华蟾酥毒基抑制人前列腺癌PC3细胞体外增殖研究

The inhibitory effects of cinobufagin on the in vitro proliferation of human castration-resistant prostate cancer PC3 cells

目的探讨华蟾酥毒基对人前列腺癌PC3细胞体外增殖和凋亡的影响及其可能存在的作用机制。方法细胞分为不加药对照组,5、50、100、500 nmol/L华蟾酥毒基组,共5组。各组作用于PC3细胞24、48 h后,四甲基偶氮唑蓝(MTT)法检测华蟾酥毒基对人前列腺癌PC3细胞增殖的影响。不同浓度华蟾酥毒基作用于PC3细胞48 h后,光学显微镜观察PC3细胞形态变化;克隆形成实验检测华蟾酥毒基对前列腺癌PC3细胞克隆形成能力的影响;流式细胞术检测华蟾酥毒基作用48 h后各组PC3细胞凋亡情况;蛋白质印迹法检测华蟾酥毒基对髓样细胞白血病-1(MCL-1)蛋白表达的影响。结果 24 h后华蟾酥毒基可在体外抑制PC3细胞增殖(P <0. 01),48 h后华蟾酥毒基对PC3细胞增殖抑制作用更为明显(P <0. 05),华蟾酥毒基对PC3细胞增殖能力有较好的抑制作用,且呈剂量和时间依赖性。细胞克隆形成实验发现,0. 5 nmol/L华蟾酥毒基即可抑制细胞克隆形成(P <0. 05),并且此种抑制作用与药物浓度呈正相关(P <0. 05)。流式细胞术结果显示,0. 5nmol/L以上华蟾酥毒基均可诱导PC3细胞凋亡,随浓度增加凋亡率显著增加(P <0. 01),50 nmol/L华蟾酥毒基干预48 h后,PC3细胞凋亡率为39. 667%;同时光学显微镜下细胞增殖抑制明显,并呈凋亡形态改变。蛋白质印迹法结果显示,不同浓度华蟾酥毒基均可下调抗凋亡蛋白MCL-1蛋白表达(P <0. 01),以50 nmol/L华蟾酥毒基作用于PC3细胞48 h下调MCL-1蛋白表达更为显著(P<0. 01)。结论华蟾酥毒基可抑制前列腺癌PC3细胞的体外增殖,并可诱导肿瘤细胞凋亡,其机制可能与抗凋亡蛋白MCL-1表达下调有关。

Objective To explore the inhibitory effects of cinobufagin on the in vitro proliferation and apoptosis of human castration-resistant prostate cancer PC3 cells,and to explore its mechanism of action.Methods PC3 cells were divided into 5 groups: control group and 5,50,100,500 nmol/L cinobufagin groups. At 24 and 48 hours,the proliferation of PC3 cells was observed by using MTT assay. At 48 hours,the morphological changes of PC3 cells were observed under microscope; the effect of cinobufagin on the clonal formation ability of PC3 cells was detected by using clone formation assay; the apoptosis of PC3 cells was detected with flow cytometry 48 hours after the action of cinobufagin; and the effect of cinobufagin on the expression of MCL-1 protein was detected by using Western blot assay. Results At24 hours,the proliferation of PC3 cells was inhibited by cinobufagin( P < 0. 01). At 48 hours,the proliferation of PC3 cells was also inhibited by cinobufagin( P < 0. 05). The inhibiting effects of cinobufagin on the proliferation of PC3 cells was in a dose and time dependent manner. Cell clone formation assay showed that 0. 5 nmol/L cinobufagin could inhibit cell clone formation( P < 0. 05),and the inhibition was positively correlated with drug concentration( P < 0. 05). The results of flow cytometry showed that the apoptosis rate of PC3 cells was significantly increased with the increase of concentration( P< 0. 01). After the intervention of 50 nmol/L cinobufagin for 48 h,the apoptosis rate of PC3 cells was( 39. 667 +7. 636) %; cell proliferation under the light microscope also presented with apparent inhibition and the morphology of apoptosis was changed. Western blot assay showed that the expression of antiapoptotic protein MCL-1 was down-regulated by different concentrations of cinobufagin( P < 0. 01),and the expression of MCL-1 was down-regulated by 50 nmol/L cinobufagin at 48 hours( P < 0. 01).Conclusion Cinobufagin can inhibit the external proliferation of PC3 cells and induce apoptosis of prostate cancer cells. This effect may be related to down-regulating the expression of anti-apoptotic protein Mc L-1.