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目的:探讨热休克蛋白HSP90拮抗剂17-烯丙胺基-17-脱甲氧基格尔德霉素(17-aag)对体外结肠癌细胞株SW1116增值的抑制作用及其作用方式,并分析热休克因子-1(HSF-1)对17-aag诱导凋亡的影响。方法:将不同浓度的17-aag作用于野生型SW1116细胞株24、48、72 h,应用CCK-8法测定各处理组细胞的活性,计算增值抑制效应,应用流式细胞仪(FCM)检测细胞周期变化及细胞凋亡比例。构建针对HSF-1的shRNA质粒,通过瞬转质粒及药物干预的不同将肿瘤细胞分为4组,即HSF-1(-)17-aag(-)组、HSF-1(-)17-aag(+)组、HSF-1(+)17-aag(-)组、HSF-1(+)17-aag(+)组。注:HSF-1(-)代表转染了含目的干扰片段质粒,HSF-1(+)代表转染了空载质粒,17-aag(-)代表未受17-aag干预,17-aag(+)代表受17-aag干预。每组细胞经相应处理后,通过FCM测定各组在处理后的细胞凋亡比例,比较各处理因素对结肠癌细胞株SW1116的杀伤作用。通过Western印迹检测各组中HSP90、HSP70、HSP27的表达量,评价细胞凋亡比例的高低与热休克蛋白的表达量是否有关。结果:17-aag对野生型SW1116细胞增殖具有明显的抑制作用,可诱导G2/M期周期阻滞和细胞凋亡。转染组细胞后,HSF-1(-)17-aag(-)组与HSF-1(+)17-aag(-)组间细胞凋亡比例差异无统计学意义;HSF-1(-)17-aag(+)组较HSF-1(+)17-aag(+)组细胞凋亡比例高,而细胞内HSP90、HSP70、HSP27表达量均较HSF-1(+)17-aag(+)组降低。结论:17-aag可诱导体外结肠癌SW1116细胞发生凋亡和周期阻滞。在干扰HSF-1表达后,17-aag诱导细胞凋亡的作用明显增强。
OBJECTIVE: To investigate the inhibitory effect of heat shock protein HSP90 antagonist 17-allylamino-17-amethoxygeldanamycin (17-aag) on proliferation of human colon cancer cell line SW1116 in vitro and its effect Effect of shock factor-1 (HSF-1) on apoptosis induced by 17-aag. Methods: The wild-type SW1116 cells were treated with different concentrations of 17-aag for 24, 48 and 72 h. The activity of cells in each treatment group was determined by CCK-8 assay, and the proliferation inhibitory effect was calculated. Flow cytometry (FCM) Cell cycle changes and apoptosis ratio. The shRNA plasmid targeting HSF-1 was constructed. Tumor cells were divided into four groups according to the transient plasmid and drug intervention: HSF-1 (-) 17-aag (-), HSF-1 (+) Group, HSF-1 (+) 17-aag (-) group and HSF-1 (+) 17-aag group. Note: HSF-1 (-) represents the plasmid transfected with the interference fragment of interest, HSF-1 (+) represents the transfected empty vector, 17-aag +) Represents intervention by 17-aag. After corresponding treatment, the proportion of apoptotic cells in each group after treatment was determined by FCM, and the cytotoxicity against various colon cancer cell lines SW1116 was compared. Western blotting was used to detect the expression of HSP90, HSP70, HSP27 in each group to evaluate whether the ratio of apoptosis was related to the expression of heat shock protein. Results: 17-aag significantly inhibited the proliferation of wild-type SW1116 cells and induced G2 / M cycle arrest and apoptosis. There was no significant difference in the percentage of apoptotic cells between HSF-1 (-) 17-aag (-) group and HSF-1 (+) 17-aag Compared with HSF-1 (+) 17-aag (+) group, the percentage of apoptosis in 17-aag (+) group was higher than that in HSF-1 (+) 17-aag ) Group decreased. Conclusion: 17-aag can induce apoptosis and cycle arrest of SW1116 cells in vitro. The effect of 17-aag on apoptosis was significantly enhanced after interfering with HSF-1 expression.