fenretinide and Prostatic-Neoplasms

The magnitude of the problem of prostate cancer, and the failure of conventional chemotherapy and surgery to effect a marked diminution in the total number of deaths from this disease, now indicate that chemoprevention of prostatic carcinogenesis must be seriously considered. We describe the development of a new system for quantitating the process of prostatic carcinogenesis in an experimental animal, and the use of this system to demonstrate that the retinoid, 4-hydroxyphenylretinamide, the deltanoid, Ro24-5531, and the estrogen analog, tamoxifen, all are effective agents for prevention of experimental prostate cancer.

Topics: Animals; Anticarcinogenic Agents; Calcitriol; Disease Models, Animal; Drugs, Investigational; Fenretinide; Male; Neoplasm Staging; Prostatic Neoplasms; Rats; Tamoxifen; Treatment Outcome

Primary prevention of prostate cancer is a relatively new concept. Through large-scale studies it is possible that we may be able to define better the risk for prostate cancer and identify those who would benefit from an intervention to lower their risk of disease. As risk for prostate cancer is better defined, a number of interventions may eventually be tested. Several interventions are sufficiently mature that they can be implemented in large-scale trials. Diet modification is an intervention that is ready for evaluation. It may also have additional benefits by decreasing mortality from other malignancies and cardiac disease. 5 alpha-reductase inhibitors are also ready for testing. The National Cancer Institute and its clinical cooperative groups have begun a large trial to assess finasteride in the prevention of prostate cancer.

Topics: 5-alpha Reductase Inhibitors; Androgen Antagonists; Animals; Dietary Fats; Eflornithine; Fenretinide; Finasteride; Humans; Male; Prevalence; Primary Prevention; Prostatic Neoplasms; Risk Factors

Fenretinide is a synthetic retinoid with activity in prostate cancer and other cell lines. The aim of this study was to assess the efficacy and tolerability of fenretinide in chemotherapy-naïve men with hormone refractory prostate cancer.. Eligibility criteria included hormone refractory prostate cancer with a rising PSA at least 6 weeks after peripheral anti-androgen withdrawal, ECOG performance status (PS) 0-1, and no prior chemotherapy. Fenretinide was administered orally at 900 mg m(-2) twice daily for 7 of every 21 days. PSA was measured before each cycle. The primary endpoint was a > or =50% reduction in PSA maintained for at least 3 weeks; secondary endpoints included duration of PSA response, time to treatment failure (TTF: treatment stopped for progression or toxicity) and adverse events (AE).. Twenty seven pts were recruited from 7 centres over 27 months. Median age was 74 (range 49-86), median baseline PSA was 129 (range 19-1,000), and 70% had a PS of 0. The median number of cycles received was 2 (range 0-11) and 20 pts completed at least 1 cycle. One pt (4%) achieved a 50% reduction in PSA lasting 39 days and 15 pts (56%) had not progressed within 6 weeks of starting fenretinide. The median TTF was 54 days (IQR 19-73): 22 (81%) failed with tumour progression, 3 (11%) failed with toxicity and 2 (7%) never commenced the drug. Grade 3 rash occurred in 1 patient, all other AE were grade 1 or 2. The most common AE were nausea (40%), hot flushes (36%), constipation (32%) and nyctalopia (32%).. High-dose fenretinide had limited anti-tumour activity in patients with advanced hormone refractory prostate cancer: further evaluation in this setting is not warranted.

Topics: Administration, Oral; Aged; Aged, 80 and over; Antineoplastic Agents; Disease Progression; Fenretinide; Humans; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Neoplasms; Treatment Failure

Fenretinide is a synthetic retinoid that is cytotoxic to a variety of cancers. We conducted a phase II trial of oral fenretinide in patients with biochemically recurrent prostate cancer.. Eligible patients had histologically confirmed prostate cancer and a confirmed rising prostate-specific antigen (PSA) >or= 2 ng/mL following either radical prostatectomy and/or pelvic radiation therapy, without clinical or radiographic evidence of metastasis. The primary endpoint was PSA response, which was defined as a confirmed decrease by >or=50%, and >or=5 ng/mL, from the pretreatment value. Treatment comprised oral fenretinide 900 mg/m2 twice daily for 1 week, every 3 weeks, for 1 year.. After a median follow-up of 17.7 months, out of 23 patients, 7 (30%) patients had PSA stable disease (SD), 11 (48%) patients had PSA progression within 3 months, 4 patients had minimal increases over 3 months that did not qualify as SD or progression (17%), and one patient (4%) was not evaluable. Median time to PSA progression was 4.6 months (95% CI, 3.2-8.2 months). Observed grade 3 toxicities included fatigue, pain, hypermagnesemia, a rise in lipase, and nyctalopia.. Although well-tolerated, oral fenretinide did not meet prespecified PSA criteria for response in biochemically recurrent prostate cancer; however, 30% of patients had SD, which suggests modest single-agent clinical activity. The role of different formulations of fenretinide, which might allow for higher serum concentrations of the drug, is currently under investigation.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Androgen Antagonists; Antineoplastic Agents; Disease Progression; Fenretinide; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prostatectomy; Prostatic Neoplasms; Treatment Outcome

The objective of phase 2 cancer chemoprevention trials is to evaluate whether a chemopreventive agent will cause significant modulation of intermediate endpoint biomarkers (IEB) in patients at high risk for the disease. A phase 2 chemoprevention trial of 4-hydroxyphenyl retinamide (4-HPR) versus placebo was conducted in men with a histologic diagnosis of early prostate cancer and scheduled to have radical prostatectomy. A Bayesian monitoring method was used to sequentially monitor this trial for evidence of biological activity or ineffectiveness based on a single IEB variable. Different prior distributions were used and posterior distributions were obtained to calculate the probability that treatment differences are greater than or less than a predetermined clinically significant effect. The interim analysis of transforming growth factor-alpha expression indicated a high probability of insufficient biological activity of 4-HPR on this IEB. This study demonstrates the potential utility of Bayesian methods in the decision-making process in the conduct of phase 2 chemoprevention trials.

