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

摘要

OBJECTIVE:To investigate the effects of tangeretin on in vitro proliferation and invasion of human hepatocellular carcinoma HepG2 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 mRNA 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 HepG2 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 HepG2 cells;mRNA and protein expression of N-cadherin,Vimentin,Notch-1,CD44 and MMP-9 decreased 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 HepG2 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.%目的：考察桔皮素对人肝癌HepG2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）mRNA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：10～80µmol/L桔皮素对人肝癌HepG2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 mRNA及其蛋白水平表达明显降低，E-cadherin mRNA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HepG2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。