mibolerone and Prostatic-Neoplasms

Prostate cancer (PCa) is the most commonly diagnosed male cancer in the United States and is a hormone-driven disease. Androgens have been recognized as a major promoter of PCa development and progression. However, the mechanism of androgen action in PCa, especially in PCa cell invasion and metastasis remains largely unclear. SMAD ubiquitination regulatory factor-1 (SMURF1) is a C2-WW-HECT-domain E3 ubiquitin ligase that plays important roles in cancer cell metastasis. Whether there is a relationship between androgens and SMURF1 expression is not known.. The effect of androgens on the expression of SMURF1 in PCa cell lines was examined by Western blot analyses and reverse transcription-polymerase chain reaction (RT-PCR). Gene transfection was performed by electroporation to manipulate the expression levels of proteins studied. The binding of AR to the SMURF1 gene enhancer was determined by chromatin immunoprecipitation (ChIP) assay. Cell migration and invasion was measured by wound healing and Matrigel invasion assays, respectively.. We found that expression of SMURF1 is upregulated by androgens in PCa cell lines and that this effect of androgens is mediated through the androgen receptor (AR). We further showed that androgens regulate SMURF1 expression at transcriptional level and provided evidence that AR transcriptionally activates SMURF1 by binding to its enhancer that contains a canonical half androgen responsive element (ARE). Finally, we demonstrated that SMURF1 is important for androgen-induced invasion of PCa cells.. We demonstrate for the first time that SMURF1 is a bona fide target gene of the AR. Our findings also suggest a potential role of SMURF1 in PCa metastasis.

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Nandrolone; Neoplasm Invasiveness; Prostatic Neoplasms; Receptors, Androgen; Transcription, Genetic; Ubiquitin-Protein Ligases

Post-transcriptional regulation by microRNA (miRNA) is an important aspect of androgen receptor (AR) signalling in prostate cancer cells. However, the global profiling of miRNA expression in prostate cancer cells following treatment with AR ligands has not been reported so far. In this study we examined the effect of treatment with two AR agonists (mibolerone (MIB) and dihydrotestosterone (DHT)) and an AR antagonist (bicalutamide (BIC)) on miRNA expression in the human androgen-dependent LNCaP prostate cancer cell line using microarray technology and verification of selected miRNA using quantitative real-time PCR (qRT-PCR). No miRNA was identified as differentially expressed following treatment with the AR antagonist BIC. In contrast, a number of common and compound-specific alterations in miRNA expression were observed following treatment with AR agonists. Unexpectedly it was found that treatment with the AR agonists resulted in the repression of miR-221, a miRNA previously established to be involved with prostate cancer development. This observation indicates that this miRNA may have a more complex role in prostate cancer development than considered previously. Treatment with MIB led to an induction of miR-210 expression, a hypoxia-related miRNA. This miRNA is reported to be involved in cell adaptation to hypoxia and thus induction in conditions of normoxia may be important in driving metabolic changes observed in prostate cancer. Thus examining the effect of AR agonists and antagonists on miRNA expression can provide novel insights into the response of cells to AR ligands and subsequent downstream events.

Topics: Androgen Antagonists; Androgens; Anilides; Antineoplastic Agents, Hormonal; Cell Line, Tumor; Cell Proliferation; Dihydrotestosterone; Dose-Response Relationship, Drug; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kallikreins; Ligands; Male; MicroRNAs; Nandrolone; Neoplasms, Hormone-Dependent; Nitriles; Oligonucleotide Array Sequence Analysis; Prostate-Specific Antigen; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Receptors, Androgen; Time Factors; Tosyl Compounds

We previously showed that growth hormone (GH) receptors (GHR) are expressed in the most commonly studied human prostate cancer (PCa) cell lines and that GHR isoforms undergo differential, cell-type-specific hormonal regulation. We now report that human GH (hGH) can stimulate/modulate insulin-like growth factor (IGF) and β-oestradiol (E(2) ) receptor (ER(β) ) gene expressions in these cells and interact with IGF-I and E(2) to stimulate androgen-dependent LNCaP cell proliferation. We observed a cell type-dependent, differential regulation of IGF axis gene expression by GH: IGF-I was stimulated in the androgen-dependent LNCaP cells; IGF-II was stimulated in androgen-insensitive (AI) PC3 cells; the IGF-I cognate receptor, IGF-IR, was stimulated in LNCaP cells, but inhibited in PC3 cells; IGF-IIR was stimulated in both LNCaP and PC3 cells. GH also stimulated ER(β) gene expression in LNCaP and PC3 cells, but had little or no effect on any of those genes in AI DU145 cells. The potent androgen analogue, mibolerone, also stimulated IGF-I, IGF-IR and ER(β) , but reduced IGF-IIR mRNAs in LNCaP cells. Furthermore, triiodothyronine (T(3) ) and E(2) also stimulated the expression of those four genes in LNCaP cells, but co-administration of GH had almost no effect. Finally, we also studied the effects of GH, IGF-I and E(2) , alone or in combination, on LNCaP cell proliferation. Importantly, we demonstrated, for the first time, that although GH and IGF-I alone had no effect on LNCaP cell proliferation, concomitant administration for 96 h revealed a permissive role of GH on IGF-I-induced proliferation. GH also appeared to exert a synergistic effect on E(2) -stimulated LNCaP cell proliferation. Taken together, these findings indicate that GH via GHRs, most likely in concert with gonadal steroids, T(3) , IGF system axis and probably other hormones and growth factors, potentially plays an important role in the mechanisms underlying tumour cell growth in PCa.

