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目的:研究紫金牛三萜皂苷类化合物TSP02体外诱导人肝癌细胞HepG2凋亡和侵袭迁移的作用及其分子机制。方法:采用MTT比色法,检测不同时间、不同浓度的TSP02对人肝癌HepG2细胞和人正常肝HL-7702细胞的增殖抑制作用。采用流式细胞术检测药物处理后HepG2和HL-7702细胞周期和凋亡变化情况。进一步用Western blot法检测TSP02对周期调控蛋白CDK1,2和4,细胞凋亡相关蛋白Caspase-8,以及细胞迁移侵袭相关蛋白TGF-β1和E-cadherin表达水平的影响。最后通过细胞划痕实验和Transwell小室体外侵袭实验检测HepG2细胞经过TSP02处理后迁移侵袭能力的变化。结果:TSP02显著抑制人肝癌细胞HepG2的生长,抑制效果有明显的时间依赖性和浓度依赖性,而对人正常肝细胞HL-7702的作用不明显。与对照组比较,TSP02处理24 h在造成HepG2细胞S期细胞消失,细胞凋亡率明显增加的同时,还能够显著降低HepG2细胞中CDK1,2,4的表达,提高促凋亡蛋白Caspase-8的表达和活化,而对正常肝HL-7702细胞无论在周期和凋亡率上均无明显影响。此外,TSP02处理后的HepG2细胞移动和侵袭性下降的同时,分别下调和上调了肝癌侵袭相关蛋白TGF-β1和E-cad-herin的表达。结论:TSP02选择性促进人肝癌细胞HepG2凋亡并抑制肝癌细胞的迁移和侵袭能力。TSP02的这些体外抗肿瘤活性与其改变周期和凋亡调控蛋白,并影响侵袭相关TGF-β1和E-cadherin的表达有关。
AIM: To investigate the apoptosis-inducing effect and its molecular mechanism of TSP02, a triterpene saponin of Hepatocarcinoma, on the apoptosis, invasion and migration of human hepatocellular carcinoma cell HepG2. Methods: MTT colorimetric assay was used to detect the proliferation of human hepatocellular carcinoma HepG2 cells and human normal liver HL-7702 cells with different concentrations of TSPO2 at different times and different concentrations. The changes of cell cycle and apoptosis in HepG2 and HL-7702 cells treated with drug were detected by flow cytometry. The effects of TSPO2 on the expression of CDK1, 2 and 4, Caspase-8, TGF-β1 and E-cadherin in cell migration and invasion were further analyzed by Western blot. Finally, the cell invasion and invasion ability of HepG2 cells treated with TSP02 were detected by cell scratch assay and Transwell chamber in vitro invasion assay. Results: TSP02 significantly inhibited the growth of HepG2 cells in a time-dependent and concentration-dependent manner, but not in human hepatocytes HL-7702. Compared with the control group, TSP02 treatment for 24 h resulted in the disappearance of S phase cells in HepG2 cells and significantly increased the apoptosis rate of HepG2 cells, as well as significantly decreased the expression of CDK1, 2 and 4 and increased the expression of proapoptotic protein Caspase-8 The expression and activation of HL-7702 cells in normal liver have no significant effect on the cycle and apoptosis rate. In addition, TSP02-treated HepG2 cells down-regulated and up-regulated the expression of TGF-β1 and E-cad-herin, respectively, in invasion and metastasis of HepG2 cells. Conclusion: TSP02 can selectively promote the apoptosis of HepG2 cells and inhibit the migration and invasion of hepatocellular carcinoma cells. These in vitro antitumor activities of TSP02 are related to its role in altering the cycle and regulatory proteins of apoptosis and affecting the expression of invasion-associated TGF-β1 and E-cadherin.