cytochrome-c-t and Breast-Neoplasms

Cancer is a disease characterized by a very little apoptosis, ie, genetically programmed cell death. Aberrations in apoptotic pathways are central to tumorigenesis, tumor progression, and overall tumor growth and regression in response to chemotherapy. It is now increasingly accepted that chemotherapeutic drug efficacy is partially related to its ability to induce apoptosis. Apoptosis, therefore, represents not only a vital target in cancer therapy but also a unique biomarker opportunity that has thus far been largely unexploited. In response to therapy, tumor cells undergo apoptosis and release their cellular components in the circulation. As such, these materials may serve as biomarkers to assess response. Apoptosis markers in breast cancer include circulating soluble FasL, granzyme B, and cytochrome c that increase following chemotherapy. Unfortunately, there is a paucity of information in the literature with respect to this approach. As such, large-scale prospective studies are clearly needed to validate this approach and more fully elucidate clinical usefulness.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Carcinogenesis; Cytochromes c; Disease Progression; Fas Ligand Protein; Female; Gene Expression Regulation, Neoplastic; Granzymes; Humans; Neoplasm Proteins; Signal Transduction

As one of the frequent malignancies, breast cancer (BCa) is the foremost reason for cancer-related deaths among women. The role of Human papillomavirus (HPV) in chemoresistance has rarely been investigated in previous studies. The current study sets out to the possible role of HPV in BCa chemoresistance. In this research, 90 BCa tissue and 33 normal breast tissue were collected. We evaluated the presence of the HPV genome along with the viral (E2, E6, E7) and cellular gene expression associated with cell resistance to death. Statically significant differences in the prevalence of HPV between the BCa group (25.2% or 23/90) and the control group (21.8% or 7/32) were not found. HPV-16 and HPV-18 genotypes were the abundant HPV genotypes. Resistance to the Adriamycin-Cyclophosphamide (AC), paclitaxel regimen was elevated in the HPV- group (56/70) in comparison to the HPV+ group (14/70). Nevertheless, there was no significant difference in the prevalence of resistance to AC + paclitaxel + triple-negative breast cancer combination therapy between the HPV+ group (9/20) and in the HPV- group (11/20). In the BCa group in contrast to the control group, the expression level of Bcl-2, BCL-XL, and c-IAP2 demonstrated a significant decrease, while, the expression level of cytochrome C and caspase 3 was significantly increased. This study suggests that HPV infection might contribute to BCa chemoresistance through disrupt cellular genes involved in cell death.

Topics: Breast Neoplasms; Caspase 3; Cytochromes c; Drug Resistance, Neoplasm; Female; Human Papillomavirus Viruses; Humans; Oncogene Proteins, Viral; Paclitaxel; Papillomaviridae; Papillomavirus Infections; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

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

Cytochrome c (Cyt c) is a key protein that is needed to maintain life (respiration) and cell death (apoptosis). The dual-function of Cyt c comes from its capability to act as mitochondrial redox carrier that transfers electrons between the membrane-embedded complexes III and IV and to serve as a cytoplasmic apoptosis-triggering agent, activating the caspase cascade. However, the precise roles of Cyt c in mitochondria, cytoplasm and extracellular matrix under normal and pathological conditions are not completely understood. To date, no pathway of Cyt c release that results in caspase activation has been compellingly demonstrated in any invertebrate. The significance of mitochondrial dysfunctionality has not been studied in ductal carcinoma to the best of our knowledge. We used Raman spectroscopy and imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo surgically resected specimens of human breast tissues, and in vitro human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231). We showed that Raman imaging provides insight into the biology of human breast ductal cancer. Here we show that proper concentration of monounsaturated fatty acids, saturated fatty acids, cardiolipin and Cyt c is critical in the correct breast ductal functioning and constitutes an important parameter to assess breast epithelial cells integrity and homeostasis. We look inside human breast ducts by Raman imaging answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the extracellular matrix around the cancer duct during cancer development in situ. Our results show that human breast cancers demonstrate a redox imbalance compared to normal tissue. The reduced cytochrome c is upregulated in all stages of cancers development. The results of the paper shed light on a largely non-investigated issues regarding cytochromes and mitochondrial function in electron transfer chain. We found in histopathologically controlled breast cancer duct that Cyt c, cardiolipin, and palmitic acid are the main components inside the lumen of cancerous duct in situ. The presented results show direct evidence that Cyt c is released to the lumen from the epithelial cells in cancerous duct. In contrast the lumen in normal duct is empty and free of Cyt c. Our results demonstrate how Cyt c is likely to function in cancer developme

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cytochromes c; Female; Humans; In Vitro Techniques; MCF-7 Cells; Mitochondria; Oxidation-Reduction; Spectrum Analysis, Raman

Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response.. The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide.. Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin.. NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.

Topics: Antineoplastic Agents; Apoptosis; Arylamine N-Acetyltransferase; Breast Neoplasms; Caspase 8; Cell Death; Cytochromes c; Female; Humans; Isoenzymes; Necroptosis

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cytochromes c; Female; Humans; Iridoid Glucosides; Mitochondria; Protein Processing, Post-Translational; Reactive Oxygen Species

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Benzoates; Breast Neoplasms; Cell Cycle Checkpoints; Cytochromes c; Female; Gold; Humans; MCF-7 Cells; Phosphines; Reactive Oxygen Species; Sulfhydryl Compounds

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Cytochromes c; Cytosol; Female; Glycoproteins; Humans; MCF-7 Cells; Protein Binding

The main objective of this article was to evaluate the association of voltage-dependent anion channel 1 (VDAC1) with Cytochrome C (Cytc) expression, various clinicopathological features, and prognosis in breast cancer (BC) patients. Meanwhile, the correlation of Cytc expression with various clinical features and 5-year disease-free survival (5-DFS) of BC was also investigated.. Expression of VDAC1 is conversely associated with Cytc in BC (. VDAC1 was elevated in BC tissues and conversely associated with Cytc. Detection of VDAC1 may provide guidance for the poor prognosis of BC, especially TNBC.

Topics: Breast Neoplasms; Cell Movement; Cell Proliferation; Cytochromes c; Disease-Free Survival; Female; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; MCF-7 Cells; Middle Aged; Multivariate Analysis; Prognosis; Receptor, ErbB-2; Triple Negative Breast Neoplasms; Voltage-Dependent Anion Channel 1

Invasive ductal carcinoma (IDC) is the most recurrent cancer, accounting for 80% of all breast cancers worldwide. Originating from the milk duct, it eventually invades the fibrous tissue of the breast outside the duct, proliferation takes 1-2 months for each division. Quinacrine (QC), an FDA-approved small molecule, has been shown to have anti-cancer activity in numerous cancerous cell lines through diverse pathways; ultimately leading to cell death. Here, we have investigated the mode of action of QC in MCF7 cells. This study demonstrated the modulation of cellular cytoskeleton, such as the formation of distinct filopodial and lamellipodial structures and spikes, through the regulation of small-GTPases. We also observed that QC induces a signaling cascade by inducing apoptotic cell death by increasing ROS generation and altering HSP70 expression; which presumably involves ERK regulation. Our findings show that QC could be an attractive chemotherapeutic agent having a "shotgun" nature with potential of inducing different signaling pathways leading to apoptotic cell death. This opens new avenues for research on developing QC as an effective therapeutic agent for the treatment of invasive ductal carcinomas.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carcinoma, Ductal; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; HSP70 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; MCF-7 Cells; Neoplasm Recurrence, Local; Quinacrine; Reactive Oxygen Species; Signal Transduction

Despite major advances in the last 10 years, whether in terms of prevention or treatment, the 5 year survival rate remains relatively low for a large number of cancers. These therapeutic failures can be the consequence of several factors associated with the cellular modifications or with the host by itself, especially for some anticancer drugs such as cisplatin, which induces a nephrotoxicity. In the strategy of research for active molecules capable both of exerting a protective action against the deleterious effects of cisplatin and exerting a chemosensitizing action with regard to cancer cells, we tested the potential effects of Ephedra alata Decne extract (E.A.) rich in polyphenolic compounds towards a 4T1 breast cancer model in vitro and in vivo. We showed that E.A. extract inhibited cell viability of 4T1 breast cancer cells and induced apoptosis in a caspase-dependent manner, which involved intrinsic pathways. Very interestingly, we observed a synergic antiproliferative and pro-apoptotic action with cisplatin. These events were associated with a strong decrease of breast tumor growth in mice treated with an E.A./cisplatin combination and simultaneously with a decrease of hepato- and nephrotoxicities of cisplatin.

Topics: Adjuvants, Pharmaceutic; Animals; Breast Neoplasms; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cytochromes c; Drug Synergism; Enzyme Activation; Ephedra; Female; Inhibitory Concentration 50; Mammary Neoplasms, Animal; Mice, Inbred BALB C; Mitochondria; Models, Biological; Phenols; Plant Extracts; Proto-Oncogene Proteins c-bcl-2

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Proliferation; Cytochromes c; Drug Screening Assays, Antitumor; Female; HeLa Cells; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Ursolic Acid

To enhance the efficacy of nanoparticle-based cancer therapy with reduced side effects and promote its clinical translation, a biocompatible nanocomposite based on mesoporous silica-coated gold nanorods (AuNR@MSN) for triple tumor therapy is reported in this study. The gold core served as a hyperthermia agent, while the MSN shell acted as a reservoir of chemotherapeutics owing to its excellent loading capacity. Cytochrome c with the apoptosis inducing function was anchored on the surface of AuNR@MSN to prevent drug leakage through redox-responsive disulfide bonds. The successful construction of a nanocomposite was confirmed by characterization of the physicochemical properties. In vitro and in vivo studies demonstrated that the nanocomposite displayed an optimizing anti-tumor effect with a synergistic strategy of excellent photothermal therapy, chemotherapy and protein therapy. Therefore, this cooperative strategy paves the way for high-efficiency oncotherapy with reduced side effects.

Topics: Animals; Apoptosis; Breast Neoplasms; Cytochromes c; Delayed-Action Preparations; Drug Delivery Systems; Female; Gold; Humans; Hyperthermia, Induced; MCF-7 Cells; Mice, Nude; Nanotubes; Oxidation-Reduction; Porosity; Silicon Dioxide

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cytochromes c; Dammaranes; Female; Gynostemma; Humans; Hydrolysis; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Saponins; Triterpenes

Cytochrome c (Cyt c) plays an important role in cell apoptosis. However, it could be functionally compromised by interaction with anti-apoptosis proteins, known as protein-protein interactions (PPIs). Among the proteins potentially interacting with Cyt c, both HSP27 and Bcl-xL serve as pivotal anti-apoptosis proteins. Because multiple PPIs, especially those involve the same protein, could affect each other, their simultaneous and quantitative detection is highly needed. In this study, a combined approach of molecularly imprinted polymers (MIPs) and LC-MS/MS-based targeted proteomics was developed for simultaneous quantification of Cyt c-HSP27 and Cyt c-Bcl-xL interactions. Surrogate peptides of Cyt c, HSP27 and Bcl-xL were first selected and used for the corresponding proteins quantification in targeted proteomics analysis. For MIPs, epitope approach was employed and a short peptide of Cyt c was selected as template for protein complexes recognition and enrichment. The characteristics of the synthesized MIPs including adsorption capacity, kinetics and efficiency were then evaluated. After validation, this combined assay was applied to quantitative analysis of total Cyt c including Cyt c in mitochondria and cytosol, total HSP27, total Bcl-xL and Cyt c-HSP27 and Cyt c-Bcl-xL protein complexes in breast cells. The result was also compared with that using Co-IP/Western Blotting. SIGNIFICANCE: Protein-protein interactions (PPIs) are essential for many cellular processes and the changes of PPIs are often associated with cellular dysfunction. More importantly, each protein typically has more than one interaction partner and multiple PPIs, especially those involve the same protein, could affect each other. The selectivity of these interactions determines the activities of proteins and further the developmental potential of the cell. Thus, simultaneous and quantitative detection of multiple PPIs is highly needed in biological research and related disciplines. However, it is still challenging to even qualitatively or semi-quantitatively analyze multiple PPIs because of the limitations of current experimental techniques for interaction detection. In this study, molecularly imprinted polymers (MIPs) epitope approach was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) targeted proteomics for the simultaneous and quantitative detection of Cyt c-HSP27 and Cyt c-Bcl-xL interactions in breast cancer. Given high sensitivity, high selectivity and wid

Topics: bcl-X Protein; Breast Neoplasms; Chromatography, Liquid; Cytochromes c; Epitopes; Female; Heat-Shock Proteins; Humans; MCF-7 Cells; Molecular Chaperones; Molecular Imprinting; Proteomics; Tandem Mass Spectrometry

ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with

Topics: Animals; Apoptosis; Benzoates; Blotting, Western; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Female; Flow Cytometry; Fluorine Radioisotopes; Humans; Iodine Radioisotopes; Kidney; Mice; Radiopharmaceuticals; Receptor, ErbB-2; Single-Domain Antibodies; Succinimides; Xenograft Model Antitumor Assays

Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cytochromes c; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MCF-7 Cells; Proto-Oncogene Proteins c-bcl-2; Receptors, Steroid; Receptors, Thyroid Hormone; RNA, Messenger; Survival Analysis; Transcription, Genetic; Tumor Suppressor Protein p53

Current predictive model is not developed by inflammation-related genes to evaluate clinical outcome of breast cancer patients.. With mRNA expression profiling, we identified 3 mRNAs with significant expression between 15 normal samples and 669 breast cancer patients. Using 7 cell lines and 150 paraffin-embedded specimens, we verified the expression pattern by bio-experiments. Then, we constructed a three-mRNA model by Cox regression method and approved its predictive accuracy in both training set (n = 1095) and 4 testing sets (n = 703).. We developed a three-mRNA (TBX21, TGIF2, and CYCS) model to stratify patients into high- and low-risk subgroup with significantly different prognosis. In training set, 5-year OS rate was 84.5% (78.8%-90.5%) vs 73.1% (65.9%-81.2%) for the low- and high-risk group (HR = 1.573 (1.090-2.271); P = 0.016). The predictive value was similar in four independent testing sets (HR>1.600; P < 0.05). This model could assess survival independently with better predictive power compared with single clinicopathological risk factors and any of the three mRNAs. Patients with both low-risk values and any poor prognostic factors had more favorable survival from nonmetastatic status (HR = 1.740 (1.028-2.945), P = 0.039). We established two nomograms for clinical application that integrated this model and another three significant risk factors to forecast survival rates precisely in patients with or without metastasis.. This model is a dependable tool to predict the disease recurrence precisely and could improve the predictive accuracy of survival probability for breast cancer patients with or without metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Inflammation; Male; Middle Aged; Prognosis; Repressor Proteins; RNA, Messenger; T-Box Domain Proteins

BACKGROUND Breast cancer is one of the most malignant tumors worldwide. The natural flavonoid diosmetin has been reported to exhibit various pharmacological activities, including anti-cancer effects. This study aimed to investigate the anti-breast cancer effects of diosmetin on MDA-MB-231 cells and to explore the underlying molecular mechanisms of cell apoptosis. MATERIAL AND METHODS The MDA-MB-231 cells were incubated with diosmetin for 24 h. Then, cell viability and lactate dehydrogenase (LDH) leakage were detected using CCK-8 and LDH assay kits, respectively. Inverted fluorescence microscopy and flow cytometry were used to measure the mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS). Cell apoptosis and cell cycle were determined by flow cytometry. The expressions of apoptosis and cell cycle-related genes were determined by Western blotting and qRT-PCR. RESULTS The results revealed that diosmetin exerts significant cytotoxic effects on MDA-MB-231 cells, as indicated by decreased cell viability, increased intracellular ROS accumulation and LDH release, as well as cell cycle arrest in G0/G1 phase, inducing mitochondrial dysfunction and apoptosis. Moreover, diosmetin treatment significantly downregulated the expression levels of Bcl-2 and Cyclin D1, and upregulated that of p53, Bax, caspase 3, cleaved caspase 9, and cleaved caspase 3. CONCLUSIONS These findings demonstrate that diosmetin has anti-proliferative and pro-apoptotic activities against MDA-MB-231 cells via cell cycle arrest and the mitochondria-mediated intrinsic apoptotic pathway. Our results extend the understanding of the anti-tumor mechanism of diosmetin and suggest that it may be of use as an active natural agent for the prevention or treatment of human breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Female; Flavonoids; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Oral chemotherapy using anticancer drugs would improve the clinical practice and the life quality of patients. The aim of the present study was to develop paclitaxel hybrid nanomicelles for oral administration to treat resistant breast cancer.. Evaluations were performed on human breast cancer MCF-7 cells, drug-resistant breast cancer MCF-7/Adr cells, and in MCF-7/Adr-xenografted BALB/c nude mice. The nanomicelles were composed of the polymer soluplus, d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS. The nanomicelles improved cellular uptake and anticancer efficacy in the resistant breast cancer cells and induced mitochondria-mediated apoptosis. The mechanism of the apoptosis-inducing effect was related to the co-localization of the nanomicelles with mitochondria; the activation of pro-apoptotic protein Bax, cytochrome C, and apoptotic enzymes caspase 9 and 3; and the inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1. Oral administration of paclitaxel hybrid nanomicelles had the same anticancer efficacy as the intravenous injection of taxol in resistant breast cancer-bearing mice. The oral suitability of this formulation was associated with the nanostructure and the actions of TPGS. The fabricated paclitaxel hybrid nanomicelles could provide a promising oral formulation to treat drug-resistant breast cancer.

Topics: Administration, Oral; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cytochromes c; Dequalinium; Drug Resistance, Neoplasm; Female; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Micelles; Mitochondria; Nanostructures; Paclitaxel; Polyethylene Glycols; Polyvinyls; Xenograft Model Antitumor Assays

The aim of this study was to investigate the effects of hirsutine on apoptosis of breast cancer cells and its possible mechanism. The MCF-10A, MCF-7 and MDA-MB-231 cells were treated with hirsutine at different concentrations for 48 h or incubated with 160 μmol/L hirsutine for 24, 48, and 72 h. The MCF-10A cell line is a non-tumorigenic epithelial cell line, and the MCF-7 and MDA-MB-231 are human breast adenocarcinoma cell lines. CCK-8 assay was employed to detect the cell viability. Flow cytometry was used to assay the apoptosis and mitochondrial membrane potential (MMP). The protein expressions of Bcl-2, Bax, cleaved-caspase 9, cleaved-caspase 3 and cytochrome C (Cyt C) in the MDA-MB-231 cells were detected by Western blotting. The results showed that hirsutine remarkably reduced the viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner (P < 0.05) with IC50 values of 447.79 and 179.06 μmol/L, respectively. In the MDA-MB-231 cells, hirsutine induced apoptosis and depolarization of MMP (P < 0.05), released Cyt C from mitochondria (P < 0.05), and activated caspase 9 and caspase 3 (P < 0.05). However, these effects induced by hirsutine were all inhibited by cyclosporin A (CsA) (P < 0.05), a specific inhibitor of mitochondrial permeability transition pore (MPTP). In addition, hirsutine down-regulated the protein level of Bcl-2 and up-regulated the protein level of Bax (P < 0.05). These results suggest that hirsutine may induce apoptosis of human breast cancer MDA-MB-231 cells through decreasing the ratio of Bcl-2 to Bax, opening MPTP, releasing Cyt C from mitochondria, and activating caspase 9 and caspase 3.

Topics: Alkaloids; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Proto-Oncogene Proteins c-bcl-2

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Topics: Adenocarcinoma; ADP Ribose Transferases; Animals; Ankyrin Repeat; Bacterial Toxins; Breast Neoplasms; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cytochromes c; Exotoxins; Female; Heme; Humans; Liposomes; Luminescent Proteins; Ovarian Neoplasms; Particle Size; Pseudomonas aeruginosa Exotoxin A; Receptor, ErbB-2; Red Fluorescent Protein; Virulence Factors

IR-783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR-783 has not been well demonstrated. In this study, we showed that IR-783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA-MB-231 cells to IR-783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR-783 induced dynamin-related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission-1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR-783-mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR-783 markedly inhibited tumour growth and induced apoptosis in an MDA-MB-231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR-783 induces apoptosis in human breast cancer cells by increasing Drp1-mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti-breast cancer effects of IR-783 and provided novel perspectives for the application of IR-783 in the treatment of breast cancer.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Cytochromes c; Dynamins; Female; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

Multidrug resistance (MDR) of breast cancer is the major challenge to successful chemotherapy while mitochondria-targeting therapy was a promising strategy to overcome MDR.. The particle size of HPS-DQAsomes was ~110 nm with spherical shape. In vitro cytotoxicity assay indicated that HPS-DQAsomes could increase the cytotoxicity against MCF-7/ADR cell line. Cellular uptake and mitochondria-targeting assay demonstrated that HPS-DQAsomes could target delivering therapeutical agent to mitochondria and inducing mitochondria-driven apoptosis process. In vivo antitumor assay suggested that HPS-DQAsomes could reach favorable antitumor activity due to both tumor targetability and sub-organelles' targetability. Histological assay also indicated that HPS-DQAsomes showed a strong apoptosis-inducing effect. No obvious systematic toxicity of HPS-DQAsomes could be observed.. In summary, multifunctional HPS-DQAsomes provide a novel and versatile approach for overcoming MDR via mitochondrial pathway in cancer treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cholesterol; Cytochromes c; Dequalinium; Doxorubicin; Drug Delivery Systems; Drug Liberation; Drug Resistance, Neoplasm; Endocytosis; Female; Humans; Hydrogen-Ion Concentration; Liposomes; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Particle Size; Peptides; Polyethylene Glycols; Receptor, ErbB-2; Schiff Bases; Toxicity Tests

In this study, zeolite X synthesised from coal fly ash (CFA) was examined for its anti-proliferative effects on human breast cancer MCF-7 cells by in vitro studies and allied probable molecular mechanism were also assessed. MTT assay exposed that zeolite X showed a concentration-dependent inhibitory result on the proliferation of MCF-7 cells and caused early apoptotic death and does not induce cytotoxicity in non-cancerous cells (MCF-12A). Flow cytometric study specified the accrual of cells at S phase suggest induction of apoptosis which was avowed through fluorescence and confocal microscopy following annexin V-FITC/propidium iodide (PI). MCF-7 cells treated by 10, 15 and 20 µg/ml concentrations of zeolite X showed internucleosomal DNA fragments in ladder form, thereby indicates cell death is associated with apoptosis. Mechanistically, our data support the induction of apoptosis through the activation of mitochondrial dependent pathway as indicated by an up-regulation of Bax and downregulation of Bcl-2 ratio, the expulsion of cytochrome c from the mitochondria to the cytosol and cleavage of pro-caspase-3 into activation of caspase-3 in MCF-7 cells. Based on these outcomes zeolite X induced early apoptosis and strongly provided experimental evidence for the usage of zeolite X as an essential therapeutic agent in the prevention and therapy of human breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Coal Ash; Cytochromes c; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; S Phase; Zeolites

Topics: Alkaloids; Animals; Azocines; bcl-2-Associated X Protein; Biological Mimicry; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Female; Heterocyclic Compounds, 2-Ring; Humans; MCF-7 Cells; Mice; Mitochondria; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Pterocarpans; Quinolizines; RAW 264.7 Cells

This manuscript describes a synergistic therapy for mastocarcinoma by pH and temperature dual-sensitive nanogel, and effects of microstructure, composition and properties of nanogel on the cellular response mechanism. The extracellular internalization of nanogels was obviously enhanced, due to the passive targeting function at T>VPTT. Interestingly, the increased cytotoxicity was further synergistically enhanced by an unexpected apoptosis as evoked by the 5-fluorouracil loaded nanogel (FLNG). The systemically evaluation of the effectors generated from different sub-cellular organelles including endosome, lysosome, autophagosome confirmed that it was a lysomal dependent apoptosis. Such specific apoptosis was mainly attributed to its activatable protonated PEI at low pH, which caused lysosomal membrane destruction and lysosomal enzyme cathepsin B (Cat B) leakage. This Cat B was then translocated to the mitochondria resulting in mitochondrial membrane permeability increase and mitochondrial membrane potential (MMP) decrease, followed by cytochrome c (Cyt C) release. Cyt C was the main molecule that evoked apoptosis as reflected by overexpression of caspase 9. Additionally, such lysosome dependent, apoptosis was further enhanced by the passive cellular targeting at T>VPTT. Thus, the tumor growth inhibition was synergistically enhanced by the extracellular temperature dependent passive targeting and intracellular pH activatable lysosomal dependent apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 9; Cathepsin B; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Drug Carriers; Female; Fluorouracil; Gels; Humans; Hydrogen-Ion Concentration; Imines; Lysosomes; Membrane Potential, Mitochondrial; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Molecular Targeted Therapy; Nanostructures; Particle Size; Polyethylenes

The common cancer treatment strategies like chemotherapy and radiotherapy are nonspecific and can trigger severe side effects by damaging normal cells. So, targeted cancer therapies, such as apoptosis induction, have attracted great attention in recent years. In this project, two nano-complexes, MUC1 aptamer-NAS-24 aptamer-Graphene oxide (GO) and MUC1 aptamer-Cytochrome C aptamer-GO, were designed to induce cell programmed death in MDA-MB-231 and MCF-7 cells (breast cancer cell lines) and to verify the level of apoptosis in both cell lines. MUC1 aptamer was a molecular recognition probe that led the internalization of two nano-complexes into MDA-MB-231 and MCF-7 cells (MUC1 positive cells) but not into HepG2 cell (liver cancer cell line, MUC1 negative cells). The apoptosis induction relied on binding of NAS-24 aptamer to its target, vimentin, in MDA-MB-231 and MCF-7 (target cells) with different levels of vimentin content. The function of first nano-complex was confirmed by binding of FAM-labeled cytochrome C aptamer to its target (cytochrome C) which was released from mitochondria, based on the function of the first nano-complex. Fluorometric analysis and gel retardation assay proved the formation of nano-complexes. The results of flow cytometry and fluorescence microscopy indicated efficient apoptosis induction just in target cells (MDA-MB-231 and MCF-7 cells) but not in non-target cells (HepG2 cell). The results of MTT assay also confirmed cell death process. Overall, our results proved excellent targeted apoptosis in breast cancer cells by designed nano-complexes which can be applied as an efficient cancer therapy method.