Topics: Anticarcinogenic Agents; Bayes Theorem; Biomarkers, Tumor; Biopsy; Cohort Studies; Double-Blind Method; Fenretinide; Humans; Male; Prostate; Prostatic Neoplasms; Transforming Growth Factors

To determine (1) whether nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are differentially expressed in normal and in cancerous human prostate tissues and (2) whether oral fenretinide therapy impacts the expression of these receptors in prostate cancer.. In situ hybridization with antisense riboprobes was used to probe for RAR and RXR transcripts in prostate tissues in a two-phased study: (1) expression of retinoid receptors in eight normal prostates was compared with their expression in 10 randomly picked radical prostatectomy specimens (group A); (2) expression of retinoid receptors was determined in 22 radical prostatectomy specimens from participants in a clinical study (group B). Twelve patients received oral fenretinide 200 mg/d, and 10 received placebo pills for 28 days before surgery.. RARalpha, RARgamma, RXRalpha, and RXRgamma mRNAs were detected in most normal and cancerous prostates. In group A, RARbeta mRNA was expressed in only four of 10 malignant prostates but was present in seven of eight benign prostates (P =.05). RXRbeta mRNA was expressed in four of eight benign prostates and in zero of 10 malignant prostates (P =.023). In group B prostates, RARbeta and RXRbeta mRNAs were markedly reduced in all cancers and in the adjacent, nonmalignant tissue. There were no differences between receptor expression in the fenretinide-treated group and the placebo group.. RARbeta and RXRbeta mRNAs are selectively lost in both prostate cancer and adjacent morphologically normal prostatic tissue, supporting the concept of a field of carcinogenesis. One month of oral fenretinide (200 mg/d) did not influence the expression of retinoid receptors in prostate cancer.

Topics: Aged; Anticarcinogenic Agents; Fenretinide; Humans; In Situ Hybridization; Male; Middle Aged; Nuclear Proteins; Prostate; Prostatic Neoplasms; Receptors, Retinoic Acid; Transcription Factors

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

An NCI-sponsored, phase II trial of N-(4-hydroxyphenyl)- retinamide (4-HPR) in patients with organ-confined prostate cancer in the period prior to radical prostatectomy was carried out. Thirty-seven men with the histologic diagnosis of prostate cancer planning to have radical prostatectomy entered the study after informed consent and were given 4-HPR (or matching placebo) as a single daily dose (two 100-mg capsules of 4-HPR or two capsules of placebo daily) for 3 weeks prior to surgery. Four men dropped out for unrelated reasons. Thirty-three men completed the study. At the time of surgery, repeat biopsies of the prostate were performed to study the effects of the drug on potential surrogate endpoint biomarkers (SEBs) of malignancy within the tissue. The panel of potential SEBs of malignancy include p53, cytomorphometric indices, ploidy, PNCA, erbB-2, erbB-3, EGF receptor, TGF-alpha tumor-associated glycoprotein-72, fatty acid synthetase and Lewis Y antigen. Twenty-three patients had matching pre- and posttherapy lesions and were considered informative. Results from the patients indicate significant differential expression of biomarkers in pretreatment specimens of uninvolved prostatic tissue (normal-appearing epithelia) prostatic intraepithelial neoplasia (PIN) and prostate cancer. The mean erbB-2 expression was 0.58 in uninvolved vs. 1.04 in PIN (p = 0.002); while the mean erbB-2 expression was 1.35 in prostate cancer (p = 0.0007, uninvolved vs. prostate cancer). A similar pattern of increased biomarker expression between uninvolved and PIN or prostate cancer tissues can be observed for EGF receptor (mean = 1.21, 1.87 and 1.76 for uninvolved, PIN and prostate cancer, respectively) and erbB-3 (mean = 0.81, 1.59 and 1.30 for uninvolved, PIN and prostate cancer, respectively). There were no statistically significant differences in biomarkers observed in the 4-HPR-treated patients when compared with placebo-treated control patients. There was a posttreatment up-regulation of biomarkers observed in both groups of patients. This observation is most likely explained by an effect due to the diagnostic sextant biopsy equally affecting both groups of patients. Results from this study do not demonstrate a chemoprevention effect of 4-HPR on tissue-based SEBs at the dose given.

Topics: Adenocarcinoma; Aged; Antineoplastic Agents; Biomarkers, Tumor; Biopsy, Needle; Fenretinide; Humans; Immunohistochemistry; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Neoplasms; Reference Values; Sensitivity and Specificity; Statistics, Nonparametric

Prostate cancer is the most common cancer diagnosed in American men. The need to find effective means of preventing this disease is clear. Vitamin A and its analogues (retinoids) act as transcriptional regulators within the nucleus and have been tested as both preventative and therapeutic agents in human malignancy. Fenretinide (N-4-hydroxyphenyl retinamide) (4HPR) has been found to be relatively nontoxic in preclinical experiments and early clinical trials. Its toxicity and feasibility for use as a chemoprevention agent in men at high risk for prostate cancer was evaluated in this study. Twenty-two patients were entered into a clinical trial that involved taking 4HPR for twelve 28-day cycles. Eight patients with negative prestudy biopsies had positive prostate biopsies prior to or at the time of their 12th cycle evaluation. This led to early closure of the study. 4HPR was well-tolerated, and no major toxicities were associated with its use. The significance of this study is limited due to small sample size. Chemoprevention studies such as this can be difficult to complete because of the commitment required of otherwise healthy individuals; nevertheless additional dosages and schedules for 4HPR administration merit further investigation.

Topics: Adenocarcinoma; Aged; Anticarcinogenic Agents; Biopsy; Feasibility Studies; Fenretinide; Humans; Male; Middle Aged; Prostatic Neoplasms

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

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) exhibits anticancer activity in vivo and triggers apoptosis in transformed cells in vitro. Thus, apoptosis induction is acknowledged as a mechanistic underpinning for 4HPR's cancer preventive and therapeutic effects. Apoptosis induction by 4HPR is routinely preceded by and dependent on the production of reactive oxygen species (ROS) in transformed cells. Very little evidence exists, outside the possible involvement of the mitochondrial electron transport chain or the plasma membrane NADPH oxidase complex, that would pinpoint the predominant site of 4HPR-induced ROS production in transformed cells. Here, we investigated the role of dihydroorotate dehydrogenase (DHODH; an enzyme associated with the mitochondrial electron transport chain and required for de novo pyrimidine synthesis) in 4HPR-induced ROS production and attendant apoptosis in transformed skin and prostate epithelial cells. In premalignant prostate epithelial cells and malignant cutaneous keratinocytes the suppression of DHODH activity by the chemical inhibitor teriflunomide or the reduction in DHODH protein expression by RNA interference markedly reduced 4HPR-induced ROS generation and apoptosis. Conversely, colon carcinoma cells that lacked DHODH expression were markedly resistant to the pro-oxidant and cytotoxic effects of 4HPR. Together, these results strongly implicate DHODH in 4HPR-induced ROS production and apoptosis.

