重组荞麦胰蛋白酶抑制剂通过下调己糖激酶Ⅱ抑制HepG2细胞生长  

Recombinant Buckwheat Trypsin Inhibitor Suppressed HepG2 Cell Growth by Down-regulation of Hexokinase Ⅱ Expression

作　　者：崔晓东 闫红 任荣 王转花 

机构地区：[1]山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《中国生物化学与分子生物学报》2017年第9期939-946,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31300653);山西省科技平台项目(No.2014091028)资助~~

摘　　要：糖酵解过度活跃是肿瘤细胞能量代谢的显著特征。抑制过度糖酵解已经成为一种新的癌症疗法。重组荞麦胰蛋白酶抑制剂(recombinant buckwheat trypsin inhibitor,r BTI)可以通过上调磷酸酶及张力蛋白同源基因(PTEN)进而抑制Hep G2细胞增殖。有关r BTI对肿瘤细胞能量代谢的影响仍未见报道。本研究中的MTT和ATP检测分析表明,r BTI以剂量依赖性方式抑制细胞活力及胞内ATP含量。qRT-PCR和Western印迹分析表明,r BTI处理Hep G2细胞后,己糖激酶Ⅱ转录显著下调,但是糖酵解过程中的其他酶及葡萄糖转运蛋白基因在转录水平未发生显著变化,同时己糖激酶Ⅱ蛋白水平的表达也显著下调。酶活性分析也表明,r BTI能显著降低己糖激酶的活性。进一步分析表明,r BTI使细胞内PTEN转录及表达水平明显上调,己糖激酶Ⅱ转录和p-AKT,p-m TOR、己糖激酶Ⅱ的表达下调。当PTEN抑制剂phen存在时,可阻断r BTI诱导的己糖激酶Ⅱ表达下降,表明r BTI能通过上调PTEN进而影响己糖激酶Ⅱ的表达。免疫荧光及Western印迹分析显示,r BTI作用后减弱了己糖激酶Ⅱ在线粒体的定位,导致己糖激酶Ⅱ与线粒体电压依赖性阴离子通道蛋白(voltage-dependent anion channel,VDAC)分离,促使己糖激酶Ⅱ从线粒体转位到细胞质,降低糖酵解的效率。上述结果证明,r BTI对肿瘤细胞能量代谢的调控作用主要通过抑制PI3K/AKT信号通路,下调己糖激酶Ⅱ的表达并影响空间定位,进而抑制肿瘤细胞糖酵解过程,导致癌细胞生长受到抑制。Glycolysis hyperactivity is one of the significant characteristics of energy metabolism in tumor cells. Inhibition of excessive glycolysis could be the most promising strategies that might lead to the development of novel anticancer therapy. Previous studies have shown that recombinant buckwheat trypsin inhibitor（ r BTI） could inhibit Hep G2 cells proliferation by up-regulation of phosphatase and tensin homolog（ PTEN） expression. The effect of r BTI on energy metabolism of tumor cells has not been reported. MTT and ATP assays showed that r BTI could inhibit proliferation in Hep G2 cells and decreaseATP content in a dose-dependent manner. qRT-PCR and Western blotting revealed that stimulation with r BTI significantly down-regulated the mRNA transcription and protein expression of hexokinase Ⅱ,while other metabolism enzymes in glycolysis had no statistical change. The activity of hexokinase Ⅱ was also markedly decreased. PTEN transcription was obviously increased and hexokinase Ⅱ was decreased after treatment of r BTI. Results also showed that up-regulation of PTEN and downregulation of p-AKT,pm TOR and hexokinase Ⅱ was dose and time dependent after treated with r BTI. Immunofluorescence and Western blotting results showed that r BTI weakened the hexokinase Ⅱ co-localization with voltagedependent anion channel（ VDAC） in mitochondria. It suggested that r BTI could promote translocation of hexokinase Ⅱ from mitochondria to cytoplasm,reducing the efficiency of glycolysis. Hexokinase Ⅱactivity was increased in cytoplasm but was declined in the mitochondria. The results showed that r BTI could inhibit PI3K/AKT pathway, down-regulate hexokinase Ⅱ expression, change hexokinase Ⅱlocalization,and suppress tumor cell glycolysis,which in turn influences cancer cell growth.

关 键 词：荞麦胰蛋白酶抑制剂 能量代谢 磷酸酶及张力蛋白同源基因 己糖激酶Ⅱ 

分 类 号：Q78[生物学—分子生物学]