Topics: Cell Line, Tumor; Estradiol; Gene Expression Regulation; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Nandrolone; Prostatic Neoplasms; Receptor, IGF Type 1; Receptor, IGF Type 2; Receptors, Estradiol; Triiodothyronine

Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer.

Topics: Bone Neoplasms; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Humans; Immunohistochemistry; Male; Nandrolone; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; RNA-Binding Protein FUS

We previously demonstrated the gene expression of two growth hormone (GH) receptor (GHR) isoforms in prostate cancer (PCa) patient tissues and human PCa cell lines. In that initial study, we characterized LNCaP cell GH binding characteristics to GHR and its activation of relevant signal transduction pathways. We now show that GH binding to GHR and GHR mRNA expression in the cell lines studied are hormonally regulated. In the androgen-dependent LNCaP cells, the potent, specific and stable androgen analogue, mibolerone, caused a time- and biphasic dose-dependent, stimulation of (125)I-hGH specific binding to cells cultured in serum-free medium (SFM); however, when LNCaP cells were grown in chemically defined Gc full medium, long-term mibolerone-induced inhibition was observed. This effect of Gc on the androgen response was mimicked by the triiodothyronine (T(3)) contained in GC. In contrast, oestradiol (E(2)), cortisol, and insulin-like growth factor (IGF)-I and -II all caused stimulation of GH binding. Furthermore, we also observed homologous and heterologous, isoform- and cell-type-specific regulation of GHR mRNA expression in all three cell lines. In LNCaP cells, GH caused stimulation of both GHR mRNA and of its exon 9-truncated isoform, GHR(tr); however, mibolerone, E(2) and T(3) all stimulated GHR(tr) mRNA more potently than they did GHR. In androgen-independent PC3 cells, GH stimulated GHR(tr) expression, but almost not GHR, while in contrast, in androgen-independent DU145 cells, GH caused a clear reduction in GHR and less so in GHR(tr). The differential regulation of GHR isoform gene expression in human PCa cell lines and of GHR functional capacity (GH binding), by hormones and growth factors relevant to disease progression, suggests that GHR may prove to be an additional therapeutic target to slow down/prevent progression of human prostate cancer.

Topics: Androgen Antagonists; Androgens; Cell Line, Tumor; Culture Media; Culture Media, Serum-Free; Estradiol; Gene Expression Regulation, Neoplastic; Human Growth Hormone; Humans; Hydrocortisone; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Iodine Radioisotopes; Male; Mutant Proteins; Nandrolone; Prostatic Neoplasms; Protein Isoforms; Receptors, Somatotropin; RNA, Messenger; Triiodothyronine

Perillyl alcohol is a hydroxylated monocyclic monoterpene. In animal study, monoterpene has shown to have an anti-tumor effect. The aim of this study is to evaluate whether POH plays an important role in the development and progression of prostate cancer (pCa). We treated LNCaP cells with different concentrations of perillyl alcohol (POH). First of all, we performed cell proliferation assay and prostate-specific antigen (PSA) and human glandular kallikrein (hK2) quantification assays. LNCaP cells were treated with or without POH for Western blot analysis of androgen receptor (AR) and c-Jun. Finally, we performed transient transfection assay by transfecting LNCaP cells-which were treated with or without POH-with pGL-3 luciferase vector containing PSA promoter and AR promoter. We observed inhibition of the expression and function of the AR by POH, through inhibition of androgen-induced cell growth and androgen-stimulated secretion of prostate-specific antigen and hK2, in human pCa cell line LNCaP. In addition, we demonstrated, for the first time, that POH inhibits the transcription activities of the AR gene promoter by over-expression of c-Jun protein. These novel properties of POH strongly suggest that POH could be highly useful for intervention of pCa.

Topics: Androgen Receptor Antagonists; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Genes, Reporter; Humans; Luciferases; Male; Monoterpenes; Nandrolone; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-jun; Receptors, Androgen; Tissue Kallikreins; Transcriptional Activation; Transfection

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.

