去甲斑蝥素通过JAK-STAT3途径诱导结肠癌LS-174T细胞凋亡

Norcantharidin Induced Apoptosis of Human Colon Carcinoma Cells LS-174T through Inhibiting JAK-STAT3 Signal Pathway

目的探讨去甲斑蝥素(NCTD)诱导结肠癌LS-174T细胞的凋亡效应及其可能分子机制。方法应用MTT法检测NCTD对细胞生长的抑制作用,通过AO/EB染色、透射电子显微镜观察细胞形态及超微结构的变化,AnnexinV/PI染色检测细胞凋亡率,Western blot检测stat3、survivin、Mcl-1、bax蛋白的表达情况。结果去甲斑蝥素对结肠癌LS-174T细胞具有显著的生长抑制作用,并呈时间—剂量依赖性;40μg/ml作用72 h时达到最大抑制率(72.4±3.1)%;AO/EB染色及流式细胞术显示,随着NCTD浓度的增加,细胞凋亡率明显增加;透射电子显微镜观察发现,结肠癌LS-174T细胞出现明显凋亡超微结构变化;Western blot结果显示,stat3、survivin、Mcl-1表达明显下降,bax表达明显上升。结论去甲斑蝥素可以抑制结肠癌LS-174T细胞的生长并促进其凋亡,机制可能是通过抑制stat3途径实现的。

Objective To investigate the effect of norcantharidin(NCTD) on the apoptosis of human colon carcinoma cells LS-174T and its possible molecular mechanism.Methods MTT assay was used to detect the effects of NCTD on growth inhibition in LS-174T cells. Acridine orang/ ethidium(AO/EB) bromide double staining and transmission electron microscope technique were used to observe the changes of cellular morphology and ultrastructure. AnnexinV/PI staining was used to detect the rate of apoptosis. Western blot was used to assess the protein levels of stat3, survivin, Mcl-1 and bax.Results NCTD significantly inhibited LS-174T cells growth in a time- and dose-dependent manner(5, 10, 20, 40 μg/ml). The maximum inhibition rate was(72.4±3.1)% at 40 μg/ml and 72 h. AO/EB staining and flow cytometry showed that with the increase of the concentration of NCTD, apoptosis rate was increased significantly.Transmission electron microscope found significant morphological change of apoptosis; Western blot results showed that the protein expression of stat-3, survivin and Mcl-1 was significantly reduced, whereas the expression of bax was significantly increased.Conclusion NCTD can inhibit the growth of colon cancer cells LS-174T and promote its apoptosis. The mechanism maybe through inhibiting stat3.