metribolone and Carcinoma

Androgen receptor (AR) signals have been implicated in bladder carcinogenesis and tumor progression. Activation of Wnt/β-catenin signaling has also been reported to correlate with bladder cancer progression and poor patients' outcomes. However, cross talk between AR and β-catenin pathways in bladder cancer remains uncharacterized. In radical cystectomy specimens, we immunohistochemically confirmed aberrant expression of β-catenin especially in aggressive tumors. There was a strong association between nuclear expressions of AR and β-catenin in bladder tumors (P=0.0215). Kaplan-Meier and log-rank tests further revealed that reduced membranous β-catenin expression (P=0.0276), nuclear β-catenin expression (P=0.0802), and co-expression of nuclear AR and β-catenin (P=0.0043) correlated with tumor progression after cystectomy. We then assessed the effects of androgen on β-catenin in AR-positive and AR-negative bladder cancer cell lines. A synthetic androgen R1881 increased the expression of an active form of β-catenin and its downstream target c-myc only in AR-positive lines. R1881 also enhanced the activity of β-catenin-mediated transcription, which was abolished by an AR antagonist hydroxyflutamide. Using western blotting and immunofluorescence, R1881 was found to induce nuclear translocation of β-catenin when co-localized with AR. Finally, co-immunoprecipitation revealed androgen-induced associations of AR with β-catenin or T-cell factor (TCF) in bladder cancer cells. Thus, it was likely that androgen was able to activate β-catenin signaling through the AR pathway in bladder cancer cells. Our results also suggest that activation of β-catenin signaling possibly via formation of AR/β-catenin/TCF complex contributes to the progression of bladder cancer, which may enhance the feasibility of androgen deprivation as a potential therapeutic approach.

Topics: Aged; Aged, 80 and over; Androgens; beta Catenin; Carcinoma; Cell Line, Tumor; Female; Humans; Male; Metribolone; Middle Aged; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; TCF Transcription Factors; Urinary Bladder Neoplasms

Glycoprotein transmembrane nmb (GPNMB) gene was originally identified in osteoblasts and belongs to the pmel-17/nmb family. The function or regulation of GPNMB in the human prostate remains unknown.. The expression of GPNMB in prostate carcinoma cells were determined by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and immunoblot assays. Effects of ectopic GPNMB overexpression on cell proliferation, invasion, and tumorigenesis were determined by (3) H-thymidine incorporation, matrigel invasion, soft agar cloning assays, and murine xenograft study. Effects of GPNMB, p53, and androgen on target gene were assessed using RT-PCR, immunoblotting, and transient gene expression assays.. In vitro analysis using several prostate cell lines suggested that expression of GPNMB may be relevant to the extent of neoplasia. Ectopic overexpression of GPNMB significantly attenuated cell proliferation and invasion and exerted antitumorigenic activity on PC-3 cells in vitro and in vivo. GPNMB overexpression induced the gene expressions of N-myc downstream regulated gene 1 (Ndrg1) and maspin in PC-3 cells. Doxorubicin treatment or transient overexpression of p53 increased GPNMB expression. Androgen (R1881) treatment has a divergent effect on gene expression of prostate-specific antigen (PSA) and GPNMB in LNCaP cells. Androgen treatment enhanced cell proliferation but downregulated GPNMB protein expression in stably overexpressed androgen receptor (AR) CA-HPV-10 cells.. Together these results suggest that GPNMB gene is a p53- and androgen-dysregulated gene and should be regarded as an anti-tumor gene for prostate cancer. The enhancement of Ndrg1 and maspin gene expressions may account for the anti-proliferative and anti-invasive function of GPNMB in PC-3 cells.

Topics: Animals; Carcinoma; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Metribolone; Mice; Neoplasm Invasiveness; Prostatic Neoplasms; Testosterone Congeners

Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive.. Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility.. The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.

Topics: 3T3-L1 Cells; Androgens; Animals; Carcinoma; Cell Movement; Cells, Cultured; Chlorocebus aethiops; Contractile Proteins; COS Cells; Filamins; Humans; Integrin beta1; Male; Metribolone; Mice; Microfilament Proteins; Neoplasm Metastasis; NIH 3T3 Cells; Prostatic Neoplasms; Protein Binding; Receptors, Androgen

