红花多糖对人肝癌细胞凋亡和自噬的影响及潜在机制

Effect and potential mechanism of Safflower polysaccharide on apoptosis and autophagy of human hepatocellular carcinoma cells

目的探讨红花多糖(SPS)对人肝癌细胞凋亡和自噬的影响及潜在机制。方法以人肝癌细胞HepG2、SMMC-7721、Huh-7为研究对象,以SPS为干预药物,筛选敏感细胞、药物干预浓度及干预时间。以所选敏感细胞为对象,以不同浓度的SPS进行干预,观察其凋亡、迁移、克隆形成、形态、自噬发生情况,并检测其凋亡、自噬和磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白的表达情况。结果SPS可抑制人肝癌细胞HepG2等3种细胞的增殖,其对SMMC-7721细胞的半数抑制浓度显著低于其余2种细胞(P<0.05)。经低、中、高浓度(20、40、80μmol/L)SPS干预48 h后,SMMC-7721细胞凋亡较对照组有所增加,24、48 h的细胞迁移率(24 h的SPS低浓度组除外)和24 h的细胞克隆形成率均较对照组显著降低(P<0.05或P<0.01);与对照组比较,SPS各浓度组细胞数量均明显减少,自噬水平均有所提升,细胞中剪切的胱天蛋白酶3、胱天蛋白酶9、Bax、beclin-1蛋白的相对表达量和微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)与LC3Ⅰ的比值均显著升高,Bcl-2、p62、PI3K、mTOR、Akt蛋白的相对表达量均显著降低(P<0.05或P<0.01)。结论SPS可抑制人肝癌SMMC-7721细胞的增殖并诱导其凋亡、自噬,这种作用可能与抑制PI3K/Akt/mTOR信号通路有关。

OBJECTIVE To investigate the effect and potential mechanism of safflower polysaccharide on apoptosis and autophagy of human hepatocellular carcinoma cells.METHODS Human hepatocellular carcinoma HepG2,SMMC-7721 and Huh-7 cells were selected as subjects,and safflower polysaccharide was used as intervention drug to screen sensitive cells,intervention concentration and intervention time.The sensitive cells were selected as the object and intervened with different concentrations of safflower polysaccharide;the apoptosis,migration,clone formation,morphology and autophagy of human hepatocellular carcinoma cells were observed;the expressions of apoptosis,autophagy and phosphatidyl inositol-3-kinase(PI3K)/protein kinase B(Akt)/mammals rapamycin target protein(mTOR)signaling pathway related protein were detected.RESULTS safflower polysaccharide could inhibit the proliferation of 3 kinds of human hepatocellular carcinoma cells,and the half inhibition concentration of it to SMMC-7721 cells was significantly lower than to other two kinds of cells(P<0.05).After 48 h intervened with low,medium and high concentrations of safflower polysaccharide(20,40,80μmol/L),the apoptosis of SMMC-7721 cells was increased compared with the control group,and cell migration rates at 24 and 48 h(except for safflower polysaccharide lowdose group at 24 h)and clone formation rate at 24 h were significantly decreased compared with the control group(P<0.05 or P<0.01).Compared with the control group,cell number in safflower polysaccharide groups was significantly decreased,and autophagy levels were improved to some extent;the relative expressions of cleaved caspase-3,caspase-9,Bax and beclin-1 protein and ratio of LC3Ⅱ/LC3Ⅰ were significantly increased,and the relative protein expressions of Bcl-2,p62,PI3K,mTOR and Akt were significantly decreased(P<0.05 or P<0.01).CONCLUSIONS Safflower polysaccharide could effectively inhibit the proliferation and induce apoptosis and autophagy of human hepatocellular carcinoma SMMC-7721 cells,the mechanism of which may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.