【摘要】：糖酵解過度活躍是腫瘤細(xì)胞能量代謝的顯著特征。抑制過度糖酵解已經(jīng)成為一種新的癌癥療法。重組蕎麥胰蛋白酶抑制劑(recombinant buckwheat trypsin inhibitor,r BTI)可以通過上調(diào)磷酸酶及張力蛋白同源基因(PTEN)進(jìn)而抑制Hep G2細(xì)胞增殖。有關(guān)r BTI對腫瘤細(xì)胞能量代謝的影響仍未見報道。本研究中的MTT和ATP檢測分析表明,r BTI以劑量依賴性方式抑制細(xì)胞活力及胞內(nèi)ATP含量。qRT-PCR和Western印跡分析表明,r BTI處理Hep G2細(xì)胞后,己糖激酶Ⅱ轉(zhuǎn)錄顯著下調(diào),但是糖酵解過程中的其他酶及葡萄糖轉(zhuǎn)運蛋白基因在轉(zhuǎn)錄水平未發(fā)生顯著變化,同時己糖激酶Ⅱ蛋白水平的表達(dá)也顯著下調(diào)。酶活性分析也表明,r BTI能顯著降低己糖激酶的活性。進(jìn)一步分析表明,r BTI使細(xì)胞內(nèi)PTEN轉(zhuǎn)錄及表達(dá)水平明顯上調(diào),己糖激酶Ⅱ轉(zhuǎn)錄和p-AKT,p-m TOR、己糖激酶Ⅱ的表達(dá)下調(diào)。當(dāng)PTEN抑制劑phen存在時,可阻斷r BTI誘導(dǎo)的己糖激酶Ⅱ表達(dá)下降,表明r BTI能通過上調(diào)PTEN進(jìn)而影響己糖激酶Ⅱ的表達(dá)。免疫熒光及Western印跡分析顯示,r BTI作用后減弱了己糖激酶Ⅱ在線粒體的定位,導(dǎo)致己糖激酶Ⅱ與線粒體電壓依賴性陰離子通道蛋白(voltage-dependent anion channel,VDAC)分離,促使己糖激酶Ⅱ從線粒體轉(zhuǎn)位到細(xì)胞質(zhì),降低糖酵解的效率。上述結(jié)果證明,r BTI對腫瘤細(xì)胞能量代謝的調(diào)控作用主要通過抑制PI3K/AKT信號通路,下調(diào)己糖激酶Ⅱ的表達(dá)并影響空間定位,進(jìn)而抑制腫瘤細(xì)胞糖酵解過程,導(dǎo)致癌細(xì)胞生長受到抑制。
[Abstract]:Hyperactivity of glycolysis is a significant feature of energy metabolism in tumor cells. Inhibition of excessive glycolysis has become a new cancer therapy. Recombinant buckwheat trypsin inhibitor (recombinant buckwheat trypsin inhibitor,r BTI can inhibit the proliferation of Hep G2 cells by up-regulating (PTEN). The effect of r BTI on energy metabolism of tumor cells has not been reported. In this study, MTT and ATP analysis showed that BTI inhibited cell viability and intracellular ATP content in a dose-dependent manner. QRT-PCR and Western blot analysis showed that the transcription of hexokinase 鈪，

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