinoid treatments. We have assessed 9cUAB30 retinoic acid treatment of human breast cancer cells to determine the potential of this drug as an effective telomerase inhibitor and its application to cancer therapy. 9cUAB30 was found to decrease DNA methyltransferase and telomerase expression in MDA-MB-361, T-47D, and MCF-7 human breast cancer cells and to inhibit the proliferation of these cells. This low-toxicity retinoid also reduced colony formation in soft agar assays in each of these cell types. Combination treatments of 9cUAB30 and all-trans RA proved to be synergistically more effective than either RA alone, further suggesting a possible general epigenetic mechanism that contributes to the anti-telomerase activity of the retinoids. Therefore, the novel retinoid, 9cUAB30, is effective in inhibiting the growth of human breast cancer cells, its anti-cancer effects appear to be related to telomerase inhibition and the mechanism for this process could be mediated through epigenetic modifications.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Dose-Response Relationship, Drug; Down-Regulation; Fatty Acids, Unsaturated; Gene Expression Regulation, Neoplastic; Humans; Naphthalenes; Retinoids; Telomerase; Time Factors; Tretinoin

We have shown previously that neonatal testicular gonocytes express platelet-derived growth factor receptors (PDGFR) alpha and beta. We report the expression of a novel PDGFRbeta (V1-PDGFRbeta) transcript in gonocytes of 3-d-old rat testes. V1-PDGFRbeta nucleotide sequence spans from intron 6 to exon 23 of the PDGFRbeta gene, and is predicted to encode a protein lacking part of the extracellular domain. V1-PDGFRbeta transcripts are expressed preferentially in developing gonads. The embryonic teratocarcinoma F9 cells, in which differentiation is driven by retinoic acid (RA), express V1-PDGFRbeta, but not wild-type PDGFRbeta. Green fluorescent protein-tagged V1-PDGFRbeta localized mainly in cytosol of F9, MA-10, and COS-1 cells. FLAG and green fluorescent protein-tagged V1-PDGFRbeta displayed tyrosine kinase activities and contain phosphotyrosine residues, suggesting that V1-PDGFRbeta is a cytosolic tyrosine kinase. Treatment of F9 cells with RA induced V1-PDGFRbeta gene expression, concomitant with changes in morphology and increased mRNA expression of collagen IV and laminin B1, suggesting that V1-PFGRbeta is involved in cell differentiation. Similarly, treatment of postnatal d 3 rat gonocytes with RA induced a dose-dependent increase in V1-PDGFRbeta expression together with an increase in c-kit and Stra8, markers of more differentiated germ cells and a concomitant decrease in GFRalpha1, a marker of spermatogonial stem cells. However, an excess of V1-PDGFRbeta inhibited RA-mediated collagen IV and laminin B1 expression and altered both RA-dependent and RA-independent morphological changes in F9 cells, while increasing cell survival. These results suggest that the expression of V1-PDGFRbeta is tightly regulated during differentiation and that it may play an active role in germ cell differentiation.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; COS Cells; Female; Genetic Variation; Germ Cells; Glioma; Green Fluorescent Proteins; Humans; Male; Mice; Mice, Inbred BALB C; Pregnancy; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Receptor, Platelet-Derived Growth Factor beta; Teratocarcinoma; Testis; Tretinoin

Increased expression of the sodium iodide symporter (NIS) is required for effective radioiodine treatment and reporter gene imaging of breast cancer. We investigated the effect of retinoic acid on adenovirus-mediated expression of the human NIS gene in the MCF-7 breast cancer cell line.. The MCF-7 cell line was infected with recombinant adenovirus carrying the human NIS gene (Rad-NIS). Levels of NIS messenger RNA (mRNA) and protein expression and radioiodine ((125)I) uptake were measured to evaluate adenovirus-mediated NIS gene expression in wild-type and Rad-NIS-infected MCF-7 cells after treatment with all-trans-retinoic acid (ATRA; 10(-8)-10(-6) mol/L).. The transduction efficiency of adenovirus in MCF-7 cells at a multiplicity of infection (MOI) of 50 was >60%. After incubation with 10(-6) mol/L ATRA, the mRNA level in Rad-NIS-infected MCF-7 cells increased to 118.5 times that of wild-type MCF-7 cells, whereas the mRNA level in wild-type MCF-7 cells showed only a 2.1-fold increase. Western blot, immunocytochemical staining, and flow cytometry analyses showed that NIS protein expression in MCF-7 cells infected with Rad-NIS increased after ATRA treatment. With ATRA treatment, the amount of (125)I uptake increased in a dose-dependent manner (P < 0.001). The (125)I uptake in wild-type MCF-7 cells increased 3.1-, 5.5-, and 7.6-fold with treatment with 10(-8), 10(-7), and 10(-6) mol/L ATRA, respectively. Rad-NIS-infected cells showed a 4.0-fold increase in (125)I uptake. Treatment of Rad-NIS-infected cells with 10(-8), 10(-7), and 10(-6) mol/L ATRA increased (125)I uptake by 4.9-, 8.2-, and 27.6-fold, respectively, compared with wild-type MCF-7 cells. The level of NIS expression in Rad-NIS-infected MCF-7 cells treated with 10(-6) mol/L ATRA (245.0 +/- 13.7 pmol/10(6) cells) was much greater than the sum of the expression levels seen in ATRA-treated wild-type cells and Rad-NIS-infected wild-type cells.. Retinoic acid increases adenovirus-mediated NIS expression in MCF-7 cells. Our results indicate that improved efficiency of NIS gene therapy or reporter imaging in breast cancer may be possible with retinoic acid treatment.

Topics: Adenoviridae; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Transfer, Horizontal; Genetic Therapy; Humans; Iodine Radioisotopes; Receptors, Retinoic Acid; Retinoid X Receptors; Symporters; Tretinoin

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER -ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumour xenografts. The ER +ve cell lines were more sensitive (IC(50) values between 3.0 and 609 nM) to the RAMBAs than the ER -ve MDA-MB-231 cell line (IC(50)=5.6-24.0 microM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-alpha (ER-alpha). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle; Cell Proliferation; Cyclin D1; Female; Fenretinide; Humans; Imidazoles; Mice; Neoplasm Transplantation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transplantation, Heterologous; Tretinoin

We recently established a cell line derived from pleural effusion from a 13-year-old girl with primary alveolar rhabdomyosarcoma (RMS with a chromosomal translocation t[2;13]) in the breast tissue. The cell line was designated as HUMEMS. Cases of primary alveolar RMS swelling in the breast are extremely rare (about 0.2% of all RMSs). Therefore, the HUMEMS cell line is an important material for studying therapeutics for malignant tumors in children. The HUMEMS cell line we isolated consisted of two morphological subtypes. One type (SSN cells) is small in size and has a single nucleus. Another (LMN cells) is large in size and has two or more nuclei. Both SSN cells and LMN cells were immunohistochemically positive for desmin and slightly positive for myoglobin. Our data suggested LMN cells are well-differentiated SSN cells. Moreover, in some of the LMN cells, rapid cell contractions (1-5 times/10 sec) were observed. We investigated the anticancer drug susceptibility of the HUMEMS cell line with an oxygen electrode apparatus (Daikin, DOX-10, JPN) and effect of all-trans-retinoic acid (atRA) to the cell line. The atRA-treatment inhibited proliferation of the HUMEMS cells.

Topics: Adolescent; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Desmin; Drug Screening Assays, Antitumor; Female; Humans; Immunohistochemistry; Myoglobin; Pleural Effusion, Malignant; Rhabdomyosarcoma, Alveolar; Tretinoin

The promise of retinoids as chemopreventive agents in breast cancer is based on the differentiation and apoptosis induced upon their binding to the retinoic acid (RA) receptor beta (RARbeta). We have previously shown that HOXA5 induces apoptosis in breast cancer cells. In this study, we investigated whether RA/RARbeta and HOXA5 actions intersect to induce apoptosis and differentiation in breast cancer cells. We found that HOXA5 expression can be induced by RA only in RARbeta-positive breast cancer cells. We have, for the first time, identified the RA response element in HOXA5, which was found to be located in the 3' end of the gene. Chromatin immunoprecipitation assays showed that RARbeta binds directly to this region in vivo. Overexpression of RARbeta strongly enhances RA responsiveness, and knocking down RARbeta expression abolishes RA-mediated induction of HOXA5 expression in breast cancer cells. In addition, there is coordinated loss of both HOXA5 and RARbeta expression during neoplastic transformation and progression in the breast epithelial cell model, MCF10A. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment. These results strongly suggest that HOXA5 acts directly downstream of RARbeta and may contribute to retinoid-induced anticancer and chemopreventive effects.

Topics: Apoptosis; Base Sequence; Binding Sites; Breast Neoplasms; Cell Proliferation; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Receptors, Retinoic Acid; Response Elements; Tretinoin; Tumor Cells, Cultured

Lactoferrin (Lf) is a multifunctional iron-binding protein that was first identified in mammary secretions, but is synthesized by most mammalian tissues. The protein has a signal sequence that dictates secretion; it also has a nuclear localization sequence that facilitates entry into the cell nucleus. The mechanism of the latter action is currently unknown, but is thought to occur via a Lf receptor. Lactoferrin content of mammary tissue and secretions varies with developmental state; it is synthesized in mammary tissue at high levels during both pregnancy and involution, and during mammary infections. Using fluorescent (FITC)-labeled holo-bLf, we show that bovine primary epithelial cells and MCF-7 breast cancer cells do not translocate the exogenously added Lf to the nucleus after culture in serum free media (SFM). However, the supplementation of SFM with 1microM all-trans retinoic acid (atRA) caused breast cancer cells to gain the capacity to take up labeled bLf into the cell nucleus. Primary bovine mammary cells (MeBo) exhibited similar capacity in culture. This suggests that in addition to Lf, one or more components modulated by atRA, are necessary for nuclear translocation to occur. Transfection experiments with atRA treated MCF-7 cells containing retinoic acid response element reporter constructs showed that the extracellular application of lactoferrin alters reporter gene expression. Lactoferrin increased a DR5 luciferase response element in a dose-dependent manner only when atRA was applied. Immunocytochemical markers for the cell cycle (Ki67) and apoptotic events (Caspase-3 and PARP-85) showed that lactoferrin alters the atRA-induced phenotype, blocking apoptosis and maintaining cell cycle activity in both MCF-7 and MeBo cells in the presence of 1muM atRA. We propose that nuclear lactoferrin interacts with retinoic acid signaling pathways in cells and alters/blocks the signals so that cells remain in the cell cycle and/or do not enter the apoptotic pathway.

Topics: Animals; Apoptosis; Breast Neoplasms; Cattle; Cell Cycle; Cell Division; Cell Line, Tumor; Cells, Cultured; Drug Interactions; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Ki-67 Antigen; Lactoferrin; Mammary Glands, Animal; Receptors, Retinoic Acid; Response Elements; Retinoid X Receptors; Retinoids; Signal Transduction; Transfection; Tretinoin

The sodium iodide symporter (NIS) mediates the active iodide uptake in the thyroid gland as well as lactating breast tissue. Recently induction of functional NIS expression was reported in the estrogen receptor-positive human breast cancer cell line MCF-7 by all-trans retinoic acid (atRA) treatment in vitro and in vivo, which might offer the potential to treat breast cancer with radioiodine.. In the current study, we examined the effect of dexamethasone (Dex) on atRA-induced NIS expression and therapeutic efficacy of 131-I in MCF-7 cells.. For this purpose, NIS mRNA and protein expression levels in MCF-7 cells were examined by Northern and Western blot analysis after incubation with Dex (10(-9) to 10(-7) m) in the presence of atRA (10(-6) m) as well as immunostaining using a mouse monoclonal human NIS-specific antibody. In addition, NIS functional activity was measured by iodide uptake and efflux assay, and in vitro cytotoxicity of 131-I was examined by in vitro clonogenic assay.. After incubation with Dex in the presence of atRA, NIS mRNA levels in MCF-7 cells were stimulated up to 11-fold in a concentration-dependent manner, whereas NIS protein levels increased up to 16-fold and iodide accumulation was stimulated up to 3- to 4-fold. Furthermore, iodide efflux was modestly decreased after stimulation with Dex in the presence of atRA. Furthermore, in the in vitro clonogenic assay, selective cytotoxicity of 131-I was significantly increased from approximately 17% in MCF-7 cells treated with atRA alone to 80% in MCF-7 cells treated with Dex in the presence of atRA.. Treatment with Dex in the presence of atRA significantly increases functional NIS expression levels in addition to inhibiting iodide efflux, resulting in an enhanced selective killing effect of 131-I in MCF-7 breast cancer cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Survival; Dexamethasone; Female; Humans; Immunohistochemistry; Iodides; Iodine Radioisotopes; Receptors, Estrogen; RNA, Messenger; Symporters; Tretinoin; Tumor Stem Cell Assay

The breast cancer susceptibility gene BRCA1 is involved in the repair of double-strand breaks induced by ionizing radiation and chemotherapy drugs. BRCA1 interacts with coactivators such as p300 and CREB-binding protein (CBP) to activate target gene transcription. Estrogen and retinoic acid receptors (ER and RAR) also require coactivator proteins for their ligand-dependent functions. Few studies have suggested a role for nuclear hormone receptors in DNA repair.. DNA damage and repair activity were quantified with the use of single-cell gel electrophoresis and plasmid end-joining assays. Cell cycle progression and apoptosis were determined by bromodeoxyuridine and TdT-mediated dUTP nick end labelling assays. Stable transfection was accomplished with the lipofection procedure. Protein interaction and expression were determined by immunoprecipitation and western blotting.. 17beta-estradiol (E2) and all-trans retinoic acid (RA) had opposing effects on DNA damage and breast cancer cell survival after double-strand break damage. Treatment with E2, but not with RA, resulted in complex formation between ERalpha, CBP, and BRCA1 in ER-positive cell lines. Mutant BRCA1 reduced the expression and activity of DNA damage repair proteins but did not block nuclear hormone-dependent effects. Mutant BRCA1 failed to form complexes with ERalpha and CBP, which correlated with its ability to exert E2-independent effects on DNA repair. Mutant BRCA1 inhibited cell cycle progression and produced increased survival in cells with double-strand breaks. Ectopic ERalpha expression reproduced the E2-mediated effects on DNA damage, repair, and survival.. The present study proposes a new mechanism by which ER and RAR regulate BRCA1-mediated DNA repair by means of CBP.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Survival; CREB-Binding Protein; DNA Damage; DNA Repair; Estradiol; Estrogen Receptor alpha; Female; Genes, BRCA1; Humans; Immunoprecipitation; Tretinoin; Tumor Cells, Cultured

The sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine's action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Biological Transport; Breast Neoplasms; Cell Membrane; Cells, Cultured; Colforsin; Female; Humans; Hydrocortisone; Iodides; Phosphodiesterase Inhibitors; Signal Transduction; Symporters; Thyroid Gland; Tretinoin

Breast cancer is an increasingly common malignancy. Several vitamins such as retinoic acid (RA), ascorbic acid (AA), vitamin D and vitamin E are known to prevent the development and progression of breast cancer.. We sought to determine whether RA and AA together (RA+AA) acted synergistically in blocking the proliferation of human breast cancer cells. To elucidate the mechanism by which RA+AA inhibited breast carcinoma proliferation, we then evaluated the gene expression profiles of the treated and untreated cells by radioactive cDNA microarray analysis.. We cultured the human breast cancer cell line MCF-7 for 3 days with 100 nM RA and/or 1 mM AA, counted the cell numbers and harvested the total RNAs for cDNA microarray analysis.. RA, AA and RA+AA reduced MCF-7 cell proliferation by 20.7%, 23.3% and 75.7% relative to the untreated cell proliferation, respectively. The synergistic ratio of RA and AA was 1.72. The MCF-7 gene expression profiles showed that 29 genes were up-regulated and 38 genes were down-regulated after RA+AA treatment. The nature of these genes suggests that the mechanism by which RA and AA act synergistically in inhibiting human breast cancer cell proliferation may involve the expression of genes that induce differentiation and block proliferation, and the up-regulation of antioxidant enzymes and proteins involved in apoptosis, cell cycle regulation and DNA repair.. Combined treatment with RA and AA inhibits the proliferation of human breast cancer cells by altering their gene expression related to antioxidation processes as well as the proliferation inhibitory pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotide Array Sequence Analysis; Tretinoin

Active metabolites of vitamin A and D are well known to act as growth inhibitors in hormone-related prostate and breast cancers. When various concentrations of 1alpha,25-dihydroxyvitamin D3 (vitamin D3), all-trans-retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) were examined, the androgen-stimulated growth of mouse mammary carcinoma SC-3 cells was inhibited by vitamin D3 alone in a dose-dependent manner. A flow cytometer analysis showed that vitamin D3 leads SC-3 cells to relative G1-growth arrest after 72 h. Characterization of vitamin D3-responsive genes using an oligonucleotide microarray demonstrated that 220 genes were upregulated at more than threefold, and 84 genes were downregulated to less than one-third, compared with the testosterone-stimulated SC-3 cells. Neither cyclin-dependent kinase inhibitors (CDKIs) nor the antiapoptotic bcl-2 gene were induced in vitamin D3-responsive genes, with the exception of a slight induction of p15(INK4B). Importantly, fgf8 was markedly repressed in response to vitamin D3. The exogenous addition of FGF8 canceled the growth suppression by vitamin D3 in SC-3 cells, suggesting that the repression of fgf8 is an indispensable step in vitamin D3-mediated growth inhibition. In reporter assays using the ARE-containing artificial construct and the natural androgen-regulated PSA promoter, co-transfection of the vitamin D receptor (VDR) and androgen receptor (AR) suppressed AR-stimulated promoter activity. In addition, vitamin D3 also suppressed androgen-stimulated promoter activity in the stably transfected SC-3 cells. Moreover, VDR repressed the core promoter activity of fgf8 in COS1 cells and in the SC-3 cells. All these findings strongly suggest that vitamin D3 serves as a negative regulator for both androgen-related and fgf8 transcriptions.

Topics: Androgens; Animals; Breast Neoplasms; Cell Line; Cell Proliferation; Chlorocebus aethiops; Cholecalciferol; Down-Regulation; Fibroblast Growth Factor 8; Gene Expression Regulation, Neoplastic; Mammary Glands, Animal; Mice; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Transcription, Genetic; Tretinoin

This study tests the hypothesis that the activators of peroxisome proliferator-activated receptors (PPARs) and 9-cis-retinoic acid receptor (RXR) regulate human semaphorin 6B (Sema6B) gene expression. The human MCF-7 breast adenocarcinoma cell line was chosen because it expresses Sema6B at a high level. The Sema6B mRNA level was analyzed by RT-PCR and the semaphorin 6B protein content was determined using a polyclonal antibody that we have produced and characterized. Treatments with fenofibrate (a PPARalpha activator) and troglitazone (a PPARgamma ligand) strongly decreased the Sema6B mRNA. The drop in Sema6B mRNA level and in protein content was more important when the treatment combined the action of fenofibrate or troglitazone and 9-cis-retinoic acid. On the other hand, no significant change was observed in the Sema6B mRNA and protein levels when the cells were exposed to the combined action of GW610742 (a PPARbeta activator) and 9-cis-retinoic acid. These data suggest that PPARalpha/RXR and PPARgamma/RXR heterodimers are involved in the regulation of Sema6B gene expression and open new perspectives concerning the participation of these nuclear receptors in cell recognition and migration.

Topics: Alitretinoin; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chromans; Dimerization; Fenofibrate; Gene Expression; HT29 Cells; Humans; K562 Cells; PPAR alpha; PPAR gamma; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Semaphorins; Thiazoles; Thiazolidinediones; Tretinoin; Troglitazone

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cyclin B; Cyclin B1; Drug Synergism; Female; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Paclitaxel; Ploidies; Survivin; Tretinoin

Insulin-like growth factors (IGFs) play an important role in normal and cancerous cell proliferation. Moreover, in recent studies IGF-I has been implicated as a major cancer risk factor. The tomato carotenoid lycopene and all-trans retinoic acid (atRA) have been shown to inhibit growth factor-induced proliferation of different types of cancer cells. This action is associated with inhibition of cell cycle progression in G0/G1 phase. Cyclin D1 acts as a growth factor sensor in G1 phase and is overexpressed in many breast cancer tumors. We have previously demonstrated that slowdown of serum-stimulated cell cycle progression from G1 to S phase by lycopene correlates with reduction in cyclin D1 levels, suggesting that the expression of this protein is a main target for lycopene's action.. To determine whether the reported reduction in cyclin D1 level is the key mechanism for lycopene and atRA inhibitory action on IGF-I-induced cell cycle progression.. Human breast (MCF-7) and endometrial (ECC-1) cancer cells were synchronized in G0/G1 phase by serum deprivation followed by stimulation with IGF-I. Cell treatment with lycopene and atRA inhibited IGF-I-stimulated cell cycle progression from G1 to S phase and decreased retinoblastoma protein (pRb) phosphorylation. These events were associated with a reduction in cyclin D1 and p21(CIP1/WAF1) level, but not that of p27(KIP1). To test the hypothesis that the decrease in cyclin D1 has a major role in the inhibitory effects of lycopene and atRA, we examined the ability of these two agents to suppress cell cycle progression in MCF-7.7D1.13 cells which are capable of expressing cyclin D1 under the control of the Zn-inducible metallothionein promoter. Our results showed that ectopic expression of cyclin D1 can overcome cell cycle inhibition caused by lycopene and atRA.. Our findings suggest that attenuation of cyclin Dl levels by lycopene and atRA is an important mechanism for the reduction of the mitogenic action of IGF-I.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Carotenoids; Cell Cycle; Cell Division; Culture Media, Serum-Free; Cyclin D1; Endometrial Neoplasms; Female; Humans; Insulin-Like Growth Factor I; Lycopene; Phosphorylation; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

Topics: Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Promyelocytic Leukemia Protein; Protein Structure, Tertiary; Receptors, Retinoic Acid; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transcription, Genetic; Tretinoin; Tripartite Motif Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

The function of sodium iodide symporter (Na(+)/I(-) symporter, or NIS) in mammary epithelial cells is essential for the accumulation of I(-) in milk; the newborn's first source of I(-) for thyroid hormone synthesis. Furthermore, increased mammary gland NIS expression has previously been shown in human breast cancer. Several hormones and factors including all-trans-retinoic acid (tRA) regulate the expression of NIS. In this study, using breast cancer cell lines, we established that tRA-responsive NIS expression is confined to estrogen receptor-alpha (ERalpha) positive cells and we investigated the role of ERalpha in the regulation of NIS expression. We showed that the suppression of endogenous ERalpha by RNA interference downregulates NIS expression in ERalpha positive mammary cells. Besides, in an ERalpha negative cell line, reintroduction of ERalpha resulted in the expression of NIS in a ligand-independent manner. We also identified a novel estrogen-responsive element in the promoter region of NIS that specifically binds ERalpha and mediates ERalpha-dependent activation of transcription. Our results indicate that unliganded ERalpha (apo-ERalpha) contributes to the regulation of NIS gene expression.

Topics: Base Sequence; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Estrogen Receptor alpha; Gene Expression; Humans; Ligands; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Symporters; Transcription, Genetic; Tretinoin

The homeobox gene HEX is expressed in several cell types during different phases of animal development. It encodes for a protein localized in both the nucleus and the cytoplasm. During early mouse development, HEX is expressed in the primitive endoderm of blastocyst. Later, HEX is expressed in developing thyroid, liver, lung, as well as in haematopoietic progenitors and endothelial cells. Absence of nuclear expression has been observed during neoplastic transformation of the thyroid follicular cells. Aim of the present study was to evaluate the localization and the function of the protein HEX in normal and tumoral breast tissues and in breast cancer cell lines.. HEX expression and nuclear localization were investigated by immunohistochemistry in normal and cancerous breast tissue, as well as in breast cancer cell lines. HEX mRNA levels were evaluated by real-time PCR. Effects of HEX expression on Sodium Iodide Symporter (NIS) gene promoter activity was investigated by HeLa cell transfection.. In normal breast HEX was detected both in the nucleus and in the cytoplasm. In both ductal and lobular breast carcinomas, a great reduction of nuclear HEX was observed. In several cells from normal breast tissue as well as in MCF-7 and T47D cell line, HEX was observed in the nucleolus. MCF-7 treatment with all-trans retinoic acid enhanced HEX expression and induced a diffuse nuclear localization. Enhanced HEX expression and diffuse nuclear localization were also obtained when MCF-7 cells were treated with inhibitors of histone deacetylases such as sodium butyrate and trichostatin A. With respect to normal non-lactating breast, the amount of nuclear HEX was greatly increased in lactating tissue. Transfection experiments demonstrated that HEX is able to up-regulate the activity of NIS promoter.. Our data indicate that localization of HEX is regulated in epithelial breast cells. Since modification of localization occurs during lactation and tumorigenesis, we suggest that HEX may play a role in differentiation of the epithelial breast cell.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Health; Histone Deacetylase Inhibitors; Histone Deacetylases; Homeodomain Proteins; Humans; Immunohistochemistry; Lactation; Mammary Glands, Human; Promoter Regions, Genetic; Symporters; Transcription Factors; Tretinoin

While many studies have documented tamoxifen's benefits as an adjuvant therapy in the treatment and prevention of recurrent breast cancer in estrogen receptor positive (ER+) breast carcinoma, this beneficial effect may decrease with long-term tamoxifen use. This experimental study was designed to compare the cytotoxic responses of ER+ primary breast cancer solid tumors derived from the MCF7 cell line to experimental therapeutics, including genistein, tamoxifen, all-trans retinoic acid (ATRA) and parthenolide in the presence and absence of exogenous beta-estradiol. The results of this study suggest that the growth inhibitory effects of tamoxifen, were dependent on beta-estradiol levels. In contrast, the cytotoxic effects of the isoflavone soy derivative, genistein, were observed to be independent of exogenous estrogen. Moreover, combined therapy using tamoxifen and genistein produced enhanced cytotoxic effects also independent of beta-estradiol levels. Additional studies involving the use of the novel agents all trans retinoic acid (ATRA) and parthenolide produced notable tumor responses and combined effects that were also estrogen-independent. Overall, these preclinical research findings suggest possible clinical applications suggesting that genistein might be a useful clinical adjuvant, particularly in post-menopausal women in whom breast cancer occurs more frequently. Moreover, this research suggests that combined treatment approaches involving the use of tamoxifen in conjunction with agents that inhibit NFkappaB pathway signaling, such as parthenolide and genistein, warrant further study.

Topics: Anticarcinogenic Agents; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estradiol; Female; Genistein; Humans; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Sesquiterpenes; Spheroids, Cellular; Tamoxifen; Tretinoin

Retinoic acid (RA) modulates the rates of transcription of numerous genes and thus plays key roles in multiple biological processes and is used in therapy of a number of diseases. However, RA therapy is often confounded by toxicity, raising the need for methodologies for its ready quantitation in biological samples. We describe a fluorescence-based method for quantitating RA that takes advantage of the high affinity and selectivity of the intracellular lipid-binding protein termed CRABP-I and CRABP-II and that uses them as RA sensors. L28C CRABP mutants were generated, and the inserted cysteine was covalently labeled with an environmentally sensitive fluorescent probe. The label was introduced into a region of the protein that undergoes a conformational shift on ligation. Consequently, RA binding resulted in distinct changes in the fluorescence of the protein-bound probe, allowing direct quantitation of RA. We show that the method can be used to monitor the biosynthesis of RA from its precursor retinal in cultured mammalian cells as well as the detection of exogenous RA in serum. The assay provides ease of use and sensitivity that enable quantitation of RA in biological samples of limited size, and it should prove to be useful in a variety of research and clinical applications.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Crystallography, X-Ray; Fluorescein; Fluorescence; Humans; Models, Molecular; Receptors, Retinoic Acid; Tretinoin

Retinoic acid (RA) is a master epigenetic regulator that plays a pivotal role in both breast morphogenesis and development. Here, we show for the first time that RA, via the RA receptor alpha (RARalpha), epigenetically regulates in a concerted fashion the transcription of two RA-responsive genes, the RA receptor beta2 (RARbeta2) and the cellular retinol-binding protein 1 (CRBP1). Specifically, an impaired RA signal through RARalpha in human breast epithelial cells triggers a repressive epigenetic domino effect, involving first RARbeta2 and second CRBP1. The phenotype acquired by breast epithelial cells clearly implies that the resistance to RA-mediated growth inhibition precedes the acquisition of morphological epithelial transformation, thus supporting the occurrence of sequential transcriptional silencing of first RARbeta2 and second CRBP1. The identification of this epigenetic network mechanistically linking RARbeta2 and CRBP1 transcription provides the basis for devising more accurate epigenetic tests for the prediction of breast cancer risk.

Topics: Breast; Breast Neoplasms; Cell Transformation, Neoplastic; Epigenesis, Genetic; Epithelial Cells; Female; Gene Silencing; Genetic Predisposition to Disease; Humans; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Risk; Tretinoin

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal estrogen receptor (ER)-positive breast cancer. However, the inevitable development of resistance to treatment is a concern. We investigated the effects of novel retinoic acid metabolism blocking agent, VN/14-1, in overcoming letrozole resistance in long-term letrozole cultured (LTLC) cells. Compared with MCF-7 cells stably transfected with aromatase (MCF-7Ca), LTLC cells were no longer sensitive to growth inhibition by aromatase inhibitors. The HER-2/phosphorylated mitogen-activated protein kinase (pMAPK) growth factor signaling pathways were activated, and ERalpha and coactivator amplified in breast cancer 1 (AIB1) were up-regulated approximately 3-fold in LTLC cells. VN/14-1 inhibited aromatase activity and growth values of in MCF-7Ca cells with IC(50) of 8.5 and 10.5 nmol/L, respectively. In human placental microsomes, aromatase activity was inhibited with IC(50) of 8.0 pmol/L. The IC(50) in LTLC cells was 0.83 nmol/L, similar to letrozole (IC(50), 0.3 nmol/L) in MCF-7Ca cells. LTLC cells were 10-fold more sensitive to growth inhibition by VN/14-1 than MCF-7Ca cells. VN/14-1 treatment effectively down-regulated ERalpha, AIB1, pMAPK, HER-2, cyclin D1, cyclin-dependent kinase 4 (CDK4), and Bcl2 and up-regulated cytokeratins 8/18, Bad, and Bax. Tumor growth of LTLC cells in ovariectomized nude mice was independent of estrogens but was inhibited by VN/14-1 (20 mg/kg/d; P < 0.002). Decreases in ERalpha, cyclin D1, CDK4, and pMAPK and up-regulation of cytokeratins, Bad, and Bax with VN/14-1 in tumor samples may be responsible for the efficacy of this compound in inhibiting LTLC cell growth in vitro and in vivo.

Topics: Animals; Apoptosis Regulatory Proteins; Aromatase; Aromatase Inhibitors; Blotting, Western; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Histone Acetyltransferases; Hormones; Humans; Imidazoles; Letrozole; Mice; Mice, Nude; Microsomes; Mitogen-Activated Protein Kinases; Nitriles; Nuclear Receptor Coactivator 3; Ovariectomy; Placenta; Pregnancy; Trans-Activators; Tretinoin; Triazoles; Xenograft Model Antitumor Assays

The promyelocytic leukemia protein (PML), involved in the pathogenesis of acute promyelocytic leukemia, is a coactivator of p53 tumor suppressive functions. The ability of PML to inhibit growth and induce cell death in solid tumor cells, however, has not been determined. We therefore assayed the tumor suppressor activities of PML and compared them with those of p53 in four liver cancer cell lines. Following infection of cells with replication-deficient recombinant PML adenovirus, the exogenous PML localized in the nucleus and formed abnormally enlarged PML-nuclear bodies after 24 hours. In vitro growth curve analysis showed that the overexpressed PML initially induced a substantial G1 cell cycle arrest and triggered massive cell death in all tested cell lines, irrespective of their p53 status. PML-induced cell death decreased by about 30% in the presence of a broad caspase inhibitor, zVAD. The cell death effect of PML was higher than that induced by p53 over a longer period of time. As with p53, overexpression of PML was closely related to upregulation of p21 and decrease of cyclin D1 expression. Unexpectedly, retinoic acid (RA) antagonized rather than enhanced PML-triggered cell death. RA enhanced the expression of adenovirus-cytomegalovirus-promoted PML at both transcription and protein levels within 12 hours after treatment; however, the PML protein was significantly degraded in the presence of RA at days 3-5 postinfection. PML degradation was also observed in SK-BR3 breast cancer cells treated with RA. Taken together, our findings strongly support the hypothesis that PML acts as a strong independent cell death inducer and that RA conversely abolishes the therapeutic effects of the PML proteins through proteasomal degradation of the protein.

Topics: Adenoviridae; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Genetic Therapy; Humans; Liver Neoplasms; Neoplasm Proteins; Nuclear Proteins; Promyelocytic Leukemia Protein; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Zinc Fingers

Functional significance of RARbeta2 as a putative tumor suppressor gene has been studied in breast cancer and other tumors. The long 5'-untranslated region (5'-UTR) of its transcript with multiple open-reading frames (uORFs) is considered as a regulatory unit for translation. Here, for the first time we identified RARbeta2 transcript variants with short 5'-UTRs in both normal and malignant breast epithelial cells. The 5'-RACE analysis of RARbeta2 mRNA in these cells demonstrated the existence of short RARbeta2 transcript variants that are identical to the sequence of known RARbeta2, but lack all the uORFs present in the full-length 5'-UTR. By RT-PCR analysis, we found that the expression of both transcripts with short and full-length 5'-UTR is mediated by retinoic acid, while cellular sensitivity is preferentially correlated to upregulation of short RARbeta2 transcript variants in response to retinoic acid. The transfection and in vitro translation assay indicated that the short 5'-UTR has no inhibitory effects on translation, while the presence of full-length 5'-UTR inhibited translation by 60%. In addition, no promoter activity was detectable in RARbeta2 full-length 5'-UTR region. Our data suggest that the RARbeta2 transcript variants with short 5'-UTR may serve as major transcripts for RARbeta2 protein translation as well as potential targets for retinoids in breast cancer prevention and therapy studies.

Topics: 5' Untranslated Regions; Base Sequence; Breast Neoplasms; Cell Line; Epithelial Cells; Genes, Tumor Suppressor; Humans; Mammary Glands, Human; Molecular Sequence Data; Open Reading Frames; Promoter Regions, Genetic; Protein Biosynthesis; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Transcription Initiation Site; Transcription, Genetic; Tretinoin; Up-Regulation

Testicular germ cell tumors (TGCTs) arise despite possessing high levels of wild-type p53, suggesting p53 latency. We have previously shown that p53 repression in TGCT-derived human embryonal carcinoma (EC) is relieved upon treatment with all-trans retinoic acid (RA), resulting in enhanced p53 transactivation activity. To further investigate p53 repression in EC, a series of gal4-p53 truncation constructs were generated. Deletion of the core DNA-binding region, residues 117-274, had no effect on basal or RA-induced p53 activity. Progressively, larger truncations were made in the C- or N-terminal direction. Deletion of residues toward the C-terminus of p53 as far as residue 354 did not affect either the basal or RA-inducible activity of gal4-p53. When a small region in the N-terminus was deleted (residues 105-116), relief of the basal repression of p53 activity characteristic of EC was observed. Fusion of this region to the VP16 activation domain (VPAD) resulted in a 10-20-fold repression of VPAD activity in NT2/D1 human EC cells, indicating that this region acts as a heterologous repressor. Owing to its location in the N-terminal half of p53, we have named this region the p53 N-terminal Repression Domain (p53-NRD). The p53-NRD mediated repression in a variety of cell lines, with the most prominent repression observed in human EC cells. While RA alone had no effect on p53-NRD activity, cotreatment with RA and the histone deacetylase inhibitor trichostatin-A (TSA) completely relieved p53-NRD-mediated repression. In contrast, NRD-mediated repression was not sensitive to RA and TSA in a derived RA-resistant cell line with a retinoic acid receptor gamma (RARgamma) defect, but sensitivity could be restored with transfection of RARgamma. These data indicate that a unique repressor domain resides in p53 at residues 90-116 whose activity can be modulated in the presence of 'differentiation therapy' and 'transcription therapy' agents.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Carcinoma, Embryonal; Cell Division; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetinae; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Transcription, Genetic; Transfection; Tretinoin; Tumor Suppressor Protein p53

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease Progression; Female; Humans; Leukemia, Promyelocytic, Acute; Recurrence; Scalp; Tretinoin

Epigenetic silencing of tumor suppressor genes has been established as an important process of carcinogenesis. The retinoic acid (RA) receptor beta2 (RARbeta2) gene is one such tumor suppressor gene often silenced during carcinogenesis. The combined use of histone deacetylase and DNA methyltransferase inhibitors has been shown to reverse the epigenetic silencing of numerous growth regulatory genes. Valproic acid (VPA), which has long been used in the treatment of epilepsy, was shown recently to be an effective histone deacetylase inhibitor that can induce differentiation of neoplastically transformed cells. In this study, we show for the first time that VPA, in combination with RA and the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza-dC), can overcome the epigenetic barriers to transcription of a prototypical silenced tumor suppressor gene, RARbeta2, in human breast cancer cells. Chromatin immunoprecipitation assays show that the combination of VPA, RA, and Aza-dC increases histone acetylation at the silenced RARbeta2 promoter of MCF-7 breast cancer cells. Furthermore, reverse transcription-PCR analyses reveal cell type-specific effects in the actions of VPA on RARbeta2 expression in cultured human breast cancer cells. Finally, we show that VPA, in combination with RA and Aza-dC, inhibits the proliferation of both estrogen receptor alpha-positive (MCF-7) and estrogen receptor alpha-negative (MDA-MB-231) breast cancer cell lines. These data suggest that VPA may ultimately be useful in combination therapies in the treatment of human breast cancers.

Topics: Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Decitabine; DNA Primers; Enzyme Inhibitors; Histone Deacetylases; Histones; Humans; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Time Factors; Tretinoin; Valproic Acid

The sodium/iodide symporter (NIS) mediates iodide uptake in lactating breast tissue and is expressed in some breast cancers. We have previously demonstrated that all-trans retinoic acid (tRA) stimulates NIS gene expression and the selective cytotoxic effect of beta-emitting radioiodide-131 ((131)I) in both in vitro and in vivo MCF-7 breast cancer cell systems. We studied the ability of natural and synthetic retinoids, in combination with other nuclear receptor ligands, to achieve greater and more sustained induction of NIS in MCF-7 cells and enhance (131)I-mediated cytotoxicity. Selective stimulation of retinoic acid receptor (RAR) beta/gamma produced marked NIS induction; and selective stimulation of RARalpha, RARgamma, or retinoid X receptor produced more modest induction. Maximal NIS induction was seen with 9-cis retinoic acid and AGN190168, a RAR beta/gamma-agonist. Dexamethasone (Dex), but not the other nuclear receptor ligands, in combination with tRA synergistically induced iodide uptake and NIS mRNA expression, predominantly by prolonging NIS mRNA half-life. The addition of Dex reduced the EC(50) of tRA for NIS stimulation to approximately 7%, such that 10(-7) m tRA with addition of Dex enhanced iodide uptake and selective cytotoxicity of (131)I greater than 10(-6) m tRA alone. AGN190168 combined with Dex synergistically increased iodide uptake and significantly prolonged induction (5 d) of iodide uptake compared with that induced by the combination of tRA/Dex or 9-cis retinoic acid/Dex. The addition of Dex reduced the effective dose of retinoid and prolonged the induction of NIS, especially with AGN190168, suggesting higher efficacy of (131)I after combination treatment.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Dexamethasone; Drug Administration Schedule; Drug Combinations; Female; Gene Expression; Glucocorticoids; Humans; Iodides; Iodine Radioisotopes; Ligands; Receptors, Cytoplasmic and Nuclear; Retinoids; RNA, Messenger; Symporters; Tretinoin; Tumor Stem Cell Assay

The S14 (spot 14) gene encodes a protein that is predominantly expressed in lipogenic tissues, such as the liver, white and brown adipose tissues and the lactating mammary glands. Accumulated evidence suggests that S14 could play an important role in the induction of lipogenic enzymes. In humans, the S14 locus resides in the chromosome region 11q13, which is frequently amplified in breast tumours, and as a result, it has been suggested that this protein could play a role in the metabolism and growth of these kinds of tumours. In the present study, we have examined the effects of S14 overexpression in MCF-7 human breast cancer cells. We found that S14 causes (i) an inhibition of cell proliferation and of anchorage-independent growth, (ii) a marked reduction in the number of viable cells and (iii) the induction of differentiation and cell death of these cells. The inhibition of cell growth was associated with a decrease in the expression of cyclin D1 and a reduction of cyclin D1 promoter activity. Increased expression of S14 also caused the accumulation of cytochrome c in the cytosol and loss of mitochondrial membrane potential. These findings suggest that S14 may function as an important modulator of tumorigenesis in human breast by decreasing cell growth and inducing cell death and differentiation.

Topics: Breast Neoplasms; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Cyclin D1; Gene Expression; Humans; Nuclear Proteins; Promoter Regions, Genetic; Prostaglandin D2; Proteins; Time Factors; Transcription Factors; Tretinoin

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical albeit poorly defined role in the development and progression of several cancer types including those of the breast, colon, and lung. A PPAR response element (PPRE) reporter assay was utilized to evaluate the selective transactivation of PPARgamma in 10 different cell lines including normal mammary epithelial, breast, lung, and colon cancer cells. Cells were treated with one of four compounds including rosglitizone (Ros), ciglitizone (Cig), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), or GW 9662 (GW). We observed differences in transactivation between cell lines from different tissue origin, across cell lines from a single tissue type, and selective modulation of PPARgamma within a single cell line by different ligands. Interestingly, GW, a PPARgamma antagonist in adipocytes, enhanced PPRE reporter activation in normal mammary epithelial cells while it had virtually no effect in any of the cancer cell lines tested. Within each cancer type, individual cell lines were found to respond differently to distinct PPARgamma ligands. For instance, Ros, Cig, and PGJ2 were all potent agonist of PPARgamma transactivation in lung adenocarcinoma cell lines while these same ligands had no effect in squamous cell or large cell carcinomas of the lung. Message levels of PPARgamma and retinoid X receptor alpha (RXRalpha) in the individual cell lines were quantitated by real time-polymerase chain reaction (RT-PCR). The ratio of PPARgamma to RXRalpha was predictive of how cells responded to co-treatment of Ros and 9-cis-retinoic acid, an RXRalpha agonist, in two out of three cell lines tested. These data indicate that PPARgamma can be selectively modulated and suggests that it may be used as a therapeutic target for individual tumors.

Topics: Alitretinoin; Anilides; Breast Neoplasms; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; HT29 Cells; Humans; Ligands; Lung Neoplasms; PPAR gamma; Prostaglandin D2; Retinoid X Receptor alpha; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transfection; Tretinoin

The structure of the Gene 33 protein suggests that it plays a role in intracellular signaling and Gene 33 is induced by many mitogenic and stressful stimuli. Previously, we found that Gene 33 expression is significantly induced by retinoic acid (RA), insulin and synergistically by both in a liver-derived cell line. In the present study, we investigated the basal expression and regulation of Gene 33 in multiple human breast cancer cell lines. These cell lines expressed different levels of Gene 33 protein, but Gene 33 protein was not regulated by RA or insulin, either alone, or in combination. However, epidermal growth factor (EGF) induced Gene 33 expression in SK-BR-3 cells and this induction was inhibited by co-treatment with RA. There was a strong correlation between endogenous basal Gene 33 expression and doubling time. Exogenous expression of Gene 33 in MCF-7 cells did not affect cell cycle distribution, but inhibited apoptosis and specifically increased the level of Poly(ADP-ribose) Polymerase (PARP-1) protein. This suggests that Gene 33 promotes breast cancer cell growth by an anti-apoptotic rather than a mitogenic effect, possibly involving up-regulation of PARP-1.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Breast Neoplasms; Cell Cycle; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin; Poly(ADP-ribose) Polymerases; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs.. The IL-4/GM-CSF (granulocyte-macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells.. Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR-Lin- cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-gamma production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR-Lin- cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid.. These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest.

Topics: Aged; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Carcinoma, Renal Cell; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dendritic Cells; Female; Humans; Kidney Neoplasms; Middle Aged; Phenotype; Putrescine; T-Lymphocytes; Tretinoin

Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Animals; Bacteria; Breast Neoplasms; Carcinogenicity Tests; Cell Transformation, Neoplastic; Ethanol; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Inactivation, Metabolic; Neoplasms; Polymorphism, Genetic; Rats; Tretinoin

The membrane receptor Fas (Apo-1/CD95) is an important initiator of programmed cell death induced by anti-Fas antibody or Fas ligand. MCF-7 human breast cancer cells have low levels of Fas receptor (FasR) and are resistant to anti-FasR antibody mediated apoptosis, however two naturally occurring substances, interferon and all-trans retinoic acid (AT), act synergistically to enhance antiproliferative processes in these cells, suggesting this combination may also be an effective means for enhancing FasR expression. When this was studied, it was found that IFN-gamma and AT in combination acted synergistically to induce expression of FasR mRNA and FasR protein in a time-dependent and dose-dependent manner. This induction required continuous protein synthesis, and STAT1 protein, but not PKR or TR1 protein, was induced in a manner quantitatively and temporally related to FasR protein induction, and consistent with STAT1 mediation of the synergistic effect of IFN-gamma and AT on FasR expression. FasR-induced cells were resistant to stimulation of apoptosis by anti-FasR antibody, however treatment with cycloheximide rendered these cells sensitive to antibody-induced apoptosis, suggesting endogenous blockade to signaling. These cells did not express caspase 3, or FLIP(L), but strongly expressed the endogenous inhibitor of apoptosis Bcl-2, indicating a type II Fas signaling pathway. Expression of these proteins was not modulated by IFN/AT, however treatment of Fas-induced cells with Bcl-2 specific small interfering RNA (SiRNA) downregulated Bcl-2 protein expression and rendered these cells sensitive to the cytotoxic effects of anti-Fas antibody. These findings indicate that IFN-gamma+AT in combination modulate Fas signaling and provide a novel mechanism for the promotion of cell death in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; fas Receptor; Humans; Interferon-gamma; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Statistics, Nonparametric; Time Factors; Tretinoin

Retinoic acid (RA) displays pronounced anticarcinogenic activities in several types of cancer. Whereas the mechanisms that underlie this activity remain incompletely understood, tumor suppression by RA is believed to emanate primarily from its ability to regulate transcription of multiple target genes. Here, we investigated molecular events through which RA inhibits the growth of MCF-7 mammary carcinoma cells, focusing on the involvement of the two proteins that mediate transcriptional activation by RA, the nuclear hormone receptor retinoic acid receptor (RAR) and the cellular retinoic acid-binding protein (CRABP) II, in this process. RA treatment of MCF-7 cells did not affect cell cycle distribution but triggered pronounced apoptosis. Accordingly, expression array analyses revealed that RA induces the expression of several proapoptotic genes, including caspase 7 and caspase 9. Whereas caspase 7 is an indirect responder to RA signaling, caspase 9 is a novel direct target for RAR, and it harbors a functional retinoic acid response element in its second intron. In agreement with the known role of CRABP-II in enhancing the transcriptional activity of RAR, the binding protein augmented RA-induced up-regulation of caspase 9, cooperated with RA in activating both caspase 7 and 9, and amplified the ability of RA to trigger apoptosis. Surprisingly, the data indicate that CRABP-II also displays proapoptotic activities on its own. Specifically, overexpression of CRABP-II, in the absence of RA, up-regulated the expression of Apaf1 and triggered caspase 7 and caspase 9 cleavage. These observations suggest that, in addition to its known role in direct delivery of RA to RAR, CRABP-II may have an additional, RA-independent, function.

