桔皮素对人肝癌HepG2细胞体外增殖和侵袭的影响及机制研究

Effects of Tangeretin on in vitro Proliferation and Invasion of Human Hepatocellular Carcinoma HepG2 Cells and Its Mechanism Study

目的：考察桔皮素对人肝癌Hep G2细胞体外增殖和侵袭的影响,并探讨其作用机制。方法：以0（空白对照组）、10、20、40、60、80μmol/L桔皮素分别作用于细胞24、48 h后,采用CCK-8法检测细胞增殖率,并计算其半数抑制浓度（IC50）。以0、20、30μmol/L桔皮素作用细胞48 h后,采用Transwell侵袭实验考察其对细胞侵袭的影响;并分别采用实时荧光定量聚合酶链式反应法和Western blot法考察其对细胞中E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）、Notch-1、免疫细胞表面抗原44（CD44）、基质金属蛋白酶9（MMP-9）m RNA及其蛋白表达的影响;采用Western blot法考察蛋白激酶B（Akt）、糖原合成酶激酶3β（GSK-3β）的磷酸化水平。结果：1080μmol/L桔皮素对人肝癌Hep G2细胞的体外增殖均具有明显的抑制作用,且呈时间和剂量依赖性,增殖率较空白对照组显著降低（P〈0.05）,作用24、48 h后的IC50分别为55.7、23.4μmol/L。20、30μmol/L桔皮素作用48 h后,细胞侵袭能力明显受到抑制;细胞中N-cadherin、Vimentin、Notch-1、CD44和MMP-9 m RNA及其蛋白水平表达明显降低,E-cadherin m RNA及其蛋白表达水平明显升高,且Akt、GSK-3β磷酸化水平升高,较空白对照组差异均有统计学意义（P〈0.05）。结论：桔皮素可抑制人肝癌Hep G2细胞的体外增殖和侵袭;其机制可能与下调细胞中Notch-1表达、抑制细胞的上皮-间质转化而抑制CD44和MMP-9的表达,以及抑制其上游Akt-GSK-3β信号途径有关。

OBJECTIVE：To investigate the effects of tangeretin on in vitro proliferation and invasion of human hepatocellular carcinoma Hep G2 cells,and to investigate its mechanism.METHODS：After treated with 0（blank control group）,10,20,40,60,80 μmol/L tangeretin for 24,48 h,the rate of cell proliferation was detected by CCK-8 assay,and IC50 was calculated.After treated with 0,20,30 μmol/L tangeretin for 48 h,Transwell invasion test was used to investigate the effects of tangeretin on cell invasion.The effects of tangeretin on m RNA and protein expression of E-cadherin,N-cadherin,Vimentin,Notch-1,CD44 and MMP-9 were determined by RT-PCR and Western blot assay.The phosphorylation of Akt and GSK-3β were investigated by Western blot assay.RESULTS：10-80 μmol/L tangeretin significantly inhibited in vitro proliferation of human hepatocellular carcinoma Hep G2 cells in time and dose-dependent manner;proliferation rates of them were significantly lowered,compared to blank control group（P〈0.05）;IC50 were 55.7,23.4 μmol/L after treated for 24,48 h.After treated with 20,30 μmol/L tangeretin for 48 h,cell invasion was inhibited in Hep G2 cells;m RNA and protein expression of N-cadherin,Vimentin,Notch-1,CD44 and MMP-9decreased significantly,while those of E-cadherin increased significantly;the phosphorylated levels of Akt and GSK-3β increased;there was statistical significance between them and blank control group（P〈0.05）.CONCLUSIONS：Tangeretin can inhibit in vitro proliferation and invasion of human hepatocellular carcinoma Hep G2 cell,which may be associated with inhibiting the expression of CK44 and MMP-9 and signal pathway of upstream Akt-GSK-3β through up-regulating the expression of Notch-1 and inhibiting epithelial-mesenchymal transition.