Topics: Apoptosis; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Graphite; Humans; MCF-7 Cells; Mucin-1; Oxides; Theranostic Nanomedicine

Alpha-mangostin, a natural xanthonoid, has been reported to possess the anti-cancer property in various types of human cancer. However, its effects and mechanism of α-mangostin in cervical cancer remain unclear. We found that α-mangostin effectively inhibited cell viability, resulted in loss of mitochondrial membrane potential (MMP), release of cytochrome C, increase of Bax, decrease of Bcl-2, and activation of caspase-9/caspase-3 cascade in cervical cancer cells. Alpha-mangostin elevated the contents of reactive oxygen species (ROS) to activate p38. Disrupting ASK1/p38 signaling pathway by a specific inhibitor of p38, or by the siRNAs against ASK1, MKK3/6, or p38, significantly abolished α-mangostin-induced cell death and apoptotic responses. Moreover, α-mangostin also repressed tumor growth in accordance with increased levels of p-ASK1, p-p38, cleaved-PARP and cleaved-caspase-3 in the tumor mass from the mouse xenograft model of cervical cancer. In the current study, we provided first evidence to demonstrate that dietary antioxidant α-mangostin could inhibit the tumor growth of cervical cancer cells through enhancing ROS amounts to activate ASK1/p38 signaling pathway and damage the integrity of mitochondria and thereby induction of apoptosis in cervical cancer cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Female; Humans; MAP Kinase Kinase Kinase 5; Matrix Metalloproteinases; Membrane Potential, Mitochondrial; Mice; Mitochondria; Models, Biological; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Reactive Oxygen Species; Signal Transduction; Xanthones; Xenograft Model Antitumor Assays

In the present study, the antitumor effect of n‑3 fatty acid was evaluated, and the effect of docosahexaenoic acid (DHA) on the induction of apoptosis and its underlying mechanism were examined. Flow cytometry and western blot analysis were performed to analyze apoptosis and the expression of protein factors in human breast cancer cells. The data revealed that DHA inhibited the viability of MCF‑7 breast cancer cells in vitro, and promoted cell death by the induction of apoptosis. DHA decreased the expression of B‑cell lymphoma 2 (Bcl‑2), whereas the expression of Bcl‑2‑associated X protein was increased. DHA was also shown to promote the release of Smac/Diablo and cytochrome c from the mitochondria. DHA increased the levels of cleaved caspase‑8, ‑9 and ‑3. Additionally, the protein expression of tumor necrosis factor‑related apoptosis‑inducing ligand, death receptor 4 and Fas were increased following DHA treatment. In conclusion, DHA caused apoptosis of the human breast cancer cells in vitro through the death receptor and mitochondria‑mediated pathways. The results of this study encourage further investigation of the effect of fish oil on the prevention and treatment of human breast cancer.

Topics: Apoptosis; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Docosahexaenoic Acids; Female; Gene Expression; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Receptors, Death Domain; Signal Transduction

Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression.. Among NCI60 human cancer cell line panel Cladosporol A showed least IC. Our experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death. The proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.

Topics: Acetylcysteine; Apoptosis; Autophagy; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromosomes; Cladosporium; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Drug Screening Assays, Antitumor; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Microtubules; Naphthols; Reactive Oxygen Species

Topics: Animals; Apoptosis; Berberine; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasms, Experimental

The past decades have seen significant interest in the study of polyphenolic compounds as potential therapeutic agents in medicine because they display a vast array of cellular effects beneficial to treat or manage a plethora of chronic diseases including inflammatory diseases, cardiovascular abnormalities and several types of cancer. These compounds act at different stages of carcinogenesis but deciphering their mode of action is a complex task. Live MCF-7 breast cancer cells were investigated using Raman imaging to evaluate the perturbations induced after incubating cells with four different polyphenols: EGCG, gallic acid, resveratrol and tannic acid. First, clear spectral changes could be observed between the spectra of the cytoplasm and the nucleus of live MCF-7 cancer cells demonstrating a difference in their respective global chemical composition. The treatments induced significant modifications in the cells but no clear common pattern of modifications from the 4 drugs could be observed in the cell spectra in the 1800-600 cm

Topics: Apoptosis; Breast Neoplasms; Catechin; Cytochromes c; Cytoplasm; Cytosol; Gallic Acid; Humans; Lipid Metabolism; MCF-7 Cells; Polyphenols; Resveratrol; Tannins

Chlorogenic acid (CGA) exhibits potentials towards liver, breast and skin cancer. Cancer cells stimulated with CGA exhibits differential expression of transcriptional factors and regulatory molecules but the molecular target of the molecule is not known. Superposition of biophoric elements of CGA with Curcumin gives maximum common substructure score of 0.90. Molecular modeling studies further suggest that CGA fits into the C1b domain of PKC with extensive interaction with residues lining binding site. It binds PKC in a concentration dependent manner with dissociation constant KD, 28.84±3.95 μM. PKC-CGA complex is stable with minimal distortion to the 3-D structure and maintains the hydrogen bonding between ligand and receptor during simulation period. Cells stimulated with CGA causes 12.1 ± 0.56% PKC translocation from the cytosol to the plasma membrane. It disturbs the cell cycle and arrest the cancer cell at the G1 phase with a reduction in S-phase. Chlorogenic acid exhibits killing of cancer cells in a dose-dependent manner with an IC50 of 75.88 ± 4.54μg/ml and 52.5 ± 4.72μg/ml towards MDAMB-231 and MCF-7 cells respectively. It induces apoptosis in cancer cells as evident by AO/EtBr staining and degradation of genomic DNA to give a laddering pattern. Apoptosis in cancer cells involves mitochondrial pathway as supported by a reduction in mitochondrial potentials and release of cyt-C into the cytosol. Hence, the current study has established PKC as an important signaling molecule to the observed anti-cancer effects of CGA and provides the impetus to design better CGA analogs for improved anti-cancer potential against the malignant tumor.

Topics: Antineoplastic Agents; Apoptosis; Biological Transport; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Membrane; Chlorogenic Acid; Cytochromes c; Cytosol; Female; Humans; Ligands; Mitochondria; Protein Binding; Protein Kinase C; Signal Transduction; Structure-Activity Relationship

Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis that can be applied in vitro on canine breast cancer cells. However, the exact pathway responsible for HMME-PDT in canine breast cancer cells remains unknown. CHMm cells morphology and apoptosis were analyzed using optical microscope, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescein staining and DNA ladder assays. Apoptotic pathway was further confirmed by Real-time-polymerase chain reaction and Western blotting assays. Our results showed that HMME-PDT induced significant changes in cell morphology, such as formation of cytoplasmic vacuoles and the gradual rounding of cells coupled with decreased size and detachment. DNA fragmentation and cell death was shown to occur in a time-dependent manner. Furthermore, HMME-PDT increased the activities of caspase-9 and caspase-3, and released cytochrome c from mitochondria into the cytoplasm. HMME-PDT also significantly increased both mRNA and protein levels of Bax and decreased P53 gene expression in a time-dependent manner, while the mRNA and protein expression of Bcl-2 were repressed. These alterations suggest that HMME-PDT induced CHMm cell apoptosis via the mitochondrial apoptosis pathway and had anti-canine breast cancer effects in vitro.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Dog Diseases; Dogs; Gene Expression; Helium; Hematoporphyrins; Lasers; Mitochondria; Neon; Photosensitizing Agents

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF-7 and MDA-MB-231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration-dependent manner. By upregulating Bax and downregulating Bcl-2, ramalin caused cytochrome c and apoptosis-inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase-8 and caspase-9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase-3 only in the MDA-MB-231 cells. Ramalin treatment also increased the levels of LC3-II and p62. Moreover, the inhibition of autophagy by 3-methyladenine or Atg5 siRNA significantly enhanced ramalin-induced apoptosis, which was accompanied by a decrease in Bcl-2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non-invasive or invasive breast cancer.

Topics: Adenine; Antineoplastic Agents; Antioxidants; Apoptosis; Apoptosis Inducing Factor; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Biological Products; Breast Neoplasms; Caspases; Cell Cycle; Cell Proliferation; Cytochromes c; Female; Glutamates; Humans; Lichens; Microtubule-Associated Proteins; Mitochondria; Proto-Oncogene Proteins c-bcl-2

The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers.. This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development.. We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis-associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors.. This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348-60. ©2016 AACR.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Progression; Exosomes; Female; GTP Phosphohydrolases; Humans; Liver Neoplasms; Lung Neoplasms; MCF-7 Cells; Mitochondria; Mitochondrial Proteins; Superoxides; Tumor Suppressor Proteins

Electrochemical approaches have played crucial roles in bio sensing because of their Potential in achieving sensitive, specific and low-cost detection of biomolecules and other bio evidences. Engineering the electrochemical sensing interface with nanomaterials tends to new generations of label-free biosensors with improved performances in terms of sensitive area and response signals. Here we applied Silicon Nanowire (SiNW) array electrodes (in an integrated architecture of working, counter and reference electrodes) grown by low pressure chemical vapor deposition (LPCVD) system with VLS procedure to electrochemically diagnose the presence of breast cancer cells as well as their response to anticancer drugs. Mebendazole (MBZ), has been used as antitubulin drug. It perturbs the anodic/cathodic response of the cell covered biosensor by releasing Cytochrome C in cytoplasm. Reduction of cytochrome C would change the ionic state of the cells monitored by SiNW biosensor. By applying well direct bioelectrical contacts with cancer cells, SiNWs can detect minor signal transduction and bio recognition events, resulting in precise biosensing. Our device detected the trace of MBZ drugs (with the concentration of 2nM) on electrochemical activity MCF-7 cells. Also, experimented biological analysis such as confocal and Flowcytometry assays confirmed the electrochemical results.

Topics: Biosensing Techniques; Breast; Breast Neoplasms; Cytochromes c; Drug Screening Assays, Antitumor; Electrochemical Techniques; Female; Humans; MCF-7 Cells; Mebendazole; Nanowires; Silicon; Tubulin Modulators

Ionizing radiation (IR) in cancer radiotherapy can induce damage to neighboring cells via non-targeted effects by irradiated cells. These so-called bystander effects remain an area of interest as it may provide enhanced efficacy in killing carcinomas with minimal radiation. It is well known that reactive oxygen species (ROS) are ubiquitous among most biological activities. However, the role of ROS in bystander effects has not been thoroughly elucidated. We hypothesized that gradient irradiation (GI) has enhanced therapeutic effects via the ROS-mediated bystander pathways as compared to uniform irradiation (UI). We evaluated ROS generation, viability, and apoptosis in breast cancer cells (MCF-7) exposed to UI (5 Gy) or GI (8-2 Gy) in radiation fields at 2, 24 and 48 h after IR. We found that extracellular ROS release induced by GI was higher than that by UI at both 24 h (p < 0.001) and 48 h (p < 0.001). More apoptosis and less viability were observed in GI when compared to UI at either 24 h or 48 h after irradiation. The mean effective doses (ED) of GI were ~130% (24 h) and ~48% (48 h) higher than that of UI, respectively. Our results suggest that GI is superior to UI regarding redox mechanisms, ED, and toxic dosage to surrounding tissues.

Topics: Apoptosis; Breast Neoplasms; Bystander Effect; Cell Survival; Cytochromes c; Female; Humans; MCF-7 Cells; Radiation, Ionizing; Reactive Oxygen Species; Signal Transduction

In spite of their high specificity and potency, few protein therapeutics are applied in clinical cancer therapy owing to a lack of safe and efficacious delivery systems. Here, we report that redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels (HA-NGs) show highly efficient loading and breast tumor-targeted delivery of cytochrome c (CC). HA-NGs were obtained from hyaluronic acid-graft-oligo(ethylene glycol)-tetrazole (HA-OEG-Tet) via inverse nanoprecipitation and catalyst-free photoclick cross-linking with l-cystine dimethacrylamide (MA-Cys-MA). HA-NGs exhibited a superb CC loading content of up to 40.6 wt %, intrinsic fluorescence (λem = 510 nm), and a small size of ca. 170 nm. Notably, CC-loaded nanogels (CC-NGs) showed a fast glutathione-responsive protein release behavior. Importantly, released CC maintained its bioactivity. MTT assays revealed that CC-NGs were highly potent with a low IC50 of 3.07 μM to CD44+ MCF-7 human breast tumor cells. Confocal microscopy observed efficient and selective internalization of fluorescent HA-NGs into MCF-7 cells. Interestingly, HA-NGs exhibited also effective breast tumor penetration. The therapeutic results demonstrated that CC-NGs effectively inhibited the growth of MCF-7 breast tumor xenografts at a particularly low dose of 80 or 160 nmol CC equiv./kg. Moreover, CC-NGs did not cause any change in mice body weight, corroborating their low systemic side effects. Redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels have appeared as a "smart" protein delivery nanoplatform enabling safe, efficacious, traceable, and targeted cancer protein therapy in vivo.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Drug Delivery Systems; Humans; Hyaluronic Acid; Mice; Nanoparticles; Oxidation-Reduction

This study investigated the nature and mechanism of juglone-induced apoptosis in the human breast cancer cell line MCF-7. The inhibitory effect of juglone on MCF-7 cell growth was evaluated by the dimethylthiazol tetrazolium assay. Morphological apoptotic changes were characterized using an inverted microscope, Hoechst 33258 fluorescence staining, and Giemsa staining. The rate of cell apoptosis, intracellular levels of reactive oxygen species (ROS), and mitochondrial membrane potential were detected using flow cytometry. Intracellular Ca(2+) concentrations were detected using laser scanning confocal fluorescence microscopy. Expression of the proteins Bcl-2, Bax, and cytochrome C was assessed by western blotting. Caspase-3 activity was quantified using a caspase-3 activity kit. Juglone inhibited the growth of MCF-7 cell line with an IC50 of 11.99 μM. The rates of MCF-7 cell apoptosis at 24 h after exposure to 5, 10, and 20 μM juglone were 9.29, 20.67, and 28.39%, respectively; compared to unexposed cells, juglone-exposed cells exhibited significant elevation in intracellular ROS level, decrease in mitochondrial membrane potential, and increase in intracellular Ca(2+) concentration. Juglone upregulated the expression of Bax, and downregulated the expression of Bcl-2, promoting the release of cytochrome C, thereby upregulating the activity of caspase-3. The results suggest that the mechanism of juglone-induced apoptosis in MCF-7 cells is characterized by elevated ROS levels, reduced Bcl-2 expression, increased Bax expression, decreased mitochondrial membrane potential, increased intracellular Ca(2+) concentration, outer mitochondrial-membrane rupture, cytochrome C release, and caspase-3 activation.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence.

Topics: Aldehyde Dehydrogenase; Antineoplastic Agents; Apoptosis; Biological Transport; Breast Neoplasms; Caspase 7; Caspase 9; Cell Self Renewal; Cytochromes c; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Molecular Targeted Therapy; Neoplastic Stem Cells; Organometallic Compounds; Phosphatidylserines; Resting Phase, Cell Cycle; Tin; Wnt Signaling Pathway

Chemotherapy, radiotherapy, and endocrinotherapy are documented to induce autophagy among breast cancer cells, but the role of autophagy in this disease has been attributed as cytoprotective as well as tumor-suppressing. Thus we studied MDA-MB-231 and SK-BR-3 breast cancer cell lines treated with epirubicin (EPI) to assess autophagy and apoptosis. We found out that EPI induced apoptosis and autophagy in both cell lines. The lysosomal inhibitor bafilomycin A1 inhibited cellular autophagy and enhanced EPI-triggered apoptosis, perhaps due to inhibition of autolysosome formation, which then inhibited autophagic effects of engulfing and clearing damaged mitochondria. This inhibition increased mitochondrial cytochrome C release which augmented epirubicin-induced caspase-dependent apoptosis and cytotoxicity. In addition, the lysosomal neutralizing agent ammonia chloride (AC), and Atg7 knockdown by siRNA, could inhibit epirubicin-triggered autophagy, enhance cytotoxicity, and increase caspase-9- and caspase-3-dependent apoptosis. Thus, autophagy plays a prosurvival role in EPI-treated MDA-MB-231 and SK-BR-3 cells, and autophagy inhibition can potentially reverse this effect and increase the cytotoxicity of EPI.

Topics: Ammonium Chloride; Apoptosis; Autophagosomes; Autophagy; Breast Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Epirubicin; Humans; In Situ Nick-End Labeling; Lysosomes; Macrolides; Mitochondria; Neoplastic Stem Cells

Anticancer agents used in chemotherapy for tumors induce apoptosis in malignant cells. Soluble Fas ligand, granzyme B and cytochrome c are key elements in the process of apoptosis. The objective of this preliminary study was to evaluate the changes in the serum concentrations of these parameters in breast cancer patients undergoing adjuvant chemotherapy.. Sixty patients with histopathologically proven breast cancer were included in the present study. The blood samples were taken after surgery before chemotherapy and after 3weeks of administration of the first cycle of chemotherapy. Thirty healthy female controls were selected for comparison. Soluble FasL, granzyme B and cytochrome c were estimated from serum by ELISA.. Significantly increased concentrations of soluble FasL, granzyme B and cytochrome c were found in stage II and stage III of breast cancer patients after chemotherapy compared with concentrations before chemotherapy (P<0.0001). A significant positive correlation was found between soluble FasL and cytochrome c as well as between granzyme B and cytochrome c in breast cancer patients after chemotherapy.. Serum concentrations of apoptotic markers such as soluble FasL, granzyme B and cytochrome c were increased after administration of the first cycle of chemotherapeutic drugs. The measurement of these circulating apoptotic markers may help clinicians in evaluating treatment efficacy in breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Chemotherapy, Adjuvant; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; Female; Granzymes; Humans; Middle Aged; Prospective Studies

A novel triterpenoid, named 3β-trans-cinnamoyloxy-2α-hydroxy-urs-12-en-28-oic acid (CHUA), was one of the main components of apple peels and showed potent in vitro antitumor activity against human tumor cells. In vivo antitumor experiments showed that CHUA could significantly inhibit the growth of mammary tumor in a nude mouse xenograft model at a dose of 50 mg/kg/day without body weight loss and mortality. In vitro, CHUA could induce apoptosis in MDA-MB-231 cells through the detection of DNA fragments and LDH activity. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c was increased after CHUA treatment. It also up-regulated the expression ratio of mitochondrial Bax/Bcl-2 regulated by SIRT1 and p53. Interestingly, z-VAD-fmk and z-DEVD-fmk augmented cell death after CHUA treatment. Other protease(s) different from caspase-3 might be responsible for the degradation of PARP. These results suggested that the pro-apoptotic activity of CHUA may be adjusted by mitochondrial and caspase-independent pathways.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cytochromes c; Female; Fruit; Humans; Malus; Mice; Mice, Nude; Mitochondria; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Triterpenes

The anti-carcinogenic effect of the new quinazolinone compound, named MMD, was tested on MCF-7 human breast cancer cell line. The synthesis of quinazolinone-based compounds attracted strong attention over the past few decades as an alternative mean to produce analogues of natural products. Quinazolinone compounds sharing the main principal core structures are currently introduced in the clinical trials and pharmaceutical markets as anti-cancer agents. Thus, it is of high clinical interest to identify a new drug that could be used to control the growth and expansion of cancer cells. Quinazolinone is a metabolite derivative resulting from the conjugation of 2-aminobenzoyhydrazide and 5-methoxy-2- hydroxybenzaldehyde based on condensation reactions. In the present study, we analysed the influence of MMD on breast cancer adenoma cell morphology, cell cycle arrest, DNA fragmentation, cytochrome c release and caspases activity. MCF-7 is a type of cell line representing the breast cancer adenoma cells that can be expanded and differentiated in culture. Using different in vitro strategies and specific antibodies, we demonstrate a novel role for MMD in the inhibition of cell proliferation and initiation of the programmed cell death. MMD was found to increase cytochrome c release from the mitochondria to the cytosol and this effect was enhanced over time with effective IC50 value of 5.85 ± 0.71 μg/mL detected in a 72-hours treatment. Additionally, MMD induced cell cycle arrest at G0/G1 phase and caused DNA fragmentation with obvious activation of caspase-9 and caspases-3/7. Our results demonstrate a novel role of MMD as an anti-proliferative agent and imply the involvement of mitochondrial intrinsic pathway in the observed apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Cycle Checkpoints; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drug Design; Female; Humans; Inhibitory Concentration 50; L-Lactate Dehydrogenase; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; NF-kappa B; Quinazolinones; Reactive Oxygen Species; Signal Transduction; Structure-Activity Relationship; Time Factors

Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup within the armadillo (ARM) family, which has one or two ARM repeat domains as opposed to more than six-thirteen repeats in the classical Armadillo family members.. In the study, we explore the biological functions of ALEX1 in breast cancer cells. Overexpression of ALEX1 and silencing of ALEX1 were performed with SK-BR3 and MCF-7 cell lines. Cell proliferation and colony formation assays, along with flow cytometry, were carried out to evaluate the roles of ALEX1.. ALEX1 overexpression in SK-BR3 breast cancer cells inhibited proliferation and induced apoptosis. Furthermore, depletion of ALEX1 in MCF-7 breast cancer cells increased proliferation and inhibited apoptosis. Additional analyses demonstrated that the overexpression of ALEX1 activated the intrinsic apoptosis cascades through up-regulating the expression of Bax, cytosol cytochrome c, active caspase-9 and active caspase-3 and down-regulating the levels of Bcl-2 and mitochondria cytochrome c. Simultaneouly, silencing of ALEX1 inhibited intrinsic apoptosis cascades through down-regulating the expression of Bax, cytosol cytochrome c, active caspase-9, and active caspase-3 and up-regulating the level of Bcl-2 and mitochondria cytochrome c.. Our data suggest that ALEX1 as a crucial tumor suppressor gene has been involved in cell proliferation and apoptosis in breast cancer, which may serve as a novel candidate therapeutic target.

Topics: Apoptosis; Armadillo Domain Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knock-In Techniques; Gene Knockdown Techniques; Humans; MCF-7 Cells; Oncogene Proteins; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Up-Regulation

Docetaxel (DTX) is widely used for the treatment of metastatic prostate and breast cancers. Despite the clinical success of DTX, drug-related cumulative toxicity restricts its clinical use in cancer therapy. Thus, there is an urgent need for new therapeutic options. Octreotide (OCT) is a synthetic somatostatin analog that induces apoptosis in different cancer cell lines in vitro. In this study, we investigated the possible synergistic apoptotic effects of DTX in combination with OCT in prostate and breast cancer cell lines.. The XTT cell viability assay was used to determine cytotoxicity. Apoptosis was evaluated by Cell Death Detection ELISA(Plus) Kit. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. Levels of SSTR2 and SSTR5 proteins were determined by western blot analysis.. DTX and OCT combination induced apoptosis in both breast and prostate cancer cells in a concentration- and time-dependent manner. Moreover, combination treatment resulted in inhibition of anti-apoptotic proteins such as Bcl-2 and Bcl-xL and induction of pro-apoptotic proteins Bax, Cytochrome c and IAPs in all of the tested cancer cell lines. SSTR2 and SSTR5 protein levels were induced as compared to any agent alone.. These results indicate that this combination treatment is a significant inducer of apoptosis in a synergistic manner in breast and prostate cancer cells. This strong synergism helps to lower the dose of DTX in both types of cancers, thus letting DTX to be used for longer periods by delaying resistance development and lesser side effects.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Docetaxel; Drug Synergism; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Octreotide; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Somatostatin; Taxoids

Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Calcium Chelating Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Diphosphonates; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Imidazoles; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Zoledronic Acid

Identification of targets for apoptosis induction is important to provide novel therapeutic approaches in breast cancer. Our earlier studies showed that down regulation of protein kinase C δ (PKCδ) induces death in breast cancer cells. In this study we set out to identify previously unrecognized apoptosis regulators in breast cancer cells. To identify candidates, global expression analysis with microarray was performed after down regulation of PKCδ in the basal-like breast cancer cell lines MDA-MB-231, MDA-MB-468 and BT-549. Genes that were down regulated in all cell lines were further studied for survival-supporting effects. The claudin-like CLDND1 was singled out since several independent siRNAs targeting CLDND1 induced cell death in several cell lines. The cell death induced by CLDND1 knockdown was caspase-dependent, suggesting induction of apoptosis. Nuclear fragmentation, cleavage of caspase-3 and PARP and release of cytochrome C from the mitochondria upon CLDND1 depletion demonstrated involvement of the intrinsic apoptotic pathway. Inhibition of MEK1/2 and JNK further potentiated the cell death induction by CLDND1 knockdown. However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation. A concomitant inhibition of MEK1/2 suppresses the ERK1/2 phosphorylation and markedly potentiates the cell death following CLDND1 siRNA treatment. There is today little information on the function of CLDND1. These data provide novel information on CLDND1 and highlight it as a novel survival factor in basal-like breast cancer cell lines.

Topics: Apoptosis; Base Sequence; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Claudins; Cytochromes c; Down-Regulation; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Mitochondria; Poly(ADP-ribose) Polymerases; Proteolysis; RNA, Small Interfering

2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) is a cyclohexanedione found in the roots of Averrhoa carambola L., commonly known as starfruit. Researchers have shown that DMDD has significant therapeutic potential for the treatment of diabetes; however, the effects of DMDD on human cancers have never been reported. We investigated the cytotoxic effects of DMDD against human breast, lung and bone cancer cells in vitro and further examined the molecular mechanisms of DMDD-induced apoptosis in human breast cancer cells. DMDD suppressed the growth of breast carcinoma cells, but not normal mammary epithelial cells, via induction of G1 phase cell cycle arrest, oxidative stress and apoptosis. DMDD increased the level of intracellular reactive oxygen species (ROS) and DMDD-induced ROS generation was found to be associated with the mitochondrial activity. The cytotoxicity that was induced by DMDD was attenuated by co-treatment with the antioxidant N-acetyl-L-cysteine (NAC). DMDD-induced cell apoptosis involved the activation of both the intrinsic mitochondrial pathway and the extrinsic receptor pathway. In addition, DMDD inhibited the canonical NF-κB signaling pathway at all steps, including TNF-α production, phosphorylation of NF-κB p65 and IκBα, as well as TNF-α activated NF-κB p65 nuclear translocation.Collectively, our studies indicate that DMDD has significant potential as a safe and efficient therapeutic agent for the treatment of breast cancer.

Topics: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis; Averrhoa; Bone Neoplasms; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclohexenes; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hypoglycemic Agents; Inhibitory Concentration 50; Lung Neoplasms; MCF-7 Cells; NF-kappa B; Oxidative Stress; Phosphorylation; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Recently, gold nanoparticles (AuNPs) have shown promising biological applications due to their unique electronic and optical properties. However, the potential toxicity of AuNPs remains a major hurdle that impedes their use in clinical settings. Mesoporous silica is very suitable for the use as a coating material for AuNPs and might not only reduce the cytotoxicity of cetyltrimethylammonium bromide-coated AuNPs but might also facilitate the loading and delivery of drugs. Herein, three types of rod-like gold-mesoporous silica nanoparticles (termed bare AuNPs, core-shell Au@mSiO2NPs, and Janus Au@mSiO2NPs) were specially designed, and the effects of these AuNPs on cellular uptake, toxic behavior, and mechanism were then systematically studied. Our results indicate that bare AuNPs exerted higher toxicity than the Au@mSiO2NPs and that Janus Au@mSiO2NPs exhibited the lowest toxicity in human breast cancer MCF-7 cells, consistent with the endocytosis capacity of the nanoparticles, which followed the order, bare AuNPs > core-shell Au@mSiO2NPs > Janus Au@mSiO2NPs. More importantly, the AuNPs-induced apoptosis of MCF-7 cells exhibited features that were characteristic of intracellular reactive oxygen species (ROS) generation, activation of c-Jun-N-terminal kinase (JNK) phosphorylation, an enhanced Bax-to-Bcl-2 ratio, and loss of the mitochondrial membrane potential. Simultaneously, cytochrome c was released from mitochondria, and the caspase-3/9 cascade was activated. Moreover, both ROS scavenger (N-acetylcysteine) and JNK inhibitor (SP600125) partly blocked the induction of apoptosis in all AuNPs-treated cells. Taken together, these findings suggest that all AuNPs induce apoptosis through the ROS-/JNK-mediated mitochondrial pathway. Thus, Janus Au@mSiO2NPs exhibit the potential for applications in biomedicine, thus aiding the clinical translation of AuNPs.

