3MA对Ce6-PDT诱导结肠癌SW620细胞凋亡及自噬的影响

摘要

目的 探讨自噬抑制剂3-甲基腺嘌呤(3MA)对Ce6-PDT诱导结肠癌SW620细胞凋亡及自噬的影响.方法 取对数生长期SW620细胞,分为空白对照组、单纯光照组、Ce6-PDT组和3MA-Ce6-PDT组,Ce6-PDT组加入不同浓度Ce6(0.125、0.25、0.5、1.0、2.0、4.0、8.0μg/mL),3MA-Ce6-PDT组给予1.0μg/mL的Ce6+5 mmol/mL的3MA.采用MTT法测算细胞存活率;AnnexinV-PE/7-ADD染色及罗丹明123联合流式细胞仪检测细胞凋亡率和细胞线粒体膜电位变化;吖啶橙染色联合荧光显微镜观察细胞自噬体形成;蛋白免疫印迹法检测细胞凋亡相关蛋白Caspase-3、B细胞淋巴瘤/白血病基因-2(Bcl-2)及自噬相关蛋白LC3Ⅱ/Ⅰ表达.结果 Ce6-PDT组细胞存活率与Ce6浓度浓度呈负相关(r=-0.820,P<0.01),细胞凋亡率与Ce6浓度呈正相关(r=0.845,P<0.01).选取细胞存活率和细胞凋亡率约为50%时Ce6浓度1μg/mL为后续给药浓度.在Ce6浓度达到1μg/mL时,线粒体膜电位随Ce6浓度增加而降低(P<0.01).空白对照组和单纯光照组几乎没有亮红色荧光产生,Ce6-PDT组中有大量亮红色荧光出现,3MA-Ce6-PDT组中出现亮红色荧光的数量较Ce6-PDT组减少.与Ce6-PDT组相比,3MA-Ce6-PDT组细胞存活率降低、细胞凋亡率升高、自噬相关蛋白表达降低、凋亡相关蛋白表达增加(P均<0.05).结论 Ce6-PDT可引起结肠癌SW620细胞发生凋亡和自噬,3MA会促进Ce6-PDT诱导的结肠癌细胞SW620凋亡.%Objective To investigate the effects of autophagy inhibitor 3-methyladenine (3MA)combined with Ce6-PDT on the apoptosis and autophagy of human colorectal cancer SW620 cells,and to provide theoretical and practical basis for autophagy inhibitors improving sensitivity of Ce6-PDT in treatment of colorectal cancer. Methods SW620 cells in the logarithmic phase were divided into the blank control group,negative control group,Ce6-PDT group,and 3MA-Ce6-PDT group. Cells in the Ce6-PDT group were treated with different concentrations of Ce6 (0. 125,0. 25,0. 5,1. 0,2. 0,and 8. 0 μg/ mL),and cells in the 3MA-Ce6-PDT group were treated with 1. 0 μg/ mL Ce6 + 5 mmol/ mL 3MA. Cell viability was detected by using MTT. The apoptosis and cell mitochondrial membrane potential changes were detected by using An-nexinV-PE/ 7-ADD and Rho123 combined with flow cytometry. Formation of autophagosome was observed by using fluores-cence microscopy with Acridine orange staining. The expression of apoptosis-related proteins Caspase-3,B-cell lymphoma/leukemia-2 (Bcl-2)and autophagy-related protein LC3Ⅱ/ Ⅰ was detected by Western blotting. Results In the Ce6-PDT group,the cell viability was negatively correlated with Ce6 concentration (r = - 0. 820,P < 0. 01);the apoptosis rate was positively correlated with the concentration of Ce6 (r = 0. 845,P < 0. 01);When the cell survival rate and apoptosis rate were 50%,1 μg/ mL Ce6 was taken as the concentration of the subsequent administration. When the Ce6 concentration reached 1 μg/ mL,the mitochondrial membrane potential decreased with the increase of Ce6 concentration (P < 0. 01).There was almost no bright red fluorescence in the blank control group and the negative control group. There was a large a-mount of bright red fluorescence in the Ce6-PDT group. The number of bright red fluorescence in the 3MA-Ce6-PDT group was significantly lower than that in the Ce6-PDT group. Compared with the Ce6-PDT group,the cell viability decreased (P< 0. 05),the apoptosis rate increased (P < 0. 05),mitochondrial membrane potential decreased (P < 0. 05),the expres-sion of autophagy-related protein decreased (P < 0. 05),and the expression of apoptosis-related protein increased in the 3MA-Ce6-PDT group (P < 0. 05). Conclusions Ce6-PDT induces the apoptosis and autophagy of colorectal cancer cells SW620. 3MA may promote the apoptosis of Ce6-PDT-induced colorectal cancer cells.