Topics: Antigens, Differentiation; Cell Differentiation; Cell Line; Clone Cells; Epithelial Cells; Humans; Male; Nandrolone; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Stem Cells

To identify the androgen-responsive genes in prostate and screen the molecular targets for further studying human prostate cancer.. The potential androgen-responsive gene pituitary tumor transforming gene 1 (PTTG1) was selected which had been previously screened by cDNA microarray in rat prostate and its mRNA level was detected by Northern blot in the castrated rat prostate with and without replacement of Mibolerone. Immunohistochemistry was performed to determine the expression and location of PTTG1 in human prostate tissues. Then human androgen-dependent prostate cancer cells LNCaP were used as a model to study the regulation of PTTG1 by Mibolerone.. PTTG1 mRNA was hardly detectable in the prostate of 7-day castrated rats, while it was up-regulated dramatically in the prostate of 7-day castrated rats treated with Mibolerone for 2 days. It was showed that high expression of PTTG1 was localized to the epithelial cells of human prostate cancer but not to the stromal cells with Immunohistochemistry. Northern blot analysis indicated that LNCaP cells treated with 0.1 nmol/L Mibolerone for 2 days led to the high PTTG1 mRNA expression. The basic expression of PTTG1 in human androgen-independent prostate cancer cell lines PC3 or DU145 was even higher than that in the human androgen-dependent prostate cancer cells LNCaP treated with Mibolerone.. Androgen can up-regulate the PTTG1 expression in castrated rat prostate and human prostate cancer cell LNCaP. It suggests that PTTG1 is potential to play an important role in human prostate cancer progression.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Line, Tumor; Gene Expression; Humans; Immunohistochemistry; Male; Nandrolone; Neoplasm Proteins; Orchiectomy; Prostate; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; RNA, Messenger; Securin; Testosterone Congeners

Androgens control prostate homeostasis and regulate androgen response genes. Here, we report the identification and characterization of U19, a novel testosterone-regulated apoptosis inducer with tumor suppressive activity. U19 is an evolutionarily conserved protein expressed in many human tissues, with the most abundant expression in the prostate, bone marrow, kidney, and lymph nodes. Overexpression of U19 in 12 surveyed cell lines induced apoptosis, and new protein synthesis is required for apoptosis induction. Expression of U19 in xenograft prostate tumors markedly induced apoptosis and inhibited tumor growth in vivo. Consistent with its tumor-suppressive role, U19 down-regulation was observed in all of the surveyed prostate cancer cell lines and in 19 of 23 clinical human prostate tumor specimens. Loss of heterozygosity analysis revealed U19 allelic loss in 19 of the 23 specimens. Furthermore, two of the specimens had homozygous U19 deletions, and one specimen had hypermethylated U19 promoter, indicating that U19 can be inactivated genetically or epigenetically. These observations suggest that U19 is growth inhibitory and tumor suppressive and that the disruption of androgen-dependent growth inhibition via U19 down-regulation is commonly associated with prostate cancer progression.

Topics: Amino Acid Sequence; Animals; Apoptosis; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Loss of Heterozygosity; Male; Mice; Mice, Nude; Molecular Sequence Data; Nandrolone; Neoplasm Transplantation; Promoter Regions, Genetic; Prostatic Neoplasms; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Proteins

The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCFSKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.. We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCFSKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.. Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.

Topics: Androgens; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; G1 Phase; Genes, Tumor Suppressor; Humans; Ligases; Male; Nandrolone; Prostatic Neoplasms; S-Phase Kinase-Associated Proteins; Testosterone Congeners; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Protein Ligases; Up-Regulation

The study of prostate carcinogenesis and tumor progression is made difficult by the lack of appropriate in vitro and in vivo models. High prevalence of prostatic intra-epithelial neoplasia and latent prostatic carcinoma, representing multiple steps in carcinogenesis to invasive carcinoma, are relevant targets for cancer prevention. From the RWPE-1, immortalized, non-tumorigenic, human prostate epithelial cell line, we have derived four tumorigenic cell lines with progressive malignant characteristics.. Cell lines were derived by exposure of RWPE-1 to N-methyl-N-nitrosourea (MNU), selected and cloned in vivo and in vitro, and characterized by prostatic epithelial and differentiation markers, karyotype analysis, anchorage-independent growth, invasiveness, tumorigenicity, and pathology of the derived tumors.. Cytokeratins 8 and 18, androgen receptor, and prostate-specific antigen expression in response to androgen, confirm prostatic epithelial origin. RWPE-1 cells do not grow in agar and are not tumorigenic in mice, but the growth, tumorigenicity, and tumor pathology of the MNU cell lines correlate with their invasive ability. The WPE1-NA22 (least malignant) form small, well-differentiated, and WPE1-NB26 cells (most malignant) form large, poorly differentiated, invasive tumors. Overall, loss of heterozygosity for chromosomes 7q, 13q, 18q, and 22, and gain of 5, 9q, 11q, and 20, was observed. The MNU cell lines, in order of increasing malignancy are; WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26.. This family of cell lines with a common lineage represents a unique and relevant model which mimics stages in prostatic intra-epithelial neoplasia (PIN) and progression to invasive cancer, and can be used to study carcinogenesis, progression, intervention, and chemoprevention.