Most prostate cancers escape endocrine therapy by diverse mechanisms. One of them might be growth repression by androgen. We reported that androgen represses the growth in culture of MOP cells (a sub-line of LNCaP cells) and that of MOP cell xenografts, although tumor growth becomes androgen-independent (AI). Here we explore whether AI tumors contain androgen-responsive cells. ME carcinoma cells were established from AI tumors. The responses to androgen were examined by cell counting, DAPI labeling, flow cytometry, PSA immunoassay and tumor size follow-up. Androgen receptors (AR) were analyzed by western blotting and DNA sequencing. The pattern of responses of these cells to androgen was compared to that of MOP cells and that of JAC cells established from LNCaP-like MOP cells. R1881, a synthetic androgen: (1) repressed the growth of all the six ME cell lines obtained, MOP and JAC cells, (2) augmented the secretion of PSA, (3) induced spectacular cell bubbling/fragmentation and (4) blocked the cell cycle and induced a modest increase of apoptosis. All the androgen-repressed cells expressed the same level of mutated AR as LNCaP cells. In nude mice, the growth of ME-2 cell xenografts displayed transient androgen repression similar to that of MOP cells. In culture neither fibroblasts nor extra-cellular matrix altered the effects of R1881 on cell proliferation. These results demonstrate that androgen-independent tumors contain androgen-responsive cells. The apparent discrepancy between the responses to androgen of tumors and those of carcinoma cells in culture suggests that microenvironmental factors contribute to the androgen responsiveness of tumor cells in vivo. These modifications, albeit unspecified, could be suitable targets for restoring the androgen responsiveness of AI tumors.

Topics: Androgens; Animals; Carcinoma; Cell Line, Tumor; Female; Fluorescent Antibody Technique, Indirect; Growth Inhibitors; Humans; Male; Metribolone; Mice; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Time Factors; Transplantation, Heterologous; Xenograft Model Antitumor Assays

Vitamin D has been suggested as a chemopreventive and therapeutic modality for prostate cancer. However, hypercalcemic toxicity has limited the use of 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in clinical trials, prompting the search for analogs of vitamin D with less toxicity while retaining efficacy as a modality for cancer intervention. In this study, the less hypercalcemic vitamin D analog 1alpha,24-dihydroxyvitamin D(2) (1,24-(OH)(2)D(2)) was examined for its effects on cellular growth inhibition and differentiation induction in the LNCaP human prostate carcinoma cell line.. LNCaP cell growth was determined by quantifying DNA levels. Protein levels were determined using the ELISA method and immunoblotting. Levels of mRNA were determined using real-time quantitative reverse transcriptase PCR.. LNCaP growth was decreased 50% by exposure to 0.01 nM 1,24-(OH)(2)D(2) after 96 hr in the presence of a growth stimulatory 0.1 nM dose of the androgen R1881. Prostate specific antigen (PSA) levels were increased 3.5-fold with 10 nM 1,24-(OH)(2)D(2) treatment compared to a 1.9-fold increase in PSA levels found with 10 nM 1,25-(OH)(2)D(3) under low androgen conditions. Neither 1,24-(OH)(2)D(2) nor 1,25-(OH)(2)D(3) affected the expression of cytokeratin 18 protein levels. Treatment with 10 nM 1,24-(OH)(2)D(2) alone produced a 1.3-fold increase in AR mRNA and a 2.2-fold increase in AR protein levels after 96 hr. Surprisingly, the addition of 1.0 nM R1881 alone or in combination with 10 nM 1,24-(OH)(2)D(2) produced an approximately 60% decrease in AR mRNA, whereas AR protein levels were increased 1.6-fold.. Overall, 1,24-(OH)(2)D(2) was found to be at least as effective as 1,25-(OH)(2)D(3) at inhibiting growth and inducing differentiation markers in LNCaP prostate carcinoma cells and may thus prove useful in prostate cancer treatment.

Topics: Blotting, Western; Calcitriol; Carcinoma; Cell Differentiation; Cell Division; DNA, Neoplasm; Ergocalciferols; Humans; Male; Metribolone; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Testosterone Congeners; Tumor Cells, Cultured

Heterogeneity in human androgen receptor (hAR) expression in prostate cancer is considered to be implicated in tumor progression. hAR expression was therefore studied immunohistochemically in localized and locally progressive, hormone refractory (HR) prostate cancer. Because altered functional activity of the hAR may be due to changes in the structural integrity of the hAR gene, exons 2 to 8 of the hAR gene were assessed for mutations by single-strand conformation polymorphism (SSCP) analysis and exon 1 was analyzed for the size of the CAG repeat. The hormone binding capacity, a prerequisite for ligand-regulated receptor function, was determined by a ligand binding assay. Coexpression of the hAR and prostate-specific antigen (PSA) was studied by a sequential double immunoenzymatic staining to verify whether PSA expression is a parameter of hAR function. Almost all human prostatic carcinomas revealed heterogeneous hAR expression, regardless of tumor differentiation and progression. Putative predominance of hAR-negative tumor areas in HR prostate cancer was not observed. No hAR gene mutations or major changes in the CAG repeat were found in the 18 HR carcinomas or in the 9 control samples. Moreover, all selected hAR-expressing cancers were able to bind the synthetic androgen methyltrienolone (R1881). Immunoenzymatic double staining revealed even PSA expression in hAR-negative tumor areas. PSA immunohistochemistry in human prostatic carcinomas therefore is of no use in determining hAR functional activity. Thus, most prostatic carcinomas, even when progressed to a state of hormone insensitivity, contain a structurally intact hAR gene, heterogeneously expressed with retained androgen binding capacity.

