tretinoin and Prostatic-Neoplasms

There is a need for agents that eliminate cancer stem cells, which sustain cancer and are also largely responsible for disease relapse and metastasis. Conventional chemotherapeutics and radiotherapy are often highly effective against the bulk of cancer cells, which are proliferating, but spare cancer stem cells. Therapeutics that target cancer stem cells may also provide a

Topics: Carcinoma, Hepatocellular; Child; Humans; Liver Neoplasms; Male; Necroptosis; Neoplastic Stem Cells; Prostatic Neoplasms; Receptors, Retinoic Acid; Tretinoin

A number of hormonal ligands and/or the nuclear receptors that mediate their actions have been targeted for prostate cancer therapy. Androgens, the ligands for the androgen receptor (AR), are critical for the growth of prostate cancer. Inhibition of androgen production has been the mainstay of treatment for advanced prostate cancer for decades. Other more recently tested targets include retinoid receptors (RAR and RXR), glucocorticoid receptors (GR), estrogen receptors (ER) and peroxisome proliferator-activated receptors (PPAR). Calcitriol, acting through the Vitamin D receptor (VDR), has many tumor suppressive activities in the prostate, including inhibition of proliferation, induction of apoptosis and/or differentiation, and reduction of cellular invasion. Because of these properties, calcitriol and its less hypercalcemic analogs are being evaluated as agents to prevent or treat prostate cancer. Androgens, retinoids, glucocorticoids, estrogens and agonists of PPAR directly or indirectly impact Vitamin D signaling pathways, and vice versa. In order to design the most effective strategies to use calcitriol to prevent or treat prostate cancer, the interactions of other nuclear receptors and their ligands with the Vitamin D signaling pathway need to be considered.

Topics: Alitretinoin; Androgen Receptor Antagonists; Androgens; Animals; Antineoplastic Combined Chemotherapy Protocols; Calcitriol; Drug Interactions; Glucocorticoids; Histone Deacetylase Inhibitors; Hormones; Humans; Male; PPAR gamma; Prostatic Neoplasms; Proteasome Inhibitors; Receptors, Androgen; Receptors, Cytoplasmic and Nuclear; Retinoids; Signal Transduction; Tretinoin; Vitamin D

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

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

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

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

Topics: Alitretinoin; Androgens; Angiotensin Receptor Antagonists; Animals; Benzimidazoles; Biphenyl Compounds; Cholecalciferol; DNA-Binding Proteins; Drug Design; Genistein; Humans; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Telomerase; Tetrazoles; Tretinoin

Most prostate cancers eventually develop resistance to hormonal therapy and chemotherapies. Many mechanisms for resistance to chemotherapy have been identified. Mutations or inactivation of the p53 suppressor gene and overexpression of bcl-2 are among such mechanisms. Mutations in the p53 gene can lead to resistance to certain chemotherapy agents, and such mutations are seen more often in metastatic than in primary prostate cancers. Thus, agents that are active in the setting of mutated p53 may have some advantage in prostate cancer. Overexpression of bcl-2 occurs frequently in prostate cancer and is associated with both hormonal therapy and chemotherapy resistance. In experimental systems, bcl-2 overexpression occurs after androgen deprivation and transfection of bcl-2 into sensitive cell lines makes them resistant to chemotherapy and hormonal therapies. Bcl-2 can be inactivated by phosphorylation as occurs with taxanes. The retinoids, as a class, can inhibit the growth of resistant cell lines that overexpress bcl-2, and the combination of interferon (IFN) and cis-retinoic acid (CRA) demonstrated increased antitumor activity. In our cell line model the combination of IFN and CRA greatly enhanced the cytotoxicity of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ). Based on these observations, we conducted a phase I/II trial of CRA and IFN-alpha in patients with biochemical recurrence of prostate cancer. Twenty-six percent achieved a decrease of prostate-specific antigen (PSA), which was correlated to elevated serum transforming growth factor-beta. We then conducted a phase I trial of 13-CRA, IFN-alpha, and escalating doses of paclitaxel. Eighteen patients were treated with 1 mg/kg CRA and 1x10(6) unit IFN on days 1 to 4 and paclitaxel at doses from 100 to 175 mg/m2. Eleven patients received the 175 mg/m2 paclitaxel dose. Two patients in the phase I study achieved partial responses (one cervix and one prostate cancer). We subsequently initiated a phase II study of 13-CRA, IFN-alpha, and paclitaxel in hormone refractory prostate cancer. For entry patients must show progressive disease after androgen ablation. To test the mechanism of action, we are assaying peripheral blood monocytes and, when possible, tumor tissue for bcl-2 expression. As our understanding of the mechanisms of tumor resistance to chemotherapy improves, we will be able to design better approaches in treatment targeted to overcome the mechanisms of resistance.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Drug Resistance, Neoplasm; Gene Expression; Genes, bcl-2; Genes, p53; Humans; Interferons; Male; Mutation; Paclitaxel; Prostatic Neoplasms; Tretinoin

Three new different aspects of prostate cancer have been considered in this review: the existence of an hereditary form, the role of estrogens as predisposing factors and the efficacy of differentiation therapies. Prostate cancer shows a stronger familial aggregation than colon and breast carcinoma. Hereditary prostate cancer is distinguished by early age at onset and autosomal dominant inheritance within families. However, only 2% of all prostate cancer in United States white men occur in those 55 years old or younger. Thus, the impact of hereditary prostate cancer in the population is the greatest at younger ages but this accounts for only a small proportion of the total disease burden. Using the developmentally estrogenized mouse model, an alternative role for estrogens as a predisposing factor for prostate diseases was proposed: estrogen exposure during development may initiate cellular changes in the prostate which would require estrogens and/or androgens later in life for promotion to neoplasia. A combination therapy employing both differentiation therapy and hormone therapy may be effective in the treatment of advanced prostate cancers. Recent advances in the field of differentiation therapy have resulted in the development of novel retinoic acid metabolism blocking agents. Unlike previous differentiating agents such as the retinoids, these agents increase the endogenous levels of retinoic acid by inhibiting its breakdown in cancer cells.

Topics: Animals; Cell Differentiation; Estrogens; Female; Humans; Male; Mice; Middle Aged; Neoplastic Syndromes, Hereditary; Pregnancy; Prenatal Exposure Delayed Effects; Prostate; Prostatic Neoplasms; Tretinoin

Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy. Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer. In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3beta-ol, may open new perspectives in this field. In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide+hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane. An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro, it enhances the antiproliferative and differentiation effects of RA in cell lines that express RA metabolism, such as F9 teratocarcinoma and MCF-7 breast carcinoma cells. In vivo, monotherapy with LIA increases RA plasma levels and, to a greater extent, endogenous tissue RA levels leading to retinoid-mimetic effects. In the rat Dunning prostate cancer models, it inhibits the growth of androgen-independent as well as androgen-dependent carcinomas relapsing after castration. Concurrently, changes i

Topics: Adenocarcinoma; Aldehyde-Lyases; Androgen Antagonists; Androgens; Animals; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Cattle; Clinical Trials as Topic; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Design; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Imidazoles; Ketoconazole; Male; Mice; Neoplasm Proteins; Prostatic Neoplasms; Rats; Rats, Wistar; Salvage Therapy; Steroid 17-alpha-Hydroxylase; Tretinoin; Tumor Cells, Cultured

An understanding of the natural history of changes in prostate-specific antigen (PSA) may be valuable as a surrogate view of prostate dynamics, as a method to differentiate between benign and malignant growth, and as a means to assess the use of PSA as a tool for monitoring activity of chemoprevention agents. Although PSA appears to be useful as a noninvasive marker of prostatic growth, PSA changes should not be confused with a direct measure of tumor growth. Serum PSA levels are a function of tumor volume but are also influenced by the volume of benign epithelium, grade of carcinoma (if any), inflammation, androgen levels, growth factors, and the extracellular matrix. The biological functions of PSA in the prostate and in its secretions need to be more completely elucidated in order that PSA measurements may more accurately describe prostate dynamics. The expression of PSA is androgen-regulated. It is one of the most abundant prostate-derived proteins in the seminal fluid. Seminogelin, a major protein in seminal fluid, is cleaved by PSA, and this cleavage is important in the liquefaction of semen. Less is known about other PSA substrates. Current PSA studies indicate that cancer cases exhibit an early slow linear PSA phase followed by a rapid exponential phase, and that PSA levels begin to increase exponentially approximately 7-9 years before diagnosis. The establishment of age-specific PSA reference ranges (ASRR) and of PSA velocity (PSAV) rates provide elements of a baseline from which prediction models could measure malignant potential of a prostatic carcinoma. Moreover, recent discoveries of different molecular forms of PSA in serum may allow a much more accurate differentiation of benign and malignant growth as well as a more potent measure of the impact of chemoprevention agents. If PSA doubling time is approximately 2.4-3.0 years and accurately reflects tumor doubling time, and if the average man has less than 0.5 ml of latent prostatic tumor tissue and the average stage T2 cancer is approximately 4 ml when detected, then the available PSA data suggest that the 3 doublings necessary to change from 0.5-4.0 ml, would take 7-12 years for a typical small volume tumor to reach the size of most stage T2 tumors. The findings that histologic cancers appear at much younger ages than previously known is disturbing. It indicates that disease initiation may begin sooner than ever thought likely. "Normal" PSA levels for younger men (< 40 years of age) may need

Topics: Adult; Age Factors; Aged; Chemoprevention; Clinical Trials as Topic; Humans; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Neoplasms; Reference Values; Transforming Growth Factor beta; Tretinoin; Vitamin A; Vitamin D

The clinical pharmacology of all-trans retinoic acid (RA) has distinct differences from that of its widely studied stereoisomer 13-cis retinoic acid (cRA). RA is much more rapidly cleared from plasma following oral administration; their respective half-lives are < 1 h and 13 h. There is extensive accumulation of the 4-oxo-cRA in plasma following repeated doses of cRA, while 4-oxo-RA is only a minor metabolite in plasma following RA administration. The extent of isomerization in vivo differs for the two retinoids. In contrast to cRA, where up to a 1:3 ratio of RA to cRA is observed in patient plasma following drug administration, cRA concentrations in excess of 10 ng/ml are rarely observed in plasma of patients receiving exogenous RA. RA administration produces autoinduction of its own oxidative catabolism; this effect does not occur with cRA. These pharmacokinetic differences have been observed in leukemia and solid tumor patients. Detailed analysis of the results of the population studied suggest that both constitutive and RA-induced hypercatabolism of RA occurs. Both of these hypercatabolic states can be modulated by concurrent administration of ketoconazole, an inhibitor of cytochrome P-450 and lipoxygenase-mediated oxidations.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Ketoconazole; Leukemia, Promyelocytic, Acute; Lung Neoplasms; Male; Metabolic Clearance Rate; Multiple Myeloma; Oxidation-Reduction; Prostatic Neoplasms; Tretinoin

Topics: Animals; Cell Division; Humans; Male; Prostatic Neoplasms; Tretinoin

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

The evaluation of new therapies in prostate cancer requires unique end points for agents with diverse mechanisms of action. Because retinoic acid may have a confounding effect on prostate-specific antigen, we incorporated a pathological end point into the outcome assessment of two sequential clinical trials using all-trans-retinoic acid (ATRA) and the combination of 13-cis-retinoic acid and IFN-2a (cRA¿IFN). Pre- and posttherapy tumor biopsy specimens were studied for histological changes, apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), and proliferation index (Ki67). Prostate-specific membrane antigen (PSMA) expression was also evaluated using two different monoclonal antibodies to its intracellular domain (Cytogen 7E11 and Hybritech PM2). Fourteen patients with androgen-independent disease were treated with ATRA (50 mg/m2 p.o. every 8 h daily) and 16 androgen-independent and 4 androgen-dependent patients were treated with cRA¿IFN (10 mg/kg/day cRA plus 3, 6, or 9 million units daily IFN). Both therapies were well tolerated, with fatigue and cheilitis being the most common adverse events. Clinical activity, assessed by radiographs and serum prostate-specific antigen, was minimal, and the majority of patients progressed within 3 months. One patient with androgen-dependent disease had prolonged stabilization for >1 year. The majority of cases (95%) showed no gross histological changes and no difference in apoptotic or proliferative indices. Increased PSMA immunoreactivity was seen in seven of nine (78%) cases using PM2 antibody and in two of nine (22%) cases using the 7E11 antibody. Although antitumor effects were modest, the results suggest a role for retinoids in modulating the expression of PSMA on prostate cancer cells.

Topics: Aged; Antigens, Surface; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Bone Neoplasms; Carboxypeptidases; Cheilitis; Dyspnea; Exanthema; Fatigue; Glutamate Carboxypeptidase II; Hematologic Diseases; Humans; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Liver; Male; Middle Aged; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Recombinant Proteins; Transaminases; Treatment Outcome; Tretinoin

To examine the feasibility of using fenretinide (4-HPR) for the prevention and treatment of prostate cancer.. We measured the impact of 4-HPR therapy on retinoid concentrations in vivo, in a mouse model of prostate cancer and clinically, in patients with prostate cancer who were given oral 4-HPR (200 mg/d) or placebo for 4 weeks before undergoing a radical prostatectomy.. Prostate tumors in mice treated with 4-HPR contained high levels of 4-HPR and of all-trans-retinoic acid (RA) and reduced levels of retinol (ROH). Patients given 4-HPR were found to have significantly higher concentrations of 4-HPR in the cancerous prostate as compared with the serum levels (463 nmol/L v 326 nmol/L; P =.049), but they were only 1/10 the levels found in mice and were far below the concentrations reported in human breast tissue. Serum and tissue ROH levels were reduced to less than half the concentrations found in untreated controls. RA concentrations in human serum and in cancerous prostates were not significantly affected by 4-HPR treatment, in contrast with the findings in mice.. The standard oral dose of 4-HPR proposed for breast cancer (200 mg/d) achieved only modest drug levels in the prostate and is unlikely to be effective for prostate cancer prevention or treatment. Higher doses need to be explored.

Topics: Aged; Animals; Antineoplastic Agents; Double-Blind Method; Fenretinide; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Placebos; Prostatectomy; Prostatic Neoplasms; Tretinoin; Vitamin A

We assess the efficacy and toxicity of all-trans retinoic acid administered intermittently for hormone refractory prostate cancer.. A total of 26 patients with histologically confirmed adenocarcinoma of the prostate and manifestations of progressive metastatic disease, despite at least 1 hormonal therapy, were included in the study. Patients received a single oral dose of 45 mg./m.2 all-trans retinoic acid daily for 7 days followed by 7 days of no treatment, and then resumed treatment on day 14. This schedule was continued until progression or limiting toxicity occurred. Response was assessed based on serial measurements of serum prostate specific antigen and pain scores every other week. In addition, objective responses were evaluated every 12 weeks in patients with bi-dimensionally measurable metastases.. All-trans retinoic acid was administered for 168 weeks (median 6 weeks per patient, range 2 to 21). Toxicity was mild. No patient stopped therapy because of toxicity. Of the patients 4 (15%) demonstrated a biological response of 50% or greater serum prostate specific antigen decrease. No objective response was observed among 11 patients with measurable metastases. Of 17 patients who required analgesics at the onset of all-trans retinoic acid treatment only 1 achieved a significant reduction of pain. Median survival time from the onset of all-trans retinoic acid treatment was 19 months (range 2 to 24).. All-trans retinoic acid has minimal activity in hormone refractory prostate cancer.

Topics: Adenocarcinoma; Aged; Antineoplastic Agents; Drug Administration Schedule; Humans; Male; Middle Aged; Prostatic Neoplasms; Tretinoin

All-trans-retinoic acid (ATRA) is a retinoid analogue that has been shown to be effective in acute promyelocytic leukemia. It is currently being investigated for efficacy in the treatment and prevention of various types of cancer. One of the factors limiting its use is the observed increase in ATRA clearance and elimination which occurs shortly after treatment is started, leading to reduced levels of drug in the body and loss of effectiveness. ATRA efficacy may be enhanced if this autoinduction of metabolism can be overcome, for example through the inhibition of the activity of the induced specific metabolizing enzyme(s). This requires the identification of this induced enzyme(s) and development of approaches to selectively inhibit its activity.. In the course of a phase II evaluation of ATRA in prostate cancer, we investigated the activities of five specific cytochrome P450 (CYP) (CYPs 1A2, 2C19, 2D6, 2E1 and 3A4) and N-acetyltransferase enzymes using a newly developed five-drug cocktail involving caffeine, mephenytoin, debrisoquine, chlorzoxazone and dapsone respectively. Enzyme activities were assessed in 17 patients with hormone-refractory prostate cancer before the initiation of ATRA therapy, after 14 days of continuous ATRA administration and 7 days after cessation of drug therapy.. After 14 days of ATRA therapy, the activities of CYP2E1 (chlorzoxazone hydroxylase) and N-acetyltransferase (in fast acetylators only) were increased by 83% and 29% (P < 0.05), respectively. Both activities returned to baseline by 7 days after cessation of therapy and the profiles were similar to the changes seen in the clearance of ATRA itself. There were no changes in the activities of any of the other enzymes investigated.. This study shows that ATRA selectively modulates the activities of specific metabolizing enzymes and that this approach may be useful in identifying enzymes that can be explored in an attempt to mitigate ATRA autoinduction through selective modulation of enzyme activities. Further investigations are required to determine whether the elevated enzymes are also responsible for the increased clearance and elimination of ATRA.

Topics: Adenocarcinoma; Antineoplastic Agents; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Enzyme Inhibitors; Humans; Male; Metabolic Clearance Rate; Phenotype; Prostatic Neoplasms; Tretinoin

Retinoids have been shown to have substantial anticancer activity in a number of preclinical and clinical situations. There are considerable epidemiologic, in vitro and in vivo data which indicate that retinoids may have a role in the prevention and therapy of human prostate cancer. Based on anecdotal evidence of response in one patient with hormone-refractory prostate cancer (HRPC), we conducted a phase II trial in HRPC during which we also examined changes in pharmacokinetics of all-trans-retinoic acid (ATRA) which occurred during therapy. Enrolled in the study were 17 patients with HRPC who received 50 mg/m2 ATRA three times daily orally on days 1-14, repeated every 22 days. The pharmacokinetics of ATRA were assessed with the first dose on day 1, again on day 14 and after a 7-day interruption in ATRA therapy on day 22. Patients were evaluable for response if they completed two 14-day courses of ATRA; among 13 such patients no responses were seen. Four patients were considered unevaluable for response owing to rapid disease progression in three and intercurrent illness in one. Apparent clearance of ATRA changed substantially during therapy: day 1 3779 +/- 4215 ml/min, day 14 7179 +/- 3197 ml/min, day 22 3213 +/- 2357 ml/min. Area under the curve was proportionately diminished on day 14 compared with day 1 and had returned to baseline by day 22. We conclude that ATRA is not active in HRPC. Failure of this agent in HRPC may be related to failure of drug delivery associated with enhanced mechanisms of ATRA clearance which occur within a few days of beginning ATRA treatment.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biomarkers, Tumor; Humans; Leukocytes; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Neoplasms; Tretinoin; Vitamin A

Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer.. We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes.. Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1.. The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells.