Topics: Animals; Apoptosis; Brain; Breast Neoplasms; Caspase 3; Caspase 9; Cells, Cultured; Cytochromes c; Endothelium, Vascular; Female; Gene Expression Profiling; Gold; Humans; MAP Kinase Signaling System; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Nanoparticles; Phosphorylation; Rats; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Silicon Dioxide

Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; DNA Primers; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondria; Nicotinamide N-Methyltransferase; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles

In the present study, the radiation-induced effect of cytochrome c (CytC) and vitamin C (VitC) is studied with respect to survival of MCF-7 cancer cells grown in aerated media. Both, CytC and VitC, were incubated with MCF-7 cancer cells under various concentrations individually, as well as in mixture and cells were subsequently treated with γ-ray in a dose range of 0 to 30 Gy. Generally, an increase of cell survival was observed under substance treatment up to a radiation dose of 5 Gy compared to the control group. Based on the obtained results it is believed that cell survival depends strongly on the action of free radicals produced at the given concentration of the incubated CytC and VitC as well as on the specific reaction rate constants (k: l mol(-1) s(-1)) of the involved processes.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Breast Neoplasms; Cell Proliferation; Cytochromes c; Female; Free Radicals; Gamma Rays; Humans; Radiation-Sensitizing Agents; Tumor Cells, Cultured

There is growing interest in development of natural products as anti-cancer and chemopreventive agents. Many triterpenoids have been proved as potential agents for chemoprevention and therapy of breast cancer. Ginsenosides from ginseng, which mostly belong to dammarane-type triterpenoids, have gained great attention for their anti-breast cancer activity with diverse mechanisms. However, studies of other kinds of triterpenoid saponins on breast cancer are limited. Previously, we purified and identified a novel oleanane-type triterpene saponin named D Rhamnose β-hederin (DRβ-H) from Clematis ganpiniana, a Chinese traditional anti-tumor herb. In the present study, DRβ-H showed strong inhibitory activity on the growth of various breast cancer cells and induced apoptosis in these cells. DRβ-H inhibited PI3K/AKT and activated ERK signaling pathway. PI3K inhibitor LY294002 synergistically enhanced DRβ-H-induced apoptosis whereas MEK inhibitor U0126 reduced the apoptosis rate. Moreover, DRβ-H regulated the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Furthermore, DRβ-H induced depolarization of mitochondrial membrane potential which released Apaf-1 and Cytochrome C from the inter membrane space into the cytosol, where they promoted caspase-9 and caspase-3 activation. This is the first report on the pro-apoptotic effects of DRβ-H, a novel oleanane-type triterpenoid saponin, on breast cancer cells and its comprehensive apoptosis pathways. It implied that oleanane-type triterpenoid saponin DRβ-H could be a promising candidate for chemotherapy of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 3; Caspase 9; Chromones; Cytochromes c; Drug Screening Assays, Antitumor; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Female; Humans; MAP Kinase Signaling System; MCF-7 Cells; Membrane Potential, Mitochondrial; Morpholines; Oleanolic Acid; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Saponins

Histone deacetylase inhibitors (HDACIs)-based therapies have stimulated interest via their anti-tumor activities, including apoptosis induction, cell cycle arrest, cell differentiation, and autophagy. However, the mechanisms of HDACI-associated anti-tumor activity are not yet clearly defined. The aim of this study was to explore the key events of Trichostatin A (TSA), a classic HDACI agent, against breast cancer cells.. The MCF-7, MDA-MB-231 and MCF-10A cell lines were evaluated with colony-forming and cell viability assays. Apoptosis and cell cycle distribution were detected by flow cytometry. Mitochondrial function was measured with biochemical assays, flow cytometry and transmission electron microscopy.. TSA inhibited breast cancer cell viability and proliferation, without affecting MCF-10A cell. TSA-induced breast cancer cell apoptosis was initiated by G2-M arrest and depended on mitochondrial reactive oxygen species (ROS) produced subsequent to reduced mitochondrial respiratory chain activity. The enhanced mitochondrial ROS production and apoptosis in cancer cells were markedly attenuated by antioxidants, such as N-acetyl cysteine (NAC), reduced glutathione (GSH) and Vitamin C.. The present study demonstrated that TSA-induced cell death by arresting cell cycle in G2-M phase and was dependent on production of mitochondria-derived ROS, which was derived from impaired mitochondrial respiratory chain.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Proliferation; Cytochromes c; Electron Transport; Female; G2 Phase; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species

Histone deacetylase inhibitors (HDACi) have been reported to have potent chemopreventive activity because of their effects on the inhibition of cell growth and apoptosis in human cancer cell lines. In the present study, we investigated the apoptotic effect of a novel HDACi, Ky2, and its molecular mechanism in MDA-MB-231 human breast cancer cells in vitro. The chemopreventive effects of Ky2 in MDA-MB-231 cells were evaluated using the MTS assay, anchorage-independent cell transformation assay, DAPI staining, western blot analysis, reverse transcriptase-PCR, and small interfering RNA. Ky2 enhanced histone acetylation and decreased cell viability. Ky2 induced apoptosis evidenced by nuclear condensation and fragmentation, the accumulation of sub-G1 phase, and caspase-dependent PARP cleavage. In addition, Ky2 released cytochrome c from mitochondria to cytosol through the regulation of mitochondria-related proteins (Bid, Bim, and Bcl-xL). Ky2 markedly decreased the level of Sp1 protein expression through both the decrease of Sp1 mRNA level and proteasome-dependent protein degradation. Interestingly, the apoptotic effect of Ky2 is more potent than SAHA, a well-known HDACi. Furthermore, the knockdown of Sp1 protein by Sp1-specific inhibitor, mithramycin A, and siRNA resulted in the alteration of truncated Bid and Bim to induce apoptosis. Furthermore, Ky2 significantly decreased TPA-induced or EGF-induced neoplastic cell transformation in JB6 cells. Our results suggest that Ky2 may be a potential chemopreventive and chemotherapeutic agent by modulating Sp1 in human breast cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemoprevention; Cytochromes c; Drug Therapy; Female; Histone Deacetylase Inhibitors; Humans; Peptides, Cyclic; RNA, Messenger; Sp1 Transcription Factor

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.

Topics: Antacids; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Breast Neoplasms; Calcium Carbonate; Cell Line, Tumor; Cytochromes c; Doxorubicin; Drug Delivery Systems; Female; Humans; Mitochondrial Proteins; Nanoparticles; Neoplasm Metastasis; Neoplasm Proteins

Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCɛ signaling shown in a xenograft breast cancer model.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Depsipeptides; Female; Fluorescence Recovery After Photobleaching; Hexokinase; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice; Mice, SCID; Mitochondria; Phosphatidylserines; Protein Kinase C; Signal Transduction; Transplantation, Heterologous; Voltage-Dependent Anion Channels

The present study concerns molecular mechanisms involved in induction of apoptosis by a fungal taxol extracted from the fungus Cladosporium oxysporum in T47D human breast cancer cells.. Apoptosis-induced by the fungal taxol was assessed by MTT assay, nuclear staining, DNA fragmentation, flow cytometry and pro- as well as anti-apoptotic protein expression by Western blotting.. Our results showed inhibition of T47D cell proliferation with an IC50 value of 2.5 μM/ml after 24 h incubation. It was suggested that the extract may exert its anti-proliferative effect on human breast cancer cell line by suppressing growth, arresting through the cell cycle, increase in DNA fragmentation as well as down-regulation of the expression of NF-?B, Bcl-2 and Bcl-XL and up-regulation of pro-apoptotic proteins like Bax, cyt-C and caspase-3.. We propose that the fungal taxol contributes to growth inhibition in the human breast cancer cell through apoptosis induction via a mitochondrial mediated pathway, with possible potential as an anticancer therapeutic agent.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cladosporium; Cytochromes c; DNA Fragmentation; Down-Regulation; Female; Fungal Proteins; Humans; Mitochondria; NF-kappa B; Paclitaxel; Up-Regulation

The mechanism of action of fatty acid synthase (FASN) in drug tolerance of breast cancer cells with epithelial-mesenchymal transition (EMT) features was investigated.. The breast cancer cell line MCF-7-MEK5 with stably occurring EMT and tumour necrosis factor-α (TNF-α) tolerance was used as the experimental model, whereas MCF-7 acted as the control. Tumour cells were implanted into nude mice for in vivo analysis, and cerulenin was used as a FASN inhibitor. RT-PCR, real-time quantitative PCR and Western blot were employed to detect the expression of FASN, TNFR-1, TNFR-2, Wnt-1, β-catenin and cytC at the RNA and protein levels.. Compared with MCF-7, TNFR-1 expression in MCF-7-MEK5 was slightly changed, TNFR-2 was decreased, and FASN, Wnt-1, β-catenin and cytC were increased. The expression of Wnt-1 and β-catenin in MCF-7-MEK5 decreased after cerulenin treatment, whereas cytC expression increased.. The important function of FASN in the drug tolerance of breast cancer may be due to the following mechanisms: FASN downregulated TNFR-2 expression through lipid rafts to make the cells less sensitive to TNF-α, and simultaneously activated the Wnt-1/β-catenin signalling pathway. Thus, cytC expression increased, which provided cells with anti-apoptotic capacity and induced drug tolerance.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cytochromes c; Drug Tolerance; Epithelial-Mesenchymal Transition; Fatty Acid Synthase, Type I; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Real-Time Polymerase Chain Reaction; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Tumor Necrosis Factor-alpha; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown to inhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticancer activity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPD and cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and Annexin V-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2 and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetric assay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an IC50 value of 33.3 μM at 24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysis in cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and 30.5% in MCF-7 cells treated with 0, 15, 30 and 60μM of 20(S)-PPD, respectively. Moreover, 20(S)-PPD could induce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression. These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD was blocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death. In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death by a caspase-mediated apoptosis pathway.

Topics: Antidepressive Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Proliferation; Cytochromes c; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Sapogenins; Signal Transduction; Tumor Cells, Cultured

The recurrence of breast cancer is associated with drug-resistance of cancer stem cells (CSCs), while overexpression of cell membrane ATP-binding cassette (ABC) transporters and resistance of mitochondrial apoptosis-related proteins are responsible for the drug-resistance of CSCs. The targeting berberine liposomes were developed to modulate the resistant membrane and mitochondrial proteins of breast CSCs for the treatment and prevention of breast cancer relapse. Evaluations were performed on human breast CSCs and CSC xenografts in nude mice. The targeting berberine liposomes were shown to cross the CSC membrane, inhibit ABC transporters (ABCC1, ABCC2, ABCC3, ABCG2) and selectively accumulate in the mitochondria. Furthermore, the pro-apoptotic protein Bax was activated while the anti-apoptotic protein Bcl-2 was inhibited resulting in opening of the mitochondrial permeability transition pores, release of cytochrome c, and activation of caspase-9/caspase-3 enzymes. Significant efficacy of the administrations in mice was observed, indicating that the targeting berberine liposomes are a potential therapy for the treatment and prevention of breast cancer relapse arising from CSCs.

Topics: Animals; Apoptosis Regulatory Proteins; ATP-Binding Cassette Transporters; Berberine; Breast Neoplasms; Cell Death; Cytochromes c; Diagnostic Imaging; Drug Resistance, Neoplasm; Female; Humans; Liposomes; MCF-7 Cells; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Multidrug Resistance-Associated Protein 2; Neoplastic Stem Cells; Phenotype; Signal Transduction; Treatment Outcome; Tumor Burden

Heat shock proteins (HSPs) are chaperones for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSPs (27, 70, and 90) are abundantly expressed in a wide range of cancers and are transcriptionally regulated by heat shock factor (HSF1). Most of the synthetic HSP inhibitors exhibit toxicity, therefore, searching for inhibitors with limited or no toxicity will be of help. The objective of the present study was to determine the effect of natural isothiocyanate (phenethyl isothiocyanate; PEITC) on different HSPs (27, 70, and 90) and HSF1 in 2 breast cancer cell lines, namely breast adenocarcinoma MCF-7 (with wild type p53) and highly metastatic breast cancer cell MDA-MB-231 (with mutated p53). PEITC significantly inhibited the expression of HSPs (particularly HSP 90) and HSF1. Molecular consequences due to HSP inhibition were downregulation of cell-cycle regulatory proteins like Cyclin B1, CDK1, Cdc25C, PLK-1, and upregulation of p21 irrespective of p53 status. These modulations were accompanied by cell-cycle arrest at G2/M phase and apoptosis by activation of caspases 3 and 9. PEITC therefore may be regarded as a potent HSP inhibitor and an antitumor agent in the treatment of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA Fragmentation; Heat-Shock Proteins; Humans; Isothiocyanates; MCF-7 Cells; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

RU486 (mifepristone) exerts an anticancer effect on cancer cells via induction of apoptosis. However, the molecular mechanisms are not fully understood. Here, we investigated the effect of RU486 on the apoptosis of U937 human leukemia cells. RU486 markedly increased apoptosis in U937 cells as well as in MDA231 human breast carcinoma, A549 human lung adenocarcinoma epithelial and HCT116 human colorectal carcinoma cells. RU486 increased dose-dependent release of mitochondrial cytochrome c, and reduced the mitochondrial membrane potential (MMP, Δψm) in RU486-treated U937 cells. We also found that overexpression of Bcl-2 completely blocked RU486-mediated apoptosis. However, reactive oxygen species signaling had no effect on RU486-induced apoptosis. RU486 increased the phosphorylation of p38 MAPK and JNK, but p38 MAPK only was associated with RU486-mediated apoptosis. Taken together, RU486 induces apoptosis through reduction in the mitochondrial membrane potential and activation of p38 MAPK in U937 human leukemia cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Female; HCT116 Cells; Hormone Antagonists; Humans; Leukemia; Lung Neoplasms; Lymphoma; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mifepristone; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Glucocorticoid; U937 Cells

PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo.. We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated.. We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed.. Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Antineoplastic Agents; Apoptosis; Binding Sites; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell-Penetrating Peptides; Cytochromes c; Drug Design; Female; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Molecular Sequence Data; Molecular Targeted Therapy; Protein Binding; Protein Phosphatase 2; Species Specificity; Xenograft Model Antitumor Assays

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Female; Humans; Mitochondria; Quinolines

Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins delicately regulate mitochondrial outer membrane integrity through protein-protein interactions, which makes the mitochondrion an ideal cell-free system for screening molecules targeting the Bcl-2 anti-apoptotic proteins. But assay conditions need to be optimized for more reliable results. In this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins. In this study, high ionic strength buffer was adopted during the initiation of MOMP. Mitochondria isolated from human breast cancer cell lines with distinct expression patterns of Bcl-2 anti-apoptotic proteins were permeabilized by different BH3 peptides alone or in combination, with or without the presence of recombinant anti-apoptotic Bcl-2 family proteins. Cytochrome C and Smac/Diablo were tested in both supernatants and mitochondrial pellets by Western blotting.. Sufficient ionic strength was required for optimal release of Cytochrome C. Bad and Noxa BH3 peptides exhibited their bona fide antagonistic effects against Bcl-2/Bcl-xL and Mcl-1 proteins, respectively, whereas Bim BH3 peptide antagonized all three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergized with each other in the induction of MOMP when mitochondria were dually protected by both Bcl-2/Bcl-xL and Mcl-1.. This method based on MOMP is a useful screening tool for identifying BH3 mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major Bcl-2 anti-apoptotic proteins.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell-Free System; Cytochromes c; Female; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Reproducibility of Results; Surface Plasmon Resonance

Verrucarin A (VA) is a member of the family of macrocyclic trichothecenes, which exhibit anti-cancer and immune-modulating activities. However, VA has not yet been demonstrated to be involved in the sensitization of tumor necrosis factor-alpha (TNF-α)-mediated apoptosis. In the present study, we found that VA triggers TNF-α-induced apoptosis in human breast cancer MDA-MB-231 and MCF-7 cells. In particular, activation of caspas-3 and caspase-8 as well as release of cytochrome c were significantly enhanced in response to the combined treatment with VA and TNF-α (VA/TNF-α) and the pan-caspase inhibitor z-VAD-fmk completely reversed the apoptosis, suggesting that caspases are the main effector molecules in VA/TNF-α-induced apoptosis via the intrinsic and extrinsic pathway. Moreover, we confirmed that enhanced Fas expression plays a critical role, because the Fas-blocking antibody partially inhibited VA/TNF-α-induced apoptosis. VA also increased specific DNA-binding activity of nuclear factor-kappaB (NF-κB) via nuclear translocation of p50 and p65. In addition, pretreatment with the NF-κB inhibitor MG132 blocked VA/TNF-α-induced apoptosis by suppression of NF-κB-dependent Fas expression. These results indicated that VA enhances TNF-α-induced apoptosis via NF-κB-dependent Fas overexpression.

Topics: Active Transport, Cell Nucleus; Antibodies; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 8; Caspase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; fas Receptor; Female; Humans; MCF-7 Cells; NF-kappa B; NF-kappa B p50 Subunit; Signal Transduction; Time Factors; Transcription Factor RelA; Trichothecenes; Tumor Necrosis Factor-alpha; Up-Regulation

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes. In a recent study, we showed that Pho-s has antitumor effect in the several tumor cells. In this study we evaluated the antitumor activity of synthetic Pho-s on MCF-7 breast cancer cells. Here we demonstrate that Pho-s is cytotoxic to MCF-7 cells in a dose-dependent manner, while it is cytotoxic to MCF10 only at higher concentrations. In addition, Pho-s induces a disruption in mitochondrial membrane potential (Δψm). Furthermore, Pho-s induces mitochondria aggregates in the cytoplasm and DNA fragmentation of MCF-7 cells visualized by confocal microscopy. In agreement with the reduction on Δψm, we showed that Pho-s induces apoptosis followed by an increase in cytochrome c expression and capase-3-like activity in MCF-7 cells. Our results demonstrate that Pho-s induces a cell cycle arrest in the G1 phase through an inhibition of cyclin D1 and stimulates p53. An additional highlight of this study is the finding that Pho-s inhibits Bcl-2, inducing apoptosis through the mitochondrial pathway. Taken together, these results show that Pho-s is a promising compound in the fight against cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cyclin D1; Cytochromes c; Cytoplasm; DNA Fragmentation; Ethanolamines; Female; G1 Phase; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Protein p53

Trabectedin (Yondelis, ET-743), a semi synthetic tetrahydroisoquinoline alkaloid that was originally derived from the marine tunicate Ecteinascidia turbinata. The objective of this study was to investigate whether trabectedin mediated apoptosis shows any diversity in human breast cancer cell lines with different genotypes. Trabectedin induced cytotoxicity and apoptosis in both breast cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL-R2/DR5, FAS/TNFRSF6, TNF RI/TNFRSF1A, and FADD were significantly increased by 2.6-, 3.1-, 1.7-, 11.2- and 4.0-fold by trabectedin treatment in MCF-7 cells. However, in MDA-MB-453 cells, the mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, Smac/DIABLO, and Cleaved Caspase-3 expressions were induced by 4.2-, 3.6-, 4.8-, 4.5-, and 4.4-fold, and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 4.8- and 5.2-fold in MDA-MB-453 cells. Moreover, trabectedin treatment increased the generation of ROS in both breast cancer cells. We have shown that trabectedin causes selective activation of extrinsic and intrinsic apoptotic pathways in two genotypically different breast cancer cells. This preliminary data might guide clinicians to choose appropriate combination agents with trabectedin based on different molecular subtypes of breast cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-Associated Death Protein; Breast Neoplasms; Caspase 3; Caspase 7; Cell Line, Tumor; Cytochromes c; Dioxoles; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; MCF-7 Cells; Mitochondria; Mitochondrial Proteins; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Tetrahydroisoquinolines; Trabectedin

We previously reported that extracts of an Indonesian marine sponge Haliclona sp. showed potent cytotoxicity and the induction of apoptosis against human solid cancer cell lines. In this study, we examine the cytotoxic mechanism of the major chemical compound, papuamine, on MCF-7 human breast cancer cells. Papuamine at 5 µM did not show significant cytotoxic effects after incubation for 24 h, but autophagosome vesicular formation was apparent. At 10 µM of papuamine, significant reduction in cell survival was observed at 12 h, and increases in autophagy at this concentration were time-dependent and apparent before the appearance of cytotoxic effects. Both the release of cytochrome c to the cytosol and increase in Bax in the mitochondrial fraction were found to be concentration-dependent. Moreover, mitochondrial membrane potential shows concentration- and time-dependent decreases with exposure to papuamine. The release of cytochrome c has been shown to be accompanied by an increase in JNK activation. 3-Methyladenine (MA), a classical autophagy inhibitor showed increased JNK activation by exposure to papuamine. In conclusion, our results indicate that papuamine causes earlier onset autophagy and delayed reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 cells.

Topics: Alkaloids; Apoptosis; Autophagy; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Cytochromes c; Female; Humans; MAP Kinase Kinase 4; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

To observe the expression of the C-terminal truncated human apoptosis-inducing factor (AIF) and its biological effect on MCF-7 cells.. PcDNA3.0-FDT-AIFδ1-480 was transfected into human breast carcinoma MCF-7 cells with lipofectamine. The expression of the truncated AIF gene was detected by Western blotting, and its effects on the biological behaviors of MCF-7 cells and on the expression of cytochrome c (cytC) were evaluated using flow cytometry, MTT assay, colony-forming assay, and mitochondrial membrane potential measurement.. PcDNA3.0-FDT-AIFδ1-480 enhanced AIF expression in MCF-7 cells, obviously inhibited the cell proliferation, and significantly reduced the mitochondrial membrane potentials (P<0.05). Transfection of the cells with PcDNA3.0-FDT-AIFδ1-480 promoted the expression of cytC and resulted in significantly increased apoptosis of MCF-7 cells (P<0.05).. The expression of C-terminal truncated human AIF gene can induce apoptosis of human MCF-7 cells by promoting cytC release from mitochondria.

Topics: Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Cell Proliferation; Cytochromes c; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Caspase 8; Caspases; Cell Proliferation; Cell Size; Cyclin D1; Cyclin-Dependent Kinase 4; Cytochromes c; Dose-Response Relationship, Drug; Fucus; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Microscopy, Fluorescence; Molecular Structure; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Magnolol, a small-molecule hydroxylated biphenol, isolated from the root and stem bark of Magnolia officinalis, has been shown to possess antiproliferative effect on various cancer cell lines. In the current study, we found that magnolol potently inhibited proliferation and induced apoptosis in MCF-7 human breast cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with cell cycle arrest at G2/M phase, increased generation of reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP), release of cytochrome c (Cyto c) and apoptosis inducing factor (AIF) from mitochondria to cytosol, upregulation of Bax, p21 and p53, and down-regulation of Bcl-2, cyclin B1 and cyclin-dependent kinase 1 (CDK1). Our findings indicated that magnolol induced apoptosis in MCF-7 cells via the intrinsic pathway with release of AIF from mitochondrial and G2/M phase arrest pathway. Therefore, magnolol might be a potential lead compound in the therapy of breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Biphenyl Compounds; Bisbenzimidazole; Breast Neoplasms; Caspases; Cell Division; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Cytochromes c; Female; Flow Cytometry; G2 Phase; Genes, bcl-2; Humans; Indicators and Reagents; Lignans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

The ability of a breast cancer cell to evade apoptosis has a key role in tumor progression and sensitivity to treatment. High levels of Bcl-2-associated X protein (Bax) in tumor cells have been found to promote apoptosis and sensitize cells to anti-cancer therapies. Bcl-2-associated X protein redistribution to the mitochondrial membrane results in the release of proapoptotic factors including cytochrome C, second-mitochondrial-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (Smac/DIABLO), and Ca(2+). We aimed to explore this pathway in cancerous breast cell lines treated with the naturally occurring antioxidant 3,5-dimethoxy-4-hydroxystilbene (pterostilbene).. We used whole cell lysates +/- Bax SiRNA from the cell lines MCF-7 and MDA-MB-231 in an enzyme-linked immunosorbent assay to quantify Bax, cytochrome C, Smac/DIABLO expression, and manganese superoxide dismutase (MnSOD) activity after treatment with pterostilbene. We quantified cell death using histone-related DNA complexes from cytosolic and mitochondrial fractions and used methylthiazol tetrazolium assay to analyze cell proliferation, in the presence of Bax-silencing or scrambled RNA. We measured changes in cytosolic calcium using the ratiometric calcium-sensitive dye fura-2-AM using an inverted ratiometric monochromator microscope.. Treatment of MCF-7 and MDA-MB-231 (MDA) cells with pterostilbene caused concentration-dependent increases in intracellular Bax at all doses tested. RNA silencing of Bax resulted in reduced rates of apoptosis in both cells types and increased cell survival when treated with pterostilbene. We observed an increase in cytochrome C in MDA cells after treatment with pterostilbene. The MCF-7 cells showed a net increase in cytosolic cytochrome C, with a corresponding reduction in mitochondrial cytochrome C after treatment with 50 and 75 μmol/L pterostilbene. We observed this again in Smac/DIABLO expression in both cell types. In MCF-7 cells, pterostilbene treatment caused an increase in cytosolic but a decrease in mitochondrial Smac/DIABLO protein concentrations. Pterostilbene significantly increase MnSOD activity in MDA-MB-231 cells. Finally, pterostilbene resulted in significant increases in cytosolic calcium concentrations.. The natural dietary compound pterostilbene has an anti-proliferative effect and induces apoptosis in breast cancer cells in vitro via Bax activation and overexpression, resulting in increased MnSOD, Smac/DIABLO, and cytochrome C activity and cytosolic Ca(2+) overload.

