醌茜素对人肝癌Huh7细胞的凋亡诱导作用及其抗肿瘤机制

Apoptotic Effect of Quinalizarin on Human Hepatoma Huh7 Cells and Its Antitumor Mechanism

目的探讨醌茜素诱导人肝癌Huh7细胞凋亡的机制。方法 MTT法测定醌茜素对人肝癌Hep G2、Hep3B和Huh7细胞的杀伤作用;倒置显微镜下观察醌茜素处理后Huh7细胞形态学变化;Annexin V-FITC/PI双染法及流式细胞术检测其对肝癌Huh7细胞的诱导凋亡作用;蛋白质免疫印迹法检测细胞凋亡相关蛋白的表达量变化;流式细胞术检测醌茜素处理后细胞内ROS的表达水平。结果与对照组(0 h)相比醌茜素能够明显抑制人肝癌细胞的存活率(P=0.0204)。经醌茜素处理后的实验组Huh7细胞呈现经典的细胞皱缩、核固缩等现象。实验组(24 h)Huh7细胞凋亡率为50.63%,明显高于对照组(0 h)(15.58%)(P=0.0001)。共同处理Akt抑制剂和醌茜素后,促凋亡蛋白Bad和cleaved-caspase-3表达量均逐渐增加,抗凋亡蛋白磷酸化的Akt和Bcl-2均逐渐减少(P<0.05)。醌茜素处理后,可增加Huh7细胞内活性氧的水平。结论醌茜素对人肝癌Huh7细胞具有杀伤作用,且呈浓度依赖性。其机制可能是醌茜素抑制细胞内Akt的表达及磷酸化水平或诱导活性氧的产生,进而促进Huh7细胞的凋亡。

Objective To investigate the effect of quinalizarin on the apoptosis of hepatoma Huh7 cells and to explore its possible mechanism. Methods Human hepatoma HepG2, Hep3B and Huh7 cells viabilities were detected by MTT assay. The morphological alterations of Huh7 cells were demonstrated by inverted microscope. Cell apoptosis was analyzed by Annexin V-FITC/PI double staining and flow cytometry. The expressions of apoptotic proteins were detected by Western blot. The levels of ROS were measured by flow cytometry after quinalizarin treatment. Results MTT assay results suggested that compared with control group （0 h）, quinalizarin significantly inhibited the viabilities of human hepatoma HepG2, Hep3B and Huh7 cells in a dose-dependent manner （P=-0.0204）. The apoptotic morphology of Huh7 cells in the experimental group was found under the inverted microscope, such as cell shrinkage, karyopyknosis. The apoptotic rates of Huh7 cells in 24 h experimental group was 50.63%, significantly higher than that in 0 h control group （15.58%） （P=0.0001）. Akt inhibitor pretreatment could synergize with quinalizarin to upregulate the expression levels of Bad and cleaved-caspase-3 protein, and downregulate the expression levels of p-Akt and Bcl-2 （P〈0.05）. The intracellular reactive oxygen species （ROS） level was increased after quinalizarin treatment. Conclusion Quinalizarin could inhibit the proliferation of Huh7 cells in a dose-dependent manner. Its mechanism may be related to quinalizarin down-regulating the expression of phosphorylated Akt or inducing the levels of ROS. and then to promote the Huh7 cells apoptosis.