Topics: Cell Line, Tumor; Co-Repressor Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Neoplastic Cells, Circulating; Prostatic Neoplasms; Receptors, Androgen; Tretinoin

Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer-induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer-induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer-induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, β, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer-induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer-induced bone formation to improve clinical outcomes for patients with bone metastasis.. This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

Topics: Bone Neoplasms; Endothelial Cells; Humans; Male; Osteoblasts; Prostatic Neoplasms; Receptors, Retinoic Acid; Tretinoin; Ubiquitin-Protein Ligases

We have previously shown that ATRA stimulates transactivation of RARγ at sub-nanomolar concentrations (EC. We suggest that the use of RARγ-selective antagonists may be effective in PC (and potentially other cancers), either as a single agent or in combination with cytotoxic chemotherapy.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Male; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Tretinoin

Tumor cells require increased rates of cell metabolism to generate the macromolecules necessary to sustain proliferation. They rely heavily on NAD

Topics: Acrylamides; ADP-ribosyl Cyclase 1; AMP-Activated Protein Kinase Kinases; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Cytokines; Fatty Acids; Gene Expression; Humans; Lipids; Male; Membrane Glycoproteins; Mitochondria; NAD; Nicotinamide Phosphoribosyltransferase; PC-3 Cells; Piperidines; Prostatic Neoplasms; Protein Kinases; Transcriptome; Transfection; Tretinoin

9-cis-Retinoic acid (9cRA), which binds to both retinoic acid receptors and retinoic X receptors, inhibits prostate cancer induction in rats and reduces growth of prostate cancer cells. However, the nature of this growth inhibition and the interactive influence of androgens are not well defined and are the subject of this report. LNCaP and PC-3 cells were cultured and treated with a range of 9cRA concentrations for 3-6 days in the absence or presence of 5α-dehydrotestosterone. 9cRA inhibited cell proliferation in a dose-dependent manner, plateauing at 10 mol/l. Treatment of cells with 10 mol/l 9cRA inhibited 5α-dihydroxytestosterone (DHT)-stimulated proliferation, the effect of which was maximal at 10 mol/l DHT. Treatment of DHT (10 mol/l)-exposed cells with 9cRA caused a dose-dependent increase in prostate-specific antigen in the medium after 6 days, but not 3 days. 9cRA caused a dose-dependent increase in apoptotic cells stained with H33258 after 3 days, but not 6 days; however, on using flow cytometry, apoptosis was apparent at both 3 and 6 days. Flow cytometry also revealed interference of G0/G1 to S phase transition by 9cRA. Inhibition by 9cRA of anchorage-independent growth of PC-3 cells was also found; LNCaP cells did not grow colonies in soft agar. 9cRA inhibited growth and induced differentiation of human LNCaP prostate cancer cells in vitro and inhibited anchorage-independent growth of PC-3 cells. Because 9cRA and 13-cis-retinoic acid, which is retinoic acid receptor-selective, prevent prostate carcinogenesis in rats, and 13-cis-retinoic acid also inhibits growth of human prostate cancer cells, the RAR is a potential molecular target for prostate cancer prevention and therapy.

Topics: Alitretinoin; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Interactions; Humans; Male; Prostatic Neoplasms; Testosterone; Tretinoin

The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Humans; Male; MCF-7 Cells; Mice; Mice, Nude; Mice, SCID; Molecular Structure; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptors, Adrenergic; Signal Transduction; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Androgen receptor (AR) and MNK activated eIF4E signaling promotes the development and progression of prostate cancer (PCa). In this study, we report that our Novel Retinamides (NRs) target both AR signaling and eIF4E translation in androgen sensitive and castration resistant PCa cells via enhancing AR and MNK degradation through ubiquitin-proteasome pathway. Dual blockade of AR and MNK initiated eIF4E activation by NRs in turn induced cell cycle arrest, apoptosis, and inhibited cell proliferation. NRs also inhibited cell migration and invasion in metastatic cells. Importantly, the inhibitory effects of NRs on AR signaling, eIF4E translation initiation and subsequent oncogenic program were more potent than that observed with clinically relevant retinoids, established MNK inhibitors, and the FDA approved PCa drugs. Our findings provide the first preclinical evidence that simultaneous inhibition of AR and eIF4E activation is a novel and efficacious therapeutic approach for PCa, and that NRs hold significant promise for treatment of advanced prostate cancer.

Topics: Androgen Antagonists; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Receptors, Androgen; RNA Interference; Signal Transduction; Time Factors; Transfection; Tretinoin

In this study retinoic acid (RTA) loaded solid lipid nanoparticles (SLNs) were optimized by tuning the process parameters (pressure/temperature) and using different lipids to develop nanodispersions with enhanced anticancer activity. The RTA-SLN dispersions were produced by high-pressure homogenization and characterized in terms of particle size, zeta potential, drug entrapment efficiency, stability, transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and in vitro drug release. Thermal and X-ray analysis showed the RTA to be in the amorphous state, whilst microscopic images revealed a spherical shape and uniform particle size distribution of the nanoparticles. Anticancer efficiency was evaluated by incubating RTA-SLNs with human prostate cancer (LNCap) cells, which demonstrated reduced cell viability with increased drug concentrations (9.53% at 200 ug/ml) while blank SLNs displayed negligible cytotoxicity. The cellular uptake of SLN showed localization within the cytoplasm of cells and flow cytometry analysis indicated an increase in the fraction of cells expressing early apoptotic markers, suggesting that the RTA loaded SLNs are able to induce apoptosis in LNCap cells. The RTA-SLN dispersions have the potential to be used for prostate anticancer treatment.

Topics: Antineoplastic Agents; Calorimetry, Differential Scanning; Cell Line, Tumor; Chemistry, Pharmaceutical; Dose-Response Relationship, Drug; Drug Carriers; Drug Liberation; Drug Stability; Flow Cytometry; Freeze Drying; Humans; Lipids; Male; Microscopy, Electron, Transmission; Nanoparticles; Particle Size; Prostatic Neoplasms; Solubility; Tretinoin; X-Ray Diffraction

To elucidate a hypothetical link between retinoic acid (RA) signaling and minocycline for targeting prostate carcinoma (PCA). RA signaling has been implicated in growth-inhibition of malignant PCA, and intracellular RA homeostasis has been investigated as a potential therapeutic target. Minocycline is a tetracycline antibiotic with pleiotropic actions in many tissues and reaches comparably high levels in human prostate tissue. Interestingly, minocycline exhibits the rare side effect of a pseudotumor cerebri, which is otherwise known to occur from vitamin A intoxication or in retinoid therapy. Therefore, we hypothesized minocycline to putatively interact with intracellular RA homeostasis in PCA.. Using LN-CAP, DU-145, and PC-3 cell lines, effects of minocycline on microsomal RA metabolism and on cell growth were assessed in vitro.. Minocycline was identified to potently inhibit cell growth, at concentrations within the range of tissue levels readily reached under standard therapeutic conditions. In vitro inhibition experiments revealed inhibition of RA breakdown, yet only at comparably high concentrations of minocycline. Using all trans-RA, RA metabolism inhibitor liarozole, and different retinoid receptor antagonists, the putative RA-dependent effects of minocycline were further evaluated and confirmed to be independent of RA signaling.. Our findings add to the growing body of evidence for the many pleiotropic actions of minocycline. In view of the striking effects of minocycline on cell growth in PCA cell lines in vitro and its relatively safe side effect profile, the use of minocycline for targeting PCA should be timely clinically evaluated.

Topics: Cell Line, Tumor; Cell Proliferation; Humans; Male; Minocycline; Prostatic Neoplasms; Tretinoin

Tumor-associated macrophages (TAMs) are a prominent cell type of the tumor stroma and stimulate malignant cell growth, survival and metastasis. The present manuscript demonstrates that prostate cancer cell-derived factors induce a pro-tumoral TAM-like phenotype characterized by increased proliferation and increased expression of pro-angiogenic, immunosuppressive and pro-metastatic factors. These effects were abrogated by all trans-retinoic acid (ATRA), a clinically available molecule with known immune-modulating properties. Furthermore, ATRA inhibited the cancer cell-stimulated proliferation of the pro-tumoral macrophages and restored their cytotoxic capacity towards prostate cancer cells. These findings suggest the use of ATRA as an immunomodulating agent to block the activity of prostate cancer TAMs.

Topics: Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytokines; Cytotoxicity, Immunologic; Humans; Immunosuppression Therapy; Macrophages; Male; MAP Kinase Signaling System; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappaB-Inducing Kinase; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Tretinoin

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

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

Retinoids constitute a family of organic compounds that are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelocytic leukemia. Their use however is limited due to serious adverse effects and there is a great need for analogues with better safety profile. In the present work, the effect of N(1),N(12)-bis(all-trans-retinoyl)spermine (RASP), a conjugate of all-trans-retinoic acid (atRA) with spermine, on angiogenesis in vivo and viability of human endothelial and prostate cancer cells in vitro were studied. Both atRA and RASP dose-dependently inhibited angiogenesis in the chicken embryo chorioallantoic membrane model. RASP was more effective and could be used in a wider dose range due to lower toxicity compared with atRA. Both retinoids decreased the number of human umbilical vein endothelial and prostate cancer LNCaP and PC3 cells in a concentration-dependent manner. RASP was more effective and potent compared with atRA, spermine, their combination, or conjugates of spermine with other acidic retinoids and/or psoralens in prostate cancer cells. The inhibitory effect of both atRA and RASP seems to be related to an increase of the tumour repressing gene retinoic acid receptor beta mRNA, was mediated by retinoic acid receptor alpha, and was proportional to endogenous retinoic acid receptor beta expression. These data suggest that RASP is more effective than atRA in decreasing angiogenesis and prostate cancer cell growth and identify retinoic acid receptor alpha as the receptor through which it causes retinoic acid receptor beta up-regulation and decrease of prostate cancer cell growth.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Cell Count; Cell Line, Tumor; Cell Survival; Chick Embryo; Human Umbilical Vein Endothelial Cells; Humans; Male; Neovascularization, Physiologic; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Spermine; Tretinoin

In the human prostate, expression of prostate-specific genes is known to be directly regulated by the androgen-induced stimulation of the androgen receptor (AR). However, less is known about the expression control of the prostate-restricted TGM4 (hTGP) gene. In the present study we demonstrate that the regulation of the hTGP gene depends mainly on retinoic acid (RA). We provide evidence that the retinoic acid receptor gamma (RAR-G) plays a major role in the regulation of the hTGP gene and that presence of the AR, but not its transcriptional transactivation activity, is critical for hTGP transcription. RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites. Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR. Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

Topics: Androgens; Cell Line, Tumor; Enhancer Elements, Genetic; Gene Expression Regulation, Enzymologic; Gene Regulatory Networks; Humans; Male; Metribolone; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Receptors, Androgen; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transcriptional Activation; Transglutaminases; Tretinoin

In carcinomas the expression of thrombomodulin (TM) is inversely correlated with tumour progression and metastasis. In the present study a decreased TM expression in human prostate cancer cell lines, LNCaP, DU-145, and PC-3, in relation to normal prostate epithelial cells (PrEC) is shown. Sequencing and methylation-specific high resolution melting (MS-HRM) analyses of bisulphite-modified genomic DNA indicates a high degree of methylation in DU-145 cells and lesser degrees in PC-3 and LNCaP cells, whereas in PrEC the TM promoter is unmethylated. The expression of TM is negatively regulated by NF-κB- and GSK3-β-dependent signalling pathways and positively regulated by retinoic acid and transcription factor Sp1 in PrEC, LNCaP and PC-3 cells, but not in DU-145 cells. However, exposure of DU-145 cells to the demethylating agent, 5-aza-2'deoxycytidine, restores the TM expression and its control by retinoic acid, NF-κB- and GSK3-β-dependent signalling. In conclusion, the study establishes that in prostate cancer cell lines relative to PrEC the TM is down-regulated and that the TM promoter is hypermethylated, which seems to be responsible for the down-regulation and failed regulation of TM expression in DU-145 cells.

Topics: Cell Line; CpG Islands; DNA Methylation; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; NF-kappa B; Promoter Regions, Genetic; Prostatic Neoplasms; Sp1 Transcription Factor; Thrombomodulin; Tretinoin

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

Topics: Alitretinoin; Androgens; Cell Line, Tumor; Connexins; Gap Junction beta-1 Protein; Gap Junctions; Humans; Male; Prostatic Neoplasms; Retinoids; Tretinoin

All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Enhancer of Zeste Homolog 2 Protein; Epigenetic Repression; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Models, Genetic; Polycomb Repressive Complex 1; Polycomb Repressive Complex 2; Polycomb-Group Proteins; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; Transcription, Genetic; Tretinoin; Up-Regulation

The bond between the research laboratory and the clinic is especially strong in the field of photomedicine. Much is learned in preclinical animal models, which is translated to the clinic for investigation, and then refinements in theory and technique are explored back in the laboratory. With many cancers becoming resistant to treatment, photodynamic therapy (PDT) offers a mechanistically distinct alternative. Studies have shown that PDT not only mitigates chemoresistance but also synergizes with chemotherapy and molecularly targeted therapies. From the world of biochemistry comes this unique look at 2 approaches to maximize the photodynamic effect through PDT combinations with targeted therapies: 1) using the molecular response after PDT to guide the selection of targeted agents and 2) preconditioning cancer cells to modulate nuclear molecular targets before PDT.

Topics: Aminolevulinic Acid; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cetuximab; Combined Modality Therapy; ErbB Receptors; Female; Humans; Male; Mice; Molecular Targeted Therapy; Ovarian Neoplasms; Pancreatic Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Prostatic Neoplasms; Tretinoin; Vascular Endothelial Growth Factor A; Verteporfin; Vitamin D

We report that all-trans retinoic acid (ATRA) in combination with zoledronic acid has strong synergistic cytotoxic and apoptotic effects against human hormone- and drug-refractory prostate cancer cells, PC-3 and DU-145, in a time- and dose-dependent manner. We further investigated the effect of the combination treatment on the apoptotic process by both oligoarray and protein array analysis in DU-145 cells, in which the drug combination shows much more strong synergistic effects, as compared to PC-3 cells. Moreover, we have also performed real time-PCR array analysis to validate oligoarray results. We demonstrated that the combination of ATRA and zoledronic acid is a strong inducer of apoptotic related cell death in human androgen-and drug refractory prostate cancer cells DU-145, at either transcriptional or translational levels. While expression of proapoptotic genes such as tumor necrosis factor receptor superfamily (TNFRSF), Bad, Bax, Fas, FADD are induced with the exposure of the combination, expression of antiapoptotic genes or proteins such as members of inhibitor apoptosis family (IAPs), MCL-1, LTBR, p53 and bcl-2 are reduced. Because this novel combination treatment has fewer side effects than is generally the case with conventional cytotoxic agents, this regimen may be a good option for treatment of elderly prostate cancer patients.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 7; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphosphonates; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Hormones; Humans; Imidazoles; Male; Prostatic Neoplasms; Receptors, Tumor Necrosis Factor; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tretinoin; Zoledronic Acid

We have recently isolated novel IFN-inducible gene, Gene associated with Retinoid-Interferon-induced Mortality-1 (GRIM-1), using a genetic technique. Moderate ectopic expression of GRIM-1 caused growth inhibition and sensitized cells to retinoic acid (RA)/IFN-induced cell death while high expression caused apoptosis. GRIM-1 depletion, using RNAi, conferred a growth advantage. Three protein isoforms (1α, 1β and 1γ) with identical C-termini are produced from GRIM-1 mRNA. We show that GRIM-1 isoforms interact with NAF1 and DKC1, two essential proteins required for box H/ACA sno/sca RNP biogenesis and suppresses box H/ACA RNA levels in mammalian cells by delocalizing NAF1. Suppression of these small RNAs manifests as inefficient rRNA maturation and growth suppression. Interestingly, yeast Shq1p also caused growth suppression in mammalian cells. Consistent with its growth-suppressive property, GRIM-1 expression is lost in a number of human primary prostate tumors. Our observations support a recent study that GRIM-1 might act as a co-tumor suppressor in the prostate.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Immunohistochemistry; Interferon-beta; Male; Neoplasms; Nuclear Proteins; Prostatic Neoplasms; Protein Binding; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins; RNA, Ribosomal; RNA, Small Nucleolar; RNA, Small Untranslated; Saccharomyces cerevisiae Proteins; Tretinoin

p27 is a cell cycle suppressor gene, whose protein is a negative regulator of cyclin/cdk complexes. p27 is also a potential target of retinoids in cancer prevention studies. In benign prostate hyperplasia (BPH), and in most carcinomas, p27(Kip1) is down-regulated, suggesting its potential resistance to retinoids. To test this hypothesis, we examined the efficacy of 9-cis retinoic acid (9cRA) to suppress prostate cell proliferation (PECP) and carcinogenesis in p27(Kip1) deficient mice.. p27(Kip1) deficient (-/-), heterozygous (+/-) and homozygous (+/+) mice were treated for 7 days with testosterone, 9cRA, or with both, and cell proliferation in dorsolateral prostate (DLP) was determined by BrdU labeling. Prostate carcinogenesis was induced by N-Methyl-N-Nitrosourea (MNU) and hormone stimulation.. PECP in DLP of two-month-old mice of all genotypes was similar but significantly increased in old p27-/- mice only. Testosterone treatment increased PECP in all three p27 genotypes with the highest values in p27-/- mice. p27(Kip1) deficiency did not affect the response of PEC to 9cRA and to 9cRA+testosterone. The decrease of p27(Kip1) in p27+/- and p27-/- mice progressively increased the incidence and frequency of PIN and tumors. 9cRA suppressed PIN in all three p27 genotypes and this was associated with decreased PECP and increased cellular senescence.. This data indicates that p27(Kip1) deficiency promotes prostate cell proliferation and carcinogenesis but does not affect 9cRA's potential to suppress prostate carcinogenesis, suggesting that patients with PIN and carcinomas lacking or having a low level of p27(Kip1) expression may also benefit from clinical trials with retinoids.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Genotype; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Prostatic Neoplasms; Retinoids; Tretinoin

In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with alpha-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARalpha-dependent, the effect of its active analogues was not inhibited by a selective RARalpha antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

Topics: Amino Acids; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Male; Polyenes; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Structure-Activity Relationship; Substrate Specificity; Tretinoin

