摘要
目的研究沙蟾毒精对体外人结肠癌HCT116细胞和SW620细胞凋亡与自噬的影响及其相关作用机制。方法以人结肠癌HCT116细胞和SW620细胞为靶细胞,以不同浓度(0、25、50、100、200 nM)的沙蟾毒精处理后,采用细胞计数试剂盒8(CCK-8)检测人结肠癌HCT116细胞和SW620细胞活力,采用倒置相差显微镜观察沙蟾毒精干预后HCT116细胞和SW620细胞的形态学改变,AnnexinV/PI流式细胞术检测人结肠癌细胞的凋亡情况,蛋白质免疫印迹法(WB)检测人结肠癌细胞凋亡相关蛋白、自噬相关蛋白及雷帕霉素靶蛋白(mTOR)通路相关蛋白的表达水平。结果CCK-8法显示沙蟾毒精能显著抑制人结肠癌HCT116、SW620细胞增殖,并具有浓度与时间依赖性(P<0.05,P<0.01)。流式细胞术检测结果显示,不同浓度(25、50、100 nM)的沙蟾毒精作用人结肠癌细胞48 h后,HCT116、SW620细胞凋亡率明显提高(P<0.05,P<0.01)。WB检测结果显示,100 nM沙蟾毒精作用人结肠癌HCT116、SW620细胞48 h后,沙蟾毒精组酵母Atg6同系物(Beclin-1)、微管相关蛋白1轻链3(LC3)-Ⅱ蛋白表达升高(P<0.01),P62、B淋巴细胞瘤-xl(Bcl-xl)、磷酸化雷帕霉素靶蛋白(P-mTOR)抗体蛋白表达水平降低(P<0.05)。与单独沙蟾毒精处理组比较,沙蟾毒精联合自噬抑制剂3-甲基腺嘌呤(3-MA)作用人结肠癌HCT116、SW620细胞后,LC3-Ⅱ蛋白表达水平降低(P<0.05,P<0.01),P62蛋白表达水平升高(P<0.01)。结论沙蟾毒精对人结肠癌细胞株生长具有显著的抑制作用,且具呈剂量和时间依赖性,同时沙蟾毒精可促进细胞凋亡与自噬,且可能通过mTOR通路发生自噬,可为结肠癌的治疗提供新的治疗策略。
Objective To study in vitro the effects of arenobufagin on the apoptosis and autophagy of colon cancer cells(HCT116 and SW620)and its related action mechanisms.Methods Human colon cancer cells(HCT116 and SW620)were used as target cells,treated with different concentrations of arenobufagin(0~200 nM).And then the proliferation capacity of colon cancer cells(HCT116 and SW620)was detected by the cell proliferation toxicity test kit(CCK-8).The inverted phase contrast microscope was used to observe the morphological changes of HCT116 and SW620 cells after the intervention of arenobufagin.AnnexinV/PI flow cytometry and Western Blotting(WB)test were applied to separately detect the apoptosis of colon cancer cells and the expression levels of apoptosis-related proteins,autophagy-related proteins and mTOR pathway-related protein of colon cancer cells.Results CCK-8 method showed that arenobufagin could significantly inhibit the proliferation of colon cancer cells(HCT116 and SW620),and it was concentration-and time-dependent(P<0.05,P<0.01).The results of flow cytometry showed that the apoptosis rate of colon cancer cells(HCT116 and SW620)was significantly increased(P<0.05,P<0.01)after treating with different concentrations of arenobufagin(0,25,50,100 nM)for 48 hours.WB test results showed that after 100 nM of arenobufagin acted on colon cancer cells(HCT116 and SW620)for 48 hours,the protein expression levels of homolog of arenobufagin yeast Atg6(Beclin-1)and microtubule-associated protein 1 light chain 3(LC3)-Ⅱwas increased(P<0.01).While the protein expression levels of P62,B lymphoma-xl(Bcl-xl),and phosphorylated mammalian target of rapamycin(P-mTOR)antibody were decreased(P<0.05).After arenobufagin combined with autophagy inhibitor 3-methyladenine(3-MA)treated colon cancer cells(HCT116 and SW620),the protein expression level of LC3-Ⅱwas decreased(P<0.05)while the level of P62 was increased(P<0.01).Conclusion Arenobufagin has a significant inhibitory effect on the growth of human colon cancer cell lines,and is dose-and time-dependent.At the same time,it can promote the apoptosis and autophagy,develop the autophagy through the mTOR pathway,and provide the new treatment strategies for treating colon cancer.
作者
陈进宝
徐可
邱艳艳
王海静
吴文韬
吴宏磊
贾琳琳
李炜
殷佩浩
曹亦军
CHEN Jinbao;XU Ke;QIU Yanyan(Department of General Surgery,Putuo Central Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062;不详)
出处
《河北中医》
2020年第8期1216-1221,1232,I0003,共8页
Hebei Journal of Traditional Chinese Medicine
基金
国家自然科学基金项目(编号:81603464,81873137)
2017年度上海市普陀区卫生系统科研自主创新项目(编号:ptkwws201701)。