Topics: Apoptosis; Carcinoma; Cell Line, Transformed; Cell Line, Tumor; Colonic Neoplasms; Crotonates; Dihydroorotate Dehydrogenase; Epithelial Cells; Fenretinide; Humans; Hydroxybutyrates; Male; Nitriles; Oxidoreductases Acting on CH-CH Group Donors; Prostatic Neoplasms; Reactive Oxygen Species; RNA, Small Interfering; Skin; Toluidines

Prostate cancer shows an extremely slow progression, appearing in its metastatic, hormone refractory phenotype mostly in elderly men. The chemopreventive targeting of this tumor could accordingly delay its malignancy over life expectancy. The cancer chemopreventive retinoid N-(4 hydroxyphenyl)retinamide (4HPR) has already been shown to restrain prostate cancer growth in vitro and in vivo, though its mechanisms of action are only partially explained.. We found that 4HPR impairs DU145 and PC3 prostate cancer cells migration and invasion by down-regulating FAK and AKT activation and by enhancing beta-catenin degradation, causing the downregulation of target genes like cyclin D1, survivin and VEGF. This non-migratory phenotype was similarly produced in both cell lines by stable silencing of beta-catenin. 4HPR was able to decrease AKT phosphorylation also when powerfully upregulated by IGF-1 and, consequently, to impair IGF-1-stimulated cell motility. Conversely, the expression of constitutively active AKT (myr-AKT) overcame the effects of 4HPR and beta-catenin-silencing on cell migration. In addition, we found that BMP-2, a 4HPR target with antiangiogenic activity, decreased prostate cancer cell proliferation, migration and invasion by down-regulating the pathway described involving AKT phosphorylation, beta-catenin stability and cyclin D1 expression.. These data point to 4HPR as a negative regulator of AKT phosphorylation, effectively targeting the beta-catenin pathway and inducing a relatively benign phenotype in prostate cancer cells, limiting neoangiogenesis and cell invasion.

Topics: Anticarcinogenic Agents; beta Catenin; Blotting, Western; Bone Morphogenetic Protein 2; Cell Line, Tumor; Fenretinide; Focal Adhesion Protein-Tyrosine Kinases; Gene Silencing; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR's effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca(2+)] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR's cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., rho(0) cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR's apoptogenic signaling in transformed human prostate epithelial cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line; Epithelial Cells; Fenretinide; Humans; Male; Mitochondria; Oxygen Consumption; Prostate; Prostatic Neoplasms; Reactive Oxygen Species

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR) is an aminophenol-containing synthetic retinoid derivative of all-trans-retinoic acid, which is a potent chemopreventive and antiproliferative agent against various cancers. Clinical studies of 4-HPR have shown side effects consisting of night blindness and ocular toxicity. To maintain potent anticancer activity without side effects, p-dodecylaminophenol (p-DDAP) was designed based on structure-activity relationships of 4-HPR. In our study, we investigate whether p-DDAP shows anticancer activity against human prostate cancer cell line PC-3 when compared with 4-HPR. p-DDAP inhibited PC-3 cell growth progressively from low to high concentration in a dose-dependent manner. p-DDAP was the most potent antiproliferative agent in vitro among 6 p-alkylaminophenols and 3 4-hydroxyphenyl analogs examined including 4-HPR. Cells treated with p-DDAP were shown to undergo apoptosis, based on condensation nuclei, cytofluorimetric analysis, propidium iodide staining and the expression of bcl-2 and caspase 3. p-DDAP arrested the S phase of the cell cycle, while 4-HPR arrested the G(0)/G(1) phase. In addition, both the i.v. and i.p. administration of p-DDAP suppressed tumor growth in PC-3-implanted mice in vivo. p-DDAP showed no effects on blood retinol concentrations, in contrast to reductions after 4-HPR administration. These results indicate that p-DDAP exhibits excellent anticancer efficacy against hormonal independent prostate cancer in vitro and in vivo, and it may have great potential for clinical use in the treatment of prostate cancer with reduced side effects.

Topics: Aminophenols; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Cycle; Disease Models, Animal; Fenretinide; Humans; Male; Mice; Mice, Inbred BALB C; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Vitamin A

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

Butyrates and retinoids are promising antineoplastic agents. Here we analyzed effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide (4-HPR) on prostate cancer cells as monotherapy or in combination in vitro and in vivo. Sodium butyrate and 4-HPR induced concentration-dependent growth inhibition in prostate cancer cells in vitro. The isobologram analysis revealed that sodium butyrate and 4-HPR administered together antagonize effects of each other. For the in vivo studies, a water-soluble complex (4-HPR with a cyclodextrin) was created. A single dose of sodium butyrate and 4-HPR showed a peak level in chicken plasma within 30 minutes. Both compounds induced inhibition of proliferation and apoptosis in xenografts of the chicken chorioallantoic membrane. Analysis of the cytotoxic effects of the drugs used in combination demonstrated an antagonistic effect on inhibition of proliferation and on induction of apoptosis. Prolonged jun N-terminal kinase phosphorylation induced by sodium butyrate and 4-HPR was strongly attenuated when both compounds were used in combination. Both compounds induced inhibition of NF-kappaB. This effect was strongly antagonized in LNCaP cells when the compounds were used in combination. These results indicate that combinational therapies have to be carefully investigated due to potential antagonistic effects in the clinical setting despite promising results of a monotherapy.