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

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

Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Cell Line, Tumor; Cell Nucleus; Co-Repressor Proteins; Fibroblasts; Humans; Leukemia, Promyelocytic, Acute; Male; Mice; Molecular Chaperones; Nuclear Proteins; Promyelocytic Leukemia Protein; Prostatic Neoplasms; PTEN Phosphohydrolase; Transcription Factors; Tretinoin; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Ubiquitin-Specific Peptidase 7; Ubiquitination; Ubiquitins

The management of hormone-refractory prostate cancer (HRPC) still remains as an important challenge of daily oncology practice. Docetaxel has proved to be a first line treatment choice. All-trans retinoic acid (ATRA) could potently inhibit the growth of prostate cancer cells in vitro and its combination with various anticancer agents results in increased cytotoxicity. Based on these data, our aim was to examine the synergistic/additive cytotoxic and apoptotic effects of combination of docetaxel and ATRA, in hormone- and drug refractory human DU-145 prostate cancer cells. Furthermore, we have searched for the underlying mechanisms of apoptosis by demonstrating apoptosis-related genes.. XTT cell proliferation assay was used for showing cytotoxicity. For verifying apoptosis, both DNA Fragmentation by ELISA assay and caspase 3/7 activity measurement were used. For detecting the mechanism of apoptosis induced by docetaxel-ATRA combination, OligoGeArray which consists of 112 apoptosis related genes was used.. Our results revealed that docetaxel and ATRA were synergistically cytotoxic and apoptotic in DU-145 cells, in a dose- and time dependent manner. It was also shown by our studies that apoptosis was induced in DU-145 prostate carcinoma cells with significant cytotoxicity, no matter which agent applied first. We have found out that docetaxel-ATRA combination significantly downregulates survivin (BIRC5), myeloid cell leukemia-1 (MCL-1) and lymphotoxin beta-receptor (LTbetaR) genes, which all three have pivotal roles in regulation of apoptosis and cell cycle progression.. In conclusion, we strongly suggest that docetaxel and ATRA combination is a good candidate for this challenging era of daily oncologic practice. Also, the combination of docetaxel and ATRA might allow a reduction in docetaxel doses and by this way may diminish docetaxel adverse effects while maintaining the therapeutic effect in patients with HRPC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Docetaxel; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Humans; Inhibitor of Apoptosis Proteins; Lymphotoxin beta Receptor; Male; Microtubule-Associated Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survivin; Taxoids; Tretinoin

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Imidazoles; Male; Neoplasms, Experimental; Phenylenediamines; Prostatic Neoplasms; Pyridines; Transplantation, Heterologous; Tretinoin

To investigate the enhancing effect of all-trans retinoic acid (ATRA) on the bystander effect of the herpes simplex virus thymidine kinase(HSV-TK)/ganciclovir (GCV) against androgen unresponsive prostate cancer.. The bystander effect of the HSV-TK/GCV system was measured by methyl thiazolyl tetrazolium (MTT) assay on PC-3 cells before and after ATRA treatment. The growth and the histopathology of transplant tumors were observed in 4 groups of nude mice with prostate cancer.. ATRA augmented significantly the bystander effect of the HSV-TK/GCV system by reducing TK positive PC-3 cells from 50% to 30% (P < 0.05). HSV-TK showed an inhibiting effect, while ATRA with the HSV-TK/GCV system produced significant effect on prostate cancer 1 week earlier than the former (P < 0.05).. ATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

Topics: Animals; Antineoplastic Agents; Bystander Effect; Cell Line, Tumor; Cell Survival; Ganciclovir; Genes, Transgenic, Suicide; Genetic Therapy; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Simplexvirus; Thymidine Kinase; Tretinoin; Xenograft Model Antitumor Assays

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

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

Human PCAN1 (prostate cancer gene 1) is a prostate-specific gene that is highly expressed in prostate epithelial tissue, and frequently mutated in prostate tumors. To better understand the regulation of the PCAN1 gene, a 2.6-kb fragment of its 5' flanking region was obtained by PCR. Its promoter activity was examined via the dual-luciferase reporter assay after it had been cloned into a pGL(3)-basic vector generating pGL(3)-p2.6 kb and transfected into LNCaP cells. pGL(3)-basic and pGL(3)-control were respectively used as the negative and positive controls. Sequence analysis with the MatInspector database showed that some possible binding sites for the transcriptional factors, NKX3.1, P53, SP1, cEBP and the PPAR/RXR heterodimers may locate on a 2.6-kb region upstream of the PCAN1 gene. To examine the relevant regulation of PCAN1, pGL(3)-p2.6 kb was transfected into the prostate cancer cell line LNCaP, which was treated with R1881 (10(-7) approximately 10(-9) mol/l), 17beta-estradiol (17beta-E(2), 10(-7) approximately 10(-9) mol/l), all-trans-retinoic acid (all-trans-RA, 10(-5) approximately 10(-7) mol/l) or 9-cis-retinoic acid (9-cis-RA, 10(-5) approximately 10(-7) mol/l), and eukaryotic expression plasmids of NKX3.1, p53, Sp1, Pten, PPARgamma or cEBPalpha were cotransfected with pGL(3)-p2.6 kb into LNCaP cells. pRL-TK, a Renilla luciferase reporter vector, was cotransfected into all the transfection lines as an internal control. The activities of pGL(3)-p2.6 kb (PCAN1 promoter) were analyzed via the dual-luciferase reporter assay 48 h after transfection. The results showed that 9-cis-RA enhanced the PCAN1 promoter activity in a dose-dependent manner, while R1881, 17beta-E(2) and all-trans-RA had no significant effect on PCAN1 promoter activities. Cotransfection with pGL(3)-p2.6kb and the expression plasmids of NKX3.1, p53, Sp1 or Pten respectively resulted in 1.66-, 2.48-, 2.00-and 1.72-fold 2.6 kb PCAN1 promoter activity increases relative to the controls, which were cotransfected with pcDNA3.1(+), while cotransfection of PPARgamma and cEBPalpha yielded no significant effect on PCAN1 promoter activities. These results could be applied for further study of the function and transcription regulation of the PCAN1 gene in prostate development and carcinogenesis.

Topics: Alitretinoin; Base Sequence; Cell Line, Tumor; Cloning, Molecular; Gene Expression Regulation; Humans; Luciferases; Male; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in human prostate cancer epithelial (LNCaP) cells. In the present study, we attempted to identify if any of the three ALDH1A/RA synthesis enzymes are androgen responsive and how this may affect retinoid-mediated effects in LNCaP cells. We demonstrated that exposure of LNCaP cells to the androgen dihydrotestosterone (DHT) results in a 4-fold increase in ALDH1A3 mRNA levels compared with the untreated control. The mRNA for two other ALDH1A family members, ALDH1A1 and ALDH1A2, were not detected and not induced by DHT in LNCaP cells. Inhibition of androgen receptor (AR) with both the antiandrogen bicalutamide and small interfering RNA for AR support that ALDH1A3 regulation by DHT is mediated by AR. Furthermore, specific inhibition of the extracellular signal-regulated kinase and Src family of kinases with PD98059 and PP1 supports that AR's regulation of ALDH1A3 occurs by the typical AR nuclear-translocation cascade. Consistent with an increase in ALDH1A3 mRNA, DHT-treated LNCaP cells showed an 8-fold increase in retinaldehyde-dependent NAD(+) reduction compared with control. Lastly, treatment of LNCaP with all-trans retinal (RAL) in the presence of DHT resulted in significant up-regulation of the RA-inducible, RA-metabolizing enzyme CYP26A1 mRNA compared with RAL treatment alone. Taken together, these data suggest that (i) the RA biosynthesis enzyme ALDH1A3 is androgen responsive and (ii) DHT up-regulation of ALDH1A3 can increase the oxidation of retinal to RA and indirectly affect RA bioactivity and metabolism.

Topics: Aldehyde Dehydrogenase; Androgens; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Dihydrotestosterone; Estradiol; Flavonoids; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Male; Metribolone; Prostatic Neoplasms; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Retinaldehyde; Retinoic Acid 4-Hydroxylase; RNA, Messenger; RNA, Small Interfering; src-Family Kinases; Tretinoin

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDREhPTHrP) within the human PTHrP gene and involves an interaction between nVDREhPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRalpha forms part of the nuclear protein complex that interacts with nVDREhPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9-cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9-cis-retinoic acid enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-retinoic acid. Promoter activity driven by nVDREhPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH)2D3 and EB1089 in both cell lines. Co-treatment with these compounds and 9-cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell type-specific manner in prostate cancer cells.

Topics: Alitretinoin; Calcitriol; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Male; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; Receptors, Calcitriol; Retinoid X Receptors; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured; Vitamin D Response Element

In recent studies, we have identified several highly potent all-trans-retinoic acid (ATRA) metabolism blocking agents (RAMBAs). On the basis of previous effects of liarozole (a first-generation RAMBA) on the catabolism of ATRA and on growth of rat Dunning R3227G prostate tumours, we assessed the effects of our novel RAMBAs on human prostate tumour (PCA) cell lines. We examined three different PCA cell lines to determine their capacity to induce P450-mediated oxidation of ATRA. Among the three different cell lines, enhanced catabolism was detected in LNCaP, whereas it was not found in PC-3 and DU-145. This catabolism was strongly inhibited by our RAMBAs, the most potent being VN/14-1, VN/50-1, VN/66-1, and VN/69-1 with IC50 values of 6.5, 90.0, 62.5, and 90.0 nM, respectively. The RAMBAs inhibited the growth of LNCaP cells with IC50 values in the microM-range. In LNCaP cell proliferation assays, VN/14-1, VN/50-1, VN/66-1, and VN/69-1 also enhanced by 47-, 60-, 70-, and 65-fold, respectively, the ATRA-mediated antiproliferative activity. We then examined the molecular mechanism underlying the growth inhibitory properties of ATRA alone and in combination with RAMBAs. The mechanism appeared to involve the induction of differentiation, cell-cycle arrest, and induction of apoptosis (TUNEL), involving increase in Bad expression and decrease in Bcl-2 expression. Treatment of LNCaP tumours growing in SCID mice with VN/66-1 and VN/69-1 resulted in modest but statistically significant tumour growth inhibition of 44 and 47%, respectively, while treatment with VN/14-1 was unexpectedly ineffective. These results suggest that some of our novel RAMBAs may be useful agents for the treatment of prostate cancer.

Topics: Animals; Apoptosis; Cell Cycle; Cell Differentiation; Cell Proliferation; Enzyme Inhibitors; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, SCID; Prostatic Neoplasms; Transplantation, Heterologous; Tretinoin

To study the regulatory effects of 9-cis retinoic acid (RA) on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP.. Flow cytometry, reverse transcriptase polymerase chain reaction and Western blotting were performed to evaluate the effects of 9-cis RA on NKX3.1 expression and cell cycle of LNCaP cells. To identify a regulatory region within the NKX3.1 promoter contributing to the regulation induced by 9-cis RA, we have constructed an NKX3.1 promoter-reporter plasmid, pGL3-1040bp, and its 5'-deletion mutants, which were transfected into LNCaP cells with treatment of 9-cis RA in indicated concentrations.. With the treatment of 9-cis RA, the NKX3.1 promoter activity was increased in reporter gene assay and NKX3.1 expression was enhanced at both mRNA and protein levels in LNCaP cells. We found that the region between -936 and -921 in the upstream of NKX3.1 gene involved the inducible regulation by 9-cis RA treatment. In flow cytometry, 9-cis RA treatment caused accumulation of cells in the G(1) phase of the cell cycle and a fewer cells pass through to G(2)/M.. Our results demonstrated that 9-cis RA as a differentiating agent can arrest prostate cancer cells in G(1) phase and reduce cell mitosis, and upregulate the expression of human homeobox gene NKX3.1, which is thought to play an important role in prostate differentiation and to act as a tumor suppressor gene in the prostate.

Topics: Alitretinoin; Base Sequence; Cell Cycle; Cell Differentiation; Cell Line, Tumor; DNA Primers; Flow Cytometry; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tretinoin; Up-Regulation

NKX3.1 is a homeobox gene, expression of which is largely restricted to the adult prostatic epithelium. Loss of NKX3.1 expression has been linked to prostate carcinogenesis and disease progression and occurs in the absence of mutations in the coding region of the NKX3.1 gene. In this study, we have characterized regulation of NKX3.1 expression by all-trans retinoic acid (tRA), a naturally occurring vitamin A metabolite that is accumulated at high levels in the prostate. Using the prostate cancer cell line LNCaP, Western blot analysis revealed a approximately twofold induction of NKX3.1 protein levels following tRA exposure, with sequential analysis of NKX3.1 protein levels in cycloheximide co-treated cells indicating that tRA does not alter NKX3.1 protein turnover. The approximately 1.6-fold increase in NKX3.1 mRNA levels detected in tRA-treated LNCaP cells also occurred independently of new protein synthesis and was not mediated by changes in NKX3.1 mRNA stability. In contrast, nuclear run-on assays indicated that tRA treatment increased NKX3.1 transcription. To identify retinoid responsive regions of the NKX3.1 gene, DNA sequences encompassing approximately 2 kb of the NKX3.1 promoter or the entire 3'untranslated region (UTR) were cloned into luciferase reporter plasmids. Analysis of induced luciferase activity following transfection of these constructs into prostate cancer cells did not identify tRA responsiveness, however the 3'UTR was found to be strongly androgen responsive. These studies demonstrate that the NKX3.1 gene is a direct target of retinoid receptors and suggest that androgen regulation of NKX3.1 expression is mediated in part by the 3'UTR.

Topics: 3' Untranslated Regions; Antineoplastic Agents; Cell Line, Tumor; Gene Expression Regulation; Genes, Reporter; Homeodomain Proteins; Humans; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Retinoic Acid; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tretinoin

All-trans-retinoic acid (RA) regulates cellular growth, differentiation and apoptosis in human prostate by binding to RA receptors. Non-genomic retinoid effects on signal transduction kinases in the cytoplasm are also described in several cells but they are still unknown in prostate cells.. Using an epithelial cell line derived from normal human prostate (EPN), and normal (NPEC) and malignant (CPEC) epithelial primary cultures of human prostate, we have examined effects of RA on both extracellular signal-regulated kinase 1/2 (Erk1/2) and cAMP accumulation. Then we have verified the effect of the inhibition of Erk1/2 on RA-induced growth arrest and apoptosis in malignant cells.. In NPEC and in EPN treated with RA for up to 24 h, Western blot analyses of Erk1/2 phosphorylation show that RA causes a rapid activation of Erk1/2 within 5 min, which is maintained for 30 min, followed by a return to basal levels. In CPEC, the activated phosphorylation levels persist up to 24 h. While basal cAMP levels are not affected by 30 min treatment with RA in both EPN and NPEC, levels are increased in CPEC. Forskolin-induced cAMP levels are decreased by RA in all cell types. CPEC were incubated for up to 96 h with RA with and without the inhibitor of Erk1/2, UO126. CPEC incubated with RA and UO126 for 72 h showed a significant arrest of cell growth and after 96 h apoptosis in 11% of cells.. We show rapid effects of RA on cytoplasmic messenger pathways in human prostate, and that responses can differ between normal and malignant cells. The inhibition of these pathways could improve the efficiency of RA in prostate cancer growth control.

Topics: Aged; Aged, 80 and over; Apoptosis; Butadienes; Cell Division; Cyclic AMP; Enzyme Inhibitors; Epithelial Cells; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Prostate; Prostatic Neoplasms; Tretinoin; Tumor Cells, Cultured

Treatment of cultured PANC-1, MIA PaCa-2, and BxPC-3 human pancreatic adenocarcinoma cells with 0.1 to 1.6 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 96 h inhibited the proliferation of these cells in a dose-dependent manner, and PANC-1 and MIA PaCa-2 cells were more sensitive to TPA than BxPC-3 cells. Inhibition of proliferation by TPA in PANC-1 cells was associated with an increase in the level of p21, but this was not observed in MIA PaCa-2 or BxPC-3 cells. The TPA-induced increase of p21 in PANC-1 cells was blocked by bisindolylmaleimide or rottlerin (inhibitors of protein kinase C). Studies in NCr-immunodeficient mice with well established PANC-1 tumor xenografts indicated that daily i.p. injections of TPA strongly inhibited tumor growth, increased the percentage of caspase-3-positive cells, and decreased the ratio of mitotic cells to caspase-3-positive cells in the tumors. Studies with BxPC-3 tumors in NCr mice receiving daily i.p. injections of vehicle, TPA, all-trans retinoic acid (ATRA), or a TPA/ATRA combination showed that TPA had an inhibitory effect on tumor growth, but treatment of the animals with the TPA/ATRA combination had a greater inhibitory effect on tumor growth than TPA alone. Treatment with the TPA/ATRA combination resulted in a substantially decreased ratio of the percentage of mitotic cells to the percentage of caspase-3-positive cells in the tumors compared with tumors from the vehicle-treated control animals. The inhibitory effects of TPA on tumor growth occurred at clinically achievable blood levels.