Topics: Alkylating Agents; Animals; Carcinogenicity Tests; Cell Adhesion; Cell Culture Techniques; Cell Division; Chromosome Aberrations; Chromosome Disorders; Disease Progression; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Keratins; Male; Methylnitrosourea; Mice; Mice, Nude; Nandrolone; Neoplasm Invasiveness; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Transforming Growth Factor beta; Tumor Cells, Cultured

A number of reports have shown that the polyphenolic flavonoid silymarin (SM) is an effective anticancer agent. Agents with novel mechanisms of blocking androgen receptor (AR) function may be useful for prostate cancer prevention and therapy. Previous studies showed that silibinin (SB), the major active component of SM, could inhibit cell proliferation of a human prostate cancer cell line, LNCaP, by arresting the cell cycle at the G(1) phase without causing cell death. This study further delineates the potential molecular mechanism by which SM and SB exhibit antiproliferative effects on androgen-responsive prostate cancer cells by inhibiting function of the AR. We observed that SM and SB inhibited androgen-stimulated cell proliferation as well as androgen-stimulated secretion of both prostate-specific antigen (PSA) and human glandular kallikrein (hK2). Additionally, for the first time, we show that an immunophilin, FKBP51, is androgen regulated and that this up-regulation is suppressed by SM and SB. We further demonstrate that transactivation activity of the AR was diminished by SM and SB using gene transfer of PSA promoter and hK2 androgen-responsive element constructs. However, expression and steroid-binding ability of total AR were not affected by SM in western blotting and ligand-binding assays. Intriguingly, we found that nuclear AR levels are significantly reduced by SM and SB in the presence of androgens using western blotting assay and immunocytochemical staining. This study provides a new insight into how SM and SB negatively modulate androgen action in prostate cancer cells.

Topics: Androgen Receptor Antagonists; Cell Division; Cell Nucleus; Down-Regulation; Humans; Male; Nandrolone; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Silymarin; Tacrolimus Binding Proteins; Testosterone Congeners; Tissue Kallikreins; Transcriptional Activation; Transfection; Tumor Cells, Cultured

The androgen receptor (AR) promotes growth and functionality of androgen sensitive benign and cancer tissues. The pineal hormone melatonin is an androgen protagonist in vivo and in vitro. The interference of melatonin in the AR cascade was explored.. The effects of melatonin on AR expression, level, agonist and androgen-response element (ARE) binding, reporter gene activity and intracellular localization were explored in prostate cancer LNCaP cell line.. Melatonin increased immunoreactive AR cells in the absence and presence of dihydrotestosterone. Despite this increase and maintenance of AR agonist binding capacity, the androgen-induced reporter gene activity and suppression of AR-mRNA were attenuated. Immunocytochemical analysis and subcellular fractionation studies revealed nuclear exclusion of AR by melatonin.. The melatonin-mediated nuclear exclusion of the AR may explain the attenuation of AR activity in the prostate cancer cells. This is the first demonstration of a hormone-induced mislocalization of the AR in prostate epithelial cells and may represent a novel route for regulating AR activity.

Topics: Androgen Receptor Antagonists; Blotting, Western; Cell Nucleus; Humans; Immunohistochemistry; Male; Melatonin; Nandrolone; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tumor Cells, Cultured

Human prostate-specific antigen (PSA) and human kallikrein 2 (hK2) are expressed primarily by prostate epithelial cells. PSA and hK2 both exist as free protein and complexed with protease inhibitors (e.g., alpha1-antichymotrypsin, ACT) in serum. The expression of PSA and hK2 in LNCaP cells is upregulated by androgen.. LNCaP, a prostate cancer cell line that secretes both PSA and hK2, was used as a model to study the biosynthesis and processing of PSA and hK2 upon androgen induction.. Precursor (zymogen or pro) forms of both PSA and hK2 were detected in spent media of induced and noninduced LNCaP cells, indicating that PSA and hK2 are secreted as proPSA (pPSA) and prohK2 (phK2), respectively, and are converted to the mature forms extracellularly. A 3-fold higher ratio of mature to pPSA was detected in the spent media of mibolerone-induced LNCaP cells compared to noninduced cells. In addition to the inactive proform of PSA, more than half of the mature unclipped PSA present in the spent media did not complex with exogenously added ACT. Spent media of mibolerone-induced LNCaP cells contained nearly 100% mature hK2, whereas the spent media of noninduced cells contained mostly phK2.. These results indicate that androgens not only upregulate the expression of these kallikreins, but also have a significant effect on the processing of PSA and hK2. These results also show that LNCaP cells express a heterogeneous mixture of inactive PSA and hK2 forms that may serve as a model for the genesis of these forms in physiological fluids. These findings may also provide insights into the forms and ratios of PSA and hK2 in normal and malignant breast tissues.