Topics: Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 7; Caspase 9; Caspases; Cell Growth Processes; Cell Line, Tumor; Humans; Introns; Proteins; Receptors, Retinoic Acid; RNA, Messenger; Signal Transduction; Tretinoin; Up-Regulation

To investigate the effect of all-trans-retinoic acid (ATRA) on arsenic trioxide (As(2)O(3))-induced apoptosis of human hepatoma, breast cancer, and lung cancer cells in an attempt to find a better combination therapy for solid tumors.. Human hepatoma cell lines HepG2, Hep3B, human breast cancer cell line MCF-7, and human lung adenocarcinoma cell line AGZY-83-a were treated with As(2)O(3) together with ATRA. Cell survival fraction was determined by MTT assay, cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate (FITC) and PI staining, and intracellular glutathione (GSH) and glutathione-S-transferase (GST) activities were determined using commercial kits.. Cytotoxicity of ATRA was low. ATRA (0.1, 1, and 10 micromol/L) could synergistically potentiate As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis in each of the cell lines. HepG2 and Hep3B with low intracellular GSH or GST activities were remarkably sensitive to As(2)O(3) or As(2)O(3)+ATRA, while AGZY-83-a with higher GSH or GST activities was less sensitive to As(2)O(3) or As(2)O(3)+ATRA. Treatment with 2 micromol/L As(2)O(3) for 72 h significantly decreased intracellular GSH and GST levels in each of the cell lines, and 1 micromol/L ATRA alone reduced minimal intracellular GSH and GST levels. ATRA potentiated the effect of As(2)O(3) on intracellular GSH levels, but intracellular GST levels were not significantly affected by the combination of As(2)O(3) and ATRA for 72 h as compared to As(2)O(3) alone.. ATRA can strongly potentiate As(2)O(3)-induced growth-inhibition and apoptosis in each of the cell lines, and two drugs can produce a significant synergic effect. The sensitivity to As(2)O(3) or As(2)O(3)+ATRA is inversely proportional to intracellular GSH or GST levels in each of the cell lines. The GSH redox system may be the possible mechanism by which ATRA synergistically potentiates As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Glutathione; Glutathione Transferase; Humans; Lung Neoplasms; Oxides; Tretinoin

The retinoic acid receptor beta 2 (RARbeta2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARbeta2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype.. RNA from MDA-MB-435 human breast cancer cells transduced with RARbeta2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors.. RARbeta2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 x 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARbeta2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007).. Antimetastatic RARbeta2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARbeta2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN.

Topics: Animals; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Chromosomes, Human, X; Gene Expression Profiling; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genetic Vectors; Genotype; Humans; Interferons; Ligands; Mice; Models, Statistical; Neoplasm Metastasis; Neoplasm Transplantation; Nucleic Acid Hybridization; Phenotype; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Transcription, Genetic; Tretinoin

Novel retinoic acid metabolism blocking agents (RAMBAs) have been synthesized and characterized. The synthetic features include introduction of nucleophilic ligands at C-4 of all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid, and modification of terminal carboxylic acid group. Most of our compounds are powerful inhibitors of hamster liver microsomal ATRA metabolism enzyme(s). The most potent compound is methyl (2E,4E,6E,8E)-9-(3-imidazolyl-2,6,6-trimethylcyclohex-1-enyl)-3,7-dimethylnona-2,4,6,8-tetraenoate (5) with an IC(50) value of 0.009 nM, which is 666,667 times more potent than the well-known RAMBA, liarozole (Liazal, IC(50) = 6000 nM). Quite unexpectedly, there was essentially no difference between the enzyme inhibitory activities of the two enantiomers of compound 5. In MCF-7 cell proliferation assays, the RAMBAs also enhance the ATRA-mediated antiproliferative activity in a concentration dependent manner. The novel atypical RAMBAs, in addition to being highly potent inhibitors of ATRA metabolism in microsomal preparations and in intact human cancer cells (MCF-7, T47D, and LNCaP), also exhibit multiple biological activities, including induction of apoptosis and differentiation, retinoic acid receptor binding, and potent antiproliferative activity on a number of human cancer cells. Following subcutaneous administration to mice bearing human breast MCF-7 tumor xenografts, 6 (VN/14-1, the free carboxylic acid of 5) was well-tolerated and caused significant tumor growth suppression ( approximately 85.2% vs control, p = 0.022). Our RAMBAs represent novel anticancer agents with unique multiple mechanisms of action. The most potent compounds are strong candidates for development as therapeutic agents for the treatment of a variety of cancers.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cricetinae; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Female; Humans; Male; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Microsomes, Liver; Neoplasm Transplantation; Prostatic Neoplasms; Retinoic Acid 4-Hydroxylase; Stereoisomerism; Transplantation, Heterologous; Tretinoin

All-trans retinoic acid (ATRA) affects cell proliferation, differentiation and apoptosis through its receptors, RARs and RXRs. Besides these, other receptors such as orphan receptor TR3, are also involved in the regulatory process of ATRA. However, how different receptors function in response to ATRA is still largely unknown. In the present study, we found that formation of TR3/RXRalpha heterodimers in the nucleus and their subsequent translocation into the cytoplasm, in association with regulation of apoptosis-related proteins Bcl-2, Bcl-xl and Bax, was critical for apoptosis induction by ATRA in breast cancer cells MCF-7. When such translocation was blocked by Leptomycin B (LMB), ATRA-induced apoptosis was consequently abolished. However, in ATRA-induced gastric cancer cells MGC80-3, RXRalpha heterodimerised with RARalpha but not with TR3, and remained in the nucleus exerting its effect on cell cycle regulation. When transfected with antisense-RARalpha, MGC80-3 cells changed from ATRA-sensitive to ATRA-resistant and most cells were arrested in the S phase, implying the importance of RARalpha in cell cycle regulation. Furthermore, we demonstrated that the effects of ATRA depend on the relative levels of TR3, RARalpha and RXRalpha expression in cancer cells. In ATRA-induced MCF-7 cells, highly expressed TR3 favours the formation of TR3/RXRalpha and promotes the TR3/RXRalpha signalling pathway causing apoptosis; while in ATRA-induced MGC80-3 cells, high expression of RARalpha favours the formation of RARalpha/RXRalpha and promotes the RXRalpha/RARalpha signalling pathway in mediating cell cycle regulation. In conclusion, these results reveal the novel mechanism that cellular expression and location of protein is associated with diverse signalling transduction pathways and the resultant physiological process.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Nuclear Receptor Subfamily 4, Group A, Member 1; Receptors, Retinoic Acid; Receptors, Steroid; Receptors, Thyroid Hormone; Retinoic Acid Receptor alpha; Signal Transduction; Stomach Neoplasms; Tretinoin

Insulin-like growth factor binding protein-3 (IGFBP-3) has both IGF-dependent and -independent effects on cell growth, which are frequently growth-inhibitory. Interestingly, the development of a more aggressive phenotype in breast cancer cells (BCCs) correlates positively with elevated expression of IGFBP-3 and is often associated with all-trans-retinoic acid (atRA)-resistance. IGFBP-3 was previously demonstrated to interact directly with retinoid X receptor (RXR). In this study we have shown that IGFBP-5 also interacts with RXR and that both IGFBPs interact with retinoic acid receptor (RAR). To investigate whether the presence of IGFBP-3 regulates breast cancer cell responsiveness to atRA, we immuno-neutralized the IGFBP-3 expressed by the atRA-resistant Hs578T and MDA-MB-231 BCCs (which express IGFBP-3 constitutively) and showed that they become more sensitive to the growth-inhibitory effects of atRA. Similarly, in Hs578T cells expressing a reporter gene under the control of an RAR response element (RARE), depletion of IGFBP-3 resulted in the induction of reporter gene expression in response to atRA. In investigating possible mechanisms for IGFBP-3 regulation of atRA-sensitivity, we found that IGFBP-3 blocked the formation of RAR:RXR heterodimers and disrupted the ligand-inducible receptor complex. Thus, IGFBP-3 has the potential to reduce the RARE-mediated transactivation of target genes and modulate the atRA-response in BCCs.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line, Tumor; Dimerization; Drug Resistance, Neoplasm; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Protein 5; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Transcription Factors; Tretinoin

Taxotere is a cytotoxin effective in treating breast and prostate cancer. It stabilizes microtubules and causes catastrophic cell cycle arrest in G2/M. Taxanes also initiate apoptosis by activating signal pathways, such as the jun N-terminal kinase (JNK) pathway. Strategies aimed at potentiating cell death signaling may improve their efficacy while lessening the potential side effects. We reported that all-trans retinoic acid (ATRA) potentiated taxane-mediated cell death. Here we investigated whether ATRA potentiates cell death signaling through the JNK pathway. Activation of JNK by Taxotere 0.01, 0.1 and 1.0 microM was observed at 24 h in adherent cells and increased at 48 h. Taxotere 0.001 microM-induced JNK activation started after 48 h and increased at 72 h. The timing and intensity of PARP cleavage was similar to that of JNK activation. JNK activation and PARP cleavage induced by 30 nM Taxotere at 48 h were reversed by curcumin, PD169316 and SP600125, JNK inhibitors in order of progressive specificity. None of these inhibitors had an effect on p38 or ERK phosphorylation. All three inhibitors reversed Taxotere-induced phosphorylation of Bcl-2. ATRA induced JNK activation at 24, 48 and 72 h. Incubating cells with ATRA 0.01 microM for 3 days prior to Taxotere treatment potentiated Taxotere-induced JNK activation 24 and 48 h later, an effect sustained for 72 h. Cytotoxicities from 3-day ATRA 0.01 microM incubations were synergistic with subsequent 1-h Taxotere 0.01, 0.1 and 1.0 microM incubations in breast cancer cell lines MCF-7 and MDA-MB-231 and in prostate cancer cell lines LNCaP and PC-3, and additive in breast cancer cell line SK-Br-3. These data demonstrate the potentiation of Taxotere-induced cell death by ATRA pretreatment in breast and prostate cancer cells, and support a mechanism through accentuated and sustained JNK activation.

Topics: Breast Neoplasms; Cell Death; Cell Line, Tumor; Docetaxel; Dose-Response Relationship, Drug; Enzyme Activation; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Taxoids; Tretinoin

Lactating breast tissue and some breast cancers express the sodium/iodide symporter (NIS) and concentrate iodide. We recently demonstrated that all-trans retinoic acid (tRA) induces both NIS gene expression and iodide accumulation in vitro in well-differentiated human breast cancer cells (MCF-7). In the present study, we investigated the in vivo efficacy and specificity of tRA-stimulated iodide accumulation in mouse breast cancer models. Immunodeficient mice with MCF-7 xenograft tumors were treated with systemic tRA for 5 days. Iodide accumulation in the xenograft tumors was markedly increased, approximately 15-fold greater than levels without treatment, and the effects were tRA dose dependent. Iodide accumulation in other organs was not significantly influenced by tRA treatment. Significant induction of NIS mRNA and protein in the xenograft tumors was observed after tRA treatment. Iodide accumulation and NIS mRNA expression were also selectively induced in breast cancer tissues in transgenic mice expressing the oncogene, polyoma virus middle T antigen. These data demonstrate selective induction of functional NIS in breast cancer by tRA. Treatment with short-term systemic retinoic acid, followed by radioiodide administration, is a potential tool in the diagnosis and treatment of some differentiated breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Mice; Mice, SCID; Radionuclide Imaging; Symporters; Tissue Distribution; Transplantation, Heterologous; Tretinoin

The tumor suppressor gene p53 is a transcription factor that mediates both cell cycle arrest and apoptosis. Interestingly, p53 also induces differentiation of a number of tissues, including leukemic cells. However, although p53-mediated differentiation of leukemic U-937 cells depends on the transcriptional activity of p53, a p53 target gene mediating differentiation has hitherto not been identified. To screen for novel p53 target genes in leukemic cells, a cDNA microarray analysis was performed with U-937-4/ptsp53 cells, expressing a temperature-sensitive p53 mutant. We report that transcription of the Staf50 (stimulated transacting factor of 50 kDa) gene is upregulated in response to wild-type p53 in U-937-4, K562 and MCF-7 cells. Staf50 was directly activated by p53, as determined by the independence of de novo protein synthesis. Moreover, while the proximal promoter of Staf50 was found not to be p53 responsive, a functional enhancer-like p53-response element in intron 1 of the Staf50 gene was identified that was also transactivated by the p53-family member p73. Direct binding of p53 to the response element was shown by electrophoretic mobility shift analysis. Ectopic expression of Staf50 in U-937 cells resulted in reduced clonogenic growth. Moreover, levels of endogenous Staf50 mRNA correlated to all-trans retinoic acid-induced differentiation of promyelocytic NB-4 and HL60 cells, suggesting that Staf50 could be involved in proliferation and/or differentiation of leukemic cells.

Topics: Amino Acid Motifs; Binding Sites; Breast Neoplasms; DNA-Binding Proteins; Gene Expression Regulation; Genes, Tumor Suppressor; Humans; Interferon-alpha; Minor Histocompatibility Antigens; Myeloid Cells; Nuclear Proteins; Promoter Regions, Genetic; Repressor Proteins; RNA, Messenger; Transcription Factors; Tretinoin; Tripartite Motif Proteins; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; U937 Cells

The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer.. Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event.. CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Down-Regulation; Evolution, Molecular; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Mice; Oncogenes; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Retinoids, which include vitamin A (retinol; ROL) and its derivatives, have been investigated in the treatment of bladder cancer. We have shown that expression of the enzyme lecithin:ROL acyltransferase (LRAT), which converts ROL to retinyl esters, is reduced in several human cancers. Here we evaluated expression of LRAT protein and mRNA in normal and malignant bladder tissue specimens from human patients. We also examined the effect of retinoids on LRAT expression in bladder cancer cell lines.. We evaluated 49 bladder cancer specimens for LRAT protein expression using immunohistochemistry with affinity-purified antibodies to human LRAT. LRAT mRNA expression was assessed using reverse transcription-PCR in bladder specimens from an additional 16 patients. We examined the effect of retinoic acid and ROL on LRAT mRNA expression in five human bladder cancer cell lines.. LRAT protein was detected throughout the nonneoplastic bladder epithelium in all of the specimens. In bladder tumors, LRAT protein expression was reduced compared with the nonneoplastic epithelium or was completely absent in 7 of 32 (21.9%) superficial tumors versus 16 of 17 (94.1%) invasive tumors (P < 0.001). All of the non-neoplastic bladder specimens tested (11 of 11) showed LRAT mRNA expression, compared with 5 of 8 (62%) superficial tumors and 0 of 5 (0%) invasive tumors (P = 0.001). Three of five human bladder cancer cell lines expressed LRAT mRNA independent of retinoid exposure, whereas in two cell lines LRAT mRNA expression was induced by retinoid treatment.. We report a significant reduction in LRAT expression in bladder cancer. Moreover, we demonstrate an inverse correlation of LRAT mRNA and protein expression with increasing tumor stage. These data suggest that loss of LRAT expression is associated with invasive bladder cancer.

Topics: Acyltransferases; Adult; Aged; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Disease Progression; Esters; Extracellular Matrix; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Prognosis; Proportional Hazards Models; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; Risk; Risk Factors; RNA, Messenger; Tretinoin; Urinary Bladder Neoplasms

Retinoic acid receptor alpha (RARalpha) plays an important role in mediating all-trans retinoic acid (ATRA) signals. In this study, we found that ATRA up-regulated RARalpha mRNA and protein expression in gastric cancer BGC-823 cells. However, in breast cancer MCF-7 cells it down-regulated RARalpha protein expression with no effect on its RARalpha mRNA. Immunoprecipitation/Western blot analysis showed that, although sumoylated and ubiquitinated RARalpha existed simultaneously in both cancer cell lines, ATRA exerted different regulatory effects on sumoylation and ubiquitination of RARalpha. In MCF-7 cells, ATRA treatment enhanced the ubiquitination of RARalpha and the subsequent degradation of RARalpha through the ubiquitin/proteasome pathway. This resulted in a reduction in the DNA binding activity of RARalpha/retinoid X receptor alpha (RXRalpha) heterodimer, the separation of RXRalpha from RARalpha and the translocation of RXRalpha from the nucleus to the cytoplasm. By contrast, in BGC-823 cells, ATRA augmented sumoylation, not ubiquitination, of RARalpha. The stability of sumoylated RARalpha was significantly stronger than in non-sumoylated RARalpha. These results also showed an increase in the DNA binding activity of the RARalpha/RXRalpha heterodimer and the stability of nuclear localization of this heterodimer, which normally facilitates the ATRA signal transduction. In conclusion, our results reveal a novel mechanism for the regulation of RARalpha-dependent signal transduction through the ubiquitin/proteasome pathway in breast cancer cells and the sumoylation pathway in gastric cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Dimerization; Female; Humans; Polyubiquitin; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; Stomach Neoplasms; SUMO-1 Protein; Tretinoin

Acute promyelocytic leukemia (APL) is characterized by the presence of the t(15;17) translocation, resulting in the PML-RAR fusion protein. Standard treatment consists of the combination of all-trans retinoic acid (ATRA) with an anthracycline that results in complete remission (CR) rates in excess of 90%. Recently, several new agents have been shown to have clinical activity in APL. These include a liposomal formulation of ATRA (lipo-ATRA), and gemtuzumab ozogamicin (GO). Herein, we report a patient with APL who relapsed with extramedullary disease 2.5 years after lipo-ATRA therapy and was successfully treated with the sequence of A2O3, ATRA, and GO and we summarize our experience with patients with isolated extramedullary relapse in APL.

Topics: Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Breast Neoplasms; Female; Gemtuzumab; Humans; Leukemia, Promyelocytic, Acute; Leukemic Infiltration; Middle Aged; Oxides; Recurrence; Tretinoin

Retinoids and interferons are signaling molecules with pronounced anticancer activity. We show that in both acute promyelocytic leukemia and breast cancer cells the retinoic acid (RA) and interferon signaling pathways converge on the promoter of the tumoricidal death ligand TRAIL. Promoter mapping, chromatin immunoprecipitation and RNA interference reveal that retinoid-induced interferon regulatory factor-1 (IRF-1), a tumor suppressor, is critically required for TRAIL induction by both RA and IFNgamma. Exposure of breast cancer cells to both antitumor agents results in enhanced TRAIL promoter occupancy by IRF-1 and coactivator recruitment, leading to strong histone acetylation and synergistic induction of TRAIL expression. In coculture experiments, pre-exposure of breast cancer cells to RA and IFNgamma induced a dramatic TRAIL-dependent apoptosis in heterologous cancer cells in a paracrine mode of action, while normal cells were not affected. Our results identify a novel TRAIL-mediated tumor suppressor activity of IRF-1 and suggest a mechanistic basis for the synergistic antitumor activities of certain retinoids and interferons. These data argue for combination therapies that activate the TRAIL pathway to eradicate tumor cells.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Humans; Interferon Regulatory Factor-1; Interferon-gamma; Leukemia; Membrane Glycoproteins; Phosphoproteins; Promoter Regions, Genetic; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tretinoin; Tumor Necrosis Factor-alpha

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Antagonism; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Mice; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tretinoin; Up-Regulation

Certain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment.. We determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro.. PPAR-alpha and PPAR-gamma ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-alpha-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound.. RXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; Drug Resistance, Neoplasm; Fatty Acids, Unsaturated; gamma-Linolenic Acid; Gene Expression; Humans; Ligands; Nuclear Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Tetrahydronaphthalenes; Transcription Factors; Tretinoin

The sodium/iodide symporter (NIS) is a plasma membrane protein that mediates active iodide transport in thyroid and mammary cells. It is a prerequisite for radioiodide treatment of thyroid cancer and a promising diagnostic and therapeutic tool for breast cancer. We investigated the molecular mechanisms governing NIS expression in mammary cells. Here we report that Nkx-2.5, a cardiac homeobox transcription factor that is also expressed in the thyroid primordium, is a potent inducer of the NIS promoter. By binding to two specific promoter sites (N2 and W), Nkx-2.5 induced the rNIS promoter (about 50-fold over the basal level). Interestingly, coincident with NIS expression, Nkx-2.5 mRNA and protein were present in lactating, but not virgin, mammary glands in two human breast cancer samples and in all-trans retinoic acid (tRA)-stimulated MCF-7 breast cancer cells. A cotransfected dominant-negative Nkx-2.5 mutant abolished tRA-induced endogenous NIS induction, which shows that Nkx-2.5 activity is critical for this process. Remarkably, in MCF-7 cells, Nkx-2.5 overexpression alone was sufficient to induce NIS and iodide uptake. In conclusion, Nkx-2.5 is a novel relevant transcriptional regulator of mammary NIS and could thus be exploited to manipulate NIS expression in breast cancer treatment strategies.

Topics: Animals; Base Sequence; Binding Sites; Breast Neoplasms; Cell Line, Tumor; DNA, Neoplasm; Female; Gene Expression; HeLa Cells; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Humans; Lactation; Mutagenesis, Site-Directed; Pregnancy; Promoter Regions, Genetic; Rats; RNA, Messenger; RNA, Neoplasm; Symporters; Thyroid Gland; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin

We showed that the HER2/Grb2/Akt pathway induces all-trans retinoic acid (ATRA) resistance in breast cancer cells by suppressing the DNA binding activity of retinoic acid receptors (RAR). AP-1 activation was shown to induce ATRA resistance. Here, we determined whether AP-1 binding activity is correlated with ATRA resistance in HER2-overexpressing cells. Inhibition of HER2/Grb2/Akt decreased AP-1 binding activity in HER2-transfected cells, but increased AP-1 activity in cells that are naturally HER2-overexpressing. Since HER2/Grb2/Akt inhibition sensitized both cell types to ATRA, our results indicate that, unlike RAR, AP-1 binding activity is not correlated with ATRA sensitivity in HER2-overexpressing breast cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Breast Neoplasms; Electrophoretic Mobility Shift Assay; Female; Genes, jun; GRB2 Adaptor Protein; Humans; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Retinoic Acid; src Homology Domains; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

Vitamin D3 (VD) and all-trans-retinoic acid (ATRA) have been postulated as a novel treatment option for breast carcinoma. Since the combined effects of retinoids and VD derivatives are attributed to heterodimeric interactions between members of the nuclear receptor family, the expression patterns of the heterodimers formed by vitamin D3 receptor (VDR) and the retinoid receptors RARs (RAR-alpha, RAR-beta and RAR-gamma) and RXRs (RXR-alpha, RXR-beta and RXR-gamma) have been studied by immunohistochemistry in benign and malignant breast tissues. Present results revealed that immunoexpressions to all receptor types studied were higher in both in situ and infiltrative carcinomas than in benign breast diseases. In a variable number of cases of infiltrative carcinoma, immunostaining appeared in the nucleus, whereas in the other two disorders immunostaining was only cytoplasmic. The correlation established between VDR and the different isoforms of retinoid receptors revealed that VDR seems to select mainly RAR-alpha to form heterodimers and to exert their properties as transcription factor. The results of this study suggest that this heterodimer plays a critical role in cancer malignancy, and its presence indicates those patient groups presenting a better response to adjuvant therapies based on the combination of vitamin D and ATRA.

Topics: Adult; Aged; Blotting, Western; Breast Neoplasms; Dimerization; Female; Humans; Middle Aged; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Tretinoin; Vitamin D

The ability of retinoids to inhibit breast cancer cell growth correlates with estrogen receptor (ER) alpha status, as shown by the antiproliferative effects of retinoids in ERalpha-positive breast cancer cells and their use as chemopreventive agents in premenopausal women. The discovery of ERbeta, also present in breast cancer cells, has added a new level of complexity to this malignancy. To determine the retinoid response in ERbeta-expressing breast cancer cells, we used retroviral transduction of ERbeta in ER-negative MDA-MB-231 cells. Western blot and immunofluorescence confirmed expression and nuclear localization of ERbeta, whereas functionality was shown using an estrogen response element-containing reporter. A significant retinoic acid (RA)-mediated growth inhibition was observed in the transduced ERbeta-positive cells as shown by proliferation assays. Addition of estradiol, tamoxifen, or ICI 182,780 had no effect on cell growth and did not alter RA sensitivity. We observed that retinoids altered ERbeta-mediated transcriptional activity from an estrogen response element, which was confirmed by decreased expression of the pS2 gene, and from an activator protein response element. Conversely, the expression of ERbeta altered RA receptor (RAR) beta expression, resulting in greater induction of RARbeta gene expression on RA treatment, without altered expression of RARalpha. Our data provide evidence of transcriptional crosstalk between ERbeta and RAR in ERbeta-positive breast cancer cells that are growth inhibited by RA.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; Humans; Receptors, Retinoic Acid; Response Elements; Transcription Factor AP-1; Transcriptional Activation; Transfection; Tretinoin

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogen Receptor Modulators; Female; Humans; Isotretinoin; Ki-67 Antigen; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Thymidine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Suppressor Protein p53

The synthetic retinoid N-(4-hydroxphenyl) retinamide (4HPR) has manifold actions, which may contribute to its chemopreventive effects on breast cancer cell growth and progression. A role for ceramide as a stress-response signal is investigated here during the cytotoxic action of 4HPR in MCF-7 cells. N-(4-hydroxphenyl) retinamide induced a dose-dependent decline in cell growth and survival associated with a maximal 10-fold increase in ceramide production at 10 microM. N-(4-hydroxphenyl) retinamide exhibited a greater potency than all-trans retinoic acid (ATRA) on growth inhibition and ceramide production. The synthetic peroxisome proliferator-activated receptors agonist troglitazone (TGZ), but not the native ligand 15-deoxy-delta 12,14-prostaglandin J2, abrogated both these actions of 4HPR but not that of ATRA. The antioxidant N-acetylcysteine mimicked the abrogative effect of TGZ on 4HPR action, while the exogenous oxidant H2O2 also stimulated ceramide production. The inhibitors of de novo ceramide synthesis, fumonisin B1 and myriocin, blocked the ceramide response to 4HPR and partially reversed the apoptotic response, but did not prevent the overall decline in cell survival. The pancaspase inhibitor Z-VAD fmk reduced the decrease in cell survival caused by 4HPR, but did not affect the ceramide response. These findings describe a novel redox-sensitive elevation of ceramide levels associated with the cytotoxic response of breast cancer cells to 4HPR. However, a major mediatory role for this sphingolipid in this context remains equivocal.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Division; Cell Survival; Ceramides; Fenretinide; Glucosylceramides; Humans; Oxidation-Reduction; PPAR gamma; Tretinoin; Tumor Cells, Cultured

The efficacy of MDI-301, a non-toxic novel synthetic retinoid, was found to be equivalent to the natural 9-cis-retinoic acid (RA) in vitro against estrogen-dependent MCF7 and T47D breast cancer cell lines which express RA receptor (RAR) alpha. Both retinoids also showed similar efficacy against established PC-3 prostate carcinoma xenografts. MCF7 tumor xenografts showed a reduction in tumor growth of 48% without systemic side-effects upon treatment with MDI-301 compared with MCF7 controls. Tumor xenografts derived from MDA-MB-231, an estrogen-independent breast cancer cell line that expresses low levels of RARalpha, were unresponsive. This study demonstrates that MDI-301 is as efficacious as 9-cis-RA against cancer cells with RARalpha, with no signs of toxicity in vivo, making it a potential candidate for cancer therapy.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Tretinoin; Xenograft Model Antitumor Assays

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 3; Protein Kinase C; Protein Kinase C-alpha; Receptor Protein-Tyrosine Kinases; Retinoids; Signal Transduction; Tretinoin

The anti-estrogen 4-hydroxytamoxifen (TAM) and vitamin A-related compounds, the retinoids, in combination act synergistically to inhibit growth of breast cancer cells in vitro and in vivo. To clarify the mechanism of this synergism, the effect of TAM and all trans-retinoic acid (AT) on proliferation of MCF-7 breast cancer cells was studied in vitro. TAM and AT acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. In a temporally related manner, TAM+AT acted synergistically to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis. TAM and AT each blocked cell cycle progression throughout 7 days of treatment but without any synergistic or additive effect on this process, indicating a selective synergism for apoptosis. The negative growth factor-transforming growth factor beta (TGFbeta) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM+AT on apoptosis. TAM+AT acted synergistically to induce a fivefold increase in TGFbeta1 secretion over 72 h. TGFbeta1 alone had no apoptotic effects on these cells; however, TGFbeta1 in combination with AT acted synergistically to inhibit growth, to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner comparable with that noted for TAM+AT. The synergism of both TAM+AT and TGFbeta1+AT for apoptosis was suppressed by estradiol. Co-incubation of TAM+AT with anti-TGFbeta antibody did not block down-regulation of Bcl-2 protein expression or stimulation of apoptosis. The synergistic effects of TAM+AT on apoptosis therefore occur independently of TGFbeta, although TGFbeta may interact with AT in a novel manner to provide another important anti-proliferative mechanism for breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Estrogen Receptor Modulators; Female; Humans; Stimulation, Chemical; Tamoxifen; Time Factors; Transforming Growth Factor beta; Tretinoin

Metastasis, the main reason for high mortality of cancer, is a multistep process. One important step in this process is the adhesion of tumor cells to vascular endothelium at sites distant from primary tumors during hematogenous dissemination. In order to investigate and quantify the adhesion of tumor cells to endothelial cells we developed an in vitro model using MCF-7 breast cancer cells and monolayers of human umbilical vein endothelial cells (HUVEC). The tumor cells were specifically labeled with a fluorescent dye for quantification; for increasing the amount of adherent cells, HUVEC monolayers were stimulated with phorbol ester before the addition of the tumor cells. Due to previous reports that products of several P450 enzymes contribute to the progression of certain kinds of cancer, inhibitors of CYP5 (thromboxane A(2) synthase), CYP17 (17alpha-hydroxylase-C17, 20-lyase), and CYP19 (aromatase) were tested in this in vitro model for their potency to reduce cancer cell adhesion. Within each series of P450 inhibitors, compounds with high inhibitory activity on tumor cell adhesion were identified. At an initial concentration of 100 microM, BW26, a potent inhibitor of CYP5, reduced tumor cell adhesion of MCF-7 to HUVECs to 15%, BW40 (CYP17) to 29%, and SU5a (CYP19) to 11% of the corresponding controls (no inhibitor). Reduction of tumor cell adhesion was shown to occur in a concentration-dependent manner. In addition to these inhibitors of CYP5, CYP17, and CYP19, liarozole, known to be a potent inhibitor of CYP26 (retinoic acid-4-hydroxylase) and ATRA (all-trans-retinoic acid) metabolism, was able to reduce tumor cell adhesion to 51% of the initial rate. Experiments elucidating the mode of action of these compounds revealed that inhibition of the mentioned CYP enzymes is not responsible for their ability to reduce tumor cell adhesion.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Inhibitors; Humans; Models, Biological; Molecular Structure; Tretinoin

Retinoids regulate gene transcription through activating retinoic acid receptors (RARs)/retinoic X receptors (RXRs). Of the three RAR receptors (alpha, beta, and gamma), RARbeta has been considered a tumor suppressor gene. Here, we identified a novel RARbeta isoform-RARbeta5 in breast epithelial cells, which could play a negative role in RARbeta signaling. Similar to RARbeta2, the first exon (59 bp) of RARbeta5 is RARbeta5 isoform specific, whereas the other exons are common to all of the RARbeta isoforms. The first exon of RARbeta5 does not contain any translation start codon, and therefore its protein translation begins at an internal methionine codon of RARbeta2, lacking the A, B, and part of C domain of RARbeta2. RARbeta5 protein was preferentially expressed in estrogen receptor-negative breast cancer cells and normal breast epithelial cells that are relatively resistant to retinoids, whereas estrogen receptor-positive cells that did not express detectable RARbeta5 protein were sensitive to retinoid treatment, suggesting that this isoform may affect the cellular response to retinoids. RARbeta5 isoform is unique among all of the RARs, because a corresponding isoform was not detectable for either RARalpha or RARgamma. RARbeta5 mRNA was variably expressed in normal and cancerous breast epithelial cells. Its transcription was under the control of a distinct promoter P3, which can be activated by all-trans-retinoic acid (atRA) and other RAR/RXR selective retinoids in MCF-7 and T47D breast cancer cells. We mapped the RARbeta5 promoter and found a region -302/-99 to be the target region of atRA. In conclusion, we identified and initially characterized RARbeta5 in normal, premalignant, and malignant breast epithelial cells. RARbeta5 may serve as a potential target of retinoids in prevention and therapy studies.

Topics: Amino Acid Sequence; Base Sequence; Breast; Breast Neoplasms; Cell Line, Tumor; Cloning, Molecular; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Precancerous Conditions; Promoter Regions, Genetic; Protein Isoforms; Receptors, Retinoic Acid; RNA, Messenger; Transcription, Genetic; Transfection; Tretinoin

Doxorubicin (Adriamycin) is the most active drug in the treatment of breast cancer. The aim of this study was to investigate the interaction of doxorubicin and retinoids in the inhibition of proliferation of hormone sensitive (ER+) human breast cancer cell line MCF-7 and to find out whether this combination can result in the enhancement of its therapeutic effect. As a comparison we also used estradiol and tamoxifen. We also made an attempt to elucidate the effect of these compounds on the stimulation of the apoptotic pathway in breast cancer cells. Cell proliferation in a 24-hour culture was assessed by [3H] thymidine incorporation into cancer cells and by immunocytochemical analysis of cellular cycle-related PCNA and Ki-67 antigens expression, after the incubation of the cell culture with 10, 20 and 50 nM doxorubicin (DOX), 2 nM estradiol (E2), 10 microM tamoxifen (TAM) and 1 nM, 0.01, 0.1, 1 and 10 microM of all-trans retinoid acid (ATRA). The assessment of cell viability and analysis of apoptotic and necrotic cells were performed after the 72-hour incubation of the culture with the examined substances and following apoptosis induction using acridine orange and ethidine bromide. Of the doxorubicin concentrations used in the study, 20 nM inhibited thymidine incorporation to 84.83 +/- 10.00% (control=100%). In the same culture conditions, 2 nM E2 stimulated cancer cells to 157.09 +/- 8.84%. Concentrations of 10 microM TAM and 10 microM ATRA inhibited the proliferation to 63.16 +/- 7.85% and 52.19 +/- 3.21%, respectively. A statistically significant reduction of these values was observed when 20 nM DOX was added to medium with E2 - 39.24 +/- 7.6%, TAM - 48.34 +/- 2.05% and ATRA - 21.98 +/- 1.69%, respectively; the percentage of PCNA- and Ki-67-positive cells was also reduced. Despite high antiproliferative efficacy of 20 nM DOX and 10 microM ATRA combination, the percentage of apoptotic cells was only 25 +/- 0.81%, being similar to that obtained in the culture with 20 nM DOX. The concentrations of 10, 20 and 50 nM DOX that were used to inhibit the proliferation of MCF-7 cell line were not particulary effective. The inhibitory effect was obtained when 20 nM of DOX and E2, TAM or ATRA were used simultaneously. The use of E2 caused a two-fold decrease in the percentage of proliferating cells. It was also shown that the effectiveness of DOX in combination with ATRA is significantly higher than that of DOX combined with TAM, which might suggest a valuable appro

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Doxorubicin; Estradiol; Estrogen Antagonists; Female; Humans; Immunohistochemistry; Necrosis; Tamoxifen; Tretinoin

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.

Topics: Breast Neoplasms; Calcitriol; Calcium; Cell Movement; Cells, Cultured; Chemotaxis; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Lysophospholipids; Male; Membrane Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; Signal Transduction; Sphingosine; Tretinoin

The human retinoic acid receptor beta (RARbeta) has three isoforms (beta1, beta2, and beta4), which play important, distinct roles in mediating the effects of retinoic acid on cell growth and apoptosis. Whereas RARbeta2 is a potent inhibitor of breast cancer cell proliferation, RARbeta4 can act as a dominant-negative repressor of RARbeta2-mediated growth suppression. In this study we investigated the effects of all-trans-retinoic acid (ATRA) on two clones derived from the breast cancer cell line MDA-MB-435: a non-metastatic clone (NM-2C5) and a metastatic clone (M-4A4). ATRA treatment of the NM-2C5 cells resulted in growth inhibition and apoptosis, whereas the M-4A4 cells were resistant to ATRA. Analyses of the expression of RARbeta isoforms revealed that the sensitive NM-2C5 clone expressed only RARbeta2, whereas the resistant M-4A4 cells expressed both RARbeta2 and RARbeta4 mRNA and protein. ATRA treatment increased RARbeta2 mRNA level in NM-2C5 cells, whereas the same treatment of the M-4A4 cells resulted in an increase in RARbeta4 and a decrease in RARbeta2 mRNA. ATRA treatment of NM-2C5 cells increased the protein levels of the histone acetyl transferases p300 and CBP, suppressed the level of histone deacetylase and increased the level of acetylated histone H4. ATRA also decreased Bcl-2 and increased Bax and decreased VEGF. In contrast, the same treatment of the M-4A4 cells resulted in opposite effects. These results suggest that the effects of ATRA on the growth of the metastatic and non-metastatic breast cancer cell lines depend on the expression of RARbeta isoforms and that the expression of RARbeta4 may contribute to metastatic properties.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Division; Cell Line, Tumor; Clone Cells; Drug Resistance, Neoplasm; Enzyme Induction; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Histone Deacetylases; Histones; Humans; Neoplasm Metastasis; Neoplasm Proteins; Nuclear Proteins; Protein Isoforms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Trans-Activators; Tretinoin; Vascular Endothelial Growth Factor A

Luciferase genes are widely used as reporters to analyze promoter and regulatory elements. We found that a luciferase reporter gene vector with a modified firefly luciferase gene (luc+), but not Renilla luciferase (Rluc), was induced by all-trans retinoic acid (tRA) in the MCF-7 breast cancer cell line. tRA (5 x 10(-6) M) increased luciferase activity of the pGL3 promoter vector (containing luc+) up to approximately 3.8-fold in MCF-7 cells, but not in LNCaP prostate cancer cells or JEG-3 choriocarcinoma cells. Chimeric plasmids were constructed and showed that tRA-induction required the luc+ gene, but not any specific promoter or vector sequence. Time course and dose-response studies of tRA-induction indicated that longer treatment (> 24h) and higher tRA dose (> 10(-6) M) were required for luc+ induction compared with those for a positive retinoic acid response element (maximum induction at 6 h and 10(-8) M tRA). Studies with the translation inhibitor, cycloheximide, indicated the half-life of the luc+ protein was increased from 9.7 +/- 1.5 to 22.1 +/- 3.1 h with tRA treatment. Other retinoids, TTNPB, a retinoic acid receptor beta/gamma-specific ligand, and a retinoid X receptor ligand, did not significantly increase luc+ expression. Caution is needed in analysis of retinoid responsive gene regulation with the luciferase reporter system in MCF-7 cells, especially at high retinoid concentrations.

Topics: Animals; Breast Neoplasms; Coleoptera; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Humans; Luciferases; Tretinoin; Tumor Cells, Cultured

Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cadherins; Cell Communication; Connexin 26; Connexins; Gap Junctions; Humans; Immunohistochemistry; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Tamoxifen; Telomerase; Tretinoin; Tumor Cells, Cultured

Nuclear retinoid receptors mediate retinoid effects through tissue-specific, ligand-receptor interactions and subsequent transcriptional regulation of secondary target genes. Retinoic acid receptor beta (RARbeta) is itself a retinoid target gene with a retinoic acid response element (betaRARE) in the 5' untranslated region of the RARbeta2 gene. Altered transcriptional regulation of RARbeta may play a role in human carcinogenesis and the retinoid-responsiveness of malignant cells. Here we used retinoid X receptor-specific antibodies in electrophoretic mobility shift assays to show that the retinoid X receptor beta (RXRbeta) protein was recruited to the betaRARE, after retinoid treatment of retinoid-sensitive neuroblastoma (NB), lung and breast cancer cell lines, but not retinoid-resistant lung and breast cancer cell lines. RXRbeta selectively enhanced retinoid-induced transcriptional activation of the betaRARE. Stable overexpression of RXRalpha and RXRbeta in NB cells resulted in marked growth inhibition and cell death, which increased after retinoid treatment. However, only proteins from the RXRbeta transfectants exhibited specific RXRbeta binding to the betaRARE in vitro and in vivo, enhanced histone acetylation and increased endogenous RARbeta expression. These data indicate that recruitment of RXRbeta to the betaRARE, and consequent induction of endogenous RARbeta expression, is an important component in the retinoid anticancer signal. RXRalpha may also participate in the retinoid signal, but through mechanisms that do not involve RARbeta.

Topics: Acetylation; Antineoplastic Agents; Breast Neoplasms; Cell Division; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Histones; Humans; Lung Neoplasms; Nervous System Neoplasms; Neuroblastoma; Promoter Regions, Genetic; Protein Transport; Receptors, Retinoic Acid; Response Elements; RNA, Messenger; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

To analyze patient cases of therapy-related acute promyelocytic leukemia (tAPL), occurring after chemotherapy (CT), radiotherapy (RT) or both for a prior disorder, diagnosed during the last 20 years in three European countries.. The primary disorder and its treatment, interval from primary disorder to tAPL, characteristics of tAPL, and its outcome were analyzed in 106 patients.. Eighty of the 106 cases of tAPL were diagnosed during the last 10 years, indicating an increasing incidence of tAPL. Primary disorders were predominantly breast carcinoma (60 patients), non-Hodgkin's lymphoma (15 patients), and other solid tumors (25 patients). Thirty patients had received CT alone, 27 patients had received RT alone, and 49 patients had received both. CT included at least one alkylating agent in 68 patients and at least one topoisomerase II inhibitor in 61 patients, including anthracyclines (30 patients), mitoxantrone (28 patients), and epipodophyllotoxins (19 patients). Median interval from primary disorder to tAPL diagnosis was 25 months (range, 4 to 276 months). Characteristics of tAPL were generally similar to those of de novo APL. With treatment using anthracycline-cytarabine-based CT or all-trans-retinoic acid combined with CT, actuarial survival was 59% at 8 years.. tAPL is not exceptional, and develops usually less than 3 years after a primary neoplasm (especially breast carcinoma) treated in particular with topoisomerase II-targeted drugs (anthracyclines or mitoxantrone and less often etoposide). Characteristics and outcome of tAPL seem similar to those of de novo APL.

Topics: Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Belgium; Breast Neoplasms; Child; DNA Topoisomerases, Type II; Female; France; Humans; Leukemia, Promyelocytic, Acute; Leukemia, Radiation-Induced; Lymphoma; Male; Middle Aged; Retrospective Studies; Spain; Treatment Outcome; Tretinoin

In the MCF-7 breast cancer cell line, insulin-like growth factors (IGFs) are known to elicit antiproliferative actions via the insulin receptor substrate-1 (IRS-1)/PI 3-kinase/AKT pathway. All-trans retinoic acid (RA) is a potent inhibitor of MCF-7 cell proliferation, but the mechanism by which growth regulation is achieved remains unclear. We investigated the effects of RA on the regulation of the IGF-IR and its key signaling elements: IRS-1, IRS-2, and SHC. Treatment of MCF-7 cells with RA caused a significant reduction in IRS-1 protein and tyrosine phosphorylation levels at a concentration and time consistent with RA-mediated growth inhibition. IRS-1 regulation is selective, as RA did not influence IRS-2 or SHC levels. Downstream signaling events were also selectively reduced, as RA abrogated IGF-I-stimulated AKT activation but did not alter erk1/2 activation. To confirm the importance of IRS-1 regulation by RA, we examined the response to RA in MCF-7 cells overexpressing IGF-IR and IRS-1. RA resistance was observed in MCF-7 cells overexpressing IRS-1 but not IGF-IR. This suggests that RA-mediated growth inhibition requires the selective downregulation of IRS-1 and AKT. Therapeutic agents targeting the IRS-1/PI 3-kinase/AKT pathway may enhance the cytostatic effects of RA in breast cancer, since overexpression of IRS-1 and AKT have been reported in primary breast tumors.

