柴胡皂甙d对肝癌细胞VEGF和Ang-2表达的影响

Effect of saikosaponin-d on expression of VEGF and angioprotein-2 in human hepatocellular carcinoma cells

目的观察柴胡皂甙d(saikosaponins-d,SSd)对人肝癌SMMC-7721细胞血管内皮生成因子(vascular endo-thelial growth factor,VEGF)和血管生成素-2(angiopoietin-2,Ang-2)表达的影响,探讨其抗癌作用机制。方法肝癌SMMC-7721细胞株经LPS诱导,加入不同终浓度的SSd(2.5、5、10、20 mg/L)作用48 h,并以300μmol/L终浓度的Nimesulide(COX-2选择性抑制剂)作为阳性对照,采用MTT法检测细胞增殖,免疫细胞化学法测定肝癌细胞内VEGF和Ang-2蛋白表达,RT-PCR法检测VEGF mRNA表达,ELISA法检测培养上清中VEGF含量。结果 SSd可显著抑制肝癌SMMC-7721细胞的生长、增殖。与LPS诱导组比较,10 mg/L SSd作用组肝癌细胞VEGF蛋白表达率、mRNA表达水平和培养上清中VEGF含量均显著降低[VEGF蛋白:80.0%vs 60.0%;mRNA表达:(0.83±0.07)vs(0.09±0.03);培养上清中VEGF含量:(655.00±5.56)vs(446.15±5.05)pg/ml,P<0.05,P<0.01],同时Ang-2蛋白表达亦显著降低(86.67%vs 53.33%,P<0.05)。在2.5～10 mg/L作用终浓度范围内,SSd对培养上清中VEGF含量降低的影响呈现一定的剂量依耐性。结论 SSd可能通过VEGF和Ang-2信号通路,发挥其抗癌作用。

Objective To observe the effect of saikosaponin-d（SSd） on expression of VEGF and angioptrotein-2（Ang-2） in hepatocellular carcinoma（HCC） SMMC-7721 cells.Methods Human HCC SMMC-7721 cells were induced by LPS and treated with SSd at different concentrations（2.5,5,10,or 20 mg/L） for 48 h.Nimesulide（a COX-2 selective inhibitor） at the concentration of 300 μmol/L served as a positive control.Proliferation of HCC SMMC-7721 cells was measured by MTT assay.Expressions of VEGF and Ang-2 protein as well as VEGF mRNA in HCC SMMC-7721 cells were detected by immunocytochemistry and RT-PCR,respectively.VEGF level in cultured supernatant was measured by ELISA.Results SSd significantly inhibited the growth and proliferation of HCC SMMC-7721 cells.The expression rates of VEGF and Ang-2 were significantly higher in 10 mg/L SSd group than in LPS induction group（80.0% vs 60.0%,86.67% vs 53.33%,P〈0.05）,while the expression level of VEGF mRNA in HCC SMMC-7721 cells and the VEGF level in cultured supernatant were also significantly higher in 10 mg/L SSd group than in LPS induction group（0.83±0.07 vs 0.09±0.03,655.00±5.56 vs 446.15±5.05 pg/ml,P〈0.01）,indicating that SSd inhibits the VEGF level in cultured supernatant in a dose-dependent manner.Conclusion SSd exerts its anti-tumor effect through the VEGF and Ang-2 signaling pathways.