Moderate DNA damage promotes metabolic flux into PPP via PKM2 Y-105 phosphorylation: a feature that favours cancer cells
Moderate DNA damage promotes metabolic flux into PPP via PKM2 Y-105 phosphorylation: a feature that favours cancer cells
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DOI：
10.1007/s11033-015-3876-8
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发表时间：
2015-08-01
影响因子：
2.8
通讯作者：
Bamezai, Rameshwar N. K.
中科院分区：
文献类型：
--
作者：
Kumar, Bhupender;Bamezai, Rameshwar N. K.
丙酮酸激酶M2是一种重要的代谢酶，通过促进有氧糖酵解（瓦尔堡效应）促进癌细胞增殖。揭示这种重要的糖酵解途径酶在亚致死剂量的依托泊苷下的状态是相关的，依托泊苷是一种常用的抗增殖遗传毒性药物，用于在HeLa细胞中诱导轻度/中度DNA损伤，作为模型系统，并识别其对PKM 2表达、磷酸化、二聚体：四聚体比率、活性和相关效应的影响。蛋白质印迹法检测HeLa细胞PKM 2蛋白表达和磷酸化水平。还使用相同的蛋白质裂解物来估计总丙酮酸激酶活性，并使用甘油梯度超离心来评估总二聚体：四聚体含量。细胞内PEP用标准曲线手工估算，NADPH用NADPH估算试剂盒估算。统计分析采用非配对t检验和双因素方差分析。观察到PKM 2表达相对减少，随后Y105磷酸化呈剂量和时间依赖性增加。PKM 2二聚体含量和Y105-磷酸化的伴随增加导致PKM 2活性降低，促进PEP积累和NADPH产生，代表增加的PPP代谢通量，这是有利于癌细胞的特征。很明显，亚致死剂量的依托泊苷诱导培养的癌细胞中DNA损伤不足，促进了促生存条件，这是由于PKM 2的Y105磷酸化、其稳定的二聚化和失活，这是一种以前未知的独特关联，表明肿瘤复发或复发中可能发生的情况。
Pyruvate kinase M2, an important metabolic enzyme, promotes aerobic glycolysis (Warburg effect) to facilitate cancer cell proliferation. Unravelling the status of this important glycolytic pathway enzyme under sub-lethal doses of etoposide, a commonly used anti-proliferative genotoxic drug to induce mild/moderate DNA damage in HeLa cells as a model system and discern its effect on: PKM2 expression, phosphorylation, dimer: tetramer ratio, activity and associated effects, was pertinent. Protein expression and phosphorylation of PKM2 from HeLa cells was estimated using Western blotting. Same protein lysate was also used to estimate total pyruvate kinase activity and the total dimer: tetramer content evaluated using glycerol gradient ultra-centrifugation. Intracellular PEP was estimated manually using standard curve; while NADPH was assessed by NADPH estimation kit. Unpaired t test and two-way-ANOVA was used for statistical analysis. A relative decrease in PKM2 expression and a subsequent dose and time dependent increase in Y105-phosphorylation were observed. A concomitant increase in PKM2 dimer content and Y105-phosphorylation responsible for reduced PKM2 activity promoted PEP accumulation and NADPH production, representing increased metabolic flux into PPP, a feature that favours cancer cells. It was apparent that the sub-lethal doses of etoposide induced inadequate damage to DNA in cancer cells in culture promoted pro-survival conditions due to Y105-phosphorylation of PKM2, its stable dimerization and inactivation, a unique association not known earlier, indicating what might happen in tumour revivals or recurrences.