白杨素对人肝癌BEL-7402细胞表面超微结构和蛋白质磷酸化及相关通路的影响

Influence of chrysin on cell surface ultrastructures,protein phosphorylation and related pathways in human hepatocellular carcinoma BEL-7402 cells

背景:白杨素是保健食品和中草药中常见的具有防癌抗癌活性的食源黄酮类天然产物。白杨素是否有助于防治肝癌?其作用机制为何?是否促进肝癌干细胞的分化?对此尚缺乏系统研究。目的:以人肝癌细胞为对象,揭示白杨素的亚细胞和分子作用机制。方法:应用酸性磷酸酶实验、扫描电镜、免疫印迹技术探讨白杨素对BEL-7402细胞的作用及其机制。结果与结论:白杨素显著抑制BEL-7402细胞的体外增殖,半数抑制浓度IC50为24.9mol/L或6.3mg/L。白杨素与PI3K-AKT途径特异性抑制剂LY294002联用具有显著的协同抗癌效应。但白杨素本身并不改变AKT、磷酸化pAKT、AKT下游分子GSK-3和磷酸化pGSK-3的表达水平,虽然高剂量下β-catenin略有下调。白杨素导致细胞表面微绒毛样突起密度明显增加并形成纳米膜粒;细胞回缩,间隙加宽;出现形态上不同于凋亡的死亡细胞。白杨素显著改变多种蛋白质的丝/苏氨酸磷酸化水平;对酪氨酸磷酸化亦有广谱下调作用,接近于表柔比星对照。白杨素对CDK1、磷酸化pCDK1和pCDK2、CDC25A、CDC25B和CD133均无明显影响,但可剂量依赖性诱导PARP-1降解。总之,白杨素对BEL-7402细胞的抑制作用并非起因于PI3K-AKT和Wnt/β-catenin通路阻断或细胞周期核心调控因子阻断,而更可能起因于生物膜功能障碍所致细胞表面超微结构和蛋白质磷酸化的大范围改变,并以非经典方式诱导肝癌细胞死亡。

BACKGROUND：Chrysin,a dietary flavonoid or natural product,widely distributed in health food and Chinese herbal medicine,has promising cancer-preventive and cancer-therapeutic activities.Some problems remain poorly understood：whether chrysin is beneficial to liver cancer prevention and treatment？ What are the underlying mechanisms？ Whether chrysin promotes the differentiation of liver cancer stem cells？ OBJECTIVE：By using human hepatocellular carcinoma cells as the subject,the study aimed to uncover the subcellular and molecular mechanisms of actions for chrysin.METHODS：The acid phosphatase assay,scanning electron microscopy and immunoblotting were used to investigate the effects of chrysin on the BEL-7402 cells,and to reveal the underlying anticancer mechanisms.RESULTS AND CONCLUSION：Chrysin has potent antiproliferative activity against cultured BEL-7402 cells,with an IC50 of 24.9 mol/L or 6.3 g/mL.When administered together with LY294002,a specific inhibitor of the PI3K-AKT pathway,chrysin cooperatively and significantly augmented the anticancer effect.However,chrysin alone did not lead to changes in the expression levels of AKT,phosphorylated AKT,and AKT＇s downstream molecule GSK-3 and phospho-GSK-3,though β-catenin reduced slightly under a higher dosage.In chrysin-treated cells,the density of the microvillus-like protrusions on the cell surfaces was drastically increased;Membrane nanoparticles were emerging from the protrusions.Cells were severely retracted,leaving wide gaps between cultured cells.There were also dying or dead cells that were morphologically different from apoptotic cells.Numerous phospho-Ser/Thr bands were changed in chrysin-treated cells.Chrysin also caused extensive dephosphorylation of tyrosine on proteins,which was also seen in positive control or epirubicin-treated cells.No significant changes were observed in the levels of CDK1,phospho-CDK1/2,CDC25A,CDC25B and CD133.However,dose-dependent induction of PARP-1 fragmentation was observed upon chrysin treatment.Taken together,the antiproliferative effect of chrysin on the BEL-7402 cells was not resulted from blocking of the PI3K-AKT or Wnt/β-catenin pathway,nor from blocking of the key regulatory factors in cell cycle,but was likely resulted from ultrastructural alterations on the cell surfaces due to malfunction of biomembranes,and widespread modifications of protein phosphorylation.As a consequence,non-classical cell death was likely induced in chrysin-treated liver cancer cells.