Soliman Ghada A, Steenson Sharalyn M, Etekpo Asserewou H
Department of Health Promotion, Social and Behavioral Health College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, 68198 USA.
Metabolomics (Los Angel). 2016;6(3). doi: 10.4172/2153-0769.1000183. Epub 2016 Aug 20.
Pancreatic Cancer (PC) is a devastating lethal disease. Therefore, there is an urgent need to develop new intervention strategies. The mammalian Target of Rapamycin (mTOR) is a conserved kinase and master regulator of metabolism and cell growth. mTOR is dysregulated in chronic diseases including diabetes and pancreatic cancer. Recent reports indicate that 50% of Pancreatic Ductal Adenocarcinoma (PDAC) patients are diabetic at the time of diagnosis. Furthermore, the anti-diabetic drug, metformin, which indirectly inhibits mTOR, has emerged as a potential therapeutic target for PC. The objective of this study is to determine the targeted-metabolomics profile in PDAC cell line (HPAF-II) with mTOR inhibition and the interaction between mTOR ATP-competitive inhibitor (Torin 2) and metformin as potential combined therapy in PC. HPAF-II cell lines were cultured in the presence of either Torin 2, metformin, both, or control vehicle. We utilized targeted LC/MS/MS to characterize the alterations in glycolytic and tricarboxylic acid cycle metabolomics, and employed Western Blot analysis for cell signaling activation by phosphorylation. Comparisons between groups were analyzed using one-way Analysis of Variance followed by secondary post-hoc analysis. After 1 h incubation with metformin, AMP concentration was significantly increased compared to other groups (p<0.03). After 24 h, Torin-2 significantly decreased glycolysis intermediates (fructose 1,6-bisphosphate (FBP), and 2-phosphoglycerate/3-phosphoglycerate), TCA intermediate metabolites (citrate/isocitrate, and malate), as well as Nicotinamide Adenine Dinucleotide (NAD) and Flavin Adenine Dinucleotide (FAD), and ATP levels. When HPAF-II cells were incubated with both Torin-2 and metformin, there was a significant reduction in NAD and FAD, suggesting decreased levels of the energy equivalents that are available to the electron transport chain. Targeted metabolomics data indicate that mTOR complexes inhibition by Torin 2 reduced glycolytic intermediates and TCA metabolites in HPAF- II and may synergize with metformin to decrease the electron acceptors NAD and FAD which may lead to reduced energy production.
胰腺癌（PC）是一种极具毁灭性的致命疾病。因此，迫切需要开发新的干预策略。哺乳动物雷帕霉素靶蛋白（mTOR）是一种保守的激酶，也是代谢和细胞生长的主要调节因子。mTOR在包括糖尿病和胰腺癌在内的慢性疾病中失调。最近的报告表明，50%的胰腺导管腺癌（PDAC）患者在诊断时患有糖尿病。此外，间接抑制mTOR的抗糖尿病药物二甲双胍已成为PC的潜在治疗靶点。本研究的目的是确定mTOR抑制的PDAC细胞系（HPAF-II）中的靶向代谢组学特征，以及mTOR ATP竞争性抑制剂（Torin 2）与二甲双胍作为PC潜在联合治疗的相互作用。HPAF-II细胞系在Torin 2、二甲双胍、两者或对照载体存在的情况下进行培养。我们利用靶向液相色谱/串联质谱法（LC/MS/MS）来表征糖酵解和三羧酸循环代谢组学的变化，并采用蛋白质免疫印迹分析来检测磷酸化引起的细胞信号激活。使用单因素方差分析进行组间比较，随后进行二次事后分析。与二甲双胍孵育1小时后，与其他组相比，AMP浓度显著增加（p<0.03）。24小时后，Torin-2显著降低了糖酵解中间体（果糖1,6-二磷酸（FBP）和2-磷酸甘油酸/3-磷酸甘油酸）、三羧酸循环中间代谢物（柠檬酸/异柠檬酸和苹果酸）以及烟酰胺腺嘌呤二核苷酸（NAD）、黄素腺嘌呤二核苷酸（FAD）和ATP水平。当HPAF-II细胞与Torin-2和二甲双胍一起孵育时，NAD和FAD显著减少，表明可用于电子传递链的能量等效物水平降低。靶向代谢组学数据表明，Torin 2对mTOR复合物的抑制降低了HPAF-II中的糖酵解中间体和三羧酸循环代谢物，并且可能与二甲双胍协同作用，降低电子受体NAD和FAD，这可能导致能量产生减少。