Topics: Animals; Butyrates; Cell Line, Tumor; Cell Proliferation; Chickens; Chorioallantoic Membrane; Dose-Response Relationship, Drug; Fenretinide; Humans; Male; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Prostatic Neoplasms; Transplantation, Heterologous

Classical isobologram analysis offers a way for analysing combined drug effects in dose-response experiments statistically. The aim is to determine as to whether two agents or drugs can be considered synergistic or antagonistic in their effect.. We describe a MATLAB-based software tool for automated isobologram analysis and computation of combination indices. Statistical issues like estimation together with respective confidence intervals are of key interest. Additional predictive values are computed to facilitate a more easy interpretation of obtained results.. Analysis of an experimental and a real in vitro data set demonstrates the approach and the way of interpreting results. Results are summarized in two ways: tables and graphical displays containing classical isobolograms.. Our package supplements the clinical software-equipment and is a tool for automatic evaluation of combined dose-response experiments in experimental oncology in the urologic clinic.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Data Interpretation, Statistical; Docetaxel; Dose-Response Relationship, Drug; Drug Interactions; Fenretinide; Humans; Inhibitory Concentration 50; Logistic Models; Male; Models, Statistical; Prostatic Neoplasms; Software; Taxoids

Prostate cancer, the most commonly diagnosed cancer among American men, develops slowly over many years. The long latent period of 20 to 30 years, involved in the multistep process of carcinogenesis, provides an important opportunity to block or reverse progression to a malignant state. Vitamin A (retinoids) and vitamin D not only have the ability to block steps in the process of carcinogenesis but they can also modulate or reverse some malignant characteristics of cancer cells. However, at high levels, vitamins A and D have undesirable side effects, thus, limiting effective dose levels and efficacy. Therefore, combination treatment at low doses, to increase efficacy and avoid toxicity, is of special interest. This study examines the effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in combination with cholecalciferol (vitamin D3) on growth, and on the expression of vimentin, matrix metalloproteinase-2 (MMP-2), and retinoid and vitamin D receptor expression, using the non-tumorigenic, human prostate epithelial cell line RWPE-1. Treatment with 4-HPR and cholecalciferol resulted in synergistic growth inhibition when compared to that caused by each agent alone. A decrease in vimentin expression and MMP-2 activity, and up-regulation of vitamin D receptor (VDR) and some of the retinoid-X (RXRs) and retinoic acid receptor (RARs) subtypes, was observed. These results suggest that combined treatment with 4-HPR and cholecalciferol, at doses lower than what might be effective with single agents, increases their efficacy and suggest that this may serve as an effective strategy for chemoprevention and treatment of prostate cancer.

Topics: Anticarcinogenic Agents; Cholecalciferol; Drug Synergism; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 2; Prostatic Neoplasms; Retinoids; Up-Regulation; Vimentin; Vitamins

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenoviridae; Animals; Apoptosis; Benzimidazoles; Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Densitometry; Disease Progression; Enzyme Activation; Epithelial Cells; Fenretinide; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; JNK Mitogen-Activated Protein Kinases; Luciferases; Male; MAP Kinase Kinase 4; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Neoplasms; Neurons; Neurosecretory Systems; Phenotype; Plasmids; Prostate; Prostatic Neoplasms; Rats; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; Synaptophysin; Tetrazolium Salts; Thiazoles; Tissue Distribution; Transcription Factors; Transcription, Genetic; Up-Regulation

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

Androgen stimulation strongly affects the sensitivity to anticancer drug-induced apoptosis in prostate cancer cells. We investigated the influence of androgen stimulation with testosterone on N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis in the androgen-sensitive prostate cancer cell line LNCaP. Overexpression of a dominant negative form of mitogen-activated protein kinase kinase 7, a specific kinase of c-jun NH(2)-terminal kinase (JNK), significantly inhibited 4-HPR-induced JNK activation and apoptosis and canceled the hormone-dependent sensitization. Testosterone activated extracellular signal-regulated kinase (ERK), activating protein-1, subsequently increased the expression of c-jun. In addition, testosterone significantly enhanced in vivo phosphorylation of c-jun by 4-HPR as well as JNK activation. Transfection with an antisense oligonucleotide of c-jun blocked 4-HPR-induced apoptosis and the testosterone-induced sensitization, suggesting a major contribution of the JNK/c-jun mediated pathway in androgen-dependent sensitization. Interestingly, inhibition of testosterone-induced activation by PD98059 also canceled an upregulation of c-jun and increased apoptosis. These results suggested that modulation of JNK activation and expression of c-jun through ERK might have been essentially involved in androgen-mediated sensitization to 4-HPR-induced apoptosis in prostate cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Enzyme Inhibitors; Fenretinide; Flavonoids; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Signal Transduction; Testosterone; Tumor Cells, Cultured

To investigate the feasibility of targeting ceramide metabolism to enhance chemotherapy cytotoxicity in prostate cancer. Discovering new targets for cancer treatment is an important endeavor, especially in prostate malignancies, which often revert to hormone- and chemotherapy-refractory disease states.. Ceramide metabolism was measured in human prostate cancer cell lines using [(3)H]palmitic acid as the tracer. Cellular lipids were analyzed by thin-layer chromatography and liquid scintillation counting. Cell viability in response to drug exposure was measured spectrophotometrically using commercial cell proliferation reagents.. LNCaP cells were five times more sensitive to N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid, compared with PC-3 cells. Ceramide levels increased only twofold in PC-3 cells versus 10-fold in LNCaP cells in response to 10 microM 4-HPR. PC-3 resistance to 4-HPR could be reversed by the addition of tamoxifen or other agents that block the metabolism of ceramide to glucosylceramide, and with tamoxifen this was marked by a ninefold increase in cellular ceramide levels. The influence of 4-HPR on ceramide metabolism was shown to be through activation of serine palmitoyltransferase, the rate-limiting enzyme in the ceramide synthesis pathway. Blocking the ceramide generated by 4-HPR reduced the extent of apoptosis.. Increasing intracellular concentrations of ceramide may be an avenue to enhance the cytotoxic response to chemotherapy in human prostate cancer.