Topics: Animals; Apoptosis; Body Weight; Cell Cycle; Cell Proliferation; Humans; Immunohistochemistry; Male; Mice; Neoplasm Transplantation; Paclitaxel; Pancreatic Neoplasms; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Retinoblastoma Protein; Sulindac; Tetradecanoylphorbol Acetate; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The human prostate gland is a well known source of H1 and H2 relaxin but information is lacking on the expression and potential role of the INSL3 peptide hormone within the prostate gland. In the present study we have investigated the expression of human INSL3 in patients with benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma tissues. Of the prostate epithelial cells, strongest INSL3 expression was detected in the basal epithelial cell compartment. Weaker INSL3 mRNA expression and immunoreactive INSL3 production were observed in secretory epithelial cells and in interstitial smooth muscle cells. Prostate epithelial cells were also a source for transcripts encoding the INSL3 receptor LGR8 suggesting the presence of an autocrine/paracrine INSL3-LGR8 ligand-receptor system within the human prostate. Three human prostate carcinoma cell lines displayed differential gene activity for INSL3 and LGR8. While LNCaP was devoid of INSL3, the androgen-insensitive PC-3 and the stromal prostate cell line hPCP co-expressed INSL3 and LGR8 transcripts. In addition to expressing INSL3 mRNA, the LGR8-negative DU-145 also expressed an INSL3 splice form formerly demonstrated in thyroid carcinoma cells. When incubated with recombinant INSL3, PC-3 cells showed significantly increased intracellular cAMP levels indicating functional LGR8 receptors. INSL3 did not alter the proliferative or metabolic activity of PC-3 carcinoma cells. Instead, PC-3 responded to INSL3 with significantly enhanced tumor cell motility and a transcriptional down-regulation of ErbB receptors and EGF. All-trans-retinoic acid was demonstrated in PC-3 to up-regulate LGR8 gene activity in a dose- and time-dependent manner while having no effect on INSL3 gene activity. In conclusion, we have identified a functional INSL3-LGR8 ligand-receptor system in human prostate carcinoma cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic AMP; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Insulin; Male; Prostatic Hyperplasia; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Proteins; Receptors, G-Protein-Coupled; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tretinoin

Prostate cancer is the most common cancer among men in the United States, and aberrant DNA methylation is known to be an early molecular event in its development. Here, we have used expression profiling to identify novel hypermethylated genes whose expression is induced by treatment of prostate cancer cell lines with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-dC). Of the 271 genes that were induced by 5-aza-dC treatment, 25 also displayed reduced expression in primary prostate tumors compared with normal prostate tissue, and the decreased expression of only one gene, aldehyde dehydrogenase 1 family, member A2 (ALDH1a2), was also associated with shorter recurrence-free survival. ALDH1a2 encodes an enzyme responsible for synthesis of retinoic acid (RA), a compound with prodifferentiation properties. By immunohistochemistry, we observed that ALDH1a2 was expressed in epithelia from normal prostate but not prostate cancer. Using bisulfite sequencing, we determined that the ALDH1a2 promoter region was significantly hypermethylated in primary prostate tumors compared with normal prostate specimens (P = 0.01). Finally, transfection-mediated reexpression of wild-type ALDH1a2 (but not a presumptive catalytically dead mutant) in the prostate cancer cell line DU145 resulted in decreased colony growth (P < 0.0001), comparable with treatment with either 5-aza-dC or RA. Taken together, our findings implicate ALDH1a2 as a candidate tumor suppressor gene in prostate cancer and further support a role of retinoids in the prevention or treatment of prostate cancer.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Azacitidine; Base Sequence; Cell Line, Tumor; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Isoenzymes; Male; Molecular Sequence Data; Neoplastic Stem Cells; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Retinal Dehydrogenase; Tretinoin

25-Hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) is an important inactivating enzyme and its expression is induced by 25-hydroxyvitamin D3 (25OHD3) and 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) through action of heterodimers of vitamin D receptor (VDR) and retinoid X receptor (RXR). RXRs also act as heterodimer partners for retinoic acid receptors (RARs), mediating the action of all-trans-retinoic acid (ATRA). Prostate stroma plays a crucial role in prostate cancer development and benign prostatic hyperplasia. We demonstrate here that ATRA markedly reduced the expression of 24-hydroxylase mRNA induced by 25OHD3 and 1alpha,25-(OH)2D3 in human prostatic stromal cells P29SN and P32S but not in epithelial cells PrEC or cancer cells LNCaP. By using transfection and RAR-selective ligands, we found that the inhibitory effect of ATRA on 24-hydroxylase expression in stromal cells was mediated by RARalpha but not by RARbeta. Moreover, the ATRA-induced expression of RARbeta was also mediated by RARalpha. The combined treatment of 1alpha,25-(OH)2D3 and RARalpha agonist Am80 at 10 nM exhibited strong growth-inhibitory effect whereas either alone had no effect. Our data suggest that ATRA suppresses 24-hydroxylase expression through RARalpha-dependent signaling pathway and can enhance vitamin D action in suppression of cell growth.

Topics: Benzoates; Calcitriol; Cell Line; Cell Line, Tumor; Cells, Cultured; Dibenzazepines; Epithelial Cells; Gene Expression Regulation; Humans; Male; Prostate; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Steroid Hydroxylases; Tetrahydronaphthalenes; Transfection; Tretinoin; Vitamin D3 24-Hydroxylase

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

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

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

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

Clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.16-0.32 nM) and all-trans-retinoic acid (ATRA; 0.5-1 micro M) had a synergistic inhibitory effect on the growth of cultured LNCaP prostate cancer cells, and apoptosis was markedly stimulated. In additional studies, NCr immunodeficient mice received s.c. injection with LNCaP cells in Matrigel. After 4-6 weeks, mice with well-established tumors received i.p. injection with vehicle, TPA (0.16 nmol/g body weight), ATRA (0.5 nmol/g body weight), or TPA+ATRA in vehicle once a day for 46 days. Tumor growth occurred in all of the vehicle-treated control mice. The percentage of animals with some tumor regression after 21 days of treatment was 0% for the control group, 31% for the ATRA group, 62% for the TPA group, and 100% for the TPA+ATRA group (13 mice/group). Although treatment of the mice with TPA or TPA+ATRA continued to inhibit tumor growth for the duration of the 46-day study, treatment of the mice with ATRA alone did not inhibit tumor growth beyond 28 days of daily injections (6 mice/group). Mechanistic studies indicated that treatment of the mice with TPA or TPA+ATRA for 46 days increased apoptosis in the tumors, and treatment with TPA+ATRA also decreased the mitotic index. Because the dose of TPA used in this study was effective and resulted in clinically achievable blood levels, clinical trials with TPA alone or in combination with ATRA in patients with prostate cancer may be warranted.

Topics: Animals; Apoptosis; Cell Division; Cell Line, Tumor; Drug Therapy, Combination; Male; Mice; Prostatic Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Human Kruppel-like factor 5 (hKLF5) is a transcription factor with a potential tumor suppressor function in prostate and breast cancers. In the majority of cancer samples examined, a significant loss of expression for KLF5 has been detected. Whereas hemizygous deletion appears to be responsible for KLF5's reduced expression in about half of the cases, the mechanism for reduction is unknown in the remaining half; gene promoter methylation does not appear to be involved. In this report, we studied the regulation of KLF5 and cloned and functionally characterized a 1944-bp fragment of the 5'-flanking region of the hKLF5 gene. Several mitogens as well as global demethylation induced the expression of KLF5, implicating multiple factors in the regulation of KLF5. KLF5's promoter lacks a TATA box and has a GC-rich region. Deletion mapping in combination with promoter activity assay showed that multiple cis-elements are involved in the transcriptional regulation of KLF5, some of which may play a repressor role whereas some others play an enhancer role. The Sp1 site between position -239 and -219 is essential for a basal promoter activity. Deletion or mutations of this Sp1 site significantly reduced promoter activity in several epithelial cell lines. Electrophoretic mobility shift assays (EMSAs) revealed that the Sp1 site binds Sp1 protein in nucleic extracts of different cell lines. In addition, overexpression of Sp1 protein transactivates KLF5 promoter activity. These findings suggest that Sp1 is a key transcription factor in KLF5's dynamic transcriptional regulation.

Topics: Angiotensin II; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cloning, Molecular; Conserved Sequence; DNA-Binding Proteins; Early Growth Response Protein 1; Electrophoretic Mobility Shift Assay; Epidermal Growth Factor; Epithelial Cells; Fibroblast Growth Factor 2; Gene Expression; HeLa Cells; Humans; Immediate-Early Proteins; Kruppel-Like Transcription Factors; Luciferases; Male; Metribolone; Mice; Molecular Sequence Data; Oligonucleotides; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; Rats; Recombinant Fusion Proteins; Rodentia; Sequence Deletion; Sequence Homology, Nucleic Acid; Sp1 Transcription Factor; Tetradecanoylphorbol Acetate; Trans-Activators; Transcription Factors; Transfection; Tretinoin; Tumor Necrosis Factor-alpha

All-trans retinoic acid (ATRA) promotes terminal differentiation in epithelial cells and anti-angiogenesis and thus, may have beneficial effects in an intervention therapy for prostate cancer.. We used the autochthonous spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model system to test the ability of ATRA to prevent initiation and progression of prostate cancer in a pre-clinical setting.. Initial studies demonstrated that exposure of TRAMP-derived C2N prostate tumor cells to ATRA in vitro decreased total viable cell numbers with a concomitant decrease in the fraction of cells in S phase. When TRAMP mice were treated in vivo with ATRA for either 6 or 8 weeks at low, medium, or high dose, mice on average presented with lower grade and more differentiated tumors. However, ATRA therapy conferred no significant protection on incidence of tumors or frequency of metastasis at any dose. Nevertheless, we were able to observe a significant decrease in the expression of synaptophysin, a marker of neuroendocrine differentiation, in tumors of mice receiving the highest dose of ATRA. As well, expression of the cell cycle inhibitor p21 was found to be elevated only in well-differentiated tumors of mice, treated with ATRA while expression of p27, was found to be elevated only in the poorly differentiated tumors.. Collectively, our in vitro and in vivo data demonstrates that ATRA was able to slow prostate tumor cell proliferation, induce apoptosis, and block the emergence of the neuroendocrine phenotype. Furthermore, our study suggests the differential regulation of p21 and p27 as a molecular mechanism whereby ATRA intervention therapy can inhibit the natural history of spontaneous prostate cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Disease Progression; Male; Mice; Prostatic Neoplasms; Tretinoin

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

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

To observe the synergistic effects of docetaxol and retinoic acid on prostate cancer cell line PC-3 in vitro and in vivo.. Cell morphology, MTT, flow cytometry, immunocytochemical method were used to observe the effects of 10(-6) mol/L, 10(-7) mol/L, 10(-8) mol/L docetaxol and 10(-5) mol/L, 10(-6) mol/L, 10(-7) mol/L retinoic acid on prostate cancer cell line PC-3 in single or synergistic administration ways for 24 and 48 hours in vitro. Male BALB/C-nu mice with PC-3 prostate cancer cell lines were treated by docetaxol and retinoic acid singly or synergistically in vivo. Serum PSA of mice, weights of mice and PSA expression in xenograft tumors of mice by immunohistochemical method were measured.. Above 10(-6) mol/L retinoic acid enhanced the growth suppression (suppression ratio > or = 69.2%, P<0.01), apoptosis (Apoptosis ratio > or = 23.8%, P<0.05) and down-regulation of the expression of cyclin D1 (expression ratio < or = 14.2%, P<0.05) induced by above 10(-7) mol/L docetaxol in PC-3 cells. Retinoic acid changed the ratio of G2/M phase induced by docetaxol from 70.3% to 54.6%, and reversed the G2/M arrest partially (P<0.05). Mean serum PSA of mice [(43+/-11) ng/ml], weight of xenograft tumors [(2.8+/-0.4) g] and PSA expression in tumors [(26+/-3.2)%] with PC-3 prostate cancer cell lines of nude mice were decreased significantly in docetaxol combined with retinoic acid group than in control group except weight of mice [(20.3+/-1.1) g].. Retinoic acid can enhance the growth suppression and apoptosis induced by docetaxol in synergistic way in vitro and in vivo, thus showing their great potential in the treatment of androgen-independent carcinoma of prostate.

Topics: Adult; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Docetaxel; Drug Synergism; Drugs, Chinese Herbal; Humans; Male; Mice; Mice, Inbred BALB C; Prostatic Neoplasms; Taxoids; Tretinoin

1,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is an effective agent for inhibiting the growth of prostate cancer cells including LNCaP and PC-3 cell lines. However, the extent of growth inhibition in these cell lines differs because LNCaP cells are much more responsive than PC-3 cells. Previous studies in LNCaP cells have shown that 1,25-(OH)(2)D(3) treatment results in G(0)/G(1) cell cycle accumulation, loss of Ki67 expression, and induction of apoptosis. One difference between the two cell lines is that PC-3 cells lack functional p53, a protein that plays roles both in cell cycle regulation and induction of apoptosis. In this study, the role of p53 in 1,25-(OH)(2)D(3) action was examined using the p53-negative PC-3 cells and a line of LNCaP cells, called LN-56, in which p53 function was shut off using a dominant negative p53 fragment. We found that treatment with 1,25-(OH)(2)D(3) extensively inhibits growth of LN-56 prostate cancer cells lacking p53, but in contrast to the parental LNCaP cells, the LN-56 cells recover rapidly. Moreover, in prostate cancer cells, the synergism between 1,25-(OH)(2)D(3) and 9-cis retinoic acid appears to be dependent on the presence of functional p53; however, 1,25-(OH)(2)D(3)-mediated induction of G(1) cell cycle accumulation and induction of apoptosis is not.

Topics: Alitretinoin; Apoptosis; Calcitriol; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; G1 Phase; Humans; Ki-67 Antigen; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Elevated expression levels of the bcl-2 proto-oncogene have been extensively correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t(14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer because there are no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2-expressing, androgen-independent prostate cancer cell line, by all-trans-retinoic acid treatment (ATRA), but this did not occur in the androgen-dependent cell line expressing low levels of Bcl-2. The decrease in the Bcl-2 protein and transcript following all-trans-retinoic acid treatment was accompanied by changes in localization of Par-4 and an induction in the expression of WT1 protein. In stable clones expressing ectopic Par-4 and in ATRA-treated cells, we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. The involvement of WT1 in the Par-4-mediated down-modulation of Bcl-2 was further defined by blocking endogenous WT1 expression, which resulted in an increase in Bcl-2 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1-binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1-binding site on the bcl-2 promoter. These data also identify Par-4 nuclear localization as a novel mechanism for ATRA-mediated bcl-2 regulation.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Base Sequence; Binding Sites; Carrier Proteins; DNA Primers; Down-Regulation; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; WT1 Proteins

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. The generation of immortalized primary prostate cancer cells that will accurately reflect the in situ characteristics of malignant epithelium is greatly needed. We have successfully established a neoplastic immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor. The RC-9 cells transduced through infection with a retrovirus vector expressing the E6 and E7 genes (E6E7) of human papilloma virus-16 (HPV-16) are currently growing well at passage 40, whereas RC-9 cells senesced at passage 7. RC-9/E6E7 cells exhibit epithelial morphology and high level of telomerase activity. More importantly, these immortalized cells produced tumors (SCID5038D) when inoculated into SCID mice. RC-9/E6E7 cells and SCID-5038D cells exhibit a high level of telomerase activity and androgen-responsiveness when treated with R1881. Expression of prostate specific antigen (PSA), androgen receptor (AR), prostate stem cell antigen (PSCA), an androgen-regulated prostate specific gene (NKX3.1), p16, cytokeratins 8, 15 and HPV-16 E6 gene was detected in both of these cells. RC-9/E6E7 and SCID5038D cells also showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1, potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes 2p, 3p, 8p, 13, 14, 16, 17, 18, 21 and the gain of 7 and 20 in the tumor cell line (SCID5038D). These results demonstrate that this primary tumor-derived HPE cell line retained its neoplastic phenotypes and its prostate-specific markers and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of a malignant AR and PSA positive established human prostate cancer cell line from a primary tumor of a prostate cancer patient.

Topics: Animals; Cell Culture Techniques; Cell Division; Epithelial Cells; Humans; Karyotyping; Male; Mice; Mice, SCID; Middle Aged; Prostate; Prostatectomy; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Telomerase; Transplantation, Heterologous; Tretinoin

PC-3 prostate cancer cell growth is inhibited by 1alpha,25-dihydroxyvitamin D(3) (1,25 D) and retinoids, but not to the same extent as the androgen receptor (AR) positive LNCaP prostate cancer cells. Previous reports suggest a role for AR in growth inhibition of LNCaP cells by 1,25 D and retinoids. PC-3 cells do not express AR. We therefore asked whether re-expression of AR would enhance the response of PC-3 cells to 1,25 D and retinoids.. PC-3 cells were stably transfected with wild type human AR cDNA. Pooled cells expressing AR protein at levels comparable to LNCaP cells were used to analyze response to 1,25 D, retinoids, androgens, and anti-androgens.. AR re-expression in PC-3 cells restored response to androgens and anti-androgens, but growth inhibition by 1,25 D was not significantly altered. However, cells were sensitized to low levels of retinoids, and, in contrast to the parental PC-3 cells, sub-optimal levels of 1,25 D and retinoids caused additive growth inhibition.. Restoring AR expression and activity in PC-3 cells results in enhanced sensitivity to low levels of retinoids while the response to 1,25 D remains unaltered. Thus, the involvement of AR in prostate cancer growth inhibition by 1,25 D appears to be cell line specific.

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Receptors, Androgen; Tretinoin; Tumor Cells, Cultured; Vitamin D

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.

Topics: Adenocarcinoma; Blotting, Northern; Blotting, Western; Cell Death; Gene Expression; Genetic Therapy; Humans; Iodides; Iodine Radioisotopes; Kinetics; Male; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Symporters; Transfection; Tretinoin; Tumor Cells, Cultured

We have developed a nontumorigenic epithelial cell line, DP-153, from the dorsal prostate of a Lobund/Wistar rat treated with N-methyl-N-nitrosourea and testosterone propionate. DP-153 cells express cytokeratins 5 and 14, but not cytokeratin 18, consistent with a basal epithelial cell phenotype. Similar to the nontumorigenic NRP-152 prostatic cell line, DP-153 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic cells. They express prostatic acid phosphatase and androgen receptors and require several mitogens (epidermal growth factor, insulin, dexamethasone, and cholera toxin) for sustained growth in culture under serum-containing conditions. DP-153 cells are also growth-stimulated by keratinocyte growth factor and basic fibroblast growth factor and growth-inhibited by all-trans-retinoic acid, 1,25-dihydroxyvitamin D(3), and transforming growth factor (TGF)-beta1. We demonstrate that expression of dominant-negative TGF-beta receptor type II by retroviral transduction of DP-153 cells leads to complete loss of TGF-beta1-induced growth inhibition. When transplanted s.c. in athymic mice, DP-153 cells expressing dominant-negative TGF-beta receptor type II form tumors as early as 4 weeks, in contrast to the vector control and parental cell line, which do not form tumors even 8 months after transplantation, supporting the observation that TGF-beta functions as a tumor suppressor in these cells. Our data further support that DP-153 is a suitable cell line for analysis of normal prostatic growth and carcinogenesis.