Topics: alpha 1-Antichymotrypsin; Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, High Pressure Liquid; Gene Expression Regulation, Neoplastic; Humans; Male; Nandrolone; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Isoforms; Sequence Analysis, Protein; Testosterone Congeners; Tissue Kallikreins; Tumor Cells, Cultured

Peroxisome proliferator-activated receptor (PPAR) alpha is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARalpha is activated by peroxisome proliferators and fatty acids and has been shown to be involved in the transcriptional regulation of genes involved in fatty acid metabolism. In rodents, the PPARalpha-mediated change in such genes results in peroxisome proliferation and can lead to the induction of hepatocarcinogenesis. Using the mRNA differential display technique and Northern blot analysis, we have shown that chronic exposure of the prostate cancer epithelial cell line LNCaP to the synthetic androgen mibolerone results in the down-regulation of PPARalpha mRNA. Levels of PPARalpha mRNA are reduced to approximately 40% of control levels in LNCaP cells exposed to 10 nM mibolerone for 96 h. PPARalpha-responsive reporter plasmids derived from human ApoA-II and muscle carnitine palmitoyl-transferase I genes were stimulated by the PPARalpha-activating ligand Wy-14,643 in LNCaP cells. In situ hybridization and immunohistochemical analyses showed that PPARalpha expression in prostate is confined to epithelial cells. In benign prostatic tissue, PPARalpha mRNA was either absent or only weakly expressed in the basal epithelial cells. In 11 of 18 (61%) poorly differentiated (Gleason score, 8-10) prostatic carcinoma specimens, there was strong expression of PPARalpha compared with 4 of 12 Gleason score 7 tumors and 2 of 11 Gleason score 3-6 tumors (P < 0.01). These results suggest that PPARalpha is found and functional in human prostate and is down-regulated by androgens. The role of PPARalpha may be to integrate dietary fatty acid and steroid hormone signaling pathways, and its overexpression in advanced prostate cancer may indicate a role in tumor progression with the potential involvement of dietary factors.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Male; Nandrolone; Prostate; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Testosterone Congeners; Transcription Factors; Tumor Cells, Cultured

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study r

Topics: Adenocarcinoma; Androgens; Carrier Proteins; Cell Membrane; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Iodides; Male; Membrane Proteins; Nandrolone; Neoplasms, Hormone-Dependent; Organ Specificity; Prostate-Specific Antigen; Prostatic Neoplasms; Recombinant Fusion Proteins; Symporters; Transfection; Tumor Cells, Cultured

Androgens are required for the development and function of the prostate. In a normal human prostate, androgens control the synthesis of proteins such as prostate-specific antigen and human glandular kallikrein. The prostate secretes these proteins as well as a number of other compounds to form the prostatic fluid. Using differential display PCR to detect novel androgen-regulated genes, clathrin heavy chain expression was identified as potentially being up-regulated by androgens in the prostate cancer cell line LNCaP. We report here that the clathrin heavy chain and light chain genes are regulated by androgens. Clathrin heavy chain messenger RNA was up-regulated by androgens in a concentration- and time-specific manner in the LNCaP cell line. Translation of clathrin heavy chain messenger RNA was stimulated by androgens. Steady state levels of clathrin light chains a and b were up-regulated in the presence of androgen in LNCaP cells. Clathrin gene expression was examined in normal rat prostates, and similar results were found. Clathrin heavy chain protein levels in the rat prostate are also affected by the androgen status of the animal. We hypothesize that clathrin may be involved in the exocytosis of androgen-regulated secretory proteins such as prostate-specific antigen and human glandular kallikrein.

Topics: Androgens; Animals; Blotting, Northern; Clathrin; Gene Expression Regulation; Humans; Kinetics; Male; Nandrolone; Polymerase Chain Reaction; Prostate; Prostatic Neoplasms; Rats; RNA, Messenger; Testosterone; Testosterone Congeners; Tumor Cells, Cultured

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Topics: Adenocarcinoma; Androgen Antagonists; Androgens; Anilides; Dihydrotestosterone; Dose-Response Relationship, Drug; Enzyme Induction; Fatty Acid Synthases; Gene Expression Regulation, Neoplastic; Humans; Lipids; Male; Metribolone; Nandrolone; Neoplasm Proteins; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Testosterone; Testosterone Congeners; Tosyl Compounds; Tumor Cells, Cultured