Topics: Acyltransferases; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Survival; Ceramides; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Fenretinide; Glucosylceramides; Humans; Intracellular Fluid; Male; Oxidoreductases; Prostatic Neoplasms; Serine C-Palmitoyltransferase; Tamoxifen; 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

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to induce apoptosis in various types of tumors, including prostate cancer. We sought to examine the key mechanisms affecting the resistance to 4-HPR-induced apoptosis in three human prostate cancer cell lines, PC-3, DU145, and LNCaP. Concentrations of more than 40 microM 4-HPR produced apoptosis to almost the same extent in all cell lines; however, only the LNCaP line remained highly sensitive to concentrations less than 10 microM. These differing sensitivities at low concentrations correlated well with the level of constitutive activation of nuclear factor kappa B (NFkappaB) in the individual cell lines. We found that NFkappaB activation inhibited c-jun NH(2)-terminal kinase and caspase 3 activation induced by 4-HPR and that NFkappaB inhibition by the I kappa B alpha phosphorylation inhibitor compound Bay 117082 resulted in increasing sensitization of both PC-3 and DU145 lines to apoptosis induced by 4-HPR at low concentrations. Furthermore, we found that inhibition of extracellular signal-regulated kinase (ERK) enhanced the suppression of NFkappaB by 4-HPR and also resulted in sensitization to apoptosis in the DU145 cell line, in which ERK is activated constitutively. It thus appears that mitogen-activated protein kinase associated with the activity of NFkappaB plays an important role in the degree of resistance to 4-HPR-induced apoptosis in human prostate cancer cells.

Topics: Antineoplastic Agents; Apoptosis; beta-Galactosidase; Caspase 3; Caspases; Cell Survival; Drug Resistance, Neoplasm; Electrophoretic Mobility Shift Assay; Fenretinide; Humans; I-kappa B Kinase; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 6; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Organic Chemicals; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Sulfones; Tumor Cells, Cultured

To explore the mechanisms underlying the pro-apoptotic effects of the synthetic retinoid N-4-(hydroxyphenyl)retinamide (4-HPR) on LNCaP human prostate cancer cells, we used the differential display-polymerase chain reaction (DD-PCR) technique to identify 4-HPR-responsive genes. RNA extracted from LNCaP cells that had been treated for 24 h with 4-HPR at a dose (2.5 microM) optimal for apoptosis induction was used for DD-PCR analysis using random primers. A differentially expressed 115 bp fragment was cloned and sequenced and then identified in GenBank as having a high degree of homology with several members of the cyclophilin gene family. Northern blot analyses using specific probes for cyclophilin A, cyclophilin D, and the cloned 115-bp fragment were performed on RNA extracted from LNCaP cells and MCF-7 human breast cancer cells treated with 4-HPR, N-acetylcysteine (NAC, an anti-oxidant), 4-HPR plus NAC, cyclosporin A, R-1881 (a synthetic androgen), dehydroepiandrosterone, all-trans retinoic acid, or prednisone. 4-HPR downregulated the transcript detected by the 115-bp fragment. Expression patterns detected by the 115-bp fragment and cyclophilin D probes were identical in response to each treatment; none of these treatments affected cyclophilin A expression. Furthermore, expression of mRNA transcripts detected by the 115-bp fragment and cyclophilin D probes correlated with the generation of reactive oxygen species (ROS), as detected by measurement of 2,7-dichlorofluorescein oxidation. Therefore, members of the cyclophilin gene family, such as cyclophilin D (a component of the mitochondrial permeability transition pore previously linked with oxidative stress and apoptosis), may play a role in the ROS-mediated apoptotic effects of 4-HPR.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Northern; Breast Neoplasms; Cyclophilins; Fenretinide; Gene Expression Profiling; Gene Expression Regulation; Humans; Male; Neoplasm Proteins; Polymerase Chain Reaction; Prostatic Neoplasms; Reactive Oxygen Species; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The development of prostate cancer through a multistep process of carcinogenesis may have a long latent period of 20-30 years. It is possible that progression to a malignant state could be blocked or reversed during this time. This study focuses on the ability of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide (4-HPR), to reverse changes associated with malignant transformation and tumor progression, towards a normal phenotype. To examine the responsiveness of cells at different steps of prostate carcinogenesis, three immortalized, but non-tumorigenic (RWPE-1, WPE1-7 and WPE1-10), and one human prostate carcinoma cell line (DU-145), were used. The effects of 4-HPR on cell proliferation, expression of intermediate filament proteins cytokeratin 18 and vimentin, and tumor suppressor proteins p53 and pRb were examined by immunostaining and compared. Results show that 4-HPR caused inhibition of growth in all cell lines in a dose-dependent manner. 4-HPR induced an increase in staining for cytokeratin 18, a marker of differentiation for prostate epithelial cells. While all cell lines showed strong immunostaining for vimentin, treatment with 4-HPR for 8 days caused a marked decrease in staining for vimentin in all cell lines. In an in vitro assay, 4-HPR also caused inhibition of invasion by DU-145 cells in a dose-dependent manner. Furthermore, 4-HPR treatment was effective in significantly decreasing the abnormal nuclear staining for the tumor suppressor proteins p53 and pRb. Because 4-HPR decreased invasion-associated vimentin expression, inhibited invasion, and normalized p53 and pRb immunostaining, we propose that 4-HPR may be an effective agent for secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

Topics: Anticarcinogenic Agents; Cell Count; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fenretinide; Humans; Immunoenzyme Techniques; Keratins; Male; Neoplasm Invasiveness; Phenotype; Prostate; Prostatic Neoplasms; Retinoblastoma Protein; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vimentin

