RESUMO
BACKGROUND: Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). METHODS: Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. RESULTS: The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. CONCLUSIONS: Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.
Assuntos
Antineoplásicos/farmacologia , Compostos Azo/farmacologia , Doadores de Óxido Nítrico/farmacologia , Piperazinas/farmacologia , Pró-Fármacos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Glutationa Transferase/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Óxido Nítrico/biossíntese , Neoplasias da Próstata/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: A recently identified mechanism allowing prostate cancer (PCa) cells to grow in the absence of androgens is the expression of constitutively active, C-terminally truncated androgen receptor (AR) variants lacking vast parts of the ligand-binding domain. These AR variants termed ARΔLBD are either products of alternative splicing, point mutations leading to premature stop codons or proteolytic cleavage of the AR. Some controversies exist about the requirement of additional full-length AR for the full transcriptional activity of the ARΔLBD. On basis of a mutated, C-terminally truncated AR termed Q640X, we developed an experimental model for the study of ARΔLBD in PCa cells. METHODS: Activation of AR-dependent promoters was analyzed by reporter gene assays. Dimerization studies were conducted using a mammalian two-hybrid system. RESULTS: Although Q640X/Q640X homodimers were able to induce the expression of certain AR target genes, Q640X/AR heterodimers were necessary to activate the full panel of androgen-dependent genes under androgen-deprived conditions. CONCLUSIONS: The following study supports the hypothesis that castration-resistant prostate cancer (CRPC) cells are able to activate specific androgen-dependent genes by selective modulation of the ratio between ARΔLBD and their putative dimerization partners like the full-length AR or other ARΔLBD in the absence of androgens. The present data suggest that AR-mutant Q640X is a powerful experimental tool for the functional analysis of ARΔLBD in CRPC.
Assuntos
Variação Genética/genética , Modelos Biológicos , Mutação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/fisiopatologia , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Androgênios/genética , Androgênios/fisiologia , Castração , Linhagem Celular Tumoral , Dimerização , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Neoplasias da Próstata/patologia , Transfecção , Falha de TratamentoRESUMO
The molecular mechanisms leading to castration-resistant prostate cancer (CRPC) are poorly understood. Among several mechanisms leading to CRPC growth a dysregulation of androgen receptor (AR) co-regulators (i.e. up-regulation of co-activators or down-regulation of co-repressors) is discussed. There are numerous reports demonstrating an increased expression of co-activators during prostate cancer progression. On the contrary, the impact of co-repressors on tumor growth and development is less clear. In this study we compared the effects of two known co-repressors, NCoR and SMRT, on AR transcriptional activity in prostate cancer (PCa) cell lines and compared them to that in COS-1 cells. Interestingly, we found that NCoR/SMRT overexpression did not repress AR-dependent gene expression in the PCa cell lines, but rather activated it. This finding is probably due to an impaired AR-co-repressor interaction in the prostate cancer cell lines. In conclusion, we provide evidence that up-regulation of NCoR or SMRT may increase transcriptional activity of the AR in a cell type-specific context.