含溴结构域和ET域蛋白抑制剂对结肠癌细胞哺乳动物雷帕霉素靶蛋白通路和有氧糖酵解的影响

Inhibition of bromodomain and extra-terminal bromodomain influences Warburg effect of colorectal cancer cell via suppressing mammalian target of rapamycin activation

目的观察抑制含溴结构域和ET域(BET)蛋白能否影响结肠癌细胞的有氧糖酵解,并进一步探讨其影响机制是否通过下调哺乳动物雷帕霉素靶蛋白(mTOR)通路活性实现。方法选择人结肠癌RKO、LS174T、HCT116细胞株。实验分成DMSO组和JQ1组。DMSO组分别于RKO、LS174T、HCT116细胞株中加入溶媒DMSO处理,JQ1组分别于RKO、LS174T、HCT116细胞株中加入BET蛋白抑制剂JQ1(将1μmol/L JQ1溶于DMSO中)处理,每组每个细胞株设3个副孔。测定两组各细胞株的乳酸含量和计算糖耗量,通过细胞计数观察RKO细胞的增殖密度。为研究JQ1能否协同低糖环境发挥其效应,另取未经处理的HCT116细胞株加入低糖DMEM培养基(葡萄糖浓度6.1 mmol/L)中培养,进行细胞计数。采用Western印迹法测定mTOR下游蛋白表达情况。结果JQ1组RKO、LS174T、HCT116细胞株的乳酸含量和糖耗量均显著低于DMSO组同细胞株(P值均<0.01)。JQ1组RKO细胞株和LS174T细胞株的细胞计数均显著少于DMSO组(P值均<0.01)。高糖或低糖培养基中,JQ1组HCT116细胞株的细胞计数均显著少于DMSO组(P值均<0.05)。光学显微镜下见,JQ1组RKO细胞增殖密度明显减少。Western印迹法结果显示,JQ1组RKO细胞株mTOR下游蛋白(抗磷酸化的核糖体S6蛋白)、HCT116细胞株mTOR下游蛋白(抗磷酸化的核糖体S6蛋白、抗4E结合蛋白1、抗S6蛋白激酶1蛋白)表达的灰度值均显著低于DMSO组(P值分别<0.05、0.01)。结论抑制BET蛋白可能通过抑制mTOR通路影响结肠癌细胞有氧糖酵解,从而抑制结肠癌细胞增殖。

Objective To evaluate the effect of JQ1, a newly identified bromodomain and extra-terminal （BET） bromodomain inhibitor, on the aerobic glycolysis of colorectal cancer cells, and to explore whether or not the achievement of the influence via suppressing mammalian target of rapamycin （mTOR） signaling activation. Methods Colorectal cancer cell lines （RKO, LS174T and HCT116） were treated by DMSO and JQ1 （1μmol/L）, respectively. There were 3 holes in each cell lines. The lactate production was measured and glucose consumption was calculated. Cell counting and crystal violet staining were used to observe the proliferation of RKO. In order to observe the effect of JQ1 on cells cultured in the low glucose environment, HCT116 were cultured in the low glucose DMEM （6.1 mmol/L glucose）, and the cells were counted. Western blotting was used to determine the expression level of mTOR downstream proteins. Results JQ1 treatment induced a significant decrease in lactate production and glucose consumption in LS174T, HCT116 and RKO as compared with DMSO treatment （all P〈 0.01 ）. Cell counting of JQ1 group were significantly less than those in DMSO group in RKO and LS174T （both P〈 0.01）. Both in high and low glucose medium, cell counting of JQ1 group in HCT116 were significantly less than DMSO group （both P〈0.05）. Under the crystal violet staining and optical microscope, the proliferation of JQ1 group in RKO was significantly decreased. Western blot showed that the downstream protein expression of mTOR signaling （p-S6） in RKO and roTOR signaling （p-S6, 4E-BP1 and S6K1） in HCT116 in JQ1 group were significantly lower than those in DMSO group （P〈0.05 or 0.01）. Conclusion Inhibition of BET protein probably influences the a erobic glycolysis of colorectal cancer cell via suppressing mTOR signaling activation.