Previous investigations have demonstrated that a synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR), inhibits the invasion of prostate adenocarcinoma in vitro. Urokinase-type plasminogen activator (uPA) is a prerequisite for tumor invasion. The purpose of this study was to evaluate the effects of 4-HPR on uPA and plasminogen activator inhibitor-1 (PAI-1) in prostate cancer. Human prostate adenocarcinoma cell lines, TSU-PR1 and PC3, were grown in serum-free media containing 4-HPR. Cellular mRNA and protein were subsequently extracted. Northern blot analysis, enzyme-linked immunosorbent assay (ELISA) and chromogenic functional analysis were performed on the samples. Administration of 10(-6) M 4-HPR for 3 days resulted in an increase in uPA mRNA expression (TSU-PR1: 391%, PC3: 356%), and a simultaneous increase in PAI-1 mRNA expression (TSU-PR1: 217%, PC3: 235%) was observed. ELISA concomitantly demonstrated a significant increase (p < 0.05) in uPA protein in the conditioned media (TSU-PR1: 134%, PC3: 139%) and cell lysates (TSU-PR1: 284%, PC3: 255%). Both cell lines demonstrated a significant increase (p < 0.05) in PAI-1 protein in the conditioned media (TSU-PR1: 152%, PC3: 167%) and cell lysates (TSU-PR1: 170%, PC3: 222%). Concentrations below 10(-6) M failed to alter the protein production of either uPA or PAI-1. The functional uPA assay demonstrated a reduction of the proteolytic activity of uPA (TSU-PR1: 13%, PC3: 7%) in cell lysates of 10(-6) M 4-HPR (p < 0.05), while there was minimal uPA activity in the conditioned media. 4-HPR stimulates a paradoxical increase in uPA and PAI-1, but the anti-invasive effects of 4-HPR are consistent with the increase in both uPA and PAI-1, resulting in an overall reduction of functional uPA activities.

Topics: Adenocarcinoma; Antineoplastic Agents; Cycloheximide; Fenretinide; Gene Expression; Humans; Male; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; Protein Synthesis Inhibitors; RNA Stability; RNA, Messenger; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/-) or nullizygous (-/-) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/- or p53 -/- MPRs. For, p53 +/- MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P = 0.0477, t-test). Notably, in p53 +/- MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P = 0.035, chi2-test). In p53 -/- MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 -/- animals harbored bone metastases (P = 0 < 0.05, chi2-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/- and p53 -/- MPRs revealing a novel and potentially clinically useful activity of this retinoid.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Division; Diet; Disease Models, Animal; Fenretinide; Male; Mice; Prostatic Neoplasms; Survival Rate; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been shown to induce apoptosis in various malignant cells including human prostate carcinoma cells (HPC). We examined several possible mechanisms by which 4HPR could induce apoptosis in HPC cells. 4HPR exhibited concentration- and time-dependent decrease in cell number both in androgen-dependent (LNCaP) and -independent (DU145 and PC-3) cells. The 4HPR concentrations causing 50% decrease in cell number in LNCaP, DU145, and PC-3 cultures were 0.9 +/- 0.16, 4.4 +/- 0.45, and 3.0 +/- 1.0 microM, respectively, indicating that LNCaP cells were more sensitive to 4HPR than the other cells. 4HPR-induced apoptosis in all three cell lines was evidenced by increased enzymatic labeling of DNA breaks and formation of a DNA ladder. 4HPR increased the level of reactive oxygen species, especially in LNCaP cells. 4HPR-induced apoptosis could be suppressed in LNCaP cells by antioxidant and in DU145 cells by a nuclear retinoic acid receptor-specific antagonist, suggesting the involvement of reactive oxygen species or retinoic acid receptors in mediating apoptosis induced by 4HPR in the different HPC cells. Furthermore, 4HPR modulated the expression levels of some apoptosis-related gene (p21, c-myc, and c-jun), which may also contribute to the induction of apoptosis by 4HPR in HPC cells.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Fenretinide; Gene Expression; Humans; Male; Oncogenes; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Retinoic Acid; Tumor Cells, Cultured; Tumor Suppressor Protein p53

To explore the mechanisms underlying the chemopreventive effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in prostate cancer, we evaluated the anti-proliferative and apoptosis-inducing effects of 4-HPR in the androgen-sensitive human prostate cancer cell line LNCaP. 4-HPR decreased the number of viable LNCaP cells (as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in a dose-dependent manner. Although 4-HPR exerted a modest G1 cell-cycle block (as determined by flow cytometry), its effect on reduced cell number appeared to result primarily from induction of apoptosis (as measured by an enzyme-linked immunosorbent assay and flow-cytometric assays). The mitogenic effects of R1881, a non-metabolizable androgen that potently induces LNCaP cell proliferation, was completely blocked by greater than 0.5 microM 4-HPR. Furthermore, increasing the R1881 concentration in the presence of 2.0 microM 4-HPR increased apoptotic cell death. 4-HPR decreased prostate-specific antigen (PSA) protein levels in conditioned medium and decreased PSA mRNA expression. 4-HPR also decreased the ratio of bcl-2 to bax mRNA expression in LNCaP cells by approximately 45%, indicating that the apoptotic effects of 4-HPR may be mediated, at least in part, by alterations in the bcl-2/bax-regulated apoptotic pathway. N-acetylcysteine (4 mM) completely blocked the anti-proliferative and apoptotic-inducing effects of 4-HPR, suggesting that an oxidative mechanism may be involved. We concluded that (i) 4-HPR exerts growth-suppressive and apoptotic effects on LNCaP cells, (ii) 4-HPR can interact with androgen to suppress proliferation and induce apoptosis, (iii) the apoptotic effects of 4-HPR may be mediated in part by the bcl-2/bax pathway, and (iv) a pro-oxidant mechanism may contribute to the anti-proliferative and apoptotic-inducing effects of 4-HPR.

Topics: Androgens; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Cycle; DNA, Neoplasm; Drug Screening Assays, Antitumor; Fenretinide; Gene Expression Regulation; Genes, bcl-2; Humans; Male; Metribolone; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