Topics: Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Female; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Stilbenes; Superoxide Dismutase

Dihydroartemisinin (DHA), a semi-synthetic derivative and active metabolite of artemisinin, has been shown to have profound anticancer potential in addition to its strong anti-malarial activity. The purpose of the present study was to thoroughly investigate the anti-neoplastic effects induced by DHA and to provide a molecular basis for the use of DHA in the treatment of breast cancer. Our results demonstrated that DHA could significantly inhibit the cell proliferation of breast cancer in a dose- and time-dependent manner that was associated with induced apoptosis and G0/G1 cell cycle arrest, and the half maximal inhibitory concentrations (IC50) of DHA treatment were 60.03, 33.86 and 17.18 µM for 24, 48 and 72 h, respectively. Moreover, the DHA treatment dramatically increased the protein expression of caspase-8, cleaved caspase-9, activated Bid and induced the release of cytochrome c from mitochondria into the cytosol. In addition, the apoptotic action of DHA was associated with the increased expression of the pro-apoptotic gene Bim and a decreased expression of the anti-apoptotic gene Bcl-2. Therefore, the mitochondrial pathway is involved in the apoptosis of breast cancer cells induced by DHA and the imbalance of the Bim/Bcl-2 interaction may promote the beneficial effect against breast cancer cells. Overall, our study provides the scientific rationale for the clinical usage of DHA for breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Artemisinins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Breast Neoplasms; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Female; Humans; Membrane Proteins; Microbial Sensitivity Tests; Mitochondria; Proto-Oncogene Proteins

Tautomycetin (TMC), originally isolated from Streptomyces griseochromogenes, has been suggested as a potential drug retaining specificity toward colorectal cancer. However, we found that TMC exhibited inhibitory effects on cell proliferation of many cancer cell lines including adriamycin-resistant human breast adenocarcinoma. We investigated its anti-tumor activity and mechanisms in human breast cancer cells for the first time. In this study, we showed that TMC effectively inhibited breast cancer cell proliferation, migration, and invasion. TMC also induced apoptosis in MCF-7 cells. This apoptotic response was in part mediated by Bcl-2 cleavage, leading to the release of cytochrome c, which facilitates binding of Apaf-1 to caspase-9 in its presence and subsequent activation of caspase-7 in apoptosis induction signaling pathways. Furthermore, we identified that TMC induced apoptosis by suppressing Akt signaling pathway activation, which is independent of protein phosphatase PP1 inhibition. The levels of downstream targets of Akt, including phospho-forkhead transcription factor and Bad, were also reduced after TMC treatment. Overall, our results indicate that TMC could be used as a potential drug candidate for breast cancer therapy. More importantly, our study provides new mechanisms for the anticancer effects of TMC.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 7; Caspase 9; Cell Movement; Cell Proliferation; Cytochromes c; Female; Furans; Humans; Lipids; MCF-7 Cells; Oncogene Protein v-akt; Proto-Oncogene Proteins c-bcl-2; Receptors, Neuropeptide Y; Signal Transduction; Tumor Cells, Cultured

Fucoidan, a fucose-rich polysaccharide extracted from brown seaweed, has antitumor, anticoagulant, antiviral, anti-inflammatory, and antibacterial activities. Several studies have shown that a fucoidan treatment of cancer cells induced cytotoxicity and apoptosis and inhibited angiogenesis and invasion. We investigated in the present study the effect of low-molecular-weight fucoidan (LMWF) on apoptosis in estrogen receptor-negative MDA-MB-231 human breast cancer cells. The LMWF treatment of MDA-MB-231 cells was associated with the activation of caspases and mitochondrial dysfunction, including dissipation of the mitochondrial membrane potential (ΔΨm), alteration of Ca(2+) homeostasis, cytochrome c release, and decreased expression of antiapoptotic Bcl-2 family proteins. Understanding the molecular events that mediated LMWF-induced MDA-MB-231 cell death will contribute to a more rational approach to cancer chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Gene Expression; Humans; Membrane Potential, Mitochondrial; Mitochondria; Molecular Weight; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Seaweed; Signal Transduction

Breast cancer stem cells play a crucial role in the relapse of breast cancers because they are resistant to a standard chemotherapy and the residual cancer stem cells are able to proliferate indefinitely. The objectives of present study were to construct a kind of mitochondrial targeting daunorubicin plus quinacrine liposomes for treating and for preventing the recurrence of breast cancer arising from the cancer stem cells. MCF-7 cancer stem cells were identified as CD44(+)/CD24(-) cells and cultured in free-serum medium. Evaluations were performed on MCF-7 cancer stem cells, MCF-7 cancer stem cell mammospheres, and the relapsed tumor by xenografting MCF-7 cancer stem cells into female NOD/SCID mice. The particle size of mitochondrial targeting daunorubicin plus quinacrine liposomes was approximately 98 nm. The mitochondrial targeting liposomes evidently increased the mitochondrial uptake of drugs, were selectively accumulated into mitochondria, activated the pro-apoptotic Bax protein, dissipated the mitochondrial membrane potential, opened the mitochondrial permeability transition pores, released cytochrome C by translocation, and initiated a cascade of caspase 9 and 3 reactions, thereby inducing apoptosis of MCF-7 cancer stem cells. The mitochondrial targeting liposomes showed the strongest efficacy in treating MCF-7 cancer cells in vitro, in treating MCF-7 cancer stem cells in vitro, and in treating the relapsed tumor in mice. Mitochondrial targeting daunorubicin plus quinacrine liposomes would provide a new strategy for treating and preventing the relapse of breast cancers arising from cancer stem cells.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Daunorubicin; Female; Humans; Liposomes; Membrane Potential, Mitochondrial; Mice; Mice, SCID; Mitochondria; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Quinacrine

Breast cancer is conventionally treated by surgery and radiotherapy, with adjuvant chemotherapy and hormonotherapy as supplementary treatments. However, such treatments are associated with adverse side effects and drug resistance. In this study, Pheophorbide a (Pa), a photosensitizer isolated from Scutelleria barbata, was analysed for its antiproliferative effect on human breast tumour cells. The IC (inhibitory concentration)(50) of the combined treatment of Pa and photodynamic therapy (Pa-PDT) on human breast tumour MCF-7 cells was 0.5 µm. Mechanistic studies in MCF-7 cells demonstrated that Pa was localized in the mitochondria, and reactive oxygen species were found to be released after Pa-PDT. Apoptosis was the major mechanism responsible for the tumour cell death, and mitochondrial membrane depolarization and cytochrome c release highlighted the role of mitochondria in the apoptotic mechanism. Up-regulation of tumour suppressor protein p53, cleavage of caspase-9 and poly (ADP-ribose) polymerase suggested that the caspase-dependent pathway was induced, while the release of apoptosis-inducing factors demonstrated that the apoptosis was also mediated by the caspase-independent mechanism. In vivo study using the mouse xenograft model showed a significant inhibition of MCF-7 tumour growth by Pa-PDT. Together, the results of this study provide a basis for understanding and developing Pa-PDT as a cure for breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Cytochromes c; DNA Fragmentation; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Photochemotherapy; Photosensitizing Agents; Plant Extracts; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Scutellaria; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Plant-derived polyhenols inhibit cancer cell proliferation and induce apoptosis. Recently, prenylflavonoids and alkyl-phloroacetophenones have been reported for their in vitro antitumor activity. In the present study, we examined the cytotoxic activity of prenyl (3-PAP) and geranyl (3-GAP) derivatives of phloroacetophenone, and xanthohumol (XN), a prenyl-chalcone, in human breast cancer (MCF-7) and human sarcoma (HT1080) cell lines in vitro. 3-GAP showed the strongest cytotoxicity in these cell lines with IC(50) values of less than 10 µM. In addition, we report that 3-GAP is a more potent anti-cancer agent for breast cancer than XN which is a well-known anticancer flavonoid. Moreover, 3-GAP did not induce cytotoxicity in the normal cell line, TCMK-1, whereas 3-PAP and XN significantly reduced TCMK-1 cell viability. In 3-GAP-treated MCF-7 cells, nuclear accumulation and transcriptional activity of p53 were increased. In addition, pro-apoptotic Bax but not B-cell lymphoma 2 (Bcl-2) expression was increased by 3-GAP. In accordance with the Bax increase, 3-GAP induced mitochondrial cytochrome c release and activated caspase-9, an initiator of the caspase cascade. In the MCF-7 cell line which does not express caspase-3, activation of caspase-7, a member of the caspase-3 subfamily, was increased by 3-GAP. The present results indicate that synthetic 3-GAP is a safe and effective anti-cancer agent, and the Bax-mediated mitochondrial pathway is the main apoptosis signaling pathway of 3-GAP in MCF-7 cells.

Topics: Acetophenones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Cell Line; Cell Line, Tumor; Cytochromes c; Female; Flavonoids; Humans; Inhibitory Concentration 50; Mitochondria; Phytotherapy; Plant Extracts; Propiophenones; Proto-Oncogene Proteins c-bcl-2; Sarcoma; Tumor Suppressor Protein p53

The phosphoinositol-3-kinase (PI3K)/Akt signal transduction pathway is critically important for tumor cell growth, proliferation and apoptosis. Apoptosis activation has been reported to be a good target in cancer therapies. In this study, we have found that jolkinolide B (JB), a diterpenoid from the traditional Chinese medicinal herb Euphorbia fischeriana Steud, strongly inhibited the expression of the PI3K p85 subunit and the phosphorylation of Akt. Furthermore, we evaluated the effects of JB on the proliferation and apoptosis of MDA-MB-231 human breast cancer cells. Our results show significant induction of apoptosis in MDA-MB-231 cells incubated with JB. This effect was enhanced by combination with LY294002. In addition, treatment with JB could induce downregulation of the Bcl-2/Bax ratio, and subsequent promotion of mitochondrial release of cytochrome c and activation of caspase-3. Taken together, JB-induced apoptosis of MDA-MB-231 cells occurs through the mitochondrial pathway. Further, the PI3K/Akt signaling cascade plays a role in the induction of apoptosis in JB-treated cells. These observations suggest that JB may have therapeutic applications in the treatment of cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chromones; Cytochromes c; Diterpenes; Drugs, Chinese Herbal; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Morpholines; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Strobilanthes crispus has been traditionally used as antidiabetic, anticancer, diuretic, antilytic and laxative agent. However, cytotoxicity and antiproliferative effect of S. crispus is still unclear.. Strobilanthes cripus was able to reduce cell viability and proliferation in MTT and BrdU assays. Both cell cycle progression and Tunel assay suggested that IC50 of S. crispus ethanol extract induced sub-G1 cell cycle phase, and DNA fragmentation. On the other hand, translocation of mitochondria cytochrome c release, induction of caspase 3/7 and p53 while suppress XIAP on treated MCF-7 cell were also observed in this study.. Our findings suggest that S. crispus ethanol extract induced apoptosis and DNA fragmentation on hormone dependent breast cancer cell line MCF-7 via mitochondria dependent p53 apoptosis pathway.

Topics: Acanthaceae; Adenocarcinoma; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Female; G1 Phase; Hormones; Humans; Inhibitory Concentration 50; Mitochondria; Phytotherapy; Plant Extracts; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

The development of molecularly targeted drugs has greatly advanced cancer therapy, despite these drugs being associated with some serious problems. Recently, increasing attention has been paid to the anticancer effects of natural products. α-Mangostin, a xanthone isolated from the pericarp of mangosteen fruit, has been shown to induce apoptosis in various cancer cell lines and to exhibit antitumor activity in a mouse mammary cancer model. In this study, we investigated the influence of α-mangostin on apoptosis and cell cycle in the human breast cancer cell line MDA-MB231 (carrying a p53 mutation, and HER2, ER, and PgR negative) in order to elucidate its anticancer mechanisms. In α-mangostin-treated cells, induction of mitochondria-mediated apoptosis was observed. On cell-cycle analysis, G1-phase arrest, increased p21(cip1) expression and decreases in cyclins, cdc(s), CDKs and PCNA were observed. In conclusion, α-mangostin may be useful as a therapeutic agent for breast cancer carrying a p53 mutation and having HER2- and hormone receptor-negative subtypes.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Shape; Cell Survival; Cytochromes c; Female; Fruit; Garcinia mangostana; Humans; Plant Extracts; Xanthones

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Topics: Acetylation; Adenine; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzamides; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Histone Deacetylase Inhibitors; Humans; Microtubule-Associated Proteins; Naphthols; Sirtuins; Tumor Suppressor Protein p53; Up-Regulation

The anti-tumor effect of Icariside II (IcaS), a natural prenylated flavonol glycoside, was studied on human breast cancer MCF7 cells to unveil the underlying mechanisms involved. IcaS in MCF7 cells produced a loss of mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9 revealed the involvement of the intrinsic apoptosis pathway. In contrast, IcaS enhanced the expression level of Fas and the Fas-associated death domain (FADD), and activated caspase-8, suggesting the involvement of the extrinsic apoptosis pathway. IcaS also increased the expression of Bax and BimL without affecting the expression status of Bcl-2 and Bid, suggesting that the apoptosis induced by IcaS was related to Bcl-2 family protein regulation. IcaS thus induced apoptosis in MCF7 cells involving both the intrinsic and extrinsic signaling pathways. Its potential as a candidate for an anti-cancer agent warrants further investigation.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Carcinoma; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; fas Receptor; Fas-Associated Death Domain Protein; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Resveratrol (3,4',5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. Several studies were undertaken to obtain synthetic analogues of resveratrol with potent anticancer activity. The aim of the present study was to investigate the effect of HS-1793 as a new resveratrol analog on apoptosis via the mitochondrial pathway in murine breast cancer cells. A pharmacological dose (1.3-20 µM) of HS-1793 exerted a cytotoxic effect on murine breast cancer cells resulting in apoptosis. HS-1793-mediated cytotoxicity in FM3A cells by several apoptotic events including mitochondrial cytochrome c release, activation of caspase-3 and PARP occurred. In addition, HS-1793 induced collapse of ∆Ψm and enhanced AIF and Endo G release from mitochondria while undergoing apoptosis. These results demonstrate that the cytotoxicity by HS-1793 in FM3A cells can mainly be attributed to apoptosis via a mitochondrial pathway by caspase activation or contributions of AIF and Endo G.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; G1 Phase; Membrane Potential, Mitochondrial; Mice; Mitochondria; Naphthols; Poly(ADP-ribose) Polymerases; Resorcinols; Signal Transduction

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Breast Neoplasms; Caspases; Cell Survival; Cytochromes c; Fas Ligand Protein; Female; Humans; Limonins; MCF-7 Cells; Membrane Potential, Mitochondrial; Neoplasms, Hormone-Dependent; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand

Gambogic acid (GA) is considered a potent anti-tumor agent for its multiple effects on cancer cells in vitro and in vivo. Low concentrations of GA (0.3-1.2 µmol/L) can suppress invasion of human breast carcinoma cells without affecting cell viability. To get a whole profile of the inhibition on breast cancers, higher concentrations of GA and spontaneous metastatic animal models were employed. Treatment with GA (3 and 6 µmol/L) induced apoptosis in MDA-MB-231 cells and the accumulation of reactive oxygen species (ROS). Furthermore, GA induced PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, as well as an increased ratio of Bax/Bcl-2. Moreover, the translocation of apoptotic inducing factor (AIF) and the release of cytochrome c (Cyt c) from mitochondria were observed, indicating that GA induced apoptosis through accumulation of ROS and mitochondrial apoptotic pathway. GA also inhibited cell survival via blocking Akt/mTOR signaling. In vivo, GA significantly inhibited the xenograft tumor growth and lung metastases in athymic BALB/c nude mice bearing MDA-MB-231 cells. Collectively, these data provide further support for the multiple effects of GA on human breast cancer cells, as well as for its potential application to inhibit tumor growth and prevent metastasis in human cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Reactive Oxygen Species; Signal Transduction; Xanthones; Xenograft Model Antitumor Assays

The present study was designed to investigate the abnormalities in the expression of apoptosis-associated proteins that lead to the progression of breast cancer. Sixty breast cancer patients histologically categorized as grade I, II and III, and as pre- and post-menopausal were chosen for the study. We analyzed the expression of the anti-apoptotic and pro-apoptotic Bcl-2 family proteins as well as cytochrome C, Apaf-1 and caspases in tumour and adjacent tissues by immunohistochemical and Western blot analyses. The breast tumours analyzed in the present study were characterized by increased expression of Bcl-2, Bcl-xL and Mcl-1, associated with downregulation in the expression of Bax, cytosolic cytochrome C, Apaf-1 and caspases. The magnitude of the changes was however more pronounced in premenopausal patients and in grade III tumours. The results of the present study confirm that differential expression patterns of Bcl-2 family proteins and caspases are involved in evasion of apoptosis and in the progression of breast cancer.

Topics: Adenocarcinoma; Adult; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; bcl-X Protein; Breast; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Disease Progression; Female; Humans; Middle Aged; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Postmenopause; Premenopause; Proto-Oncogene Proteins c-bcl-2

TMS (2,3',4,5'-tetramethoxystilbene), a stilbene analog derived from rhapontigenin, was previously demonstrated to induce apoptosis in hormone-resistant breast cancer cells. Therefore, this study investigated the anticancer effect of a new stilbene analog, HTMS ((E)-2-hydroxy-3',4,5'-trimethoxystilbene), and its mechanism in various breast cancer cell lines.. The effect of HTMS on cell proliferation of MDA-MB-231, MCF-7, and LTED cells was evaluated using MTT assays. Cell apoptosis was detected by FITC-annexin V staining and flow cytometry analysis, changes in mitochondrial potential were determined by fluorescence microscopy using TMRE staining, and the expression of cleaved PARP and release of cytochrome c were assessed by Western blot analysis.. HTMS significantly decreased the cell viability of various types of breast cancer cells in a dose- and time-dependent manner, characterized by G2/M arrest of the cell cycle and the induction of apoptosis. In particular, HTMS disturbed the mitochondrial membrane potential, causing a release of cytochrome c during apoptosis. Furthermore, HTMS was superior to TMS in inhibiting cancer cell growth in a pilot comparison study.. HTMS is an effective apoptotic agent for breast cancer cells, making it a candidate therapeutic agent for the treatment of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Aryl Hydrocarbon Hydroxylases; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochrome P-450 CYP1B1; Cytochromes c; Enzyme Inhibitors; Female; Fluorescent Dyes; G2 Phase; Humans; Membrane Potential, Mitochondrial; Mitochondria; Organometallic Compounds; Peptide Fragments; Pilot Projects; Poly(ADP-ribose) Polymerases; Stilbenes

Intrinsic resistance of cancers is a major cause of failure in chemotherapy. We proposed here a strategy to overcome intrinsic resistance by constructing cancer cell mitochondria-specifically targeting drug-loaded liposomes, namely, mitosomal daunorubicin plus amlodipine. Anticancer agent daunorubicin and apoptotic inducer amlodipine were loaded together into the mitosomes, and targeting molecule dequalinium was modified on the surface. Evaluations were performed on the breast cancer MCF-7 and resistant MCF-7/adr cells and in animals. Mitosomal daunorubicin plus amlodipine were about 97 nm, selectively accumulated in mitochondria, induced the swelling and disruption of mitochondria, dissipated the mitochondrial membrane potential, released a large amount of cytochrome C by translocation, cleaved Bid, and initiated a cascade of caspase 8 and 3 reactions. A robust anticancer effect was evidenced in vivo. Mitochondria-specifically targeting drug-loaded liposomes would provide a new strategy for treating resistant cancers.

Topics: Amlodipine; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Daunorubicin; Drug Carriers; Drug Resistance, Neoplasm; Female; Humans; In Situ Nick-End Labeling; Liposomes; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria

The aim of the present study was to evaluate the underlying apoptotic mechanisms of celastrol, a major biologically active component of Tripterygium regelii, in human breast adenocarcinoma MCF-7 cells. Celastrol was isolated from T. regelii chloroform extract by silica gel column chromatography, and its chemical structure was identified via (1)H NMR and (13)C NMR. Celastrol significantly inhibited cell growth in dose- and time-dependent manners. Celastrol induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in MCF-7 cells. Celastrol triggered the activation of caspase family proteins. Celastrol caused activation of caspase-7, -8, and -9, PARP cleavage, caspase-8-mediated bid cleavage, and release of cytochrome c and AIF. In addition, celastrol decreased the expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein. These results suggest that celastrol inhibits the proliferation of MCF-7 cells through induction of apoptosis, which is mediated by a mitochondrial-dependent caspase pathway.

Topics: Actins; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Structure; Pentacyclic Triterpenes; Tripterygium; Triterpenes

Involvement of activities of mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) remains unsolved in norcantharidin-associated breast cancer cell apoptosis. This study investigated the anti-cancer effect of norcantharidin and its underlying mechanism in two human breast cancer cell lines, estrogen receptor (ER)- HS-578T and ER+ MCF-7 cells. Norcantharidin induced potent cytotoxicity and arrested cell growth through increasing phosphorylation of Chk1, Chk2 and total p21(Waf1/Cip1) and reducing cyclin B and cdc25c expression. It also induced apoptosis through extrinsic death receptor and intrinsic mitochondrial pathways by cytochrome c release, caspase activation, oligonucleosome appearance, PARP cleavage, and aberration of Bcl-2 family protein expression and phosphorylation. Although norcantharidin did not affect STAT1, STAT3, and STAT5 protein expression, it suppressed STAT3 and STAT5 phosphorylation in HS-578T cells, whereas it up-regulated STAT1 phosphorylation and down-regulated STAT5 phosphorylation in MCF-7 cells. Moreover, norcantharidin activated MAPK family member proteins, extracellular signal-regulated kinase (ERK), p38(MAPK) and c-Jun N-terminal kinase (JNK), were all phosphorylated by treatment. Pretreatment with selective kinase inhibitors significantly attenuated the norcantharidin-induced cytotoxicity in breast cancer cells. These findings suggest the potential involvement of MAPK and STAT pathways in norcantharidin-induced apoptogenesis. Norcantharidin may be an effective anti-cancer drug against breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Female; Humans; Mitogen-Activated Protein Kinases; STAT Transcription Factors

Breast cancer is the second most frequent cause of death by cancer. In Romania, its incidence is around 4200 new cases per year and the mortality is around 2500 cases per year. During the past few years a great number of anti-neoplastic therapies with higher specificity and efficiency were developed. Monitoring the effects of these polychemotherapies is of great importance together with the study of their influence on cellular metabolism. The purpose of the study was to establish the changes in the cellular energy metabolism and apoptotic potential in the anti-neoplastic therapy with bevacizumab. The results obtained show that the apoptotic potential of malignant cells increases significantly during the anti-angiogenic treatment, with reduction of tumor vasculature and that the mitochondrial apoptotic pathway is activated by releasing cytochrome c from the mitochondrial inter-membrane space.

Topics: Adenosine Triphosphatases; Angiogenesis Inhibitors; Apoptosis; Breast Neoplasms; Case-Control Studies; Cytochromes c; Cytosol; Female; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Middle Aged; Neovascularization, Pathologic; Succinate Dehydrogenase

Traditional medicinal herbs are an untapped source of potential pharmaceutical compounds. This study aims to determine whether the proliferation of breast cancer cell lines could be inhibited by germacrone, a natural product isolated from Rhizoma curcuma. Germacrone treatment significantly inhibited cell proliferation, increased lactate dehydrogenase (LDH) release, and induced mitochondrial membrane potential (ΔΨm) depolarization in both MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Germacrone induced MDA-MB-231 and MCF-7 cell cycle arrest at the G0/G1 and G2/M phases respectively and induced MDA-MB-231 cell apoptosis. Furthermore, germacrone treatment significantly increased Bok expression and cytochrome c release from mitochondria without affecting Bcl-2, Bcl-xL, Bax, and Bim protein expressions. In addition, germacrone treatment induced caspase-3, 7, 9, PARP cleavage. We concluded that germacrone inhibited the proliferation of breast cancer cell lines by inducing cell cycle arrest and apoptosis through mitochondria-mediated caspase pathway. These results might provide some molecular basis for the anti-tumor activity of Rhizoma curcuma.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Female; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Sesquiterpenes, Germacrane; Signal Transduction

Neogambogic acid (NGA), an active ingredient in garcinia, can inhibit the growth of some solid tumors and result in an anticancer effect. We hypothesize that NGA may be responsible for the inhibition of proliferation of human breast cancer cell line MCF-7 cells. To investigate its anticancer mechanism in vitro, MCF-7 cells were treated with various concentrations of NGA. Results of MTT (methyl thiazolyl tetrazolum) assay showed that treatment with NGA significantly reduced the proliferation of MCF-7 cells in a dose-dependent manner. NGA could increase the expression of the apoptosis-related proteins FasL, caspase-3, caspase-8, caspase-9, and Bax and decrease the expression of anti-apoptotic protein Bcl-2 accompanied by the mitochondrial transmembrane damage. The antiproliferative effect of NGA on MCF-7 cells is due to the G(0)/G(1) arrest, increased apoptosis and activation of Fas/FasL and cytochrome C pathway. These results provide an important insight into the cellular and molecular mechanisms through which NGA impairs the proliferation of breast cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Fas Ligand Protein; fas Receptor; Female; G1 Phase; Garcinia; Humans; Membrane Potential, Mitochondrial; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle; Terpenes; Xanthenes; Xanthones

Ursolic acid (3-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid derived from leaves, berries, fruits, and flowers of medicinal plants, such as Rosemarinus officinalis. Ursolic acid has been shown to inhibit tumorigenesis, tumor promotion, and suppress angiogenesis. In our present study, we found that ursolic acid decreased cell proliferation rate and induce apoptosis in human breast cancer cell line, MDA-MB-231. When we checked the expression levels of proteins associated with apoptosis signal by using immunoblotting, we found that ursolic acid induces various apoptotic molecules related to either extrinsic or intrinsic apoptosis signal pathway in MDA-MB-231 cells. In our study, we found that ursolic acid induced the appearance of Fas receptor and cleavage of caspase-8, -3 and PARP. We also found that ursolic acid induced Bax up-regulation and Bcl-2 down-regulation and release of cytochrome C to the cytosol from mitochondria. Moreover, ursolic acid cleaved caspase-9 and decreased mitochondrial membrane potential (ΔΨm) as shown with JC-1 staining. These data indicate that ursolic acid induce apoptosis through both mitochondrial death pathway and extrinsic death receptor dependent pathway in MDA-MB-231 cells. Our data clearly indicate that ursolic acid could be used as a potential anticancer drug for breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Down-Regulation; Drug Evaluation, Preclinical; Drug Screening Assays, Antitumor; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Receptors, Death Domain; Signal Transduction; Triterpenes; Up-Regulation; Ursolic Acid

Radix Stephaniae tetrandrae, which contains tetrandrine (Tet) and fangchinoline, is traditionally used as an analgesic, antirheumatic, and antihypertensive drug in China. In this study, we investigated its effect on breast cancer cell proliferation and its potential mechanism of action in vitro. Treatment of cells with fangchinoline significantly inhibited MDA-MB-231 cell proliferation in a concentration- and time-dependent manner. To define the mechanism underlying the antiproliferative effects of fangchinoline, we studied its effects on critical molecular events known to regulate the apoptotic machinery. Specifically, we addressed the potential of fangchinoline to induce apoptosis of breast cancer cells. Fangchinoline induced internucleosomal DNA fragmentation, chromatin condensation, activation of caspases-3, -8, and -9, and cleavage of poly(ADP ribose) polymerase, as well as enhanced mitochondrial cytochrome c release. Furthermore, fangchinoline increased the expression of the proapoptotic protein B cell lymphoma-2 associated X (Bax) and decreased the expression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). In addition, the proliferation-inhibitory effect of fangchinoline was associated with decreased levels of phosphorylated Akt. Our results indicate that fangchinoline can inhibit breast cancer cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and decreasing phosphorylated Akt. Thus fangchinoline may be a novel agent that can potentially be developed clinically to target human malignancies.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Benzylisoquinolines; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Drugs, Chinese Herbal; Female; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-bcl-2; Stephania tetrandra

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits anti-carcinogenic effect. The results of present study showed that NFD inhibited the proliferation of breast cancer MDA-MB-231 cells through the induction of S-phase arrest and apoptosis. NFD-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B, and cyclin-dependent kinase (Cdk)2. NFD-induced apoptosis was characterized by increase of sub-G1 population, phosphatidylserine (PS) externalization, and activation of caspases. Moreover, up-regulation of Bad and down-regulation of Bcl-2, Bcl-X(L), and survivin led to the loss of mitochondrial membrane potential (DeltaPsim), the release of cytochrome c and sequential activation of caspase-9 and caspase-3. NFD activated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in MDA-MB-231 cells. Inhibitors of JNK (SP600125) and ERK (PD98059), but not p38 MAPK (SB203580) suppressed NFD-induced S-phase arrest and apoptosis in MDA-MB-231 cells. Both SP600125 and PD98059 attenuated Bad up-regulation, and reversed down-regulation of Bcl-2, Bcl-X(L), survivin, cyclin A, cyclin B, and Cdk2 in NFD-treated cells. Taken together, our data show that JNK and ERK-signaling pathways play important roles in NFD-mediated S-phase arrest and apoptosis of MDA-MB-231 cells.