Topics: Animals; Calcitriol; Cell Division; Cell Transformation, Neoplastic; Growth Substances; Isoenzymes; Karyotyping; Keratins; Male; Mice; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type II; Receptors, Androgen; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The vitamin D(3) receptor, which is the nuclear receptor for 1alpha,25-dihydroxyvitamin D(3) (VD(3)), forms a heterodimer with the retinoid X receptor (RXR), which is the nuclear receptor for 9-cis-retinoic acid (9-cis-RA). The heterodimer binds to a specific response element consisting of two directly repeated pairs of motifs, AGGTGA, spaced by three nucleotides [direct repeat (DR) 3] and modulates the expression of VD(3)-responsive genes. Telomerase activity, which is seen in most immortal cells and germ cells, is a complex of enzymes that maintain the length of telomeres. One of the major components of human telomerase, human telomerase reverse transcriptase (hTERT), is the catalytic subunit, and the expression of hTERT might correlate most strongly with telomerase activity. We found that the sequence of 5'-AGTTCATGGAGTTCA-3' (DR3') is similar to that of DR3 in the promoter region of hTERT. Our results showed that the combination of VD(3) and 9-cis-RA inhibited telomerase activity through direct interaction of the heterodimer of vitamin D(3) receptor and RXR with the DR3' sequence in the hTERT promoter as well as the combination of VD(3) and selective RXR ligand did. Also, in vivo data showed that the growth of xenografts in nude mice was inhibited by VD(3) and 9-cis-RA. The results of the present study provide evidence on the molecular mechanism of the inhibition of cell growth by these agents, and they could be novel therapeutic agents for prostate cancer.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Calcitriol; DNA-Binding Proteins; Drug Synergism; Humans; Male; Mice; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Telomerase; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In most previous studies, the incidence and multiplicity of chemically induced prostate tumors have been used as end points for assessing the efficacy of various chemopreventive agents. In this study, we used prostate intraepithelial neoplasia (PIN) in Noble rats as an intermediate end point to examine the chemopreventive efficacy of two retinoids, 9-cis-retinoic acid (9cRA) and 4-(hydroxyphenyl)retinamide, which in previous studies have shown promising inhibitory effects on various carcinogenesis models. We found that 80-100% of Noble rats treated for 36 weeks with testosterone + 17beta-estradiol developed multiple PIN lesions predominantly in the dorso-lateral prostate, which appears relevant to the place of origin of PIN and carcinoma in the human prostate. 9cRA at 50 or 100 mg/kg diet significantly decreased the multiplicity of PIN, whereas 4-(hydroxyphenyl) retinamide at 392 or 784 mg/kg diet, did not have an inhibitory effect on PIN. Thus, we provide for the first time evidence that the testosterone + 17beta-estradiol-induced PIN in Noble rats could be used as a potential intermediate end point in assessing the efficacy of retinoids and possibly of other agents on prostate carcinogenesis, and that 9cRA alone or in combination with other agents may have clinical promise in preventing the development of prostate cancer in men.

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Body Weight; Estradiol; Fenretinide; Male; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Rats; Testosterone; Tretinoin

Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may be applied to the treatment of neoplasms in a variety of organs. In order to enhance existing regimens of photodynamic therapy, we investigated the effects of adding differentiation therapy to photodynamic therapy in human prostate cancer cells in vitro. The objective of differentiation therapy per se is to reverse the lack of differentiation in cancer cells using pharmacological agents. The motivation for this study was to exploit the differentiation-dependent expression of some heme enzymes to enhance tumour cell toxicity of ALA-photodynamic therapy. A short course of differentiation therapy was applied to increase PpIX formation during subsequent ALA exposure. Using the synthetic androgen R1881, isomers of retinoic acid, and analogues of vitamin D for 3 to 4 days, exogenous ALA-dependent PpIX formation in LNCaP cells was increased, along with markers for growth arrest and for differentiation. As a consequence of higher PpIX levels, cytotoxic effects of visible light exposure were also enhanced. Short-term differentiation therapy increased not only the overall PpIX production but also reduced that fraction of cells that contained low PpIX levels as demonstrated by flow cytometry and fluorescence microscopy. This study suggests that it will be feasible to develop protocols combining short-term differentiation therapy with photodynamic therapy for enhanced photosensitisation.

Topics: Aminolevulinic Acid; Cell Differentiation; Flow Cytometry; Humans; Male; Metribolone; Photochemotherapy; Photosensitizing Agents; Prostatic Neoplasms; Protoporphyrins; Testosterone Congeners; Tretinoin; Tumor Cells, Cultured; Vitamin D

Recent studies from our laboratory have indicated that the metabolism of vitamin A (retinol) to retinyl esters, carried out primarily by the enzyme lecithin:retinol acyltransferase (LRAT), is greatly reduced in human carcinoma cell lines of the oral cavity, skin, breast, and kidney as compared with their normal epithelial counterparts. These studies suggest that human carcinoma cells are retinoid-deficient relative to normal epithelial cells. In this study, we examined the metabolism of [(3)H]retinol and [(3)H]retinoic acid (RA) in human prostate cancer lines and in primary cultures of human prostate epithelial cells. Normal cells esterified all of the [(3)H]retinol added to the cultures. In contrast, all seven prostate cancer cell lines and four primary cultures derived from prostatic adenocarcinomas metabolized only trace amounts of [(3)H]retinol to [(3)H]retinyl esters. Correlated with this relative lack of esterification of [(3)H]retinol by the cancer cells was loss of expression of LRAT protein, whereas normal cells expressed abundant levels of LRAT protein by Western analysis. The metabolism of [(3)H]RA was also examined in these prostatic cells. Two of the prostate cancer tumor lines, DU 145 and PJ-1, exhibited rapid metabolism of [(3)H]RA; in contrast, the other tumor lines or primary cultures metabolized [(3)H]RA at a much slower rate. We also found that the immortalization of normal human prostatic epithelial cells by SV40 T antigen led to a reduction in LRAT protein expression and esterification of [(3)H]retinol. Further transformation to tumorigenicity with the ras oncogene resulted in loss of detectable LRAT expression. Finally, we analyzed LRAT protein expression in tissue sections from six prostatectomy specimens by immunohistochemistry. LRAT protein was predominantly expressed in the basal cells of normal prostatic epithelium, whereas its expression was lost in prostate cancer. Collectively, these data implicate aberrant retinoid metabolism in the process of prostatic carcinogenesis.

Topics: Acyltransferases; Antineoplastic Agents; Cell Division; Epithelial Cells; Humans; Male; Prostatic Neoplasms; Tretinoin; Tumor Cells, Cultured; Vitamin A

Modulation of the expression of retinoic acid receptors (RAR) alpha and gamma in adult rat prostate by testosterone (T) suggests that RAR signaling events might mediate some of the androgen effects on prostate cells.. In this study, we examined the interactions between T and retinoic acid (RA) in cell growth of human prostate carcinoma cells, LNCaP, and their relationship with the expression of RAR and epidermal growth factor receptor (EGF-R).. Both T and RA, when administered alone, stimulated 3H-thymidine incorporation in LNCaP cells in a dose-dependent manner; the effect of each agent was reciprocally attenuated by the other agent. Testosterone treatment of LNCaP cells also resulted in dose dependent, biphasic increases in RAR alpha and gamma mRNAs; increases paralleled that of 3H-thymidine incorporation and were attenuated by the presence of 100 nM RA. These results suggest a link between RAR signaling and the effect of T on LNCaP cell growth. Gel electrophoretic mobility shift assays revealed the presence of putative androgen responsive element (ARE) in the promoter region of RAR alpha gene, suggesting that a direct AR-DNA interaction might mediate the effects of T on RAR alpha gene. Furthermore, treatment of LNCaP cells with 20 nM T resulted in an increase in EGF-R. In contrast, EGF-R was suppressed by 100 nM RA that also suppressed the effect of T.. Current results demonstrate interactions between T and RA in the expression of RARs and cell growth in LNCaP cells. The presence of putative ARE in the promoter of the RAR alpha gene suggests that AR-DNA interaction might mediate the effects of T on RAR alpha gene. The opposite effects of T and RA on the expression of RAR and EGF-R suggest that signal events of these receptors might be involved in the interaction between T and RA in the control of LNCaP cell growth.

Topics: Dose-Response Relationship, Drug; Drug Interactions; Electrophoretic Mobility Shift Assay; ErbB Receptors; Humans; Male; Prostate; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; RNA, Messenger; Testosterone; Thymidine; Tretinoin; Tumor Cells, Cultured

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

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

We report that all- trans retinoic acid (ATRA) enhanced the toxicity of docetaxel against DU145 and LNCaP prostate cancer cells, and that the nature of the interaction between ATRA and docetaxel was highly synergistic. Docetaxel-induced apoptotic cell death was associated with phosphorylation and hence inactivation of Bcl-2. ATRA enhanced docetaxel-induced apoptosis and combined treatment with ATRA and docetaxel resulted in down-regulation of Bcl-2. Docetaxel caused phosphorylation and hence inactivation of cdc2 kinase result ing in G2/M arrest. ATRA inhibited docetaxel-induced phosphorylation of cdc2 resulting in activation of cdc2 kinase and partial reversal of the G2/M arrest. ATRA also inhibited docetaxel-induced activation of MAPK indicating that the effects of docetaxel and ATRA on cdc2 phosphorylation are dependent on MAPK. We conclude that ATRA synergistically enhances docetaxel toxicity by down-regulating Bcl-2 expression and partially reverses the docetaxel-induced G2/M arrest by inhibiting docetaxel-induced cdc2 phosphorylation in a pathway that is dependent on MAPK.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cyclin D1; Docetaxel; Down-Regulation; Drug Interactions; Humans; Male; Paclitaxel; Prostatic Neoplasms; Taxoids; Tretinoin; Tumor Cells, Cultured

Novel synthetic antagonists of retinoic acid receptors (RARs) have been developed. To avoid interference by serum retinoids when testing these compounds, we established serum-free grown sub-lines (>3 years) of the prostate carcinoma lines LNCaP, PC3 and DU145. A high affinity pan-RAR antagonist (AGN194310, K(d) for binding to RARs = 2-5 nM) inhibited colony formation (by 50%) by all three lines at 16-34 nM, and led to a transient accumulation of flask-cultured cells in G1 followed by apoptosis. AGN194310 is 12-22 fold more potent than all-trans retinoic acid (ATRA) against cell lines and also more potent in inhibiting the growth of primary prostate carcinoma cells. PC3 and DU145 cells do not express RARbeta, and an antagonist with predominant activity at RARbeta and RARgamma (AGN194431) inhibited colony formation at concentrations (approximately 100 nM) commensurate with a K(d)value of 70 nM at RARgamma. An RARalpha antagonist (AGN194301) was less potent (IC(50) approximately 200 nM), but was more active than specific agonists of RARalpha and of betagamma. A component(s) of serum and of LNCaP-conditioned medium diminishes the activity of antagonists: this factor is not the most likely candidates IGF-1 and EGF. In vitro studies of RAR antagonists together with data from RAR-null mice lead to the hypothesis that RARgamma-regulated gene transcription is necessary for the survival and maintenance of prostate epithelium. The increased potencies of RAR antagonists, as compared with agonists, suggest that antagonists may be useful in the treatment of prostate carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Benzoates; Cell Cycle; Humans; Male; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoids; Thiophenes; Tretinoin; Tumor Cells, Cultured

Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells. 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5. 957E/hTERT cells exhibit epithelial morphology. Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells. Prostatic stem cell antigen and p16 were also expressed in this line. 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth. Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range. However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q. The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20. The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes. This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient.

Topics: Adenocarcinoma; Adult; Cell Division; DNA-Binding Proteins; Growth Inhibitors; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Male; Prostatic Neoplasms; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Telomerase; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

We report the first case of granulomatous tubulointerstitial nephritis induced by all-trans retinoic acid (ATRA) in a patient with acute promyelocytic leukemia (APL). Acute renal failure during treatment with ATRA has been previously reported as a part of an ATRA syndrome or a thrombotic complication of a hypercoagulable state. This case indicates an alternative mechanism of acute renal failure occurring during ATRA therapy.

Topics: Aged; Benzamidines; Dalteparin; Drug Administration Schedule; Gabexate; Guanidines; Humans; Idarubicin; Kidney Neoplasms; Leukemia, Promyelocytic, Acute; Male; Nephritis, Interstitial; Prostatic Neoplasms; Thrombophilia; Tretinoin

CD178 (CD95-ligand) is expressed on several tumor cells and likely influences the interaction of the tumor with the host immune system. However, little is known about the mechanisms that regulate its expression on the cell surface. We have evaluated the ability of various compounds and cytokines to regulate cell surface expression and release of soluble CD178 in various carcinoma cell lines. Vitamin E succinate (VES) and retinoic acid (RA) were found to reduce CD178 surface expression, whereas interferon-gamma stimulated a slight upregulation. At 48 h, the regulation of surface CD178 by VES and RA arose from a small decrease in CD178 mRNA and to a greater extent due to an increase in the release of soluble CD178; the latter was blocked by addition of a metalloproteinase inhibitor. Accordingly, VES and RA treatment diminished the ability of tumor cells to kill CD95-sensitive cells and this effect was markedly reduced by the presence of a metalloproteinase inhibitor. Our results indicate that, in vitro, CD178 expression on the cell surface of tumor cells can be regulated by agents that alter both expression and release of the ligand. In vivo, such treatments may play an important role in the outcome of tumor sensitivity or resistance to host immune mechanisms.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Coculture Techniques; Fas Ligand Protein; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Immune System; Jurkat Cells; Lung Neoplasms; Male; Membrane Glycoproteins; Ovarian Neoplasms; Prostatic Neoplasms; RNA, Messenger; Tretinoin; Vitamin E

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. We have successfully established an immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor with telomerase. The actively proliferating early passaged RC-58T cells were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase was detected in RC-58T/hTERT cells but not RC-58T cells. RC-58T/hTERT cells are currently growing well at passage 50, whereas RC-58T cells senesced at passage 7. RC-58T/hTERT cells exhibit transformed morphology. More importantly, these immortalized cells showed anchorage-independent growth as they formed colonies in soft agar and grew above the agar layer. Expression of androgen-regulated prostate specific gene NKX3.1 and epithelial specific cytokeratin 8 (CK8) but not prostate specific antigen (PSA) and androgen receptor was detected in RC-58T/hTERT cells. Prostate stem cell antigen (PSCA) and p16 were also expressed in this cell line. RC-58T/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1 known potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes Y, 3p, 10p, 17p, 18q and the gain of chromosomes 16 and 20. These results demonstrate that this primary tumor-derived HPE cell line retained its transformed phenotypes and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of an established human prostate cancer cell line from a primary tumor of a prostate cancer patient with telomerase.

Topics: Agar; Cell Culture Techniques; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA-Binding Proteins; Epithelial Cells; Humans; Karyotyping; Male; Models, Biological; Prostatic Neoplasms; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Telomerase; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.

Topics: Actins; Adenocarcinoma; Animals; Antineoplastic Agents; Cell Differentiation; Cell Movement; Cytoskeleton; Eflornithine; Male; Microscopy, Confocal; Neoplasm Metastasis; Phenotype; Prostatic Neoplasms; Rats; Rats, Inbred Strains; Tretinoin; Tumor Cells, Cultured

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

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

Retinoic acid (RA) is described as an inhibitor of prostate cancer cell growth. We utilized reverse transcription-polymerase chain reaction (RT-PCR) to analyze expression of different isoforms of fibroblast growth factor 8 (FGF8) in response to RA. Results in the prostate cancer cell line LNCaP show that whereas overall expression levels of FGF8 appear to remain constant, RA addition induces an inversion of the ratio between FGF8a and -b mRNAs. Along with this observed "isoform switch," unexpected expression of retinoic acid receptor alpha was detected. Although preliminary, these data allow one to hypothesize on the existence of a possible link between the morphogenic hormone RA and the regulation of the potent mitogen FGF8.

Topics: Base Sequence; DNA Primers; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression; Humans; Male; Prostatic Neoplasms; Protein Isoforms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Onconase (Onc) is a ribonuclease from amphibian oocytes that is cytostatic and cytotoxic to many tumour lines. It shows in vivo antitumour activity in mouse tumour models and is currently in Phase III clinical trials. The present study was designed to test whether cytotoxic effects of ONC can be modulated by differentiating agents. Human leukaemic HL-60 and prostate cancer LNCaP and JCA-1 cells were treated with Onc in the absence and presence of several inducers of differentiation and frequency of apoptosis was assessed using three different cytometric methods and confirmed by analysis of cell morphology. A moderate degree of apoptosis observed after 48-72 h incubation of HL-60 cells in the presence of 0.42 microM Onc alone was markedly potentiated by administration of retinoic acid (all trans), sodium butyrate or dimethylsulfoxide at concentrations known to induce differentiation but be minimally cytotoxic. Likewise, the frequency of apoptosis of LNCaP and JCA-1 cells treated with Onc was increased in the cultures to which phenylbutyrate was added. Although cell treatment with Onc alone, with each of the differentiating agents alone or with Onc in combination with the differentiating agents led to an increase in the proportion of G1 cells, no specific cell cycle phase preference in induction of apoptosis was observed. The data suggest that cells undergoing differentiation are particularly vulnerable to Onc; a combination of Onc and differentiating agents should be considered for further in vivo tests to assess its possible usefulness in the clinic.

Topics: Antineoplastic Agents; Apoptosis; Butyrates; Cell Differentiation; Dimethyl Sulfoxide; DNA; Drug Synergism; Egg Proteins; HL-60 Cells; Humans; Male; Phenylbutyrates; Prostatic Neoplasms; Ribonucleases; RNA; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and its natural and synthetic analogs, the retinoids, regulate many biological processes, including development, differentiation, cell growth, morphogenesis, metabolism and homeostasis. Retinoid effects are mediated by specific nuclear receptors, the RARs and RXRs. Because of their ability to control cell growth and induce differentiation, retinoids are being examined for the prevention and treatment of several cancers. The majority of retinoids so far analyzed and available inhibit primarily cell proliferation and tumor progression but cannot eliminate cancer cells. In addition, the beneficial effects of the natural retinoids are undermined by undesirable side effects, possibly due to indiscriminate activation of all retinoid receptor subtypes and response pathways. Here, we show that a synthetic retinoid, CD-271, that activates selectively the RAR gamma subtype in a given context, shows increased anti-proliferative activity against certain carcinoma cells over all-trans-retinoic acid (tRA). CD-271 exhibits enhanced activity against DU-145 prostate adenocarcinoma cells through apoptosis-inducing activity, while tRA does not. The selective anti-cancer cell action appears to be receptor-mediated as an RAR antagonist reverses the inhibition. This profile was not seen with other selective retinoids, such as RAR alpha-selective agonists, anti-AP-1 compounds and a non-apoptosis inducing RAR gamma agonist. Our data point to a specific role for RAR gamma in controlling the growth of the prostate, consistent with previous RAR gamma gene knockout data. The identified retinoid represents a new class of compounds with potential for the treatment of prostate cancer.

Topics: Androgens; Apoptosis; Cell Division; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Response Elements; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

A chemoprevention study was conducted to evaluate the activity of 9-cis-retinoic acid (9-cis-RA) as an inhibitor of prostate carcinogenesis in male Wistar-Unilever (HsdCpb:Wu) rats. After pretreatment with a sequential regimen of cyproterone acetate (50 mg/kg/day for 21 days) and testosterone propionate (100 mg/kg/day for 3 days), groups of 40 rats received a single i.v. injection of N-methyl-N-nitrosourea (MNU; 30 mg/kg body weight). Beginning 2 weeks after carcinogen administration, rats received chronic exposure to testosterone administered in s.c. implanted silastic capsules. The study was terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. Continuous dietary administration of 9-cis-RA at 100 mg/kg diet or 50 mg/kg diet beginning 1 week before MNU administration reduced cancer incidence in the dorsolateral + anterior prostate from 65% in dietary controls to 18 and 20%, respectively (P < 0.001 for both comparisons). Similarly, these dose levels of 9-cis-RA reduced the incidence of cancer in all accessory sex glands from 79% in dietary controls to 48 and 33% (P < 0.01 for both comparisons), respectively. Chronic dietary administration of 9-cis-RA induced no gross or organ-specific toxicity in any animal and did not suppress group mean body weight gain. The potent anticarcinogenic activity of 9-cis-RA in the rat prostate, when considered with its apparent lack of toxicity in rodents, suggests that this and other ligands for the retinoid X receptor merit consideration for evaluation in clinical prostate cancer chemoprevention trials.

