Araki Chie, Okahashi Nobuyuki, Maeda Kousuke, Shimizu Hiroshi, Matsuda Fumio
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University.
Mass Spectrom (Tokyo). 2018;7(1):A0067. doi: 10.5702/massspectrometry.A0067. Epub 2018 Jun 14.
Cancer cells often respond to chemotherapeutic inhibitors by redirecting carbon flow in the central metabolism. To understand the metabolic redirections of inhibitor treatment on cancer cells, this study established a C-metabolic flux analysis (C-MFA)-based method to evaluate metabolic redirection in MCF-7 breast cancer cells using mass spectrometry. A metabolic stationary state necessary for accurate C-MFA was confirmed during an 8-24 h window using low-dose treatments of various metabolic inhibitors. Further C-labeling experiments using [1-C]glucose and [U-C]glutamine, combined with gas chromatography-mass spectrometry (GC-MS) analysis of mass isotopomer distributions (MIDs), confirmed that an isotopic stationary state of intracellular metabolites was reached 24 h after treatment with paclitaxel (Taxol), an inhibitor of mitosis used for cancer treatment. Based on these metabolic and isotopic stationary states, metabolic flux distribution in the central metabolism of paclitaxel-treated MCF-7 cells was determined by C-MFA. Finally, estimations of the 95% confidence intervals showed that tricarboxylic acid cycle metabolic flux increased after paclitaxel treatment. Conversely, anaerobic glycolysis metabolic flux decreased, revealing metabolic redirections by paclitaxel inhibition. The gap between total regeneration and consumption of ATP in paclitaxel-treated cells was also found to be 1.2 times greater than controls, suggesting ATP demand was increased by paclitaxel treatment, likely due to increased microtubule polymerization. These data confirm that C-MFA can be used to investigate inhibitor-induced metabolic redirection in cancer cells. This will contribute to future pharmaceutical developments and understanding variable patient response to treatment.
癌细胞通常通过重新引导中心代谢中的碳流来对化疗抑制剂作出反应。为了了解抑制剂处理对癌细胞的代谢重定向，本研究建立了一种基于碳代谢通量分析（C-MFA）的方法，使用质谱来评估MCF-7乳腺癌细胞中的代谢重定向。在使用各种代谢抑制剂的低剂量处理的8-24小时窗口期间，确认了准确的C-MFA所需的代谢稳态。使用[1-C]葡萄糖和[U-C]谷氨酰胺进行的进一步C标记实验，结合对质量同位素异构体分布（MID）的气相色谱-质谱（GC-MS）分析，证实在用紫杉醇（一种用于癌症治疗的有丝分裂抑制剂）处理24小时后，细胞内代谢物达到了同位素稳态。基于这些代谢和同位素稳态，通过C-MFA确定了紫杉醇处理的MCF-7细胞中心代谢中的代谢通量分布。最后，95%置信区间的估计表明，紫杉醇处理后三羧酸循环代谢通量增加。相反，无氧糖酵解代谢通量降低，揭示了紫杉醇抑制导致的代谢重定向。还发现紫杉醇处理的细胞中ATP的总再生与消耗之间的差距比对照大1.2倍，这表明紫杉醇处理增加了ATP需求，可能是由于微管聚合增加所致。这些数据证实C-MFA可用于研究抑制剂诱导的癌细胞代谢重定向。这将有助于未来的药物开发以及理解患者对治疗的不同反应。