Topics: Anhydrides; Annexin A5; Apoptosis; Breast Neoplasms; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Cytosol; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Flow Cytometry; Humans; MAP Kinase Kinase 4; Mitochondria; Mitogen-Activated Protein Kinases; Naphthalenes; S Phase; Signal Transduction

A synthetic amphiphilic block copolymer, Pluronic, is a potent chemosensitizer of multidrug resistant (MDR) cancers that has shown promise in clinical trials. It has unique activities in MDR cells, which include a decrease in ATP pools and inhibition of P-glycoprotein (Pgp) resulting in increased drug accumulation in cells. This work demonstrates that Pluronic rapidly (15min) translocates into MDR cells and co-localizes with the mitochondria. It inhibits complex I and complex IV of the mitochondria respiratory chain, decreases oxygen consumption and causes ATP depletion in MDR cells. These effects are selective and pronounced for MDR cells compared to non-MDR counterparts and demonstrated for both drug-selected and Pgp-transfected cell models. Furthermore, inhibition of Pgp functional activity also abolishes the effects of Pluronic on intracellular ATP levels in MDR cells suggesting that Pgp contributes to increased responsiveness of molecular "targets" of Pluronic in the mitochondria of MDR cells. The Pluronic-caused impairment of respiration in mitochondria of MDR cells is accompanied with a decrease in mitochondria membrane potential, production of ROS, and release of cytochrome c. Altogether these effects eventually enhance drug-induced apoptosis and contribute to potent chemosensitization of MDR tumors by Pluronic.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Mitochondria; Nitrogen Oxides; Oxygen Consumption; Phosphorylation; Poloxamer; Reactive Oxygen Species

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.

Topics: Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspase 6; Cell Line, Tumor; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Female; Humans; JNK Mitogen-Activated Protein Kinases; Lipopeptides; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Peptides, Cyclic; Phosphorylation; Reactive Oxygen Species

The most common alterations found in breast cancer are inactivation or mutation of tumor suppressor gene p53. The present study revealed that theaflavins induced p53-mutated human breast cancer cell apoptosis. Pharmacological inhibition of caspase-8 or expression of dominant-negative (Dn)-caspase-8/Fas-associated death domain (FADD) partially inhibited apoptosis, whereas caspase-9 inhibitor completely blocked the killing indicating involvement of parallel pathways that converged to mitochondria. Further studies demonstrated theaflavin-induced Fas upregulation through the activation of c-jun N-terminal kinase, Fas-FADD interaction in a Fas ligand-independent manner, caspase-8 activation and t-Bid formation. A search for the parallel pathway revealed theaflavin-induced inhibition of survival pathway, mediated by Akt deactivation and Bcl-xL/Bcl-2-associated death promoter dephosphorylation. These well-defined routes of growth control converged to a common process of mitochondrial transmembrane potential loss, cytochrome c release and activation of the executioner caspase-9 and -3. Overexpression of either constitutively active myristylated-Akt (Myr-Akt) or Dn-caspase-8 partially blocked theaflavin-induced mitochondrial permeability transition and apoptosis of p53-mutated cells, whereas cotransfection of Myr-Akt and Dn-caspase-8 completely abolished theaflavin effect thereby negating the possibility of existence of third pathways. These results and other biochemical correlates established the concept that two distinct signaling pathways were regulated by theaflavins to induce mitochondrial death cascade, eventually culminating to apoptosis of p53-mutated human breast cancer cells that are strongly resistant to conventional therapies.

Topics: Antioxidants; Apoptosis; bcl-Associated Death Protein; Biflavonoids; Blotting, Western; Breast Neoplasms; Caspase 8; Caspase 9; Caspase Inhibitors; Catechin; Cytochromes c; fas Receptor; Fas-Associated Death Domain Protein; Female; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunoprecipitation; Membrane Potential, Mitochondrial; Mitochondria; Mutation; Phosphorylation; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Hormonal, targeted and chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by intolerable toxicities. Effective and less toxic therapies for estrogen receptor negative (ER-) breast cancers are urgently needed. Here, we present the potential molecular mechanisms mediating the selective pro-apoptotic effect induced by BN107 and its principle terpene, oleanolic acid (OA), on ER- breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in ER- breast lines. Apoptosis was the major cellular pathway mediating the cytotoxicity of BN107. We demonstrated that sensitivity to BN107 was correlated to the status of ERalpha. Specifically, the presence of functional ERalpha protected cells from BN107-induced apoptosis and absence of ERalpha increased the sensitivity. BN107, an extract rich in OA derivatives, caused rapid alterations in cholesterol homeostasis, presumably by depleting cholesterol in lipid rafts (LRs), which subsequently interfered with signaling mediated by LRs. We showed that BN107 or OA treatment in ER- breast cancer cells resulted in rapid and specific inhibition of LR-mediated survival signaling, namely mTORC1 and mTORC2 activities, by decreasing the levels of the mTOR/FRAP1, RAPTOR and RICTOR. Cotreatment with cholesterol abolished the proapoptotic effect and restored the disrupted mTOR activities. This is the first report demonstrating possible concomitant inhibition of both mTORC1 and mTORC2 activities by modulating the levels of protein constituents present in these signaling complexes, and thus provides a basis for future development of OA-based mTOR inhibitors.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cholesterol; Cytochromes c; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Fluorescent Antibody Technique; Gleditsia; Humans; Mechanistic Target of Rapamycin Complex 1; Membrane Microdomains; Membrane Potential, Mitochondrial; Multiprotein Complexes; Oleanolic Acid; Plant Extracts; Proteins; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Cells, Cultured

The aim of this study was to investigate the pro-apoptotic mechanism of C-phycocyanin (C-PC)-mediated photodynamic therapy (PDT) in a murine tumor model and cultured MCF-7 cells. The mice were divided into four groups: control, He-Ne laser radiation, C-PC treatment, and C-PC treatment + He-Ne laser radiation. The effects of C-PC and/or laser on immune organs, immunocyte proliferation, tumor genesis, and apoptosis-related proteins expressions were investigated by immunohistochemistry, in situ hybridization, MTT, electron microscope, western blot, and immunofluorescence assay. The results showed that He-Ne laser treatment alone showed marginal effects. In C-PC-treated mice, the weight of immune organs, proliferation of immunocytes, and expression of pro-apoptotic Fas protein were increased, whereas the tumor weight and the expressions of anti-apoptotic proteins (NF-kappaB and P53) and CD44 mRNA were comparatively decreased. In vitro, C-PC was able to inhibit MCF-7 cell proliferation and cause ultrastructural changes including microvilli loss, formation of membrane blebs, and chromatin condensation. Moreover, C-PC treatment could activate caspase-9 expression, induce cytochrome c release, and downregulate Bcl-2 expression. When combined with He-Ne laser irradiation, the effects of C-PC treatment were further enhanced. Facilitating the apoptosis signals transduction and finally leading to the apoptosis of MCF-7 cells may be the mechanism of the anti-tumor activities of C-PC-mediated PDT.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 9; Cell Proliferation; Cell Survival; Cytochrome c Group; Cytochromes c; Humans; K562 Cells; Male; Mice; NF-kappa B; Photochemotherapy; Phycocyanin; Tumor Suppressor Protein p53

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Inducing Factor; Aromatase; Aromatase Inhibitors; Benzenesulfonates; Breast Neoplasms; Caspase 7; Caspase 9; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Female; Humans; Letrozole; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Niacinamide; Nitriles; Phenylurea Compounds; Phosphoproteins; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-myc; Pyridines; Retinoblastoma Protein; Ribosomal Protein S6 Kinases, 70-kDa; Sorafenib; Testosterone; Time Factors; TOR Serine-Threonine Kinases; Transfection; Triazoles

Although the anti-tumor effects of carvacrol have been demonstrated earlier, the exact underlying molecular mechanisms involved in its action have not been defined and in the present study an attempt has been made to identify the mechanism of carvacrol induced cell death in human metastatic breast cancer cells, MDA-MB 231.. Apoptosis induced by carvacrol was determined based on different assays like MTT assay, Annexin V, mitochondrial membrane potential assay, multicaspase activation assay and cell cycle analysis by flow cytometer. Cleavage of PARP, cytochrome c release and modulation of Bax and Bcl2 ratio by Western blot analysis were also studied.. The study clearly showed induction of apoptosis by carvacrol in MDA-MB 231 cells dose dependently at an IC(50) of 100 microM with a decrease in the mitochondrial membrane potential of the cells resulting in release of cytochrome c from mitochondria, caspase activation and cleavage of PARP.. The data in the present study clearly demonstrated anti-tumor effects of carvacrol on human metastatic breast cancer cells, MDA-MB 231, and that the compound could have a potential therapeutic significance in treating cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cymenes; Cytochromes c; Dose-Response Relationship, Drug; Female; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mitochondria; Monoterpenes; Origanum; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Thymus Plant

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. Exposure of MDA-MB-231 cells with 0.03, 0.09, and 0.15 microM of CTX III for 18 h, CTX III-induced cell apoptosis, as evidenced by accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (DeltaPsim) with subsequent release of cytochrome c, and activation of both capases-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X(L), and survivin in CTX III-treated cells. Mechanistic studies showed that CTX III suppressed the phosphorylation of JAK2, STAT3, Akt, and activation of PI3K. Moreover, the PI3K inhibitor wortmannin blocked activation of STAT3 and Akt without affecting the JAK2 activation, whereas JAK2 inhibitor AG490 suppressed the levels of phospho-STAT3, phospho-Akt, and PI3K, suggesting that PI3K activation occurs after JAK2 phosphorylation, and both PI3K and JAK2 kinases cooperate to mediate STAT3 and Akt phosphorylation. Both AG490 and wortmannin also led to up-regulation in Bax and Bad, and down-regulation of Bcl-2, Bcl-X(L), and survivin in MDA-MB-231 cells. Taken together, these results indicate that CTX III induces apoptosis in MDA-MB-231 cells via concomitant inactivation of the JAK2, STAT3, PI3K, and Akt signaling pathways.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Survival; Cobra Cardiotoxin Proteins; Cytochromes c; Cytosol; Down-Regulation; Elapid Venoms; Enzyme Activation; Female; Flow Cytometry; Genes, bcl-2; Humans; Janus Kinase 2; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Taiwan

In the present study, the antiproliferative mechanism of action of 1 microM 2-methoxyestradiol (2ME) was investigated in the MCF-7 cell line. Measurement of intracellular cyclin B and cytochrome c protein levels, reactive oxygen species formation, cell cycle progression and apoptosis induction were conducted by means of flow cytometry. Morphological changes were evaluated using transmission electron microscopy and fluorescent microscopy by employing Hoechst 33342 and acridine orange. Gene expression changes were conducted by means of microarrays. 2ME-treated cells demonstrated an increase in cyclin B protein levels, hydrogen peroxide formation, intracellular levels of cytochrome c, as well as an increase in early and late stages of apoptosis. In addition, morphological data revealed the presence of autophagic processes. Fluorescent microscopy showed an increase in acridine orange staining and electron microscopy revealed an increase in vacuolar formation in 2ME-treated cells. The gene expression of several genes associated with mRNA translation, autophagy-related processes and genes involved in microtubule dynamics were affected. The study contributes to the mechanistic understanding of 2ME's growth inhibition in MCF-7 cells and highlights the possibility of both apoptotic and autophagic processes being activated in response to 2ME treatment in this cell line.

Topics: 2-Methoxyestradiol; Adenocarcinoma; Antineoplastic Agents; Apoptosis; Autophagy; Breast Neoplasms; Caspase 3; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Shape; Cyclin B1; Cytochromes c; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotide Array Sequence Analysis; Reactive Oxygen Species; Receptors, Estrogen; Vacuoles

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.. The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.. We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.. Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.. Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Ceramides; Cytochromes c; Cytoplasm; Female; Glutathione; Humans; Lipid Peroxidation; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction

beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis.. The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting.. Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells.. Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Time Factors; Uterine Cervical Neoplasms

N(6)-isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose-dependent manner by arresting the cells at G(0)/G(1) phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl-2 and Bcl-xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase-3). We observed that i6A suppressed the nuclear factor kappaB (NF-κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF-κB cell survival pathway.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 2; Cytochromes c; Female; Humans; Isopentenyladenosine; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction

Bioactive compounds found in many plant species have been used in Chinese, Unani, and Indian ayurvedic medicine. Accumulative evidences in both in vitro and in vivo studies using berberine demonstrated anti-cancer and anti-inflammatory properties in different cancer cells. In the present study, a putative compound from commercial sample was purified by chromatographic techniques. The structure of the pure compound was confirmed by spectroscopic studies. The purified berberine was tested against breast cancer (MCF-7) and normal human breast epithelial (MCF-12F) cells for 24, 48 and 72 h at various concentrations. Using MTT assay, berberine exhibited a significant cytotoxic effect on the MCF-7 cells (P<0.01) without affecting the breast normal epithelial cell growth at 25 microM concentration. Based on these results, MCF-7 cells were treated with 25 microM berberine for 48 and 72 h for further studies to illustrate induction of apoptosis through cell cycle distribution and DNA fragmentation with agarose gel electrophoresis. Western blotting with treated cells revealed that berberine induces apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytoplasmic cytochrome c, caspase-9 activity and cleavage of PARP while decreasing levels of Bcl-2. Furthermore, immunoblotting results demonstrated that p53 and p27 were up-regulated suggesting that barberine seems to play a pro-apoptotic role in cancer cells. In conclusion, berberine inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway. It is possible that berberine may serve as a potential naturally occurring compound for breast cancer therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Blotting, Western; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Female; Humans; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) induces apoptosis in MCF-7 and HCC-1954 human breast cancer cells in a dose- and time-dependent manner. α-TEA induces increased levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor-5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE-like inhibitory protein (c-FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c-FLIP (L), caspase-8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase-9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α-TEA to enhance DR5 protein levels, downregulate c-FLIP(L) protein levels and induce apoptosis. Combination of α-TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c-FLIP (L) protein levels. siRNA knockdown of c-FLIP produces a low level of spontaneous apoptosis and enhances α-TEA- and TRAIL-induced apoptosis. Taken together, these studies show that α-TEA induces TRAIL/DR5 mitochondria-dependent apoptosis in human breast cancer cells, and that TRAIL/DR5-dependent increases in DR5 and decreases in c-FLIP expression are triggered by TRAIL or α-TEA treatments.

Topics: alpha-Tocopherol; Antioxidants; Apoptosis; Blotting, Western; Breast Neoplasms; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspase 9; Cytochromes c; Cytosol; Female; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Receptors, TNF-Related Apoptosis-Inducing Ligand; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Vitamin E

Metastatic breast cancers are hard to treat and almost always fatal. Chloroquine diphosphate, a derivative of quinine, has long been used as a potent and commonly used medicine against different human diseases. We therefore investigated the effects of chloroquine diphosphate on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of 4T1 mouse breast cancer cells with chloroquine diphosphate resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated that induction of apoptosis was associated with the loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. The effect of chloroquine diphosphate was then examined using a mice model in which 4T1 cells were implanted subcutaneously. Chloroquine diphosphate (25mg/kg and 50mg/kg, respectively) significantly inhibited the growth of the implanted 4T1 tumor cells and induced apoptosis in the tumor microenvironment. Moreover, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggested that chloroquine diphosphate might have chemotherapeutic efficacy against breast cancer including inhibition of metastasis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cytochromes c; Female; Mammary Neoplasms, Experimental; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Poly Adenosine Diphosphate Ribose; Xenograft Model Antitumor Assays

Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage-inducible gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53-independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl-2 ratio resulted in enhanced apoptosis. Caspase-8 and tBid (truncated Bid) expression also increased in a time-dependent manner. The enzymatic activities of caspase-3 and caspase-9 increased approximately 13-fold and 7.8-fold, respectively. In addition, the proteolytic cleavage of poly-(ADP-ribose)-polymerase (PARP) and DNA fragmentation factor-45 (DFF-45) increased in dose- and time-dependent manners. Our data suggest a promising therapeutic application of garcinol in p53-independent apoptosis in cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Arabidopsis Proteins; Breast Neoplasms; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Female; Hep G2 Cells; Humans; Intramolecular Transferases; Liver Neoplasms; Mitochondrial Diseases; Plant Extracts; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Reactive Oxygen Species; Terpenes; Transcription Factor CHOP

The role of selenium as potential cancer chemopreventive and chemotherapeutic agents has been supported by epidemiological, preclinical and clinical studies. Although cell apoptosis has been evidenced as a critical mechanism mediating the anticancer activity of selenium, the underlying molecular mechanisms remain elusive. In the present study, we showed that selenocystine (SeC), a naturally occurring selenoamino acid, induced caspase-independent apoptosis in MCF-7 breast carcinoma cells, which was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage, caspase activation, DNA fragmentation, phosphatidylserine exposure and nuclear condensation. Moreover, SeC induced the loss of mitochondrial membrane potential (DeltaPsi(m)) by regulating the expression and phosphorylation of Bcl-2 family members. Loss of DeltaPsi(m) led to the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF) which subsequently translocated into the nucleus and induced chromatin condensation and DNA fragmentation. MCF-7 cells exposed to SeC shown increase in total p53 and phosphorylated p53 on serine residues of Ser15, Ser20, and Ser392 prior to mitochondrial dysfunction. Silencing and attenuating of p53 activation with RNA interference and pifithrin-alpha treatment, respectively, partially suppressed SeC-induced cell apoptosis. Furthermore, generation of reactive oxygen species and subsequent induction of DNA strand breaks were found to be upstream cellular events induced by SeC. The thiol-reducing antioxidants, N-acetylcysteine and glutathione, completely blocked the occurrence of cell apoptosis. Taken together, these results suggest that SeC, as a promising anticancer selenocompound, induces MCF-7 cell apoptosis by activating ROS-mediated mitochondrial pathway and p53 phosphorylation.

Topics: Acetylcysteine; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspases; Cell Line, Tumor; Chromatin Assembly and Disassembly; Cystine; Cytochromes c; Female; Humans; Membrane Potential, Mitochondrial; Organoselenium Compounds; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Suppressor Protein p53

Mitochondria and associated oxidative stress have been shown to play critical roles in apoptotic death induced by various stress agents. Previously, we reported the antitumor property of diospyrin (D1), a plant-derived bisnaphthoquinonoid, and its diethylether derivative (D7), which was found to cause apoptotic death in human cancer cell lines. The present study aims to explore the relevant mechanism of apoptosis involving generation of cellular reactive oxygen species (ROS) by D7 in human breast carcinoma (MCF-7) cells. It was found that while D7 inhibited the proliferation of tumor cells, the associated apoptosis induced by D7 was prevented by treating the cells with N-acetyl-L-cysteine (NAC), an antioxidant, and cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition (MPT). Experiments using suitable inhibitors also demonstrated that D7 could alter the electron flow in mitochondrial electron transport chain by affecting target(s) between complex I and complex III, and indicated the probable site of D7-induced generation of ROS. These results were further supported by confocal microscopic observation on changes in mitochondrial organization and shape in cells treated with D7. Taken together, the results of our study clearly suggested that the apoptosis induced by D7 would involve alteration of MPT, cardiolipin peroxidation, migration of Bax from cytosol to mitochondria, decreased expression of Bcl-2, and release of cytochrome c, indicating oxidative mechanism at the mitochondrial level in the tumor cells.

Topics: Acetylcysteine; Allopurinol; Animals; Antimycin A; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cardiolipins; Cell Line, Tumor; Cyclosporine; Cytochromes c; Female; Free Radical Scavengers; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Rotenone; Uncoupling Agents; Vitamin K 3

This study investigated the role of the caspase activation cascade in extrinsic and intrinsic apoptosis induced by equol in human breast cancer MDA-MB cells. First, the antiproliferative effect of equol was determined in cells treated with 1-100 microM equol for 24, 48, and 72h. Equol significantly inhibited cell proliferation in a dose-dependent manner (p<0.05). Exposure to 50 or 100 microM equol for 72h strongly promoted apoptosis. Under the same conditions, remarkable cytochrome c release was observed. Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by equol at high concentrations, but caspase-8 activation of receptor-mediated apoptosis was not observed. At both equol concentrations, the caspase-8 and -9 activity assays showed similar patterns. In addition, equol treatment activated caspase-3, which is downstream from caspase-9, and this was accompanied by the cleavage of capase-6 and -7. Activation of these caspases leads to increased activation of PARP, lamin, and ICAD. This study suggests that equol induces the intrinsic pathway of apoptosis via caspase-9 and cytochrome c, independent of caspase-8, in human breast cancer MDA-MB-453 cells.

Topics: Apoptosis; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Equol; Humans; Isoflavones

This study investigated apoptotic mechanisms of down-expression vascular endothelial growth factor (VEGF) by short interfering RNA (siRNA) in human breast cancer MCF-7 cells. Human breast cancer cells were evaluated for the expression of VEGF and VEGF receptor 2 (VEGFR-2). siRNA targeting VEGF mRNA were chemically synthesized and transfected into cells with Lipofectamine2000. In vitro assessments were then made of the ability of anti-VEGF siRNA to knock down expression of VEGF and the subsequent effect this decreased expression had on breast cancer cell apoptosis. Growth curve construction and nude mice experimentation in vivo were performed to assess the effects of VEGF silencing on tumor growth. Those cells transfected with siRNA targeting VEGF showed a 65% knockdown in VEGF expression and a marked increase in cell apoptosis. The expression of Bcl-2 protein in MCF-7 cells was decreased, the level of Bax protein was kept the same, cytochrome c was released from mitochondria into cytosol, and the cleaved Caspase-3 protein rose after siRNA transfection. The siRNA targeting human VEGF could induce apoptosis in MCF-7 cells and the mechanism of apoptosis is possibly related with changing Bcl-2/Bax expression ratio, releasing cytochrome c from mitochondria into cytosol, and up-regulation of Caspase-3 protein, but also could suppress the growth of breast cancer cells in vivo. VEGF might be a potential therapeutic target for human breast cancer.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Apoptosis is a key mechanism in numerous pathologies. However, there are no effective noninvasive means available for an early detection and quantitative assessment of evolution dynamics of the apoptotic process. Here, we have characterized the ability of the novel PET voltage sensor (18)F-fluorobenzyl triphenyl phosphonium ((18)F-FBnTP) to quantify the time-dependent apoptotic action of the taxanes paclitaxel and docetaxel in vitro and in vivo.. The duration-dependent treatment effect of paclitaxel on (18)F-FBnTP uptake was assayed in human MDA-MB-231 breast carcinoma cells. The expression of the proapoptotic Bax and antiapoptotic Bcl-2 mitochondrial proteins, release of the apoptogen cytochrome c, and activation of executioner caspase-3 were determined by Western blotting. The fraction of viable cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The effect of docetaxel on (18)F-FBnTP and (18)F-FDG uptake in orthotopic prostate tumors in mice was compared.. (18)F-FBnTP cellular uptake in viable cells declined linearly with the increasing duration of paclitaxel treatment, from 3 to 24 h, and plateaued at 48 h. The extent of decrease of (18)F-FBnTP correlated strongly with the Bax-to-Bcl-2 ratio (R(2) = 0.83) and release of cytochrome c (R(2) = 0.92), but preceded in time the caspase-3 cleavage. The P-glycoprotein blocker verapamil did not interfere with (18)F-FBnTP cellular uptake. (18)F-FBnTP prostate tumor contrast was greater than (18)F-FDG prostate tumor contrast. Docetaxel caused a marked decrease (52.4%) of (18)F-FBnTP tumor uptake, within 48 h, whereas (18)F-FDG was much less affected (12%).. The voltage sensor (18)F-FBnTP is a viable means for quantification of paclitaxel pharmacodynamics. (18)F-FBnTP permits the detection of paclitaxel apoptotic action in vivo earlier than does (18)F-FDG. (18)F-FBnTP may afford a novel approach for early detection and quantitative assessment of the cumulative-effect kinetics of proapoptotic drugs and conditions using PET.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Docetaxel; Genes, bcl-2; Humans; Membrane Potential, Mitochondrial; Organophosphorus Compounds; Paclitaxel; Positron-Emission Tomography; Radiopharmaceuticals; Taxoids; Tubulin Modulators

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced apoptosis in human breast MCF-7 cancer cells was confirmed by sub-G1 formation, phosphatidylserine (PS) externalization, and poly (ADP-ribose) polymerase (PARP) cleavage with an IC50 of 2 microg/ml at 48 h. Effects of CTX III on proliferation and apoptosis correlated with upregulation of Bax, and downregulation of Bcl-XL, Bcl-2, and XIAP, with no appreciable alteration on the protein levels of Bid, Bim, and survivin. CTX III treatment also caused release of mitochondrial cytochrome c to the cytosol, which led to subsequent activation of capase-9. Moreover, CTX III inhibited the nuclear factor-kappaB (NF-kappaB) activation through inhibition of IkappaB kinase (IkappaK) activity. Overall, our results indicate that CTX III downregulates NF-kappaB in MCF-7 cells, leading to the suppression of proliferation and induction of apoptosis. These findings suggest the molecular basis for CTX III-induced apoptotic death of MCF-7 cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cobra Cardiotoxin Proteins; Cytochromes c; Cytosol; Elapid Venoms; Female; Humans; I-kappa B Kinase; Mitochondria; NF-kappa B; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; X-Linked Inhibitor of Apoptosis Protein

Epidemiological and animal model studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anti-cancer effect in different type of cancers. In the current study, five breast carcinoma cell lines were used to explore the anti-cancer mechanisms of a nimesulide derivative compound 76. The compound dose dependently suppressed SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC(50) of 0.9microM, 2.2microM and 4.0microM, respectively. However, it needs much higher concentrations to inhibit MCF-7 and MDA-MB-231 breast cancer cell growth with IC(50) at 22.1microM and 19.6microM, respectively. Further investigation reveals that compound 76 induced apoptosis in SKBR-3 and BT474 cells. Since these cells are Her2 overexpressing cells, the Her2 intracellular signaling pathways were examined after the treatment. There was no significant changing of kinase activity. However, the cytochrome c release assay indicated that the apoptosis induced by the compound was mediated by the mitochondria. These results suggest that compound 76 selectively induce apoptosis in Her2 overexpressing breast cancer cells through the mitochondria, and could be used as a lead to design more potent derivatives.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2 Inhibitors; Cytochromes c; Female; Humans; Inhibitory Concentration 50; Mitochondria; Receptor, ErbB-2; Sulfonamides

Doxorubicin (Dox) is a commonly used drug to treat various types of cancers. Previously, we demonstrated that coupling Dox to cell-penetrating peptides (CPPs) represent a valuable strategy to overcome drug resistance in MDA-MB 231 breast cancer cells. In the present study, we evaluated the properties of these Dox conjugates (Dox-CPPs) in terms of apoptosis induction. Dox-CPPs were found to induce apoptotic death in MDA-MB 231 cells at a lower dose than that needed for unconjugated Dox. Cell death induction was associated with Bax oligomerisation, release of cytochrome c, caspase activation, chromatin condensation and internucleosomal degradation. However, whereas Bcl-2 overexpression was very potent in inhibiting apoptosis triggered by Dox, this anti-apoptotic protein was largely inefficient in preventing Dox-CPPs-induced apoptosis. These observations suggest that mitochondrial disruption is the main event in Dox-induced apoptotic signaling but that Dox-CPPs are probably able to trigger additional apoptotic pathways independent of mitochondrial events. Thus, the higher efficacy of Dox conjugated to CCPs in apoptosis induction might not be due exclusively to increased drug accumulation but also to the activation of multiple apoptotic pathways.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; CHO Cells; Chromatin Assembly and Disassembly; Cricetinae; Cricetulus; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Carriers; Enzyme Activation; Female; Humans; Mice; Mitochondria; Peptides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Time Factors

Gene therapy and virotherapy are among the approaches currently being used to treat lung cancer. The success of cancer gene therapy depends on treatments where different types of tumors can be selectively targeted and destroyed without affecting normal cells and tissue. Previously, we described a promoter system (TTS) that we designed that is specifically targeted to lung cancer cells but which does not affect other types of cells including stem cells. In our study, we have enhanced the utility of the TTS system by inserting the pro-apoptotic gene BH3 domain interacting death agonist (Bid) into the TTS promoter system (TTS/Bid) to create a drug regulatable lung cancer-specific gene therapy. A recombinant adenoviral vector was used to introduce TTS/Bid (Ad-TTS/Bid) into lung cancer cells. BID expression and apoptosis occurred in A549 pulmonary adenocarcinoma cells but little Bid expression or apoptosis occurred in MCF7 breast cancer cells or in normal human lung fibroblasts. The use of cisplatin enhanced the processing of full length BID to t-BID which significantly increased lung cancer-specific cell death. In in vivo experiments, intraperitonal injection of cisplatin enhanced the antitumor effects of the vector in a lung cancer xeno-graft mouse model. Moreover, dexamethasone effectively suppressed exogenous BID expression and the antitumor effect of Ad-TTS/Bid both in vitro and in vivo. Here, we describe the efficacy of the use of cisplatin and dexamethasone with the anti lung cancer promoter system (Ad-TTS/Bid) for a safe and effective gene therapy against advanced lung cancer.