A noninvasive method for detecting and quantifying androgen receptors (AR) in metastatic prostate cancer may be helpful in choosing the method of treatment and in better understanding the pathophysiology of this disease. Nine previously synthesized fluorinated androgens exhibited high affinity binding to AR and showed AR-mediated uptake in the ventral and dorsal prostate of the rat. Further evaluation of these agents for PET imaging is needed since sex hormone binding globulin (SHBG), a glycoprotein which binds androgens with high affinity, is absent in rat blood but is present at high levels in the blood of primates. We chose to study three of the nine fluoro-androgens by PET in the baboon.. In this study, 16beta-[18F]fluoro-5 alpha-dihydrotestosterone (I), 16beta-[18F]fluoromibolerone (II) and 20-[18F]fluoromibolerone (III) were synthesized and studied in both a young and old male baboon using PET. Blood samples were withdrawn in three of the 10 studies and analyzed for total radioactivity and percent unmetabolized radioligand. Tissue radioactivity was evaluated semiquantitatively, using prostate absolute, standard and target to nontarget uptake values.. Prostate uptake was observed with all three 18F-androgens. At 60 min postinjection, compound I gave the highest prostate to soft tissue ratios in both baboons and prostate uptake was shown to be AR-mediated by blocking uptake through the coadministration of testosterone. Compound I gave the highest level of unmetabolized radioligand present in blood up to 45 min postinjection, and gave a 37-fold greater prostate-to-bone ratio at 2 hr postinjection in baboons compared to rats. The favorable behavior of this compound in the baboon may be related to its high affinity for SHBG.. All three compounds can be used to determine AR-positive tissue in primates. Compound I was selected for the evaluation of AR in men with prostate cancer using PET.

Topics: Animals; Dihydrotestosterone; Drug Evaluation, Preclinical; Fluorine Radioisotopes; Male; Nandrolone; Papio; Prostate; Prostatic Neoplasms; Receptors, Androgen; Testosterone Congeners; Tomography, Emission-Computed

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Carrier Proteins; Cloning, Molecular; Cycloheximide; Diazepam Binding Inhibitor; Dihydrotestosterone; DNA, Complementary; Humans; Immune Sera; Male; Metribolone; Nandrolone; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Testosterone; Tumor Cells, Cultured

The human prostate-specific kallikreins, human glandular kallikrein-1 (hKLK2) and prostate-specific antigen (hKLK3), have been shown to be regulated by androgens. To determine whether the androgen induction of these genes is transcriptionally regulated via an androgen response element, an hKLK2 promoter DNA fragment was linked to a promoterless chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression vector in an androgen receptor-less human prostate cell line, PC-3. Dose response and steroid specificity experiments showed that the hKLK2 promoter confers androgen receptor-mediated gene induction in a ligand-specific manner. Moreover, 5' deletion constructs of the hKLK2 promoter DNA and internal deletion constructs devoid of the 5' half-site of the putative androgen responsive element (ARE) were used to show that the putative ARE is indeed acting as a functional ARE in prostate cells. In addition, multiple AREs from both hKLK2 and hKLK3 were able to reconstitute androgenic induction, further strengthening the argument that the AREs are functional. Although previous studies have shown that hKLK3 mRNA is expressed at a higher level than that of hKLK2, our results suggest that the hKLK2 ARE may have higher androgenic inducibility than the hKLK3 ARE. These results suggest that other cis-acting elements may be involved in coordinating in vivo androgenic induction of hKLK2 and hKLK3 genes.

Topics: Androgens; Base Sequence; Chloramphenicol O-Acetyltransferase; Dihydrotestosterone; DNA-Binding Proteins; Enzyme Induction; Gene Expression Regulation, Enzymologic; Humans; Kallikreins; Kinetics; Male; Molecular Sequence Data; Nandrolone; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Sequence Deletion; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Topics: Androgens; Antibodies; Base Sequence; Blotting, Northern; Breast Neoplasms; Cells, Cultured; Down-Regulation; Fibroblasts; Gene Expression Regulation; Humans; Immunohistochemistry; Male; Molecular Sequence Data; Nandrolone; Prostatic Neoplasms; Protein Processing, Post-Translational; Receptors, Androgen; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.

Topics: Alkaloids; Antigens, Neoplasm; Biomarkers, Tumor; Calcimycin; Colforsin; Dose-Response Relationship, Drug; Down-Regulation; Humans; Male; Nandrolone; Phorbols; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Staurosporine; Testosterone Congeners; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This paper presents an approach for the assessment of the androgen receptor (AR) status in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues. Evaluation of AR was carried out in both soluble and nuclear fractions by a standard competition method, using tritiated mibolerone as radioligand. Based on our experience with breast and endometrial cancer, this approach focused on both type I (high affinity, low capacity) and type II (reduced affinity, higher capacity) binding sites, aiming mainly at establishing a putative "functional" receptor mechanism, i.e., the presence of type I AR in both cytosol and nucleus. Ancillary studies were carried out to exclude a potential overestimation of the AR content by interference with other steroid receptors, namely, progesterone (PgR) or glucocorticoid (GcR) receptors. Results showed that the interaction by PgR or GcR upon AR measurement was not relevant. The distribution of AR, namely the percent of positivity either in a single or in both cell compartments, was not significantly different in BPH (N = 32) or PCa (N = 24) tissues. For type I binding, the percent of positivity in both soluble and nuclear fractions (i.e., the "functional" AR status) was very close to that observed for other endocrine-related tumors, like breast cancer. Concentrations of type I AR appeared significantly higher in PCa than in BPH tissues; this was true for both soluble and nuclear fractions. In contrast, no significant difference was found in type II AR concentrations in either cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Binding Sites; Humans; Male; Nandrolone; Predictive Value of Tests; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Radioligand Assay; Receptors, Androgen; Testosterone Congeners; Tritium

