紫金牛三萜皂苷TSP02抑制人肝癌细胞增殖和侵袭作用机制研究

Study on inhibitory effect of triterpenoid saponin from Ardisia japonica TSP02 on proliferation and metastasis of human hepatocellular carcinoma cells and its mechanism

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。

Objective： To study the effect of TSP02, a triterpenoid saponin compound from Ardisia japonica, on proliferation and metastasis of in vitro induced human hepatoma HepG2 cells and its molecular mechanism. Method： MqT assay was performed to detect the inhibitory effect of TSP02 of different concentrations on proliferation of human hepatoma HepG2 and normal human hepatic cell HL-7702 cells. The changes in cell cycle and apoptotic of processed HepG2 and HL-7702 cells were detected by using flow cytometry. The effect of TSP02 on expression levels of CDK1, 2, 4, apoptosis-related protein Caspase-8, metastasis-related TGF-β1 and E-eadherin was measured by western blot. Wound-healing assay and transwell assay were performed to detect the changes in the metastasis of TSPO2-processed HepG2. Result： TSP02 significantly inhibited the proliferation of HepG2 cells, with notable time dependence and concentration dependence, but without remarkable effect on normal human liver HL-7702 cells. Compared with the control group, TSP02 processed for 24 h could eliminate HepG2 cells in S stage, significantly increase the cell apoptotic rate. Furthermore, TSP02 was capable of down-regulating the expression of multiple CDK1, 2, 4, and TGF-β1, and up-regulating the expression and activity of Caspase-8, without significant effect on cycle and apoptotic rate of normal human liver HL-7702 cells. Additionally, TSP02 caused metastasis and invasiveness HepG2 cells, while down-regulating liver cancer invasiveness-related TGF-β1 and E-cadherin. Conclusion： TSP02 selectively promotes apoptosis of liver cancer cell HepG2, and inhibits its metastasis and invasiveness. TSP02＇s in vitro antineo- plastic activity is related to the changes in cycle and apoptosis proteins, and the regulation in the expression of invasiveness-related TGF-βl and E-eadherin.