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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。
Overexpression of glycolysis is a significant feature of the energy metabolism of tumor cells. Suppression of excessive glycolysis has become a new cancer therapy. Recombinant buckwheat trypsin inhibitor (rBTI) can inhibit the proliferation of Hep G2 cells by upregulating phosphatase and tensin homolog (PTEN). The impact of rBTI on the energy metabolism of tumor cells has not been reported yet. The MTT assay and ATP assay in this study showed that rBTI inhibited cell viability and intracellular ATP content in a dose-dependent manner. qRT-PCR and Western blot analysis showed that the transcription of hexokinase II was significantly down-regulated in rBTI treated Hep G2 cells, but no significant changes were observed in transcription levels of other enzymes and glucose transporters during glycolysis, The expression of kinase II protein was also significantly down-regulated. Enzyme activity analysis also showed that rBTI significantly reduced the activity of hexokinase. Further analysis showed that rBTI significantly up-regulated the transcription and expression of PTEN, and decreased the expression of hexokinase II, p-AKT, p-mTOR and hexokinase II. When PTEN inhibitor phen existed, it could block the decrease of hexobitokinase II induced by rBTI, indicating that rBTI can affect the expression of hexokinase II by up-regulating PTEN. Immunofluorescence and Western blot analysis showed that rBTI attenuated the localization of hexokinase Ⅱ in mitochondria and led to the separation of hexokinase Ⅱ from the mitochondrial voltage-dependent anion channel (VDAC) Kinase II translocates from the mitochondria to the cytoplasm, reducing the efficiency of glycolysis. The above results demonstrate that rBTI regulates the energy metabolism of tumor cells mainly by inhibiting the PI3K / AKT signaling pathway, down-regulating the expression of hexokinase II and affecting the spatial localization, thereby inhibiting the tumor cell glycolytic process and leading to the inhibition of the growth of cancer cells .