Topics: Breast Neoplasms; Cell Division; Down-Regulation; Female; Humans; In Vitro Techniques; Insulin Receptor Substrate Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Invasion and metastasis are the main causes of death in breast cancer patients. Increased expression of matrix metalloproteinases (MMPs), especially gelatinases (MMP-2 and -9), has been closely associated with tumor progression. One of the nuclear hormone receptors (NHR), peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-activated transcriptional factor that regulates cell proliferation, differentiation and apoptosis in both normal and cancer cells. Recent data indicate that PPARgamma activation by its ligands can also lead to the inhibition of gelatinase B (MMP-9) and the blockage of migration in macrophages and muscle cells, implying the possibility that PPARgamma ligands may possess anti-invasive activities on tumor cells. In this study, we showed that treatment of the highly aggressive human breast cancer cell line MDA-MB-231 with the synthetic PPARgamma ligands pioglitazone (PGZ), rosiglitazone (RGZ), GW7845 or its natural ligand 15-deoxy-delta 12, 14-prostaglandin J2(15d-PGJ2), at concentrations at which no obvious cytotoxicity was observed in vitro, led to a significant inhibition of the invasive capacities of this cell line through a reconstituted basement membrane (Matrigel) in a Transwell chamber model. All-trans-retinoic acid (ATRA), a ligand for retinoic acid receptor (RAR), was also studied and showed a similar inhibitory effect on invasion. Although no change was observed in the expression of MMP-9 after challenge with PPARgamma ligands and/or ATRA on this cell line, the natural tissue inhibitor of gelatinases, namely the tissue inhibitor of MMP 1 (TIMP-1) was upregulated by these treatments and the gelatinolytic activities of gelatinases in the conditioned media were decreased. Since MMP-2 was not detectable in the conditioned media of MDA-MB-231 cells, and the gelatinolytic activities of the conditioned media were reduced only by MMP-9 neutralizing antibodies, it is most likely that the reduction of gelatinolytic activities by PPARgamma ligands and/or ATRA was due to the decrease of MMP-9 activities. Because MMP-9 was absolutely required in the transmigration of this cell line through Matrigel in our in vitro model as demonstrated by neutralizing antibodies against MMP-2 and -9, we concluded that down-regulation of gelatinase activities is, at least in part, responsible for the reduction of the invasive capacities of MDA-MB-231 cell line in vitro. Our results, for the first time, indicate that PPARgamma ligands may

Topics: Breast Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nuclear Proteins; Pioglitazone; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazolidinediones; Tissue Inhibitor of Metalloproteinases; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The POU (representing a homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86) homeodomain protein OCT3/Oct-3 (where OCT stands for octamer-binding protein) is an embryonic transcription factor expressed in oocytes, embryonic stem and embryonic carcinoma cells. We have demonstrated previously that human breast cancer cells regain the ability to express OCT3 mRNA [Jin, Branch, Zhang, Qi, Youngson and Goss (1999) Int. J. Cancer 81, 104-112]. Antibodies against human OCT3 were not available when this study was conducted. By using a human OCT3-glutathione S-transferase fusion protein to affinity purify a polyclonal antibody against the mouse Oct-3, we obtained an antibody that enabled us to detect OCT3 in human breast cancer cells by Western-blot analysis. Thus we have now confirmed that OCT3 is expressed in human breast cancer cells but not in normal human breasts and in three other organs. When breast cancer cell lines were treated with all- trans -retinoic acid, OCT3 expression was repressed, associated with decreased cell proliferation. Although another POU protein Brn-3 has been shown to be a repressor for BRCA1 (breast-cancer susceptibility gene 1), OCT3 does not repress human or mouse BRCA1/Brca-1 promoters. However, OCT3 is capable of activating a fusion promoter containing the fibroblast growth factor-4 (FGF-4) enhancer element. In addition, we documented for the first time that human breast cancer cells express FGF-4 protein, and its expression could be inhibited by all- trans -retinoic acid. Furthermore, overexpressing OCT3 stimulated endogenous FGF-4 expression in MCF7 breast cancer cell line. These observations indicate that OCT3 protein is selectively expressed in human breast cancer cells, and its expression may be implicated in mammary gland tumorigenesis via up-regulating FGF-4 expression.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line; DNA-Binding Proteins; Down-Regulation; Female; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Humans; Octamer Transcription Factor-3; Promoter Regions, Genetic; Proto-Oncogene Proteins; Response Elements; Thymidine Kinase; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Iodide uptake by normal and cancerous thyroid cells is an active process mediated by the sodium/iodide symporter (NIS). Using quantitative real-time RT-PCR, we found that all 22 fresh human breast cancer samples had very low NIS expression similar to levels in untreated MCF-7 breast cancer cells. 9-cis retinoic acid (9-cis RA), a ligand for both retinoic acid receptor (RAR)/retinoic X receptor (RXR) heterodimers as well as RXR/RXR homodimers, markedly induced NIS mRNA expression in MCF-7 breast cancer cells in a dose- and time-dependent fashion, with maximal levels occurring at 12 h. All-trans retinoic acid, ATRA, a RAR specific ligand had a similar potency. Among eight breast cancer cell lines, three out of four estrogen receptor (ER)-positive and zero of four ER-negative cell lines responded to 9-cis RA by increasing their expression of NIS. Combining a RAR with a RXR selective ligand enhanced both NIS mRNA expression and iodide uptake in MCF-7 cells. Similarly, a ligand for proliferator-activated receptor gamma (PPARgamma) when combined with 9-cis RA synergistically increased both NIS mRNA levels and iodide uptake in these MCF-7 cells. The iodide uptake was blocked by KClO4. In conclusions, these findings suggest that selected combinations of NHR ligands should be examined in a limited trial to determine if their administration to patients allows the use of radioactive iodine for diagnosis and possibly treatment of metastatic breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Humans; Iodine; Iodine Radioisotopes; Ligands; Neoplasm Metastasis; Radionuclide Imaging; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Symporters; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The synthetic retinoid N-(4-hydroxyphenyl)retinamide [4-HPR (or fenretinide)] has preclinical and clinical preventive activity in breast carcinogenesis. 4-HPR and its metabolites have been shown to accumulate in the mammary tissue of rodents. We assessed levels of 4-HPR and its major metabolite, N-(4-methoxyphenyl)retinamide (4-MPR), in plasma and in normal and neoplastic breast tissue obtained from women treated with 4-HPR.. We randomly assigned 14 women with suspected or very recently diagnosed breast cancer to receive 100, 200, or 300 mg of 4-HPR daily for 3-12 days before scheduled biopsy, lumpectomy, or mastectomy. Using high-performance liquid chromatography, we measured post-4-HPR-treatment concentrations of 4-HPR and 4-MPR in plasma and breast tissue obtained during surgery.. Breast tissue and plasma retinamide (4-HPR plus 4-MPR) concentrations increased significantly with short-term oral administration of 4-HPR. Retinamide levels increased in a linear and dose-related fashion in plasma, whereas they peaked and plateaued at 200 mg/day in breast tissue. The total retinamide concentration in breast tissue exceeded that in plasma at each 4-HPR dose. The highest mean tissue:plasma retinamide ratios were achieved at 200 mg/day: 639.5 +/- 253.8 to 190.6 +/- 91.9 ng/ml (4.8:1) for 4-HPR and 594.4 +/- 201.9 to 130.5 +/- 37.8 ng/ml (6.6:1) for 4-MPR. Plasma retinol levels decreased in association with increasing 4-HPR doses. Two patients experienced grade 1 toxicity at the 300 mg/day dose.. These findings indicate that retinamides preferentially accumulate in human breast tissue (versus plasma). 4-HPR tissue concentrations at 200 mg/d were equivalent to those that inhibit growth and induce apoptosis of breast cancer cells in vitro. Previous clinical and correlative laboratory results suggest that 4-HPR may reduce risk in premenopausal women, who are more prone (than are postmenopausal women) to estrogen receptor (ER)-negative breast cancer development. The present results and previous data (including in vitro 4-HPR activity against ER-negative breast cancer) support further study of 4-HPR in the setting of premenopausal/ER-negative breast cancer prevention.

Topics: Administration, Oral; Adult; Aged; Apoptosis; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Fenretinide; Humans; Kinetics; Middle Aged; Random Allocation; Time Factors; Tretinoin

Transcriptional cross-talk exists between the estrogen receptor (ERalpha) and retinoic acid receptor (RAR) pathways in human breast cancer cells. We have previously shown that re-expression of ERalpha in ER-negative cells stimulates the transcriptional and growth inhibitory effects of all-trans-retinoic acid (tRA) by a mechanism that is independent of the ER ligands estradiol and tamoxifen. In this study, we generated cell lines stably expressing ERalpha-deletion mutants to elucidate the mechanism whereby ERalpha modulates RAR transcriptional activity. Using RT-PCR and RNAse protection assays, we observed that expression of ERalpha suppresses basal expression of the RA-responsive gene RARbeta2, while allowing it to be strongly induced by tRA. Repression of basal RARbeta2 transcription was confirmed by transient expression of the reporter plasmid betaRE-tk-CAT, containing the RARbeta2 promoter. In the ERalpha-negative cells, on the other hand, transcription was only weakly induced by RA. We further determined that this effect of ERalpha on RARbeta induction required the N-terminal AF-1-containing region, including the DNA-binding domain, but was independent of the C-terminal ligand-binding domain. Consistent with these results, the ER agonist estradiol and the AF-2 antagonist 4-hydroxytamoxifen had no significant effect on betaRARE activity. Conversely, the full ER antagonist ICI 182,780, which blocks ERalpha AF-1 activity, was able to completely relieve repression of basal betaRARE activity. The effect of ERalpha is specific for RAR-mediated transcription and does not occur on promoters containing typical response elements for the Vitamin D or thyroid hormone receptors. Moreover, the cross-talk between ERalpha and RAR does not seem to be mediated by sequestration of a number of common co-regulators, suggesting a novel mechanism whereby the N-terminal region of ERalpha modulates the transcriptional activity of RAR.

Topics: Animals; Breast Neoplasms; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Mice; Plasmids; Protein Structure, Tertiary; Receptor Cross-Talk; Receptors, Estrogen; Receptors, Retinoic Acid; Recombinant Proteins; Sequence Deletion; Tamoxifen; Trans-Activators; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (Herceptin) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Humans; Immunohistochemistry; Nitric Oxide; Nitric Oxide Synthase; Receptor, ErbB-2; Trastuzumab; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA), a synthetic derivative of vitamin A, inhibits the growth of breast cancer cells. To elucidate the mechanism by which ATRA causes cell growth inhibition, we examined changes in cell cycle and intracellular signaling pathways, focusing on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Using the estrogen receptor-negative, retinoid receptor-positive breast cancer cell line SKRB-3, we found that treatment with ATRA significantly decreased the expression of PKCalpha, as well as reducing ERK MAPK phosphorylation. ATRA treatment leads to dephosphorylation of Rb, and consequently to G(1) arrest. Marked changes in the expression of cyclins (particularly cyclins A and E) were observed in SKBR-3 cells treated with ATRA. Using a series of pharmacological and molecular approaches, we found evidence that ATRA-induced SKBR-3 cell growth inhibition involves the deregulation of the PKCalpha-MAPK pathway. These data suggest that retinoids interfered with signal transduction pathways that are crucial for cell cycle progression, and highlight the complexities of the biological effects of retinoid derivatives.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Flow Cytometry; Gene Expression Regulation; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.

Topics: Administration, Oral; Animals; Apoptosis; Body Weight; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Fenretinide; Humans; Hydrolysis; Male; Rats; Rats, Sprague-Dawley; Tretinoin; Vitamin A; Vitamin A Deficiency

Retinoids are analogues of all-trans-retinoic acid, a powerful hormone that mediates many fundamental biological processes. Cancer and other serious hyperproliferative diseases are attractive therapeutic targets for retinoids, but the therapeutic use of retinoids is limited due to severe toxicity. We report here the design of retinoid receptor-alpha specific ligands with growth inhibitory activity in breast cancer cell lines, and which do not cause the cutaneous toxicity associated with the currently available nonselective retinoid agonists.

Topics: Amides; Antineoplastic Agents; Breast Neoplasms; Female; Humans; Hydrogen Bonding; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Structure-Activity Relationship

Melatonin in the in vitro conditions inhibits cell growth and proliferation of estrogen sensitive (ER+) cell line MCF-7 in culture. In the present study, during a 48-hour incubation melatonin at a concentration of 10(-5) M inhibited [3H]thymidine incorporation into cancer cells at the level of 69.52% +/- 10.99. Melatonin had no inhibitory effect on the physiological stimulatory action of estradiol. Tamoxifen added to the medium modulated the melatonin action only when the latter was added 24 hours after tamoxifen (46.45% +/- 4.40, p < 0.05). Tretinoin added to the culture caused a statistically significant reduction in [3H]thymidine incorporation into the cancer cells, compared to the melatonin and tretinoin groups, when treatment with retinoid was synergistic (39.05% +/- 5.44, p < 0.05) or sequential (tretinoin and after 24 h melatonin) (39.96% +/- 1.55, p < 0.05). This was confirmed by immunocytochemical investigations, which showed a statistically significant reduction in the percentage of PCNA- and Ki67-positive cells. Apart from the inhibitory effect on MCF-7 cell proliferation retinoids induce the apoptotic pathway in a dose-dependent manner. Melatonin added to the culture enhances this effect, which may indicate the potential for the use of both substances in the treatment of breast cancer in women.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Count; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Estradiol; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Melatonin; Necrosis; Proliferating Cell Nuclear Antigen; Tamoxifen; Tretinoin; Tumor Cells, Cultured

We observed that all-trans retinoic acid (ATRA) inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transactivating ligand HEREGULIN. This suggests that Her2/neu causes breast cancer cells to be resistant to the growth inhibitory effects of ATRA. To confirm this observation, MDA-MB-453 and BT-474 cells, which have high levels of Her2/neu and are resistant to ATRA, were incubated with the trastuzumab (Herceptin) antibody so that we could determine whether inhibition of the expression and function of Her2/neu would resensitize these cells to ATRA. Indeed, we found that MDA-MB-453 and BT-474 cells treated with trastuzumab were growth inhibitory by ATRA. We then determined whether Her2/neu uses Grb2 and Akt proteins to induce ATRA resistance. Liposome-incorporated Grb2 antisense oligonucleotides (L-Grb2) and a dominant negative (DN) AKT mutant were used to down-regulate Grb2 expression and inhibit Akt activity, respectively. When incubated with L-Grb2 or transfected with the DN AKT mutant, ATRA-resistant, Her2/neu-overexpressing cells became sensitive to ATRA. Our results indicate that Her2/neu utilizes Grb2 and Akt proteins to induce ATRA resistance in breast cancer cells. ATRA sensitivity was also correlated with RARalpha protein levels since higher RARalpha protein levels were observed in cells in which the Her2/neu pathway was inhibited.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Survival; DNA Primers; Down-Regulation; Drug Resistance, Neoplasm; Female; Humans; Ligands; Neuregulin-1; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The use of combination chemotherapy is the accepted standard for most human malignancies but little attention has been paid to drug interactions. A combination of drugs may be synergistic, additive, or antagonistic in cytotoxic activity. This study evaluated combinations of agents with docetaxel, one of the most active agents in human breast cancer, using a median effects model to look at synergy or antagonism in vitro as a potential predictor of clinical outcome. Three human breast cancer cell lines, MCF7/wt, MCF7/adr (multiply drug resistant), and BT474 were grown to confluence, plated into 96 well dishes, and incubated with combinations of drugs for 72h. Cytotoxic effect was measured by the MTT assay. Median effect analysis was used to calculate the combination index (CI) with values less than 1 indicating synergism, 1 additive effects, and greater than 1 antagonism. Potentially useful combinations for clinical study which were identified included docetaxel with vinorelbine, docetaxel with dexrazoxane, docetaxel with cis-retinoic acid, docetaxel with disulfiram and either doxorubicin or epirubicin, and docetaxel with dexrazoxane and epirubicin.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disulfiram; Docetaxel; Drug Interactions; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Epirubicin; Female; Humans; Paclitaxel; Razoxane; Taxoids; Tretinoin; Tumor Cells, Cultured; Vinblastine; Vinorelbine

Retinoic acid (RA) receptor (RAR) beta2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RAR beta2 promoter and 5'-UTR is the underlying cause. Here we show that RAR alpha2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR alpha2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR alpha2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR alpha2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR alpha2 and RAR beta2. On the basis of a clonogenic assay, RAR alpha2 displayed ligand-dependent growth-suppressive activity similar to that of RARb eta2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR alpha2-transfected cells compared with 75 and 77%, respectively, in RAR beta2-transfected cells. We conclude that the silencing of the RAR alpha2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.

Topics: Breast Neoplasms; Carcinoma; Cell Division; DNA Methylation; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

4HPR, an analogue of ATRA, effectively induces growth inhibition and apoptosis in breast cancer cell lines and animal models but is ineffective against advanced human breast tumors. Different compounds, including tamoxifen, are currently being tested to increase 4HPR efficacy in the clinic. Here, we report that cyclosporin A selectively increases the ability of 4HPR, but not ATRA, to induce growth inhibition and apoptosis in ER(+) and ER(-) breast cancer cell lines. Increased apoptosis by the 4HPR and cyclosporin A combination was correlated with increased production of the free radical nitric oxide. Thus, the 4HPR and cyclosporin A combination may potentially be a novel therapeutic modality against breast tumors.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Division; Cyclosporine; Drug Synergism; Female; Fenretinide; Humans; Immunosuppressive Agents; Nitric Oxide; Receptors, Estrogen; Tretinoin; Tumor Cells, Cultured

The biochemical mechanisms of apoptosis-induction by all-trans-retinoic acid (atRA) and N-(4-hydroxyphenyl)retinamide (4HPR) in cultured MCF7 cancer cells were studied by multiparameter flow cytometry. Retinoid treatment induced formation of two biochemically distinct cell subpopulations, which preceded the appearance of cells with fragmented nuclei. Exposure to atRA led to a transient increase in NADH level and mitochondrial oxidative turnover and a slow decline in reduced thiol level and mitochondrial membrane potential, suggesting that atRA treatment induces a transient defense mechanism. The synthetic retinoid 4HPR, in contrast, caused a gradual decrease in mitochondrial oxidative turnover and cardiolipin level together with a small decline in mitochondrial membrane potential, suggesting that 4HPR induces oxidation of cardiolipin and subsequent leakage of the mitochondria. Co-incubation with cyclosporin A, an inhibitor of the mitochondrial permeability transition, did not prevent formation of fragmented nuclei or induction of changes in mitochondrial parameters by retinoids. Thus, the mitochondrial permeability transition does not appear to be involved in retinoid induction of apoptosis in MCF7 cells. Retinoid exposure of diploid human mammary epithelial cells induced mild oxidative stress but did not lead to formation of two cell subpopulations. We conclude that atRA and 4HPR induce apoptosis in MCF7 cells by two distinct and novel biochemical mechanisms.

Topics: Antineoplastic Agents; Apoptosis; Breast; Breast Neoplasms; Cell Division; Cell Line; Cell Survival; DNA Fragmentation; DNA, Mitochondrial; Epithelial Cells; Female; Fenretinide; Humans; Ion Channels; Kinetics; Membrane Potentials; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; NAD; Oxidative Stress; Tretinoin; Tumor Cells, Cultured

WNT signaling molecules, playing key roles in embryogenesis and carcinogenesis, are potent targets for regenerative medicine and clinical oncology. We have previously cloned and characterized the human orthologue of mouse proto-oncogene Wnt-10b using bioinformatics and cDNA-PCR. Human WNT10B is moderately expressed in MKN45 and MKN74 cells derived from human gastric cancer, and is up-regulated by tumor necrosis factor alpha (TNFalpha) in MKN45 cells. Here, expression and regulation of WNT10B in human cancer other than gastric cancer were investigated using cDNA-PCR. WNT10B mRNA was expressed in the majority of squamous cell carcinoma cell lines derived from esophageal cancer and cervical cancer. WNT10B mRNA was relatively highly expressed in TE3, TE6, TE10, TE11 (esophageal cancer), Hs700T (pancreatic cancer), SKG-IIIa, HeLa S3 (cervical cancer), and T-47D (breast cancer). Expression of WNT10B mRNA was up-regulated by beta-estradiol in MCF-7 cells expressing estrogen receptors. Expression of WNT10B mRNA was down-regulated by all-trans retinoic acid in NT2 cells with the potential of self renewal and neuronal differentiation. WNT10B might be implicated in self renewal of stem cells as well as in carcinogenesis through activation of the WNT - beta-catenin pathway.

Topics: Breast Neoplasms; Cell Line; Down-Regulation; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Neoplasms, Germ Cell and Embryonal; Proto-Oncogene Mas; Proto-Oncogene Proteins; Tretinoin; Up-Regulation; Wnt Proteins

AIB1 (amplified in breast cancer 1) is a nuclear receptor coactivator gene amplified and overexpressed in breast cancer. However, the mechanisms by which AIB1 is regulated are unclear. Here we show that 17beta-estradiol represses AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing AIB1 gene transcription. Estrogen levels present in fetal calf serum are sufficient to maintain AIB1 mRNA and protein at low basal levels, and this repression is reversed by the addition of antiestrogens or all-trans retinoic acid. Interestingly, cycloheximide inhibition experiments revealed that secondary protein synthesis was necessary to induce AIB1 expression by antiestrogens and retinoids. Experiments with TGF-beta and TGF-beta blocking antibodies demonstrated that this growth factor modulates AIB1 expression and showed that the antiestrogen and retinoid induction of AIB1 gene expression is mediated at least in part through TGF-beta. These data reveal a mechanism of estrogen-induced down-modulation of the overall hormone sensitivity of cells through feedback inhibition of coactivator gene expression. These data also suggest that antiestrogens can shift the sensitivity of cells to non-estrogenic proliferative signaling by increasing cellular levels of AIB1. This effect may play a role in breast cancer progression and resistance to drug treatment.

Topics: Breast Neoplasms; Cell Cycle; Estradiol; Fulvestrant; Gene Expression Regulation, Neoplastic; Half-Life; Humans; Nuclear Receptor Coactivator 3; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and its derivatives inhibit the proliferation of normal human mammary epithelial cells (HMEC) and some breast carcinoma lines by mechanisms which are not fully understood. To identify genes that mediate RA-induced cell growth arrest, an HMEC cDNA library was synthesized and subtractive screening was performed. We identified the interleukin-1beta (IL-1beta) gene as an RA induced gene in HMEC. Northern blot analyses showed that the IL-1beta gene was up-regulated as early as 2 h after RA treatment. Results from the treatment of HMEC with cycloheximide and actinomycin D indicated that the regulation of the IL-1beta gene by RA occurred at the transcriptional level and that the IL-1beta gene is a direct, downstream target gene of RA. To evaluate the effects of IL-1beta on cell proliferation, the proliferation of HMEC was measured in the presence of RA or IL-1beta, or both. Either RA or IL-1beta could significantly inhibit the proliferation of HMEC. However, the addition of soluble IL-1 receptor antagonist (sIL-1ra) to the cell culture medium did not block RA-induced HMEC growth inhibition, whereas sIL-1ra did block the growth inhibition of HMEC by IL-1beta. IL-1beta expression was not observed in the three carcinoma cell lines, MCF-7, MDA-MB-231, and MDA-MB-468, as compared to the HMEC. Growth curves of the breast carcinoma cell lines showed strong inhibitory effects of RA and IL-1beta on the growth of the estrogen receptor (ER) positive MCF-7 cell line, but only a small effect on the ER negative MDA-MB-231 cells. The expression of the IL-1beta gene was also transcriptionally activated by RA in normal epithelial cells of prostate and oral cavity. Our results suggest that: (a) the IL-1beta gene is a primary target of RA receptors in HMEC; (b) the enhanced expression of the IL-1beta gene does not mediate the RA-induced growth arrest of HMEC; and (c) the expression of the IL-1beta gene is low or absent in all three human breast carcinoma cell lines examined, but the defect in the IL-1beta signaling pathway may be different in ER positive versus ER negative carcinoma cells.

Topics: Breast; Breast Neoplasms; Carcinoma; Cell Division; Cycloheximide; Dactinomycin; Drug Synergism; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Kinetics; Male; Prostate; Protein Synthesis Inhibitors; Receptors, Estrogen; Receptors, Retinoic Acid; Recombinant Fusion Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.

Topics: Adenocarcinoma; Alcohol Dehydrogenase; Alcohol Oxidoreductases; Aldehyde Dehydrogenase; Amino Acid Sequence; Animals; Base Sequence; Breast Neoplasms; Cells, Cultured; Cloning, Molecular; Cytochrome P450 Family 2; Epithelium; Estrous Cycle; Female; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tretinoin; Uterus; Vitamin A

Retinoic acid receptors (RARs) are ligand-dependent transcription factors which are members of the steroid/thyroid hormone receptor gene family. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. PCR-amplified subtractive hybridization was used to identify candidate retinoid-regulated genes that may be involved in growth inhibition. One candidate gene identified was SOX9, a member of the high mobility group (HMG) box gene family of transcription factors. SOX9 gene expression is rapidly stimulated by RAR-agonists in T-47D cells and other retinoid-inhibited breast cancer cell lines. In support of this finding, a database search indicates that SOX9 is expressed as an EST in breast tumor cells. SOX9 is known to be expressed in chondrocytes where it regulates the transcription of type II collagen and in testes where it plays a role in male sexual differentiation. RAR pan-agonists and the RARalpha-selective agonist Am580, but not RXR agonists, stimulate the expression of SOX9 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not stimulate SOX9 in breast cancer cell lines which were not growth inhibited by retinoids. Expression of SOX9 in T-47D cells leads to cycle changes similar to those found with RAR-agonists while expression of a dominant negative form of SOX9 blocks RA-mediated cell cycle changes, suggesting a role for SOX9 in retinoid-mediated growth inhibition.

Topics: Animals; Benzoates; Breast Neoplasms; Cell Cycle; Cell Division; Estradiol; Estrogens; Expressed Sequence Tags; Female; Gene Expression Regulation, Neoplastic; Gene Targeting; Genes, Dominant; Growth Substances; High Mobility Group Proteins; Humans; Kidney; Male; Mammary Glands, Animal; Mice; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Organ Specificity; Receptors, Estrogen; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor alpha; Retinoids; SOX9 Transcription Factor; Testis; Tetrahydronaphthalenes; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Overexpression of Her2/neu is implicated in the development of resistance to the antiestrogen tamoxifen (TAM) that exerts its inhibitory effect through interaction with estrogen receptor (ER). Whereas Her2/neu and ER are believed to be important cell survival/death factors in human breast cancer cells, if and how they interact to confer resistance to hormone therapy is not known. This prompted us to investigate whether modulation of the effect of TAM occurs via the Her2/neu pathway and whether targeting the interaction between the Her2/neu pathway and the ER pathway is beneficial. There are 2 forms of ER that are localized to the cell membrane and to the nucleus. For the first time, we found that Her2/neu directly interacts with ER at the cell membrane. We then investigated the role of Her2/neu overexpression in the regulation of the cell membrane ER pathway in TAM-resistant breast cancer cells and the nature of this interaction in apoptotic signaling. Relief of TAM resistance was associated with Her2/neu downregulation and ER upregulation. TAM-induced apoptosis occurred immediately after dissociation of Her2/neu from cell membrane ER. These results demonstrate a novel mechanism by which Her2/neu regulates the cell membrane ER-coupled apoptosis and the possible involvement of the Her2/neu in TAM resistance of breast cancer cells. Moreover, the antiproliferative activity of TAM should rely on the integration between the signal transduction from the cell membrane ER and the gene regulation by the nuclear ER. Coordinated modulation on the cell membrane ER/Her2/neu pathway and the nuclear ER/RAR pathway may provide a new approach for treatment of ER-positive, Her2/neu overexpressing breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Caspases; Cell Membrane; Cell Nucleus; Cell Separation; Cytosol; DNA Fragmentation; Down-Regulation; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Immunohistochemistry; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Phenylbutyrates; Precipitin Tests; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Retinoic Acid; Subcellular Fractions; Tamoxifen; Time Factors; Tretinoin; Tumor Cells, Cultured

We have shown that Cyr61, an angiogenic regulator, is overexpressed in invasive and metastatic human breast cancer cells and tumor biopsies. We have further demonstrated that Cyr61 promotes acquisition of estrogen-independence and anti-estrogen resistance in vivo in breast cancer cells. Moreover, we have demonstrated that Cyr61 induces tumor formation and tumor vascularization in vivo, events mediated through the activation of the MAPK and the Akt signaling pathways. Here we investigate how Cyr61 expression is regulated in both estrogen receptor (ER)-positive and ER-negative breast cancer cells. We demonstrate that Cyr61 mRNA and protein expression is inducible by estrogen and anti-estrogens in ER-positive breast cancer cells. We show that a labile protein as well as a negative regulator might be involved in Cyr61 expression in estrogen-dependent breast cancer cells. Other important regulators of Cyr61 expression in breast cancer cells that we found are the phorbol ester TPA, vitamin D, and retinoic acid. TPA causes positive regulation of Cyr61 expression in ER-positive MCF-7 cells. Vitamin D induces a transient stimulatory effect on Cyr61 gene expression. Lastly, retinoic acid has a negative effect on Cyr61 expression and downregulates its expression in MCF-7 cells. Interestingly, most of these effects are not seen in aggressive breast cancer cells that do not express ER and express high levels of Cyr61, such as the MDA-MB-231 cells. Our results are in agreement with our knowledge that Cyr61 promotes tumor growth, and that tumor-promoting agents have a positive impact on cells that express low levels of Cyr61, such as the ER-positive breast cancer cells; however, these agents have no significant effect on cells that express high levels of Cyr61. Our findings suggest an association between increased Cyr61 expression and an aggressive phenotype of breast cancer cells.

Topics: Active Transport, Cell Nucleus; Adenocarcinoma; Breast Neoplasms; Calcitriol; Cysteine-Rich Protein 61; Drug Resistance; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Genes, Immediate-Early; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phenotype; Protein Structure, Tertiary; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Selective Estrogen Receptor Modulators; Tamoxifen; Tetradecanoylphorbol Acetate; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

Topics: Animals; Antineoplastic Agents; Benzothiazoles; Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Female; Humans; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Inbred Strains; Mixed Function Oxygenases; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Thiazoles; Tretinoin; Tumor Cells, Cultured

Cellular retinoic acid-binding protein II (CRABP-II) is an intracellular lipid-binding protein that associates with retinoic acid with a subnanomolar affinity. We previously showed that CRABP-II enhances the transcriptional activity of the nuclear receptor with which it shares a common ligand, namely, the retinoic acid receptor (RAR), and we suggested that it may act by delivering retinoic acid to this receptor. Here, the mechanisms underlying the effects of CRABP-II on the transcriptional activity of RAR and the functional consequences of these effects were studied. We show that CRABP-II, a predominantly cytosolic protein, massively undergoes nuclear localization upon binding of retinoic acid; that it interacts with RAR in a ligand-dependent fashion; and that, in the presence of retinoic acid, the CRABP-II-RAR complex is a short-lived intermediate. The data establish that potentiation of the transcriptional activity of RAR stems directly from the ability of CRABP-II to channel retinoic acid to the receptor. We demonstrate further that overexpression of CRABP-II in MCF-7 mammary carcinoma cells dramatically enhances their sensitivity to retinoic acid-induced growth inhibition. Conversely, diminished expression of CRABP-II renders these cells retinoic acid resistant. Taken together, the data unequivocally establish the function of CRABP-II in modulating the RAR-mediated biological activities of retinoic acid.

Topics: Animals; Biological Transport, Active; Breast Neoplasms; Cell Division; COS Cells; Female; Humans; In Vitro Techniques; Ligands; Mice; Models, Biological; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Transcription, Genetic; Tretinoin

Glucuronidation, mediated by UDP-glucuronosyltransferases (UGTs), affects the actions and disposition of diverse endo- and xenobiotics. In the case of catecholestrogens (CEs), glucuronidation is likely to block their oxidation to quinone estrogens that are the putative mediators of CEs' actions as initiators of cancers. The goal of this study was to determine whether UGT2B7, the isoenzyme with a high affinity for 4-hydroxyestrone, is expressed in human breast parenchyma. Glucuronidation of 4-hydroxyestrone has relevance to breast carcinogenesis because quinone metabolites of 4-hydroxylated CEs can form potentially mutagenic depurinating DNA adducts, and because in breast tissue estrone is likely to be the predominant estrogen available for 4-hydroxylation. Using reverse transcriptase-polymerase chain reaction, immunocytochemistry, immunoblot analyses, and assays of glucuronidation of 4-hydroxyestrone, we show that UGT2B7 is expressed in human mammary epithelium, and that its expression is dramatically reduced in invasive breast cancers. In many in situ carcinomas, however, 4-hydroxyestrone immunostaining was not only preserved but even more intense than in normal mammary epithelium. The finding of reduced UGT2B7 protein and glucuronidation of 4-hydroxyestrone in invasive cancers suggests a tumor-suppressor function for the enzyme. Recent identification of all-trans retinoic acid as a substrate of UGT2B7 suggests that this function includes the generation of retinoyl-beta-glucuronide, a potent mediator of actions of retinoids important for maintaining epithelia in a differentiated state. Current knowledge does not provide any ready explanation for the apparent increase in UGT2B7 expression in carcinomas in situ. However, this finding, together with reduced immunostaining at loci showing breach of the basement membrane (microinvasion), suggests involvement of UGT2B7-catalyzed reaction(s) in protection against invasion of surrounding tissue by cancer cells.

Topics: Breast; Breast Neoplasms; Female; Glucuronosyltransferase; Humans; Hydroxyestrones; Immunoblotting; Immunohistochemistry; Neoplasm Invasiveness; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Distribution; Tretinoin

Xenopus wnt-8 (Xwnt-8) is one of the most potent Wnts to activate the WNT - beta-catenin - TCF signaling pathway. We have previously cloned and characterized WNT8A and WNT8B, two human homologues of Xwnt-8. Here, we investigated expression and regulation of WNT8A and WNT8B mRNAs in human tumor cell lines by using cDNA-PCR. WNT8A mRNA was undetectable in 7 pancreatic cancer cell lines, but WNT8B mRNA was detected in pancreatic cancer cell lines PSN-1, BxPC-3, MIA PaCa-2. Both WNT8A and WNT8B mRNAs were undetectable in 7 brain tumor cell lines. Although WNT8A mRNA was undetectable in 3 breast cancer cell lines, WNT8B mRNA was detected in the breast cancer cell line MCF-7. WNT8B mRNA, but not WNT8A mRNA, was significantly up-regulated by beta-estradiol in MCF-7 cells. WNT8A mRNA was detected in embryonal tumor cell lines NEC-14, NCC-IT, and NT2, while WNT8B mRNA was detected in embryonal tumor cell lines NEC-8, NEC-14, and NT2. Because NT2 cells differentiate into neuronal cells after all-trans retinoic-acid treatment, effects of all-trans retinoic acid on mRNA expression of WNT8A and WNT8B were next investigated. WNT8A and WNT8B mRNAs were down-regulated together in NT2 cells after all-trans retinoic-acid treatment. WNT8A and WNT8B might play key roles in embryonal tumors and embryonic stem cells through synergistic activation of the beta-catenin - TCF signaling pathway.

Topics: Breast Neoplasms; Cytoskeletal Proteins; DNA, Complementary; Down-Regulation; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Poly A; Polymerase Chain Reaction; Protein Biosynthesis; Proteins; RNA, Messenger; Signal Transduction; Time Factors; Tretinoin; Tumor Cells, Cultured; Up-Regulation; Wnt Proteins; Zebrafish Proteins

Loss of expression of retinoic acid receptor beta2 (RARbeta2), a potent tumor suppressor gene, is commonly observed during breast carcinogenesis. RARbeta2 silencing can be traced to epigenetic chromatin changes affecting the RARbeta P2 promoter. Here we show that retinoic acid therapy fails to induce RARbeta2 in primary breast tumors, which carry a methylated RARbeta P2 promoter. DNA methylation leads to repressive chromatin deacetylation at RARbeta P2. By inducing an appropriate level of histone reacetylation at RARbeta P2 we could reactivate endogenous RARbeta2 transcription from unmethylated as well as methylated RARbeta P2 in breast cancer cell lines and xenograft tumors, and obtain significant growth inhibition both in vitro and in vivo. This study may have translational implications for breast cancer and other cancers carrying an epigenetically silenced RARbeta P2 promoter.

Topics: Acetylation; Antineoplastic Agents; Breast Neoplasms; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Retinoic Acid; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Our laboratory has demonstrated that treatment of MCF-7 breast cancer cells with melatonin (Mlt) followed 24h later with physiological concentrations of all-trans retinoic acid (atRA) results in apoptosis. These studies were extended into trials using the N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model. Initial studies conducted by feeding the animals 9-cis-retinoic acid (9cRA in the chow) and administering melatonin by subcutaneous injection in the late afternoon demonstrated that the combination of Mlt and 9cRA was able to significantly prevent tumor development, and that the combination was more efficacious that either Mlt or 9cRA alone. In this report, we conducted studies to determine if lower doses of 9cRA could be used in combination with Mlt while still maintaining anti-tumor activity and if the route of administration of 9cRA (bolus (gavage) v.s. chronic (chow) routes) affected its interaction with Mlt. The studies presented here demonstrate that significantly reduced doses of 9cRA can be used in combination with Mlt while maintaining anti-tumor efficacy. Furthermore, our studies demonstrate that 9cRA is equally effective when it is administered chronically (chow) or as a bolus (gavage). These data demonstrate that the combined use of Mlt and 9cRA produces additive or synergistic effects, which are more efficacious than 9cRA alone. This combination of Mlt and 9cRA could be a potentially useful clinical treatment regimen for breast cancer since it allows the use of lower doses of retinoic acid, thus, avoiding the toxic side effects associated with the use of high dose retinoids.

Topics: Administration, Oral; Alitretinoin; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Female; Injections, Subcutaneous; Mammary Neoplasms, Experimental; Melatonin; Methylnitrosourea; Rats; Rats, Sprague-Dawley; Tretinoin

The present study was undertaken to compare ROH growth responsiveness between normal human mammary epithelial cells (HMECs), estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cells, and assess whether this responsiveness is correlated with differences in ROH metabolism, particularly RA synthesis. HMECs were markedly more growth sensitive to a physiological dose of ROH than breast cancer cells, exhibiting a significant decrease in cell number by 48h and >70% decrease by 144h. In comparison, numbers of MCF-7s were only decreased 32% by 144h. MDA-MB-231 cells were not affected. However, HMECs and MCF-7 cells displayed similar growth responsiveness to 1 microM RA, while MDA-MB-231 cells were minimally affected. Although the initial rates and extent of ROH uptake were comparable among cell types, ROH levels in HMECs progressively decreased to 20% of the peak by 24h and < or = 10% by 72h. In contrast, ROH levels in the cancer cells remained relatively constant through 48 h. The decrease in HMEC ROH was attributable to greater metabolism as evidenced by rapid and predominant retinyl ester formation. HMECs also produced approximately 5 times more RA from ROH than MCF-7s and approximately 10 times more than MDA MB-231 cells. Our results demonstrate that normal HMECs are markedly more responsive to the growth inhibitory effects of ROH than breast cancer cells, and that this responsiveness is associated with greater ROH metabolism including greater RA synthesis. These data suggest that altered ROH metabolism may be a factor in breast cancer progression.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Division; Cell Line; Epithelial Cells; Female; Growth Inhibitors; Humans; Receptors, Estrogen; Tretinoin; Tumor Cells, Cultured; Vitamin A

Aberrant proliferation is an early-occurring event in vitro prior to tumorigenesis in vivo in the multistep process of carcinogenesis. Inhibition of aberrant proliferation therefore may represent a useful biomarker to evaluate the efficacy of chemopreventive agents. Retinoids have exhibited preventive efficacy in vitro and in vivo predominantly through the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Clinically relevant biochemical and cellular mechanistic endpoints for chemopreventive effects of retinoids should provide novel biomarkers. The present study was designed to examine the preventive efficacy of natural retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9cisRA), and to identify the possible mechanisms for their effects using the HER-2/neu oncogene expressing preneoplastic human mammary epithelial 184-B5/HER cells. Seven-day treatment with ATRA and 9cisRA exhibited a dose-dependent growth inhibition. Long-term (21 days) treatment with IC20 doses of 50 nM ATRA and 100 nM 9cisRA inhibited anchorage-dependent colony forming efficiency by about 75.4% (p<0.01) and 84.9% (p<0.01), respectively. Cell cycle analysis revealed that a 24-h treatment with IC90 doses of 2 microM ATRA and 3 microM 9cisRA accumulates cells in the G0/G1 phase and inhibit S and/or G2/M phase of the cell cycle. ATRA and 9cisRA induced an 11-fold (p=0.03) and a 9-fold (p=0.04) increase in subG0/G1 (apoptotic) population relative to the solvent control, respectively. ATRA and 9cisRA induced 77% (p=0.01) and 51% (p=0.02) decrease in tyrosine kinase immunoreactivity, respectively. Similarly, the two retinoids caused almost a 50% (p=0.01) down-regulation of Bcl-2 immunoreactivity. Western blot analysis revealed that ATRA induced an increase in RARbeta expression and a decrease in RARgamma expression, while 9cisRA down-regulated RXRalpha expression. These data demonstrate that ATRA and 9cisRA may inhibit HER-2/neu induced aberrant proliferation in part by retarding cell cycle progression, down-regulating HER-2/neu-mediated signal transduction and inducing Bcl-2-dependent apoptosis through a retinoid receptor-mediated mechanism.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast; Breast Neoplasms; Cell Adhesion; Cell Cycle; Cell Division; Dose-Response Relationship, Drug; Epithelial Cells; Female; Gene Expression; Humans; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Retinoic Acid; Tretinoin

Topics: Antineoplastic Agents; Antioxidants; Benzamides; Breast Neoplasms; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Genes, BRCA1; Genes, BRCA2; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lung Neoplasms; Male; Mastectomy; Ovarian Neoplasms; Ovariectomy; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Receptors, Retinoic Acid; Smoking; Tobacco Smoke Pollution; Tretinoin; Vitamins

Activation of telomerase is an early event in the development of breast and other cancers that may lead to cell immortalization, a critical and rate-limiting step in cancer progression. Breast epithelial cells from women with Li-Fraumeni syndrome (LFS) immortalize spontaneously and reproducibly in culture. We, therefore, tested whether immortalization of these cells could be prevented by treating them with chemopreventive agents and by inhibiting telomerase activity.. Noncancerous, preimmortal breast epithelial cells derived from a patient with LFS were treated for 3 months with nontoxic concentrations of the chemopreventive agents oltipraz, difluoromethylornithine, tamoxifen, and retinoic acid or with two different telomerase inhibitors. The frequency of spontaneous immortalization of LFS-derived cells was estimated by an approach based on fluctuation analyses. Statistical analyses were two-sided.. The frequency of spontaneous immortalization events of LFS-derived breast epithelial cells was reduced by long-term treatment with retinoic acid (P<0.001) or tamoxifen (P<0.05) compared with solvent-treated cells. The frequency of immortalization was also reduced by treating LFS-derived cells with an antitelomerase antisense oligonucleotide (P<0.001) or by inducing the cells to express a dominant negative mutant of telomerase (P<0.025) compared with cells treated with a control oligonucleotide or with empty vector, respectively.. Treatment of preimmortal LFS breast epithelial cells with chemopreventive and antitelomerase agents decreased the frequency of spontaneous immortalization in vitro. These studies validate the application of a new cell culture model system to screen the effects of novel chemopreventive agents by use of cell immortalization as an end point. The results also suggest that the telomerase ribonucleoprotein complex may be an important molecular target for breast cancer prevention.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Disease Progression; DNA, Complementary; Eflornithine; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Female; Humans; Li-Fraumeni Syndrome; Oligodeoxyribonucleotides, Antisense; Point Mutation; Pyrazines; Reverse Transcriptase Inhibitors; Tamoxifen; Telomerase; Thiones; Thiophenes; Transformation, Genetic; Tretinoin

The active form of vitamin D(3), 1,25(OH)(2)D(3), inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D(3) analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3) (Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)(2)D(3) in inhibiting 50% clonal growth (ED(50)) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10(-8) M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)(2)D(3) (10(-8) M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)(2)D(3) induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10(-8) M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)(2)D(3) at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G(1)/G(0) phase and expression level of p27(kip1). Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphoric Monoester Hydrolases; Prostatic Neoplasms; PTEN Phosphohydrolase; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all-trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10(-6) M RA induced a rapid breakdown of both RARalpha and RARgamma in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARalpha and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor alpha (RXRalpha) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.

Topics: Antineoplastic Agents; Breast Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA; Humans; Leupeptins; Multienzyme Complexes; Nuclear Proteins; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Tretinoin; Tumor Cells, Cultured; Ubiquitins

Through the use of microarray analysis it was discovered that the nuclear receptor coregulator, receptor interacting protein 140 (RIP140), was induced early during all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells. A rapid, fourfold induction of RIP140 mRNA was detected within 3 h of RA treatment in human embryonal carcinoma and MCF-7 human breast cancer cells. RIP140 protein levels were induced within 6 h of RA treatment. The RA induction of RIP140 mRNA did not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors. Promoter/enhancer elements directly upstream of the RIP140 coding region supported RA-induced transcription of a luciferase gene. In addition the ability of overexpressed RIP140 to repress ligand activated retinoid receptors was confirmed. The finding that RIP140 is a direct transcriptional target of RA is one of the first examples of acute transcriptional regulation of a nuclear receptor coactivator or corepressor. These data are consistent with a model by which RA induction of RIP140 supplies a negative feedback signal toward ligand-activated retinoid receptors.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Carcinoma, Embryonal; Feedback; Female; Gene Expression Regulation, Neoplastic; Humans; Models, Genetic; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Receptors, Cytoplasmic and Nuclear; Teratocarcinoma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Vitamin D3 derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)2D3 and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)2D3 10(-10) and 10(-9) M were synergistic with ATRA 10(-8) M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)2D3 and ATRA individually induced an accumulation of MCF-7 cells in the G1 phase of the cell cycle and an associated increase in p21WAFI/CiP1, p27KiP1 and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)2D3 but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)2D3, a noncalcemic analogue 1,25(OH)2-16-ene-23-yne-D3, ATRA, AGN195183, an RARalpha-specific agonist, and AGN190168 (tazarotene), an RARbeta/gamma-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)2-16-ene-23-yne-D3 combined with ATRA or with tazarotene at 10(-9) M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)2D3, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Calcium Channel Agonists; Cell Cycle; Cell Differentiation; Female; Gene Expression Regulation, Neoplastic; Humans; Phenotype; Tretinoin; Tumor Cells, Cultured

Retinoic acid activation of retinoic acid receptor alpha (RARalpha) induces protein kinase Calpha (PKCalpha) expression and inhibits proliferation of the hormone-dependent T-47D breast cancer cell line. Retinoic acid has no effect on proliferation or PKCalpha expression in a hormone-independent, breast cancer cell line (MDA-MB-231). To test the role of PKCalpha in retinoic acid-induced growth arrest of human breast cancer cells we established MDA-MB-231 cell lines stably expressing PKCalpha. Constitutive expression of PKCalpha did not affect proliferation of MDA-MB-231 cells but did result in partial retinoic acid sensitivity. Retinoic acid treatment of PKCalpha-MDA-MB-231 cells decreased proliferation (by approximately 40%) and inhibited serum activation of MAP kinases and induction of c-fos. Similar results were seen in MDA-MB-231 cells in which transcription of the transfected PKCalpha cDNA was reversibly induced by isopropyl beta-d-thiogalactoside. Expression of RARalpha in PKCalpha expressing MDA-MB-231 cells resulted in even greater retinoic acid responses, as measured by effects on cell proliferation, inhibition of serum signaling, and transactivation of an RARE-CAT reporter plasmid. In summary, PKCalpha synergizes with activated RARalpha to disrupt serum growth factor signaling, ultimately arresting proliferation of MDA-MB-231 cells.

