sincalide and Prostatic-Neoplasms--Castration-Resistant

To establish castration-resistant prostate cancer (CRPC) - Lncap androgen-independent (AI) cell line from Lncap androgen-dependent (AD) cell line, and explore the different molecular biological between these two cell lines.. The Lncap-AD cell line was cultured and passaged 60 times over 16 months. The morphology of the Lncap-AI cell line was observed. AR levels identification were detected in qRT-PCR and Western Blot assay. CCK-8, EdU assay, wound healing assay and cell adhesion assays were used to observe the ability of proliferation, migration, and adhesion. SEM and TEM were used to observe microculture structure. At last, the PSA secrete ability was evaluated by Elisa assay.. The Lncap-AD cell line was cultured and passaged 60 times over 16 months. The Lncap-AI cell line showed a morphologic change at the end stage of culture, the cells turned slender and cell space turned separated compared to the Lncap-AD cell line. The relative levels of AR-related genes in the Lncap-AI cell line were up-regulation compared to the Lncap-AD cell line both in mRNA and protein levels. The expression of AR and HK2 proteins were influenced and down-regulation by Enzalutamide in the Lncap-AD cell line, but no obvious difference in Lncap-AI cell lines. Lncap-AI cell line showed strong viability of proliferation, migration, and adhesion by CCK-8, EdU assay, wound healing assay, and adhesion assay. The microstructure of Scanning Electron Microscopy (SEM) showed many synapses in the Lncap-AI cell line and PC3 cell line, but not in the Lncap-AD cell line. At last, the PSA secrete ability was evaluated by Elisa assay, and PCa cell lines showed no significant difference.. Simulation of CRPC progression, Lncap-AD cell line turned to Lncap-AI cell line with androgen deprivation therapy.

Topics: Androgen Antagonists; Androgens; Cell Line; Flutamide; Humans; Male; Prostate-Specific Antigen; Prostatic Neoplasms, Castration-Resistant; Sincalide

Androgen receptor (AR) is critical for prostate tumorigenesis and is frequently overexpressed during prostate cancer (PC) progression. However, few studies have addressed the epigenetic regulation of AR expression.. We analyzed SMYD3 expression in human PC with Western blot and immunohistochemistry. SMYD3 expression was knocked down using short hairpin RNA (shRNA) or small interfering RNA (siRNA). Cell proliferation, colony formation, and apoptosis analyses and xenograft transplantation were performed to evaluate the impact of SMYD3 depletion on PC cells. AR expression and promoter activity were determined using real-time quantitative polymerase chain reaction, western blot, and luciferase reporter assay. AR promoter association with Sp1, SMYD3, and histone modifications was assessed by chromatin immunoprecipitation. Differences in AR mRNA abundance and promoter activity were analyzed using Wilcoxon signed-rank tests, SMYD3 expression was analyzed using with Mann-Whitney U tests for unpaired samples, and tumor weight was analyzed with Student t test. All statistical tests were two-sided.. The upregulation of SMYD3 protein expression was observed in seven of eight prostate tumor specimens, compared with matched normal tissues. Immunohistochemical analysis showed a strong SMYD3 staining in the nuclei of PC tissues in eight of 25 (32%) cases and in the cytoplasm in 23 out of 25 (92%) cases, whereas benign prostate tissue exhibited weak immunostaining. Depletion of SMYD3 by siRNA or shRNA inhibited PC cell proliferation (72 hours relative to 24 hours: control shRNA vs SMYD3 shRNA 1: mean fold change = 2.76 vs 1.68; difference = 1.08; 95% confidence interval = 0.78 to 1.38, P < .001), colony formation, cell migration, invasion, and xenograft tumor formation. Two functional SMYD3-binding motifs were identified in the AR promoter region.. SMYD3 promotes prostate tumorigenesis and mediates epigenetic upregulation of AR expression.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; Immunoblotting; Immunohistochemistry; Male; Neoplasm Invasiveness; Promoter Regions, Genetic; Prostatic Neoplasms, Castration-Resistant; Real-Time Polymerase Chain Reaction; Receptors, Androgen; RNA, Small Interfering; Sincalide; Transcription, Genetic; Transfection; Up-Regulation; Xenograft Model Antitumor Assays