A long latent period of 20 to 30 years may be involved in the multistep process of carcinogenesis represented by prostatic intraepithelial neoplasia (PIN) in the prostate. It is, therefore, possible that progression to a malignant state could be blocked or reversed during this time. Retinoids not only have the ability to block steps in the process of carcinogenesis but they may also modulate or reverse some malignant characteristics of cancer cells. This study focuses on the ability of N-(4-hydroxyphenyl)-retinamide (4-HPR), a synthetic retinoid, to reverse malignant characteristics towards a normal phenotype, using the human prostate carcinoma cell line DU-145. These malignant characteristics include abnormal cell proliferation, intermediate filament expression, motility, invasion, and cell survival. Results show that 1 microM and 10 microM 4-HPR caused 31% and 96% inhibition of growth, while all-trains retinoic acid (ATRA) produced similar effects at 10 and 100 microM, making 4-HPR ten times more effective than ATRA. While DU-145 cells show strong immunostaining for vimentin, treatment with 1 microM 4-HPR for eight days caused a marked decrease in vimentin staining. This was accompanied by a change from an elongated to an epithelial cell morphology. Densitometric analysis of Western blots for vimentin showed a 53% decrease in vimentin expression in 1 microM 4-HPR treated cells. Concomitant with the decrease in vimentin expression, cell motility and invasive ability also decreased by 32% and 52%, respectively. Growth inhibition was accompanied by DNA fragmentation and apoptosis. Exposure of cells to 1 microM 4-HPR caused a marked upregulation of nuclear retinoid receptors RARalpha and a detectable expression of RARgamma. These results suggest that inhibition of growth and vimentin expression, and induction of apoptosis by 4-HPR in prostate cancer cells may occur via a receptor-mediated mechanism involving transrepression of AP-1 by retinoid receptors. We propose that vimentin may serve as a useful intermediate marker for early detection of prostate cancer in biopsy specimens and that 4-HPR may be effective in blocking several steps in prostate carcinogenesis as well as the progression of PIN to invasive carcinoma.

Topics: Apoptosis; Carcinoma; Cell Division; Cell Movement; Epithelial Cells; Fenretinide; Humans; Male; Neoplasm Invasiveness; Phenotype; Prostatic Neoplasms; Receptors, Retinoic Acid; Tumor Cells, Cultured; Up-Regulation; Vimentin

The influence of chemical carcinogen, hormonal stimulation, and chronic dietary administration of the synthetic retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), on the induction of prostate cancer in male Wistar-Unilever rats was determined. Three different tumor induction regimens were used: (a) a single i.v. dose of 50 mg of N-methyl-N-nitrosourea (MNU) per kg body weight, followed by chronic androgen stimulation via s.c. implantation of two silastic capsules containing 40 mg testosterone each; (b) a single i.v. dose of 50 mg of MNU per kg body weight (no testosterone treatment); and (c) chronic androgen stimulation with implanted testosterone capsules (no MNU treatment). In a fourth series of animals, the incidence of spontaneous prostate tumors was determined in groups of rats receiving neither carcinogen nor hormone stimulation. Within each series, parallel groups of animals were fed a control (vehicle-supplemented) diet or control diet supplemented with 4-HPR beginning 1 day after carcinogen administration; retinoid administration was continuous until termination of the study at 450 days. The incidence of accessory sex gland cancer in rats treated sequentially with MNU + testosterone was >60%, in comparison with cancer incidences of <20% in rats receiving MNU only and <5% in rats treated with testosterone only. No spontaneous accessory sex gland tumors were observed in rats receiving no carcinogen and no testosterone. Tumor induction in the accessory sex glands by MNU + testosterone was relatively specific for the prostate: the incidence of carcinoma of the dorsolateral/anterior prostate was more than 5-fold greater than the incidence of cancer present only in the seminal vesicle. 4-HPR conferred no protection against cancer induction in the prostate by any regimen of MNU and/or testosterone. These results demonstrate the importance of both carcinogen exposure and hormone stimulation on the induction of neoplasia in the prostate of Wistar-Unilever rats.

Topics: Animals; Anticarcinogenic Agents; Body Weight; Carcinogens; Cocarcinogenesis; Fenretinide; Male; Methylnitrosourea; Prostatic Neoplasms; Rats; Rats, Wistar; Testosterone

N-(4-Hydroxyphenyl)retinamide (4-HPR, Fenretinide) is a retinoid derivative with antineoplastic activity in various tumor types including prostate carcinoma. The mechanism of action of 4-HPR toxicity is unknown. 4-HPR induces apoptosis in leukemia- and lymphoma-derived cells, neuroblastoma, and small cell lung cancers. The present study was designed to investigate: (a) the mechanism of 4-HPR cytotoxicity in prostate cancer cells; and (b) correlate increased expression of transforming growth factor beta 1 (TGF beta 1) with induction of apoptosis. 4-HPR exposure to PC-3 cells in vitro was associated with apoptosis as evidenced by increased incidence of hypodiploid nuclei in propidium iodide fluorescence histograms and DNA fragmentation. An increase in the percentage of nuclei in the G1 phase of the cell cycle preceded induction of apoptosis. TGF beta 1-increased expression was noted in mRNA levels and in secretion of active TGF beta 1 into culture media. TGF beta 1 and TGF-beta receptor type II detected immunohistochemically were increased in 4-HPR-treated PC-3 cells. Furthermore, 4-HPR-induced cytotoxicity in PC-3 cells was abrogated by the addition of anti-TGF beta 1 antibody. In BT-20 cells, a 4-HPR-resistant breast carcinoma cell line, apoptosis was not observed after exposure to 4-HPR nor was TGF beta 1 expression enhanced in stained cells or in conditioned media. It is concluded that 4-HPR induces the expression of TGF beta 1 in association with the induction of apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Female; Fenretinide; Flow Cytometry; Humans; Immunohistochemistry; Male; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Although fenretinide (4-HPR) is currently being evaluated in a phase II clinical study for the chemoprevention of prostate cancer [Greenwald et al.: CA 45:31-49, 1995], the mechanism underlying its antineoplastic activity has not been elucidated.. Androgen-dependent human prostatic LNCaP cells cultured with fetal bovine serum (FBS) were treated with 4-HPR and evaluated for effects on cell growth and cell cycle phase distribution, induction of apoptosis, and changes in proliferating cell nuclear antigen (PCNA), prostate-specific antigen (PSA), and androgen receptor (AR) levels.. LNCaP cells treated with 4-HPR for 6 days showed 82-95% suppression of cell growth, with accompanying time- and dose-dependent downregulation of PCNA, a partial arrest in G1 phase of the cell cycle, and a marked increase in the percentage of apoptotic cells. Apoptosis was demonstrated by the characteristic DNA fragmentation pattern seen on agarose gels, and by flow cytometric analysis. 4-HPR-induced prostate-specific phenotype changes included significant downregulated expression of both intracellular and secreted forms of PSA, which were preceded by a reduction of AR expression.. These data suggest that 4-HPR acts as a pleiotropic effector of prostate cell growth and specific gene expression.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cattle; Cell Cycle; Cell Division; Cell Survival; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Down-Regulation; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Male; Proliferating Cell Nuclear Antigen; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Tumor Cells, Cultured

A significant reduction in JCA-1 cell proliferation was observed as a result of treatment with 4-HPR. This was accompanied by the down-regulated expression of the proliferating cell nuclear antigen PCNA, cyclins D and E, and p34cdc2. Unexpectedly, p53 and pRB was also suppressed in response to 4-HPR. A prolonged exposure to 4-HPR (6 days) promoted the appearance of non-adherent cells showing morphological features and DNA fragmentation patterns indicative of apoptosis.