Topics: Antineoplastic Agents; Blood Proteins; Breast Neoplasms; Calcium; Cell Division; Drug Interactions; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Isoenzymes; Isopropyl Thiogalactoside; Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; RNA, Messenger; Signal Transduction; Transfection; Tretinoin; Tumor Cells, Cultured

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Topics: Agar; Apoptosis; Breast; Breast Neoplasms; Carrier Proteins; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Chromones; Contact Inhibition; Enzyme Activation; Enzyme Inhibitors; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Female; Humans; Morpholines; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Signal Transduction; Simian virus 40; Tretinoin; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Vitamin A

Human SEC14L1 shows partial sequence homology to the budding yeast SEC14 protein and the Japanese flying squid retinal-binding protein and was previously generally localized to 17q25. We more precisely mapped SEC14L1 within a discrete region of 17q25 that likely harbors at least one putative breast and ovarian tumor suppressor gene. We determined that this gene consists of 18 exons ranging in size from 70 bp (exon 11) to 3088 bp (exon 17) and spanning at least 58 kb of DNA. Exon 17 contained a highly polymorphic variable number of tandem repeats (VNTR) and was present only in the larger ubiquitously expressed 5.5-kb transcript. The 3.0-kb ubiquitously expressed transcript included sequences at the beginning of exon 17 (designated exon 17a) and the end of exon 17 (designated exon 18), but lacked the internal 2439 bp of exon 17, including the VNTR. This alternative splicing resulted in a predicted protein of 719 residues from the smaller transcript with four more terminal amino acids than the 715 residue protein predicted from the larger transcript. EST H49244 spanned exon 11 of SEC14L1 and was specifically expressed in human peripheral blood leukocytes. One intragenic single nucleotide polymorphism (SNP) was confirmed. SEC14L1 contained the CRAL/TRIO domain also found in alpha-tocopherol transfer protein (TTPA) and cellular retinaldehyde-binding protein (CRALBP). As retinoids have been shown to inhibit the growth of breast cancer cells, loss of the proposed SEC14L1 retinal-binding function may contribute to breast tumorigenesis. As TTPA and CRALBP have been implicated in retinitis pigmentosa (RP), altered SEC14L1 expression may contribute to RP in previously unlinked families. Coding exon-specific PCR primers were designed to aid in future expression and mutational analyses.

Topics: Adult; Breast; Breast Neoplasms; Carrier Proteins; Cell Line; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 17; Contig Mapping; DNA, Neoplasm; Exons; Expressed Sequence Tags; Female; Fetal Proteins; Genes, Tumor Suppressor; Glycosylation; Golgi Apparatus; Humans; Leukocytes; Minisatellite Repeats; Molecular Sequence Data; Multigene Family; Organ Specificity; Polymorphism, Genetic; Protein Processing, Post-Translational; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing; Tretinoin; Tumor Cells, Cultured

Retinoids are a group of compounds which inhibit cell proliferation and induce cellular differentiation. The aim of this study was to compare the antiproliferative activity of various concentrations of 13-cis retinoic acid (isotretinoin) and all-trans retinoic acid (tretinoin) in a culture of the estrogen-sensitive human breast cancer cell line MCF-7. Evaluation was based on [3H]thymidine incorporation into the cancer cells and through immunocytochemical analysis of cell cycle-associated PCNA and Ki-67 protein expression. Both retinoids inhibited [3H]thymidine incorporation into the cancer cells most effectively at a concentration of 3x10(-3) M. Two basic substances used for line MCF-7 culture experiments, one stimulating - estradiol - and the other inhibiting - tamoxifen - were applied. Estradiol added to a culture containing decreasing concentrations of isotretinoin (from 3x10(-3) to 3x10(-8) M) caused a statistically significant reduction in the percentage of [3H]thymidine incorporation into the cancer cell line MCF-7, compared to the 17 beta estradiol group (189.25%+/-62.64, control=100%, p<0.05). In the group of decreasing tretinoin concentrations, statistically significant differences were found only at 3x10(-3), 3x10(-4) and 3x10(-8) M. Following culture supplementation with tamoxifen (1 microM), statistically significant differences were observed only at the highest concentrations of both retinoids (3x10(-3) and 3x10(-4) M). The evaluation of breast carcinoma cells with a positive immunocytochemical reaction to PCNA and Ki-67 has revealed that isotretinoin reduces their percentage in the most determined and statistically significant way (38.00%+/-2.58 and 39.25%+/-3.09), compared to the control group (86.50%+/-9.20 and 100%+/-3.87, p<0.001 and p<0.0001) and to the estradiol group (87.00%+/-6.79 and 86.10%+/-7.0, p<0.001). Apart from their blocking effect on the cell cycle, retinoids also induce the apoptotic pathway.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Cell Survival; Estradiol; Female; Humans; Immunohistochemistry; Isotretinoin; Ki-67 Antigen; Neoplasms, Hormone-Dependent; Proliferating Cell Nuclear Antigen; Tamoxifen; Thymidine; Tretinoin; Tumor Cells, Cultured

To clone apoptosis-related genes induced by all-trans retinoic acid (ATRA) from human breast cancer cell line MCF-7, and to analyze the association between the cloned genes and apoptosis.. An apoptotic cell model of MCF-7 cells was constructed with ATRA induction. The apoptosis-related genes were cloned by improved PCR-based subtractive hybridization.. Twelve differentially expressed clones were screened out. After exclusion of false positive clones by reverse dot blotting, 5 clones containing fragments of 0.5-1.5 kb were sequenced. The results of sequencing were compared with BLAST. A novel gene, named apmcf-1, coding for 47 amino acids was identified. This gene was accepted by GenBank, (Accession number: AF141882). The result of reverse dot blotting showed that it was related to apoptosis. The other 4 genes were already known. Three of them were related to apoptosis as previously reported. Two of them, hsp-90 and rb-L3, had little information about their relationship with apoptosis.. ATRA-induced tumor cell apoptosis is a complex process with multiple genes involved.

Topics: Apoptosis; Breast Neoplasms; Cloning, Molecular; GTP-Binding Proteins; Humans; Nucleic Acid Hybridization; Polymerase Chain Reaction; Proto-Oncogene Proteins; Tretinoin; Tumor Cells, Cultured

Retinoic acid receptor beta (RARbeta) plays a critical role in mediating the anticancer effects of retinoids. Expression of RARbeta is highly induced by retinoic acid (RA) through a RA response element (betaRARE) that is activated by heterodimers of RARs and retinoid X receptors (RXRs). However, RARbeta induction is often lost in cancer cells despite expression of RARs and RXRs. In this study, we provide evidence that orphan receptor COUP-TF is required for induction of RARbeta expression, growth inhibition, and apoptosis by RA in cancer cells. Expression of COUP-TF correlates with RARbeta induction in a variety of cancer cell lines. In addition, stable expression of COUP-TF in COUP-TF-negative cancer cells restores induction of RARbeta expression, growth inhibition, and apoptosis by RA, whereas inhibition of COUP-TF by expression of COUP-TF antisense RNA represses the RA effects. In a transient transfection assay, COUP-TF strongly induced transcriptional activity of the RARbeta promoter in a RA- and RARalpha-dependent manner. By mutation analysis, we demonstrate that the effect of COUP-TF requires its binding to a DR-8 element present in the RARbeta promoter. The binding of COUP-TF to the DR-8 element synergistically increases the RA-dependent RARalpha transactivation function by enhancing the interaction of RARalpha with its coactivator CREB binding protein. These results demonstrate that COUP-TF, by serving as an accessory protein for RARalpha to induce RARbeta expression, plays a critical role in regulating the anticancer activities of retinoids.

Topics: Animals; Apoptosis; Base Sequence; Binding Sites; Breast Neoplasms; Cell Division; Cell Line; COUP Transcription Factor I; Dimerization; DNA-Binding Proteins; Female; HeLa Cells; Humans; Kinetics; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Receptors, Glucocorticoid; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The organic arsenical known as melarsoprol (Mel-B) is used to treat African trypanosomiasis. Recently, another arsenical, As2O3 was shown to be effective in treatment of acute promyelocytic leukaemia. We have investigated the anti-tumour activities of Mel-B either with or without all-trans-retinoic acid (ATRA) using the MCF-7 human breast cancer cells, as well as the PC-3 and DU 145 human prostate cancer cells both in vitro and in vivo. The antiproliferative effects of Mel-B and/or ATRA against breast and prostate cancer were tested in vitro using clonogenic assays and in vivo in triple immunodeficient mice. Furthermore, the mechanism of action of these compounds was studied by examining the cell cycle, levels of bcl-2, apoptosis and antiproliferative potency using a pulse-exposure assay. Clonogenic assays showed that the cancer cell lines were sensitive to the inhibitory effect of Mel-B (effective dose that inhibited 50% clonal growth [ED50]: 7 x 10(-9) M for MCF-7, 2 x 10(-7) M for PC-3, 3 x 10(-7) M for DU145 cells. Remarkably, the combination of Mel-B and ATRA had an enhanced antiproliferative activity against all three cancer cell lines. Furthermore, the combination of Mel-B and ATRA induced a high level of apoptosis in all three cell lines. Treatment of PC-3 and MCF-7 tumours growing in triple immunodeficient mice with Mel-B and ATRA either alone or in combination markedly retarded tumour size and weight of the tumours without major side-effects. In conclusion, our results suggest that either Mel-B alone or with ATRA may be a useful, novel therapy for breast and prostate cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Division; Female; Humans; Male; Melarsoprol; Mice; Prostatic Neoplasms; Tretinoin; Trypanocidal Agents; Tumor Cells, Cultured

The effect of retinoic acid and dexamethasone on alkaline phosphatase (AP) expression was investigated in human breast cancer MCF-7 cells. Cellular AP activity was induced significantly by retinoic acid or dexamethasone in a time-dependent and dose-dependent fashion. A marked synergistic induction of AP activity was observed when the cells were incubated with both agents simultaneously. Two AP isozymes, tissue-nonspecific (TNAP) and intestinal (IAP), were shown to be expressed in MCF-7 cells as confirmed by the differential rate of thermal inactivation of these isozymes and RT-PCR. Based on the two-isozyme thermal-inactivation model, the specific activities for TNAP and IAP in each sample were analyzed. TNAP activity was induced only by retinoic acid and IAP activity was induced only by dexamethasone. Whereas dexamethasone conferred no significant effect on TNAP activity, retinoic acid was shown to inhibit IAP activity by approximately 50%. Interestingly, TNAP was found to be the only isozyme activity superinduced when the cells were costimulated with retinoic acid and dexamethasone. Northern blot and RT-PCR analysis were then used to demonstrate that the steady-state TNAP mRNA level was also superinduced, which indicates that the superinduction is regulated at the transcriptional or post-transcriptional levels. In the presence of the glucocorticoid receptor antagonist RU486, the dexamethasone-mediated induction of IAP activity was blocked completely as expected. However, the ability of RU486 to antagonize the action of glucocorticoid was greatly compromised in dexamethasone-mediated superinduction of TNAP activity. Furthermore, in the presence of retinoic acid, RU486 behaved as an agonist, and conferred superinduction of TNAP gene expression in the same way as dexamethasone. Taken together, these observations suggest that the induction of IAP activity by dexamethasone and the superinduction of TNAP by dexamethasone were mediated through distinct regulatory pathways. In addition, retinoic acid plays an essential role in the superinduction of TNAP gene expression by enabling dexamethasone to exert its agonist activity, which otherwise has no effect.

Topics: Alkaline Phosphatase; Base Sequence; Breast Neoplasms; Cell Division; Dexamethasone; DNA Primers; Enzyme Induction; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

The biologic activity of vitamin A depends, in part, on its metabolism to active nuclear receptor ligands, chiefly retinoic acid. The cellular retinol-binding protein (CRBP) binds vitamin A with high affinity and is postulated to regulate its uptake and metabolism. In this report, we analyze the expression of CRBP in normal and malignant breast tissues.. We evaluated CRBP expression by in situ hybridization in six reduction mammoplasty specimens and 49 human breast carcinoma specimens by use of digoxigenin-labeled RNA probes and in nine cultured mammoplasty specimens by northern or western blot analysis. Statistical significance was evaluated with the chi(2) test or Fisher's exact test if the sample sizes were small. All P values are from two-sided tests.. CRBP was expressed in all 15 mammoplasty specimens (normal breast tissue) and in 33 of 35 available specimens of normal tissue adjacent to carcinoma. In contrast, 12 (24%) of 49 carcinoma lesions were uniformly negative for CRBP (P =.023 for comparison with adjacent normal breast tissue). The loss of CRBP expression was as frequent in ductal carcinoma in situ (six [27%] of 22) as in invasive lesions (six [22%] of 27), suggesting that it is a relatively early event in carcinogenesis and not associated with patient age, tumor grade, and expression of steroid receptors or c-Myc. Preliminary experiments did not find an association between CRBP and retinoic acid receptor beta loss, but most (four of five) CRBP-negative tumors were also retinoic acid receptor beta negative.. CRBP is underexpressed in 24% (95% confidence interval = 12.5%-36.5%) of human breast carcinomas, implying a link between cellular vitamin A homeostasis and breast cancer. We hypothesize that the loss of CRBP restricts the effects of endogenous vitamin A on breast epithelial cells.

Topics: Blotting, Northern; Breast; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; In Situ Hybridization; Mammaplasty; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Neoplasm; Signal Transduction; Tretinoin; Vitamin A

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast; Breast Neoplasms; Cell Line; Chromatin; CpG Islands; Decitabine; DNA Methylation; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Cell Survival; Doxorubicin; Drug Synergism; Female; Humans; Paclitaxel; Receptors, Calcitriol; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

It was previously reported that 8701-BC breast tumour cells express the gene for parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor (PTHrP-R) and release immunoreactive PTHrP (iPTHrP) into the extracellular medium. Since the regulation of PTHrP and PTHrP-R by breast cancer cells has been poorly investigated so far, we have chosen the 8701-BC cell line as a model system to investigate whether alterations in the extracellular Ca2+ concentration ([Ca2+]e) and treatment with some well-known differentiation agents for breast cells, such as dimethyl sulfoxide, hydrocortisone, progesterone, prolactin, all-trans retinoic acid and transforming growth factor-beta1 might (i) modulate quantitatively the release of iPTHrP, (ii) affect the PTHrP promoter usage and mRNA splicing patterns, and (iii) modify the expression of PTHrP-R. The data obtained indicate that 8701-BC cells are potentially able to utilise different start sites and mRNA splicing patterns for PTHrP transcription, and respond to variations of [Ca2+]e and to the addition of two hormones, hydrocortisone and progesterone, with modifications in the extracellular amount of iPTHrP. Moreover, expression of PTHrP-R is also modulated by changes of [Ca2+]e or treatment with hydrocortisone. This indicates that the 8701 -BC cell line is a suitable in vitro model for further studies on the complex molecular regulation of the PTHrP/PTHrP-R pair in breast cancer.

Topics: Breast Neoplasms; Calcium; Codon, Initiator; Extracellular Space; Gene Expression Regulation; Hormones; Humans; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Protein Isoforms; Proteins; Receptors, Parathyroid Hormone; RNA Splicing; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (alpha, beta, and gamma). Recent studies have demonstrated that RARbeta plays a critical role in mediating anticancer effects of retinoids. However, how RARbeta exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARbeta. In a transient transfection assay, all three RAR subtypes, RARalpha, RARbeta, and RARgamma, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARbeta showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARalpha and RARgamma was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH2-terminal portion of RARbeta, we demonstrated that the NH2-terminal portion of RARbeta, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARbeta, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARbeta. In cancer cells, stable transfection of RARbeta exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARbeta. In studying the antitransforming activity of RARbeta, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARbeta but not RARalpha. Together, our results demonstrate that RARbeta may exert its potent anticancer effect in part through its unique anti-AP-1 activity.

Topics: Breast Neoplasms; Carcinogens; Cell Division; Collagenases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Reporter; HeLa Cells; Histone Deacetylases; Humans; Hydroxamic Acids; Mutagenesis; Plasmids; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to approximately 9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein ( approximately 68 kDa). tRA stimulated NIS gene transcription approximately 4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t(1/2) = 24 min), compared with that in FRTL-5 thyroid cells (t(1/2) = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with (131)I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Breast Neoplasms; Carrier Proteins; Cell Line; Colforsin; Down-Regulation; Female; Humans; Iodides; Iodine Radioisotopes; Kinetics; Membrane Proteins; Oxytocin; Prolactin; Rats; Sodium Iodide; Symporters; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

To gain insight into the molecular regulation of the human vitamin D3 receptor (hVDR), we have cloned and sequenced the 5' flanking region of exon 1c and examined promoter activity of this region in breast cancer cells. Sequence analysis of the first 1300 bp upstream of exon 1c reveals several characteristics of a class II promoter, including GC-rich regions and the presence of a TATA box at -29 bp. Putative transcription factor binding sites identified in this potential hVDR promoter include AP-2, Sp-1, and glucocorticoid response elements. No consensus vitamin D3 (VDRE) or estrogen (ERE) responsive elements were identified in the promoter sequence. Primer extension analysis performed with a primer specific for exon 1c confirms that transcription initiated in the 5' flanking region of exon 1c occurs in MCF-7 cells. Transient transfection of MCF-7 cells with this putative promoter region cloned into the pRLnull luciferase reporter vector generates significant reporter gene activity that is enhanced by treatment with forskolin, retinoic acid, and 17beta-estradiol. The enhancement of exon 1c promoter activity by 17beta-estradiol is blocked by the selective estrogen response modifier (SERM) tamoxifen and is not observed in estrogen receptor-negative breast cancer cells. In summary, we have cloned and characterized a TATA containing promoter upstream of exon 1c of the hVDR and provide evidence that this region represents a hormonally regulated hVDR promoter.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Cholecalciferol; Colforsin; Estradiol; Estrogen Antagonists; Estrogens; Exons; Gene Expression Regulation; Hormones; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Calcitriol; Response Elements; Tamoxifen; TATA Box; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

We show here that the combination of interferon-beta (IFN-beta) and all-trans-retinoic acid (RA) induces the death of tumor cells. To understand the molecular basis for synergistic growth-suppressive action and to identify the gene products that participate in this process, we have employed an antisense knock-out technique. This approach permits the isolation of cell death-associated genes based on their selective inactivation by overexpression of antisense cDNAs. Because the antisense mRNA inactivates gene expression of death-specific genes, transfected cells survive in the presence death inducers. Several Genes associated with Retinoid-IFN-induced Mortality (GRIM) were identified using this approach. Here we report the isolation of a novel GRIM gene, GRIM-19. This 552-base pair cDNA encodes a 16-kDa protein. Antisense expression of GRIM-19 confers a strong resistance against IFN/RA-induced death by reducing the intracellular levels of GRIM-19 protein. Overexpression of GRIM-19 enhances cell death in response to IFN/RA. GRIM-19 is primarily a nuclear protein whose expression is induced by the IFN/RA combination. Together, our studies identify a novel cell death-regulatory molecule.

Topics: Amino Acid Sequence; Apoptosis; Breast Neoplasms; DNA, Antisense; Drosophila Proteins; Female; Gene Expression Regulation; Humans; Interferon-beta; Molecular Sequence Data; Neuropeptides; RNA, Messenger; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured

Apoptosis is a tightly regulated mechanism that controls the proliferation of cells in metazoans. In mammals, multiple genes are required to regulate cell death. We have employed a gene expression knockout technique to isolate cell death-related genes. One such gene, gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), is essential for tumor cell death induced by interferon-beta (IFN-beta) and retinoic acid (RA). Here, we describe the localization of GRIM-19 to human chromosome 19p13.2. This region is essential for prostate tumor suppression. Together with its death-inductive role in the IFN-retinoid-regulated pathways and the tumor-suppressive function of this locus, the data suggest that GRIM-19 may be a novel tumor suppressor.

Topics: Breast Neoplasms; Cell Death; Chromosome Mapping; Chromosomes, Human, Pair 13; DNA, Antisense; Female; Genes, Tumor Suppressor; Genomic Library; Humans; In Situ Hybridization, Fluorescence; Interferon-beta; Karyotyping; Tretinoin; Tumor Cells, Cultured

The rates of glucose transport and of glycolysis and the expression of the glucose transporters GLUT-1 through GLUT-4 were measured in T47D human breast cancer cells that underwent differentiation by retinoic acid. Glucose transport was found to be the rate-limiting step of glycolysis in control and differentiated cells. The transporters GLUT-1, GLUT-3, and GLUT-4 were present in the cell membrane and in the cytoplasm, and GLUT-2 was present solely in the cytoplasm. Differentiation led to a reduction in GLUT-1 and to an increase in cytoplasmic GLUT-2 and GLUT-3 with no change in GLUT-4. Differentiation also caused a reduction in the maximal velocity of glucose transport by approximately 40% without affecting the Michaelis-Menten constant of glucose transport. These changes did not alter the steady-state concentration of the phosphate metabolites regulating cell energetics but increased the content of phospholipid breakdown phosphodiesters. In conclusion, differentiation of human breast cancer cells appears to be associated with decreased glycolysis by a mechanism that involves a reduction in GLUT-1 and a slowdown of glucose transport.

Topics: Algorithms; Breast Neoplasms; Cell Cycle; Cell Differentiation; Flow Cytometry; Fluorescent Antibody Technique; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 2; Glucose Transporter Type 3; Humans; Keratins; Kinetics; Magnetic Resonance Spectroscopy; Microscopy, Electron; Monosaccharide Transport Proteins; Nerve Tissue Proteins; Phosphates; Tretinoin; Tumor Cells, Cultured

Oestrogen is important in the development of breast cancer. Oestrogen receptor positive breast cancers are associated with a better prognosis than oestrogen-receptor negative breast cancers since they are more responsive to hormonal treatment. Oestrone sulphate acts as a huge reservoir for oestrogens in the breast. It is converted to the potent oestrogen, oestradiol (E(2)) by the enzymes oestrone sulphatase and oestradiol-17beta hydroxysteroid dehydrogenase (E(2)DH). Retinoic acid and carotenoids have been shown to have chemopreventive activity against some cancers. The aim of our study was to determine and compare the effects of retinoic acid and palm oil carotenoids on growth of and oestrone sulphatase and E(2)DH activities in the oestrogen receptor positive, MCF-7 and oestrogen receptor negative, MDA-MB-231 breast cancer cell lines. Retinoic acid and carotenoids inhibited MCF-7 cell growth but had no effect on MDA-MB-231 cell growth. Both retinoic acid and carotenoids stimulated oestrone sulphatase activity in the MCF-7 cell line. E(1) to E(2) conversion was inhibited by 10(-7) M carotenoids but was stimulated at 10(-6) M in the MCF-7 cell line. Retinoic acid had no effect on E(1) to E(2) conversion at 10(-7) M but stimulated E(1) to E(2) conversion at 10(-6) M. Retinoic acid and carotenoids had no effect on E(2) to E(1) conversion in the MCF-7 cell line. Retinoic acid stimulated E(1) to E(2) conversion in the MDA-MB-231 cell line but had no effect on oestrone sulphatase activity or E(2) to E(1) conversion in this cell line. Both oestrone sulphatase and E(2)DH activity were not affected by carotenoids in the MDA-MB-231 cell line. In conclusion, retinoic acid and carotenoids may prevent the development of hormone-dependent breast cancers since they inhibit the growth of the MCF-7 cell line.

Topics: Antineoplastic Agents; Breast Neoplasms; Carotenoids; Cell Division; Dose-Response Relationship, Drug; Estradiol Dehydrogenases; Female; Humans; Palm Oil; Plant Oils; Receptors, Estrogen; Sulfatases; Tretinoin; Tumor Cells, Cultured

We investigated the mechanism of retinoic acid receptor (RAR) beta2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous beta2 promoter (-1470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARbeta2. This was true even in the context of replicated chromatin, suggesting a cis-acting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modification, has been implicated in RARbeta2 silencing in cancer cells. Upon bisulfite genomic sequencing, we found that 3 CpG sites in the beta2 RARE region were variably methylated in MCF-7 cells but were not methylated in MTSV1.7 cells or in 2 MDA-MB-231 subclones that differed in RARbeta2 expression (high in clone A2, low in clone A4). However, the 5'-UTR region was hypermethylated in clone A4 relative to clone A2 cells. Following 5-azacytidine treatment, RA and trichostatin A markedly induced RARbeta2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A beta2 RARE reporter construct in which the methylation-susceptible cytosines in the sense strand were replaced by thymine displayed marked loss of activity in a replicated chromatin-dependent manner. We conclude that cytosine methylation contributes to RARbeta2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important. Oncogene (2000) 19, 4066 - 4070.

Topics: Adenocarcinoma; Azacitidine; Base Sequence; Breast Neoplasms; Clone Cells; CpG Islands; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Reporter; Humans; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Receptors, Retinoic Acid; Sequence Analysis, DNA; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

It has been established that melatonin (Mlt) and retinoic acid, individually, inhibit the proliferation of the estrogen receptor-alpha (ER alpha)-positive MCF-7 breast cancer cell line. Our laboratory has previously demonstrated that Mlt and all-trans-retinoic acid (atRA) not only inhibit the proliferation, but also induce apoptosis of MCF-7 cells when used in a sequential regimen of Mlt followed 24 h later by atRA. Using this same MCF-7 breast cancer cell line, we investigated the potential pathways through which apoptosis is being induced. We found that treatment of MCF-7 cells with Mlt for 24 h before the addition of atRA decreased the protein levels of the death suppressor, Bcl-2, and increased, although with different time courses, the levels of the death promoters, Bax and Bak; however, there was no change in the levels of the tumor suppressor gene, p53. MCF-7 cells treated sequentially with Mlt and atRA also demonstrated an enhanced sensitivity to the apoptotic effects of atRA, which did not appear to be due to increased expression of the retinoic acid receptors, RAR alpha or RXR alpha, but rather to enhanced transcriptional activity of the RAR alpha. These data suggest that the sequential treatment regimen of Mlt and atRA may induce apoptosis by modulation of members of the Bcl-2 family of proteins. Thus, this combinatorial regimen, which reduces the concentration of atRA needed for clinical efficacy while enhancing its anti-tumorigenic activity, could be of great therapeutic benefit, and may, in fact, specifically induce the regression of established breast tumors due to its apoptosis-promoting effects.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Luciferases; Melatonin; Membrane Proteins; Microscopy, Phase-Contrast; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Receptors, Retinoic Acid; Signal Transduction; Time Factors; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Our previous work had shown that retinoic acid (RA) inhibits cell growth and induces apoptosis in estrogen receptor-positive (ER-positive) MCF-7 and T-47D human breast carcinoma cells, but not in ER-negative human breast carcinoma cells MB-231 and MB-453. The purpose of this work was to determine whether these differences might be due to differences in uptake and metabolism of the drug between ER-positive and ER-negative cells.. We measured RA uptake in cultured human breast cancer cells and determined its metabolism by high-pressure liquid chromatographic analysis.. The two ER-positive cell lines reached maximum RA uptake at about 2 h, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 h and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 h there was more RA in these cells and their culture medium than in the RA-responsive ER-positive cells, even though at 2 h the ER-negative cells had taken up less RA than the ER-positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin-bound retinoid to the cells.. This study is the first to demonstrate profound differences in RA accumulation and confirms previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. The findings reported here, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy.

Topics: Biological Transport; Blood Proteins; Breast Neoplasms; Chromatography, High Pressure Liquid; Culture Media; Female; Humans; Kinetics; Receptors, Estrogen; Tretinoin; Tritium; Tumor Cells, Cultured

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.

Topics: Breast; Breast Neoplasms; Carrier Proteins; Cell Line, Transformed; Cells, Cultured; Cytochrome P-450 Enzyme System; Female; Humans; Mixed Function Oxygenases; Reference Values; Retinoic Acid 4-Hydroxylase; Tretinoin; Vitamin A

Although retinoids are known to be inhibitory to breast cancer cell growth, a key remaining question is whether they would remain effective if administered long-term. We describe here the long-term effects of all- trans retinoic acid on two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1. Although both cell lines were growth inhibited by retinoic acid in the short-term in either the absence or the presence of oestradiol, prolonged culture with 1 microM all- trans retinoic acid resulted in the cells acquiring resistance to the growth inhibitory effects of retinoic acid. Time courses showed that oestrogen deprivation of the cell lines resulted in upregulation of the basal non-oestrogen stimulated growth rate such that cells learned to grow at the same rate without as with oestradiol, but the cells remained growth inhibited by retinoic acid throughout. Addition of 1 microM all- trans retinoic acid to steroid deprivation conditions resulted in reproducible loss of growth response to both retinoic acid and oestradiol, although the time courses were separable in that loss of growth response to retinoic acid preceded that of oestradiol. Loss of growth response to retinoic acid did not involve loss of receptors, ER as measured by steroid binding assay or RARalpha as measured by Northern blotting. Function of the receptors was retained in terms of the ability of both oestradiol and retinoic acid to upregulate pS2 gene expression, but there was reduced ability to upregulate transiently transfected ERE- and RRE-linked reporter genes. Despite the accepted role of IGFBP3 in retinoic acid-mediated growth inhibition, progression to retinoic acid resistance occurred irrespective of level of IGFBP3, which remained high in the resistant MCF7 cells. Measurement of AP1 activity showed that the two cell lines had markedly different basal AP1 activities, but that progression to resistance was accompanied in both cases by a lost ability of retinoic acid to reduce AP1 activity. These results warn of potential resistance which could arise on long-term treatment with retinoic acid in a clinical situation and echo the problems of progression to endocrine resistance. It seems that whatever the constraints imposed on growth, these cells have a remarkable ability to escape from growth inhibition. However, the ability of retinoic acid to delay progression to oestrogen resistance is encouraging for endocrine therapy, and the concentration-dependence of retinoic acid

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Drug Resistance, Neoplasm; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Receptors, Estrogen; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor alpha; RNA, Messenger; Time Factors; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 microM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days of RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.

Topics: Adenocarcinoma; Alitretinoin; Antineoplastic Agents; Breast Neoplasms; Enzyme Induction; Female; Gene Expression Regulation, Neoplastic; Humans; Isotretinoin; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Telomerase; Tretinoin; Tumor Cells, Cultured

Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5-8-fold) of com1 mRNA. Furthermore, clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)2D3 (10(-7) M), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Calcitriol; Cell Cycle; Cell Division; Clone Cells; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dexamethasone; DNA-Binding Proteins; Estradiol; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Breast cancer is the most common cancer in women worldwide. The growth of breast cancer cells is either hormone-dependent or hormone-independent. Both types are represented in vitro by the estrogen-receptor positive (ER+) MCF-7 and the estrogen-receptor negative (ER-) MDA-MB-231 cell lines, respectively. The pS2 gene is an estrogen-regulated gene and serves as a marker for the ER+ tumours. Carotenoids are pigments with anti-cancer properties besides having pro-vitamin A, antioxidant and free-radical quenching effects. This study was designed firstly, to compare the effect of palm oil carotene concentrate with retinoic acid on the growth of the ER+ MCF-7 and the ER- MDA-MB-231 cells; and secondly to evaluate the effect of the palm oil carotene concentrate on the regulation of pS2 mRNA. The growth experiments were performed with monolayer cells seeded in phenol red free RPMI 1640 culture media and subsequently treated with varying concentrations of either retinoic acid or palm oil carotenoids. The cell numbers were determined at the start of each experiment and then at successive time intervals. The results showed that the palm oil carotene concentrate caused dose-dependent inhibition of estradiol-stimulated growth of MCF-7 cells but did not affect the proliferation of MDA-MB-231 cells. Retinoic acid caused similar, albeit more potent effects, as significant inhibition was observed at lower concentrations than the palm oil carotenoids. In the pS2 gene expression experiment, cell monolayers were treated with the carotene concentrate (10(-6) M), either with or without supplemented estradiol (10(-8) M), and subsequently the RNA was extracted. Northern blotting was performed and the regulation of pS2 mRNA determined using a 32P-labelled pS2 cDNA probe. The results showed that the palm oil carotene concentrate did not affect the expression of pS2 mRNA and are therefore independent of the estrogen-regulated pathway.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Carotenoids; Cell Count; Cell Division; Female; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Palm Oil; Plant Oils; Proteins; Receptors, Estrogen; RNA, Neoplasm; Trefoil Factor-1; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Treatment of estrogen receptor (ER)-positive MCF-7 human breast cancer cells with retinoic acid (RA) inhibited cell growth and increased cell adhesion to fibronectin. In contrast, ER- MDA-MB-231 cells failed to respond. Western blot analysis showed that tyrosine phosphorylation of two major bands at Mr 125,000 and Mr 68,000 was induced by RA in ER+ MCF-7 human breast carcinoma cells. However, this induction was a late phenomenon detectable at 12 and 24 h, but not within 3 h. A similar increase of tyrosine phosphorylation by RA was observed in ER+ human breast cancer cell lines T-47D and ZR-75-1, but not in the ER- cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468. Focal adhesion kinase and paxillin, which localize in focal adhesion plaques and may play important roles in the integrin signaling pathway, were identified as the major proteins showing RA-induced tyrosine phosphorylation. The retinoid X receptor-selective compound SR11237 failed to induce tyrosine phosphorylation, indicating that retinoid X receptor activation is not involved in this phenomenon. In contrast, stable overexpression of a truncated RA receptor (RAR) alpha cDNA, RARalpha403, with strong RAR dominant negative activity prevented the increase in tyrosine phosphate, suggesting that RAR signaling is involved in RA-induced tyrosine phosphorylation. Tyrosine phosphorylation was induced the most by the RAR-alpha (193836), followed by RAR-gamma (194433), but was not significantly induced by RAR-gamma (193174)-selective retinoids. This study demonstrates a coordinated albeit relatively late effect of RA on cell adhesion and tyrosine phosphorylation in ER+ human breast cancer cells and suggests RAR-alpha as the major responsible retinoid receptor.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Adhesion Molecules; Cytoskeletal Proteins; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Tyrosine

Four candidate retinoid antagonists (LE135, LE511, LE540, and LE550) were designed on the basis of the ligand superfamily concept and synthesized. Analysis of these related retinoids by transient transfection assay demonstrated that LE135, LE540, and LE550 are effective retinoic acid receptor (RAR) antagonists, whereas LE511 selectively induced RARbeta transcriptional activity. Both LE135 and LE540 inhibited retinoic acid (RA)-induced transcriptional activation of RARbeta, but not RARalpha, RARgamma or retinoid X receptor alpha (RXRalpha), on a variety of RA response elements. The retinoid antagonists also inhibited all-trans-RA-induced transcriptional activation of RARbeta/RXRalpha heterodimers, although they did not show any effect on transactivation activity of RXR/RXR homodimers. In ZR-75-1 human breast cancer cells, cotreatment of LE135 and LE540 with all-trans-RA inhibited all-trans-RA-induced apoptosis of the cells, further demonstrating that RARbeta plays a role in RA-induced apoptosis of breast cancer cells. We also evaluated the effect of these retinoids on AP-1 activity. Our data showed that LE135 and LE540 strongly repressed 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity in the presence of RARbeta and RXRalpha. Interestingly, LE550 induced AP-1 activity when RARbeta and RXRalpha were expressed in HeLa cells but not in breast cancer cells. These results demonstrate that LE135 and LE540 were a novel class of RARbeta-selective antagonists and anti-AP-1 retinoids and should be useful tools for studying the role of retinoids and their receptors.

Topics: Animals; Apoptosis; Benzoates; Benzodiazepines; Breast Neoplasms; Collagenases; Dimerization; Drug Design; Genes, Reporter; HeLa Cells; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Neither retinoic acid receptor-beta (RARbeta) nor insulin-like growth factor-binding protein-3 (IGFBP-3) is expressed in breast cancer cell line MCF-7. The expression of both proteins can be induced in response to all-trans-retinoic acid (atRA). By using an RARalpha-selective antagonist (Ro 41-5253), we demonstrated that RARbeta expression was induced by atRA through an RARalpha-dependent signaling pathway and that RARbeta induction was correlated with IGFBP-3 induction. However, MCF-7 cells transfected with sense RARbeta cDNA expressed IGFBP-3 even in the presence of the RARalpha-selective antagonist Ro 41-5253. On the other hand, antisense RARbeta cDNA transfection of MCF-7 cells blocked atRA-induced IGFBP-3 expression, indicating that RARbeta is directly involved in the mediation of IGFBP-3 induction by atRA. Induction of IGFBP-3 expression by atRA occurs at the transcriptional level, as measured by nuclear run-on assays. Finally, we showed that atRA-induced IGFBP-3 is functionally active in modulating the growth-promoting effect of IGF-I. These experiments indicate that RARalpha and RARbeta, both individually and together, are important in mammary gland homeostasis and breast cancer development. By linking IGFBP-3 to RARbeta, our experiments define the signal intersection between the retinoid and IGF systems in cell growth regulation and explain why loss of RARbeta might be critical in breast cancer carcinogenesis/progression.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Signal Transduction; Tretinoin; Tumor Cells, Cultured

In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective, even at doses that are toxic to the host. In late stages of breast cancer, patients do not respond to RA because the expression of RA receptor beta (RARbeta) is lost. In the present study, the intracellular mechanism(s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) analogue, 8-chloro-adenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined. Our data demonstrated that hormone-dependent MCF-7 cells, but not hormone-independent MDA-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis. Introduction of the RARbeta gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with all-trans-RA in inducing and activating RARbeta gene expression that correlates with the reduction in mitochondrial membrane potential, redistribution of cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase and DNA-dependent protein kinase (catalytic subunit), and induction of apoptosis. Mutations in the cAMP response element-related motif within the RARbeta promoter resulted in loss of synergy in RARbeta transcription. In addition, inhibition of RARbeta expression by an antisense construct also blocked the antitumor effects of RA + 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RARbeta expression may contribute to the tumorigenicity of human mammary epithelial cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion and may have potential for combination biotherapy for the treatment of malignant diseases.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Survival; Cytochrome c Group; DNA-Activated Protein Kinase; DNA-Binding Proteins; Drug Synergism; Gene Expression Regulation, Neoplastic; Hormones; Humans; Mitochondria; Nuclear Proteins; Poly Adenosine Diphosphate Ribose; Protein Serine-Threonine Kinases; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and metastasis. These processes require the dissolution of the basement membrane and invasion of the stromal matrix (ECM), and are mediated by MMPs. Consequently, MMP inhibitors may be attractive as new anticancer agents. To examine the potential contribution of collagenase-1 (MMP-1) in invasion of stromal matrix, we used the highly invasive and metastatic breast cancer cell line MDA-MB-231 as a model system. These cells express procollagenase-1 constitutively and this expression can be repressed by all-trans retinoic acid. Invasion of these cells into a collagen type I matrix was assessed by scanning electron microscopy (SEM), and was quantitated with a computer program and confocal laser scanning microscopy (CLSM). We found that MDA-MB-231 cells freely invaded the collagen type I matrix, suggesting that these cells possess a mechanism for activating the latent collagenase-1. In contrast, down-regulation of collagenase-1 expression by all-trans retinoic acid caused these cells to become less invasive. To confirm a role for collagenase-1 in mediating collagen type I invasion, assays were carried out in the presence of FN-439, an inhibitor of collagenase-1 enzyme activity. We found that in the presence of the proteinase inhibitor, invasion of type I collagen by MDA-MB-231 cells was also reduced. These results indicate that collagenase-1 produced by the breast tumor cells may enhance stromal matrix degradation by enabling the tumor cells to modulate their own invasive behavior, and suggest that decreasing collagenase-1 levels may be effective in breast cancer therapy.

Topics: Blotting, Northern; Breast Neoplasms; Collagen; Collagenases; Culture Media, Conditioned; Enzyme Inhibitors; Extracellular Space; Fibroblasts; Gelatinases; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Microscopy, Electron, Scanning; Neoplasm Invasiveness; Oligopeptides; Tretinoin; Tumor Cells, Cultured

Exposure of human tumor cell lines to different chemotherapeutic drugs, ionizing radiation, and differentiating agents induced morphological, enzymatic, and ploidy changes resembling replicative senescence of normal cells. Moderate doses of doxorubicin induced this senescence-like phenotype (SLP) in 11 of 14 tested cell lines derived from different types of human solid tumors, including all of the lines with wild-type p53 and half of p53-mutated cell lines. SLP induction seemed to be independent from mitotic cell death, the other major effect of drug treatment. Among cells that survived drug exposure, SLP markers distinguished those cells that became terminally growth-arrested within a small number of cell divisions from the cells that recovered and resumed proliferation. SLP induction in breast carcinoma cells treated with retinoids in vitro or in vivo was found to correlate with permanent growth inhibition under the conditions of minimal cytotoxicity, suggesting that this response may be particularly important for the antiproliferative effect of differentiating agents. The senescence-like program of terminal proliferation arrest may provide an important determinant of treatment outcome and a target for augmentation in cancer therapy.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Division; Cellular Senescence; Doxorubicin; Female; Fibrosarcoma; Gamma Rays; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Phenotype; Ploidies; Tretinoin; Tumor Cells, Cultured

This study examines how accessibility to cisplatin on various genomic regions in T47D breast cancer cells, including the retinoic acid receptor beta gene promoter and coding region and the dihydrofolate reductase gene promoter and coding region, is affected by treatment of the cells with 9-cis retinoic acid, a treatment that activates the retinoic acid receptor beta gene promoter in these cells. A PCR-based assay was used to measure cisplatin adduct density based on the inhibition of PCR amplification of templates from cisplatin treated versus untreated cells. Treatment of cells with 9-cis retinoic acid enhanced accessibility to cisplatin on the retinoic acid receptor beta gene promoter region, but not on the coding regions of that gene nor on the dihydrofolate reductase gene promoter or coding regions, where accessibilities to cisplatin remained 2-4 times lower than on the activated retinoic acid receptor beta gene promoter. Examination of smaller regions within this promoter region showed a repression of platination in the 500 bp region surrounding the TATA box in cells prior to 9-cis retinoic acid treatment, which was abolished following promoter activation. Differences in sequence composition between the various regions could not fully account for differences in platination, suggesting that structural features such as bends in retinoic acid receptor beta gene promoter DNA following gene activation, create energetically favorable sites for platination, and contribute to the cytotoxicity of the drug.

Topics: Alitretinoin; Antineoplastic Agents; Breast Neoplasms; Cisplatin; DNA Adducts; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tetrahydrofolate Dehydrogenase; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

We showed previously that the selenium-dependent glutathione peroxidase, GPX-GI, encoded by the Gpx2 gene, is highly expressed in the epithelium of the gastrointestinal (GI) tract and sporadically in breast tissue. To investigate whether Gpx2 gene expression is epithelium specific, we used in situ hybridization to show that Gpx2 mRNA is highly expressed in the crypt epithelium of human intestine. We also used Northern analysis to study human breast cells and found Gpx2 mRNA in human mammary epithelial cell lines as well as freshly isolated normal breast epithelial cells. Because we identified three putative retinoic acid response elements (RARE) in the Gpx2 gene, we examined the regulation of the Gpx2 gene expression by all-trans retinoic acid (RA) in RA-sensitive MCF-7 cells and RA-resistant HT29 cells. Without RA, MCF-7 cells had very low levels of Gpx2 mRNA and a low level of glutathione peroxidase (GPX) activity (17 mU/mg protein), whereas HT29 cells had a high level of Gpx2 mRNA and GPX activity (200 mU/mg protein). RA treatment increased Gpx2 mRNA level 3- to 11-fold and resulted in a fourfold increase of GPX activity (80 mU/mg protein) in MCF-7 cells. Neither Gpx2 mRNA level nor GPX activity was increased in HT29 cells. These results show that the Gpx2 gene is expressed in both breast and intestinal epithelium cells, and suggest that its expression can be highly regulated by retinoic acid, a known differentiation agent.