Topics: Adenocarcinoma; Adenoviridae; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Caspases; Cisplatin; Colony-Forming Units Assay; Cytochromes c; Dexamethasone; Female; Fibroblasts; Flow Cytometry; Humans; Immunoblotting; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; Xenograft Model Antitumor Assays

We tested the anticarcinogenic effect of alpha-linolenic acid (ALA) as a single compound. To test the role of ALA in breast cancer cells (MCF-7), we analyzed the antiproliferative pathway and the proapoptotic pathway. ALA exhibited growth inhibition on MCF-7 cells dose-dependently of ALA in 24, 48, and 72 h, without possible cytotoxicity per se. ALA enhanced the cell growth-inhibitory activity in a dose-dependent manner. Second, the proapoptotic pathway showed a sub-G(1) accumulation with concomitant upregulation of proapoptotic Bax expression, as well as a downregulation of antiapoptotic Bcl-2 expression dose-dependently, causing the Bcl-2/Bax ratio to decrease by about 50%. Subsequent cytochrome c release and proteolytic activation of caspase-3 followed by proteolytic cleavage of poly(ADP-ribose) polymerase all suggest ensuing progression to apoptosis. This finding suggests that ALA alone might also be responsible for growth-inhibitory and proapoptotic effects on estrogen-positive breast cancer cells.

Topics: alpha-Linolenic Acid; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen

Fucoidan is an active component of seaweed that has been shown to inhibit proliferation and induce apoptotic cell death in several tumor cells. However, the detailed mechanisms underlying this process have not yet been elucidated. In the present report, we investigated the effect of fucoidan on the induction of apoptosis in human breast cancer MCF-7 cells. Our data demonstrated that fucoidan reduced the viable cell number of MCF-7 cells in a dose- and time-dependent manner. In contrast, fucoidan did not affect the viable cell number of normal human mammary epithelial cells. Results from the apoptosis assay demonstrated that fucoidan induced internucleosomal DNA fragmentation, chromatin condensation, activation of caspase-7, -8, and -9, and cleavage of poly(ADP ribose) polymerase. Furthermore, expression of Bid was decreased, whereas truncated Bid was increased by fucoidan treatment. There was also a decline in cytosolic Bax and a striking increase of cytosolic cytochrome c. Caspase-8-specific inhibitor, z-ITED-fmk, canceled the cytotoxicity of fucoidan, activation of caspase-7, -8, and -9, and a series of changes in Bax, Bid, and cytochrome c. However, caspase-9-specific inhibitor exerted a moderate inhibitory effect on the cytotoxicity of fucoidan. These data indicated that fucoidan could induce apoptotic cell death through a caspase-8-dependent pathway in MCF-7 cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Humans; Poly(ADP-ribose) Polymerases; Polysaccharides

The physical manifestations of aging reflect a loss of homeostasis that effects molecular, cellular and organ system functional capacity. As a sentinel homeostatic pathway, changes in apoptosis can have pathophysiological consequences in both aging and disease. To assess baseline global apoptosis balance, sera from 204 clinically normal subjects had levels of sFas (inhibitor of apoptosis), sFasL (stimulator of apoptosis), and total cytochrome c (released from cells during apoptosis) measured. Serum levels of sFas were significantly higher while sFasL and cytochrome c levels were lower in men compared to women. With increasing age there was a decrease in apoptotic markers (cytochrome c) and pro-apoptotic factors (sFasL) and an increase in anti-apoptotic factors (sFas) in circulation. The observed gender differences are consistent with the known differences between genders in mortality and morbidity. In a separate cohort, subjects with either breast (n = 66) or prostate cancer (n = 38) exhibited significantly elevated sFas with reduced sFasL and total cytochrome c regardless of age. These markers correlated with disease severity consistent with tumor subversion of apoptosis. The shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation.

Topics: Adult; Aged; Aged, 80 and over; Aging; Apoptosis; Biomarkers; Body Mass Index; Breast Neoplasms; Cytochromes c; Fas Ligand Protein; fas Receptor; Female; Humans; Male; Middle Aged; Neoplasm Staging; Neoplasms; Prostatic Neoplasms; Sex Characteristics; Young Adult

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration-and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 microM and 50 microM for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cytochromes c; Dose-Response Relationship, Drug; Emodin; Female; Flow Cytometry; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors

The growth inhibitory effect of a mixture of trans, trans conjugated linoleic acid isomers (t, t CLA) was investigated in a human breast cancer cell line, MCF-7, with references to c9, t11 CLA, t10, c12 CLA, and linoleic acid. The t, t CLA treatment effectively induced a cytotoxic effect in a time-dependent (0-6 days) and concentration-dependent (0-40 microM) manner, as compared to the reference and control treatments. The apoptotic parameters were measured on cells treated with 40 microM t, t CLA for 4 days. The occurrence of the characteristic morphological changes and DNA fragmentation confirmed apoptosis. The t, t CLA treatment led to an increase in the level of p53 tumor suppressor protein and Bax protein, but suppressed the expression of Bcl-2 protein. In addition, cytochrome c was released from the mitochondria into the cytosol, and the activation of caspase-3 led to the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, the composition of the linoleic and arachidonic acids was decreased in cellular membranes. These findings suggest that incorporation of t, t CLA in the membrane induces a mitochondria-mediated apoptosis that can enhance the antiproliferative effect of t, t CLA in MCF-7 cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Humans; Linoleic Acids, Conjugated; Mitochondria; Proto-Oncogene Proteins c-bcl-2

Sanguinarine is a benzophenanthridine alkaloid that is known to have antimicrobial, anti-inflammatory, antioxidant and anticancer properties. In this study, we examined the effects of this compound on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death using a human breast carcinoma MDA-MB-231 cell line.. Cytotoxicity was evaluated by trypan blue exclusion methods. Apoptosis was detected using DAPI staining, agarose gel electrophoresis and flow cytometer. The expression levels of proteins were determined by Western blot analyses and caspase activities were measured using colorimetric assays. The ROS production and mitochondrial membrane potential (MMP, Delta Psi(m)) changes were measured fluorimetrically.. The results of this study demonstrate that sanguinarine mediates ROS production, and that this mediation is followed by a decrease in MMP, the release of cytochrome c, activation of caspase-9 and caspase-3, and downregulation of antiapoptosis factor XIAP and cIAP-1. Sanguinarine also promoted the activation of caspase-8 and truncation of Bid (tBid). Moreover, the quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the sanguinarine-induced apoptosis effects via inhibition of ROS production, MMP collapse, tBid expression and the subsequent activation of caspases. These observations clearly indicate that ROS are involved in the early molecular events in the sanguinarine-induced apoptotic pathway.. Our data imply that sanguinarine-induced ROS are key mediators of MMP collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in apoptosis.

Topics: Apoptosis; Benzophenanthridines; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Humans; Isoquinolines; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Reactive Oxygen Species

Normal breast development is controlled by a balance between cell proliferation and apoptosis. The balance between the two parameters is crucial for determining the growth or regression of breast tumours in response to therapies and treatments. Therefore, it is necessary to understand the role of apoptosis in tumour progression. Active caspases participate as essential elements in the execution of apoptotic mechanisms. In the present study, we analysed the activities of caspase-3, -8 and -9 as well as cytochrome c release in N-methyl-nitrosourea (NMU)-induced rat mammary tumours, in order to establish the apoptotic events that occur in tumour growth in this animal model. Forty female virgin Wistar rats were randomly divided into two groups. One group was injected intraperitoneally with three doses of 50 mg/kg body weight of NMU. The control group received the vehicle only. After 122 days of NMU injection, the rats were sacrificed and the tumours were excised and processed. Results showed that in mammary tumours induced by NMU, the apoptotic death receptor-mediated pathway is activated through caspase-3 and -8, but the apoptotic mitochondrial pathway is suppressed through a non-activating process of caspase-9 activity, despite the release of cytochrome c. In conclusion, these findings have demonstrated a suppression of the apoptotic mitochondrial pathway through a non-activating process of caspase-9 activity, despite the release of cytochrome c in mammary tumours induced by NMU. Although the apoptotic death receptor-mediated pathway is activated, it is not enough to maintain the balance between proliferation and apoptosis, and thus determine the overall growth of the tumour.

Topics: Alkylating Agents; Animals; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Female; Mammary Neoplasms, Animal; Methylnitrosourea; Mitochondria; Rats; Rats, Wistar; Tumor Cells, Cultured

Polyamine analogue treatment results in growth inhibition and sometimes in cell death. Therefore, polyamine analogues are considered in the treatment of cancer; however, the cellular properties that govern sensitivity are not known. The objective of this study was to elucidate molecular mechanisms behind apoptosis induced by the polyamine analogue N, N-diethylnorspermine (DENSPM). Four different breast cancer cell lines were treated with DENSPM. Cell death was evaluated with flow cytometry and a caspase 3 assay. The levels of a number of proapoptotic and antiapoptotic proteins in subcellular compartments were evaluated with western blot. In the most sensitive cell line, DENSPM treatment induced the release of cytochrome c from mitochondria, resulting in activation of caspase 3 but without decreasing the mitochondrial transmembrane potential. However, in the three other cell lines DENSPM treatment did not induce extensive cell death. This is partly explained by the high levels of antiapoptotic proteins Bcl-2 and Bad and low levels of proapoptotic proteins Bax and procaspase 3 in these three cell lines. The results are also partly explained by the degree of activation of the catabolic enzyme spermidine/spermine-N-acetyltransferase and polyamine pool reduction achieved by DENSPM treatment. Our results show that the protein profile of proapoptotic and antiapoptotic proteins may contribute to the outcome to treatment with the polyamine analogue DENSPM. The results also indicate that it should be possible to find molecular markers for sensitivity to DENSPM that could be used in the clinic to predict sensitivity to a polyamine analogue.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Spermine

Tamoxifen has efficacy as a breast cancer therapy and chemoprevention agent. However, toxicity and resistance to tamoxifen limit its clinical application. There is an urgent need to develop compounds that may be combined with tamoxifen to improve efficacy and overcome toxicity and resistance. We showed previously that the organoselenium compound methylseleninic acid (MSA) increased the growth-inhibitory effect of tamoxifen and reversed tamoxifen resistance in breast cancer cells. In this study, we examined the mechanism for induction of apoptosis by MSA combined with tamoxifen in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. 4-hydroxytamoxifen (TAM; 10(-7) mol/L) alone resulted in cell cycle arrest but no apoptosis, whereas MSA alone (10 micromol/L) induced apoptosis in tamoxifen-sensitive cells. Combination of MSA with TAM resulted in a synergistic apoptosis in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells compared with either agent alone. MSA and MSA combined with TAM induced apoptosis through the intrinsic, mitochondrial apoptotic pathway. MSA induced a sequential activation of caspase-9 and then caspase-8. These results indicate that the growth inhibition synergy and reversal of tamoxifen resistance by combination of selenium with tamoxifen occurs via a tamoxifen-induced cell cycle arrest, allowing more cells to enter the intrinsic apoptotic pathway elicited by selenium.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Blotting, Western; Breast Neoplasms; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Female; G1 Phase; Humans; Membrane Proteins; Mitochondria; Models, Biological; Organoselenium Compounds; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Tamoxifen

A water-soluble tetra-S-glycosylated porphyrin (P-Glu(4)) is absorbed by MDA-MB-231 human breast cancer cells whereupon irradiation with visible light causes necrosis or apoptosis depending on the concentration of the porphyrin and the power of the light. With the same amount of light irradiation power (9.4 W m(-2)), at 10-20 microM concentrations necrosis is predominantly observed, while at <10 microM concentrations, apoptosis is the principal cause of cell death. Of the various possible pathways for the induction of apoptosis, experiments demonstrate that calcium is released from the endoplasmic reticulum, cytochrome c is liberated from the mitochondria to the cytosol, pro-caspase-3 is activated, poly-(ADP-ribose) polymerase is cleaved, and the chromatin is condensed subsequent to photodynamic treatment of these cells. Confocal microscopy indicates a substantial portion of the P-Glu(4) is located in the endoplasmic reticulum at <10 microM. These data indicate that the photodynamic treatment of MDA-MB-231 cells using low concentrations of the P-Glu(4) porphyrin and low light induces apoptosis mostly initiated from stress produced to the endoplasmic reticulum.

Topics: Animals; Apoptosis; Breast Neoplasms; Calcium; Caspases; Cattle; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum; Enzyme Activation; Glycosylation; Hydrolysis; Indoles; Light; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Poly(ADP-ribose) Polymerases; Porphyrins; Staining and Labeling; Stress, Physiological

Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.. Estrogen deprived MCF-7:2A cells were treated with 1 nM 17beta-estradiol (E2), 100 microM BSO, or 1 nM E2 + 100 microM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model.. Exposure of MCF-7:2A cells to 1 nM E2 plus 100 microM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E2 significantly reduced tumor growth of MCF-7:2A cells.. Our data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.

Topics: Animals; Annexin A5; Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Buthionine Sulfoximine; Cell Proliferation; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Estrogens; Female; Forkhead Transcription Factors; Glutathione; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br-C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over-expressed the anti-apoptotic protein Bcl-2 in L56Br-C1 cells and investigated the effect of DENSPM treatment. DENSPM-induced cell death was significantly reduced in Bcl-2 over-expressing cells. Bcl-2 over-expression reduced DENSPM-induced release of the pro-apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl-2 over-expression reduced the DENSPM-induced activation of caspase-3. Bcl-2 over-expression also prevented DENSPM-induced Bax cleavage and reduction of Bcl-X(L) and survivin levels. The DENSPM-induced activation of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase was reduced by Bcl-2 over-expression, partly preventing polyamine depletion. Thus, Bcl-2 over-expression prevented a number of DENSPM-induced apoptotic effects.

Topics: Acetyltransferases; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Microtubule-Associated Proteins; Mitochondrial Proteins; Neoplasm Proteins; Polyamines; Proto-Oncogene Proteins c-bcl-2; Spermine; Survivin; Transfection

Goal of this study was to investigate the pro-apoptotic properties of RRR-gamma-tocopherol (gammaT) in human breast cancer cells. gammaT was shown to induce cancer cells but not normal cells to undergo apoptosis, sensitize cancer cells to Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced apoptosis, and increase death receptor 5 (DR5) mRNA, protein and cell surface expression. Knockdown of DR5 attenuated gammaT-induced apoptosis. Investigations of post-receptor signaling showed: caspase-8, Bid and Bax activation, increases in mitochondria permeability, cytochrome c release and caspase-9 activation. Thus, gammaT is a potent pro-apoptotic agent for human breast cancer cells inducing apoptosis via activation of DR5-mediated apoptotic pathway.

Topics: alpha-Tocopherol; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Female; gamma-Tocopherol; Humans; Mammary Glands, Human; Mitochondria; Mitochondrial Membranes; Permeability; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Vitamins

Recent studies suggested that boron has a chemo-preventive role in prostate cancer. In the present report, we investigated the effects of calcium fructoborate (CF) and boric acid (BA) on activation of the apoptotic pathway in MDA-MB-231 human breast cancer cells. Exposure to BA and CF inhibited the proliferation of breast cancer cells in a dose-dependent manner. Treatment with CF but not BA resulted in a decrease in p53 and bcl-2 protein levels. Furthermore, after the treatment with CF, augmentation of pro-caspase-3 protein expression, cytosolic cytochrome c level, and caspase-3 activity were observed, indicating apoptotic cell death induction. This was also demonstrated by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end-labeling assay. In conclusion, our data provide arguments to the fact that both BA and CF inhibited the growth of breast cancer cells, while only CF induced apoptosis. Additional studies will be needed to identify the underlying mechanism responsible for the observed cellular responses to these compounds and to determine if BA and CF may be further evaluated as chemotherapeutic agents for human cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Borates; Boric Acids; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Female; Fructose; Humans; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

In the present study, we tested the significance of mitochondria for apoptosis upon exposure to tamoxifen and etoposide using two human breast cancer cell lines, MCF-7 and MDA-MB-231. We showed that both tamoxifen and etoposide induced apoptosis, increased intramitochondrial calcium and nitric oxide, and decreased mitochondrial transmembrane potential in both cell lines. Both drugs increased mitochondrial protein tyrosine nitration and caused release of cytochrome c from the mitochondria of both cell lines. This study suggests that tamoxifen and etoposide utilize a common mechanism to induce apoptosis in MCF-7 and MDA-MB-231 cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Etoposide; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Nitric Oxide; Tamoxifen; Time Factors; Tyrosine

Novel dietary agents for prevention and therapy of prostate cancer (PCa) are desired. The aim of this study was to determine the effect of fisetin, a tetrahydroxyflavone, on inhibition of cell growth and induction of apoptosis in human PCa cells. Treatment of fisetin (10-60 microM, 48 h) was found to result in a decrease in the viability of LNCaP, CWR22Rupsilon1 and PC-3 cells but had only minimal effects on normal prostate epithelial cells as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide assay. Treatment of LNCaP cells with fisetin also resulted in G(1)-phase arrest that was associated with a marked decrease in the protein expression of cyclins D1, D2 and E and their activating partner cyclin-dependent kinases 2, 4 and 6 with concomitant induction of WAF1/p21 and KIP1/p27. Fisetin treatment also resulted in induction of apoptosis, poly (ADP-ribose) polymerase (PARP) cleavage, modulation in the expressions of Bcl-2 family proteins, inhibition of phosphatidyl inositol 3-kinase and phosphorylation of Akt at Ser(473) and Thr(308). There was also induction of mitochondrial release of cytochrome c into cytosol, downregulation of X-linked inhibitor of apoptosis protein and upregulation of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI on treatment of cells with fisetin. Treatment of cells with fisetin also resulted in significant activation of caspases-3, -8 and -9. Pretreatment of cells with caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced activation of caspases. These data provide the first evidence that fisetin could be developed as an agent against PCa.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Caspase 7; Caspases; Cell Cycle; Cell Line, Tumor; Cytochromes c; Diet; Enzyme-Linked Immunosorbent Assay; Flavonoids; Flavonols; Flow Cytometry; Humans; Male; Neoplasm Proteins

IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-X(L) to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-X(L) expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-X(L) expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-X(L), a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development.

Topics: Antineoplastic Agents, Phytogenic; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor II; Intracellular Signaling Peptides and Proteins; Mitochondrial Membranes; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Resveratrol; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Stilbenes

mAtNOS1 is a novel gene recently reported in mammalian genome with functions that are not fully understood. The present study shows that in human mammary adenocarcinoma MCF-7 cells, mAtNOS1 expression increases mitochondrial nitric oxide and calcium. Our study further shows that overexpression of mAtNOS1 induces apoptosis in MCF-7 cells by increasing mitochondrial protein tyrosine nitration and cytochrome c release. The present study suggests a novel function for mAtNOS1 in regulating mitochondrial nitric oxide and calcium and inducing apoptosis of MCF-7 cells.

Topics: Adenocarcinoma; Apoptosis; Arabidopsis Proteins; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Female; Humans; Immunochemistry; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Transfection; Tyrosine

25-Hydroxy-3-oxoolean-12-en-28-oic acid (Amooranin-AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A herbal preparation used for the treatment of cancer by the Ayurvedic system of medicine contains the stem bark of Amoora rohituka as one of the ingredients. In this paper, we show that AMR displays a strong inhibitory effect on survival of human breast carcinoma MDA-468, breast adenocarcinoma MCF-7 cells compared to breast epithelial MCF-10A control cells. A 50% decrease in cells (IC50) ranged from 1.8 to 14.6 microM and cell growth was suppressed by arresting cell cycle at G2 + M phase. AMR effectively induces apoptosis and triggered a series of effects associated with apoptosis including cleavage of caspase-8, -9, -3, Bid and ER stress in MDA-468 cells and caspase- 8, -9, -6 and Bid in MCF-7 cells, release of cytochrome c from the mitochondria, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation with a concomitant upregulation of p53, Bax and down-regulation of Bcl-2 in MDA-468 cells, but Bax unchanged in MCF-7 cells. The use of caspase blocking peptides and acridine orange staining confirmed the involvement of primarily caspase-9 and -3 in MDA-468 cells with mutated p53 and primarily caspase-8, -9 and -6 in MCF-7 cells expressing wt p53. We also observed in MCF-7/p53siRNA cells AMR treatment caused reduced expression of Bcl-2 without affecting levels of Bax similar to MCF-7 cells treated with AMR and proteolytic activation of Bax in MDA-468 cells. These results suggest that AMR induces apoptosis in human breast carcinoma cells via caspase activation pathway and likely it is a p53-independent apoptosis.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Caspase 7; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Female; Flow Cytometry; Humans; Molecular Structure; RNA, Small Interfering; Signal Transduction; Time Factors; Triterpenes; Tumor Suppressor Protein p53

Screening mammography is the most effective tool available for breast cancer detection. While screening mammography saves lives, it has intrinsic problems that limit further improvement. We hypothesize that protein biomarkers in nipple aspirate fluid (NAF) may separate the cancer from the non-cancer state, and therefore can be used for breast cancer detection. In this study the proteins in NAF were analyzed by surface-enhanced laser desorption ionization coupled to time-of-flight mass spectrometry (SELDI-TOF) in the m/z 5,000-85,000 range. Two methods were used to normalize spectra. Then differentially expressed signals that separate cancer from non-cancer conditions were selected by two specifically developed statistical algorithms. Proteins of interest were identified by combined liquid chromatography-tandem mass spectrometry. A set of 8 markers were identified which collectively gave 63% sensitivity, 89% specificity and 76% accuracy for distinguishing cancer from non-cancer. Further improvements in the specificity and sensitivity of this strategy could come from the development of methods for more precise quantification of the biomarkers of interest and also from focusing on the low abundant components that are not evident when unfractionated NAF is analyzed directly.

Topics: Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Chromatography, Liquid; Cytochromes c; Female; Humans; Mass Spectrometry; Multivariate Analysis; Myoglobin; Nipples; Sensitivity and Specificity

Zinc is an essential trace element with cofactor functions in a large number of proteins of intermediary metabolism, hormone secretion pathways, immune defence mechanisms, and as a cofactor of transcription factors it is also involved in the control of gene expression. Our study demonstrates that the modulation of intra and extracellular zinc alone is sufficient to induce metabolic changes or even apoptosis in two model human breast cancer cell lines MCF-7 and MDA-MB468. Treatment of breast cancer cells with different concentrations of a cell membrane permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the membrane impermeable zinc chelator, diethylenetriaminepentacetic acid, (DTPA) resulted in a significant increase of cell death. Features of apoptosis, such as chromatin condensation and nuclear fragmentation accompanied the DTPA and TPEN-induced cell death. A significant increase in the activity of caspase-9 was observed in both cell lines; whereas, caspase-3 activity was only increased in MDA-MB468 cells since caspase-3 is not expressed in MCF-7 cells. Caspase-8 activation was negligible in both cell lines. Addition of Zn(2+) or Cu(2+) prevented DTPA and TPEN-induced cytotoxicity, indicating that both bivalent cations can be replaced functionally to a certain extent in our experimental system. Interestingly, addition of Ca(2+), or Mg(2+) had no effect. The antioxidant N-Acetyl-L-Cysteine inhibited the cytotoxic effect of DTPA and TPEN, indicating that oxidative stress is the likely mediator of Zn-deficiency-related cell death.