We have prepared nine androgens substituted with fluorine at C-16 or C-20 to evaluate their potential, as positron emission tomographic (PET) imaging agents for prostatic cancer when labeled with the positron emitting radionuclide fluorine-18 (t1/2 = 110 min). These compounds represent members from the following classes of androgens: testosterone (T), 5 alpha-dihydrotestosterone (DHT), 7 alpha-methyl-19-nortestosterone (MNT), mibolerone (Mib), and metribolone (R1881). All of these compounds were prepared by functionalization of suitable androgen precursors, and the synthetic routes were developed to allow the introduction of fluorine by a fluoride ion displacement reaction late in the synthesis, as is required for the preparation of these compounds in fluorine-18 labeled form. We have also prepared four androgens in which the C-3 carbonyl or 17 beta-hydroxyl groups are replaced by fluorine. Most of the fluorine-substituted androgens show high affinity for the androgen receptor (AR), although fluorine substitution lowers their affinity by a small factor. None of the androgens where fluorine replaces oxygen functions at C-3 or C-17 have substantial affinity for AR. Derivatives of the natural androgens (T and DHT) as well as MNT have little affinity for other steroid hormone receptors (progesterone and mineralocorticoid receptors), whereas the Mib and R1881 derivatives have somewhat greater heterologous binding. With sex steroid binding protein, a human serum binding protein, the pattern of binding affinities is nearly the reverse, with derivatives of Mib, R1881 and MNT having low affinity, and DHT and T, high affinity. From these fluorine-substituted compounds, we can select several whose preparation in fluorine-18 labeled form for further tissue distribution studies is merited.

Topics: Androgens; Animals; Dihydrotestosterone; Estrenes; Fluorine; Male; Metribolone; Molecular Conformation; Molecular Structure; Nandrolone; Prostate; Prostatic Neoplasms; Rats; Receptors, Androgen; Structure-Activity Relationship; Testosterone; Tomography, Emission-Computed

The present study was undertaken to compare the relationship between response to exogenous epidermal growth factor (EGF) and the expression of the EGF-receptor (EGF-R) in an androgen sensitive (LNCaP) and insensitive (DU145) prostate cancer cell line. Although both cell lines demonstrated a single EGF-R binding site of similar high affinities (mean dissociation constant (Kd) +/- S.D. for DU145 = 1.0 +/- 0.6 nmol l-1; LNCaP = 2.8 +/- 2.2 nmol l-1) the number of binding sites (RT) for the hormone insensitive DU145 cells (mean +/- S.D. = 2.5 +/- 1.0 x 10(5) sites/cell) and 10-fold greater than that expressed in the androgen responsive LNCaP cell line (mean +/- S.D. = 2.0 +/- 1 x 10(4) sites/cell). Additionally exogenous EGF only minimally affected the growth and DNA synthesis of DU145 cells whereas LNCaP cells showed a significant response which was dose dependent. The autologous production of EGF-like molecules by DU145 cells is believed to reduce the cells needs for exogenous mitogens, thereby rendering the cells autostimulatory. Treatment of LNCaP cells with Mibolerone--a synthetic androgen--did not affect either the expression of the EGF receptor or the proliferative response observed with EGF. Western blot analysis, using monoclonal antibodies directed against the EGF receptor revealed a band of approximately 170 kD with DU145 cell lysates but the LNCaP EGF receptor was not detected using this technique.

Topics: Blotting, Western; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Nandrolone; Prostatic Neoplasms; Thymidine; Tumor Cells, Cultured

We have examined the effect of androgen treatment on androgen receptor mRNA and protein expression in the LNCaP human prostate carcinoma cell line. Incubation with androgen caused a decrease in cellular androgen receptor mRNA content that was concentration and time dependent. Maximal suppression to approximately 35% of control level was observed after 49 h of exposure to androgen. By contrast, incubation of LNCaP cells with androgen resulted in a 2-fold increase in the cellular content of androgen receptor protein at 24 h. At 49 h androgen receptor protein increased 30% as assayed by immunoblots and 79% as assayed by ligand binding. These results suggest that ligand-induced changes in androgen receptor stability and/or the translational efficiency of androgen receptor mRNA account for the phenomenon of androgen receptor upregulation observed in cultured LNCaP cells. Furthermore, the suppression of androgen mRNA and protein that is caused by prolonged incubation with androgen is incomplete and is reversible upon removal of ligand.