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Carcinogens; Cyproterone Acetate; Dose-Response Relationship, Drug; Male; Methylnitrosourea; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Rats; Rats, Wistar; Testosterone; Tretinoin

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.

Topics: Alitretinoin; Calcitriol; Cell Division; Cycloheximide; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Protein Synthesis Inhibitors; Receptors, Androgen; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Androgen ablation-induced prostate cancer regression is transient and ends with the regrowth of androgen-independent (AI) tumors. To mimic this evolution in culture, we chronically deprived an androgen-dependent (AD) prostate cancer cell line (LNCaP) of androgen, generating an AI derivative which retained limited hormone proliferative responsiveness and a barely detectable prostate-specific antigen (PSA) mRNA level. While the cytokeratin 8 (CK8) level was low, the androgen receptor (AR) protein in AI cells was on average tenfold greater than in AD cells. When challenged for susceptibility to undergo apoptosis, the AI cells were more resistant than AD cells to all-trans retinoic acid (tRA) and two chemotherapeutic agents, Taxol and Adriamycin, requiring higher doses and longer periods of treatment to achieve similar effects. Compared to AD cells, the partially apoptosis-resistant AI cells expressed four times more Bcl-2 protein and undetectable levels of p21/WAF1. Induction of apoptosis by tRA in both cell types did not affect their expression but was preceded by the activation of Rb and a pronounced reduction of AR protein level. The kinetics of the Rb activation and AR downmodulation in both cell types matched their tRA sensitivity, suggesting that these events may be required for tRA-induced apoptosis. The results show that the apoptotic pathway in AI cells, although more difficult to induce, is not irrevocably lost and that targeted reduction of the AR protein level with retinoids in combination with androgen ablation therapy may prolong remissions in advanced prostate cancer patients.

Topics: Antigens, Surface; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carboxypeptidases; Cell Differentiation; Cell Division; Dihydrotestosterone; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glutamate Carboxypeptidase II; Growth Inhibitors; Humans; Keratins; Male; Paclitaxel; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Retinoblastoma Protein; Tretinoin; Tumor Cells, Cultured

The high incidence and long latent period of prostate cancer make it an ideal target for chemoprevention. We have evaluated a series of agents for chemopreventive efficacy using a model in which hormone-dependent prostate cancers are induced in the Wistar-Unilever (WU) rat by sequential treatment with antiandrogen (cyproterone acetate), androgen (testosterone propionate), and direct-acting chemical carcinogen (N-methyl-N-nitrosourea), followed by chronic androgen stimulation (testosterone). This regimen reproducibly induces prostate cancers in high incidence, with no gross toxicity and a low incidence of neoplasia in the seminal vesicle and other non-target tissues. Dehydroepiandrosterone (DHEA) and 9-cis-retinoic acid (9-cis-RA) are the most active agents identified to date. DHEA inhibits prostate cancer induction both when chronic administration is begun prior to carcinogen exposure, and when administration is delayed until preneoplastic prostate lesions are present. 9-cis-RA is the most potent inhibitor of prostate carcinogenesis identified; a study to determine the efficacy of delayed administration of 9-cis-RA is in progress. Liarozole fumarate confers modest protection against prostate carcinogenesis, while N-(4-hydroxyphenyl)retinamide (fenretinide), alpha-difluoromethylornithine, oltipraz, DL-alpha-tocopherol acetate (vitamin E), and L-selenomethionine are inactive. Chemoprevention efficacy evaluations in the WU rat will support the identification of agents that merit study for prostate cancer chemoprevention in humans.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Dehydroepiandrosterone; Disease Models, Animal; Immunohistochemistry; Male; Neoplasms, Hormone-Dependent; Prostate; Prostatic Neoplasms; Rats; Rats, Wistar; Treatment Outcome; Tretinoin

1,25 dihydroxyvitamin D3 (1,25D) and retinoids may play an important role in preventing progression of prostate cancer.. We examined the ability of four novel 20-epi-vitamin D3 analogs (CB1093, KH1060, KH1266, and CB1267), either alone or in combination with 9-cis retinoic acid (RA) to inhibit colony growth of a human prostate cancer cell line, LNCaP, using soft agar as well as bone marrow stroma. Also, the effect of these analogs on the cell cycle and expression of Ki-67, p21(waf-1), and p27(kip1) in LNCaP cells was examined.. The analog CB1267 was the most potent, with 8 x 10(-10) M of the analog inhibiting 50% colony growth (ED50) of LNCaP. 9-cis-RA also inhibited colony growth of LNCaP (ED50, 5 x 10(-7) M). Combined, CB1267 and 9-cis-RA synergistically inhibited colony growth and significantly increased the number of LNCaP cells in G0/G1 phase. Cell cycle arrest was associated with increased levels of p21(waf-1) and p27(kip1) and decreased expression of Ki-67 protein. Pulse-exposure to this combination (5 x 10(-8) M) irreversibly inhibited colony growth, both in soft agar and on normal human bone marrow stroma.. Combination of a new vitamin D3 analog (CB1267) and a retinoid (9-cis-RA) potently inhibited colony formation of LNCaP prostate cancer cells in vitro, suggesting further studies in animal models. This combination may afford an interesting therapeutic approach to low-burden prostate cancer.

Topics: Alitretinoin; Antineoplastic Agents; Bone Marrow Cells; Calcitriol; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Drug Synergism; Humans; Male; Microtubule-Associated Proteins; Prostatic Neoplasms; Stromal Cells; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

The secosteroid hormones, all-trans- and 9-cis-retinoic acid and vitamin D3, have demonstrated significant capacity to control proliferation in vitro of many solid tumour cell lines. Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti-AP-1 retinoid [2-(4,4-dimethyl-3,4-dihydro-2H-1 benzopyran-6-yl)carbonyl-2-(4-carboxyphenyl)-1,3,-dithiane; SR11238], which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3 analogue [1alpha,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3, code name LH] together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Cadherins; Cell Division; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Male; Prostatic Neoplasms; Retinoids; Tretinoin; Tumor Cells, Cultured

Recent in vivo and in vitro studies suggest that retinoic acid receptor (RAR)-mediated processes may be involved in androgen regulation of prostate cells in a manner that may be altered during prostatic carcinogenesis. We tested this hypothesis in the newly established carcinoma and non-carcinoma rat prostate epithelial cell lines, NRP-154 and NRP-152, respectively. In DMEM/F-12 medium supplemented with 10% charcoal stripped fetal calf serum (cFCS), the number of both NRP-152 and NRP-154 cells were stimulated by testosterone (T), with a 4-fold greater effect in NRP-152 than in NRP-154 cells. Retinoic acid (RA) alone also stimulated the growth of NRP-152 cells, but failed to induce cell growth of NRP-154 cells. Importantly, the level of RAR alpha mRNA was elevated whereas the levels of RAR gamma and androgen receptor (AR) mRNA were lower in NRP-154 cells compared to those in NRP-152 cells. Treatment of NRP-152 cells with increasing doses of T resulted in a dose-dependent decrease and rebound of the level of RAR alpha and gamma mRNA in NRP-152 cells; these effects were not apparent, if not reversed, in NRP-154 cells. Both ligand binding and Western blot analyses revealed that epidermal growth factor receptor (EGF-R) was stimulated by 20 nM T but was suppressed by 0.1 microM RA, which also attenuated the stimulating effects of T on EGF-R in NRP-152 and to a lesser extent in NRP-154 cells. The differences in the level and androgen regulation of RAR mRNAs and reciprocal regulation of EGF-R expression by T and RA between NRP-154 and NRP-152 cells suggest that variations in the EGF-R and RAR signal events may contribute to differences in growth rate between these two cell lines.

Topics: Animals; Blotting, Western; Cell Division; Cell Line; Culture Media; DNA; ErbB Receptors; Gene Expression; Male; Prostate; Prostatic Neoplasms; Rats; Receptors, Retinoic Acid; RNA, Messenger; Testosterone; Tretinoin; Tumor Cells, Cultured

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Topics: Adenocarcinoma; Calcitriol; Cell Division; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Male; Metribolone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Retinoids; Testosterone Congeners; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Hormonally insensitive prostate cancer is a relatively slow-growing, but usually fatal, disease with no long-term treatment options. Transformation of normal prostate cells to a malignant phenotype often involves corruption of the apoptotic machineries. Bcl-2 protein is one of the key inhibitors of apoptosis and is often unregulated in advanced prostate cancer. The prostate cancer cell line DU-145 was used as a model of a hormonally insensitive, advanced prostate cancer. Cell growth in liquid culture was significantly inhibited by antisense Bcl-2 oligonucleotides compared with control sense oligonucleotides; inhibition by these oligonucleotides was significantly enhanced on combination with the synthetic retinoid N-(2-hydroxyphenyl)all-trans-retinamide (2-HPR). Interestingly, growth inhibition occurred in the absence of apoptosis as measured using two assay techniques. We hypothesize that in these recalcitrant cells the apoptotic pathway is compromised at several levels, and Bcl-2 may play another role in promoting cell growth. The use of Bcl-2 antisense oligonucleotides plus 2-HPR may provide a novel approach to therapy of hormone-resistant prostate cancer.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Growth Inhibitors; Humans; Male; Neoplasm Proteins; Oligonucleotides, Antisense; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

Troglitazone, a thiazolidinedione derivative, is a widely used antidiabetic drug that binds and activates peroxisome proliferator-activated receptor gamma (PPARgamma) and enhances insulin sensitivity. It induces differentiation of adipocytes, which highly express PPARgamma. We report that human prostate cancer cells expressed PPARgamma at prominent levels and normal prostate tissues had very low expression. Dose-response clonogenic assays of the PC-3 prostate cancer cell line with troglitazone showed an antiproliferative effect (ED50, 3 x 10(-7) M) and other PPARgamma ligands (BRL49653: ED50, 8 x 10(-8) M; 15-deoxy-delta12,14-prostaglandin J2: ED50, 2 x 10(-6) M; ciglitizone: ED50, not reached; indomethacin: ED50, not reached) showed similar effects. Combinations of troglitazone and a ligand specific for either retinoid X receptor or retinoic acid receptor did not show a synergistic effect. Pulse-exposure to troglitazone (10(-5) M) for different durations showed that 4 days of pulse-exposure to the agent irreversibly inhibited 50% clonal growth of PC-3 cells. Interestingly, PC-3 cells cultured with troglitazone (10(-5) M) showed dramatic morphological changes both by light and electron microscopy, suggesting that the cells became less malignant. Nevertheless, troglitazone did not affect either the cell cycle or several markers of differentiation. LNCaP cells constitutively produced prostate-specific antigen, and levels were markedly enhanced by all-trans-retinoic acid. Troglitazone (10(-5) M, 4 days) decreased by 50% the levels of prostate-specific antigen produced by these cells. In vivo treatment of PC-3 tumors growing in male BNX triple immunodeficient mice with oral troglitazone (500 mg/kg/day) produced significant inhibition of tumor growth (P = 0.01). The only objective side effect of troglitazone in mice was the elevation of serum transaminases. Short-term culture of four surgically obtained human prostate cancer tumors with troglitazone (10(-5) M, 4 days) produced marked and selective necrosis of the cancer cells (about 60%) but not the adjacent normal prostate cells. Taken together, these results suggest that troglitazone may be a useful therapeutic agent for the treatment of prostate cancer, especially in the setting of low disease burden.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Differentiation; Chromans; Drug Synergism; Humans; Ligands; Male; Mice; Mice, Nude; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin; Troglitazone; Tumor Cells, Cultured

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.

Topics: Alitretinoin; Calcitriol; Cell Division; DNA-Binding Proteins; Drug Synergism; G1 Phase; Humans; Male; Nuclear Proteins; Prostate; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) is recognized as an inhibitor of tumorigenesis, but conversely, has also been shown to act as a tumor enhancer, therefore its role in prostate tumor cell growth was investigated.. The response of two human prostate tumor cell lines (PC-3 and DU-145), and cell lines derived from a well-differentiated canine prostate adenocarcinoma (CPA) and normal canine prostate epithelium (CAPE) to all-trans RA was determined using growth curve analysis. Additionally, the constitutive expression and RA-challenged expression of retinoic acid receptors (RARs) -alpha, -beta, and -gamma mRNA was examined using Northern blotting techniques.. In response to all-trans RA, the PC-3 and DU-145 cell lines showed considerable growth promotion, while CAPE and CPA cell growth was dramatically inhibited. Each cell line expressed RAR alpha and RAR gamma, with either negligible or no RAR beta transcripts being detected. RAR alpha and -gamma mRNAs detected in the four cell lines were variably regulated in response to RA, and no distinct patterns of RAR regulation that could be related to cell growth responses were observed.. The data indicates that no simple association exists between the expression or regulation of RAR subtype mRNAs and the divergent growth responses to RA displayed by the prostate cell lines.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Line; DNA Probes; Dogs; Dose-Response Relationship, Drug; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Prostate; Prostatic Neoplasms; Receptors, Retinoic Acid; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

Management of prostate cancer that either is detectable by prostate specific antigen (PSA) measurements after curative intent or has spread outside of its capsule is a serious problem. Innovative, nontoxic approaches to the disease are required. One approach might be therapy with retinoids. Retinoid activities are mediated by two distinct families of transcription factors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 that usually mediates cellular proliferative signals. The RARs require heterodimerization with RXRs. RXRs can form either heterodimers or homodimers; and the latter can bind to DNA response elements that are distinct from those bound by the RAR/RXR heterodimers.. A series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers or RAR/RXR heterodimers, or that selectively inhibit AP-1 activity without activating transcription were evaluated for their ability to inhibit clonal growth of three human prostate cancer cell lines (PC-3, DU-145, and LNCaP).. Several notable findings were: 1) RXR-selective retinoids, such as SR11246, were able to inhibit the clonal growth of prostate cancer cells. In contrast, SR11246 had little effect on clonal growth of myeloid leukemic cells. 2) RAR-selective retinoids also inhibited clonal growth of prostate cancer cells. 3) The retinoid (SR11238) with potent anti-AP-1 activity had no effect on the clonal growth of prostate cancer cells.. This study shows that both RXR- and RAR-selective retinoids are worthy of further study and may be candidates for future clinical trials in prostate cancer.

Topics: Antineoplastic Agents; Cell Division; Dimerization; Humans; Kinetics; Leukemia, Myeloid; Male; Molecular Structure; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The family of insulin-like growth factor binding proteins (IGFBPs) can affect cell proliferation by modulating the availability and bioactivity of insulin-like growth factors (IGFs), or by mechanisms independent of IGFs. To understand better the role(s) of IGFBPs in prostate growth and malignancy, we examined the regulation of IGFBPs in PC-3 cells, a human prostatic adenocarcinoma epithelial cell line that is androgen-insensitive. Both transforming growth factor-beta (TGF-beta) and retinoic acid (RA), known inhibitors of cellular proliferation, significantly changed the IGFBP profile in PC-3 cells. In cells that were treated with transforming growth factor beta-2 (TGF-beta 2) (0.5-10 ng/mL), IGFBP-3, and IGFBP-5 protein and mRNA increased in a time- and dose-dependent manner. At 10 ng/mL TGF-beta, IGFBP-3, and IGFBP-5 protein concentrations were 14- and 9-fold, respectively, over that of controls. Cells treated with RA (0-1 microM) also showed a time- and dose-dependent increase in IGFBP-3 protein and mRNA levels. However, in contrast to TGF-beta 2, high concentrations of RA (1 microM) negatively regulated IGFBP-5 expression, with IGFBP-5 mRNA levels downregulated to 20% of that of the control, and protein levels were decreased by 50%. Since both TGF-beta and RA increased IGFBP-3 expression and both are known to inhibit prostate cell growth, we speculate that the inhibition of growth is mediated, at least in part, by IGFBP-3.

Topics: Adenocarcinoma; Blotting, Northern; Blotting, Western; Culture Media, Serum-Free; Densitometry; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Protein 5; Male; Prostatic Neoplasms; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In the present paper, two strains of LNCaP cells derived from the same source (American Type Culture Collection), but studied either at a low passage number (LP) or at a high passage number (HP), were compared in their response to R1881 (a synthetic androgen), all-trans-retinoic acid (atRA), and 1alpha,25-dihydroxycholecalciferol (VD3). [3H]Thymidine incorporation and epidermal growth factor receptor (EGF-R) binding were measured as parameters related to the proliferative response of the cells. The secretion of prostate-specific antigen (PSA) and the mRNA expression of PSA, prostatic acid phosphatase (PAP), and diazepam-binding inhibitor (DBI) were used as parameters reflecting differentiated function. Marked differences were noted in the response of LP and HP cells to androgens. [3H]Thymidine incorporation displayed a bell-shaped dose-response curve in both strains. The amplitude of the response, however, was much higher in HP cells and growth inhibition at high levels of R1881 was only observed in LP cells. On the contrary, androgen induction of PSA secretion and PSA mRNA expression, as well as the expression of PAP was much more pronounced in LP cells, whereas DBI expression was not altered according to passage number. LP cells and HP cells also displayed striking differences in their response to atRA. An up to 6-fold stimulation of [3H]thymidine incorporation was observed in LP cells, whereas in HP cells the only significant effect was growth inhibition. VD3, on the contrary, inhibited [3H]thymidine incorporation to a comparable degree in LP and HP cells. Only marginal effects of atRA and VD3 were observed on PSA secretion. In both LP and HP cells EGF-R levels were increased by androgens and to a slight extent also by atRA and VD3. It is concluded that LP and HP LNCaP cells display markedly divergent responses not only to androgens but also to atRA. The proliferative rather than antiproliferative effects of atRA in some strains of LNCaP should caution against the uncontrolled use of these agents, or of drugs affecting their metabolism, in patients with prostate cancer.