Topics: Acetylcysteine; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Chelating Agents; Cytochromes c; Ethylenediamines; Humans; Membrane Potentials; Mitochondria; Pentetic Acid; Receptors, Estrogen; Zinc

Bioflavanoids are the major pigments in plants with multitude of biological activities including inhibition of proliferation or induction of apoptosis in tumor cells. Even though the safety records of most flavanoids are exceptional, its therapeutic use is still in its infancy. We have isolated pinocembrin (5,7-dihydroxyflavanone) from Alpinia galanga that showed cytotoxicity against a variety of cancer cells including normal lung fibroblasts with relative nontoxicity to human umbilical cord endothelial cells. The compound induced loss of mitochondrial membrane potential with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116. Processing of caspase-8 was minimal. The initial trigger for mitochondrial apoptosis appears to be by the translocation of cytosolic Bax protein to mitochondria. Overexpression of proapoptotic Bax protein sensitized the colon cancer cells to pinocembrin-induced apoptosis and Bax knockout cells were resistant to pinocembrin-induced apoptosis. Antiapoptotic protein Bcl-X(L) only partially prevented apoptosis induced by this compound. The Bax-dependent cell death involving classical cytochrome c release and processing of caspase-9 and -3 suggests that pinocembrin is a classical mitochondrial apoptosis inducer. But the failure of Bcl-X(L) overexpression to completely prevent apoptosis induced by this compound suggests that pinocembrin is capable of triggering mitochondrial-independent cell death that needs to be clarified. The existence of cell death upon Bcl-X(L) overexpression is a promising feature of this compound that can be exploited against drug resistant forms of cancer cells either alone or in combination with other drugs.

Topics: Alpinia; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Breast Neoplasms; Caspase Inhibitors; Caspases; Chromatin; Colonic Neoplasms; Cytochromes c; Endothelial Cells; Fibroblasts; Flavanones; Fluorescent Antibody Technique; HCT116 Cells; Humans; Lung; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Transfection; Umbilical Cord

Tamoxifen is an anticancer drug that induces oxidative stress and apoptosis via mitochondria-dependent and nitric oxide (NO)-dependent pathways. The present report shows that tamoxifen increases intramitochondrial ionized Ca(2+) concentration and stimulates mitochondrial NO synthase (mtNOS) activity in the mitochondria from rat liver and human breast cancer MCF-7 cells. By stimulating mtNOS, tamoxifen hampers mitochondrial respiration, releases cytochrome c, elevates mitochondrial lipid peroxidation, increases protein tyrosine nitration of certain mitochondrial proteins, decreases the catalytic activity of succinyl-CoA:3-oxoacid CoA-transferase, and induces aggregation of mitochondria. The present report suggests a critical role for mtNOS in apoptosis induced by tamoxifen.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Female; Humans; Lipid Peroxidation; Mitochondria; Mitochondria, Liver; Nitric Oxide Synthase; Oxidative Stress; Oxygen Consumption; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Tamoxifen

Sulforaphane, an isothiocyanate found in cruciferous vegetables, has been shown to induce phase 2 detoxication enzymes and inhibit the growth of chemically induced mammary tumors in rats, although the exact mechanisms of action of sulforaphane are not understood. In this study, we evaluated the effects of sulforaphane on cell growth and death in several human breast cancer cell lines and examined the hypothesis that sulforaphane acts as a histone deacetylase (HDAC) inhibitor in these cell lines. Sulforaphane treatment inhibited cell growth, induced a G(2)-M cell cycle block, increased expression of cyclin B1, and induced oligonucleosomal DNA fragmentation in the four human breast cancer cell lines examined, MDA-MB-231, MDA-MB-468, MCF-7, and T47D cells. Activation of apoptosis by sulforaphane in MDA-MB-231 cells seemed to be initiated through induction of Fas ligand, which resulted in activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, whereas apoptosis in the other breast cancer cell lines was initiated by decreased Bcl-2 expression, release of cytochrome c into the cytosol, activation of caspase-9 and caspase-3, but not caspase-8, and poly(ADP-ribose) polymerase cleavage. Sulforaphane inhibited HDAC activity and decreased the expression of estrogen receptor-alpha, epidermal growth factor receptor, and human epidermal growth factor receptor-2 in each cell line, although no change in the acetylation of H3 or H4 was seen. These data suggest that sulforaphane inhibits cell growth, activates apoptosis, inhibits HDAC activity, and decreases the expression of key proteins involved in breast cancer proliferation in human breast cancer cells. These results support testing sulforaphane in vivo and warrant future studies examining the clinical potential of sulforaphane in human breast cancer.

Topics: Anticarcinogenic Agents; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Cycle; Cell Proliferation; Cyclin B; Cyclin B1; Cytochromes c; Cytosol; ErbB Receptors; Estrogen Receptor alpha; Fas Ligand Protein; Humans; Isothiocyanates; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfoxides; Thiocyanates; Tumor Cells, Cultured

Galectin-3 (GAL3), a beta-galactoside-binding lectin, confers chemoresistance to a wide variety of cancer cell types. It may exhibit anti- or pro-apoptotic activity depending on the nature of the stimulus. We report here that introducing phosphorylated galectin-3 (P-GAL3) into GAL3-null, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant human breast carcinoma cells promotes TRAIL-induced apoptotic cell death by stimulating the phosphorylation/inactivation of the pro-apoptotic molecule Bad resulting in the inhibition of mitochondrial depolarization and the release of cytochrome c. Exposure of the transfectant cells to TRAIL leads to the recruitment of the initiator capase-8 followed by activation of the effector caspase-9, independent of cytochrome c, and subsequently the processing of the executioner caspase-3. P-GAL3 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were coordinately expressed, with concomitant dephosphorylation of Akt in TRAIL-sensitive cells. In contrast, overexpression of phospho-mutant GAL3 (incapable of phosphorylation) failed to elicit similar responses. Depletion of PTEN using small interference RNAs reinstated Akt phosphorylation and conferred TRAIL resistance. In addition phosphatidylinositol 3-kinase inhibitors rendered the phospho-mutant GAL3-resistant cells sensitive to TRAIL. These findings suggest a pivotal role for P-GAL3 in promoting TRAIL sensitivity through activation of a nonclassic apoptotic pathway and identify P-GAL3 as a novel regulator of PTEN.

Topics: Apoptosis; Breast Neoplasms; Carcinoma; Chromosomes, Human, Pair 10; Cytochromes c; Enzyme Inhibitors; Galectin 3; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Models, Biological; Phosphorylation; PTEN Phosphohydrolase; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

There is an increasing interest in identifying potent cancer-preventive and therapeutic agents against breast cancer. A great number of reports have in recent years dealt with anticancer characteristics of Semecarpus anacardium nut extract (SA). The majority of these studies has been targeted on the protective effect rendered to the living system rather than the preventive effect on cancer cells. SA was tested for its inhibitory effect on human breast cancer cells (T47D). Cytotoxicity analyses suggested that these cells had become apoptotic. SA was discovered to induce rapid Ca(2+) mobilization from intracellular stores of T47D cell line, and its cytotoxicity against T47D was well correlated with altered mitochondrial transmembrane potential. At the molecular level, these changes are accompanied by decrease in bcl(2) and increase in bax, cytochrome c, caspases and PARP cleavage, and ultimately by internucleosomal DNA fragmentation. Taken together, our results provide unprecedented evidence that SA triggers apoptotic signals in T47D cells.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Caspases; Colony-Forming Units Assay; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Nitric Oxide Synthase; Nuts; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Semecarpus; Tumor Cells, Cultured

This study is the first to investigate the anticancer effect of isoobtusilactone A (IOA) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. IOA exhibited effective cell growth inhibition by inducing cancer cells to undergo G(2)-M phase arrest and apoptosis. Further investigation revealed that IOA's inhibition of cell growth was also evident in a nude mice model. Cell cycle blockade was associated with increased levels of p21 and reduced amounts of cyclin B1, cyclin A, cdc2, and cdc25C. IOA also enhanced the levels of inactivated phosphorylated cdc2 and cdc25C. IOA triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that the generation of reactive oxygen species (ROS) is a critical mediator in IOA-induced cell growth inhibition. Enhancement of ROS by IOA activated apoptosis signal-regulating kinase 1 (ASK1) resulted in the increased activation of c-Jun NH(2)-terminal kinase and p38. Antioxidants EUK8 and N-acetyl cystenine significantly decreased apoptosis by inhibiting the ASK1 dephosphorylation at Ser(967) and subsequently increased the interaction of ASK1 with thioredoxin or 14-3-3 proteins. Moreover, blocking ASK1 by small interfering RNA inhibition completely suppressed IOA-induced apoptosis. Taken together, these results imply a critical role for ROS and ASK1 in IOA's anticancer activity.

Topics: Alkanes; Animals; Antioxidants; Apoptosis; Breast Neoplasms; Caspases; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Female; Humans; Immunoblotting; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Lactones; MAP Kinase Kinase Kinase 5; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

23,24-Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound known to posses anticancer and anti-inflammatory activities. In this study, DHCB, isolated from roots of Trichosanthes kirilowli which is a traditional Chinese herb medicine used as treatments for cancer and other diseases, has been found to inhibit the proliferation of human cancer cell lines Bcap37, HeLa, SW620, SMMC-7721, K562 and MCF-7 in a dose- and time-dependent manner, and induce apoptosis in human breast cancer cell line Bcap37 at low concentration. DHCB-induced Bcap37 apoptosis was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase-like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol. The cell death was partly prevented by a caspase-family inhibitor Z-VAD-FMK. The results suggest that DHCB-induced Bcap37 apoptosis through mitochondrial dependent pathway. Flow cytometric analysis revealed that at the lower dose of 1.8 and 3.6muM, DHCB-induced cancer cell lines death via an apoptotic process rather than necrotic one; whereas, the higher dose of 8.9, 17.9 and 35.7muM induced cell death via the necrotic process. Cell-cycle analysis demonstrated DHCB induction of G(2)/M phase cell-cycle arrest and apoptosis. The overall results suggest that DHCB might have the therapeutic value against human cancer cell lines, especially the breast cancer cell lines.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspases; Cell Division; Cell Membrane; Cell Proliferation; Cell Shape; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Female; G2 Phase; HeLa Cells; Humans; K562 Cells; Mitochondria; Necrosis; Plant Roots; Poly(ADP-ribose) Polymerases; Prohibitins; Trichosanthes; Triterpenes

Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy-HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy-HDI combinations have potential for treating advanced stage breast cancer.

Topics: Apoptosis Regulatory Proteins; Breast Neoplasms; Clusterin; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Mitochondria; Models, Biological; Protein Processing, Post-Translational; Signal Transduction; Tumor Cells, Cultured

Genistein is a soy isoflavone with anti-tumor properties. Genistein-induced apoptosis involves Bcl-2 downregulation. However, overexpression of Bcl-2 in breast cancer has been associated with better prognosis and response to hormonal therapy. To examine genistein's effect on breast cancer cells with different Bcl-2 levels, we established control (MCF-7/PV) and Bcl-2 overexpressing MCF-7 (MCF-7/Bcl-2) cell lines and characterized genistein regulated apoptosis and cell cycle progression in these cells. Our results demonstrate that overexpression of Bcl-2 rendered MCF-7 cells more sensitive, rather than resistant, to genistein. We found that genistein induces enhanced cytochrome c release and mitochondrial membrane depolarization in MCF-7/Bcl-2 cells, as compared to control. We also found that genistein increases Bcl-2 levels and Bcl-2/Bax ratio in the mitochondrial fractions of MCF-7/Bcl-2 cells, suggesting that disturbed Bcl-2/Bax distribution may cause cytochrome c release and apoptosis in these cells. Cell cycle analysis indicated that genistein induces G0/G1 arrest in MCF-7/PV cells but increases in G2/M arrest in MCF-7/Bcl-2 cells. This was accompanied by modified responses of several cell cycle regulators, such as p21 and cyclin B1. Taken together, our results indicate that genistein-Bcl-2 interaction switches Bcl-2 from an anti-apoptotic protein into a proapoptotic protein, which involves disturbed Bcl-2/Bax distribution in mitochondria, increased cytochrome c release and modified cell cycle regulation.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Female; Flow Cytometry; Genistein; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Chemotherapeutic drugs are usually designed to induce cancer cell death via cell cycle arrest and/or apoptosis pathways. In this study, we used the chemical drug 15,16-dihydrotanshinone I (DHTS) to inhibit breast cancer cell proliferation and tumor growth, and investigate the underlying molecular mechanisms. Human breast cancer cell lines MCF-7 and MDA-MB-231 were both used in this study, and DHTS was found to significantly decrease cell proliferation by a dose-dependent manner in both cells. Flow cytometry indicated that DHTS induced G1 phase arrest in synchronous MCF-7 and MDA-MB-231 cells. When analyzing the expression of cell cycle-related proteins, we found that DHTS reduced cyclin D1, cyclin D3, cyclin E, and CDK4 expression, and increased CDK inhibitor p27 expression in a dose-dependent manner. In addition, DHTS inhibited the kinase activities of CDK2 and CDK4 by an immunocomplex kinase assay. In addition, DHTS also induced apoptosis in both cells through mainly mitochondrial apoptosis pathways. We found that DHTS decreased the anti-apoptotic protein Bcl-xL level and increased the loss of mitochondria membrane potential and the amount of cytochrome c released. Moreover, DHTS activated caspase-9, caspase-3, and caspase-7 and caused cell apoptosis. The fact that DHTS-induced apoptosis could be blocked by pretreating cells with pan-caspase inhibitor confirmed that it is mediated through activation of the caspase-3-dependent pathway. In a nude mice xenograft experiment, DHTS significantly inhibited the tumor growth of MDA-MB-231 cells. Taken together, these results suggest that DHTS can inhibit human breast cancer cell proliferation and tumor growth, and might have potential chemotherapeutic applications.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Furans; G1 Phase; Humans; Male; Mammary Neoplasms, Experimental; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Phenanthrenes; Quinones; Xenograft Model Antitumor Assays

Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

Topics: Amaryllidaceae Alkaloids; Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma; Caspases; Cytochromes c; DNA Fragmentation; Drug Resistance, Neoplasm; Enzyme Activation; fas Receptor; Female; Fibroblasts; Humans; Male; Mitochondria; Narcissus; Neoplasm Proteins; Phenanthridines; Prostatic Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor

We investigated the molecular mechanisms involved in the anti-proliferative activity exerted by conjugated linoleic acid (CLA) on the estrogen unresponsive MDA-MB-231 human breast cancer cell line. The effects on cell proliferation, cell cycle progression and induction of apoptosis were examined. CLA caused the reduction of cell proliferation along with the accumulation of cells in the S phase of the cycle. The occurrence of apoptosis in these cells was indicated by flow cytometry data and further confirmed by the onset of cells with morphological features typical of apoptosis. ERK1/2 reduction and upregulation of pro-apoptotic protein Bak were induced. These events were associated with: (a) reduced levels of the anti-apoptotic protein Bcl-x(L), (b) the translocation of cytochrome c from the mitochondria to the cytosol, (c) the cleavage of pro-caspase-9 and pro-caspase-3. From the above data, we are induced to think that CLA may trigger apoptosis in the estrogen unresponsive MDA-MB-231 cell line via mechanisms involving above all the mitochondrial pathway.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Female; Flow Cytometry; Humans; Linoleic Acids, Conjugated; Mitochondria; Protein Transport; Signal Transduction

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

Certain estradiol metabolites may play a pivotal role in breast carcinogenesis. Of special interest are the metabolites 2-hydroxyestradiol (2-OHE2), which can react anti-carcinogenically, and 4-hydroxyestradiol (4-OHE1) and 16a-hydroxyestrone (16-OHE1), which may have procarcinogenic potential. In the present study, we have compared for the first time the effect of these metabolites and their parent substance 17beta-estradiol (E2) on proliferation, apoptosis, apoptosis markers and markers of metastatic property of human breast cancer cells.. MCF-7 cells (human estrogen-receptor positive metastatic breast cancer cell line) were incubated with the estrogens at concentrations of 0.1-100 nM. Cell proliferation rate was measured by the ATP-assay. Apoptosis was measured by cell death assay and the apoptosis markers cytochrome C, Bcl-2, Fasl and p53 were determined in cell lysates by ELISAs. The markers of metastatic property of the cell line, VEGF and MCP-1 were measured in the cell supernatant by ELISAs.. The estrogens E2, 4-OHE2 and 16-OHE1 display a proliferative effect on MCF-7 cells which is accompanied by a down-regulation of apoptosis. Various markers of apoptosis such as Bcl-2, cytochrome C and p53 appear to be involved. No significant effect was found for the metabolite 2-OHE2. VEGF and MCP-1 were up-regulated by E2 and 16-OHE1, whereas 2-OHE2 and 4-OHE2 did not show any effect.. The most potent estrogen regarding proliferation, apoptosis and metastasis of breast cancer cells seems to be estradiol. However, the estradiol metabolites 4-OHE2 and 16-OHE1 elicit similar properties on cell proliferation, apoptosis and metastasis as compared to estradiol but only at higher concentrations. In contrast 2-OHE2 did not show any significant effect on these parameters. Thus, intracellular estradiol metabolism may determine an individual's risk for breast carcinogenesis.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Estradiol; Estriol; Estrogens; Estrogens, Catechol; Fas Ligand Protein; Female; Humans; Membrane Glycoproteins; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factors; Tumor Suppressor Protein p53; Up-Regulation

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-X Protein; Breast Neoplasms; Capsid Proteins; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Female; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, B-Cell; Male; Mitochondria; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Cell-Free System; Cytochromes c; Deoxyadenine Nucleotides; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Molecular Weight; Proteins; X-Linked Inhibitor of Apoptosis Protein

Antrodia camphorata (A. camphorata) is well known in Taiwan as a traditional Chinese medicine, and it has been shown to exhibit antioxidant and anticancer effects. In this study, therefore, its ability to induce apoptosis in cultured MCF-7 breast cancer cells was studied. Treatment of the MCF-7 cells with a variety of concentrations of the fermented culture broth of A. camphorata (25-150 microg/ml) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation, and sub-G1 phase accumulation. Furthermore, apoptosis in the MCF-7 cells was accompanied by the release of cytochrome c, activation of caspase 3, and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). Although, the A. camphorata-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Interestingly, A. camphorata induced dose-dependent reactive oxygen species (ROS) generation in MCF-7 cells. Analysis of the data suggests that A. camphorata exerts antiproliferative action and growth inhibition on MCF-7 cells through apoptosis induction, and that it may have anticancer properties valuable for application in drug products.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; Flow Cytometry; G1 Phase; Humans; Poly(ADP-ribose) Polymerases; Polyporales; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured

Apoptotic signaling defects both promote tumorigenesis and confound chemotherapy. Typically, chemotherapeutics stimulate cytochrome c release to the cytoplasm, thereby activating the apoptosome. Although cancer cells can be refractory to cytochrome c release, many malignant cells also exhibit defects in cytochrome c-induced apoptosome activation, further promoting chemotherapeutic resistance. We have found that breast cancer cells display an unusual sensitivity to cytochrome c-induced apoptosis when compared with their normal counterparts. This sensitivity, not observed in other cancers, resulted from enhanced recruitment of caspase-9 to the Apaf-1 caspase recruitment domain. Augmented caspase activation was mediated by PHAPI, which is overexpressed in breast cancers. Furthermore, cytochrome c microinjection into mammary epithelial cells preferentially killed malignant cells, suggesting that this phenomenon might be exploited for chemotherapeutic purposes.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Nuclear Proteins; Protein Structure, Tertiary; Proteins; RNA-Binding Proteins

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERalpha and ERbeta. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) delta, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells.

Topics: bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cytochromes c; Estradiol; Estrogens; Humans; JNK Mitogen-Activated Protein Kinases; Mitochondria; Protein Transport; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Receptors, Estrogen

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Ceramides; Cytochromes c; Enzyme Activation; Fatty Acids, Monounsaturated; Female; Gene Targeting; Humans; Mitochondria; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering; Sphingolipids

Lamellarin D is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cell lines and is a potent inhibitor of topoisomerase I. However, lamellarin D maintains a marked cytotoxicity toward cell lines resistant to the reference topoisomerase I poison camptothecin. We therefore hypothesized that topoisomerase I is not the only cellular target for the drug. Using complementary cell-based assays, we provide evidence that lamellarin D acts on cancer cell mitochondria to induce apoptosis. Lamellarin D, unlike camptothecin, induces early disruption of the inner mitochondrial transmembrane potential (Deltapsi(m)) in the P388 leukemia cell line. The functional alterations are largely prevented by cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), but not by the inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone. Deltapsi(m) disruption is associated with mitochondrial swelling and cytochrome c leakage. Using a reliable real-time flow cytometric monitoring of Deltapsi(m) and swelling of mitochondria isolated from leukemia cells, we show that lamellarin D has a direct MPT-inducing effect. Furthermore, mitochondria are required in a cell-free system to mediate lamellarin D-induced nuclear apoptosis. The direct mitochondrial effect of lamellarin D accounts for the sensitivity of topoisomerase I-mutated P388CPT5 cells resistant to camptothecin. Interestingly, a tumor-active analogue of lamellarin D, designated PM031379, also exerts a direct proapoptotic action on mitochondria, with a more pronounced activity toward mitochondria of tumor cell lines compared with nontumor cell lines. Altogether, this work reinforces the pharmacologic interest of the lamellarins and defines lamellarin D as a lead in the search for treatments against chemoresistant cancer cells.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Camptothecin; Cell Line, Tumor; Cell Membrane Permeability; Cell-Free System; Coumarins; Cytochromes c; Heterocyclic Compounds, 4 or More Rings; Humans; Isoquinolines; Leukemia P388; Lung Neoplasms; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membranes; NIH 3T3 Cells; Rats

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cerulenin; Chromones; Cytochromes c; Cytosol; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Mitochondria; Morpholines; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.

Topics: Antioxidants; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Iodine; Leukocytes, Mononuclear; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Reactive Oxygen Species

Combined treatment modalities using tumor necrosis factor related apoptosis-inducing ligand L (TRAIL) and cytotoxic drugs revealed highly additive effects in some tumor cell lines. Little is known about the efficacy and underlying mechanistic effects of the modalities in chemoresistant tumor cells. The purpose of this study is to investigate the possible role of JNK in the synergistic effect in Doxorubicin (Adriamycin, ADM) resistant MCF-7/ADM cells. Here we showed that the JNK pathway was activated slightly by TRAIL in MCF-7/ADM cell lines and was enhanced by the combination of the two treatments. Inhibition of JNK activity by transfection with dominant-negative JNK blocks TRAIL plus ADM induced-apoptosis significantly, and selective stimulation of the JNK pathway sensitizes ADM resistant breast cancer cells to ADM and TRAIL co-treatment through activation of mitochondria-regulated apoptotic pathway. We conclude that the JNK pathway plays an important role in mediating TRAIL plus ADM induced-apoptosis in breast cancer cells.

Topics: Anthracenes; Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Female; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Glycoproteins; Mitochondria; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.

Topics: Amino Acid Sequence; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; ErbB Receptors; Humans; Mitochondria; Models, Molecular; Molecular Sequence Data; Neuregulin-1; Peptide Fragments; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-4; Sequence Homology, Amino Acid

Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 mumol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is approximately 10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G2-M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Membrane; Collagen Type XI; Cytochromes c; Dioxoles; Female; Humans; In Situ Nick-End Labeling; Isoquinolines; Membrane Potentials; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondrial Membranes; Neoplasms, Hormone-Dependent; Noscapine; Proto-Oncogene Proteins c-bcl-2; Spindle Apparatus; Xenograft Model Antitumor Assays

Photodynamic therapy (PDT) is a novel and promising cancer treatment which employs a combination of a photosensitizing chemical and visible light to induce apoptosis in cancer cells. Singlet oxygen has been recognized as the main origin of oxidative stress in PDT. However, the precise mechanism of PDT-induced apoptosis is not well characterized, especially the dualistic role of nitric oxide (NO). To dissect the apoptosis pathways triggered by PDT, the intracellular free radicals in MCF-7 cells were investigated by examining a novel photosensitizer 2-butylamino-2-demethoxyhypocrellin B (2-BA-2-DMHB)-mediated PDT. It was found that exposure of the cells to 2-BA-2-DMHB and irradiation resulted in a significant increase of intracellular ROS in minutes, and then followed by cytoplasmic free calcium enhancement, mitochondrial nitric oxide synthase (mtNOS) activation, cytochrome c release, and apoptotic death. Scavengers of singlet oxygen or NO could attenuate PDT-induced cell viability loss, nucleus morphology changes, cytochrome c release, mitochondria swelling, and apo-apoptosis gene p53 and p21 mRNA levels. The results suggested that both ROS and NO played important roles in the apoptosis-induced by PDT.

Topics: Apoptosis; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Nucleus; Cyclic N-Oxides; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Female; Humans; Imidazoles; Mitochondria; Mitochondrial Swelling; Nitric Oxide; Perylene; Photochemotherapy; Photosensitizing Agents; Quinones; Reactive Oxygen Species; Sodium Azide; Tumor Suppressor Protein p53

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been found to induce cell death in a variety of cells. In this regard, we reported recently that 15-deoxy-Delta-(12,14)-prostaglandin J2 (15dPG-J2), a specific ligand of the nuclear receptor PPARgamma, inhibits proliferation and induces cellular differentiation and apoptosis in the breast cancer cell line MCF-7. In addition to PPARgamma activation other proteins, such as NF-kappaB and AP1, have been shown to be targets of 15dPG-J2. However, the mechanism by which 15dPG-J2 triggers cell death is still elusive. Our results demonstrate that 15dPG-J2 initiates breast cancer cell death via a very rapid and severe impairment of mitochondrial function, as revealed by a drop in mitochondrial membrane potential (DeltaPsi(m)), generation of reactive oxygen species (ROS) and a decrease in oxygen consumption. In addition, 15dPG-J2 can also activate an intrinsic apoptotic pathway involving phosphatidyl serine externalization, caspase activation and cytochrome c release. Bcl-2 over-expression and zVADfmk, albeit preventing caspase activation, have no effect on 15dPG-J2-mediated mytochondrial dysfunction and loss of cell viability. In contrast, the addition of radical scavengers or rotenone, which prevent 15dPG-J2-induced ROS production, block the loss of cell viability induced by this prostaglandin. Finally, 15dPG-J2-induced cell death appears to involve disruption of the microtubule cytoskeletal network. Together, these results suggest that PG-J2-induced mitochondrial dysfunction and ROS production inevitably leads to death, with or without caspases.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Microscopy, Confocal; Microtubules; Mitochondria; Oxygen Consumption; Peroxisome Proliferator-Activated Receptors; Prostaglandins; Reactive Oxygen Species

We have shown that exposure of the HER2/neu-overexpressing breast cancer cells to apigenin resulted in induction of apoptosis by depleting HER2/neu protein and, in turn, suppressing the signaling of the HER2/HER3-PI3K/Akt pathway. Here, we examined whether inhibition of this pathway played a role in the anti-tumor effect. The results revealed that treatment with apigenin induced apoptosis through cytochrome c release and caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of DFF-45. Furthermore, apigenin downregulated cyclin D1, D3 and Cdk4 and increased p27 protein levels. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in HER2/neu-overexpressing breast cancer cells in the presence of apigenin. In addition, a structure-activity relationship study indicated that (1) the position of B ring; and (2) the existence of the 3', 4'-hydroxyl group on the 2-phenyl group were important for the depletion of HER2/neu protein by flavonoids. These results provided new insights into the structure-activity relationship of flavonoids.