Topics: Antineoplastic Agents; Base Sequence; Breast Neoplasms; Enzyme Induction; Female; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Intestinal Mucosa; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Mitogen-Activated Protein Kinases; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

Abnormal expression of c-fms proto-oncogene, which encodes for the macrophage colony-stimulating factor-1 (CSF-1) receptor, has been observed in a variety of carcinomas of epithelial origin, including those of the breast. Here, we have investigated the effect of retinoic acid (RA), an important regulator of normal differentiation of mammary epithelial tissues, on the expression of the c-fms gene and CSF-1/CSF-1 receptor-induced invasion and anchorage-independent growth in breast carcinoma cells. We have demonstrated that all-trans-RA (atRA) significantly increases levels of c-fms transcripts in the estrogen receptor-negative but RA receptor alpha-positive breast carcinoma cell lines BT20 and SKBR3. The atRA-induced increase in fms transcript levels was completely abolished by RO41-5253, a synthetic RA receptor alpha antagonist. Our results indicate that atRA could enhance fms expression by up-regulating the activity of the first promoter of the fms gene. DNase I protection, mobility shift, and mutational analysis revealed that a potential activator protein 1 (AP-1) site in the first fms promoter sequence could mediate the observed atRA effect on fms transcription. Our results also showed that atRA, by itself and in the presence of CSF-1, can increase the ability of breast carcinoma cells to invade in vitro. Furthermore, we demonstrated that atRA is able to abolish the CSF-1-induced increase in anchorage-independent growth of breast carcinoma cells without affecting the anchorage-dependent growth. In summary, our findings suggest that retinoids may play conflicting roles throughout breast cancer progression, depending on the stage of cancer development. Although retinoids might suppress growth at the early stages of tumor formation, they might promote malignant transformation at later stages by stimulating the invasive capacity of certain cell variants in the breast tumor population.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Blotting, Northern; Breast Neoplasms; Cell Nucleus; Dexamethasone; DNA Footprinting; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Macrophage Colony-Stimulating Factor; Mutation; Plasmids; Promoter Regions, Genetic; Proto-Oncogene Mas; Receptor, Macrophage Colony-Stimulating Factor; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid receptor-beta (RAR beta) and signal transducer and activator of transcription 1 (STAT1) are important mediators of the antiproliferative and apoptotic actions of retinoids and cytokines/growth factors, respectively. Expression of both RAR beta and STAT1 is lost in most breast cancer cell lines but it can be induced by retinoids in estrogen receptor-positive cells. We investigated a possible functional connection between these two mediators and present evidence supporting RAR beta as a tumor suppressor. First, by using different receptor-selective retinoids, we demonstrated that RAR beta induction in MCF-7 cells by all-trans-retinoic acid (atRA) was associated with the activation of STAT1 gene transcription. The direct involvement of RAR beta in atRA-induced STAT1 gene activation was further demonstrated by showing that transfection with an anti-sense RAR beta construct blocked atRA-induced STAT1 expression in MCF-7 cells whereas introduction of a sense-RAR beta construct resulted in STAT1 induction by atRA in MDA-MB 231 cells. In addition, we showed that STAT1 was phosphorylated/activated under atRA treatment of MCF-7 cells; this process required the involvement of RAR beta and protein synthesis. STAT1 phosphorylation/activation was accompanied by increased tyrosine kinase activity that was not due to the activation of JAK1, JAK2 or Tyk 2, suggesting the possible involvement of an unidentified tyrosine kinase.

Topics: Base Sequence; Breast Neoplasms; DNA Primers; DNA-Binding Proteins; Dose-Response Relationship, Drug; Humans; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Retinoic Acid; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Tretinoin; Tumor Cells, Cultured

We examined the effects of all-trans retinoic acid (RA) on the insulin-induced cell growth, cell cycle progression and cyclin D1 gene expression in breast cancer cells. RA exerted a dose-dependent growth inhibition on insulin-induced proliferation in T47D and MCF-7 hormone-dependent cell lines, whereas MDA-MB231 hormone-independent cells were not affected. The RA antagonism of insulin growth effect was associated with an inhibition of cell cycle progression and a suppression of insulin-induced cyclin D1 mRNA. The effect of RA on cyclin D1 mRNA was dose-dependent and was observed within 5 h of treatment when insulin response was maximal.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Gene Expression; Genes, bcl-1; Humans; In Vitro Techniques; Insulin; Insulin Antagonists; RNA, Messenger; Tretinoin

All-trans-retinoic acid (ATRA) is well known to inhibit the proliferation of human breast cancer cells. Much less is known about the antiproliferative activity of the naturally occurring metabolites and isomers of ATRA. In the present study, we investigated the antiproliferative activity of ATRA, its physiological catabolites 4-oxo-ATRA and 5,6-epoxy-ATRA and isomers 9-cis-RA and 13-cis-RA in MCF-7 human breast cancer cells by bromodeoxyuridine incorporation. MCF-7 cells were grown in steroid- and retinoid-free medium supplemented with growth factors. Under these culture conditions, ATRA and its naturally occurring catabolites and isomers showed significant antiproliferative activity in MCF-7 cells in a concentration-dependent manner (10[-11] M to 10[-6] M). The antiproliferative activity of ATRA catabolites and isomers was equal to that of the parent compound ATRA at concentrations of 10(-8) M and 10(-7) M. Only at 10(-6) M were the catabolites and the stereoisomer 13-cis-RA less potent. The stereoisomer 9-cis-RA was as potent as ATRA at all concentrations tested (10[-11] M to 10[-6] M). In addition, we show that the catabolites and isomers were formed from ATRA to only a limited extent. Together, our findings suggest that in spite of their high antiproliferative activity the catabolites and isomers of ATRA cannot be responsible for the observed growth inhibition induced by ATRA.

Topics: Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; Estrogens; Female; Humans; Retinoids; Tretinoin; Tumor Cells, Cultured

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

Topics: Annexin A5; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; DNA, Neoplasm; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Growth Inhibitors; Humans; Ki-67 Antigen; Peptide Elongation Factor 1; Phosphatidylserines; Proteins; Tretinoin

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called telomeres to the growing ends of chromosomal DNA. The enzyme telomerase activity is present in a vast majority of tumors but is repressed in most normal tissues. Recently, two groups have reported the molecular cloning of the putative catalytic subunit (hEST2/hTRT) of the telomerase gene. We investigated the expression of this gene in diverse tumor-derived cell lines and tumors as well as in various normal tissues. The expression of hEST2/hTRT was detectable in tumor-derived cell lines, primary breast tumors, pancreatic tumors, and kidney tumors. Furthermore, the expression of hEST2/hTRT was down-regulated in response to a differentiation inducer. However, several normal tissues also expressed varying levels of hEST2/hTRT. Early passage cultures of endothelial fibroblasts and some epithelial cells also expressed the telomerase gene, albeit at low levels. In contrast, the expression of TLP1/TP1, the human homologue of Tetrahymena p80 telomerase subunit, was similar in all of these samples. Our results indicate that the differences in expression of hEST2/hTRT in tumor versus normal cells are relative and are not absolute.

Topics: Breast Neoplasms; Cell Differentiation; Cells, Cultured; Down-Regulation; Gene Expression; HL-60 Cells; Humans; Neoplasms; Polymerase Chain Reaction; Telomerase; Tissue Distribution; Tretinoin; Tumor Cells, Cultured

Vesicles, shed in the extracellular medium by several kinds of normal and tumoral cells, are known to play important roles in cell-cell and cell-matrix interactions and to participate in mechanisms by which tumoral cells acquire metastatic capability and evade immune surveillance. Regulation of the shedding phenomenon and molecular mechanisms involved in extracellular vesicle production are not known and are the subject of this investigation. Fetal calf serum stimulated shedding short after its addition and its stimulatory effect was dose dependent. This effect was reduced after gelatin-Sepharose adsorption indicating a possible involvement of gelatinases on its stimulatory effect. This conclusion was confirmed by the inhibitory effect of bathophenanthroline. Shedding of membrane vesicles decreased after treatment with all trans retinoic acid, a molecule known for its capability to induce cell differentiation. Brefeldin A, an inhibitor of intracellular vesicle movements, and methylamine, an inhibitor of exocytosis, did not abolish shedding. Quercetin, an inhibitor of phosphatidyl inositol 4 kinase and 1,4 phosphatidyl inositol 5 kinase, and 8-Cl-cAMP, a site selective cAMP analogous which induces growth inhibition and differentiation, significantly decreased the amount of shed vesicles.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Membrane; Extracellular Matrix; Female; Humans; Microscopy, Electron; Neoplasm Invasiveness; Phospholipases A; Quercetin; Second Messenger Systems; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; DNA Replication; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Imidazoles; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.

Topics: Base Sequence; Benzoquinones; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Dactinomycin; DNA, Complementary; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Genistein; Humans; Hydroquinones; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; Okadaic Acid; Phenols; Proto-Oncogene Proteins c-raf; Quinones; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Blotting, Northern; Blotting, Western; Breast Neoplasms; DNA, Neoplasm; Drug Administration Schedule; Electrophoresis; Humans; Melatonin; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

We investigated the effects of all-trans retinoic acid (ATRA) and fenretinide (4-HPR) on c-erbB-2 expression in SK-BR-3, BT-474 and MCF-7 breast cancer cells and on the growth, differentiation, apoptosis and cisplatin (CDDP) sensitivity of SK-BR-3 cells. It has been reported that oestrogen inhibits c-erbB-2 in oestrogen receptor-positive breast cancer cells. Using ELISA, Western and Northern analysis we have demonstrated that ATRA and 4-HPR exert similar effects down-regulating c-erbB-2 protein and mRNA in c-erbB-2-overexpressing SK-BR-3 and BT-474 and in normally expressing MCF-7 cells. Both retinoids inhibit SK-BR-3 cell growth. ATRA induces cellular enlargement and flattening, suggesting epithelial differentiation. 4-HPR causes nuclear and cytoplasmic condensation, DNA fragmentation and externalization of phosphatidylserine, indicating apoptosis. c-erbB-2 expression/activity has been linked to sensitivity against CDDP. Therefore, combinations of ATRA or 4-HPR with CDDP were tested for their anti-proliferative activity. Retinoid-conditioned cells were either exposed to retinoid and CDDP (schedule I, 'continuous retinoid treatment') or to CDDP alone (schedule II, 'retinoid pretreatment'). This retinoid-conditioning followed by CDDP +/- retinoid yields stronger growth inhibition compared with unconditioned cells, which were exposed to CDDP +/- retinoid (schedule III, 'no retinoid pretreatment'). The inefficacy of schedule III indicates that retinoid-conditioning is essential for the improvement of the antiproliferative effect. The interactions in schedules I and II are synergistic for ATRA and CDDP, but slightly antagonistic for 4-HPR and CDDR However, 4-HPR + CDDP is more effective in growth inhibition than each drug alone.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cisplatin; DNA Fragmentation; DNA, Neoplasm; Down-Regulation; Female; Fenretinide; Humans; Neoplasm Proteins; Receptor, ErbB-2; Tretinoin; Tumor Cells, Cultured

We report on the isolation of a cytochrome P450 (CYP)-like retinoic acid (RA) 4-hydroxylase cDNA from T-47D human breast cancer cells that is identical to the recently cloned hCYP26, which is involved in the metabolic breakdown of RA. Northern analysis showed that this novel human CYP26 is induced within 1 h upon RA treatment in RA-sensitive T-47D breast carcinoma cells but not in RA-resistant MDA-MB-231 breast cancer cells and HCT 116 colon cancer cells. Stable introduction of different RA receptor (RAR) subtypes in HCT 116 cells showed that CYP26 expression is dependent on RARalpha and RARgamma and, to a lesser extent, on RARbeta and closely paralleled RA metabolism, suggesting that it represents the major RA 4-hydroxylase in these human cells. Furthermore, stable introduction of all three RAR subtypes in HCT 116 cells resulted in restored RA sensitivity as assayed by growth inhibition. Interestingly, CYP26 activity was efficiently inhibited by liarozole, an inhibitor of RA metabolism, leading to enhanced growth inhibition by RA. The RA-induced CYP26 was shown to be highly specific for the hydroxylation of all-trans-RA and did not recognize the 13-cis and 9-cis isomers. This substrate specificity is promising for finding retinoids that are not recognized by this enzyme and, therefore, could be more effective in growth inhibition of susceptible cancer cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Chromatography; Colonic Neoplasms; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; DNA; Enzyme Induction; Female; Humans; Hydroxylation; Imidazoles; Microsomes; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Substrate Specificity; Transfection; Tretinoin; Tumor Cells, Cultured

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

Topics: Amino Acid Sequence; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Division; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Interferons; Molecular Sequence Data; Neoplasm Proteins; Oligonucleotides, Antisense; Sequence Analysis; Thioredoxin-Disulfide Reductase; Tretinoin; Tumor Cells, Cultured

Nuclear retinoid and membrane c-erbB receptors participate in signal transduction systems that control mammary epithelial cell proliferation and differentiation. Recently, we demonstrated that c-erbB receptor activation stimulates retinoic acid receptor-alpha expression. We now report that retinoids reduce SK-BR-3 breast cancer cell growth by inhibiting the cell cycle and by inducing apoptosis. This is accompanied with reduced c-erbB expression as determined by FACS, Western, Northern, RT-PCR, and reporter assays. All-trans (ATRA) and 9-cis retinoic acid (9cRA) reduce c-erbB-1 protein to 50-100%, c-erbB-2 to 20-30%, and c-erbB-3 to 10-50% of control, depending on the concentration, respectively, without influencing the tyrosine phosphorylation status. Down-regulation of c-erbB-2 and -3 was seen at all levels analyzed, whereas c-erbB-1 mRNA remained unchanged. Retinoic acid-mediated down-regulation of growth and c-erbB-2 and -3 expression was also seen in MCF-7 cells. We conclude that retinoic acids are efficient repressors of c-erbB-2 and -3 gene expression, whereas c-erbB-1 is not markedly affected.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, erbB; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Receptors, Growth Factor; Retinoids; Tretinoin

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.

Topics: Antineoplastic Agents; Breast Neoplasms; DNA, Neoplasm; Down-Regulation; Drug Synergism; Female; Humans; Interferon-alpha; Proto-Oncogene Proteins c-myc; Transfection; Tretinoin; Tumor Cells, Cultured

Four breast carcinoma cell lines (T47D, ZR-75-1, MDA-MB-231, and MCF-7) were tested for regulation of the expression of vasoactive intestinal polypeptide receptor type-1 (VIP-R1). In all four cell lines, retinoic acid (RA) treatment caused a fast and marked decrease in VIP-R1 mRNA level as examined by Northern blots. Cycloheximide pretreatment attenuated the effect from 3- to 2-fold, indicating that existing proteins can mediate the decreasing effect of RA, but to attain the maximal effect new protein synthesis might be needed. Transcriptional inhibition with Actinomyocin D showed that RA did not influence the VIP-R1 mRNA half-life, indicating that the decreasing effect of RA on the mRNA level is due to transcriptional inhibition. In agreement with the observations on mRNA level, we found that the VIP receptor number was reduced 3-fold from 88 to 32 fmol/10(6) cells in T47D cells and from 222 to 73 fmol/10(6) cells in MDA-MB-231 cells upon RA treatment for 72 h. The promoter and 5'-flanking region of the VIP-R1 gene were cloned from a human placental cosmid library, and 2.5 kb were sequenced to search for regulatory elements. Our results, therefore, imply that the regulation of VIP-R1 gene expression by RA could have a role in human mammary tumor biology.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Cloning, Molecular; Down-Regulation; Female; Gene Expression Regulation; Humans; Molecular Sequence Data; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Female; Humans; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Heparan sulfate proteoglycans (HSPG) are involved in the regulation of cellular proliferation, differentiation, and migration. We have studied the effect of three inhibitors of proliferation on 35S incorporation into HSPG of the breast cancer cell lines MCF-7 and MDA-MB-231 and the normal breast epithelial cells (NBEC). Transforming growth factor beta-1 (TGFbeta-1), which inhibits the proliferation of NBEC, but not of MCF-7 and MDA-MB-231, cells induced an increase in 35S incorporation of HSPG in NBEC, but had no effect on cancer cells. Sodium butyrate (NaB), which inhibits NBEC as well as cancer cell proliferation, induced an increase in 35S incorporation into HSPG in all cell types studied. In contrast, retinoic acid had no effect on HSPG of breast epithelial cells. Modification of HSPG induced by TGFbeta-1 or NaB treatments in normal and breast cancer epithelial cells resulted in an increase in 125I-fibroblast growth factor-2 (FGF-2) binding on HSPG. More importantly, NaB pretreatment resulted in an inhibition of the MCF-7 cell responsiveness to FGF-2, even though these cells remained sensitive to growth stimulation induced by serum or epidermal growth factor. These results indicate that changes in HSPG production are a key process involved in the mechanism of breast epithelial cell growth regulation.

Topics: Binding Sites; Breast Neoplasms; Butyrates; Cell Division; Cell Membrane; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Female; Fibroblast Growth Factor 2; Growth Inhibitors; Heparan Sulfate Proteoglycans; Humans; Mitogens; Sodium; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 microM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-sensitive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcinoma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TT

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Apoptosis; Benzoates; Bexarotene; Breast Neoplasms; Cell Division; Disease Models, Animal; Female; Fenretinide; Hormone Antagonists; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred C57BL; Mifepristone; Retinoids; Tamoxifen; Tetrahydronaphthalenes; Tretinoin; Tumor Cells, Cultured

Breast cancer cell growth inhibition was not synergistically enhanced by trans-retinoic acid (RA) or 9-cis-RA plus 1alpha,25-(dihydroxy)vitamin D3 (DHVD). The retinoid/DHVD combinations did lower their 50% effective concentrations for inhibiting retinoid-sensitive MCF-7, but not retinoid-refractory BT-20, breast cancer cell growth. In contrast, the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its analog SR11389 inhibited the growth of both cell lines. Unlike RA, 9-cis-RA and DHVD, AHPN and SR11389 also potently inhibited human umbilical vascular endothelial cell growth. These results on AHPN and SR11389 suggest that angiogenesis of tumor microvasculature should also be an effective therapeutic target for this new compound class.

Topics: Alitretinoin; Apoptosis; Breast Neoplasms; Calcitriol; Cell Division; Endothelium, Vascular; Female; Humans; Tretinoin; Tumor Cells, Cultured

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.

Topics: 17-Hydroxysteroid Dehydrogenases; Breast Neoplasms; Choriocarcinoma; Cyclic AMP; Drug Synergism; Epidermal Growth Factor; Female; Humans; Isoenzymes; Pregnancy; RNA, Messenger; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

Interest has been increasingly focused on all-trans-retinoic acid (tRA) and 13-cis-retinoic acid (13cRA) in cancer chemoprevention and treatment. We have examined the in vitro effects of these 2 retinoic acids (RAs) on human breast-cancer cell lines MCF-7 and ZR-75.1 (both estrogen-receptor-positive, ER+) and MDA-MB-231 (estrogen-receptor-negative, ER-), in terms of inhibition of proliferation and induction of apoptosis. Both retinoic acids exerted an evident dose-dependent growth inhibition, although in the ER- cell line the anti-proliferative effect was obtained only with the highest concentration used; the anti-proliferative activity of tRA was more evident than 13cRA on all 3 tested cell lines. tRA and 13cRA induced apoptosis in MCF-7 and MDA-MB-231 cell lines, but not in ZR-75.1. The apoptotic phenomenon was clearly time-dependent, and in our experience it was not related to the arrest in a specific phase of cell cycle. After treatment with RAs the levels of bcl-2 were reduced in MCF-7, while in ZR-75.1 and in MDA-MB-231 no treatment-related modifications were observed. An analysis of estrogen-receptor status, used as a marker of differentiation, demonstrated that after treatment with RAs the levels of estrogen receptor (ER) decreased in ZR-75.1 only. Our study indicates that the anti-proliferative effects of RAs are sustained by induction of apoptosis in MCF-7 and MDA-MB-231 cells, while in ZR-75.1 cells an induction of differentiation without apoptosis was the prevalent mechanism of growth inhibition. Our results encourage further studies on in vivo effects of these retinoids in breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Division; DNA Fragmentation; DNA, Neoplasm; Flow Cytometry; Humans; Isotretinoin; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Tretinoin; Tumor Cells, Cultured

NOR-1, NGFI-B, and Nurr1 are closely related orphan nuclear receptors implicated in diverse biological processes including cell growth and differentiation. We examined the effect of retinoic acids on the expression of these putative transcription factor genes in the breast cancer cell line MCF-7 by a quantitative reverse transcription and polymerase chain reaction. Both all-trans and 9-cis retinoic acids markedly induced NOR-1 mRNA and slightly increased Nurr1 mRNA. In contrast, NGFI-B mRNA was decreased. In the presence of cycloheximide, all-trans retinoic acid superinduced NOR-1 mRNA, whereas all-trans and 9-cis retinoic acids strongly suppressed the NGFI-B mRNA accumulation. The differential effects of retinoic acids on the expression of these genes are in contrast with the effects of forskolin and 12-O-tetradecanoylphorbol-13-acetate, both of which induced mRNAs of all three genes. These findings suggest that NOR-1, NGFI-B, and Nurr1 play distinct roles in the retinoic acid signaling in MCF-7 cells.

Topics: Alitretinoin; Breast Neoplasms; Colforsin; Cycloheximide; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Nerve Tissue Proteins; Nuclear Receptor Subfamily 4, Group A, Member 1; Nuclear Receptor Subfamily 4, Group A, Member 2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Thyroid Hormone; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytosol; Female; Humans; Imidazoles; Keratolytic Agents; Oxidation-Reduction; Reproducibility of Results; Retinoids; Retinol-Binding Proteins; Tretinoin; Tumor Cells, Cultured

The role of the cellular retinoic acid binding protein type II (CRABPII) in the retinoic acid (RA) signaling pathway is poorly understood. Northern blot analysis of 12 breast cell lines showed that CRABPII mRNA content correlated with growth inhibition by RA, suggesting that this binding protein enhances cellular response to RA. Ectopic CRABPII expression supported dose-dependent growth inhibition by RA in SC115-resistant but not MDA-MB-231-resistant cells, indicating that CRABPII is sufficient to rescue RA antiproliferation in a permissive background. In both cell lines, ectopic binding protein enhanced gene activation by RA. Thus, induction of tissue transglutaminase by all-trans-RA and, surprisingly, 9-cis-RA was enhanced 5-fold over and above the level of induction in control cells (SC115), and activation of a RA response element reporter was enhanced 3-fold (MDA-MB-231). A 5-fold enhancement of RA induction of RA receptor beta expression as a result of ectopic binding protein expression was also demonstrated (SC115). These findings indicate that CRABPII is a positive regulator of RA signaling in breast cells.

Topics: Breast Neoplasms; Cell Division; Female; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Receptors, Retinoic Acid; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transcriptional Activation; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Retinoids modulate several cell functions and especially inhibit the growth of a wide variety of cells including breast cancer. Retinoic acid receptor-gamma (RAR-gamma) has been shown to mediate the antiproliferative activity of retinoids. To further test this hypothesis we examined the effects of different RAR-gamma selectively binding retinoids (CD2325, CD2247, CD666 and CD437) on breast cancer cell lines. With exception of CD2247, all retinoids inhibited proliferation of MCF-7, SKBR-3, T47D and ZR-75-1 breast cancer cell lines, similar to the natural compound all-trans retinoic acid (ATRA). In addition, all 4 compounds were able to act synergistically with interferon-gamma (IFN-gamma) in all breast cancer cell lines including the retinoid-resistant BT-20 and 734-B lines. In functional transactivation assays we demonstrated that only in the MCF-7 cell line, TPA-mediated AP-1 activity was suppressed only by ATRA and CD2325, whereas in SKBR-3, another RA-sensitive breast cancer cell line, it was not. The synergistic antiproliferative activity involving retinoids and IFN-gamma could not be explained by an enhanced anti-AP-1 activity. No correlation was found between expression of RARs and cellular retinoic acid binding proteins (CRABPs) and antiproliferative effects of the retinoids. RAR-gamma selectively binding retinoids are potent inhibitors of breast cancer cell proliferation, alone and in combination with IFN-gamma. For this reason and because of a possible low toxicity, as compared with retinoic acid, we speculate that these RAR-gamma selective binding retinoids might be of clinical importance.

Topics: Binding, Competitive; Breast Neoplasms; Cell Division; Cell Line; Chloramphenicol O-Acetyltransferase; Female; Humans; Interferon-gamma; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor gamma; Retinoids; RNA, Messenger; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

We have recently found a novel retinoid, 6-[3-(1-adamantyl)-4-hydroxphenyl]-2-naphthalene carboxylic acid (CD437), which induces G1 cell cycle arrest and apoptosis in human breast carcinoma (HBC) cells (Oncogene 11, 493-504, 1995). CD437 downregulates the expression of a number of proteins which antagonize apoptosis. bcl-X(L), a homologue of bcl-2, antagonizes apoptosis, while bcl-X(S) enhances apoptosis. We have found that estrogen receptor (ER)-negative HBCs express higher levels of bcl-X(L) and significantly lower levels of bcl-2 than their ER-positive counterparts. Neither cell type expresses bcl-X(S). The addition of CD437 (1 microM) results in a fourfold downregulation of bcl-X(L) mRNA and protein levels followed by apoptosis in MDA-MB-231 and MDA-MB-468 cells. CD437 concentrations as low as 10 nM cause a significant reduction in both bcl-X mRNA and bcl-X(L) protein expression. CD437-dependent downregulation of bcl-X mRNA and bcl-X(L) protein expression occurs within 24 h of CD437 addition to the cells. Retinoic acid does not effect bcl-X mRNA or bcl-X(L) protein expression. CD437 is a potent inducer of apoptosis in a number of breast carcinoma cells lines and downregulates the expression of a number of proteins which antagonize apoptosis.

Topics: bcl-X Protein; Breast Neoplasms; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoids; Tretinoin; Tumor Cells, Cultured

Treatment of estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced formation of a nuclear aryl hydrocarbon (Ah) receptor complex as determined by ligand-binding and gel electrophoretic mobility shift assays. TCDD also induced CYP1A1-dependent activity in MDA-MB-468 cells, which represents the first ER-negative Ah receptor-positive human breast cancer cell line that has been identified. Treatment of this cell line with TCDD and related compounds also caused a 50% inhibition of cell growth, which resembled the growth inhibitory effects previously reported for epidermal growth factor (EGF). However, EGF expression is minimal in this cell line and is not induced by TCDD; moreover, EGF and TCDD induced a different pattern of oncogene expression and apoptosis in MDA-MB-468 cells. In contrast, TCDD caused a rapid and sustained induction of transforming growth factor alpha (TGF alpha) gene expression and secreted protein (nearly 2-fold); moreover, the growth-inhibitory effects of TCDD could be blocked by antibodies to the EGF receptor. In a separate experiment, it was shown that TGF alpha also inhibited growth of MDA-MB-468 cells. The results of this study indicate that the mechanism of growth inhibition of MDA-MB-468 cells by TCDD is due to induction of TGF alpha, which is a potent antimitogen in this cell breast cancer line.

Topics: Aryl Hydrocarbon Receptor Nuclear Translocator; Breast Neoplasms; Cell Division; Cell Nucleus; Cytochrome P-450 CYP1A1; Diethylstilbestrol; DNA-Binding Proteins; DNA, Neoplasm; Estradiol; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Growth Inhibitors; Humans; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

The effect of doxorubicin (DOX) used in combination with a low dose (10(-7) M) of all-trans-retinoic acid (tRA) was tested on MCF-7 breast carcinoma cell line. Both drugs are able to inhibit cell proliferation in these cells in a dose-dependent way. The combined treatment with DOX and tRA was more effective in inhibiting cell growth than each of the two compounds alone. This was evidenced in the following experimental conditions: pre-treatments with tRA, for 72 h or 18 h, before DOX incubation; post-treatment with tRA for 18 h after DOX incubation. A consistent synergism was reached by 72 h pre-treatment with tRA and also with brief pre- and post-treatments, but only if tRA was also present during DOX incubation (co-treatments). The mechanisms involved in this interaction between chemotherapeutics and differentiating agents are as yet unclear and should be evaluated further.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Female; Humans; Receptors, Estrogen; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoids mediate the normal growth of a variety of epithelial cells and may play an important role in the chemoprevention of certain malignancies. Loss of retinoic acid (RA) receptor-beta function may be an important event in mammary carcinogenesis, because the majority of breast cancers, in contrast to normal mammary epithelial cells, fail to express this receptor. We previously reported that all-trans-RA mediates G1 arrest as well as apoptosis in certain RAR beta-transduced breast cancer cell lines. We now report the effect of RA on normal human mammary epithelial cells (HMECs), which express functionally active retinoid receptors. We observe that RA induces growth suppression and G1 arrest of these HMECs but find no evidence that RA mediates apoptosis in these normal cell strains. This RA-induced G1 arrest is temporally associated with decreased levels of hyperphosphorylated retinoblastoma protein without any significant changes in c-myc, p53, p21, or p27 expression. Expression of cyclin D1, cyclin-dependent kinase 4, and cyclin E proteins, however, decreased in association with RA-mediated G1 arrest. Our studies suggest that growth inhibition, rather than apoptosis, may be a mechanism by which RA and RA receptors act to prevent the malignant transformation of normal mammary epithelial cells. The molecular target(s) of the activated RA receptors that mediate this G1 arrest in HMECs appear to be associated with a retinoblastoma-dependent pathway.

Topics: Apoptosis; Blotting, Northern; Breast; Breast Neoplasms; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA; Epithelial Cells; Epithelium; G1 Phase; Gene Expression Regulation; Growth Inhibitors; Humans; Oncogene Proteins; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoblastoma Protein; RNA, Messenger; Transcriptional Activation; Transfection; Tretinoin

Retinoic acid receptor (RAR) alpha has been shown to play a role in retinoid-induced growth inhibition of human breast cancer cell lines that express the estrogen receptor (ER). The dogma in the field has been that ER-positive breast cancer cell lines respond to retinoid treatment because they express RAR alpha, whereas ER-negative breast cancer cell lines are refractory to retinoid treatment and have been thought to express little or no RAR alpha. We set out to test several ER-negative breast cancer cell lines for expression of RAR alpha protein and responsiveness to retinoids in growth inhibition assays. Of six ER-negative breast cancer cell lines that were tested, one (SK-BR-3) had high levels of RAR alpha protein as measured by ligand-binding immunoprecipitation (approximately 55 fmol/mg protein) and also displayed sensitivity to growth inhibition by retinoids (9-cis-retinoic acid; EC50, approximately 3 nM). These cells were more sensitive than an ER-positive cell line, T-47D, which expressed approximately 35 fmol RAR alpha/mg total protein (9-cis retinoic acid; EC50, approximately 50-100 nM). Another ER-negative cell line, Hs578T, also expressed RAR alpha (approximately 23 fmol/mg) and was sensitive to retinoid-induced growth inhibition, albeit to a lesser extent than SK-BR-3 or T-47D cells. In contrast, the other ER-negative cell lines tested expressed low (<10 fmol/mg) or no detectable levels of RAR alpha protein and also did not respond to retinoids in growth inhibition assays. A RAR alpha agonist displayed 100 times greater potency than a RARgamma agonist in growth inhibition of both T-47D and SK-BR-3 cells, suggesting RAR alpha involvement in the process. Furthermore, a RAR alpha antagonist completely abolished the growth inhibition induced by RAR agonists, implying that the activity of the agonists is exerted solely through RAR alpha, not RARgamma, which is also expressed in both cell lines. Additionally, although retinoid X receptor (RXR) compounds are weakly active in growth inhibition of the RAR alpha-positive cell lines, they markedly increased the growth-inhibitory activity of RAR ligands. RXR compounds also potentiated the action of the antiestrogen 4-hydroxytamoxifen to inhibit the growth of T-47D cells. These findings have clinical ramifications in that patients with ER-negative tumors that are RAR alpha positive may be candidates for retinoid therapy. Additionally, combinations of RXR ligands with RAR ligands (especially RAR alpha agon

Topics: Alitretinoin; Aminobenzoates; Antineoplastic Agents; Benzoates; Bexarotene; Breast Neoplasms; Cell Division; Chromans; Female; Humans; Nicotinic Acids; Nuclear Proteins; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-alpha expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid.

Topics: Antineoplastic Agents; Biotransformation; Breast Neoplasms; Cell Division; Cell Polarity; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Female; Humans; Tretinoin; Tumor Cells, Cultured

Transfection and transgenic mouse experiments supported an oncogenic role for cyclin D1 in breast cancer. We recently reported that noninvasive carcinoma in situ lesions of the human breast overexpress cyclin D, suggesting that this molecular event may represent a valuable target for chemoprevention. The purpose of the present series of investigations was to identify agents which could reduce the cyclin D expression of breast cells. We report that 9-cis retinoic acid (9-cis RA) and all trans retinoic acid (tRA) inhibited the cyclin D1 and D3 expression levels of human MCF-7, ZR-75 and T-47D breast carcinoma cells in vitro. Where detectable, similar trends were observed in the immortalized, HBL-100 and MCF-10A breast cell lines. Cyclin D2 was undetectable. The effect of retinoids was both dose- and time-dependent, and correlated with altered cell cycle kinetics and proliferative status. Retinoids were also found to inhibit the expression levels of other cell cycle related proteins, including Cdk2 and Cdk4, resulting in lower kinase activities. In contrast to other breast prevention studies, no synergistic effect was observed with retinoids and tamoxifen. The data indicate that retinoids can potently reduce cyclin D expression levels in a variety of breast cell lines in vitro, and suggest further consideration of this mechanism for the chemoprevention of breast cancer.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D; Cyclins; Dose-Response Relationship, Drug; Humans; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) inhibition of breast cancer cell growth is associated with an accumulation of cells in G1 phase of the cell cycle. We have investigated the effects of RA on the expression and activity of cell cycle-regulatory proteins in MCF-7 human breast cancer cells. Flow cytometry analysis of MCF-7 cells treated with RA revealed a decrease in the percentage of cells in S phase by 48 h, which was maximal by 72 h. Phosphorylation of the retinoblastoma protein (pRb) was partially reduced in RA-treated cells accompanied by a decrease in the level of retinoblastoma protein. Expression of the cyclin D1 transcript was reduced by 48 h and cyclin-dependent kinase 2 (cdk2) mRNA levels declined within 8 h posttreatment followed by a decrease in cyclin D1 and cdk2 protein levels. Message and protein levels of cdk4 and cdc2 were not affected by RA. While cdk4 activity was similar in control and RA-treated cells, cdk2 activity began to decrease within 48 h of exposure to RA and was profoundly reduced after 72 h. This reduced activity was associated with decreased phosphorylation of cdk2. The decrease in cdk2 activity occurred in the absence of RA-mediated increases in the levels of the cdk inhibitors p21 and p27. However, assays of cdk2 from pooled lysates from RA-treated and control cells showed that RA-treated cells contain a cdk2-inhibitory activity. Our results show that RA inhibits cell cycle progression of MCF-7 cells by inhibiting cdk2 mRNA and protein production and by decreasing cdk2 activity.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Oncogene Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cell cycle analysis indicates that retinoic acid (RA) inhibition of MCF-7 cell growth occurs through induction of G1 arrest with a concomitant reduction in the proportion of cells in S and G2 + M phases. RA did not affect cyclins D1, A, and E and cyclin-dependent kinase 2 (CDK2) expression, but significantly reduced cyclin D3 and CDK4 expression after 24 h. RA also inhibited cyclin B1 and CDC2 expression, possibly responsible for the reduction of the proportion of cells in G2 + M and S phases. RA did not induce p16 and p27 expression, but obviously reduced p21 level in MCF-7 cells. The retinoid markedly reduced pRB protein level and abrogated pRB phosphorylation after 48 h; it also reduced transcription factor E2F1 expression at both the mRNA and protein levels. E2F1 promoter activity was reduced by 60%, which is probably responsible, at least in part, for the reduction of E2F1 expression in RA-treated MCF-7 cells. These observations demonstrate a marked effect of RA on some of the key cell cycle regulatory proteins in MCF-7 cells. Cyclin D3 and CDK4 are likely the early targets of RA, followed by reduced pRB expression and phosphorylation, as well as by the inhibition of the E2F1 transcription factor which controls progression from G1 to S phase. Most of these events precede the observed reduction in MCF-7 cell growth, which begins at Day 3 of RA treatment.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Phosphorylation; Promoter Regions, Genetic; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; Transcription Factor DP1; Transcription Factors; Tretinoin

Retinoic acid inhibits proliferation of hormone-dependent, but not hormone-independent breast cancer cells. Retinoic acid-induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKC delta, epsilon, and zeta are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKC alpha expression and concomitantly repressed PKC zeta expression. The changes in PKC alpha and PKC zeta reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKC alpha, but not the reduction in PKC zeta, resulted from selective activation of RAR alpha. In total, these results support an important role for PKC alpha in mediating the anti-proliferative action of retinoids on human breast carcinoma cells.

Topics: Benzoates; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Neoplasms, Hormone-Dependent; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; RNA, Messenger; Tetrahydronaphthalenes; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Collagen; Drug Combinations; Extracellular Matrix; Female; Fibroblasts; Humans; Laminin; Microscopy, Confocal; Microscopy, Electron, Scanning; Neoplasm Invasiveness; Proteoglycans; Sarcoma, Experimental; Tretinoin; Tumor Cells, Cultured

Genomic actions of the hormone 1alpha,25-dihydroxy-vitamin D3(VD) are mediated by the transcription factor VDR, which is a member of the nuclear receptor superfamily. VDR acts in most cases as a heterodimeric complex with the retinoid X receptor (RXR) from specific DNA sequences in the promoter of VD target genes called VD response elements (VDREs). This study describes a mutation (K45A) of the VDR DNA binding domain that enhances the affinity and ligand responsiveness of VDR-RXR heterodimers on some VDREs. In analogy to a homologous mutation in the glucocorticoid receptor (K461A), this lysine residue appears to function as an allosteric 'lock'. Interestingly, overexpression of RXR was found to reduce the responsiveness and sensitivity of wild type VDR to VD, but enhance the response of VDRK45A. Moreover, the transactivation domains of both VDR and RXR were shown to be essential for obtaining responsiveness of the heterodimers to VD and 9- cis retinoic acid (the RXR ligand). This indicates that RXR is an active rather than silent partner of the VDR on the VDREs tested. Taken together, transactivation by VDR-RXR heterodimers can be triggered individually by all components of the protein-DNA complex, but full potency appears to be reached through allosteric interaction.

Topics: Alitretinoin; Allosteric Regulation; Animals; Atrial Natriuretic Factor; Breast Neoplasms; Calcitriol; COS Cells; Dimerization; DNA; Humans; Ligands; Point Mutation; Promoter Regions, Genetic; Rats; Receptors, Calcitriol; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoid X Receptors; Sequence Deletion; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Binding, Competitive; Breast Neoplasms; Cell Division; DNA-Binding Proteins; Drug Resistance; Evaluation Studies as Topic; Female; Gene Expression Regulation, Neoplastic; Humans; Isomerism; Nuclear Receptor Subfamily 4, Group A, Member 1; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Receptors, Steroid; Retinoid X Receptors; Retinoids; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

To investigate the role of AP-1 transcription factors in mediating retinoid-induced growth suppression of breast cells, we studied the sensitivity of MCF7 breast cancer cells with different levels of AP-1 activity to all-trans retinoic acid (atRA). AP-1 activity was increased in MCF7 cells by stably transfecting c-jun cDNA into these cells. Parental and vector-transfected MCF7 cells, which were sensitive to the growth-inhibitory effects of atRA, exhibited atRA-dependent retinoic acid receptor (RAR) transactivation and transrepression of 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity. The c-jun-transfected MCF7 cells had increased basal AP-1 transactivation activity and increased expression of AP-1-regulated genes but were resistant to the antiproliferative effects of atRA. However, MCF7 cells transfected with a deletion mutant of c-jun, TAM-67, which lacks most of the amino-terminal transactivation domain of cJun and is unable to activate AP-1-dependent gene expression, were sensitive to the growth-inhibitory effects of atRA. These results suggest that the transactivation domain of cJun is required for induction of retinoid resistance in these breast cancer cells. atRA did not activate RAR-dependent gene transcription or transrepress 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity in these cJun-overexpressing cells. Investigation of the RAR and retinoic acid X receptor expression level demonstrated that RAR alpha and RAR gamma RNA expression was reduced in the c-jun-transfected MCF7 cells, whereas RAR beta expression was up-regulated. However, retinoic acid responsive element DNA binding activity was intact in c-jun-transfected cells. Therefore, the mechanism by which cJun overexpression induces resistance to the growth-inhibitory effect of atRA may be through interference with atRA-dependent RAR transactivation or AP-1 transrepression, possibly through titration of essential coactivators. These results suggest that the antiproliferative effects of retinoids can be overcome by cJun overexpression.

Topics: Breast Neoplasms; Cell Division; Drug Resistance, Neoplasm; Female; Genes, jun; Genes, Reporter; Humans; Kinetics; Polymerase Chain Reaction; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Tetradecanoylphorbol Acetate; Time Factors; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

The treatment of breast cancer by retinoic acid (RA) may be mediated by lipid peroxidation. Expression of metallothionein (MT) in cancer cells, however, can protect against lipid peroxidation by scavenging hydroxyl radicals. In this study, a two-by-six factorial design was used to investigate the interactive effects of all-trans-RA and zinc (Zn)-induced MT on the growth of two human breast cancer cell lines differing in basal expression of MT and estrogen receptors; MCF7 cells express estrogen receptor, BT-20 cells do not. Cells were treated with Zn to induce MT and then treated with six RA concentrations. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly increased by Zn treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. Elevated RA concentrations increased lipid peroxidation as measured by thiobarbituric acid reactive substances. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by Zn pretreatment in BT-20 cells but not in MCF7 cells. RA increased MT levels in both cell lines, which suggests that RA may generate free radicals which will induce MT mRNA expression. Glutathione did not appear to be a significant factor. Therefore, induction of MT by Zn may modulate the growth inhibitory effects of RA in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation. Induction of MT by RA may be one explanation for acquired RA resistance in cancer.

Topics: Breast Neoplasms; Cell Division; Female; Glutathione; Humans; Lipid Peroxidation; Metallothionein; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Zinc

All-trans-Retinoic acid (RA) induces differentiation and inhibits growth of many tumor types. Whereas the RA nuclear receptors mediate genomic effects of RA, there also are many nongenomic effects that do not have defined mechanisms. Some nongenomic effects of RA may involve retinoylation (RA acylation), a posttranslational modification of proteins occurring in many eukaryotic cell lines including the human breast cancer cell line MCF-7. To gain further knowledge of the role(s) of retinoylation, we studied the effects of tunicamycin (TM), an inhibitor of both protein N-glycosylation and palmitoylation, on growth and retinoylation in MCF-7 cells. We found that RA or TM alone inhibited growth of MCF-7 cells. Combinations of RA and TM inhibited growth synergistically. TM increased retinoylation and decreased palmitoylation. These results suggest that increased retinoylation and decreased glycosylation and palmitoylation may play a role in the synergistic inhibition of cell growth by combinations of TM and RA in MCF-7 cells. Furthermore, our results suggest that combinations of TM and RA may have clinical utility.

Topics: Acylation; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Division; Drug Synergism; Humans; Neoplasm Proteins; Palmitic Acid; Tretinoin; Tumor Cells, Cultured; Tunicamycin

Solid tumors are relatively resistant to growth inhibition by IFNs. To enhance sensitivity, we assessed combinations of IFNs with all-trans-retinoic acid (RA). Antiproliferative studies in vitro suggested that the growth of three human breast carcinomas (MCF-7, MDA-MB-231, and MDA-MB-468), an ovarian carcinoma (NIH-OVCAR-3), and a malignant melanoma (SK-MEL-1) was inhibited to a greater degree by combination treatment with human IFN-beta and RA compared to single agents. Some of these cell lines were resistant to 10-100 IU/ml human IFN-alpha2b or IFN-beta or to 0.1-1.0 microM RA. Growth was inhibited significantly by combinations of IFNs and RA in all cell lines tested, and in some cases, cytotoxicity was observed. Sequential treatment of MCF-7 cells with RA followed by IFN-beta was more effective at inhibiting growth than treatment with IFN-beta followed by RA, suggesting that RA modulated the anticellular response of IFN-beta rather than the converse. In nude mice, the growth of MCF-7 and NIH-OVCAR-3 tumors was suppressed completely when combination treatment was started 2 days after tumor inoculation. Established, 6-week-old NIH-OVCAR-3 tumors underwent regression when treated with the combination of IFN-beta and RA but not with single-agent therapy. Together with our recent studies that demonstrated enhancement of IFN-stimulated gene expression by RA pretreatment in IFN-resistant cells, these data suggest that combination treatment with RA and IFNs may increase IFN-stimulated gene expression in IFN-resistant tumors, leading to augmented antitumor effects.

Topics: Animals; Breast Neoplasms; Cell Division; Combined Modality Therapy; Drug Synergism; Estradiol; Female; Humans; Interferon beta-1a; Interferon beta-1b; Interferon-beta; Melanoma; Mice; Mice, Nude; Ovarian Neoplasms; Receptors, Estrogen; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; DNA; Female; Flow Cytometry; Humans; Immunohistochemistry; Ki-67 Antigen; Tretinoin

To evaluate whether the growth inhibition by retinoic acid and RAR alpha mRNA expression levels were affected by the change of ER expression.. The ER-negative breast cancer cell line MDA-MB-231 was transfected with the ER gene by stable transfection.. In ER-transfected cells not only was the RAR alpha mRNA expression increased, but their growth was inhibited by retinoic acid as well. Estrogen could greatly stimulate the RAR alpha gene expression not only in established ER-positive cell lines but also in ER-transfected MDA-MB-231 cells.. Our data strongly suggest that ER-mediated enhancement of RAR alpha levels play an important role in RA inhibition of human breast cancer cell growth.

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Gene Expression Regulation; Humans; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Transcriptional activation is thought to be mediated by DNA-bound activators through interaction with a basal transcription factor thereby stabilizing the pre-initiation complex. For such interaction cofactors such as TAFs, bridging proteins, mediators or intermediary proteins are required by binding simultaneously to the activator and the target. We have investigated the activation functions (AFs) of both RARbeta and RXRalpha and show that both activators contain two homologous AFs. By comparing the capacity to activate transcription by these AFs on several promoters, both as full-length receptors and as fusion-proteins of AFs with the DNA-binding domain of the yeast transcription factor GAL-4, we were able to show that these AFs function by different mechanisms. We found that the activity of these AFs is cell-type specific, as they are more active in certain cell lines than in others. Furthermore we observed that the AFs of RARbeta and RXRalpha can activate transcription synergistically both as GAL-fusion protein and with full-length receptors. For AF-2 of RAR beta we observed cell type-dependent difference in synergistic activation and we show that the E1A protein, which functions as a cofactor for RAR beta, permits synergistic activation in cell lines in which in the absence of this cofactor transcription occurs non-synergistically. We propose a model in which several non cell type specific cofactors and cell-specific cofactors act together to form a more stable pre-initiation complex explaining the observed cell-specific synergistic activation.

