眼镜蛇毒细胞毒素对人肝癌细胞增殖抑制活性及作用机制的研究

Growth inhibition activity of cytotoxin from cobra venoms on BEL-7404 cells and its mechanism

目的研究眼镜蛇毒细胞毒素(CTX)在体外诱导人肝癌BEL-7404细胞凋亡作用及其可能机制。方法不同浓度CTX作用于人肝癌BEL-7404细胞,采用噻唑蓝(MTT)比色法检测肿瘤细胞增殖情况;TUNEL法和透射电镜观察检测肿瘤细胞凋亡;并对caspase-3、-9活性、细胞色素C分布变化进行检测。结果眼镜蛇毒细胞毒素对人肝癌BEL-7404细胞具有明显的抑制增殖作用,12、24和48 h的IC50分别为2.56、1.94和1.35 mg·L-1,TUNEL法和透射电镜均观察到肿瘤细胞的凋亡;caspase-3,-9酶活性增高,Western blot检测到caspase-3,-9酶的表达增强,线粒体内Cytochrome C表达减少,而细胞质内Cytochrome C表达增强。结论 CTX首先通过线粒体细胞色素C释放到细胞质内,进一步诱导效应性caspase-3,-9酶激活,可能是其诱导人肝癌细胞凋亡而产生抗肿瘤作用的机制之一。

Aim To investigate the effects of CTX from cobra venoms on inducing BEL-7404 apoptosis and its mechanisms.Methods The antitumor effect of CTX in BEL-7404 cells in vitro was detected by MTT;the apoptotic inducing effect was observed using TUNEL and transmission electron microscope;the enzyme activity of caspase-9 and caspase-3 was measured with biochemistry method;the expression of caspase-9,caspase-3 and cytochrome C was detected by Western blot.Results The IC50 values of CTX affected on BEL-7404 cell for 12,24 and 48 h were 2.56,1.94 and 1.35 mg·L-1,respectively.CTX could induce apoptotic morphological changes,such as condensed chromatin and swelling mitochondria.Both enzyme activity and expression level of caspase-9,caspase-3 increased,and the content of cytochrome C decreased in mitochondrion but increased in cytoplasm.Conclusion The results suggest that CTX from cobra venoms evokes the release of cytochrome C from the mitochondria into the cytoplasm,and precedes the activation of caspase-9 and caspase-3 to leading to the apoptosis of BEL-7404 cells,which may be one of the antitumor mechanisms.