羟基红花黄色素A通过PI3K通路抑制人肝癌细胞增殖、迁移并促进其凋亡

Hydroxysafflor yellow A inhibits proliferation and migration of human hepatocellular carcinoma cells and promotes apoptosis via PI3K pathway

目的:探讨羟基红花黄色素A(hydroxysa or yellow A,HSYA)对人肝癌细胞增殖、凋亡及迁移的影响,并判断是否通过磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/丝氨酸苏氨酸蛋白激酶(protein kinase B,PKB,又称Akt)信号通路发挥作用。方法:HSYA和PI3K抑制剂LY294002处理人肝癌HepG2、Hep3B和SMMC7721细胞后,分别采用CCK-8法、克隆形成实验和划痕愈合实验检测细胞增殖、克隆形成和迁移能力。FCM法检测HSYA和LY294002对人肝癌HepG2细胞凋亡的影响,蛋白质印迹法检测HSYA和LY294002对人肝癌HepG2细胞中基质金属蛋白酶2(matrix metalloprotein 2,MMP2)、caspase 3、cleaved-caspase 3和磷酸化Akt(phospho-Akt,p-Akt)蛋白表达的影响。结果:50μmol/L HSYA和10μmol/L LY294002均可抑制人肝癌HepG2、Hep3B和SMMC7721细胞的增殖(P值均<0.05)。50μmol/L HSYA、10μmol/LLY294002和50μmol/LHSYA联合10μmol/LLY294002处理组HepG2、Hep3B和SMMC7721细胞克隆形成率和迁移率均低于未处理的对照组(P值均<0.05),HSYA联合LY294002处理组HepG2、Hep3B和SMMC7721细胞克隆形成率和迁移率低于HSYA和LY294002单独处理组(P值均<0.01)。HSYA、LY294002和HSYA联合LY294002处理组HepG2细胞凋亡率均高于未处理的对照组(P值均<0.05),HSYA联合LY294002处理组HepG2细胞凋亡率高于HSYA和LY294002单独处理组(P值均<0.01)。HSYA、LY294002和HSYA联合LY294002处理组HepG2细胞中p-Akt、MMP2和caspase 3蛋白表达水平均低于未处理的对照组(P值均<0.01),cleaved-caspase 3蛋白表达水平高于未处理的对照组(P <0.01),HSYA联合LY294002处理组的效果均好于HSYA和LY294002单独处理组(P值均<0.01)。结论:HSYA可以抑制肝癌HepG2、Hep3B和SMMC7721细胞的增殖和迁移,并可促进HepG2细胞凋亡。HSYA可能通过抑制PI3K/Akt信号通路诱导人肝癌HepG2细胞凋亡。

Objective： To investigate the effects of hydroxysafflor yellow A （HSYA） on the proliferation, apoptosis and migration of hepatocellular carcinoma cells, and to explore whether HSYA plays a role through phosphatidylinositol 3-kinase （PI3K）/protein kinase B （PKB, Akt） signaling pathway. Methods： After the treatment with HSYA and PI3K inhibitor LY294002, CCK-8 assay, clone formation assay and scratch healing assay were used to detect the proliferation, clone formation and migration abilities of human hepatoma HepG2, Hep3B and SMMC7721 cells, respectively. FCM was used to detect the effect of HSYA on the apoptosis of HepG2 cells. Western blotting was used to detect the expressions of matrix metalloprotein 2 （MMP2）, caspase 3, cleaved-caspase 3 and phospho-Akt （p-Akt）. Results： Both 50 μmol/L HSYA and 10 μmol/L LY294002 inhibited the proliferation of human hepatoma HepG2, Hep3B and SMMC7721 cells （all P 〈 0.05）. The clone formation rate and migration rate of HepG2, Hep3B and SMMC7721 cells treated with 50 μmol/L HSYA, 10 μmol/ L LY294002 and 50 μmol/L HSYA in combination with 10 μmol/L LY294002 were lower than those in the untreated control group （all P 〈 0.05）. The clone formation rate and migration rate of HepG2, Hep3B and SMMC7721 cells in HSYA combined with LY294002 treatment group were lower than those in HSYA or LY294002 alone treatment group （all P 〈 0.01）. The apoptosis rates of HepG2 cells in HSYA, LY294002 and HSYA combined with LY294002 treatment groups were higher than those in the untreated control group （all P 〈 0.05）. The apoptosis rate of HepG2 cells in HSYA combined with LY294002 treatment group was higher than that in HSYA or LY294002 alone treatment group （P 〈 0.01）. After the treatment with HSYA, LY294002 and HSYA combined with LY294002, the expression levels of p-Akt, MMP2 and caspase 3 in HepG2 cells were lower than those in the untreated control group （all P 〈 0.01）, and the expression level of cleaved-caspase 3 was higher than that in the untreated control group （P 〈 0.01）. The effects of HSYA combined with LY294002 treatment on the expressions of p-Akt, MMP2, caspase 3 and cleaved-caspase 3 were better than those of HSYA or LY294002 alone treatment group （all P 〈 0.01）.