Topics: Acid Phosphatase; Calcitriol; Carrier Proteins; Cell Division; Diazepam Binding Inhibitor; ErbB Receptors; Humans; Male; Metribolone; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Testosterone Congeners; Thymidine; Tretinoin; Tumor Cells, Cultured

In a previous report we demonstrated that androgens markedly stimulate accumulation of lipid droplets in LNCaP cells. The effects were already evident at low concentrations of androgens optimal for proliferation but became much more pronounced at high concentrations optimal for differentiation. In the present report we explored whether other agonists acting by nuclear receptors and modulating LNCaP growth and differentiation also affect lipid accumulation. The agonists investigated were 1alpha,25-dihydroxycholecalciferol (VD3), all-trans-retinoic acid (atRA), and triiodothyronine (T3). Lipid accumulation was evaluated by Oil Red O staining followed by image analysis of Oil Red O-stained cells or by extraction and measurement of absorbency. Only marginal effects were noted for VD3 and T3. The atRA, on the contrary, increased lipid staining 5-12-fold. This effect required high concentrations of retinoids (10[-6] M) and was accompanied by growth stimulation. Lipid accumulation was less pronounced than that observed with maximally effective concentrations of androgens (10[-3] M R1881). Thin layer chromatography (TLC) and enzymatic determination of the various lipid fractions demonstrated that retinoids increase triacylglycerides and an unidentified lipid fraction with a slightly higher mobility. In contrast with androgens, however, they did not stimulate the accumulation of cholesterol esters. Incorporation studies with [2-14C]acetate revealed that the increased accumulation of the mentioned lipids is related both to increased synthesis and to decreased secretion. Retinoid-induced lipid accumulation is accompanied by increased steady-state levels of the mRNA encoding fatty acid synthase (FAS), a key enzyme involved in lipid synthesis, while the expression of HMG-CoA-reductase, an enzyme controlling cholesterol synthesis is only marginally affected. It is concluded that retinoids share the ability of androgens to increase lipid accumulation in LNCaP cells. The nature of the lipids affected by both agonists, however, differs at least in part suggesting that the underlying mechanisms may also be different. For the studied compounds (androgens, VD3, atRA, and T3) no simple and consistent relationship could be observed between their ability to decrease proliferation and increase differentiation on the one hand and their ability to promote lipid accumulation on the other hand.

Topics: Acetates; Adenocarcinoma; Azo Compounds; Calcitriol; Coloring Agents; Fatty Acid Synthases; Humans; Hydroxymethylglutaryl CoA Reductases; Lipids; Male; Metribolone; Prostatic Neoplasms; RNA, Messenger; Testosterone Congeners; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Genomic methylation patterns of Dunning R-3327 cell lines anaplastic tumor-1 (AT-1), anaplastic tumor-3 (AT-3), metastasis-lymph and lung (Mat-LyLu) and metastasis-lung (Mat-Lu), and Mat-LyLu cells treated with difluoromethylornithine (DFMO), and retinoic acid (RA) have been analyzed. Each cell line was digested with HpaII and MspI restriction endonuclease enzymes to characterize methylation patterns, at the interior cytosine of the sequence CmCGG. Identical molecular weight banding patterns were found for both HpaII and MspI digests in normal dorsal prostate (NDP) used as a control. Both the treated and non-treated Dunning R-3327 cells digested with HpaII and MspI, displayed similar banding profiles from those seen in NDP solid tissues, indicative of a progressive loss of methylation at CCGG sites.

Topics: Adenocarcinoma; Antineoplastic Agents; DNA, Neoplasm; Eflornithine; Humans; Male; Methylation; Prostate; Prostatic Neoplasms; Reference Values; Tretinoin; Tumor Cells, Cultured

There is increasing evidence that growth and differentiation of prostatic carcinoma cells may be modulated not only by androgens and growth factors but also by vitamin D, retinoids, and phenylacetate (PA). The latter agonists may have a role in the prevention and therapy of prostate cancer but their exact therapeutic potential remains unclear. Since both retinoids and vitamin D act via nuclear receptors, the same way androgens do, we studied the interactions of these compounds with androgen-induced proliferation and differentiation using LNCaP cells as a model of androgen-responsive tumor cells. PA was included because of its suspected different mode of action [H3]-thymidine incorporation was used as a measure of proliferative activity, secretion of prostate-specific antigen (PSA) as a measure of differentiated function. The present data show that 1alpha,25-dihydroxycholecalciferol (VD3), all-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and PA stimulated LNCaP cell-differentiated function in the presence or absence of androgens. The effects on cell growth were more complicated. In the absence of androgens growth stimulatory effects were observed for the retinoids and under some conditions for VD3. These effects were limited, however, and tended to be more pronounced at low cell densities. In the presence of androgens nearly exclusively growth inhibitory effects were observed. On a molar basis VD3 was the most effective antiproliferative agonist (ED50 = 10(-9) M). It completely neutralized the stimulatory effects of androgens. Growth inhibition was not due to a decrease in the concentration of androgen receptor: whereas atRA, 9cRA, and PA did not alter androgen receptor levels, VD3 provoked a twofold increase. Neither in the presence nor in the absence of androgens did we observe any cooperativity in the growth stimulatory or inhibitory effects of VD3, atRA, or 9cRA. To test whether treatment with any of the studied agonists resulted in a phenotypic reversion and sustained growth arrest, LNCaP cells were pretreated with VD3, atRA, 9cRA, or PA for 6-12 days and reseeded at equal densities as untreated cells. In all cases tested [3H]-thymidine incorporation was restored within 6 days suggesting that none of these compounds caused irreversible growth inhibition.

Topics: Androgens; Antimetabolites, Antineoplastic; Calcitriol; Cell Differentiation; Cell Division; Humans; Male; Phenylacetates; Prostate-Specific Antigen; Prostatic Neoplasms; Thymidine; Tretinoin; Tumor Cells, Cultured

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Topics: Bone Marrow; Brain Neoplasms; Carcinoma; Cell Division; Culture Media; Dihydrotestosterone; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We studied the enzymatic characteristics of the oxidative catabolism of retinoic acid (RA) and its inhibition by liarozole-fumarate in homogenates of rat Dunning R3327G prostate tumors. Homogenates of rat liver were used as reference material. Both tumor and liver homogenates were able to catabolize retinoic acid. HPLC analysis revealed only very polar metabolites in tumors, while in the liver both metabolites with intermediate polarity and more polar metabolites were found. Kinetic analysis of retinoic acid catabolism showed a K(m) of 1.7 +/- 0.7 microM and a Vmax of 4.2 +/- 4.4 pmol polar RA metabolites/mg protein/hr for Dunning G tumor homogenates. In liver homogenates a K(m) value of 4.3 +/- 0.5 microM and a Vmax value of 290 +/- 120 pmol polar RA metabolites/mg protein/hr were obtained. Liarozole-fumarate inhibited retinoic acid catabolism in Dunning tumors and liver with IC50 values of 0.26 +/- 0.16 microM and 0.14 +/- 0.05, respectively. The results suggest that rat Dunning R3327G tumors are able to metabolize retinoic acid in a manner similar to that found in rat liver but with a lower metabolizing capacity.

Topics: Animals; Antineoplastic Agents; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Female; Imidazoles; Kinetics; Liver; Male; NADP; Oxidation-Reduction; Prostatic Neoplasms; Rats; Rats, Wistar; Tretinoin

Previous studies indicate that keratinocyte growth factor (KGF) acts as a paracrine factor in the prostatic epithelium and epidermal growth factor (EGF) acts as an autocrine factor. In serum-free medium, KGF or EGF promoted similar growth of human prostatic epithelial cells. Response to two growth-inhibitory factors (suramin and transforming growth factor-beta), and expression of keratins and prostate-specific antigen (PSA), were similar with either mitogen. However, colonies in medium with KGF were very compact with extensive intercellular bonds, whereas colonies with EGF consisted of widely-separated cells. Growth was decreased to a greater extent by deletion of growth factors from medium with KGF versus EGF, and retinoic acid was 10-fold more potent at inducing growth inhibition and differentiation-associated keratin with KGF compared with EGF. We conclude that regulation of growth and differentiation in the prostate might vary depending on the availability of KGF versus EGF.

Topics: Cell Division; Cells, Cultured; Clone Cells; Epidermal Growth Factor; Epithelial Cells; Epithelium; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Humans; Keratins; Male; Phenotype; Prostate; Prostatic Neoplasms; Recombinant Proteins; Tretinoin

Beta-carotene, canthaxanthin and retinoic acid (RA) inhibited growth of human DU145 prostate cancer cells by 45, 56 and 18%, respectively. Lycopene was also found to inhibit cell growth. Other carotenoids including xanthophyll (lutein), cryptoxanthin and zeaxanthin were less effective. Liarozole (a novel imidazole-derived inhibitor of intracellular RA catabolism) had a modest effect upon cell growth, this drug significantly amplified the pro-apoptotic actions of beta-carotene and RA. RA-induced expression of thymosin beta-10, an apoptotic accelerant, was associated with increased nuclear DNA nicking as measured using TUNEL. Liarozole enhanced the proapoptotic actions of RA upon DNA fragmentation in a dose-dependent manner. These actions were accompanied by inhibition of the cell survival factor bcl-2. Liarozole may thus prove useful as a novel chemotherapeutic/chemopreventive agent by boosting retinoid-induced apoptosis in the prostate.

Topics: Antineoplastic Agents; Apoptosis; beta Carotene; Blotting, Northern; Cell Division; DNA Fragmentation; Drug Synergism; Genes, bcl-2; Humans; Imidazoles; Male; Prostatic Neoplasms; Retinoids; RNA, Messenger; Thymosin; Tretinoin; Tumor Cells, Cultured

UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirement for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-alpha- and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cytoplasm; DNA Damage; Enzyme Activation; Glycogen Synthase Kinase 3; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mitogen-Activated Protein Kinases; Okadaic Acid; Phorbol Esters; Prostatic Neoplasms; Signal Transduction; Time Factors; Tretinoin; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Vitamin A (retinol) and its derivatives, the retinoids, have been implicated as chemopreventive and differentiating agents in a variety of cancers, including that of the prostate. Very little is known about the physiological role of retinoids in the prostate. Here we show that normal prostate, benign prostate hyperplasia (BPH), and prostate carcinoma tissues contain endogenous retinol and its biologically active metabolite retinoic acid. In our studies, the concentration of retinol was 2-fold elevated in BPH compared with the other two tissues. In contrast, prostate carcinoma tissue contained five to eight times less retinoic acid than normal prostate or BPH. Moreover, we found that prostate tissue expresses dehydrogenases capable of converting retinol to retinoic acid through retinaldehyde as an intermediate. Formation of retinal from retinol takes place in microsomes, and the conversion of retinal to retinoic acid occurs in the cytosol. Furthermore, we found that the nuclear retinoic acid receptors alpha, beta, and gamma are expressed in normal and tumor samples. These studies establish a role for retinoids in the physiology of the prostate and possibly also in the pathophysiology of prostate cancer.

Topics: Base Sequence; Chromatography, High Pressure Liquid; Humans; Male; Molecular Probes; Molecular Sequence Data; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Retinoic Acid; Reference Values; Retinaldehyde; Retinoids; RNA, Messenger; Tretinoin; Vitamin A

Among the more interesting studies at the 1996 annual meeting of the American Society of Clinical Oncology that related to hormone-refractory prostate cancer were several that reported on the use of cis-retinoic acid both alone and in combination with interferon-alpha. Interferon-alpha and interferon-alpha plus cis-retinoic acid have antiproliferative effects in vitro against both PC3 and D-145 prostate cancer cells in culture. BCL2 expression is increased in androgen-independent cells, which may block apoptosis, and retinoids induce transforming growth factor-beta and apoptosis in prostate cancer cell lines. This regimen raises many questions. For example, it is difficult to determine what prostate-specific antigen (PSA) level one should expect from cis-retinoic acid, 4HPR, or any of the other differentiating agents. Should there be an increase in PSA1 Should one expect a slower decline in PSA when giving additional agents that are in fact cytotoxic? What is the significance of a changing level of PSA after this and other types of treatment? These and other questions remain to be determined in future studies.

Topics: Antineoplastic Agents; Apoptosis; Humans; Male; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; Tretinoin

Overproduction of uPA by prostate cancer cells in vivo results in tumor invasiveness and osteoblastic skeletal metastasis due to its mitogenic actions in osteoblasts. In the present study we have examined the effect of several growth factors and steroid hormones on regulating uPA gene expression in the human prostate cancer cell line (PC-3). Treatment of these cells with dexamethasone (Dex) caused a decrease, whereas epidermal growth factor (EGF) and fetal bovine serum (FBS) increased uPA expression in a dose-dependent manner. Trans retinoic acid (RA) also induced uPA mRNA and protein production in a dose-dependent manner (10(-6) to 10(-9) M). This increase was seen as early as 2 hr of treatment until 48 hr. Dex treatment resulted in decreased tumor cells invasiveness, whereas exposure to EGF and RA caused an increase in the invasive capacity of PC-3 cells. These studies should help to better understand the control mechanism of uPA expression in prostate cancer, where uPA has been implicated as a major pathogenetic factor.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Cattle; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Invasiveness; Prostatic Neoplasms; RNA, Messenger; Serum Albumin, Bovine; Time Factors; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

There is increasing evidence that the course of prostatic carcinoma is determined by a complex interplay between genetic events, paracrine interactions, and hormonal and dietary factors. These latter factors include several ligands of the nuclear receptor family such as androgens, vitamin D3 and retinoids. To test whether thyroid hormones also influence the growth and differentiated function of prostatic carcinoma cells, LNCaP cells were treated with or without triiodothyronine (T3) in the absence or in the presence of other regulatory factors. Exposure of LNCaP cells to T3 for 6 days in the absence of androgens caused a dose-dependent increase in [3H]-thymidine incorporation with a maximal stimulation of 2.5-fold at 10(-9) M T3. Secretion of prostate-specific antigen (PSA) was also stimulated 2-3-fold. The observed effects may well be mediated by a nuclear T3 receptor as evidenced by displaceable T3 binding studies. Combined treatment of LNCaP cells with androgens and T3 revealed intriguing interactions between these two pathways. Below and up to 10(-10) M of the synthetic androgen R1881, the concentration that evokes optimal proliferative responses, T3 stimulated [3H] thymidine incorporation. At higher concentrations of androgens, T3 displayed antiproliferative effects. No androgen-dependent effects on T3 receptor levels were observed. Conversely, T3 increased androgen receptor levels up to twofold. Androgen as well as T3 stimulation of proliferation was abolished by high concentrations of the retinoid 9-cis-retinoic acid. These data add T3 to the list of factors that influence growth and differentiation of prostatic tumor cells and contribute to our understanding of the intricate pathways that ultimately determine the course of prostatic carcinoma.

Topics: Androgens; Cell Division; Cell Nucleus; Humans; Male; Metribolone; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Receptors, Thyroid Hormone; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Liarozole showed antitumoral activity in the Dunning AT-6sq, an androgen-independent rat prostate carcinoma. To investigate its potential mechanism of action, the effects of the drug doses (ranging from 3.75 to 80 mg/kg b.i.d.) on endogenous plasma and tissue all-trans-retinoic acid levels and on the differentiation status of the tumor cells were evaluated. To follow modulation of differentiation, cytokeratins were localized in the (un)treated tumors by immunocytochemistry and quantitatively determined by immunoblotting. Results showed that liarozole statistically significantly reduced tumor weight from 30 mg/kg upwards and induced accumulation of all-trans-retinoic acid both in plasma and tumors. In the tumors, a statistically significant accumulation was already noted from 7.5 mg liarozole/kg upwards. Concomitantly, the differentiation status shifted from a keratinizing towards a non-keratinizing squamous carcinoma, which was further confirmed by the cytokeratin profile of the carcinoma (presence of CK 8, 10, 13, 14, 18, 19). Immunoblotting revealed an overall decrease in cytokeratin content, except for CK 8. These findings suggest that the antitumoral properties of liarozole might be related to an increase in the degree of tumor differentiation through accumulation of all-trans-retinoic acid.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Imidazoles; Immunoblotting; Immunohistochemistry; Keratins; Male; Neoplasm Transplantation; Organ Size; Prostate; Prostatic Neoplasms; Random Allocation; Rats; Rats, Inbred F344; Tretinoin; Tumor Cells, Cultured; Vimentin

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

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

Effects of all-trans retinoic acid (RA) on the net enzymatic activity of secreted, extracellular urokinase-type plasminogen activator (u-PA) in DU-145 human prostatic carcinoma cells were examined to assess the potential use of retinoids in human prostate cancer prevention and treatment. u-PA is associated with tumor progression involving invasion and metastasis. Based on a chromogenic substrate assay, results show that DU-145 cells secrete five times more u-PA than normal human prostatic epithelium. DU-145 cells were treated with 0.1 to 10 micrometer RA for 48 h. This short treatment of cells with RA did not inhibit growth. After a 48-h treatment of cultures with RA, serum-free conditioned medium was analyzed for u-PA activity by SDS-PAGE zymography. Two major bands of u-PA with Mr of approximately 54,000 (high molecular weight u-PA) and approximately 33,000 (low molecular weight u-PA) were detected. Plasminogen-dependent catalytic activity of these bands could be specifically inhibited with antibody to u-PA, confirming that these bands represent u-PA. A 48-h treatment with 1.0 micrometer RA reduced u-PA activity in conditioned medium to 51.6% of control. A 50% reduction in free u-PA antigen level, as compared to control, was further demonstrated at 1.0 micrometer RA by Western blot analysis and densitometry. These results show that RA can decrease the net extracellular urokinase activity produced by prostatic carcinoma cells. It is proposed that these effects of RA may have important implications not only in the chemoprevention of prostate cancer, by inhibition of promotion of prostatic intraepithelial neoplasia to invasive carcinoma, but also in tumor progression during invasion and metastasis, by decreasing extracellular matrix degradation, as shown in our accompanying article (M. M. Webber and A. Waghray, Clin. Cancer Res., 1: 755-761, 1995).

Topics: Cell Division; Cells, Cultured; Culture Media, Conditioned; Culture Media, Serum-Free; Disease Progression; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epithelial Cells; Humans; Kinetics; Male; Molecular Weight; Neoplasm Invasiveness; Neoplasm Metastasis; Prostate; Prostatic Neoplasms; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Both normal and malignant prostatic epithelial cells in culture secrete urokinase-type plasminogen activator (u-PA) into the culture medium. u-PA has been shown to have a direct association with invasive and metastatic potential of many types of cancers. We propose that prostate cancer has the intrinsic ability to invade and metastasize because of its inherent ability to secrete the serine protease u-PA. We further propose that in prostate cancer, u-PA is the key enzyme which occupies a place at the apex of the proteolytic cascade and initiates the degradative process. Subsequently, collagenases are recruited after activation of procolla-genases by another serine protease plasmin formed by the activation of plasminogen by u-PA. Extracellular proteolysis involving plasmin can cause massive degradation of the extracellular matrix. We show that u-PA alone can use fibronectin as a substrate and degrade it, but u-PA alone did not degrade laminin. Serum-free conditioned medium from DU-145 human prostatic carcinoma cells has the ability to degrade both fibronectin and laminin. However, treatment of cultures with 1 microM all-trans retinoic acid (RA) for 48 h reduced the ability of serum-free conditioned medium to cause u-PA-mediated degradation of fibronectin and laminin. Thus, RA had a protective effect on these extracellular matrix glycoproteins. Treatment of cells with RA also decreased their ability to invade Matrigel in the in vitro invasion assay in a dose-dependent manner. RA at the 0.5, 1, and 10 microM level reduced invasion to 65.7%, 46.7%, and 34.3% of control, respectively. RA reduced extracellular proteolysis and thus inhibited extracellular matrix degradation and invasion. These results may also explain one mechanism by which retinoids inhibit invasion and metastasis in vitro and in vivo. These studies have important translational value in the chemoprevention of progression of prostatic intraepithelial neoplasia to invasive carcinoma.

