Establishment Of AIPC Cell Model And Mechanism Of LSD1in The AI-Phenotypic Transition

Objective:To establish an cell model of androgen-independent prostate cancer, and then initially investigate the mechanism of action of LSD1for the acquisition of androgen-independent phenotype by prostate cancer.Methods:LNCaP cells were continuously cultured in Phenol Red-negative RPMI-1640medium supplemented with10%charcoal stripped fetal bovine serum (CS-FBS) for3months. In the three-month period, the changes of LNCaP cells growth were dynamically observed. CCK-8assay was used to detect the proliferation of LNCaP-AI cells and LNCaP cells. The differences in transcription levels of AR, prostate-specific antigen(PSA), LSD1, p53, p21, Bax, and Bcl-2genes between LNCaP-AI cells and LNCaP cells were measured via RT-PCR. Protein expression levels of AR, prostate-specific antigen(PSA), LSD1, p53, p21, Bax, and Bcl-2genes between LNCaP-AI and LNCaP cells were examined via western blot. Secretion of PSA protein of LNCaP-AI cells and LNCaP cells was tested by ELISA. Co-Immunoprecipitation (Co-IP) was used to analyze LSD1interactions with AR and p53in LNCaP-AI cells.Results:The growth of LNCaP cells, induced in the androgen-free medium, was inhibited, and LNCaP cells showed a neuroendocrine-like status. After3months, LNCaP cells thoroughly got rid of the neuroendocrine-like status and restored fast proliferation. CCK-8assay confirmed that LNCaP-AI cells could proliferate quickly in the androgen-deprived medium, but the growth of LNCaP cells was sustainedly inhibited in the same circumstance. RT-PCR and western blot indicated that the expression of PS A gene was obviously down-regulated and the expression of AR gene was up-regulated in LNCaP-AI cells, compared with LNCaP cells. ELISA showed that the total amount of PSA protein secreted by LNCaP-AI cell colony in the androgen-ablated medium was apparently higher than that secreted by LNCaP cell colony in the same condition. RT-PCR and western blot showed that the expression of LSD1gene was obviously up-regulated in LNCaP-AI cells, compared with that in LNCaP cells. Co-IP indicated that LSD1directly interacted with AR and p53in LNCaP-AI cells. Furthermore, RT-PCR and western blot assays both revealed that the gene expressions of p53, p21, Bax were obviously down-regulated and Bcl-2gene was over-expressed in LNCaP-AI cells, in contrast with those in LNCaP cells.Conclusions:After LNCaP cells were induced in androgen-deprived medium for3months, the cell model of androgen-independent prostate cancer(AIPC) was successfully established. The process of establishing the cell model well imitated the clinical passage of androgen-dependent prostate cancer, after endocrine therapy, progressing to androgen-independent prostate cancer. LSD1gene expression levels were markedly different in androgen-independent prostate cancer and androgen-dependent prostate cancer. In the androgen-deprived condition, LSD1gene was over-expressed, which, on the one hand, might facilitate adaption of prostate cancer cells to the androgen-deprived circumstance by activating AR-dependent pathway, and which, on the other hand, possibly promoted prostate cancer cell survival from low androgen-induced apoptosis by inhibiting p53-mediated apoptotic pathway. LSD1, by positively regulating AR pathway and negatively regulating P53pathway, probably plays important roles in the AI-phenotypic transition of prostate cancer.