Topics: 3T3 Cells; Animals; Base Sequence; Breast Neoplasms; Cell Line, Transformed; Chlorocebus aethiops; DNA-Binding Proteins; Female; Gene Expression Regulation; Genes, Reporter; Humans; Kidney; Macromolecular Substances; Mice; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoid X Receptors; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

Retinoic acid (RA) is a potent in vitro inhibitor of cell proliferation in various malignant cell lines. The exact mechanisms of its actions, however, are not fully understood. To further elucidate the nature of this inhibition, we investigated the effects of RA in an estrogen receptor-negative human breast cancer cell line, MDA-MB-231. RA (0.01-5 microM) significantly inhibited MDA-MB-231 cell growth by 35-40% as compared with untreated controls. Similar growth inhibitory actions were observed when cells were treated with transforming growth factor beta2 (TGF-beta2), another factor with antiproliferative actions in breast cancer cells. Both RA and TGF-beta2 increased the levels of insulin-like growth factor binding protein (IGFBP) 3 (2-3-fold) and mRNA (1.5-2-fold), whereas IGFBP-4 levels remained essentially unchanged. The direct involvement of IGFBP-3 in cell growth inhibition was further confirmed by its action on cell growth: exogenous IGFBP-3 directly and significantly inhibited MDA-MB-231 cell number by 40%. These results provided circumstantial evidence that IGFBP-3 may mediate RA and TGF-beta2 growth inhibitory actions in human breast cancer cells. To test this hypothesis, we used an antisense IGFBP-3 oligodeoxynucleotide (ODN) which specifically inhibits IGFBP-3 expression. The antisense IGBP-3 ODN dramatically blocked both RA- and TGF-beta2-induced increases in IGFBP-3 protein (90%) and mRNA levels (90%). This effect was not observed when RA- or TGF-beta2-exposed cells were treated with sense IGFBP-3 ODN. Moreover, antisense ODN did not significantly affect IGFBP-4 protein or mRNA levels, strongly supporting the specificity of the antisense IGFBP-3 ODN effect on IGFBP-3 mRNA. This specific effect of antisense IGFBP-3 ODN on IGFBP-3 protein and mRNA levels was accompanied by significant attenuation of the inhibition of cell proliferation attained with RA or TGF-beta2 (approximately 40% of either RA- or TGF-beta2-induced inhibition). The control sense IGFBP-3 ODN did not reduce the growth inhibition observed with either RA or TGF-beta2. These results indicate that IGFBP-3 is an important mediator of RA- and TGF-beta2-induced cell growth inhibition in human breast cancer cells.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Protein 4; Molecular Sequence Data; Oligonucleotides, Antisense; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoids are known to inhibit the growth of hormone-dependent but not that of hormone-independent breast cancer cells. We investigated the involvement of retinoic acid (RA) receptors (RARs) in the differential growth-inhibitory effects of retinoids and the underlying mechanism. Our data demonstrate that induction of RAR beta by RA correlates with the growth-inhibitory effect of retinoids. The hormone-independent cells acquired RA sensitivity when the RAR beta expression vector was introduced and expressed in the cells. In addition, RA sensitivity of hormone-dependent cells was inhibited by a RAR beta-selective antagonist and the expression of RAR beta antisense RNA. Introduction of RAR alpha also restored RA sensitivity in hormone-independent cells, but this restoration was accomplished by the induction of endogenous RAR beta expression. Furthermore, we show that induction of apoptosis contributes to the growth-inhibitory effect of RAR beta. Thus, RAR beta can mediate retinoid action in breast cancer cells by promoting apoptosis. Loss of RAR beta, therefore, may contribute to the tumorigenicity of human mammary epithelial cells.

Topics: Apoptosis; Base Sequence; Breast Neoplasms; Female; Gene Transfer Techniques; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; Signal Transduction; Tretinoin

Interferons (IFN) and retinoids failed to inhibit the growth of a number of breast tumor cell lines. However, a combination of these two biological response modifiers significantly suppressed the cell growth at pharmacologically achievable doses. The molecular basis for such enhancement was investigated in MCF-7, a breast tumor cell line resistant to growth inhibition by IFN-beta. Pretreatment of cells with retinoic acid (RA) for 16 h followed by IFN-beta, but not the converse, induced cytotoxic effects in the cells. Continuous presence of RA was not necessary, although it enhanced the degree of cell death when present. Further analyses revealed that IFN-beta failed to activate IFN-stimulated gene transcription. However, IFN-beta strongly up-regulated the gene expression in RA-pretreated cells. Both IFN-beta- and IFN-gamma-inducible gene expression were enhanced via a modulation of the transcriptional factor IFN-stimulated gene factors-3 and GAF binding to respective cognate regulatory elements. STAT1 was undetectable in these cells prior to RA treatment. RA increased the levels of this crucial regulator, thereby restoring IFN responses. Thus, RA augmentation of STAT1 may be an early step in the cooperative anti-tumor effects of IFN and RA.

Topics: Animals; Breast Neoplasms; Cell Death; Cell Division; DNA-Binding Proteins; Gene Expression Regulation; Humans; Interferons; Mice; STAT1 Transcription Factor; Trans-Activators; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration-dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect.

Topics: Base Sequence; Breast Neoplasms; Cathepsin D; DNA Primers; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Nuclear Proteins; Promoter Regions, Genetic; Receptors, Retinoic Acid; Restriction Mapping; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The activities of N-(4-hydroxyphenyl)retinamide [(4-HPR), Fenretinide] and all-trans-retinoic acid (RA) were determined for (a) the inhibition of cell proliferation; (b) the activation of human retinoid receptor-mediated target gene expression; (c) the inhibition of estradiol- and progesterone-induced gene activation in breast cancer cell lines; and (d) the regulation of the expression of tumor suppressor retinoblastoma protein. Similar to RA, both 4-HPR and its active metabolite N-(4-methoxyphenyl)retinamide (4-MPR) effectively impeded the growth of MCF7 and T-47D human breast cancer cell lines, except that 4-HPR also inhibited the proliferation of RA-resistant BT-20 cells. However, when tested in human recombinant retinoic acid receptor (RAR-alpha, RAR-beta, and RAR-gamma)-induced reporter gene assays, RA was much more potent (>100-fold) than either 4-HPR or 4-MPR. 4-HPR induced transcriptional activation through all three RAR subtypes at 1-10microM, while RA showed comparable activity at 10-100microM. Despite the apparent weak interaction at the RAR level, 4-HPR was comparable to RA in the inhibition of both estrogen receptor- and progesterone receptor-mediated transcriptional activation in MCF7 and T-47D cells, respectively. Moreover, similar to RA, 4-HPR and 4-MPR caused marked up-regulation of tumor suppressor retinoblastoma protein in both MCF7 and T-47D cells. Since RA and 4-HPR showed comparable activity in the inhibition of estrogen recptor- and progesterone receptor-induced gene transcription and in the stimulation of retinoblastoma protein expression in MCF7 and T-47D cells, the reduced RAR activation by 4-HPR may result in the lack of hepatic toxicity and therefore the improved therapeutic efficacy relative to RA.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Female; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Keratolytic Agents; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The mamary gland is chiefly composed of luminal epithelial cells expressing cytokeratins (K) 8, 18 and 19, and basal/myoepithelial cells expressing cytokeratins 5 and 14. Human breast cancer T47D cells have a luminal phenotype and are growth-inhibited by retinoids, a class of compounds known to regulate cytokeratin expression. To extend our knowledge of retinoid action in breast cancer, we have studied the retinoid regulation of cytokeratin expression in the T47D model. We found that retinoid inhibition of T47D cell growth was accompanied by increases in K8, K18 and K19 mRNA steady-state levels (Northern blot analysis). The effect on K8 was studied in greater detail. This effect was seen with as low as 1 nM all-trans retinoic acid (tRA) and was maximal (up to 7 fold over control) with 1 microM tRA (the highest dose tested). Time-course studies revealed a detectable effect at 1 h and a maximal effect at 8-24 h. Non-retinoidal growth inhibitors (tamoxifen, BrcAMP and genistein) did not modulate K8 expression, demonstrating that the effect of tRA was specific, K8 mRNA upregulation was blocked by actinomycin D and cycloheximide, suggesting, in accordance with other studies, that tRA exerted a transcriptional effect that was secondary to de novo protein synthesis. Five retinoids known to activate retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) - tRA; 9-cis-retinoic acid, 9cRA; 13-cis RA, 13cRA; retinyl acetate; and N-(4-hydroxyphenyl) retinamide 4HPR - inhibited T47D cell growth and increased K8 expression, whereas an arotinoid (Ro-40-8757) that is not a RAR activator caused growth inhibition but did not upregulate K8. Activation of RAR alpha contributed to K8 upregulation, since this effect was partially blocked by the RAR alpha-selective antagonist Ro-41-5253. Analogous results were obtained throughout when blots were reprobed with K18 cDNA. Western blot and immunocytochemistry experiments demonstrated that protein levels of K8 and K18 increased by 2 days of treatment with 1 microM tRA. These results show that retinoids enhance the expression of cognate cytokeratin markers of luminal differentiation in T47D breast cancer cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Dactinomycin; Female; Gene Expression Regulation; Humans; Keratins; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Up-Regulation

Interferons (IFNs) and retinoic acid (RA) are known to possess antiproliferative effects in various human cancer cell lines, including squamous cervix carcinoma and breast cancer cell lines. Frequently synergistic effects could be observed by the combination of both, and in clinical trials high response rates could be achieved by IFN-alpha in combination with 13-cis-RA in patients with squamous carcinoma of the uterine cervix. Since radiation-potentiating effects of IFNs and RA were described, we evaluated the additional impact of irradiation on three human breast cancer cell lines and investigated the antiproliferative effects of single, double, or triple treatments with IFN-gamma, 9-cis-RA, and irradiation. Antiproliferative effects were observed in all cell lines by any single treatment. When combining IFN-gamma with 9-cis-RA, synergism could be observed in all experiments. The combination of either IFN-gamma or 9-cis-RA with irradiation resulted mostly in additive effects. Irradiation contributed additive or further synergistic effects to the already synergistic effects of the combination of these two drugs. Thus synergism of IFN-gamma and 9-cis-RA always at least persisted fully. These results suggest that a regimen of IFN, RA, and radiotherapy (RT) might be a promising combination in the therapy of solid tumors, where RT is part of the conventional treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Chemotherapy, Adjuvant; Drug Synergism; Drug Therapy, Combination; Female; Humans; Interferon-gamma; Keratolytic Agents; Radiation Tolerance; Radiation-Sensitizing Agents; Radiotherapy Dosage; Radiotherapy, Adjuvant; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Retinoids have shown great promise as chemopreventive against the development of squamous cell carcinomas of the upper aerodigestive tract. However, the exact mechanism by which they block new tumors from arising is unknown. Here, we report that 13-cis- and all-trans-retinoic acid, used at clinically achievable doses of 10(-6) mol/L or less, can directly and specifically affect cell lines cultured from oral squamous cell carcinomas, inducing them to switch from an angiogenic to an anti-angiogenic phenotype. Although retinoic-acid-treated and untreated tumor cells make the same amount of interleukin-8, the major inducer of neovascularization produced by such tumor lines, they vary in production of inhibitory activity. Only the retinoic-acid-treated cells produce a potent angio-inhibitory activity that is able to block in vitro migration of endothelial cells toward tumor cell conditioned media and to halt neovascularization induced by such media in the rat cornea. Anti-angiogenic activity is induced in the tumor cells by low doses of retinoids in the absence of toxicity with a kinetics that suggest that it could be contributing to the effectiveness of the retinoids as chemopreventive agents.

Topics: Animals; Breast Neoplasms; Carcinoma, Squamous Cell; Colonic Neoplasms; Cornea; Endothelium, Vascular; Female; Fibrosarcoma; Humans; Interleukin-8; Keratinocytes; Neovascularization, Pathologic; Neovascularization, Physiologic; Neutralization Tests; Phenotype; Rats; Rats, Inbred F344; Tongue Neoplasms; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In this study we show that a breast cancer cell line (SKBR3) that expresses no E-cadherin and very low levels of beta-catenin protein and exhibits a poorly adhesive phenotype in Matrigel responds to retinoic acid (RA) by a marked increase in epithelial differentiation. Specifically, treatment of cells with all-trans-RA, 9-cis-RA, or a RA receptor alpha-specific ligand resulted in a large increase in cell-cell adhesive strength and stimulated the formation of fused cell aggregates in Matrigel. A retinoid X receptor-specific ligand was ineffective. Exposure of cells to 9-cis-RA for as little as 4 h was sufficient to maintain the adhesive phenotype for at least 4 days. The effects of 9-cis-RA required protein and RNA synthesis, but were not mediated by factors secreted by stimulated cells or by direct cell contact and did not require serum. These 9-cis-RA-induced morphological effects were completely reversed by growing cells in 50 microM Ca2+, suggesting a mechanism involving a 9-cis-RA-induced increase in Ca(2+)-dependent adhesion. Consistent with this, beta-catenin protein levels were markedly elevated in the 9-cis-RA-treated cells, and beta-catenin became localized to a Triton-insoluble pool at regions of cell-cell contact. No change could be detected in beta-catenin steady state messenger RNA levels, but 9-cis-RA did increase beta-catenin protein stability. Treatment of cells with low calcium medium did not prevent the 9-cis-RA-induced increase in total beta-catenin protein, but did prevent its movement to a Triton-insoluble pool at the cell membrane. Among several kinase inhibitors, only the broad spectrum kinase inhibitor staurosporine and the protein kinase C inhibitor bisindoylmaleimide reversed the morphological changes induced by 9-cis-RA. Like treatment with low calcium medium, these inhibitors did not prevent the 9-cis-RA-induced increase in total beta-catenin protein levels, but completely prevented the movement of beta-catenin to the cell membrane. These results point to a role for beta-catenin and serine kinase activity in mediating the action of 9-cis-RA in epithelial differentiation.

Topics: beta Catenin; Breast Neoplasms; Cadherins; Calcium; Cell Adhesion; Cell Differentiation; Cell Membrane; Culture Media; Cytoskeletal Proteins; Drug Stability; Epithelium; Female; Humans; Indoles; Maleimides; Protein Serine-Threonine Kinases; RNA, Messenger; Staurosporine; Tissue Distribution; Trans-Activators; Tretinoin; Tumor Cells, Cultured

DNase I hypersensitivity regions correlate with genetic regulatory loci and binding sites for sequence-specific DNA-binding proteins. We present data supporting the presence of novel DNase 1 hypersensitive sites (which we have designated sites VI-IX) in both the body of the human c-myc gene downstream from exon 2 and the 3'-flanking region of the c-myc gene in HL-60 cells. All of these novel DH sites are markedly decreased when HL-60 cells are treated with either dimethyl sulfoxide (DMSO) or retinoic acid. Moreover, a similar pattern of DNase I hypersensitive sites in this region of c-myc was present in MCF-7 human breast cancer cells growing in culture. Our results suggest a potential role for these sites in transcriptional regulation of the human c-myc gene.

Topics: Base Sequence; Breast Neoplasms; Deoxyribonuclease I; Dimethyl Sulfoxide; DNA; DNA-Binding Proteins; Exons; Female; Gene Expression Regulation; Genes, myc; Humans; Introns; Molecular Sequence Data; Promoter Regions, Genetic; Sp1 Transcription Factor; Transcription, Genetic; Tretinoin

Because the retinoic acid (RA) signaling pathway regulates cell proliferation and differentiation, inactivation of genes integral to the pathway represents a potential mechanism of carcinogenesis. We have studied in human breast cancer cells (T47D, MCF-7, ZR75-1, MDA-MB-231, and BT20) the expression of a subset of retinoid signaling genes that are themselves transcriptionally up-regulated by RA, the cellular retinol binding protein type I (CRBPI) and the RA receptors (RARs) alpha2, beta2, and gamma2. We find that constitutive expression of these genes is low or undetectable, and that expression levels are seldom responsive to 24 h treatment with 1 microM all-trans or 9-cis RA (Northern blot analysis). This is in contrast to breast fibroblasts, which show RA-dependent expression of all four genes under the same conditions. Moreover, normal human breast epithelial cells express CRBPI and RARbeta2 at the mRNA level, suggesting that loss of expression of these genes is tied to malignant transformation. RARbeta2, but not CRBPI, was also expressed in RA-treated MTSV1-7 cells, an immortalized but nontumorigenic luminal epithelial cell line. Lack of CRBPI and RARbeta2 expression in cancer cells was not due to general impairment of RA signaling, as shown by RA activation of a RARE3-tk-CAT reporter in a subclone of MDA-MB-231 cells that did not express either CRBPI or RARbeta2. These results suggest that at least two independent defects in the expression of proteins that function in retinoid signaling may be involved in breast carcinogenesis.

Topics: Breast Neoplasms; Clone Cells; Epithelium; Female; Humans; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Breast Neoplasms; Cell Division; Drug Resistance; Estrogens; Female; Humans; Insulin-Like Growth Factor Binding Proteins; Neoplasms, Hormone-Dependent; Time Factors; Tretinoin; Tumor Cells, Cultured

Liarozole inhibits cytochrome P-450-dependent enzymes that play a key role in all-trans-retinoic acid (ATRA) catabolism. In MCF-7 cells, liarozole potentiates the antiproliferative effects of ATRA. The present study demonstrates this synergistic effect on cell differentiation of MCF-7 cell cultures as measured by immunocytochemistry for cytokeratins 8, 18, and 19, actin, E-cadherin, desmoglein and desmoplakins I & II. ATRA concentration-dependently (10(-8) M-10(-6) M) induced changes in actin stress fibers and cytokeratin intermediate filaments. These changes were accompanied by a more obvious interaction of these filaments with junctional complexes. Surface area and volume of the MCF-7 cells increased markedly after ATRA exposure, with extensive filopodia formation. Liarozole (10(-6) M) alone had no effect on cell morphology, cytokeratin or actin organization, or on cellular junctions. In combination with ATRA (10(-9) M and 10(-8) M), liarozole potentiated the ATRA-induced effects. The MCF-7 cell cultures used showed morphological heterogeneity, consisting of at least two cellular subpopulations. This was reflected in the staining for E-cadherin, desmoglein and desmoplakins I & II. ATRA increased E-cadherin staining at cell-cell contact sites, but had no influence on the staining patterns of desmoglein and desmoplakins I & II. Similar to what has been observed for the cytoskeletal differentiation parameters, liarozole alone had no influence on E-cadherin, desmoglein or desmoplakins I & II expression, but in combination with ATRA again intensified the effects on E-cadherin distribution. These effects on MCF-7 cells agree with previously obtained observations concerning the inhibition of ATRA catabolism by liarozole. Furthermore, our data support the hypothesis that the antiproliferative properties of the drug are accompanied by induction of differentiation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Adhesion Molecules; Cell Division; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Imidazoles; Neoplasm Proteins; Tretinoin

Apolipoprotein D (apoD) is a human plasma protein, belonging to the lipocalin superfamily, that is produced by a specific subtype of highly differentiated breast carcinomas and that is strongly up-regulated by retinoic acid (RA) in breast cancer cells. In this work, we have examined the molecular mechanisms mediating the induction of apoD gene expression by retinoids in T-47D human breast cancer cells. Northern blot analysis revealed that Ro40-6055, a synthetic retinoid that selectively binds and activates the retinoic acid receptor RARalpha, induced the accumulation of apoD mRNA in breast cancer cells in a time- and dose-dependent manner. The time course analysis demonstrated that apoD mRNA was induced 14-fold over control cells after 48 h of incubation with 10(-8) M Ro40-6055. As little as 10(-11) M of this retinoid induced apoD mRNA 5-fold over the control, whereas incubation with 10(-7) M Ro40-6055 induced maximally 15-fold over control cells. RARalpha-selective antagonists counteracted the inductive effects of all-trans-RA, 9-cis-RA, and Ro40-6055 on the expression of apoD, when present at the same concentration as the retinoid agonists. By contrast, RARbeta-, RARgamma-, and RXR-selective retinoids did not affect apoD gene expression. The retinoid agonist Ro40-6055 had an antiproliferative effect on T-47D cells, with maximal growth inhibition of approximately 60% obtained after 7 days of incubation with 10(-7) M. This antiproliferative effect could be counteracted by a 100-fold excess of the antagonist Ro41-5253. Treatment of the cells with retinoids that do not bind the nuclear retinoic acid receptors did not affect apoD expression, despite the fact that they did have a strong antiproliferative effect on T-47D cells. On the basis of these results, a role for RARalpha on apoD gene expression induction by retinoids in breast cancer cells is proposed.

Topics: Antineoplastic Agents; Apolipoproteins; Apolipoproteins D; Benzoates; Breast Neoplasms; Cell Division; Chromans; Female; Humans; Morpholines; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Signal Transduction; Tetrahydronaphthalenes; Tretinoin

When human breast cancer T47D cells were treated with all-trans-retinoic acid (RA), the RA 4- and 18-hydroxylase activities were induced in microsomes in a time-dependent manner, indicating that these cells readily metabolized RA into more polar compounds, such as all-trans-4-hydroxy-RA and all-trans-18-hydroxy-RA. In contrast, T47D cells treated for 12 h with xenobiotics, such as phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, and dimethylsulfoxide, showed lower levels of catalytic activities for 4- and 18-hydroxylases. The induction of 4- and 18-hydroxylase activities appears to be regulated at the level of transcriptional control (basal level). Competitive assays demonstrated that inhibitors and substrates for 1A, 2A, 3A, 2B, and 2C cytochrome P450 (P450 subfamilies), all-trans-retinol, and all-trans-retinal showed no inhibition of RA metabolism, but other retinoic acid derivatives competed highly with RA. The RA-inducible 4- and 18-hydroxylases showed high specificity for RA and high levels of catalytic activities, with Km and maximum velocity values for 4-hydroxylase equal to 99 nmol/L and 0.26 pmol/min.mg protein, respectively, and those for 18-hydroxylase equal to 65 nmol/L and 0.18 pmol/min.mg protein. Cell-free metabolism of RA required microsomes from RA-treated cells and NADPH, and was inhibited by liarozole, an inhibitor of P450. These data suggest that RA-inducible 4- and 18-hydroxylases may be novel P450 isozymes.

Topics: Breast Neoplasms; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; Female; Humans; Kinetics; Mixed Function Oxygenases; Retinoids; Substrate Specificity; Tretinoin; Tumor Cells, Cultured

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Breast Neoplasms; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Approval; Female; Humans; Neoplasms, Experimental; Rats; Research Design; Tretinoin; Uterine Cervical Neoplasms

We have shown earlier that surgical human breast cancer tissue can be maintained in culture as in culture as intact tissue slices (organ culture). Because tumor organ culture ostensibly preserves the interacting network of tumor cells, stromal fibroblasts, endothelial cells and extracellular matrix, it represents a rather complex culture system. Such a system may be especially useful in preclinical trials, where the objective is to make extrapolations to the even more complex in vivo situation. A classical therapeutic target in breast cancer is the estrogen receptor, and we showed earlier that human breast cancer slices retain expression of this receptor in culture. Retinoic acid, the active form of vitamin A, is also an important (negative) growth regulator in breast cancer. In the present communication, we used in situ hybridization to monitor the expression of retinoic acid receptors in tumor slices cultured for 4 days. We show that both members of the all-trans retinoic acid and 9-cis retinoic acid receptor family (RAR and RXR, respectively) are expressed. Moreover, RNase protection analysis showed that expression of the cellular retinoic acid-binding protein type II gene, a known retinoic acid target gene, is upregulated by treatment with 1 microM all-trans retinoic acid for 2 days. These findings attest to the feasibility of using tumor organ cultures as a preclinical model for the evaluation of synthetic vitamin A derivatives (retinoids).

Topics: Breast Neoplasms; Humans; Organ Culture Techniques; Receptors, Retinoic Acid; Ribonucleases; Tretinoin

Retinoids are capable of modulating cellular differentiation and proliferation. Retinoids mediate gene function through a series of nuclear receptors. The retinoic acid receptor beta (RAR beta) has been shown to play an important role in the differentiation of a number of cell types. RAR beta is either absent or expressed at extremely low levels in a number of tumor types including breast carcinoma. It was demonstrated that transfection of RAR beta gene in breast carcinoma cell with its subsequent expression resulted in inhibition of cell growth. Retinoic acid significantly inhibited monolayer growth of the breast carcinoma cells expressing RAR beta, while it had no effect on the growth of the control cells. The RAR beta expressing cells formed much smaller and fewer colonies in soft agar and were significantly less tumorigenic in nude mice than the controls. These results suggest that RAR beta may function as a tumor suppressor in breast carcinoma cells.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Female; Humans; Mice; Mice, Nude; Receptors, Retinoic Acid; Transfection; Tretinoin; Tumor Cells, Cultured

The relationship between the effect of retinoic acid (RA) on the growth of breast cancer cell and their estrogen receptor (ER) status as well as the relationship between RA effect and the expression of retinoic acid receptorsd (RAR alpha) were studied by cell growth assay, Northern Blot and gene transfection. It was found that RA could only inhibit the growth of ER-positive but not ER-negative breast cancer cells. RAR alpha mRNA level was significantly higher in ER-positive breast cancer cell lines than that in ER-negative breast cancer cell line. The expressions of other subtypes of RAR in ER-positive cells were not significantly different from those in ER-negative ones. When, RAR alpha cDNA was introduced and ixpressed in RA-resistant, ER-negative MDA-MB-231 breast cancer cell line, its growth was strongly inhibited by RA. These results indicate that RAR alpha plays a major role in the retinoi-dmediated inhibition of growth in human breast cancer cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Female; Humans; Plasmids; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids mediate their actions via retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Each of class of these nuclear retinoid receptor is further subdivided into three species namely alpha, beta and gamma. Recently studies demonstrated that estrogen receptor (ER)-positive human breast cancer (HBC) cell lines are sensitive and ER-negative cell lines are resistant to growth inhibitory effects of retinoic acid (RA). In this study, we found that only RAR alpha mRNA levels was strongly correlated with ER-status. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA into two RA-resistant ER-negative HBC cell lines. Analysis of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Our results demonstrated that RAR alpha plays a major role in mediating retinoids inhibition of growth in HBC cells and adequate levels of RAR alpha are required for such an effect.

Topics: Breast Neoplasms; Cell Division; Humans; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Coordinated expression of genes involved in development, differentiation and malignant transformation is regulated by transcription factors including homeodomain-containing proteins. However, most of their cDNA sequences are still unknown. We report here the molecular characterization of three newly cloned HOXA1 transcripts from human breast cancer cells. In addition, we provide evidence that these alternatively spliced transcripts encode one homeodomain-containing protein and two products lacking the conserved DNA-binding domain. Moreover, we demonstrate that all three HOXA1 transcripts are induced by retinoic acid in MCF7 cells. Taken together, our results suggest that HOXA1 gene may be a key element in the establishment of the breast cancer cell phenotype.

Topics: Actins; Alternative Splicing; Amino Acid Sequence; Base Sequence; Breast Neoplasms; Cell Line; Cloning, Molecular; Conserved Sequence; DNA Primers; Female; Genes, Homeobox; Homeodomain Proteins; Humans; Molecular Sequence Data; Multigene Family; Polymerase Chain Reaction; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have studied the role of the AP1 transcription factor in the progression of human breast carcinomas. This progression is characterized by a loss of dependence for proliferation on mitogenic hormones, and is also linked to loss of responsiveness to the growth inhibitor retinoic acid (RA). In the hormone-dependent breast tumor cell line MCF7 mitogenic stimulation was found to be linked to an enhancement of AP1 transcriptional activity, while growth inhibition by RA was parallelled by decreased AP1 activity. AP1 binding activity to its consensus DNA sequence was rapidly reduced in RA treated cells, in the absence of any noticeable change in expression of AP1 constituents. AP1 overexpression abrogated RA repression in MCF7 cells. In hormone-independent cell lines (BT20, Hs578T, MDA-MB231, MDA-MB468) autonomous proliferation was associated with an increased background AP1 activity. Interestingly, these cells are refractory to growth inhibition by RA, which can only be partly explained by underexpression of RA receptors. In these cells RA did not repress AP1 transactivation unless RA receptors were overexpressed by means of cotransfection with an expression vector. This suggests that the high background levels of AP1 activity in the autonomously growing cells are associated with prevention of RA inhibition of AP1 activity to occur. Therefore, increased AP1 activity may not only play a role in progression of breast tumors towards hormone-insensitivity but may also contribute to the RA resistance of such cells.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Cell Division; Consensus Sequence; DNA, Neoplasm; Hormones; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer.

Topics: Breast Neoplasms; Carrier Proteins; Cyclic AMP; Drug Synergism; Estradiol; Humans; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor Binding Proteins; Receptors, Estrogen; Somatomedins; Tretinoin; Tumor Cells, Cultured

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

Topics: Amino Acid Sequence; Breast; Breast Neoplasms; Carrier Proteins; Cells, Cultured; Cellular Senescence; Chromosome Mapping; Chromosomes, Human, Pair 4; Consensus Sequence; DNA Replication; Epithelial Cells; Epithelium; Female; Fenretinide; Gene Expression; Humans; Insulin-Like Growth Factor Binding Proteins; Kinetics; Molecular Sequence Data; RNA, Messenger; Sequence Homology, Amino Acid; Somatomedins; Time Factors; Tretinoin

Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I, IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (> or = 10(-6) M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10(-7) M RA, and that 10(-9) - 10(-7) M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10(-9) - 10(-7) M RA. Treatment with the IGF-I receptor blocking antibody, alpha IR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10(-9) - 10(-7) M RA enhancing cell proliferation and > or = 10(-6) M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies.

Topics: Breast Neoplasms; Cell Division; Growth Inhibitors; Growth Substances; Humans; In Vitro Techniques; Insulin; Insulin-Like Growth Factor I; Radioligand Assay; Receptors, Estrogen; Receptors, Progesterone; Tretinoin; Tumor Cells, Cultured

This study examined the growth inhibitory effects of combining 1,25-dihydroxyvitamin D3 (calcitriol) with retinoic acid or dexamethasone against cultured breast and ovarian carcinoma cells. Retinoic acid (12.5-50 nM) increased the effectiveness of calcitriol (12.5-50 nM) against MCF-7 and NIH:OVCAR3 cells, with synergistic interactions at two of the three ratios tested. Dexamethasone augmented calcitriol effects, with synergism at 0.05 and 0.1 nM dexamethasone in MCF-7 cells and 5 nM in Caov-4 ovarian cells. This study showed favorable interactions for calcitriol-retinoic acid and calcitriol-dexamethasone combinations in breast and ovarian cancer cell lines.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Calcitriol; Cell Division; Dexamethasone; Drug Synergism; Female; Humans; Ovarian Neoplasms; Tretinoin; Tumor Cells, Cultured

Retinoid response pathways involve retinoic acid receptors (RARs) and retinoid X receptors. N-(4-hydroxyphenyl) retinamide (4-HPR), a derivative of all-trans-retinoic acid (RA) is currently in clinical trials as a chemopreventive agent for breast cancer. The issue whether 4-HPR mediates its biological actions via classical retinoid receptor pathways remains to be investigated. In this study, we provide several lines of evidence that 4-HPR mediates its biological actions via a novel pathway(s) that does not involve the classical retinoid receptor pathways. For example, 4-HPR was more potent than RA as an antiproliferative agent and inhibited growth of otherwise RA-resistant human breast carcinoma cells. Exposure to 4-HPR resulted in the generation of DNA fragmentation with subsequent cell death in both RA-positive estrogen receptor (ER)-positive as well as RA-refractory ER-negative breast carcinoma cell lines. N-(4-Methoxyphenyl)retinamide (4-MPR), which is the major 4-HPR metabolite in circulation, was biologically inert in this system. 4-HPR and 4-MPR bound poorly to the RAR alpha, beta and gamma in vitro and only minimally activated the retinoic acid receptor element (RARE) and retinoid X receptor response elements (RXREs) in human breast carcinoma cells. Neither 4-HPR nor 4-MPR are metabolized to any of the known conventional retinoids. In addition, 4-HPR or 4-MPR transactivation of RAREs or RXREs transfected into MCF-7 and MDA-MB-231 cells was not noted at 48 h. Nevertheless 4-HPR-mediated cell death was observed at 48 h, further suggesting that neither 4-HPR nor 4-MPR are metabolized to retinoids which activate the RAREs or RXREs in breast carcinoma cells. Furthermore, unlike RA, which exhibited anti-AP1 activity, 4-HPR inhibition of growth did not involve anti-AP1 activity. These results suggest that 4-HPR acts by a unique pathway that is not mediated by retinoid receptors.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Cell Division; Cell Line; Drug Resistance, Neoplasm; Female; Fenretinide; Humans; Kinetics; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoids modulate cellular proliferation and mediate gene function through a series of nuclear receptors. The retinoic acid nuclear receptor beta (RAR beta) plays an important role in the differentiation of a number of cell types. We now demonstrate that RAR beta expression is confined to normal mammary tissue and is not expressed in either immortalized normal or malignant cell lines. Treatment of RAR beta-transfected MDA-MB-231 cells with 1 microM all-trans-retinoic acid (RA) significantly inhibited monolayer growth of the cells which express recombinant RAR beta. RAR beta-expressing MDA-MB-231 cells formed significantly smaller and fewer colonies in soft agar than the mock-transfected cells. Addition of 1 microM RA stimulated colony size and number in the RAR beta-transfected MDA-MB-231 cells. In contrast to the RAR beta-expressing cells, colony formation by the RAR alpha-expressing cells was similar to the mock-transfected controls and the addition of 1 microM RA to the RAR alpha-transfected cells inhibited colony formation. While demonstrating decreased colony formation in agar, RAR beta-expressing MDA-MB-231 cells failed to exhibit decreased growth in SCID mice. Our results show that RAR beta functions as a negative regulator of growth in breast epithelial cells. In addition, the growth of these cells is differentially regulated by RAR alpha and RAR beta which is most likely the result of the modulation of different genes.

Topics: Agar; Animals; Base Sequence; Breast Neoplasms; Cell Adhesion; Cell Division; Cloning, Molecular; Female; Humans; Mice; Mice, SCID; Molecular Sequence Data; Receptors, Retinoic Acid; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

A number of studies have demonstrated the ability of retinoic acid (RA) to inhibit the growth of estrogen receptor-positive (ER+) human breast cancer cell lines. The precise mechanism of growth inhibition is not known. However, the biological effects of RA in other model systems have been shown to be mediated via the nuclear retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). While several laboratories have examined the expression of RARs in various breast cancer cell lines, no information is available concerning the role of the RXRs and 9-cis-RA, the natural ligand of RXRs, in the response of breast cancer cells to RA. Using a representative panel of breast cancer cell lines, we determined the effect of 9-cis-RA on growth and cell cycle stage distribution, analyzed steady-state mRNA levels of RXR-alpha, -beta, and -gamma, and determined the effect of all-trans-RA and 9-cis-RA on RXR expression. Our results show that: (1) the growth of ER+/RA-sensitive breast cancer cells is inhibited by treatment with 9-cis-RA by blocking entry into S phase; (2) both ER+/RA-sensitive and ER-/RA-resistant breast cancer cell lines express RXR-alpha and RXR-beta mRNAs but not RXR-gamma; however, levels of these transcripts did not correlate with the RA response; and (3) levels of RXR-alpha and RXR-beta mRNA were not significantly altered following treatment with either all-trans-RA or 9-cis-RA. These results suggest that the mechanism responsible for the retinoid sensitivity of breast cancer cells does not involve transcriptional modulation of the RXRs by RA.

Topics: Breast Neoplasms; Cell Division; Cell Line, Transformed; Female; Humans; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The effects of the novel vitamin D analogue, EB1089 alone, or in combination with the retinoid, 9-cis retinoic acid (9-cis RA) on indices of apoptosis in MCF-7 breast cancer cells have been examined. EB1089 was capable of reducing bcl-2 protein, a suppressor of apoptosis, and increasing p53 protein levels in MCF-7 cell cultures following 96h treatment. In the presence of 9-cis RA, EB1089 acted to further enhance the down-regulation and up-regulation of bcl-2 and p53 respectively. Furthermore, EB1089 induces DNA fragmentation in MCF-7 cells, a key feature of apoptosis, alone and in combination with 9-cis RA in situ. The observation that EB1089 and 9-cis RA act in a cooperative manner to enhance induction of apoptosis in these cells may have therapeutic implications.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Calcitriol; DNA Damage; DNA, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Retinoids and gamma-interferon (IFN-gamma) have been demonstrated to synergistically amplify growth inhibition in cultured breast cancer cells. Since IFN-gamma enhances effects mediated by retinoids more than vice versa, we focused our investigations on the mRNA expression of cellular retinoic acid-binding proteins (CRABPs) and retinoic acid receptors (RARs), since these are the key molecules that mediate retinoid action. Synergistic inhibition of cell proliferation under treatment with 9-cis-retinoic acid and IFN-gamma was detected in the three breast cancer cell lines MCF-7, SKBR-3, and even in the RA-resistant BT-20 cells. RAR-alpha and RAR-gamma mRNA were observed in all cell lines, whereas RAR-beta was not detectable. CRABP I message was expressed in MCF-7 cells only, but CRABP II was found in all three breast cancer cell lines. IFN-gamma (10 ng/ml) increased RAR-gamma expression but had no influence on RAR-alpha, whereas RAR-beta was not detectable in any of the cell lines. RA (1 microM)-mediated CRABP II increase was suppressed by IFN-gamma (10 ng/ml). These observations indicate that IFN-gamma-mediated increase in RAR-gamma and suppression of RA-mediated CRABP II activation may play a role in synergistic inhibition of proliferation in breast cancer cell lines.

Topics: Breast Neoplasms; Cell Division; Cycloheximide; Drug Synergism; Humans; Interferon-gamma; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Adult; Age Factors; Aged; Breast Neoplasms; Female; Fenretinide; Humans; Insulin-Like Growth Factor I; Middle Aged; Tretinoin

Retinoic acid (RA) inhibits proliferation of estrogen receptor (ER)-positive human breast cancer cells, but not the growth of ER-negative cells. We have shown previously that ER-positive cells express higher levels of retinoic acid receptor (RAR) alpha, suggesting that RAR alpha gene expression may be regulated in breast cancer cells by estrogens. We here report that estradiol (E2) increases RAR alpha mRNA in a time- and concentration-dependent manner resulting in a marked increase in RAR alpha protein expression, and present evidence that RAR alpha 1 is the only known isoform of RAR alpha regulated by E2 in breast cancer cells. In parallel we demonstrate that ER-positive cells exhibit greater RA sensitivity in the presence of E2, suggesting that E2-induced expression of RAR alpha 1 is involved in growth inhibition by RA. To directly investigate the role of RAR alpha 1 in RA-mediated growth inhibition, we introduced RAR alpha 1 expression vectors into RA-resistant and ER-negative MDA-MB-231 cells. The RAR alpha 1-transfected cells were growth inhibited by RA, while mock- and untransfected cells were unresponsive. Together, our data indicate that adequate levels of RAR alpha 1, either generated by introduction of expression vectors or endogenously induced by estrogens, are required for growth inhibition of breast cancer cells by RA.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Drug Resistance; Estradiol; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; RNA Probes; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

The properties and regulation of the mammalian polyamine transport system are still poorly understood. In estrogen-responsive ZR-75-1 human breast cancer cells, which display low polyamine biosynthetic activity, putrescine and spermidine were internalized with high affinity (Km = 3.7 and 0.5 microM, respectively) via a single class of saturable transporter shared by both substrate types, or via distinct but closely similar carriers. The Vmax, but not the Km of polyamine transport was rapidly and synergistically up-regulated by estrogens and insulin. The steady decay in transport activity observed in hormone-deprived cells was accelerated by retinoic acid. The enhancement of uptake activity resulting from polyamine depletion was amplified 3-fold by estrogens and insulin despite profound growth inhibition, indicating that the cooperative hormonal induction of polyamine transport is dissociated from cell growth status. Polyamine uptake was under feedback inhibition by at least three distinct mechanisms in these cells, namely (i) the induction of a short-lived protein not actively synthesized without ongoing uptake or upon polyamine deletion; (ii) a more latent, protein synthesis-independent "trans-inhibition" mechanism; and (iii) a post-carrier, cycloheximide-sensitive mechanism limiting substrate accumulation. The complexity of these multiple levels of feedback transport inhibition is in keeping with the cytotoxicity of excessive polyamine content.

Topics: Biological Transport; Breast Neoplasms; Cell Division; Cell Line; Cycloheximide; Drug Synergism; Eflornithine; Estradiol; Feedback; Humans; Insulin; Kinetics; Putrescine; Spermidine; Spermine; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (RA), like insulin-like growth factor I (IGF-I) and tamoxifen, inhibit invasion of human MCF-7/6 mammary cancer cells in vitro. For tamoxifen and for IGF-I, activation of the invasion-suppressor function of the E-cadherin/catenin complex was shown to be the most probable mechanism of the anti-invasive action. We did a series of experiments to determine whether the anti-invasive effect of RA also implicated the invasion-suppressor E-cadherin/catenin complex. Human MCF-7/6 mammary and HCT-8/R1 colon cancer cells, both with a dysfunctional E-cadherin/catenin complex, were treated with RA and the function of the complex was evaluated through Ca(2+)-dependent fast aggregation. Fast aggregation of both MCF-7/6 and HCT-8/R1 cells was induced by 1 microM RA. This effect was abolished by antibodies against E-cadherin. RA-induced fast aggregation was not sensitive to cycloheximide, tyrosine kinase inhibitors or antibodies against IGF-I or against the IGF-I receptor. RA did not stimulate IGF-I receptor phosphorylation or alter the E-cadherin/catenin complex, as evidenced by immunoprecipitation. RA up-regulates the function of the invasion-suppressor complex E-cadherin/catenin. Its action mechanism is different from that of IGF-I. RA may act as an anti-invasive agent with unique mechanisms of action.

Topics: Breast Neoplasms; Cadherins; Cell Adhesion; Cell Aggregation; Humans; Neoplasm Invasiveness; Tretinoin; Tumor Cells, Cultured

The expression of the retinoic acid receptor beta (RAR beta) mRNA is absent or down-regulated in most human breast cancer cell lines. To investigate the role RAR beta may have in regulating the proliferation of breast cancer cells, we used retroviral vector-mediated gene transduction to introduce the human RAR beta gene into two RAR beta-negative breast tumor cell lines, MCF-7 and MDA-MB-231. RAR beta-transduced clones underwent growth inhibition associated with G1 arrest when treated with 1 microM all-trans-retinoic acid (RA). Moreover, the MCF7-RAR beta transduced clones also underwent apoptosis after 4 to 6 days of RA treatment. The RA-induced growth arrest in MDA231-RAR beta transduced cells is associated with c-myc mRNA down-regulation, whereas the RA-mediated apoptosis of MCF7-RAR beta transduced cells is not associated with c-myc down-regulation. These observations suggest a critical role for RAR beta in mediating growth arrest and apoptosis in breast cancer cells.

Topics: Apoptosis; Base Sequence; Breast Neoplasms; Cell Cycle; Cell Division; Down-Regulation; Flow Cytometry; Genetic Vectors; Growth Inhibitors; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; Retroviridae; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes primarily the reductive reaction of estrone to the biologically more active form, estradiol. The enzyme is highly expressed in the human placenta and the ovary and, in addition, in certain estrogen target cells, such as breast epithelial cells. To elucidate the transcriptional control of the EDH17B2 gene, the gene encoding 17HSD type 1, we fused a series of 5'-deletion mutants of the EDH17B2 gene into chloramphenicol acetyl transferase reporter gene vectors. An enhancer region was identified within the bases -661 to -392 and it increased, in both orientations, thymidine kinase promoter activity more than 200-fold in JEG-3 choriocarcinoma cells. This enhancer region was also functional in another choriocarcinoma cell line, JAR, although to a lesser extent. In BT-20 and T-47D breast cancer cells the enhancer region increased thymidine kinase promoter activity to some degree but not as efficiently as expected on the basis of endogenous enzyme expression. No such enhancer activity was observed in 17HSD type 1 nonexpressing cell lines. The retinoic acid responsive element, which was located between bases -503 and -487 in the EDH17B2 enhancer, bound retinoid acid receptor alpha retinoid X receptor alpha complex and transmitted retinoic acid induction on transcription in JEG-3 and T-47D cells. Finally, a silencer, functional in all the cell lines tested, was localized in the region from -392 to -78. Deletion of the region lad to a 4-fold increase in reporter gene expression. Altogether, our findings suggest that transcriptional control of the EDH17B2 gene is coordinated by the cell-specific enhancer and the silencer.