Topics: Androgens; Antineoplastic Agents; Apoptosis; Cell Division; Cyclins; Down-Regulation; Fenretinide; Humans; Male; Mitogens; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Tumor Cells, Cultured

We have developed a grading system for the evaluation of the histogenesis of neoplastic lesions of the prostate and seminal vesicle of the laboratory rat. Prostatic and seminal vesicle carcinomas were induced in Lobund-Wistar rats by initiation with 30 mg/kg N-nitroso-N-methylurea i.v., followed by promotion with 40 mg testosterone propionate implants 1 week later and at 3-month intervals thereafter. Experimental and control groups were sacrificed at various time points between 5 and 11 months after dosing with N-nitroso-N-methylurea in order to visualize progressive stages of carcinogenesis of the dorsolateral prostate, the anterior prostate, and the seminal vesicle. A system of staging was created which allows three different categories (in situ change, invasion, desmoplasia) of tumor development to be ranked progressively in a manner conducive to nonparametric analysis. Each category was then further subdivided to create a total of six stages. This system can be used to evaluate agents which modify tumor induction or suppression. The application of this staging system to the measurement of the effects of the synthetic retinoid, 4-hydroxyphenyl retinamide, on prostatic carcinogenesis in the Lobund-Wistar rat is described.

Topics: Animals; Evaluation Studies as Topic; Fenretinide; Genital Neoplasms, Male; Male; Methylnitrosourea; Neoplasm Staging; Prostatic Neoplasms; Rats; Rats, Wistar; Seminal Vesicles; Testosterone

The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) has been demonstrated to inhibit the development of primary and metastatic neoplasms in several animal models. In order to investigate the effect of 4-HPR on human prostate adenocarcinoma, we designed a series of in vitro experiments with the PC3 cell line to evaluate effects on proliferation, cell cycle kinetics, and c-myc mRNA expression. 4-HPR demonstrated cytotoxicity only at the supraphysiologic concentration of 10.0 microM. However, asynchronously growing cells exposed to 1 microM 4-HPR demonstrated a 51% reduction in proliferation rate, associated with an accumulation of cells in the G0/G1 phase of the cell cycle. PC3 cells synchronized with serum deprivation or aphidicolin exhibited significant decreases in DNA synthesis when treated with 1 microM 4-HPR. Additionally, these cells were found to accumulate in G0/G1 and S phase. Northern blots indicated a significant decrease in c-myc mRNA expression in asynchronously growing cells with continuous administration of 1 microM 4-HPR for 6 days. These data suggest that 4-HPR can inhibit growth of PC3 cells as a consequence of a block in cell cycle transition from G1 to S phase at a concentration of 1 microM, and that this inhibition is associated with a suppression of c-myc gene expression.

Topics: Adenocarcinoma; Antineoplastic Agents; Blotting, Northern; Cell Cycle; Cell Division; Cell Line; DNA, Neoplasm; Fenretinide; Flow Cytometry; Gene Expression; Genes, myc; Humans; Male; Prostatic Neoplasms; RNA, Messenger; Thymidine; Time Factors; Tumor Cells, Cultured

N-4-Hydroxyphenylretinamide (fenretinide or 4HPR), a derivative of retinoic acid, has been demonstrated to decrease the development of prostate cancer in a rat carcinogenesis model. This study was undertaken to determine if 4HPR is an effective agent for the treatment of established prostate cancer. In vitro, 4HPR was cytotoxic to rat and human prostate cancer cells as well as endothelial cells. Utilizing three different angiogenesis inhibition assays, it was demonstrated that 4HPR inhibited angiogenesis as well as endothelial cell motility and tubule formation. In vivo, 4HPR inhibited prostate cancer growth in a significant manner. These findings suggest that 4HPR may be a potent inhibitor of early prostate cancer growth.

Topics: Adenocarcinoma; Animals; Cell Division; Endothelium, Vascular; Fenretinide; Humans; In Vitro Techniques; Male; Neoplasm Transplantation; Neovascularization, Pathologic; Prostatic Neoplasms; Rats; Tumor Cells, Cultured

Several epidemiological studies have implicated low dietary and serum levels of retinol with an increased risk for the development of human prostate cancer. In a recent report, dietary fenretinide [N-[(4-hydroxyphenyl)] retinamide], a synthetic retinoid with low toxicity, decreased the incidence of experimentally induced prostate cancer. Fenretinide is currently being evaluated in phase I and phase II clinical trials as an agent for both the treatment and chemoprevention of human prostate cancer. Because of these findings, we investigated whether dietary fenretinide could alter the incidence of phenotype of oncogene-induced prostate cancer in the mouse prostate reconstitution model system. When compared to control-fed animals, dietary fenretinide reduced the tumor incidence by 49% and the tumor mass by 52% of ras+myc-induced cancers in the mouse prostate reconstitution model system, which was modified to prolong the latency period before cancer development. Retinoids have a wide ranging effect on cellular differentiation, growth factor synthesis, and immune function. While its mechanism of action in this system remains unclear, fenretinide is an effective agent for the chemoprevention and growth modulation of oncogene-induced prostate cancer in the mouse prostate reconstitution model system and may be effective for the chemoprevention of human prostate cancer.

Topics: Animals; Anticarcinogenic Agents; Cell Transformation, Neoplastic; Diet; Fenretinide; Fetus; Genes, myc; Genes, ras; Genetic Vectors; Male; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Transfection

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