Topics: Androgens; Base Sequence; Blotting, Western; Cell Line; Dihydrotestosterone; DNA; Down-Regulation; Humans; Ligands; Male; Molecular Sequence Data; Nandrolone; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Testosterone Congeners; Tumor Cells, Cultured

Mibolerone (dimethylnortestosterone) or 5 alpha-dihydrostestosterone (DHT) increase intracellular calcium (Ca2+i) of human prostate cancer cells (LNCaP) as early as 2 min after treatment. These effects were concentration-dependent (10(-6)-10(-12) M) and they were blocked by preincubation with hydroxyflutamide (10(-6) M). Verapamil (10(-6) M) also suppressed the mibolerone (10(-6) M)-induced increase in Ca2+i, in cells which were previously exposed to 1 mM CaCl2 introduced in a Ca(2+)-free media. The results indicate that androgens elicit changes in Ca2+i in LNCaP cells as a result of Ca2+ influx through L-type channels in the plasma membrane. Since androgens are involved in the regulation of prostate cell division and growth, these findings suggest that calcium is involved in metabolic and mitogenic responses to steroid hormone in target cells.

Topics: Calcium; Cell Line; Dihydrotestosterone; Fura-2; Humans; Kinetics; Male; Nandrolone; Prostatic Neoplasms; Spectrometry, Fluorescence; Testosterone Congeners; Time Factors; Verapamil

The synthetic androgen mibolerone elicits a set of distinct changes in the behaviour of an androgen responsive human prostatic carcinoma cell line (LNCaP). Inhibition of cell proliferation, induction of morphological change and of a prostate specific mRNA, and inhibition of colony formation in soft agar are induced by very low concentrations of mibolerone. The natural androgen dihydrotestosterone is much less effective. The changes in growth characteristics and morphology are reverted by excess antiandrogen, i.e. cyproterone acetate or hydroxyflutamide. Cell lines lacking androgen receptors (PC-3, DU 145 and MRC-5) are completely unresponsive to mibolerone. Taken together, our results indicate androgen receptor mediated suppression of the transformed phenotype in LNCaP cells.

Topics: Androgen Antagonists; Androgens; Cell Division; Cell Transformation, Neoplastic; Cyproterone; Cyproterone Acetate; Dihydrotestosterone; Flutamide; Humans; Male; Nandrolone; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Tumor Cells, Cultured

The effect of steroidal and nonsteroidal "anti-androgens" on the proliferative capacity of androgen-sensitive LNCaP-FGC human prostate tumor cells in culture was studied using charcoal-dextran stripped human serum-supplemented media. Cyproterone and medroxyprogesterone acetates, flutamide, hydroxyflutamide, and anandron (R23908) were administered alone at concentrations between 3 X 10(-12) and 3 X 10(-6) M. Results indicated that although medroxyprogesterone induced maximal proliferation at 3 X 10(-9) M, the other "anti-androgens" (with the exception of flutamide that was ineffective) were effective at 3 X 10(-8) M and higher concentrations; the amplitude of the proliferative response by these compounds was comparable to that elicited by estradiol-17 beta (3 to 5-fold over control). None of the anti-androgens tested triggered the shutoff effect characteristic of androgen action. When 3 X 10(-10) M DHT and the above mentioned anti-androgens were administered simultaneously, a synergistic pattern was seen; on the contrary, 3 X 10(-8) M DHT cancelled the proliferative effect of each of the anti-androgens when administered simultaneously. The relative binding affinity of these anti-androgens to androgen receptors present in LNCaP-FGC cells did not correlate well with their proliferative efficiency. The data collected were interpreted within the premises of the negative control hypotheses for the regulation of cell proliferation in metazoans. Within those premises, results became compatible with the notion that first, "anti-androgens" elicited the proliferation of androgen-sensitive cells by neutralizing the effect of a serum-borne inhibitor (androcolyone-I); this event seems not to be mediated by androgens receptors. Second, anti-androgens did not trigger a proliferative shutoff response like androgens do, i.e. the proliferative pattern induced by anti-androgens was comparable to that elicited by estrogens and progestins. Third, when administered simultaneously with 3 X 10(-10) M DHT, anti-androgens behaved synergistically. Fourth, the DHT-induced shutoff effect consistently overrode the proliferative effect generated by anti-androgens and estrogens when added alone. Finally, taken together these results raise important questions regarding the therapeutic role of anti-androgens in prostate cancer.

Topics: Androgens; Antibodies; Cell Division; Dihydrotestosterone; Estradiol; Flutamide; Humans; Male; Nandrolone; Prostatic Neoplasms; Receptors, Androgen; Testosterone Congeners; Tumor Cells, Cultured