Topics: 17-Hydroxysteroid Dehydrogenases; Base Sequence; Breast Neoplasms; Chloramphenicol O-Acetyltransferase; Choriocarcinoma; Enhancer Elements, Genetic; Female; Gene Deletion; Gene Expression Regulation, Enzymologic; Humans; Male; Molecular Sequence Data; Promoter Regions, Genetic; Prostatic Neoplasms; Recombinant Fusion Proteins; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

It has previously been shown that, in the estrogen-receptor-positive breast-tumor cell lines T47D and ZR75.1, the erbB-2 protein and mRNA content are controlled negatively and positively by, respectively, estrogens and anti-estrogens. Since estrogens have a positive effect on cell proliferation, while anti-estrogens inhibit cell growth, the results suggested that there may be an inverse correlation between growth and erbB-2 expression. We have now examined this matter further. The effect of various growth-modulatory agents including estrogen (E2), progesterone (Pg), retinoic acid (RA), epidermal growth factor (EGF), insulin (Ins), prolactin (Prl), 12-O-tetradecanolyl-phorbol-13-acetate (TPA) and dibutyryl-3':5'-cyclic-AMP (cAMP) on c-erbB-2 promoter activity, RNA and protein expression have been examined. The growth stimulators E2 and EGF both reduced the level of erbB-2 protein. However, while E2 clearly repressed erbB-2 transcription, in the case of EGF, neither mRNA nor transcription were decreased. Of the agents which inhibit the growth of T47D and ZR75.1 cells--Pg, Prl, cAMP, RA and TPA--only Pg and cAMP caused an increase in the erbB-2 protein level. Pg and cAMP positively influenced c-erbB-2 promoter activity and RNA amount. TPA and RA also increased promoter activity but neither erbB-2 mRNA nor protein level was enhanced. The erbB-2 protein expression in cultures of T47D and ZR75.1 cells at different densities was also analyzed. Both the level of erbB-2 protein and c-erbB-2 promoter activity rose markedly in confluent cultures, suggesting a transcriptional mechanism of control. In conclusion, the data suggest that the effects of various agents on erbB-2 expression are complex and cannot be explained simply as reflecting the growth state of the cells.

Topics: Base Sequence; Breast Neoplasms; Bucladesine; Cell Count; Epidermal Growth Factor; ErbB Receptors; Estradiol; Gene Expression; Growth Substances; Humans; Insulin; Molecular Sequence Data; Progesterone; Prolactin; Promoter Regions, Genetic; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptors, Estrogen; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured

We have examined the regulation by retinoic acid of the gene encoding apolipoprotein D (apoD), a human plasma protein belonging to the superfamily of the lipocalins that is produced by a specific subtype of highly differentiated breast carcinomas. Northern blot analysis revealed that all-trans-retinoic acid (RA) strongly induced the accumulation of apoD mRNA in T-47D and ZR-75-1 estrogen receptor-positive human breast cancer cells in a time- and dose-dependent manner, while no inductive effect was observed in estrogen receptor-negative cell lines, including MDA-MB-231 and MDA-MB-435. The effect of RA on apoD expression by T-47D cells was at least 12-fold more potent than the effect of the steroids dihydrotestosterone and dexamethasone, which had been previously described as hormonal up-regulators of apoD expression in these cells. A time course study demonstrated that the induction of apoD mRNA reached a level of 15-fold over the untreated control cells after 48 h of incubation in the presence of a 10(-7) M concentration of RA. A dose-response analysis showed that as little as 10(-13) M RA produced an accumulation of 5-fold over the control, while incubation of the cells in the presence of 10(-5) M RA induced a maximal accumulation of 24-fold over the control untreated cells. The induction of apoD mRNA was independent of the synthesis of proteins de novo, as demonstrated by the fact that the induction was also detected in the presence of cycloheximide. The incubation of the cells in the presence of RA did not affect significantly the stability of apoD mRNA. By contrast, treatment of the T-47D cells with RA produced an increase of approximately 8-fold in the rate of transcription of the apoD gene. Furthermore, treatment of the T-47D cells with RA induced the synthesis and secretion to the culture medium of apoD. This increased expression of apoD was accompanied by an inhibition of cell proliferation and a progression through a more differentiated phenotype, suggesting that the mechanisms controlling RA-induced growth arrest, cell differentiation, and apoD synthesis may be directly coordinated in human breast cancer cells.

Topics: Apolipoproteins; Apolipoproteins D; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Nucleus; Cell-Free System; Cycloheximide; Dexamethasone; Dihydrotestosterone; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Isomerism; Lactoferrin; RNA, Messenger; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.

Topics: Alkaline Phosphatase; Blotting, Northern; Breast Neoplasms; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Placenta; Pregnancy; Protein Synthesis Inhibitors; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Lippman and coworkers recently reported striking response rates achieved in patients with cervical cancer using a combined treatment with 13-cis-retinoic acid and interferon-alpha. In vitro studies demonstrated a synergistic amplification of proliferation inhibition by retinoids and interferon in malignant cells. Comparing all-trans- with 9-cis-retinoic acid, the latter was more effective in inhibiting tumor cell growth and in inducing synergism with interferon-gamma. Combination of retinoic acid with interferon-gamma increased the down-regulation of specific binding sites of interferon-gamma. Expression of MHC class II antigens induced by interferon-gamma was not further enhanced by retinoic acid. These results indicate that interferon seems to modulate the retinoic acid system.

Topics: Breast Neoplasms; Cell Division; Cell Line; Cell Survival; Drug Synergism; Female; Humans; Interferon-gamma; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (tRA) inhibits growth of estrogen receptor-positive (ER+) breast cancer cells in vitro, and a variety of retinoids inhibit development of breast cancer in animal models. 9-cis retinoic acid (9-cis RA) is a naturally occurring high affinity ligand for the retinoid X receptors, as well as the retinoic acid receptors (RARs). Whether 9-cis RA has a different spectrum of biological activity from tRA, which only binds RARs with high affinity, is largely unknown. We studied the effects of 9-cis RA on growth and gene expression in ER+ and ER- human breast cancer cells. 9-cis RA inhibited the growth in monolayer culture of several ER+, but not ER-, cell lines in a dose-dependent manner. Growth inhibition and morphological changes by 9-cis RA were similar to those of tRA, suggesting that the ability to bind both RAR and retinoid X receptors did not significantly augment growth inhibition or confer sensitivity to tRA-resistant lines. MCF-7 cells exposed to 9-cis RA showed a dose-dependent accumulation in G1. Northern analyses showed that RAR-alpha and RAR-beta were not significantly regulated, while RAR-gamma was up-regulated and retinoid X receptor alpha was down-regulated by 9-cis RA. Since interactions between tRA and ER-dependent transcription have recently been reported, we investigated whether these retinoids regulate expression of ER itself or estrogen-responsive genes. Both 9-cis RA and tRA induce down-regulation of ER mRNA and protein in MCF-7 cells. 9-cis RA down-regulates expression of the estrogen-responsive genes PR and pS2 in MCF-7 cells as reported previously for tRA. In several ER-positive subclones, we found that the degree of ER expression and regulation, but not always estrogen-sensitivity, correlates with the growth-inhibitory effects of 9-cis RA. Further, in an ER-, retinoid-unresponsive breast cancer cell line, induced ER expression confers responsiveness to retinoid growth inhibition. These data, combined with reports of additive growth inhibition of tRA and tamoxifen in vitro, suggest that 9-cis RA might augment the ability of tamoxifen to inhibit growth of ER+ breast cancer cells in vivo.

Topics: Breast Neoplasms; Cell Division; Down-Regulation; Humans; Insulin-Like Growth Factor II; Receptors, Androgen; Receptors, Estrogen; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Administration of the synthetic retinoid Fenretinide lowers circulating retinol and may thus affect night vision. We have recently shown that plasma retinol levels below 100 ng/ml are associated with moderate alterations of the dark adaptometry test. To identify which patients are more likely to experience a decrease of plasma retinol under this threshold, we measured plasma levels of retinol, Fenretinide, and its metabolite 4-MPR in a cohort of 28 women receiving Fenretinide at the daily dose of 200 mg and studied their relationship with clinical characteristics such as age, menstrual status, body mass index, and time on treatment. Our results show that patients aged over 55 years with a higher percentage of adipose tissue had higher plasma concentrations of 4-MPR, which turned out to be the major determinant of the retinol decrease. This subgroup may thus deserve careful ophthalmological surveillance.

Topics: Adipose Tissue; Adult; Age Factors; Aged; Body Mass Index; Breast Neoplasms; Cohort Studies; Dark Adaptation; Female; Fenretinide; Humans; Middle Aged; Population Surveillance; Postmenopause; Premenopause; Time Factors; Tretinoin; Vision, Ocular; Vitamin A

Estradiol 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol in endocrine-responsive tissues such as the breast. The control of 17 beta HSD expression by all-trans-retinoic acid (RA) in T47D breast cancer cells was examined using a specific 17 beta HSD complementary DNA probe. Two main 17 beta HSD messenger RNA (mRNA) transcripts of 2.2 and 1.3 kilobases (kb) were detected, of which only the 1.3-kb mRNA was regulated. RA increased expression of the 17 beta HSD 1.3-kb mRNA in a dose- and time-dependent manner, and the increased expression of this mRNA by RA was inhibited by a 10-fold excess of a RA antagonist Ro 41-5253. Insulin-like-growth factor-I, interleukin-1, and estradiol, previously shown to increase 17 beta HSD activity in breast cancer cells, had little effect on 17 beta HSD gene expression. To relate the effect of increased 17 beta HSD 1.3-kb mRNA expression to 17 beta HSD activity, the conversion of estrone to estradiol (reductive) and that of estradiol to estrone (oxidative) were measured in intact T47D cell monolayers. Whereas RA increased 17 beta HSD reductive activity, it had no effect on oxidative activity. The addition of excess NAD increased 17 beta HSD oxidative activity in control and RA-treated cells, but the addition of NADH had no effect on 17 beta HSD reductive activity. These results suggest that the increased expression of the 17 beta HSD 1.3-kb mRNA induced by RA is associated with an increase in 17 beta HSD reductive activity, but that endogenous cofactor levels may determine the direction in which this enzyme acts in T47D cells.

Topics: Breast Neoplasms; Cycloheximide; Cytokines; Estradiol Dehydrogenases; Female; Growth Substances; Hormones; Humans; Pregnancy; Proteins; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Both retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicit a number of common biochemical and toxic responses including teratogenic effects in mice and inhibition of cell proliferation of numerous cell lines. In mice, RA plus TCDD interact synergistically as teratogens, suggesting a link between the RA and aryl hydrocarbon receptor signal transduction pathways. In this study, it was shown that both RA and TCDD elicit a number of common responses in MCF-7 human breast cancer cells, including inhibition of estrogen-induced cell proliferation and [3H]thymidine uptake, inhibition of nuclear estrogen receptor (ER) ligand binding, and interactions with a consensus [32]estrogen-responsive element in a gel mobility shift assay. RA and TCDD also decrease steady-state ER mRNA levels in a time-dependent manner. Moreover, interactive studies in which cells were treated with both RA plus TCDD also resulted in decreased responses.

Topics: Analysis of Variance; Base Sequence; Blotting, Northern; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Estradiol; Humans; Molecular Sequence Data; Polychlorinated Dibenzodioxins; Receptors, Estrogen; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The effect of 13-cis-retinoic acid (cRA) and all-trans-retinoic acid (tRA) used alone or in combination with interferon alpha-2a (alpha-IFN 2a) was tested on three established human cell lines: KB (epidermoid carcinoma of the oral cavity), SCC-25 (tongue squamous cell carcinoma) and MCF-7 (mammary carcinoma). Both retinoids significantly decreased cell proliferation (growth curves) and colony forming efficiency (CFE) in all cell lines, in a dose-dependent way (at a concentration ranging from 10(-5) to 10(-9) M) and differing from line to line, following the pattern: MCF-7 > SCC-25 > KB. Retinoids at any concentration (already at 10(-7) M) combined with alpha-IFN 2a (ranging from 100 to 500 IU/ml) were more effective in inhibiting cell proliferation than each of the two compounds alone. This was particularly evident with SCC-25 cells. Concerning MCF-7 cells, on the contrary, the effects produced by the association suggested a possible additive more than synergistic amplification of growth inhibition.

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Interferon-alpha; Isotretinoin; Mouth Neoplasms; Tretinoin; Tumor Cells, Cultured

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.

Topics: Amino Acid Sequence; Animals; Base Sequence; Breast Neoplasms; Cells, Cultured; Chlorocebus aethiops; Choline O-Acetyltransferase; Choriocarcinoma; Enzyme Induction; Female; Humans; Molecular Sequence Data; Neoplasms, Hormone-Dependent; Neuroblastoma; Neurons; Organ Specificity; Proto-Oncogene Proteins c-jun; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Steroids; Transcriptional Activation; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Uterine Neoplasms

We show that 9-cis-retinoic acid (9cRA) is a potent inhibitor of mammary carcinogenesis induced by N-nitroso-N-methylurea in Sprague-Dawley rats. Rats were first treated with a single dose of N-nitroso-N-methylurea (50 mg/kg body weight) and then fed non-toxic levels of 9cRA (120 or 60 mg/kg of diet). 9cRA was highly effective in reducing tumor incidence, average number of tumors per rat, and average tumor burden, as well as extending tumor latency. The combination of 9cRA with low levels of tamoxifen (TAM; fed at either 1.0 or 0.5 mg/kg of diet) was particularly effective; addition of 9cRA to a TAM regimen doubled the number of animals that were tumor-free at autopsy and significantly diminished tumor number and tumor burden. For suppression of carcinogenesis in vivo, 9cRA was much more potent than all-trans-retinoic acid, both as a single agent or in combination with TAM, although both retinoids had equivalent inhibitory effects on DNA synthesis in cultured human breast cancer cell lines. Both 9cRA and all-trans-retinoic acid induce the expression of the adhesion molecule, E-cadherin, in the SK-BR-3 cell line. We suggest that clinical evaluation of the combination of 9cRA and TAM, either for chemoprevention or for adjuvant therapy, should be considered.

Topics: Animals; Breast Neoplasms; Drug Screening Assays, Antitumor; Humans; Mammary Neoplasms, Experimental; Methylnitrosourea; Rats; Rats, Sprague-Dawley; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each class of these nuclear retinoid receptors is further subdivided into three species, namely alpha, beta, and gamma. Recent studies demonstrate that estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines and tumor samples exhibit significantly higher levels of RAR alpha than their ER-negative counterparts. ER-positive HBC cell lines are sensitive to, and ER-negative cell lines are resistant to, growth inhibitory effects of retinoic acid (RA). We previously demonstrated that the expression of functional ERs in an established ER-negative cell line resulted in higher levels of RAR alpha and sensitivity to growth inhibition by RA. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA in two RA-resistant ER-negative HBC cell lines. Analyses of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Utilizing RAR- and RXR-class selective retinoids, we further demonstrated that only the RAR alpha-selective retinoids mediated the growth inhibition in these cells, while the RXR-selective retinoids were biologically inert. We thus provide evidence that the molecular mechanisms of retinoid inhibition of HBC proliferation predominantly involve RAR alpha.

Topics: Base Sequence; Breast Neoplasms; Carcinoma; Cell Division; DNA, Complementary; Female; Genetic Vectors; Humans; Molecular Sequence Data; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Signal Transduction; Transfection; Tretinoin

Retinoic acid (RA) inhibits insulin-like growth factor I (IGF-I)-stimulated growth of the human breast carcinoma cell line MCF-7. RA-mediated inhibition of IGF-I-stimulated growth is not associated with either a decrease in IGF-I receptor number or affinity. Since IGF-I modulation of c-fos gene expression appears to be an important step in IGF-I-mediated cellular proliferation, we investigated whether RA inhibits IGF-I stimulation of c-fos mRNA levels. Treatment of MCF-7 cells with IGF-I resulted in an approximately 10-fold increase in c-fos mRNA levels. Pretreatment of MCF-7 cells with 1 microM RA blocked IGF-I-mediated enhancement of c-fos mRNA levels by approximately 70%. The maximal RA effect (80% inhibition) on IGF-I stimulation of c-fos mRNA levels was noted within 2 h of exposure to RA. IGF-I did not modulate the c-fos gene promoter and appears to increase the stability of the c-fos mRNA. Preexposure of cells to RA results in a significant decrease (P < 0.05) in c-fos mRNA stability. The half-life of c-fos mRNA in the IGF-I-treated cells is 53 +/- 6 6 min while that in RA-pretreated cells is 27 +/- 0.4 min. We conclude that RA-mediated inhibition of IGF-I stimulation of c-fos mRNA may represent a potential mechanism by which RA inhibits IGF-I stimulation of growth.

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunoblotting; Insulin-Like Growth Factor I; Proto-Oncogene Proteins c-fos; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Most breast tumors show estrogen-dependent growth and are thus susceptible to antiestrogenic therapy. MCF-7 cells, obtained from a human estrogen-dependent breast carcinoma, are widely used for studying the modulation of estrogenic responses by different effectors. All-trans-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (Vit D3) inhibited estrogen-induced growth of MCF-7 cells and their effect was potentiated by the classical antiestrogen, hydroxytamoxifen. In MCF-7 cells, we found that RA and Vit D3 also inhibited estrogen-induced transcription; this was shown both for an endogenous gene (pS2) and for various exogenous transfected genes. Their inhibitory effect could not be reversed by increasing estradiol concentrations, showing that contrary to classical antiestrogens, they did not compete with estradiol to bind the estrogen receptor (ER). Analysis of the inhibitory mechanisms indicates that RA and Vit D3 receptors can directly or indirectly impair the binding of ER to the estrogen responsive element. The antagonist effect of RA would be found especially at DNA level since it seems to essentially involve an estrogen responsive element. The antagonist effect of Vit D3 would be found especially at the ER level since it seems to concern estrogen binding and dimerization domains of ER. We conclude that the antiestrogenic effects of RA and Vit D3 are similar since they can, via their receptors, interfere with estrogenic action at the estrogen responsive element level but that they are not identical since different molecular mechanisms are involved.

Topics: Breast Neoplasms; Calcitriol; Cell Division; Chimera; Estrogen Antagonists; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proteins; Receptors, Calcitonin; Receptors, Retinoic Acid; RNA, Messenger; Transcription, Genetic; Trefoil Factor-1; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Retinoids are important cellular, dietary factors that regulate differentiation and cellular growth. They serve as ligands for specific nuclear receptors, the retinoic acid receptors (RARs). Ligand-activated receptors regulate gene transcription through target retinoic acid-responsive elements (RAREs) found in promoter regions. We have investigated the expression of retinoic acid receptor genes (alpha, beta, gamma) and retinoid X receptor beta in normal, senescing, and tumorigenic human mammary epithelial cells. We find that most tumor cells show a loss of RAR-beta expression, but that RAR-alpha and -gamma as well as retinoid X receptor beta are variably expressed in both normal and tumor cells. RAR-beta gene expression is induced both by retinoic acid and by fenretinide in normal cells, but tumor cells fail to respond to either. In contrast, RAR-beta expression increases with serial passage in senescing cells. Paradoxically, both normal and tumor cells can trans-activate an exogenous beta-RARE, as demonstrated by reporter gene assays. Oligonucleotide mobility shift assays with the beta-RARE show a single discrete complex in normal cells, whereas tumor cells exhibit a heterogeneous set of larger complexes, which indicates that tumor cells utilize a different array of factors within the beta-RARE. Reporter gene assays with extended promoter regions indicate the presence of negative regulatory elements and/or factor binding sites that reside between -1500 and the RARE located at -59, and that the promoter is down-regulated in MCF-7 tumor cells. Our findings reveal a dichotomy: RAR-beta transcription is down-regulated in tumor cells compared with normal human mammary epithelial cells, and up-regulated in senescence.

Topics: Base Sequence; Blotting, Northern; Breast; Breast Neoplasms; Cell Line; Cellular Senescence; Chloramphenicol O-Acetyltransferase; Down-Regulation; Epithelial Cells; Epithelium; Female; Fenretinide; Humans; Luciferases; Molecular Sequence Data; Mutagenesis, Insertional; Nuclear Proteins; Oligodeoxyribonucleotides; Oligonucleotide Probes; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines.

Topics: Antigens, CD; Breast Neoplasms; Calcitriol; Carcinoma; CD59 Antigens; Cell Membrane; Cytokines; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Flow Cytometry; Fluorescent Antibody Technique; Glioma; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Membrane Glycoproteins; Ovarian Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The T47D human breast carcinoma cell line has been shown to synthesize insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) and IGF-I receptors, and to exhibit a mitogenic response to exogenous IGF-I. We have used T47D cells to investigate the regulation of IGFBPs by IGF-I and retinoic acid (RA), agents that affect cell proliferation and have been shown to regulate IGFBP levels in other cell types. Exposure of T47D cells to IGF-I resulted in the appearance of IGFBP-2, -4, and -5 in conditioned medium but had no effect on the levels of IGFBPs in Triton X-100-extracted cells. This effect was most pronounced for IGFBP-5 and was also elicited by an IGF-I analog that retains affinity for IGFBPs but not by insulin or IGF analogs that have decreased affinity for IGFBPs. Additionally, this effect was not associated with a change in IGFBP-5 messenger RNA (mRNA) levels; however, the appearance of IGFBP-5 in the conditioned medium was inhibited by an anti-IGF-I receptor antibody (alpha IR-3). RA decreased IGFBP-5 mRNA levels and cell-associated IGFBP-5 in both the presence and absence of IGF-I and inhibited the IGF-I-stimulated secretion of IGFBP-5 into T47D cell conditioned medium. These results suggest that IGF-I increases IGFBP-5 levels in the T47D cell line both through direct interaction with IGFBP-5 as well as through a receptor-mediated process that does not require direct interaction with IGFBPs. The latter results are consistent with an effect of IGF-I on a factor that may modulate an IGFBP protease activity. The inhibitory effect of RA, on the other hand, appears to be due primarily to regulation of IGFBP-5 mRNA levels. Thus, IGFBP-5 accumulation appears to be positively regulated by IGF-I, potentially at the level of susceptibility to proteolysis, and negatively regulated at the level of gene expression by RA.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Culture Media; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Receptors, Somatomedin; Somatomedins; Tretinoin; Tumor Cells, Cultured

Topics: Breast Neoplasms; Carrier Proteins; Cell Division; Culture Media, Conditioned; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Tretinoin; Tumor Cells, Cultured

Insulin-like growth-factors I and II (IGF-I, II) are potent mitogens for breast carcinoma proliferation. In extracellular fluids, most of the IGF-I and II is associated with specific IGF-binding proteins (IGFBPs). The role of these IGFBPs in IGF action is still not clear, but it has been demonstrated that these proteins may either enhance or inhibit IGF-mediated cellular effects. Synthesis and secretion of IGFBPs have been demonstrated in breast carcinoma cells. In this study, we examined retinoic acid (RA) and IGF-I modulation of IGFBP mRNA and IGFBP levels in two ER-negative human breast carcinoma cell lines. Treatment of MDA-MB-231 and MDA-MB-468 cells with RA increased the levels in conditioned media of a M(r) 42-46-kDa IGFBP, which was immunoprecipitated by an IGFBP-3 antibody. IGF-I also increased the accumulated levels of IGFBP-3 in the conditioned media of both cell lines. Both cell lines expressed high basal levels of IGFBP-3 mRNA; the addition of RA increased IGFBP-3 mRNA levels by 1.5-fold, whereas the addition of IGF-I had no effect on IGFBP-3 mRNA levels in either cell line. The difference in the magnitude of the RA enhancement of IGFBP-3 mRNA levels (1.5-fold) and RA stimulation of IGFBP-3 levels in conditioned media (3.5-4-fold) suggests that some of the effect of RA is at a posttranscriptional level. IGF-I increased the levels of IGFBP-2 and IGFBP-5 in conditioned media by greater than tenfold but had no effect on IGFBP-2 and IGFBP-5 mRNA levels, again suggesting the involvement of posttranscriptional controls. Pretreatment of MDA-MB-468 and MDA-MB-231 cells with IGF-I receptor antibody (alpha IR3) blocked the IGF-I effect on IGFBP-3 levels in the media in both cell lines and IGFBP-2 and IGFBP-5 secreted levels in MDA-MB-468 cell conditioned media. The addition of RA also blocked IGF-I stimulation of IGFBP-2 and IGFBP-5 levels. Cycloheximide treatment completely blocked the RA and/or IGF-I-mediated modulation of these binding proteins, suggesting that these agents enhance IGFBP-3, IGFBP-2, and IGFBP-5 synthesis and consequent secretion. MDA-MB-468 cells expressed IGFBP-5 mRNA, whereas both MDA-MB-231 and MDA-MB-468 expressed IGFBP-6 mRNA. RA enhanced IGFBP-6 gene expression by threefold in MDA-MB-231 cells, whereas IGF-1 had no effect on IGFBP-6 gene expression in either cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Blotting, Western; Breast Neoplasms; Carrier Proteins; Cycloheximide; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunosorbent Techniques; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and estrogen modulation of insulin like-growth factor binding protein-4 (IGFBP-4) gene expression was investigated in a number of human breast carcinoma (HBC) cell lines. Our results indicate that RA and/or estrogen modulation of IGFBP-4 mRNA levels appear to correlate with the ER-status of HBC cell lines. RA and estrogen enhanced IGFBP-4 mRNA levels in ER-positive MCF-7, RRO-I and ZR-75 cell lines and the effect of both of these agents in combination was additive. Although both individually also enhanced IGFBP-4 mRNA levels in ER-positive T47D cells, their effect in combination was antagonistic in this cell line. Transfection of human ER into ER-negative MDA-MB-231 cells conferred RA and estrogen the ability to enhance IGFBP-4 mRNA but both agents failed to concomitantly modulate IGFBP-4 levels in the CM suggesting a dual regulation by these agents at transcription/post-transcription and translation/secretion levels.

Topics: Blotting, Northern; Breast Neoplasms; Carrier Proteins; Estrogens; Female; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 4; Poly A; Receptors, Estrogen; Recombinant Proteins; RNA; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

The DNA topoisomerase enzymes are targets for the cytotoxic effects of a number of anticancer agents termed topoisomerase inhibitors. We have analysed breast cancer biopsy specimens for genetic alterations at and around topoisomerase loci in order to obtain molecular insight into factors which may determine how tumours respond to chemotherapy. We show that of 50 tumours examined, the topoisomerase II alpha locus is co-amplified in 3 cases out of 6 with erbB2 amplification and that amplification can be accompanied by high expression of topoisomerase II alpha. In our attempts to distinguish amplification from aneuploidy and define the limits of amplification, we also observed co-amplification of the retinoic acid-alpha receptor with erbB2 and topoisomerase II alpha in the same three samples. At the topoisomerase I locus on chromosome 20, we observed allelic loss in two out of 17 samples. Genetics abberations at topoisomerase loci, therefore, appear to be relatively common in breast cancer.

Topics: Aged; Aged, 80 and over; Alleles; Breast Neoplasms; Chromosomes, Human, Pair 17; DNA Topoisomerases, Type II; DNA, Neoplasm; ErbB Receptors; Female; Gene Amplification; Humans; Middle Aged; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptors, Retinoic Acid; Tretinoin

Combination of all-trans-retinoic acid (RA) with either interferon-alpha or -gamma resulted in a synergistic amplification of the anti-proliferative effect on cultured breast cancer cells. RA could be replaced by other biologically active retinoids. The synergism was also observed for the induction of 2'-5'-oligoadenylate synthetase, an enzyme which is involved in anti-viral activity of interferons and possibly in growth regulation of tumor cells. Combination of RA with interferon-gamma increased the down-regulation of specific binding sites for [125I]interferon-gamma. On the other hand interferons had no effect on the cytoplasmic binding protein for RA. Comparing all-trans- with 9-cis-RA, the latter was more effective in inhibiting tumor cell growth and in inducing synergism with interferon-gamma. This would indicate that retinoic X receptors are more important in mediating these effects than the RA receptors (RARs). This assumption is also supported by the failure of Ro-415253, a specific RAR-alpha antagonist, to reduce the synergistic interaction of RA with interferon with respect to growth inhibition.

Topics: Breast Neoplasms; Cell Division; Drug Resistance; Drug Synergism; Humans; Interferon-gamma; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Stereoisomerism; Transcription Factors; Tretinoin; Tumor Cells, Cultured

We and others have shown previously that retinoic acid (RA) selectively inhibits the growth of estrogen receptor (ER)-positive human breast carcinoma (HBC) cells and ER-negative cells are refractory to RA inhibition of growth. The ER-negative cells inherently express lower levels of RAR alpha and retinoic acid response element (RARE)-mediated RA-induced CAT activity. In this study we report that when ER-negative MDA-MB-231 cells were transfected with the ER gene they not only expressed higher levels of RAR alpha and RARE-mediated RA-induced CAT gene expression, but their growth was not inhibited by RA. Estrogen enhanced RAR alpha gene expression not only in established ER-positive cell lines but also in ER-transfected MDA-MB-231 cells. The estrogen effect appears to be direct and at the gene transcription level since it did not alter the stability of RAR alpha mRNA and cycloheximide failed to block estrogen-mediated enhancement of RAR alpha gene expression. Our data strongly suggest that ER-mediated enhancement of RAR alpha levels plays an important role in RA inhibition of HBC growth. In addition, we also report here that HBC cells appear to express a unique isoform(s) of RAR alpha which was detected only when the full-length RAR alpha cDNA was used as a probe; the RAR alpha 1 and RAR alpha 2 specific probes failed to hybridize with the HBC specific RAR alpha message.

Topics: Base Sequence; Blotting, Northern; Breast Neoplasms; Cell Division; Chloramphenicol O-Acetyltransferase; DNA Probes; Estrogens; Gene Expression; Humans; Molecular Sequence Data; Receptors, Estrogen; Receptors, Retinoic Acid; RNA, Messenger; Transfection; Tretinoin

Expression of matrix Gla protein (MGP) gene and its regulation by retinoic acid (RA) and estrogen was investigated in eight human breast cancer cell lines. The promoter region of the MGP gene contains a consensus retinoic acid response element (RARE) and the MGP gene expression has been shown to be strongly induced by RA in other systems. Our results suggest that RA negatively regulates MGP mRNA expression in human breast cancer cells that have high levels of estrogen receptors (ER), i.e. MCF-7, ZR-75 and BT474 and positively regulates its expression in cells with either no ERs, i.e. MDA-MB-468 or very low levels of ERs, i.e. T47D. This indicates that ER levels may affect RA modulation of MGP gene expression in human breast cancer cells. The inhibitory effect of RA on MGP gene expression was abolished in RRO-I, the RA-resistant MCF-7 subline. We also demonstrate for the first time that estrogen strongly induces MGP gene expression in ER-positive cells and that estrogen-mediated induction of MGP is blocked by RA even in otherwise RA-resistant cells.

Topics: Amino Acid Sequence; Breast Neoplasms; Calcium-Binding Proteins; Carcinoma; Cycloheximide; Estradiol; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Matrix Gla Protein; Molecular Sequence Data; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Tretinoin

The effect of the differentiating agent all-trans-retinoic acid on the expression of components of the milk fat globule membrane and HLA-DR by breast cancer cell lines has been examined. Effects on proliferation were also considered, to determine whether any cell surface changes were related to or independent of proliferation effects. No significant differences were observed in the expression of components detected by the milk fat globule membrane antibodies HMFG1 and HMFG2 over 8 days culture with 10(-7)-10(-9)M retinoic acid for the cell lines MCF-7, T-47 D, ZR-75, MDA-MB-231, and BT-20. In contrast , there was enhanced expression of HLA-DR by two oestrogen receptor-positive cell lines T-47 D and ZR-75 and the oestrogen receptor-negative line MDA-MB-231, with differing sensitivities. These effects were independent of inhibition of proliferation, which was only observed for oestrogen receptor-positive cell lines and for different durations of exposure. The finding of enhanced HLA-DR expression after retinoic acid treatment has not previously been reported and is of interest regarding clinical potential for the induction of tumour immunity.

Topics: Antigens, Neoplasm; Breast Neoplasms; Cell Division; Female; HLA-DR Antigens; Humans; In Vitro Techniques; Membrane Glycoproteins; Mucin-1; Tretinoin; Tumor Cells, Cultured

The dihydroxylated form of vitamin D3 (1,25-dihydroxy-D3)mediates a biological response by binding to intracellular receptors which belong to the steroid receptor superfamily. These receptors act as ligand-dependent transcription factors that bind to specific DNA sequences (reviewed in refs 6-9). We have identified two classes of vitamin D response elements that are activated either by the vitamin D receptor (VDR) alone or by heterodimers of VDR and the retinoid-X receptor-alpha (RXR-alpha). The motif GGGTGA arranged as a direct repeat with a spacing of six nucleotides or as a palindrome without spacing, or as an inverted palindrome with a 12-nucleotide spacing, confers vitamin D inducibility mediated by VDR alone. A second class of response elements, composed of directly repeated pairs of motifs (GGTCCA, AGGTCA, or GGGTGA) spaced by three nucleotides, is synergistically activated by RXR and VDR, but only in the presence of both ligands. Thus, the RXR ligand and the nature of the response element determine whether a nuclear receptor is co-regulated by RXR.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Calcitriol; DNA; Humans; Molecular Sequence Data; Osteocalcin; Receptors, Calcitriol; Receptors, Cell Surface; Receptors, Retinoic Acid; Receptors, Steroid; Repetitive Sequences, Nucleic Acid; Retinoid X Receptors; Signal Transduction; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin D

Retinoic acid (RA) strongly inhibits proliferation of the estrogen (E2)-dependent human breast cancer cell lines MCF7, T47D, and ZR75-1, but not the E2-independent and E2 receptor (ER)-negative lines MDA-MB231, MDA-MB468, BT20 and Hs578T. The specific sensitivity of the E2-dependent cell lines seems not to be caused by an inhibitory effect of RA on ER functioning since RA inhibited the proliferative response not only to E2 but also to insulin. Furthermore, endogenous RA receptors (RARs) hardly impaired transcriptional activation of an E2 responsive element-tk-CAT reporter construct. RAR alpha mRNA was highly expressed in the RA-responsive lines, but not in the unresponsive lines, except BT20. With the exception of Hs578T, also RAR beta mRNA expression was low in the unresponsive lines. While in the dependent lines and Hs578T RA activated RA responsive element-dependent transcriptional activity, this response was very low in MDA-MB231, MDA-MB468, and BT20, suggesting that the RA resistance of these latter three ER-negative lines is due to underexpression of functional RARs. Our results suggest that the loss of functional RARs may be a frequent event, leading to RA unresponsiveness of ER-negative breast cancer cells. This implies that both the steroid and retinoid receptor status of breast tumors may be used to predict a successful treatment with retinoids.

Topics: Base Sequence; Breast Neoplasms; Carrier Proteins; Cell Death; Cell Division; Drug Resistance; Estradiol; Gene Expression; Humans; Insulin; Molecular Sequence Data; Receptors, Estradiol; Receptors, Retinoic Acid; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

A novel arotinoid with a morpholine structure in the polar end group Ro 40-8757 (4-[2-[p-[(E)-2(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2- naphthyl)propenyl]phenoxy]ethyl]-morpholine) was tested for its anti-proliferative activity against nine human cancer cell lines in vitro. The lines included two estrogen receptor positive breast cancer lines (MCF-7 and ZR-75-1), two estrogen receptor negative breast cancer lines (MDA-MB-231 and BT-20), one cervix carcinoma line (KB-3-1), two lung adenocarcinoma lines (A549 and HLC-1), one large cell lung cancer line (LXFL 529) and two colorectal lines (CXF 243 and CXF 280). Proliferation of all the lines, except the two lung adenocarcinoma lines, was inhibited by lower concentrations of Ro 40-8757 than those of all-trans retinoic acid (RA) or 13-cis RA giving the same level of inhibition. The degree of inhibition of RO 40-8757 was concentration and time dependent. The arotinoid was not cytotoxic and morphological signs by differentiation were not evident in cultures treated with Ro 40-8757 for up to 2 weeks. Because this compound is active on cells such as KB-3-1 that are not inhibited by all-trans RA and because it does not bind to nuclear retinoic acid receptors, it may represent a novel class of anti-proliferative agents.

Topics: Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Division; Cell Survival; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Humans; Kinetics; Lung Neoplasms; Morpholines; Neoplasms; Receptors, Estrogen; Retinoids; Tetrazolium Salts; Thiazoles; Tretinoin; Tumor Cells, Cultured

The nuclear signaling pathways for retinoids and vitamin D differ in the specificity of the respective receptors for response elements. Two pathways for the action of both retinoic acid receptors (RARs) and vitamin D receptors (VDRs) have been identified, one being retinoid X receptor (RXR)-dependent and the other being RXR-independent. Moreover, RXRs were found to function as homodimers. In several steps we converted the retinoid specific response element of the human retinoic acid receptor beta promoter into the vitamin D/retinoic acid response element of the human osteocalcin promoter. We found that VDR homodimers only bind to the motif RGGTGA. The extended osteocalcin element also contains an imperfect direct repeat based on the motif RGGTGA spaced by three nucleotides, which is bound by RXR homodimers and activated by 9-cis-retinoic acid. The responsiveness of the osteocalcin element to all-trans-retinoic acid is mediated neither by RAR homodimers nor by RAR-RXR heterodimers. However, a VDR-RAR heterodimer binds to the osteocalcin response element and mediates activation by all-trans-retinoic acid. This heterodimer also binds to pure retinoid response elements, but it does not mediate activation by vitamin D alone. In combination with all-trans-retinoic acid, however, vitamin D enhances VDR-RAR heterodimer-mediated gene expression. This finding suggests a direct interaction between nuclear signaling by retinoic acid and vitamin D increasing the combinatorial possibilities for gene regulation by the nuclear receptors involved.

Topics: Base Sequence; Breast Neoplasms; Carrier Proteins; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Female; Humans; Liposomes; Macromolecular Substances; Molecular Sequence Data; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Protein Biosynthesis; Receptors, Calcitriol; Receptors, Cell Surface; Receptors, Retinoic Acid; Receptors, Steroid; Recombinant Proteins; Retinoid X Receptors; Retinoids; Signal Transduction; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin D

The role and regulation of the c-myc protooncogene in breast and ovarian neoplasms is receiving increased attention. The downregulation of the c-myc protooncogene by 1,25-dihydroxyvitamin D3 (calcitriol), retinoic acid (RA) and dexamethasone (Dex) is closely associated with growth inhibition in leukemic cells. Calcitriol, RA and Dex have anti-proliferative activity in breast and gynecologic carcinoma cells; however, the regulation of c-myc by these agents in breast and ovarian cancers is mostly unknown. We have addressed the regulation of c-myc in these cancers using an adaptation of a novel method which employs an immunohistochemical procedure to detect c-myc protein followed by quantification of c-myc staining with computerized image analysis. This system represents an alternative to protein product assay by Western blotting and is straightforward, rapid (1 day), can be carried out on a small scale and provides a sample size that readily facilitates statistical analysis of assay data. In MCF-7 human breast cancer cells, c-myc was suppressed 29% by 0.5 nM Dex, 45% by 0.01 nM RA and 54% by 100 nM calcitriol after 24 h of drug treatment. At the same hormone concentrations, growth was inhibited 18% by Dex, 18% by RA and 39% by calcitriol after 3 days of treatment (p < 0.05 for all hormones). Similar patterns of growth and c-myc inhibition were seen in T47D human breast cancer cells and NIH:OVCAR3 human ovarian cancer cells, with the exception of Dex in T47D cells, which caused no inhibition of c-myc or growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blotting, Western; Breast Neoplasms; Calcitriol; Cell Division; Depression, Chemical; Dexamethasone; Female; Gene Expression; Genes, myc; Humans; Immunohistochemistry; Ovarian Neoplasms; Tretinoin; Tumor Cells, Cultured

Antiproliferative activities of all-trans-retinoic acid (RA, CAS 302-79-4) and some retinoid derivatives (all-trans-retinyl-beta-D-glucuronide (RYG), methyl-(1-O-retinoyl-beta-D-glucopyranoside) uronate (MRG), all-trans-retinoic acid beta-D-galactopyranosyl ester (RGA), and all-trans-retinoic acid beta-D-glucopyranosyl ester (RGU)) were determined by microculture tetrazolium assay (MTT assay) and cell counting by Coulter Counter (CC) in breast (MCF7, MDA MB 231) and colon (SW 948) cancer cell lines of human origin. RA, MRG, RGU, and RGA (5, 25 mumol/l) were significantly more growth-inhibitory in MCF7 cells, which are known to be cellular retinoic acid-binding protein (cRABP) and cellular retinol binding protein (cRBP) positive, than in MDA MB 231 cells which are cRABP and cRBP negative. RYG was active only in MDA MB 231 cells. RA, MRG, RGA and RGU (25 mumol/l) stimulated the proliferation of SW 948 cells as determined by CC, whereas the MTT assay indicated an inhibition of cell growth.

Topics: Breast Neoplasms; Cell Division; Colorectal Neoplasms; Humans; Nitroblue Tetrazolium; Retinoids; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factor binding protein (IGFBP) gene expression and IGFBP secretion were investigated in a retinoic acid (RA)-resistant subline (RROI) of MCF-7 human breast carcinoma cells. Our results demonstrate that RRO-I cells constitutively secrete higher levels of IGFBP-3 and IGFBP-5. In addition, we found that a 5-fold increase in IGFBP-5 mRNA levels observed in RRO-I cells, which eventually leads to a similar increase in the secreted levels of IGFBP-5, was partly due to an increase in the stability of IGFBP-5 mRNA. Actinomycin D and cycloheximide differentially stabilized IGFBP-5 mRNA in MCF-7 cells but not in RRO-I cells, indicating a difference in the control of IGFBP-5 gene regulation at the level of mRNA stability in these cell lines.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Dactinomycin; Drug Resistance; Gene Expression Regulation, Neoplastic; Insulin-Like Growth Factor Binding Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; Somatomedins; Time Factors; Tretinoin; Tumor Cells, Cultured

Exposure of MCF-7 breast carcinoma cells to estradiol results in an increase in transforming growth factor alpha (TGF-alpha) synthesis and secretion. Since TGF-alpha is a potent inducer of proliferation in MCF-7 cells, the increase in TGF-alpha production by estradiol is thought to play an important role in the estrogen stimulation of growth of these cells. Retinoic acid inhibits the proliferation of MCF-7 cells and antagonizes the estrogen stimulation of growth. Addition of retinoic acid resulted in a greater than 70% inhibition of estradiol-induced TGF-alpha synthesis and secretion in MCF-7 cells. The increase in TGF-alpha mRNA expression by estradiol was also inhibited by exposure of the cells to retinoic acid. Pretreatment of the cells with retinoic acid for 24 or 72 h caused more than 50 and 90% inhibition, respectively, of the estradiol-enhanced expression of TGF-alpha mRNA. Expression of pS2 mRNA in MCF-7 cells was stimulated approximately 8-fold by estradiol. Retinoic acid treatment suppressed by greater than 80% both the basal and estradiol-induced pS2 mRNA expression. Retinoic acid modulation of the estrogen receptor gene mRNA was not responsible for the retinoic acid inhibition of the stimulation of pS2 and TGF-alpha gene expression by estradiol, since estrogen receptor gene expression was increased rather than decreased in the presence of retinoic acid. The nuclear retinoic acid receptors alpha and gamma mRNA were expressed in MCF-7 cells and its retinoic acid-resistant derivative RROI. Addition of estradiol to MCF-7 cells resulted in a decreased expression of retinoic acid receptor gamma mRNA; this reduction is prevented by the presence of retinoic acid. These results indicate that retinoic acid can inhibit estradiol-induced TGF-alpha and pS2 mRNA expression in MCF-7 cells. The suppression of TGF-alpha expression may represent one possible mechanism by which retinoic acid antagonizes the stimulation of MCF-7 proliferation by estradiol.

Topics: Breast Neoplasms; Carrier Proteins; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Humans; Receptors, Retinoic Acid; RNA, Messenger; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number of orphan receptors whose biological roles have not yet been determined. Although much has been learned in recent years about RA receptor (RAR) functions, little is known about how specific RA response programs are restricted to certain tissues and cell types during development and in the adult. It has been recently shown that RAR activities are regulated by retinoid X receptors (RXR) through heterodimer formation. In an effort to isolate and further characterize nuclear receptors that modulate RAR and/or RXR activities, we have screened cDNA libraries by using a RXR alpha cDNA probe. Two clones, COUP alpha and COUP beta, identical and closely related to the orphan receptor COUP-TF, were obtained. We show that COUP proteins dramatically inhibit retinoid receptor activities on certain response elements that are activated by RAR/RXR heterodimers or RXR homodimers. COUP alpha and -beta bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell- or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Carrier Proteins; COUP Transcription Factor I; DNA; DNA-Binding Proteins; Gene Expression Regulation; Humans; Molecular Sequence Data; Ovalbumin; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Retinoic Acid; Repetitive Sequences, Nucleic Acid; Retinoid X Receptors; Solutions; Transcription Factors; Tretinoin; Tumor Cells, Cultured

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.

Topics: Azides; Breast Neoplasms; Cyclic AMP; Estradiol; Female; Humans; Mutation; Neoplasm Proteins; Progesterone; Receptors, Estradiol; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) blocks insulin-like growth factor-I (IGF-I) stimulation of proliferation in the MCF-7 breast carcinoma cell line, and this is associated with the appearance of 42- to 46-kilodalton (kDa) IGF-binding proteins(s) (IGFBPs) in the conditioned medium (CM), in addition to the approximately 34- and 27-kDa IGFBPs present in the CM of unstimulated cells. Using immunological, biochemical, and molecular biological criteria, we have identified the 27-kDa band as IGFBP-4, the 34-kDa band as IGFBP-2, and the 42- to 46-kDa band as IGFBP-3. IGF-I alone stimulated MCF-7 cell proliferation, and this was associated with a large increase in IGFBP-2 in the CM. RA alone resulted in increased IGFBP-4 levels and the appearance of IGFBP-3 in the CM. The combination of RA and IGF-I, which resulted in decreased cellular proliferation, was associated with the appearance of IGFBP-3 in the CM at levels far exceeding those seen with RA alone. The effect of IGF-I on IGFBP-2 levels and the synergistic action of IGF-I and RA on IGFBP-3 levels in CM were blocked by alpha IR3, a monoclonal antibody to the human IGF-I receptor, indicating that these effects required signal transduction through the IGF-I receptor. IGFBP-2, -3, and -4 mRNAs were detected in unstimulated MCF-7 cells. RA increased IGFBP-3 mRNA levels, suggesting that transcriptional events contribute to the RA stimulation of IGFBP-3 appearance in CM. In contrast, the increase in IGFBP-2 protein in CM after IGF-I treatment appeared to be greater than the increase in IGFBP-2 mRNA levels. The increase in IGFBP-3 protein in CM in response to the combination of RA and IGF-I was much greater than the increase in IGFBP-3 mRNA. These results suggest that the action of RA and IGF-I in combination to increase IGFBP-3 protein in CM is principally translational or posttranslational. We speculate that RA inhibition of IGF-I-stimulated MCF-7 cell proliferation may be due to IGFBP-3, or that increased levels of IGFBP-3 in response to growth inhibition represent a compensatory response.

