野苋菜提取物抗肿瘤作用及诱导人肝癌HepG2细胞凋亡的分子机制

Anticancer activity and mechanism of apoptosis induced by Amaranthus spinosus L.extract in HepG2 cells

目的研究野苋菜提取物的抗肿瘤作用及诱导人肝癌HepG2细胞凋亡的可能机制。方法采用Alamar blue法检测野苋菜提取物对肿瘤细胞的抑制作用;光学显微镜和Hoechst 33258荧光染色观察HepG2细胞的形态变化;流式细胞术检测该提取物对HepG2细胞凋亡的影响;Western blot和caspase-3活性检测试剂盒分析野苋菜提取物诱导HepG2细胞凋亡的作用机制;caspase-9、caspase-3抑制剂(Z-LEHDFMK和Ac-DEVD-CHO)验证相关的调控信号转导通路。结果野苋菜提取物对多种肿瘤细胞均有抑制作用,其中HepG2细胞最敏感;经提取物处理过的HepG2细胞出现明显的凋亡特征,流式细胞术进一步证实野苋菜提取物能够诱导HepG2细胞的凋亡;野苋菜提取物处理48h后的HepG2细胞中Bcl-2、survivin表达下调,Bax、PARP、Apaf-1、caspase-9表达量增加,并且caspase-3、caspase-9酶活性明显提高;caspase-9和caspase-3的抑制剂能够逆转野苋菜提取物对HepG2细胞的抑制活性。结论野苋菜提取物具有抗肿瘤作用,并且能够激活caspase-9内源性凋亡信号通路,发挥其促进HepG2细胞凋亡的作用。

Aim To investigate the anticancer activity and the mechanism of the apoptosis induced by Ama-ranthus spinosus L. extract ( ASE ) in human hepatic carcinoma cell line HepG2 . Methods Alamar blue assay was used for detecting the influence of ASE on the proliferation of the cancer cells. The morphological changes of cells were observed under inverted micro-scope and Hoechst 33258 stainning. The apoptosis of HepG2 cells was detected by flow cytometry. Western blot and caspase-3 activity kit were used to detect the protein expression in HepG2 cells. The specific inhibi-tor of caspase-9 and caspase-3 ( Z-LEHD-FMK and Ac-DEVD-CHO) was used to validate the signal transduc-tion pathyway. Results The results indicated that the cell proliferation was inhibited by ASE,especicially the HepG2 cells. The HepG2 cells showed obvious apop-totic characteristics. Flow cytometry analysis further validated the apoptosis of HepG2 cells. The expression of Bcl-2 and survivin was downreagulated in HepG2 cells treated with ASE, and Bax, caspase-9, caspase-3, Apaf-1 and PARP were upregualted. Besides, the caspase-3 activity was also increased. Z-LEHD-FMK and Ac-DEVD-CHO significantly increased the cell vi-abilty of HepG2 cells induced by ASE. Conclusion These results confirm that ASE induces the apoptosis of HepG2 through mitochondria-mediated pathway.