Topics: Collagen; Culture Media, Conditioned; Culture Media, Serum-Free; Drug Combinations; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Humans; Laminin; Male; Neoplasm Invasiveness; Prostatic Neoplasms; Proteoglycans; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Expression of certain cytokeratins can be indicative of the state of differentiation of epithelial cells. The basal cells in the normal adult human prostatic epithelium are characterized by the expression of cytokeratins 5 and 14, whereas the secretory luminal cells contain cytokeratins 8 and 18. Cells cultured from the prostatic epithelium expressed cytokeratins 5, 8, and 18, and thus had features of both basal and luminal cells. Certain growth-inhibitory conditions altered keratin expression in conjunction with growth modulation. Deletion of peptide factors and hormones from the culture medium induced the expression of cytokeratins 1 and 10, associated with a squamous phenotype. These same squamous keratins were found in very dense, stratified cultures that were maintained at confluency in standard, complete medium for extended periods. Retinoic acid enhanced the expression of secretory luminal cell-associated cytokeratins 8 and 18 in semi-confluent cultures. Other growth inhibitory factors such as suramin, transforming growth factor-beta, and interferon-gamma had no effect on keratin expression. These observations indicate that the differentiation of prostatic epithelial cells can be directed toward alternate pathways, either squamous or secretory, by different growth-inhibitory conditions. However, not all growth inhibitory factors altered differentiation, demonstrating that growth inhibition in itself is not a sufficient inducer of differentiation.

Topics: Cell Division; Cells, Cultured; Epithelial Cells; Epithelium; Glucose; Growth Substances; Humans; Interferon-gamma; Keratins; Male; Phenotype; Prostate; Prostatectomy; Prostatic Hyperplasia; Prostatic Neoplasms; Reference Values; Suramin; Transforming Growth Factor beta; Tretinoin

We have established two new epithelial cell lines (NRP-152, NRP-154), with markedly different properties, from the dorsal-lateral prostate of Lobund/Wistar rats treated with N-methyl-N-nitrosourea and testosterone propionate. NRP-152 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic epithelial cells. They produce prostatic acid phosphatase, have functional androgen receptors, and require the combination of several growth factors in addition to serum for optimal growth. Their growth is stimulated by epidermal growth factor, insulin, dexamethasone, cholera toxin, dihydrotestosterone, and testosterone, and their growth is inhibited by transforming growth factor beta s and retinoic acid. These cells also respond to 1,25-dihydroxyvitamin D3 with an early growth stimulation followed by growth inhibition at later times. In contrast, tumorigenic NRP-154 cells lack detectable androgen receptor mRNA and have less stringent growth factor requirements for optimal growth. Growth of NRP-154 cells is stimulated by dexamethasone and insulin, inhibited by transforming growth factor beta 1, but not significantly altered by epidermal growth factor, cholera toxin, dihydrotestosterone, retinoic acid, or 1 alpha,25-dihydroxyvitamin D3. Our data suggest that the NRP-152 and NRP-154 cell lines are suitable systems for analysis of normal prostate growth and prostatic carcinogenesis.

Topics: Acid Phosphatase; Animals; Calcitriol; Cell Division; Cell Line; Dihydrotestosterone; Epithelium; Karyotyping; Male; Methylnitrosourea; Mice; Mice, Nude; Prostate; Prostatic Neoplasms; Rats; Rats, Wistar; Receptors, Androgen; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Recent studies have demonstrated that retinoic acid (RA) can repress the growth of human prostatic epithelial cells. Since the proliferation of prostate cells is highly dependent on androgen stimulation, presumably via its cognate receptor, we investigated the effects of RA on the expression of the androgen receptor and other androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. Using a radioligand binding assay, we found that androgen-binding activity was reduced 30-40% in cells treated with 10(-5) M RA plus 6 nM dihydrotestosterone (DHT), as compared to cells with the androgen alone. Moreover, the reduction of the androgen receptor (AR) was not accompanied by alteration of the ligand-binding affinity. Concomitant changes in the function of AR were manifested by a dramatic reduction in AR-mediated transcription activity in a transfection experiment. Androgen-induced levels of both prostate-specific antigen (PSA) and human glandular kallikrein-1 (hKLK2) mRNAs were significantly repressed by RA in a dose- and time-dependent manner. Consistent with this finding, androgen induction of PSA glycoprotein was also repressed by RA, with maximal inhibition occurring at 10(-5) M. These data suggest that the suppression of proliferation and function of prostatic cells by RA may be via modulatory effects on the AR.

Topics: Adenocarcinoma; Androgen Antagonists; Cell Division; Gene Expression; Humans; Male; Nucleic Acid Hybridization; Prostatic Neoplasms; Radioligand Assay; Receptors, Androgen; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

The addition of lipid hydroperoxides greatly accelerates the rate of oxidative catabolism of all-trans-retinoic acid (RA) in human cell microsomes; hydroperoxy metabolites of the arachidonate cascade are particularly active in the microsomal system. We have measured the plasma content of lipid peroxides in cancer patients during the course of therapy with RA, seeking to assess whether a correlation exists between the rate of oxidative catabolism of exogenously administered RA and whole body lipid peroxide levels. The assay used for plasma lipid peroxides is the capacity to react with thiobarbituric acid under specified conditions; the result is expressed as TBARS (thiobarbituric acid reactive substances). RA administration produced its own accelerated clearance RA within 72 h. Patients were considered to have "normal" or "rapid" baseline catabolism of RA if their Day 1 area under RA concentration over time curve was greater or less than 300 ng.h/ml, respectively. The mean plasma TBARS levels were: 12 normal volunteers = 0.14 microM; 19 "normal" RA catabolizers = 0.25 microM; and 14 "rapid" catabolizers = 0.82 microM. P = 0.008 (rapid catabolizers versus normal volunteers) and 0.05 (rapid catabolizers versus normal catabolizers). Repeat TBARS determinations were made during the course of therapy in 17 patients, all of whom converted to "rapid" RA catabolism on therapy. An increase in plasma TBARS levels > or = 20% of baseline was observed in 5 of 5 prostate cancer patients and 8 of 12 lung cancer patients treated with continuous RA therapy for 2 and 4 weeks, respectively. These observations support the hypothesis that high levels of lipid peroxides and rapid oxidative catabolism of RA are positively correlated.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Leukemia, Promyelocytic, Acute; Lipid Peroxides; Lung Neoplasms; Male; Metabolic Clearance Rate; Multiple Myeloma; Neoplasms; Prostatic Neoplasms; Reference Values; Thiobarbituric Acid Reactive Substances; Tretinoin

We studied the effect of retinoic acid on the growth and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. Our data showed that retinoic acid at 0.01 microM. stimulated the proliferation of LNCaP cells but inhibited their growth at 0.1 microM. under androgen-free conditions. In the presence of 0.1 nM. dihydrotestosterone (DHT), LNCaP cell proliferation was inhibited by 10 microM. retinoic acid but not by lower concentrations of retinoic acid. Retinoic acid reduced LNCaP cell growth at concentrations of 0.1 microM. in the presence of 10 nM. DHT. Retinoic acid (10 microM.) also reduced the growth response of LNCaP cells to epidermal growth factor and transforming growth factor alpha and potentiated the inhibitory effect of transforming growth factor beta. In additional studies, retinoic acid induced a dose-dependent increase in prostate specific antigen (PSA) secretion at concentrations of 0.1 to 1 microM. Dihydrotestosterone (10 nM.) also enhanced the secretion of PSA by LNCaP cells, and this effect was potentiated in a dose-dependent fashion by the addition of retinoic acid at 0.1-10 microM. Competitive binding studies showed that retinoic acid did not bind to androgen receptors. Overall, retinoic acid had a biphasic effect on LNCaP proliferation and promoted the secretion of PSA. The biphasic effect of retinoic acid on LNCaP growth should be considered in designing in vivo studies to determine the impact of retinoic acid on solid prostatic tumor growth. In addition, the ability of retinoic acid to increase PSA secretion may complicate the interpretation of serum PSA levels used for diagnostic and prognostic purposes.

Topics: Androgens; Cell Division; Dihydrotestosterone; Flow Cytometry; Humans; Male; Metribolone; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Testosterone; Tretinoin; Tumor Cells, Cultured

We report for the first time that a synthetic retinoid, N-(4-hydroxyphenyl)retinamide, can prevent the development of both primary and metastatic tumors in an animal model of metastasizing primary prostate cancer. Prostatic adenocarcinomas were induced in high incidence in Lobund-Wistar rats by initiation with methylnitrosourea i.v. and promotion with testosterone. Feeding of N-(4-hydroxyphenyl)retinamide to these rats during the latency period markedly diminished the final incidence of both primary and metastatic prostate carcinomas.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Fenretinide; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Rats, Mutant Strains; Tretinoin

Tumor cells of transplanted prostate adenocarcinoma-III (PA-III) spread with very high frequency from the extravascular implant site through ipsilateral lymphatic channels to the lungs in which they produce visible focal tumors. The latter enlarge, coalesce and eventually kill the host. This system was used to demonstrate the effect of a retinoid on metastasis. Lobund-Wistar (L-W) rats were administered 1 mmol 4-HPR/kg diet L-485, and control rats received the same diet without 4-HPR. After an interval, all rats were inoculated subcutaneously with PA-III cells. When examined at autopsy, all rats had developed an anticipated tumor at the implant site. However, the numbers of focal PA-III tumors in the lungs were significantly reduced among the 4-HPR-treated rats compared to the control rats (P = 0.002).

Topics: Adenocarcinoma; Animals; Female; Fenretinide; Male; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; Rats; Rats, Inbred Strains; Tretinoin

The effect of retinoic acid (RA) on testosterone metabolic pathway was investigated in hyperplastic and neoplastic human prostatic tissues, and also the effects of steroids on RA binding to its receptor. Steroids only had a minimal effect on the binding of RA by its receptor. The conversion of testosterone to DHT by 5 alpha-reductase was reduced in the presence of retinoic acid. The inhibition was probably due to competition with NADPH for enzyme binding sites. The degree of inhibition found with retinoic acid at a concentration of 10(-4)M was greater for hyperplastic (41%) than that for neoplastic tissue (24%). The inhibition of 5 alpha-reductase by retinoic acid was dose-dependent. The activity of 5 alpha-reductase is significantly less in neoplastic compared with hyperplastic tissue.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; 5-alpha Reductase Inhibitors; Carrier Proteins; Humans; Male; NADP; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Retinoic Acid; Tretinoin

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-beta and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.

Topics: Antibodies, Neoplasm; Biomarkers, Tumor; Calcium; Cell Differentiation; Cell Division; Cells, Cultured; Cholera Toxin; Epidermal Growth Factor; Epithelial Cells; Epithelium; Humans; Hydrocortisone; Immunohistochemistry; Insulin; Male; Prostate; Prostatic Neoplasms; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured

Prostate samples from 41 patients with acute on chronic prostatitis, benign hyperplasia, chronic prostatitis or adenocarcinoma were assayed for cytosolic retinoic acid binding protein (cRABP). Binding protein was detected in all samples. Scatchard analysis revealed dissociation constant values of 15.0-31.8 nM and maximum binding capacity of 5.2 +/- 2.0 (S.E.) pmol/mg protein. Using sucrose density gradient centrifugation, cRABP was quantitated and values varied from 1.2 to 12.5 pmol/mg protein. High levels (greater than 3.5 pmol/mg protein) were found in 28/41 samples. There was no correlation between the cRABP binding/mg protein and the volume (%) of glandular tissue (compared with stroma). While there was no significant difference in the quantities of cRABP in the different disease groups, there was a tendency for a progressive increase from acute on chronic prostatitis to chronic prostatitis, the highest levels being present in carcinoma.

Topics: Adenocarcinoma; Binding Sites; Carrier Proteins; Centrifugation, Density Gradient; Chromatography, Gel; Cytosol; Humans; Male; Neoplasm Proteins; Prostatic Hyperplasia; Prostatic Neoplasms; Prostatitis; Receptors, Retinoic Acid; Tretinoin

The effect of retinoic acid (RA) on testosterone metabolism was examined in a prostatic cancer cell line of human origin, PC-3. In cells growing as monolayers as well as in cell homogenates RA causes a dose-dependent inhibition of the 5 alpha-reductase activity, thus preventing the conversion of testosterone into its hormonally active metabolite, dihydrotestosterone. Fifty per cent inhibition of the enzyme activity occurred at an RA concentration of 2 x 10(-5)M. The pattern of inhibition was that of a non-competitive inhibitor. However, when incubations were performed in the presence of varying amounts of NADPH, it turned out that RA exerts its effect by competitive inhibition of the cofactor action. Although the severe toxicity of RA precludes its systemic use as a 5 alpha-reductase inhibitory drug in humans, the possible anti-androgenic effect of other, less toxic, retinoids should be investigated.

Topics: 5-alpha Reductase Inhibitors; Dose-Response Relationship, Drug; Humans; Male; NADP; Prostatic Neoplasms; Tretinoin; Tumor Cells, Cultured

Cells derived from the G-subline of the Dunning R-3327 rat prostatic adenocarcinoma were selected on the basis of their inducibility for alkaline phosphatase (AP) activity by retinoic acid. A p-nitrophenylphosphate-agarose overlay procedure was used to identify AP-inducible clones. The frequency of AP-inducible cells in one rapidly growing tumorigenic clone, designated 9-1C, has remained at 100% during at least 4 months of continuous culture. In culture, 9-1C cells had a mean population-doubling time in log phase of 14 hr. Retinoic acid (10 microM) did not significantly affect the rate of growth in log phase. It did, however, cause the cultures to saturate at a cell density which was 40% lower than that of control cultures. This effect on saturation density was reversible within 24 hr after removing retinoic acid from the medium. Retinoic acid-treated cells occupied greater areas on the culture dish surface, and the cross-sectional area of these cells, measured on dispersed cells by light-scatter flow cytometry, was 35 to 40% greater than that of control cells. The inducibility of 9-1C cells for AP activity decreased as the culture density increased. Cells of the 9-1C clone produced tumors when injected into male and female Fischer X Copenhagen F1 rats. No histological differences were detected between tumors grown in male and female rats. Although the tumors were poorly differentiated, primitive acinar-like structures were observed. Cells staining uniformly positive for AP activity were distributed randomly throughout the tumors. In the acinar-like structures, AP activity was localized only on the apical surfaces of the cells lining the lumens. This was also the site of enzyme activity in acini of the lateral component of the dorsolateral prostate, the source of the original R-3327 tumor. In the lateral prostatic component, AP activity was also found in the basal region of the acini, and the secretory material filling the lumens was strongly positive for the enzyme. These two regions of the tumor acini were negative for AP activity. With the exception of activity in capillaries at the basal surface, the acini of the dorsal component of the dorsolateral prostate were devoid of AP activity.

Topics: Adenocarcinoma; Alkaline Phosphatase; Animals; Cell Division; Cell Line; Clone Cells; Enzyme Induction; Histocytochemistry; Male; Prostatic Neoplasms; Rats; Tretinoin

Topics: Animals; Carrier Proteins; Castration; Cells, Cultured; Concanavalin A; Disease Models, Animal; Horseradish Peroxidase; Humans; Hydrolases; Lysosomes; Male; Neoplasm Proteins; Prostatic Neoplasms; Rats; Receptors, Retinoic Acid; Tretinoin

Topics: Adenocarcinoma; Alkaline Phosphatase; Animals; Cell Line; Clone Cells; Enzyme Induction; Kinetics; Male; Neoplasms, Experimental; Prostate; Prostatic Neoplasms; Rats; Tretinoin

The antihyperplastic activity of beta-retinoic acid (RA) and nine synthetic analogues (retinoids) was examined in organ cultures of mouse prostate made hyperplastic by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After 8 or 10 days, when most explants developed hyperplasia, the carcinogen was withdrawn and explants were incubated in control medium and medium containing different concentrations of a retinoid. The antimitotic activity of retinoids was compared with that of RA. Different retinoids produced variable degrees of mitotic inhibition in the hyperplastic prostate epithelium. The methylketo cyclopentenyl and 1-methoxyethyl cyclopentenyl analogues of RA were at least 50-fold more active than RA in reversing MNNG-induced hyperplasia. The trimethylmethoxyphenyl analogue of RA and retinyl methyl ether were significantly more active than RA. Three analogues, N-acetyiretinylamine, retinal acetyl hydrazone, and retinal oxime, were as active as RA. The chlorotrimethylphenyl analogue showed less activity than RA, and alpha-retinyl acetate was completely devoid of mitotic inhibitory activity.

Topics: Cell Division; Epithelium; Hyperplasia; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Organ Culture Techniques; Precancerous Conditions; Prostate; Prostatic Neoplasms; Structure-Activity Relationship; Tretinoin; Vitamin A

The influence of vitamin A-related compounds on hyperplasia and metaplasia induced by methylcholanthrene was studied in mouse prostate glands in organ culture. Methylcholanthrene was found to cause extensive hyperplasia and squamous metaplasia of the prostatic epithelium which persisted after withdrawal of the carcinogen. The retinoids included retinoic acid and 6 of its structural analogues synthesized in an attempt to enhance the anticarcinogenic action and reduce the toxicity of the parent compound. These where the cyclopentenyl analogus 7699, A2-retinoic acid, 13-cis-alpha-retinoic acid and 3 aromatic analogues. Administration of the compounds following the carcinogen reduced the extent and incidence of hyperplasia significantly and with the exception of one compound reversed the squamous metaplasia. Two of the aromatic analogues, one with a terminal ethylamide group (1430), and the other with a terminal ethylester group (9369), proved to be the most potent inhibitors, followed by compound 7699 and (9369), proved to be the most potent inhibitors, followed by compound 7699 and retinoic acid. A2-retinoic acid and 13-cis-alpha-retinoic acid showed the lowest activity. The inhibition of hyperplasia appeared to be mediated via a reduction of DNA synthesis. It seemed unrelated to either the biological growth-promoting activity of the compounds or their surface-active properties. It is tentatively suggested that vitamin A and its analogues may act as hormones.

Topics: Animals; DNA; Hyperplasia; In Vitro Techniques; Male; Metaplasia; Methylcholanthrene; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Prostate; Prostatic Neoplasms; Thymidine; Tretinoin; Vitamin A