Topics: Breast Neoplasms; Carrier Proteins; Culture Media; Gene Expression; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Somatomedins; Tretinoin; Tumor Cells, Cultured

Growth of the human breast cancer cell line MCF-7 is known to be inhibited both by antiestrogens such as 4-hydroxytamoxifen (OHTAM) and by retinoic acid (RA). Uncloned MCF-7 cells (UNC) and two cloned sublines, one sensitive to antiestrogens (E-3) and the other resistant to them (RR), were used in this study. Growth of UNC and E-3 was inhibited by either OHTAM (10(-7) M) or RA (10(-6) M), and this inhibition could not be overcome by the simultaneous addition of estradiol. Subline RR, which was originally selected for resistance to tamoxifen, was resistant to both OHTAM and RA as measured by either growth in culture or colony forming ability. RR was resistant to RA at all concentrations tested between 10(-9) M and 10(-6) M. The inhibition of uncloned MCF-7 cells by RA was dose dependent between 10(-9) M and 10(-6) M. Subline E-3, however, exhibited a mixed response to RA. At 10(-9) M and 10(-8) M, growth was stimulated, but at 10(-7) M and 10(-6) M it was inhibited. The level of estrogen receptor was measured in the same experiment by using a whole cell assay. In the uncloned MCF-7 cultures and in both the RR and E-3 sublines the level of estrogen receptor was increased between 50 and 200% by RA. The production of plasminogen activator by MCF-7 cells is stimulated by estrogen. RA had a dual effect on plasminogen activator production. In the absence of estrogen, RA inhibited production below the unstimulated level, but in cells stimulated by estrogen, RA increased plasminogen activator production. The results reported here support possible interactions between the mechanisms by which cells respond to estrogen, antiestrogens, and retinoids.

Topics: Breast Neoplasms; Cell Division; Clone Cells; Drug Interactions; Drug Resistance; Drug Screening Assays, Antitumor; Estrogens; Humans; Plasminogen Activators; Receptors, Estrogen; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Although the androgen receptor (AR) has been detected by ligand-binding assays, there is little known about the expression and regulation of the AR gene in human breast-cancer cells. AR mRNA, measured by Northern analysis in 18 cell lines, was found to be expressed predominantly in oestrogen- and progesterone-receptor-positive (ER+, PR+) lines as a single species of approximately 10.5 kb but was also comparatively abundant in I ER- and PR-negative cell line, MDA-MB-453. Dexamethasone (Dex), Organon 2058 (Org 2058), dihydrotestosterone (DHT), and all-trans-retinoic acid (RA) down-regulated AR mRNA levels in T-47D (ER+, PR+) cells 6 hr after treatment, whereas oestradiol (E2) had no effect. In MDA-MB-453 (ER-, PR-) cells, regulation of AR mRNA by RA differed from the other cell lines: RA increased the level of AR mRNA. DHT-binding assays indicated a corresponding increase in AR protein. Transfection of the androgen-responsive mouse mammary tumour virus long-terminal repeat (MMTV LTR) linked to a chloramphenicol acetyltransferase (CAT) reporter gene was used to examine the effect of altered AR levels on androgen action. The increased level of AR following RA pre-treatment in MDA-MB-453 cells resulted in enhanced induction of CAT activity by DHT and, conversely, a decrease in the level of AR following RA pretreatment in T-47D cells resulted in reduced induction of CAT activity by DHT. These data demonstrate that AR is expressed predominantly in ER+ and PR+ cell lines and its expression is regulated by ligands also known to regulate ER or PR, including progestins and retinoids. Androgen responsiveness measured by a transfected reporter gene was altered according to the extent of up- or down-regulation of AR expression, demonstrating that responsiveness is dependent on receptor concentration.

Topics: Breast Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Ligands; Receptors, Androgen; RNA, Messenger; RNA, Neoplasm; Steroids; Tretinoin; Tumor Cells, Cultured

We recently developed the cellular model MVLN-15 in which estrogenic action can be detected by bioluminescence. Using this cellular model, we characterized the inhibitory effect of retinoic acid on the estrogen-dependent induction of luciferase transcription. We present evidence that i) the inhibitory effect of retinoic acid is not due to a simple competition between retinoic acid and estradiol for binding to the estrogen receptor, ii) a DNA sequence restricted to an estrogen-responsive element (ERE) was sufficient for the antiestrogenic effect of retinoic acid, and iii) retinoic acid does not act via a cryptic AP-1 binding site associated with this ERE. Therefore, we conclude that the antiestrogenic effect of retinoic acid is due to an inhibition of estrogen receptor activity, for example by altering the amount of estrogen receptor protein bound to the ERE or affecting the transcriptional efficiency of this complex.

Topics: Breast Neoplasms; DNA Mutational Analysis; Estrogens; Humans; Luciferases; Receptors, Estrogen; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Liarozole is a new imidazole derivative with antitumoral properties. Effects of the compound alone and in combination with all-trans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentration dependently inhibited by all-trans-retinoic acid (drug concentration resulting in 50% growth inhibition, 2 x 10(-8) M), while liarozole at 10(-5) M inhibited cell growth by only 35%. When MCF-7 cells were incubated with a combination of all-trans-retinoic acid and liarozole, the antiproliferative effect of all-trans-retinoic acid was clearly enhanced. This enhancement was dependent on the liarozole concentration and was more than 10-fold. A combination of 10(-8) M all-trans-retinoic acid and 10(-6) M liarozole resulted in a greater antiproliferative effect than that obtained with 10(-7) M all-trans-retinoic acid alone. When MCF-7 cells were incubated for 4 h with [3H]all-trans-retinoic acid, the radioactivity in the supernatant consisted of unaltered retinoid. However, when cells had been pretreated with 10(-6) M all-trans-retinoic acid overnight, they were able to substantially metabolize [3H]all-trans-retinoic acid during a subsequent 4-h incubation. High-performance liquid chromatography analysis of the supernatants revealed that the reaction products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-trans-retinoic acid in MCF-7 cells with 10(-5) M liarozole reducing the amount of polar metabolites by 87%. It is concluded that the enhancement by liarozole of the antiproliferative effects of retinoic acid on MCF-7 human breast cancer cells is probably due to inhibition of retinoic acid metabolism. Further research into these effects in MCF-7 cells as well as in other cancer cell lines will provide more information concerning the exact mechanism of action of liarozole and the use of inhibitors of retinoid metabolism in cancer treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cytochrome P-450 Enzyme Inhibitors; Drug Synergism; Female; Humans; Imidazoles; Kinetics; Tretinoin; Tritium; Tumor Cells, Cultured

We established a simplified method for the quantitative measurement of pS2 mRNA using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Expression of the pS2 gene, which is transcriptionally induced by estrogen in breast cancer cell line MCF-7 cells, can be repressed by retinoic acid (RA) in unstimulated cells. The suppressive effect of RA on pS2 mRNA was inhibited by cycloheximide.

Topics: Breast Neoplasms; Cycloheximide; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

The invasiveness of MCF-7 human mammary carcinoma cells was tested in vitro via confronting cultures with embryonic chick heart fragments. Invasive (e.g. MCF-7/6) and non-invasive (e.g. MCF-7/AZ) variants were detected. Automated image analysis of time-lapse video-microscopy recordings showed that the plasma membrane ruffling activity of the invasive MCF-7/6 variant was higher than the ruffling activity of the non-invasive MCF-7/AZ variant. Addition of all-trans-retinoic acid to the culture medium (10(-6) M) inhibited both invasion and ruffling of MCF-7/6 cells, while MCF-7/AZ cells became invasive and acquired an increased ruffling by the same type of treatment. A similar opposite effect on MCF-7 cells was not found after treatment with other ligands of the nuclear steroid/thyroid receptor superfamily. Triiodo-l-thyronine (up to 10(-5) M) and beta-oestradiol (up to 10(-6) M) did not alter the invasiveness of the cells, while dexamethasone (10(-6) M) and the pure anti-oestrogen ICI 164,384 inhibited both invasion and ruffling. Our data show that retinoic acid can modulate invasiveness in opposite directions.

Topics: Animals; Breast Neoplasms; Cell Aggregation; Cell Division; Cell Line; Cell Membrane; Chick Embryo; Dexamethasone; Estradiol; Estrogen Antagonists; Female; Humans; Myocardium; Neoplasm Invasiveness; Polyunsaturated Alkamides; Receptors, Cell Surface; Tretinoin; Triiodothyronine

Although retinoic acid has been shown to inhibit proliferation in human breast cancer cells, the mechanisms by which these effects are mediated are not known. Since several steroid hormones and their synthetic antagonists also inhibit proliferation of human breast cancer cells, we investigated the interactions between retinoic acid, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and antioestrogens in the control of human breast cancer cell proliferation in vitro. When T-47D cells, the most sensitive of six human breast cancer cell lines to the growth inhibitory effects of retinoic acid, were treated with retinoic acid and 1,25-(OH)2D3, a synergistic inhibitory effect on cell growth was observed. Retinoic acid also enhanced the growth inhibitory effect of various antioestrogens (4-hydroxytamoxifen, 4-hydroxyclomiphene or LY117018). However, retinoic acid did not affect oestradiol-induced growth stimulation. Measurement of the cellular receptors for 1,25-(OH)2D3 and oestrogen revealed no significant change in receptor levels following treatment with concentrations of retinoic acid which modulated growth. These results indicate that retinoic acid not only has direct growth inhibitory effects on breast cancer cell proliferation but also augments the effects of some other known regulators of breast cancer cell replication including 1,25-(OH)2D3 and antioestrogens. Synergism appears to involve interactions with steroid hormone action distinct from changes in steroid hormone receptor levels.

Topics: Breast Neoplasms; Calcitriol; Cell Division; Cell Line; Clomiphene; Drug Synergism; Estradiol; Estrogen Antagonists; Female; Humans; Kinetics; Pyrrolidines; Receptors, Calcitriol; Receptors, Estradiol; Receptors, Steroid; Tamoxifen; Thiophenes; Tretinoin

Retinoids induce cellular differentiation and inhibit cellular proliferation. Proliferation of human breast carcinoma cells in vitro is markedly inhibited by these compounds. On the other hand, insulin-like growth factors (IGFs) and their receptors seem to be involved in the growth of certain breast carcinoma cells by autocrine or paracrine effects. Since the effects of both IGF-I and IGF-II may be modulated by specific binding proteins (IGF-BPs) we examined the possibility that one mechanism by which retinoic acid may inhibit cancer growth is by an alteration in these BPs, thereby blocking IGF's growth effect. Retinoic acid (RA; 1 microM) completely blocked the effect of IGF-I (50 ng/ml) on enhancing proliferation of MCF-7 cells in culture. This effect of RA was not associated with any significant change in specific IGF-I-binding sites on these cells. RA induced a 3-fold increase in IGF-binding activity in conditioned medium, measured using a polyethylene glycol-immunoglobulin precipitation assay and a charcoal absorption assay. This increase was associated with the appearance of 42- and 46-kDa IGF-BPs on ligand blotting. The effect of RA on these IGF-BPs was time and concentration dependent. In contrast, during some experiments the 27- and 36-kDa BPs actually decreased. These findings support the hypothesis that RA may inhibit the growth of certain breast carcinoma cells by increasing the secretion of certain IGF-BPs, which could directly modulate the growth effect of IGFs.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Cell Division; Culture Media; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Ligands; Retinoids; Somatomedins; Tretinoin; Tumor Cells, Cultured

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (HPR) and its glucuronide derivative on the growth of MCF-7 human breast cancer cells in vitro were compared. The results indicate that the glucuronide had slightly greater potency and much less cytotoxicity than the free retinoid. At a concentration of 10(-6) M, HPR inhibited MCF-7 cell growth by approximately 25%, whereas an equimolar concentration of the glucuronide caused a 40% growth inhibition. Higher concentrations of HPR were highly cytotoxic. At a 10(-5) M concentration of the glucuronide, cell viability was 77%, and 65% of the cells were able to resume growth. On the other hand, at 10(-5) M HPR, cell viability dropped to 49%, and only 15% of the cells were capable of resuming growth. The lower cytotoxicity and higher potency of the retinoid glucuronide compared to the parent retinamide suggest that the conjugate may have a chemotherapeutic advantage over the parent compound. The apparent higher efficacy of HPR in combination with glucarate (GT) compared to the single agents could be due to increased net formation of HPR glucuronide conjugate following conversion of GT to the beta-glucuronidase inhibitor, D-glucaro-1,4-lactone. However, HPLC analysis of the cell metabolites did not show any detectable levels of the retinoid glucuronide upon treatment of MCF-7 cells with HPR and GT.

Topics: Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Synergism; Fenretinide; Glucaric Acid; Glucuronates; Humans; Tretinoin; Tumor Cells, Cultured

Fenretinide has been used orally as a chemopreventive retinoid in a trial for women at risk of developing contralateral breast cancer. The levels of fenretinide and its metabolites were measured in the breast tissue obtained at surgery from women in the trial. Fenretinide was concentrated by breast tissue. Two major metabolites were detected in the tissue extract, one co-eluting with N-(4-methoxyphenyl)retinamide (4-MPR), and the other, more polar, eluting at 17 min under the conditions used. This metabolite remains unidentified. Division of the breast tissue into epithelial cells and fat fractions revealed that fenretinide and the metabolite at the 17 min peak were concentrated in the epithelial cells, whereas 4-MPR was principally localised in the fat compartment. Thus fat may serve as a storage compartment for the retinoid.

Topics: Adipose Tissue; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Drug Evaluation; Epithelium; Female; Fenretinide; Humans; Tretinoin

Topics: Administration, Oral; Adult; Breast Neoplasms; Female; Fenretinide; Humans; Middle Aged; Neoplasms, Second Primary; Tretinoin; Vitamin E

Retinoic acid (RA) treatment of T-47D human breast cancer cells results in a rapid decrease in the concentration of progesterone receptor (PR) mRNA which causes a slow loss of cellular PR protein (Clarke, C. L., Roman, S. D., Graham, J., Koga, M., Sutherland, R. L. (1990) J. Biol. Chem. 265, 12694-12700). The mechanisms involved are unknown and this study was undertaken to determine whether the decline in PR mRNA was due to transcriptional inhibition and to evaluate the functional consequences of the RA-mediated decrease in PR. The transcription rate of the PR gene was decreased by RA, and the effect was maximal 2-3 h after treatment. Cycloheximide cotreatment was unable to relieve the inhibitory effect of RA and PR transcription suggesting that the effect was not dependent on ongoing protein synthesis. There was no effect of RA on PR mRNA half-life at the times examined (0-6 h of RA treatment). To determine the functional consequence of PR down-regulation the progestin-responsive plasmid pMSG-CAT was expressed transiently in T-47D cells which were then exposed to RA for 24 h. RA-pretreated cells were then treated with the synthetic progestin ORG 2058 and the extent of progestin stimulation of chloramphenicol acetyltransferase (CAT) activity measured. ORG 2058 treatment resulted in an induction of CAT activity which was maximal at a progestin concentration of 1 nM. Interestingly, the ability of ORG 2058 to induce CAT activity was decreased in RA-pretreated cells. The diminished progestin responsiveness of RA-pretreated cells was confirmed in separate experiments which showed that the progestin inducibility of TGF-alpha mRNA was also decreased in cells treated with ORG 2058 following pretreatment with RA for 24 h. These data demonstrate that RA decreases PR mRNA concentrations by direct transcriptional inhibition, leading to decreased cellular PR concentrations. The decreased levels of PR result in impaired responsiveness to progestins and this suggests that RA derived from dietary vitamin A may have a role in modulating cellular sensitivity to progestins.

Topics: Breast Neoplasms; Cycloheximide; Gene Expression Regulation; Half-Life; Immunoblotting; Kinetics; Progesterone; Receptors, Progesterone; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.

Topics: Base Sequence; Breast Neoplasms; Dexamethasone; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; In Vitro Techniques; Luciferases; Molecular Sequence Data; Sequence Homology, Nucleic Acid; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

The relationship between terminal cell differentiation and changes in the subcellular levels of the HER-2/neu antigen was investigated in cultured human breast cancer cells: AU-565 cells (which overexpress the HER-2/neu gene) and MCF-7 cells (which do not overexpress this gene). Differentiation was achieved by treating the cells with mycophenolic acid (MPA), phorbol 12-myristate 13-acetate (PMA), retinoic acid (RA), or the TA-1 monoclonal antibody to the extracellular domain of the HER-2/neu protein. Ten to twenty percent of the cells in untreated, sparsely growing AU-565 cultures exhibited morphological maturation characterized by large lacy nuclei surrounded by sizable flat cytoplasms. A fraction of these cells harbored milk factors such as casein and large lipid droplets. Treatment of the AU-565 cells for 4 d with 9 microM MPA, 1.6 nM PMA, 2.5 microM RA, or 1 microgram/mL TA-1 antibody resulted in cell growth inhibition and an increase in the percentage of cells (48-97%) that exhibit a mature phenotype. MCF-7 cells were also susceptible to differentiation by MPA and RA, but to a lesser degree than the AU-565 cells. Differentiation in the AU-565 and MCF-7 cells was associated with reduced immunostaining for the HER-2/neu protein at the cell surface membrane and with a transient increased diffuse immunostaining for this protein in the cytoplasm.

Topics: Blotting, Southern; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Membrane; Cell Nucleus; Cytoplasm; Genes; Humans; Mycophenolic Acid; Proto-Oncogene Proteins; Receptor, ErbB-2; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

B-1 F cells, one of the sublines established from mouse Leydig cell tumor, have been found to be maintained as an estrogen-responsive cell line under the serum-free culture conditions. Reported results that retinoids have action mechanisms similar to those of estrogen prompted us to examine the effect of retinoids on the proliferation of B-1 F cells. Stimulation of B-1 F cell growth by retinoic acid in a dose-dependent manner was observed, whereas retinoic acid did not promote but inhibited the proliferation of MCF-7 cells (estrogen- and retinoic acid receptor-positive human breast cancer cells). To elucidate the mechanism of retinoic acid-dependent cell growth, simultaneous treatment with retinoic acid and estradiol was carried out. The result did not show the additive effect on B-1 F cell growth. Hydroxytamoxifen, a potent antiestrogen, inhibited not only estradiol-dependent but also retinoic acid-dependent cell growth. However, retinoic acid failed to be associated with estrogen receptor, suggesting that retinoic acid induced enhancement of B-1 cell growth through its interaction with retinoic acid receptor. Northern blot analyses of polyadenylated RNA with complementary DNA probes for human retinoic acid receptor alpha, beta, and gamma revealed the presence of transcripts encoded by retinoic acid receptor alpha gene in B-1 F cells. These results would suggest that enhancement of the B-1 F cell growth is mediated through interaction of retinoic acid with retinoic acid receptor alpha. This stimulatory activity is inhibited by estrogen receptor complexed with hydroxytamoxifen.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cell Division; Cells, Cultured; Culture Media; DNA Probes; Estradiol; Female; Humans; Kinetics; Leydig Cell Tumor; Male; Mice; Neoplasm Proteins; Receptors, Retinoic Acid; RNA, Messenger; Testicular Neoplasms; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.

Topics: Acetamides; Antigens, Neoplasm; Breast Neoplasms; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Colforsin; Dimethylformamide; Fluorescent Antibody Technique; Humans; Membrane Glycoproteins; Mucin-1; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) inhibits proliferation of numerous breast carcinoma cells and prevents estrogen stimulation of growth of several estrogen receptor (ER)-positive cell lines. RA inhibition of human breast carcinoma cell proliferation is associated with marked inhibition of the synthesis of a Mr 39,000 protein in the ER-positive human breast carcinoma cell lines investigated. Inhibition of the synthesis of the Mr 39,000 protein occurred within 24 h of RA addition and coincides with the onset of inhibition of cellular proliferation. Increasing the dose of RA results in increasing inhibition of Mr 39,000 synthesis. RA does not inhibit the proliferation of the ER-negative human breast carcinoma cell line MDA MB-231; synthesis and inhibition of the Mr 39,000 protein is not noted in this cell line. Tamoxifen, which inhibits ER-positive breast carcinoma proliferation, moderately inhibits Mr 39,000 synthesis, while a concentration of difluoromethylornithine which inhibits cellular proliferation by greater than 50% does not affect Mr 39,000 protein synthesis. Thus, inhibition of the Mr 39,000 protein appears not to be simply related to the cessation of cellular proliferation.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Molecular Weight; Neoplasm Proteins; Receptors, Estrogen; Time Factors; Tretinoin; Tumor Cells, Cultured

Anchorage-dependent and -independent MCF-7 cell growth was dose-dependently inhibited by retinoic acid (RA) but was insensitive to TGF-beta (from 1 to 100 pM). Growth of MCF-7 monolayer cultures was inhibited (50%) when exposed to 10(-6) M RA. RA was unable to completely inhibit MCF-7 cell proliferation, as concentrations above 10(-6) M were rapidly cytotoxic. However, the combination of TGF-beta and RA resulted an increase in RA inhibitory effect on MCF-7 monolayer growth and a 80% reduction in colony formation in soft agar. These results demonstrate that although TGF-beta does not inhibit the growth of MCF-7 cells, it potentiates the antiproliferative effect of RA, suggesting that it may play a part, albeit indirect, in the regulation of MCF-7 cell growth.

Topics: Breast Neoplasms; Cell Division; Drug Synergism; Humans; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Gene Expression; Humans; Pregnenediones; Receptors, Progesterone; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid alone has no effect on the human breast cancer cell line BT-20 but can amplify the antiproliferative action of interferon-gamma (IFN-gamma). In our system ornithine decarboxylase (ODC) activity correlates well with growth rate; it was investigated whether the antiproliferative effects of IFN-gamma and IFN-gamma plus retinoic acid could be attributed to suppression of ODC activity. The ODC inhibitor difluoromethylornithine (DFMO), which is active as a single agent did not enhance growth inhibition induced by the biological response modifiers. The substitution of the BT-20 cells with putrescine, the product of the enzymatic reaction mediated by ODC, reversed DFMO induced antiproliferative action. On the other hand putrescine did not affect the proliferation of BT-20 cells treated with interferon alone or in combination with retinoic acid.

Topics: Biogenic Polyamines; Breast Neoplasms; Cell Division; Eflornithine; Female; Humans; Interferon-gamma; Ornithine Decarboxylase Inhibitors; Tretinoin; Tumor Cells, Cultured

Concurrent with a phase-II trial of 4HPR in patients with various cancers, we studied the plasma pharmacokinetics of both 4HPR and its major metabolite 4MPR as well as the effect of 4HPR administration on plasma retinol concentrations using a simple, specific and sensitive HPLC procedure. Initial estimates of plasma pharmacokinetic parameters after oral administration of 4HPR (300 mg/day) [corrected] in 3 cancer patients were the following: 4HPR, t beta 1/2 = 13.7 hr, AUC = 3.49 micrograms.hr/ml, CL = 56.57 L/hr/m2; 4MPR, t beta 1/2 = 23.0 hr, AUC = 1.15 micrograms.hr/ml, CL = 239.29 L/hr/m2. We also found that oral administration of 4HPR resulted in a rapid, profound and significant reduction in plasma retinol concentrations. The mean plasma retinol concentrations for 9 patients decreased 60% from baseline to below 200 ng/ml within 1-2 weeks of 4HPR dosing initiation. In addition, there was a concurrent, significant reduction in plasma retinol-binding protein levels in these patients. The mechanism whereby 4HPR reduces plasma retinol levels in vivo has not been determined. The addition of 4HPR to pooled human plasma at 37 degrees C in vitro did not reduce endogenous retinol levels, suggesting no direct chemical interaction between these 2 retinoids.

Topics: Administration, Oral; Breast Neoplasms; Chromatography, High Pressure Liquid; Drug Evaluation; Fenretinide; Humans; Melanoma; Mycosis Fungoides; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Tretinoin; Vitamin A

Both retinoic acid and 17 beta-estradiol formed covalent bonds with proteins of the human breast cancer cell line MCF-7. Two-dimensional gel patterns of the labeled proteins were unique for each ligand. There were four major retinoylated proteins in MCF-7 consisting of two doublets with molecular masses of 37 kDa and 20 kDa. These proteins were designated 37a, 37b, 20c, and 20d. The extent of retinoylation was very low in a 55 kDa protein that we previously identified in the human myeloid leukemia cell line HL60 [Takahashi, N. and Breitman, T. R. (1989) J. Biol. Chem. 264, 5159-5163]. These results indicated that the protein substrates for retinoylation may vary among cell-types. About 10 proteins were labeled from 17 beta-estradiol. Two of these proteins had mobilities that were identical to the retinoylated proteins 37a and 20c. These results indicate that in MCF-7 cells there are two proteins that can be retinoylated and labeled from estradiol. The demonstration that some ligands of the steroid/thyroid receptor family are covalently linked to cellular proteins suggests new mechanisms for the many effects of these agents on cells.

Topics: Breast Neoplasms; Electrophoresis, Gel, Two-Dimensional; Estradiol; Humans; Neoplasm Proteins; Tretinoin; Tumor Cells, Cultured

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.

Topics: Breast Neoplasms; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; RNA, Messenger; Time Factors; Transforming Growth Factors; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Cultured breast cancer cells express on their surface glycoproteins which are recognized by the peanut lectin (PNA). The proliferation of these cells (ZR-75.1 and 734-B) was inhibited by PNA. A mammary carcinoma cell line (BT-20) which does not react with PNA was not affected by this lectin. The combination of PNA with either retinoic acid or 4-hydroxy-tamoxifen led to an additive amplification of the antiproliferative activity. Also interferon-gamma showed in combination with PNA an improved growth-inhibitory action.

Topics: alpha-Fetoproteins; Asialoglycoproteins; Breast Neoplasms; Cell Division; Fetuins; Humans; Interferon-gamma; Lectins; Peanut Agglutinin; Tamoxifen; Tretinoin; Tumor Cells, Cultured

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).

Topics: Breast Neoplasms; Cell Division; Female; Humans; Retinoids; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Membrane binding sites for peanut lectin or peanut agglutinin (PNA) were investigated in the established mammary carcinoma cell lines MCF-7, 734-B, ZR-75.1 and BT-20. The determination of PNA binding sites was performed in a flow cytometer after staining with fluorescein(FITC)-labeled PNA. It appeared that only the estrogen-sensitive cell lines exhibited PNA binding sites, whereas the hormone-insensitive cell line BT-20 was clearly negative. Steroid hormones, when administered singly to the cells in physiological concentrations (10(-9)-10(-8) M) had no effect on PNA binding expression. Only the combination of estradiol and progesterone together increased PNA binding sites. Pharmacological doses (10(-6) M) of medroxyprogesteroneacetate (MPA) and dexamethasone increased the number of binding sites, whereas retinoic acid decreased them. A preliminary characterization of the binding sites revealed that they have high capacity and moderate affinity for PNA (KD greater than 10(-7) M). FITC-PNA binding could be inhibited selectively by fetuin (greater than 10(-5) M) and by galactose (greater than 10(-2) M). Cytosol from MCF-7 cells and from some primary breast cancer specimens were able to decrease PNA binding to the surface of 734-B cells.

Topics: Breast Neoplasms; Cell Line; Dexamethasone; Female; Fluorescein-5-isothiocyanate; Fluoresceins; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Receptors, Mitogen; Steroids; Thiocyanates; Tretinoin

The ability of all-trans-retinoic acid, 13-cis-retinoic acid, the free acid of etretinate (RO 10-1670), the 'arotinoid' RO 13-6298 and its free acid RO 13-7410 to affect the growth of T47D human breast cancer cells in vitro was investigated. The growth of T47D cells was inhibited by all of the retinoids tested, with the arotinoids being up to 100 times more effective than all-trans-retinoic acid. The presence of cellular retinoic acid binding protein (cRABP) was indicated by the cellular uptake of [3H]all-trans-retinoic acid. Maximum binding was 460 fmol/micrograms DNA. All of the retinoids with a polar terminal free carboxyl group readily competed for the binding sites, but none of the retinoids competed for the estrogen or progesterone receptor. Co-treatment of the T47D cells with 0.1 microM all-trans-retinoic acid and either tamoxifen (1 microM) or hydroxytamoxifen (10 nM or 0.1 microM) produced an additive effect on growth inhibition. No such additive effect was observed when T47D cells were co-treated with arotinoids and antiestrogens. The results showed that the T47D cells can serve as a useful model in vitro to test the effects of the synthetic retinoids and antiestrogens on steroid receptor-positive human breast cancer.

Topics: Binding, Competitive; Breast Neoplasms; Carrier Proteins; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Estrogen Antagonists; Female; Humans; Receptors, Retinoic Acid; Retinoids; Tamoxifen; Time Factors; Tretinoin

A combination of retinoic acid (RA) and human recombinant DNA-derived interferon-gamma (Hu-IFN-gamma) was tested with respect to the growth inhibitory action on several human mammary carcinoma cell lines (ZR-75.1, 734-B, MCF-7, and BT-20), a human lung carcinoma cell line (CCL-185), and a human laryngeal carcinoma cell line (HEP-2). The mammary carcinoma cell lines were all sensitive to Hu-IFN-gamma, and 2 of them (ZR-75.1 and 734-B) were also affected by RA. The combination of both substances led to a pronounced synergistic amplification of growth inhibition in ZR-75.1 and 734-B cells. RA also increased the antiproliferative activity of Hu-IFN-gamma in the RA-resistant BT-20 cells and to a less pronounced degree in MCF-7 cells. In contrast to these findings, no synergistic effects were observed between Hu-IFN-gamma and RA in CCL-185 and HEP-2 cells. Human recombinant DNA-derived interferon-alpha 2 amplified the action of RA only in BT-20 cells, but it did not act synergistically with RA in the other cell lines tested.

Topics: Breast Neoplasms; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Drug Resistance; Drug Synergism; Female; Humans; Interferon-gamma; Recombinant Proteins; Tretinoin

The synthetic retinoid 4-hydroxyphenylretinamide (HPR) showed antiproliferative effect on cultured human breast cancer cells, which were sensitive to retinoic acid (RA) too. Investigation of the cell cycle by flow cytophotometry showed a significant increase of cells in the S-phase of the cell cycle after 24 hours of RA treatment, whereas HPR did not show this effect. After 4-7 days of retinoid treatment, the percentage of resting cells increased. The influence on the cell cycle and the antiproliferative effect were more pronounced for RA than for HPR treatment in both cell lines investigated (ZR-75.1 and 734 B).

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cells, Cultured; Female; Fenretinide; Humans; Tretinoin

After ethyl methane sulfonate mutagenesis of the mammary carcinoma cell line, MCF-7, we have isolated three clones, U-2, U-3, and U-9, resistant to retinoic acid. These three clones showed more than a 1000-fold higher level of resistance to retinoic acid than the parental MCF-7 cells when assayed by colony formation in monolayer culture system or by growth curves. The three resistant clones showed a 200-fold higher resistance to 13-cis-retinoic acid, about 10-fold higher resistance to retinol, and about 2-fold higher resistance to retinyl acetate, respectively, than MCF-7. Binding of [3H]retinoic acid or [3H]-retinol to a cellular fraction in situ showed apparent decrease of the specific binding of retinoic acid in U-2, but there was no such specific fraction bound to retinol in U-2 and MCF-7. Sucrose gradient analysis with cytoplasmic fraction showed little, if any, cellular retinoic acid-binding protein in U-2, but a significant amount of the cellular retinoic acid-binding protein could be found in MCF-7. By contrast, there was no activity of cellular retinol-binding protein in both MCF-7 and U-2. The sensitivity or resistance of mammalian cells in culture to retinoic acid is discussed in relation with cellular binding activity for vitamin A.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line; Clone Cells; Drug Resistance; Female; Humans; Karyotyping; Mutation; Receptors, Retinoic Acid; Tretinoin; Vitamin A

The cellular content of receptors for retinol (CRBP) and retinoic acid (CRABP) was measured in 148 human mammary carcinomas. High levels of CRABP were found in lobular carcinomas while those of the papillary subgroup had low levels of the receptor. Intermediate values for CRABP were observed for ductal, colloid and medullar carcinomas. The cellular levels of CRBP were high in ductal carcinomas and low in papillar carcinomas. A positive correlation was observed between the receptor for estradiol and CRABP. However, no significant correlation was found between the tumor cell DNA pattern and the content of CRABP and CRBP respectively. Recurrence of disease could be predicted by the nodal status and the level of estradiol receptor while the DNA pattern and the content of receptors for retinoic acid and retinol failed.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Papillary; Carrier Proteins; DNA, Neoplasm; Female; Humans; Middle Aged; Neoplasm Proteins; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin

Using established breast cancer cell lines in a cell culture model we studied the growth effect of retinoic acid (RA) alone or in combination with the antiestrogen 4-hydroxytamoxifen (OHT). Cytoplasmic 3HRA binding sites were determined by sucrose density gradient centrifugation analysis. Of the three cell lines Hs578T, BT 20, and 734 B only the last showed a significant amount of specific RA binding (10(5) sites/cell). This cell line showed a dose dependent decrease in proliferation after a long-term incubation with RA whereas the 3H-thymidine uptake was highly significantly increased after incubation with 10(-6)M of RA for 20 hr. Growth inhibition was not further increased by the addition of OHT (10(-6) M), but the increase in thymidine incorporation due to RA was neutralized by OHT. Hs578T and BT 20 cells were not affected by any of the treatments. The different action of RA on proliferation and thymidine incorporation suggests a cell cycle specific mechanism.

Topics: Breast Neoplasms; Carrier Proteins; Cell Division; Cell Line; Cytosol; Estrogen Antagonists; Female; Humans; Neoplasm Proteins; Receptors, Retinoic Acid; Tamoxifen; Thymidine; Tretinoin

Two sublines resistant to the growth-inhibitory effects of retinoic acid (RA) have been isolated from the parental Hs578T wild-type (W.T.) human breast cancer cell line. These sublines (Hs578T-R-1 and Hs578T-R-2) have been growing normally in 10 microM RA during more than 18 months, and their RA-resistant phenotype has remained stable after the removal of RA. The resistance is specific for RA, since their growth is still inhibited by retinol. The intracellular incorporation of [3H]RA is not deficient in the RA-resistant sublines. Cytoplasmic RA-binding protein (cRABP) is present in Hs578T-R-1 and in Hs578T-R-2 and is not different in terms of maximum binding capacity or binding affinity from cRABP in Hs578T (W.T.). These results indicate that RA resistance in these sublines is not secondary to a defect of RA uptake or of binding of RA to cRABP; the resistance may result from a defect distal to binding to cRABP, or alternatively, cRABP may not mediate this effect of RA.

Topics: Binding, Competitive; Breast Neoplasms; Carrier Proteins; Cell Division; Cell Line; Cytoplasm; Drug Resistance; Female; Humans; Neoplasm Proteins; Receptors, Retinoic Acid; Tretinoin

In vitro and in vivo investigations have shown that nontoxic treatment with vitamin A (retinol) has an inhibitory effect on the growth of malignant cells. The tumorigenic CaMa -15 cell line responds to both retinol and retinoic acid under both anchorage-dependent and anchorage-independent conditions, reducing growth or colony formation by at least 50%. To date, there have been few studies on the effects of vitamin A on xenotransplanted neoplastic cells. Twenty-five adult female nude rats (rnu/rnu) were inoculated in the inguinal fat pad with 10(6) CaMa -15 cells, a tumorigenic epithelial cell line. The rats were divided into three groups: ten high dose (3 mg retinol/day i.p.); five low dose (30 micrograms retinol/day i.p.); and ten controls (corn oil i.p.). All animals were housed in specific-pathogen-free conditions and permitted access to sterile laboratory chow (5.4 micrograms retinol/g chow) and water ad libitum. Rats were sacrificed at 21 days after inoculation. Onset of tumor development occurred between Days 9 and 13 in all groups. Tumors grew progressively and were reduced in mean diameter by 26% (p = less than 0.05) with high-dose retinol and 44% (p = less than 0.02) by low-dose treatment. No clinical signs of vitamin A toxicity were apparent. Necropsy and radiological examination revealed no evidence of toxic effects or metastases. These results indicate that vitamin A can reduce the growth of xenotransplanted tumorigenic cells at nontoxic levels in T-cell-deficient hosts. The nude rat offers a potential model to study the inhibitory effects of retinoids on xenotransplanted cancers.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line; Female; Humans; Neoplasm Transplantation; Rats; Rats, Mutant Strains; Transplantation, Heterologous; Tretinoin; Vitamin A

Studies have suggested that both natural and synthetic retinoids have extensive chemopreventive activity against a variety of carcinogens in vivo and in vitro. We have previously shown that growth of human breast cancer cells can be inhibited by retinoids, and retinoic acid-binding proteins have been demonstrated in these cell lines and tumor biopsies. We studied the activity of 13-cis-retinoic acid in the treatment of 18 patients with advanced breast cancer refractory to standard cytotoxic and/or endocrine therapy. Patients began on 0.5 mg/kg and escalated to 8 mg/kg over a one-month period unless toxicity (dry skin, dry mucosa, cheilitis, conjunctivitis) forced dose reduction. All these toxicities responded promptly to dose reduction. Four patients exhibited drug related hypercalcemia, 2 complained of severe earache and several had nausea, vomiting and abdominal cramping. There were no objective responses as defined by standard criteria. One patient with thrombocytopenia secondary to documented marrow involvement demonstrated a recovery of platelet count from 9000 to 110,000. 13-cis-Retinoic acid is not of apparent value in women with heavily pretreated breast cancer.

Topics: Breast Neoplasms; Drug Administration Schedule; Drug Evaluation; Female; Humans; Isomerism; Isotretinoin; Neoplasm Metastasis; Tretinoin

The morphological effects of treatment with the natural retinoids were studied using scanning electron microscopy (SEM) utilizing both secondary electron imaging and backscatter electron imaging. Three human cell lines originally isolated from metastatic infiltrating adenocarcinomas of the breast and one cell line isolated from human milk were treated with various concentrations of retinol and retinoic acid. Growth inhibition curves showed that all three malignant cell lines were moderately to extremely sensitive to treatment with both retinoids. The nontumorigenic cell line, HBL-100, derived from human milk was only slightly or not affected by treatment. Morphological changes with retinoid treatment varied with the cell lines examined. Two tumorigenic cell lines, MDA-MB-231 and CaMa-15, showed alteration in cell shape. CaMa-15 and MCF-7 showed a loss in number of surface microvilli while MDA-MB-231 gained microvilli. HBL-100 cells exhibited an increased number of pits or lacunae on the cell surfaces with treatment. These findings suggest that treatment with retinoids does not result in uniform morphological changes and that the changes observed vary with the cell line examined.

Topics: Breast; Breast Neoplasms; Cell Division; Cell Line; Female; Humans; Microscopy, Electron, Scanning; Tretinoin; Vitamin A

Topics: Antineoplastic Agents; Breast Neoplasms; Humans; Interferons; Neoplasms; Prognosis; Time Factors; Tretinoin

The effects of various retinoids on the proliferative capacities and on the synthesis of DNA, RNA, and protein have been investigated in MCF-7 mammary carcinoma cells in culture. Of the various retinoids tested, retinoic acid revealed maximum activity in inhibiting cell proliferation and thymidine incorporation. The degree of inhibition of cell proliferation by the various retinoids paralleled their capacity to inhibit thymidine incorporation, suggesting suppression of DNA synthesis as a primary cause of restriction of cell growth by these compounds. Two nonepithelial human cell lines were tested for sensitivity to retinoids, and showed diminished responses compared with MCF-7 cells. This suggests a correlation between the ability of retinoids to exert control of differentiation and cell proliferation for a given cell type. Reversibility of the effect of retinoid treatment, high cell viability, and lack of retinoid-induced lysosomal enzyme release, as shown in our studies, indicate that cytotoxicity may be excluded as a cause of decreased cell proliferation and inhibition of thymidine incorporation by retinoids.

Topics: Breast Neoplasms; Cell Division; Cell Line; DNA; Female; Humans; Protein Biosynthesis; RNA; Tretinoin; Vitamin A

A specific retinoic acid-binding protein was demonstrated by sucrose density gradient centrifugation and saturation binding analysis in MCF-7 human breast cancer cells. In contrast, retinol-binding protein could not be detected in this cell line. By Scatchard analysis, this retinoic acid-binding protein was found to have a dissociation constant (Kd) of 154 nM and to bind a maximum of 14 pmoles of [3H] retinoic acid per milligram of cytoplasmic protein. Experiments with intact attached cells revealed the Kd to be 125 nM, which is very close to the value obtained for cytoplasmic extract. The binding of [3H] retinoic acid was abolished by unlabeled retinoic acid. Retinal and alpha-retinoic acid competed for binding sites but were less potent than unlabeled retinoic acid. Retinol, retinyl acetate, and the analog Ro 10-9359 showed little of no competition for the retinoic acid-binding site. A specific retinoic acid-binding protein was also demonstrated by gel electrophoresis. The presence of retinoic acid binding protein in MCF-7 cells suggests that the biologic effects of retinoic acid may be mediated by this specific protein.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line; Cytosol; Female; Humans; Kinetics; Receptors, Retinoic Acid; Retinol-Binding Proteins; Structure-Activity Relationship; Tretinoin

A technique for reproduction and quantitative determination of human cellular retinoic acid-binding protein (CRABP) activity in breast tissue specimens is described. A multiphasic polyacrylamide disc gel electrophoresis system (operative at pH 10.2) was adapted for this purpose. This technique allows, after incubation with tritiated retinoic acid (RA) overnight, the separation of the specific CRABP activity from the nonspecific serum-originated binding activity and from the free RA. Previous purification of the tissue cytosols is therefore not necessary. The same assay method was also used for the determination of the molecular weight (Ferguson plot, m.w. 13,000) and the dissociation constant Kd (2.5 x 10(-7) M) of mammary CRABP. The activity in tissue cytosol, stored at -70 degrees, was found to be stable for at least 3 months. Results from 88 breast tissue specimens of different pathological degree are presented. CRABP activity was found in all tissue categories with progressively increasing amounts from normal tissue to breast cancer. The activity in the cancer tissues (14.85 +/- 12.05 pmol RA bound per mg soluble protein: N = 27) was significantly different (p less than 0.001) from the activity determined in tissue with simple dysplasia without epithelial proliferation [4.3 +/- 2.2 (S.D.) pmol RA bound per mg protein; N = 30]. It is possible that in the cases where high amounts of CRABP activity are found in dysplastic and preneoplastic tissue, a high risk for breast cancer development exists. Therefore, CRABP is tentatively proposed as a dedifferentiation and/or proliferation marker.

Topics: Adolescent; Adult; Age Factors; Aged; Breast; Breast Neoplasms; Carrier Proteins; Cytosol; Female; Humans; Kinetics; Middle Aged; Molecular Weight; Neoplasm Proteins; Receptors, Retinoic Acid; Tretinoin

Vitamin A and its analogues (retinoids) regulate the differentiation of epithelial tissues. Retinoids inhibit the induction of rat mammary cancers by carcinogens in vivo, and cellular binding proteins for retinoids have been demonstrated in some human breast cancer samples. In this study, we examined the model system of human breast cancer cell lines in long-term tissue culture for effects of retinoids on growth and for the presence of cellular retinoid binding proteins. Retinoic acid and retinol inhibit the growth of of MCF-7, Hs578T, and ZR-75-B cell lines. Retinoic acid is more potent than retinol in this regard: 50% growth inhibition is achieved by 6 nM retinoic acid in ZR-75-B and by 700 nM in MCF-7 and Hs578T, whereas 5-8 muM retinol is required in all three cell lines. The time to onset of growth inhibition varies markedly between cell lines and is not related to cell density or doubling time. Retinoic acid increases the doubling time of MCF-7 and ZR-75-B by two- to threefold, but causes cell death in Hs578T. The growth inhibition is reversible in every cell line by removal of retinoic acid. Specific and distinct binding of [(3)H]retinoic acid and [(3)H]retinol is present in cytosols of MCF-7 and Hs578T cells as assessed by sucrose density gradient centrifugation. In ZR-75-B, [(3)H]retinoic acid binding was present, but no binding of [(3)H]retinol was detectable. This study reveals that retinoids may play an important role in the regulation and treatment of human breast cancer and that human breast cancer cell lines represent a useful model to study this role.

Topics: Breast Neoplasms; Cell Division; Cytosol; Female; Humans; Retinol-Binding Proteins; Tretinoin; Vitamin A

Topics: Breast; Breast Neoplasms; Cell Division; Cell Line; Dose-Response Relationship, Drug; Humans; Melanoma; Neoplasms, Experimental; Time Factors; Tretinoin; Vitamin A

Seventy-five specimens of human breast tissue were checked for the presence of cellular retinoic acid-binding protein (cRABP). Fifty-two percent of the primary carcinomas and 43% of the dysplastic breast lesions (stage MII) contained detectable amounts of crabp, whereas no cRABP was found in normal tissue. Sucrose gradient centrifugation and electrophoresis on agarose were used for analysis of the presence of cRABP. The cRABP of human origin (normal uterus and neoplastic mammary tissue) differed in its mobility in agarose electrophoresis from that of rat testis cRABP.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Female; Humans; Male; Neoplasm Proteins; Precancerous Conditions; Rats; Testis; Tretinoin; Uterus; Vitamin A

Topics: Breast Neoplasms; Papilloma; Retinol-Binding Proteins; Skin; Structure-Activity Relationship; Trachea; Tretinoin; Vitamin A

Topics: Age Factors; Animals; Breast; Breast Neoplasms; Carcinoma; Epithelial Cells; Epithelium; Fetus; Humans; Lung; Lung Neoplasms; Molecular Weight; Neoplasm Proteins; Protein Binding; Rats; Tretinoin; Vitamin A