Topics: Apigenin; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Caspase 3; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytochromes c; Flavonoids; Humans; Phosphoinositide-3 Kinase Inhibitors; Proteins; Receptor, ErbB-2; Tumor Suppressor Proteins

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.

Topics: Alkenes; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Nucleus; Cell Survival; Coloring Agents; Cytochromes c; DNA Damage; Dose-Response Relationship, Drug; Genes, Reporter; Humans; Luciferases; Phenols; Plasmids; Receptors, Estrogen; Tetrazolium Salts; Thiazoles

Prion protein (PrP) prevents Bcl-2-associated protein X (Bax)-mediated cell death, but the step at which PrP inhibits is not known. We first show that PrP is very specific for Bax and cannot prevent Bak (Bcl-2 antagonist killer 1)-, tBid-, staurosporine- or thapsigargin-mediated cell death. As Bax activation involves Bax conformational change, mitochondrial translocation, cytochrome c release and caspase activation, we investigated which of these events was inhibited by PrP. PrP inhibits Bax conformational change, cytochrome c release and cell death in human primary neurons and MCF-7 cells. Serum deprivation-induced Bax conformational change is more rapid in PrP-null cells. PrP does not prevent active caspase-mediated cell death. PrP does not colocalize with Bax in normal or apoptotic primary neurons and cannot prevent Bax-mediated cytochrome c release in a mitochondrial cell-free system. We conclude that PrP protects against Bax-mediated cell death by preventing the Bax proapoptotic conformational change that occurs initially in Bax activation.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Caspase 6; Cell Line, Tumor; Cysteine Endopeptidases; Cytochromes c; Enzyme Inhibitors; Female; Humans; Membrane Proteins; Mitochondria; Neurons; Protein Structure, Quaternary; Protein Transport; Proto-Oncogene Proteins c-bcl-2; PrPC Proteins; Thapsigargin

Estrogen is a mitogen in most estrogen receptor-alpha (ERalpha)-positive breast cancers. We have found that Smad4, a common signal transducer in the transforming growth factor-beta superfamily, acts as an ERalpha transcriptional corepressor. Here, we show that Smad4 induces apoptosis in ERalpha-positive MCF-7 breast cancer cells, but not in ERalpha-negative MDA-MB-231 cells. Smad4 induced expression of short Bim isoforms (by alternative splicing) and Bax and release of cytochrome c in ERalpha-positive cells only, and expression of these apoptotic marker genes was reduced when ERalpha small interfering RNA was introduced. Notably, Smad4 was able to induce apoptosis in MDA-231 cells with acquired ERalpha expression. Furthermore, Smad4 inhibited ERalpha-positive tumor growth by inducing apoptosis in tumor xenografts in nude mice. The sizes of tumors expressing Smad4 were only one-tenth the size of those expressing green fluorescent protein, whereas in ERalpha-negative cells, Smad4 did not reduce the tumor size. Notably, Smad4 also promoted short Bim isoform and Bax expression and release of cytochrome c only in ERalpha-positive MCF-7 tumor xenografts. Bim was sufficient for induction of apoptosis, and the short form was the most potent inducer. Our results demonstrate that Smad4 induces apoptosis by regulating Bim splicing as an initial intrinsic signal in ERalpha-positive cells. Smad4-induced apoptosis in ERalpha-positive breast cancer cells may explain the invasive nature of ERalpha-negative breast tumors, thereby providing a potential target for breast cancer intervention.

Topics: Alternative Splicing; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA-Binding Proteins; Estrogen Receptor alpha; Female; Genetic Therapy; Humans; Membrane Proteins; Mice; Mice, Nude; Neoplasms, Experimental; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Smad4 Protein; Trans-Activators; Transplantation, Heterologous

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.

Topics: Adenosine Triphosphate; Animals; Antennapedia Homeodomain Protein; Antimetabolites, Antineoplastic; Apoptosis; Benzoquinones; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Design; Enzyme Inhibitors; Female; Fibroblasts; Gene Products, tat; HeLa Cells; Homeodomain Proteins; HSP90 Heat-Shock Proteins; Humans; Inhibitor of Apoptosis Proteins; Lactams, Macrocyclic; Male; Mice; Mice, SCID; Microtubule-Associated Proteins; Models, Molecular; Molecular Mimicry; Neoplasm Proteins; Nuclear Proteins; Peptide Fragments; Prostatic Neoplasms; Protein Binding; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Rifabutin; Stem Cells; Survivin; Telomerase; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear.. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests.. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006).. Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cardiolipins; Caspases; Coenzyme A Ligases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Female; Flavoproteins; Gene Transfer Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasms, Experimental; Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Stomach Neoplasms; Transfection; Transplantation, Heterologous; Triazenes; Tumor Suppressor Protein p53

Topics: Aromatase; Bone Density; Breast; Breast Neoplasms; Cytochromes c; Estrogens; Female; Humans; Polymorphism, Genetic; Risk Assessment; Risk Factors

Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions.. Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes.. We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability.. Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line; Cell Line, Tumor; Culture Media, Serum-Free; Cytochromes c; Cytosol; Disease Progression; DNA, Complementary; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunoblotting; Immunoprecipitation; In Situ Nick-End Labeling; Mice; Microscopy, Fluorescence; Models, Statistical; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Ribonucleases; Serpins; Staurosporine; Transcription, Genetic; Tumor Necrosis Factor-alpha

Tumor cells typically resist programmed cell death (apoptosis) induced by death receptors. Activated death receptors evoke Bax conformational change, cytochrome c release, and cell death. We report that the tumor suppressor gene RASSF1A is required for death receptor-induced Bax conformational change and apoptosis. TNFalpha or TRAIL stimulation induced recruitment of RASSF1A and MAP-1 to receptor complexes and promoted complex formation between RASSF1A and the BH3-like protein MAP-1. Normally, MAP-1 is inhibited by an intramolecular interaction. RASSF1A/MAP-1 binding relieved this inhibitory interaction, resulting in MAP-1 association with Bax. Deletion of the RASSF1A gene or short hairpin silencing of either RASSF1A or MAP-1 expression blocked MAP-1/Bax interaction, Bax conformational change and mitochondrial membrane insertion, cytochrome c release, and apoptosis in response to death receptors. Our findings identify RASSF1A and MAP-1 as important components between death receptors and the apoptotic machinery and reveal a potential link between tumor suppression and death receptor signaling.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Gene Deletion; Humans; Intracellular Membranes; Mitochondria; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Proteins

Pristimerin, a naturally occurring triterpenoid, has been shown to cause cytotoxicity in several cancer cell lines. However, the mechanism for the cytotoxic effect of pristimerin was never explored. In the present study, human breast cancer MDA-MB-231 cells treated with pristimerin (1 and 3 micromol/L) showed rapid induction of apoptosis, as indicated by caspase activation, DNA fragmentation, and morphologic changes. Pretreatment of a pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) completely prevented pristimerin-induced apoptosis. Treatment of tumor cells with pristimerin resulted in a rapid release of cytochrome c from mitochondria, which preceded caspase activation and the decrease of mitochondrial membrane potential. In addition, neither benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone nor permeability transition pore inhibitor cyclosporin A markedly prevented pristimerin-induced mitochondrial cytochrome c release. Pristimerin did not significantly alter the protein level of Bcl-2 family members (Bcl-2, Bcl-X(L), and Bax), nor did it induce Bax translocation. Moreover, Bcl-2 overexpression fails to prevent pristimerin-induced apoptosis. The generation of reactive oxygen species in MDA-MB-231 cells was also not affected by pristimerin. In a cell-free system, pristimerin induced cytochrome c release from isolated mitochondria. Taken together, these results suggested that pristimerin is a novel mitochondria-targeted compound and may be further evaluated as a chemotherapeutic agent for human cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase Inhibitors; Caspases; Cytochromes c; Female; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Triterpenes; Tumor Cells, Cultured

Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel sigma-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (alpha-tocopherol and gamma-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-L-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cathepsins; Cell Line, Transformed; Cell Line, Tumor; Cytochromes c; Female; Fibrosarcoma; Humans; Indoles; Ligands; Lysosomes; Mice; Mice, Inbred BALB C; NIH 3T3 Cells; Oxidative Stress; Receptors, sigma; Spiro Compounds; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5-7 microM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 microM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17beta-estradiol (E(2)) or in the presence of a low Tam concentration (1 microM) made the cells even more susceptible to rapid death induction by 5 or 7 microM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X(L) and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Bongkrekic Acid; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Activation; Estradiol; Estrogens; Female; Fulvestrant; Humans; Membrane Potential, Mitochondrial; Mitochondria; Onium Compounds; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Tamoxifen; Toremifene

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Epithelial Cells; G1 Phase; G2 Phase; Humans

We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process.. Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psim) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided.. MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psim, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P < .001], 85% [95% CI = 90% to 80%, P < .001], and 40% [95% CI = 45% to 35%, P < .001], respectively, whereas blockade of FasL by siRNA had no effect. Estradiol caused complete regression of MCF-7:5C tumors in vivo.. The mitochondrial pathway of apoptosis plays a critical role in estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.

Topics: Annexin A5; Antineoplastic Agents, Hormonal; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Breast Neoplasms; Caspase 8; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Estradiol; Estrogen Antagonists; Fas Ligand Protein; fas Receptor; Female; Flow Cytometry; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Membrane Glycoproteins; Microscopy, Electron; Mitochondria; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polymerase Chain Reaction; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Necrosis Factors; Tumor Suppressor Protein p53; Up-Regulation

Tocotrienols, which are Vitamin E isoforms, are known to inhibit the growth of human breast cancer cells due partly to apoptosis. However, the characterization of tocotrienol-induced apoptosis is incomplete, particularly what happens during the initiation phase that precedes execution of the cells. The objective of this study was to clarify the apoptotic effects of tocotrienols, with especial emphasis in determining if the mitochondria-mediated death pathway is activated when human breast cancer cells are incubated with a specific tocotrienol isomer. During incubation with gamma-tocotrienol, MDA-MB-231 human breast cancer cells showed membrane blebbing, and apoptotic bodies were present. Upon 4',6-diamidino-2-phenylindole staining of the cells, chromatin condensation and fragmentation were observed. Additionally, the annexin V-binding assay detected the translocation of membrane phospholipid during earlier analysis of the cells. Taken together, these results further establish that gamma-tocotrienol can induce apoptosis in human breast cancer cells. To help elucidate how gamma-tocotrienol induced the apoptosis, some important parameters related to the mitochondria-mediated death pathway were examined next. In gamma-tocotrienol-treated cells, the mitochondria were disrupted. Collapse of the mitochondrial membrane potential was detected, and cytochrome c was released later from mitochondria. However, expression of Bax and Bcl-2 (mRNA and protein) did not change. Furthermore, poly-(ADP-ribose)-polymerase cleavage was not detected, suggesting that caspases were not involved in the gamma-tocotrienol-induced apoptosis. These results imply that cytochrome c is not the critical protein released from mitochondria that triggers gamma-tocotrienol-induced apoptosis in MDA-MB-231 cells.

Topics: Annexin A5; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Chromatin; Cytochromes c; DNA Fragmentation; Humans; Hydrazines; Matrix Metalloproteinases; Membrane Potentials; Mitochondria; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tocotrienols; Tumor Cells, Cultured

Thrombospondins (TSPs) have been implicated as antitumor and antimetastasis factors in breast cancer. Although this effect has been attributed to the antiangiogenic activity of TSPs, recent observations suggest other mechanisms may be at work. The TSP receptor CD47 (integrin-associated protein) has recently been reported to mediate a novel form of apoptosis. Here, we have studied the response of breast cancer cells to CD47 ligands TSP-1, the CD47 agonist peptide 4N1K derived from TSP-1, and the anti-CD47 monoclonal antibody 1F7. All of these ligands killed four different breast cancer cell lines. This CD47-mediated cell death did not require active caspases or Bcl-2 degradation and did not cause DNA laddering or cytochrome c release. Pertussis toxin (PTX) prevented CD47-mediated death, indicating the involvement of Gi alpha. 4N1K dramatically reduced intracellular cAMP levels, an effect reversed with PTX. Forskolin, 8-bromo cAMP, and isobutylmethylxanthine (IBMX) all prevented CD47-mediated apoptosis, indicating the involvement of cAMP. H89 and protein kinase A (PKA) inhibitor peptide prevented rescue of breast cancer cells by PTX, 8-Br-cAMP, and forskolin, suggesting that the effects of cAMP are mediated via PKA-dependent phosphorylation events. Epidermal growth factor also inhibited CD47-induced apoptosis via a PKC-dependent but ERK-independent pathway. Thus, CD47-mediated killing of breast cancer cells occurs by a novel pathway involving regulation of cAMP levels by heterotrimeric Gi with subsequent effects mediated by PKA.

Topics: Antigens, CD; Apoptosis; Breast Neoplasms; Carrier Proteins; CD47 Antigen; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytochromes c; Epidermal Growth Factor; ErbB Receptors; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2

Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Division; Cytochromes c; Cytosol; DNA, Neoplasm; Ecdysone; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Mitochondria; Receptor, IGF Type 1; Receptor, IGF Type 2; Signal Transduction; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Carcinoma; Carrier Proteins; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Mutation; Oligopeptides; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Buprenorphine; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Deoxyribonucleases; DNA Fragmentation; Enzyme Activation; Female; Humans; Mitochondria; Narcotic Antagonists; Transfection; Zinc

Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC(50) of 0.03 microM in neuroepithelioma cells compared with more than 25 microM in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT. This chelator caused a marked increase of caspase-3 activity in murine Madison-109 (M109) cells. Caspase activation was at least partially mediated by the release of mitochondrial holo-cytochrome c (h-cytc) after incubation with Dp44mT. In conclusion, Dp44mT is a novel, highly effective antitumor agent in vitro and in vivo that induces apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Division; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Iron; Iron Chelating Agents; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mitochondria; Neoplasm Transplantation; Neuroectodermal Tumors, Primitive, Peripheral; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Reactive Oxygen Species; Thiosemicarbazones; Transferrin; Tumor Cells, Cultured

The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli. Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (> or =150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli. Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells. The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis. The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells. The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3. In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Calcimycin; Carcinoma; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Ionophores; Membrane Potentials; Mitochondria; Staurosporine

Inhibitors of histone deacetylase (HDAC) are considered as potential anticancer agents. We have previously demonstrated that an inhibitor of HDAC, sodium butyrate (NaB), induces apoptosis of breast cancer cells in a P53-independent and P21(waf1)-dependent manner. In this study, we showed that tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and anti-Fas agonist antibody potentiated NaB-induced growth inhibition through synergistic induction of apoptosis in breast cancer cell lines (MCF-7, T47-D, and BT-20). In MCF-7 cells, NaB increased the expression of death receptors; NaB alone or in combination with TNF-alpha, TRAIL, and anti-Fas agonist antibody increased the levels of Bid, tBid, and that of cytosolic cytochrome c. Synergistic induction of apoptosis was strongly inhibited by dominant-negative Fas-associated death domain (FADD) and inhibitors of caspases-8 and -9, indicating that potentiation of apoptosis involved key elements of death receptors' signaling pathways. Moreover, cotreatment of NaB and ligands of death receptors up-regulated the levels of P21(waf1) and that of proliferating cell nuclear antigen (PCNA) associated with P21(waf1). Transient transfections of p21(waf1) antisense or p21(waf1) deficient for its interaction with PCNA abolished synergistic induction of apoptosis. This suggested that potentiation of apoptosis by cotreatments required P21(waf1) and its interaction with PCNA. Since breast tumors contain rarely p21 mutations, our results may open interesting prospects in the fight against breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Antibodies; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Butyrates; Carcinoma; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytochromes c; Drug Synergism; fas Receptor; Fas-Associated Death Domain Protein; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Membrane Glycoproteins; Proliferating Cell Nuclear Antigen; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Up-Regulation

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Calcium; Camptothecin; Carrier Proteins; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum; Female; Humans; Microscopy, Immunoelectron; Mitochondria; Nuclear Envelope; Protein Transport; Proto-Oncogene Proteins c-bcl-2

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in a variety of human cancer cells including breast carcinoma and this property may be important for its chemopreventive and therapeutic effects. Resistance to 4HPR has been described, however, the molecular mechanisms underlying sensitivity or resistance to this retinoid are not clear. Recently, it has been shown that the carbohydrate-binding protein galectin-3, which has been implicated in tumor progression, contains the anti-death motif NWGR present in the anti-apoptotic protein Bcl-2. To determine whether galectin-3 expression can abrogate the effect of 4HPR, we tested the effects of 4HPR on apoptosis of cell clones derived from the galectin-3 deficient human BT549 breast carcinoma cells after transfection with either wild type galectin-3 (BT549Gal-3Wt), galectin-3 inactivated by a point mutation in the NWGR motif (BT549Gal-3Mu), or empty vector control (BT549Vec). Both BT549Vec and BT549Gal-3Mu cells showed a marked decrease in survival after treatment with 4HPR principally due to induction of apoptosis. 4HPR-induced apoptosis in these cells was associated with stimulation of reactive oxygen species generation, decreased levels of Bcl-2 protein, release of cytochrome c into the cytosol, increased caspase-3 activity, and poly(ADP-ribose) polymerase cleavage. In contrast, 4HPR failed to exert any of these effects in the BT549Gal-3Wt cells. The demonstration that galectin-3 suppresses 4HPR-induced apoptosis in human breast carcinoma cells suggests that the increased expression of galectin-3 during cancer progression may be associated with 4HPR resistance.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspase 3; Caspases; Cytochromes c; Female; Fenretinide; Galectin 3; Humans; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Subcellular Fractions; Tumor Cells, Cultured

Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 7; Caspase 8; Caspase 9; Caspases; Cell Survival; Cytochromes c; Drug Resistance, Multiple; Enzyme Induction; Female; Humans; Mitochondria; Neoplasm Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prodigiosin; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Calpain; Camptothecin; Carrier Proteins; Cytochromes c; Female; Green Fluorescent Proteins; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Laser Scanning Cytometry; Microscopy, Confocal; Microscopy, Immunoelectron; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured

Recent results from epidemiology, in vitro cell culture and in vivo (animal and human) studies have suggested the benefits of indole-3-carbinol (I3C) for the prevention of many types of cancer, including breast cancer. However, there are no reports, to the best of our knowledge, on the effect of I3C on isogenic nontumorigenic and tumorigenic breast epithelial cells, and there is a significant void in our understanding of the molecular mechanism(s) by which I3C induces apoptotic cell death in breast cancer cells. To fill this gap in our understanding, we conducted experiments to investigate the effects of I3C on an isogenic nontumorigenic (MCF10A) and tumorigenic (MCF10CA1a [CA1a]) breast epithelial cells. Here we show that CA1a cells are more sensitive to low concentration of I3C in terms of cell growth inhibition compared to MCF10A cells. We further report that I3C upregulates Bax/Bcl-2 ratio and downregulates Bcl-xL expression in CA1a cells but not in MCF10A cells. We also report, for the first time, that I3C induces Bax translocation to the mitochondria, causing mitochondrial depolarization, resulting in the loss of mitochondrial potential leading to the release of cytochrome c and subsequent cell death in CA1a cells but not in MCF10A cells. From these results, we conclude that I3C selectively induces apoptosis in breast cancer cells, but not in nontumorigenic breast epithelial cells, suggesting the potential therapeutic benefit of I3C against breast cancer.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Down-Regulation; Epithelial Cells; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Indoles; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

In normal cells, tumor necrosis factor-alpha (TNF-alpha) activates caspase 8 in both mitochondrion-dependent and mitochondrion-independent apoptotic pathways. It is believed that these two pathways converge, with resultant activation of effector caspases, such as caspase 6 and caspase 7. However, the precise mechanism of the activation of caspases 6 and 7 remains unknown. In this study, in order to focus on the mitochondrion-dependent pathway, we employed MCF7 human breast carcinoma cells, which do not have a functional mitochondrion-independent (caspase 3-dependent) pathway. We specifically targeted the transcript of Bid, a proapoptotic facilitator that is a substrate of caspase 8 in the mitochondrial pathway. In the TNF-alpha-treated MCF7 cells that expressed Bid-targeted ribozymes, the release of cytochrome c and the activation of caspase 9, but not of caspase 8, was delayed. Furthermore, the proteolysis of procaspase 7 was also delayed in Bid ribozyme-expressing cells. Because MCF7 cells are caspase 3 deficient, the direct cross-talk between caspase 8 and caspase 3 does not take place. Therefore, it became clear for the first time that caspase 9 by itself can activate caspase 7 in the absence of the caspase 3-dependent pathway in TNF-alpha-induced apoptosis by the use of specific ribozymes.

Topics: Apoptosis; Base Sequence; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Enzyme Precursors; Humans; Immunoblotting; Mitochondria; Models, Biological; Molecular Sequence Data; Nucleic Acid Conformation; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Catalytic; RNA, Messenger; Signal Transduction; Transfection; Tumor Necrosis Factor-alpha

Mifepristone (MIF) is an antiprogestin with potent anti-glucocorticoid and anti-androgen activity. MIF also appears to have anti-tumor activity independent of its ability to bind to nuclear receptors. In this study, we tested the ability of MIF to inhibit the growth of ER and PR negative breast cancer cells. In addition, because high-dose anti-estrogen treatment has been shown to inhibit ER and PR negative breast cancer cells, we compared the anti-proliferative activity of MIF to that of the anti-estrogen 4-hydroxytamoxifen (TAM) or combination hormonal therapy (MIF + TAM). MIF and TAM therapy induced a significant time- and dose-dependent growth inhibition and, ultimately, induced cell death in MDA-231 cells as evidenced by increased DNA fragmentation, cytochrome c release from the mitochondria, and the activation of caspase-3. The anti-proliferative activity of TAM plus MIF combination treatment was at least additive as compared to either monotherapy. The earliest indicator of TAM and MIF cytostatic and cytotoxic action on MDA-231 cells was a significant (p<0.05) induction of TGFbeta1 secretion into the growth medium within 4 h of treatment. Secreted TGFbeta1 levels at 24 and 48 h were significantly higher in the TAM plus MIF treatment group as compared to cells treated with TAM or MIF alone. TGFbeta1 neutralizing antibody or addition of mannose-6-phosphate (M6P), a reagent also used to inhibit TGFbeta1, significantly attenuated the TAM and/or MIF-induced cell growth inhibition and cell death. In summary, our results indicate that MIF used in combination with TAM can effectively kill estrogen-insensitive human breast cancer cells. Our study further implies that agents that effectively increase TGFbeta1 levels in ER negative breast cancer cells may be one treatment approach for hormone-independent breast cancers.

Topics: Apoptosis; Breast Neoplasms; Caspases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytochromes c; Drug Synergism; Drug Therapy, Combination; Estrogen Antagonists; Female; Humans; Mifepristone; Receptors, Estrogen; Retinoblastoma Protein; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Polyamine analogs have demonstrated considerable activity against many important solid tumor models including breast cancer. However, the precise mechanisms of antitumor activities of polyamine analogs are not entirely understood. The cytotoxicity of a newly developed polyamine analog compound, SL11144, against human breast cancer was assessed. Treatment of human breast cancer cell lines in culture with SL11144 decreased cell proliferation and induced programmed cell death in a time- and dose-dependent manner. SL11144 also profoundly inhibited the growth of MDA-MB-231 xenografts in host nude mice without overt toxic effects. Treatment of MDA-MB-435 cells with SL11144 led to the release of cytochrome c from mitochondria into cytosol, activation of caspase-3, and poly(ADP-ribose) polymerase cleavage. SL11144 decreased Bcl-2 and increased Bax protein levels in MDA-MB-231 cells. Furthermore, activator protein 1 transcriptional factor family member c-Jun was up-regulated by SL11144 in MDA-MB-435 and MDA-MB-231 cells, but not in MCF7 cells. In addition, significant inhibition of ornithine decarboxylase activity and a decrease in polyamine pools were demonstrated. These results demonstrate that the novel polyamine analog SL11144 has effective antineoplastic action against human breast cancer cells in vitro and in vivo and that multiple apoptotic mechanisms are associated with its cytotoxic effect in specific human breast cancer cell lines.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspases; Cell Death; Cell Division; Cell Line, Tumor; Cell Nucleus; Cell Survival; Chromatin; Cytochromes c; Cytoplasm; Cytosol; DNA Damage; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Models, Chemical; Neoplasm Transplantation; Nucleosomes; Poly(ADP-ribose) Polymerases; Polyamines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-jun; Tetrazolium Salts; Thiazoles; Time Factors; Up-Regulation

The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other down-stream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Caspase 7; Caspase 9; Caspases; Cell Line; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Activation; Humans; Insulin-Like Growth Factor I; Microscopy, Fluorescence; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Precipitin Tests; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA; Time Factors; Transfection; Ultraviolet Rays; X-Linked Inhibitor of Apoptosis Protein

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Death; Cytochromes c; Female; Immunohistochemistry; Indoles; Male; Mitochondria; Photochemotherapy; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Drug resistance is a major impediment to the successful treatment of breast cancer using chemotherapy. The photoactivatable drug calphostin C has shown promise in killing select drug-resistant tumor cells lines in vitro. To assess the effectiveness of this agent in killing doxorubicin- or paclitaxel-resistant breast tumor cells and to explore its mode of action, MCF-7 cells were exposed to increasing concentrations of either doxorubicin or paclitaxel until maximum resistance was obtained. This resulted in the creation of isogenic drug-resistant MCF-7TAX and MCF-7DOX cell lines, which were approximately 50- and 65-fold resistant to paclitaxel and doxorubicin, respectively. Interestingly, calphostin C was able to kill MCF-7TAX cells as efficiently as wildtype MCF-7 cells (IC50s were 9.2 and 13.2 nM, respectively), while MCF-7DOX cells required a 5-fold higher concentration of calphostin C to achieve the same killing (IC50 = 64.2 nM). Consistent with their known mechanisms of action, paclitaxel killed tumor cells by inducing mitotic arrest and cell multinucleation, while doxorubicin induced plasma membrane blebbing and decreased nuclear staining with propidium iodide. In contrast, cytoplasmic vacuolization accompanied cell killing by calphostin C in these cell lines, without the induction of caspase-8 or PARP cleavage or the release of cytochrome c from mitochondria. Calphostin C had little effect on the uptake of either paclitaxel or doxorubicin by the cells. Taken together, the above data suggests that calphostin C is able to potently kill drug-resistant breast tumor cells through a mechanism that may involve the induction of cytoplasmic vacuolization, without activation of typical apoptotic pathways. Consequently, calphostin C may prove useful clinically to combat tumor growth in breast cancer patients whose tumors have become unresponsive to anthracyclines or taxanes, particularly in association with photodynamic therapy.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytoplasm; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Immunoblotting; Inhibitory Concentration 50; Microscopy, Fluorescence; Naphthalenes; Paclitaxel; Poly(ADP-ribose) Polymerases; Vacuoles