苯乙基异硫氰酸酯对良性前列腺上皮细胞增殖凋亡的影响及相关机制研究

The effects and mechanism of phenylethyl isothiocyanate on cell proliferation and apoptosis in BPH-1 cells

目的探讨苯乙基异硫氰酸酯(phenethyl isothiocyanate,PEITC)对人类良性前列腺上皮细胞(benign prostatic hyperplasia cell line,BPH-1)增殖与凋亡的影响及其相关机制。方法体外培养人永生化前列腺上皮细胞,给予6、12、24μmol/L PEITC或等量溶剂二甲基亚砜,分别作用6、12、24 h。采用CCK-8检测前列腺上皮细胞活性;Annexin V及PI标记,流式细胞技术检测PEITC处理24 h后BPH-1细胞凋亡及坏死比例;Western blotting测定PEITC处理24 h后BPH-1细胞中X染色体连锁的凋亡抑制蛋白(X-linked inhibitor of apoptosis,XIAP)及自噬相关蛋白5-12(Atg5-12)表达变化;RT-PCR检测PEITC处理24 h后BPH-1细胞中XIAP mRNA表达情况。结果(1)BPH-1细胞经过6、12、24μmol/L PEITC处理6 h后,细胞相对活性分别为71.22%、48.90%、13.57%;处理12 h后分别为52.57%、42.37%、10.11%;处理24 h后分别为36.73%、25.88%、8.39%;处理36 h后分别为33.57%、25.83%、13.06%,呈现浓度依赖性和时间依赖性。(2)BPH-1细胞在6、12、24μmol/L PEITC处理24 h后,细胞凋亡率分别为19.5%、30.4%、40.1%;与对照组相比,12、24μmol/L处理组差异均具有统计学意义(P<0.05)。(3)BPH-1细胞在6、12、24μmol/L PEITC处理24 h后,XIAP蛋白表达量及mRNA表达量均下调,Atg5-12蛋白表达量下调,并呈现剂量依赖性。结论PEITC可抑制前列腺上皮细胞增殖,通过下调BPH-1中XIAP mRNA而降低XIAP蛋白表达,促进前列腺上皮细胞的凋亡并呈现出剂量依赖性;同时PEITC下调了自噬相关蛋白Atg5-12;PEITC对凋亡与自噬的调节可能存在相关性。

Objective To investigate the effects of phenethyl isothiocyanate(PEITC)on human benign prostatic epithelial cells(BPH-1).Methods Exposure of BPH-1 cells to PEITC at different concentrations(6,12,and 24μmol/L)for 6,12,24,or 36 hours.The suppression of cell proliferation was assessed with CCK-8 assay.The cell apoptosis was evaluated with Annexin V/PI staining by fluorescent flow-cytometric assay.The Western blotting analysis was applied to examine the expression of X-linked inhibitor-of-apoptosis protein(XIAP)and Atg5-12.The expression of XIAP mRNA was detected with RT-PCR.Results (1)Upon the treatment of PEITC at 6,12,and 24μmol/L,the cell relative viability was 71.22%,48.90%,and 13.57%,respectively,for 6 hours;52.57%,42.37%,and 10.11%,respectively,for 12 hours;36.73%,25.88%,and 8.39%for 24 hours,respectively,and 33.57%,25.83%,and 13.06%,respectively,for 36 hours.PEITC suppressed the proliferation of BPH-1 cells in a concentration-dependent way,compared with the control group(P<0.05).(2)The total percentages of apoptotic cells was 19.5%,30.4%,and 40.1%in BPH-1 cells upon the treatment of PEITC at 6,12,and 24μmol/L for 24 hours,respectively.PEITC promoted cell apoptosis at concentrations of 12μmol/L and 24μmol/L,compared with the control group(P<0.05).(3)The relative expression of XIAP mRNA and XIAP was suppressed in a concentration-dependent way(6,12,and 24μmol/L)upon the treatment of PEITC for 24 hours.The expression of Atg 5-12 protein was also inhibited in a concentration-dependent way(6,12,and 24μmol/L)upon the treatment of PEITC for 24 hours.Conclusions PEITC suppressed the proliferation of benign prostatic epithelial cells and induced cell apoptosis by inhibiting the expression of XIAP mRNA and XIAP in a concentration-dependent way,which was probably associated with the suppressed autophagy pathway in BPH-1 cells.