大黄酚对肝癌HCCLM3细胞增殖与转移作用研究

Effects of Chrysophanol on Proliferation and Metastasis of Hepatocellular Carcinoma HCCLM3 Cells

目的探讨大黄酚对肝癌HCCLM3细胞增殖与转移的抑制作用及其作用机制.方法采用克隆形成抑制实验和CCK-8实验检测大黄酚对HCCLM3细胞增殖的影响;通过Hoechst染色与Annexin V-FITC/PI检测大黄酚对HCCLM3细胞凋亡的影响;采用JC-1、TMRM染色检测大黄酚对HCCLM3细胞线粒体膜电位的影响;通过划痕实验与Transwell实验分析大黄酚对HCCLM3细胞转移的作用,采用Western blotting检测大黄酚对HCCLM3细胞凋亡相关蛋白PARP、cleaved caspase-3、BAX、Bcl-2及转移相关蛋白N-cadherin、MMP9、MMP2等的影响;采用DHR活性氧检测试剂盒检测大黄酚对HCCLM3细胞活性氧(ROS)产生的影响.结果大黄酚可有效抑制HCCLM3细胞增殖(P<0.05),并具有剂量效应关系;JC-1与TMRM染色结果显示:大黄酚能明显降低HCCLM3细胞线粒体膜电位(P<0.05);划痕实验与Transwell实验结果显示:大黄酚能显著抑制HCCLM3细胞转移(P<0.05);ROS实验发现:大黄酚能显著增加细胞活性氧水平(P<0.05),从而杀伤肿瘤细胞;Westen blotting结果显示:大黄酚可促进PARP、cleaved caspase-3、BAX表达,抑制抗凋亡蛋白Bcl-2及转移相关蛋白N-cadherin、MMP9、MMP2表达.结论大黄酚能有效抑制人肝癌细胞增殖和转移,通过线粒体途径导致HCCLM3细胞凋亡.

Objective To investigate the anti-tumor effect of Chrysophanol on proliferation and metastasis of liver cancer HCCLM3 cells.Method The colony forming inhibition unit assay and CCK-8 were used to detect the effect of Chrysophanol on HCCLM3 cells;The main inhibition pathways of Chrysophanol on HCCLM3 cells were analyzed by Hoechst staining,Annexin V-FITC/PI staining;The effect of Chrysophanol on HCCLM3 cells mitochondrial membrane potential was detected by JC-1 staining and TMRM staining;The effects of Chrysophanol on HCCLM3 cell metastasis were analyzed with scratch test and transwell test.The effects of Chrysophanol on apoptosis related proteins PARP,cleaved caspase-3,BAX,Bcl-2,and metastasis related proteins N-cadherin,MMP9 and MMP2 were estimated with Westen blotting;DHR reactive oxygen species assay kit was used to detect the effect of Chrysophanol on the production of reactive oxygen species(ROS)in HCCLM3 cells.Results Chrysophanol can effectively inhibit the proliferation of HCCLM3 cells in a dose dependent manner(P<0.05);JC-1 and TMRM staining showed that Chrysophanol significantly decreased the mitochondrial membrane potential of HCCLM3 cells(P<0.05);The results of scratch test and Transwell test showed that Chrysophanol significantly inhibited the metastasis of HCCLM3 cells(P<0.05);ROS experiment showed that Chrysophanol significantly increased the level of cellular reactive oxygen species(P<0.05),which can kill the tumor cells;Westen blotting results showed that Chrysophanol can promote the expression of PARP,cleaved caspase-3 and BAX,and inhibit the expression of anti-apoptotic protein Bcl-2 and metastasis related proteins N-cadherin,MMP9 and MMP2.Conclusion Chrysophanol can effectively inhibit the proliferation and metastasis of human liver cancer cells.This research provides a theoretical basis for the future application of anti-tumor drugs based on chrysophanol.