Topics: Antibodies, Monoclonal; Base Sequence; Carcinoma; DNA Primers; Exons; Gene Expression; Hormones; Humans; Hyperplasia; Immunoenzyme Techniques; Male; Metribolone; Molecular Sequence Data; Mutation; Prostate; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Receptors, Androgen; Repetitive Sequences, Nucleic Acid

An androgen-independent, transplantable prostate carcinoma line (AIT), originally derived from the dorsolateral prostate (DLP) of Noble rat, was implanted into orchiectomized Noble rats and its response to androgen stimulation was studied and compared to that of the regenerating DLP tissue in sexually ablated rats. AIT tumors carried in castrated hosts displayed a high basal level of proliferative activity (mitotic index (MI), 15.0 +/- 0.5) while DLP tissue in untreated castrates exhibited no proliferative activity. Following androgen stimulation by testosterone capsule implantation into host rats, the AIT responded with a marked increase in cell proliferation; MI values doubled to 30.0 +/- 2.9 on Day 5 following androgen stimulation. This androgen-induced increase in MI values was coincident with elevations in nuclear androgen receptor (20-fold increase) and 5 alpha-dihydrotestosterone content (3-fold increase) in the tumor. However, by Day 10 following androgen treatment, indices of cell proliferation in the AIT declined to pre-androgen-stimulated levels (MI, 14.8 +/- 1.9) despite the continued elevations in nuclear androgen receptor and tissue 5 alpha-dihydrotestosterone contents. Parallel changes in MI were also observed in the normal regenerating DLP following androgen stimulation. MI values in this tissue increased from nondetectable levels to 38.1 +/- 4.7 on Day 5 but declined to relatively low levels (4.5 +/- 0.9) by Day 10 following androgen replacement. Taken together these findings led us to conclude that the AIT carried in castrates is capable of responding to testosterone in a manner similar to that observed for androgen-stimulated DLP of sexually ablated rats. Thus, in both the neoplastic and regenerating tissues, the initial response to androgen is characterized by a marked enhancement of cell proliferation which was correlated with an increase in androgen receptor and 5 alpha-dihydrotestosterone content. However, like its tissue of origin, the AIT possesses mechanisms which act to limit androgen-induced cell division despite continued elevations in key parameters of androgen activation.

Topics: Animals; Carcinoma; Cell Division; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Male; Metribolone; Mitotic Index; Orchiectomy; Prostatic Neoplasms; Rats; Receptors, Androgen; Testosterone

Primary endometrial carcinomas from 35 non-treated patients were investigated by measurement of androgen receptors in the tumor cytosols. Receptor analysis was done with a labelled synthetic androgen, methyltrienolone 3H-R1881 and triamcinolone acetonide, and dextran-coated charcoal absorption. The concentrations of androgen receptors in the endometrial carcinomas were, in decreasing order: highly-differentiated tumors, 15.7 +/- 1.8 (fmol/mg protein, mean +/- SE) (number of cases, 21); moderately differentiated tumors, 4.6 +/- 1.6 (n = 7); poorly differentiated tumors, undetectable in 7 out of 8 cases. Highly-differentiated tumors contained a much greater concentration of receptors than the two less differentiated ones. One highly-differentiated endometrial carcinoma with pyometra virtually lacked the receptor. The moderately differentiated endometrial carcinomas contained very low levels of the receptors. The poorly differentiated tumors virtually lacked the receptors. Metastatic lymph nodes from primary endometrial carcinomas with moderate differentiation had a very low receptor level. From these results, it is concluded that human endometrial carcinomas, particularly with histologically high differentiation, contain a considerable amount of androgen receptor and that the receptor concentrations appear to correlate with the histologic grade of tumor differentiation.

Topics: Adult; Aged; Carcinoma; Cytosol; Estrenes; Female; Humans; Metribolone; Middle Aged; Receptors, Androgen; Receptors, Steroid; Triamcinolone Acetonide; Uterine Neoplasms