BackgroundThe role of anaplerotic nutrient entry into the Krebs cycle via pyruvate carboxylase has been the subject of increased scrutiny and in particular whether this is dysregulated in cancer. Here, we use a tracer-based NMR analysis involving high-resolution 1H-13C-HSQC spectra to assess site-specific label incorporation into a range of metabolite pools, including malate, aspartate and glutamate in the acute myeloid leukaemia cell line K562. We also determine how this is affected following treatment with the redeployed drug combination of the lipid-regulating drug bezafibrate and medroxyprogesterone (BaP).ResultsUsing the tracer-based approach, we assessed the contribution of pyruvate carboxylase (PC) vs. pyruvate dehydrogenase (PDH) activity in the derivation of Krebs cycle intermediates. Our data show that PC activity is indeed high in K562 cells. We also demonstrate a branched entry to the Krebs cycle of K562 cells with one branch running counterclockwise using PC-derived oxaloacetate and the other clockwise from the PDH activity. Finally, we show that the PC activity of K562 cells exclusively fuels the ROS-induced decarboxylation of oxaloacetate to malonate in response to BaP treatment; resulting in further Krebs cycle disruption via depletion of oxaloacetate and malonate-mediated inhibition of succinate dehydrogenase (SDH) resulting in a twofold reduction of fumarate.ConclusionsThis study extends the interest in the PC activity in solid cancers to include leukaemias and further demonstrates the value of tracer-based NMR approaches in generating a more accurate picture of the flow of carbons and metabolites within the increasingly inappropriately named Krebs cycle. Moreover, our studies indicate that the PC activity in cancer cells can be exploited as an Achilles heel by using treatments, such as BaP, that elevate ROS production.

中文翻译：

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丙二酸作为 ROS 产物与急性髓系白血病细胞中的丙酮酸羧化酶活性相关


背景补补营养物质通过丙酮酸羧化酶进入克雷布斯循环的作用已成为越来越多研究的主题，特别是这种作用在癌症中是否失调。在这里，我们使用基于示踪剂的 NMR 分析（涉及高分辨率 1H-13C-HSQC 谱）来评估位点特异性标签掺入一系列代谢物库的情况，包括急性髓系白血病细胞系 K562 中的苹果酸、天冬氨酸和谷氨酸。我们还确定了在重新部署调脂药物苯扎贝特和甲羟孕酮 (BaP) 的药物组合治疗后对此有何影响。结果使用基于示踪剂的方法，我们评估了丙酮酸羧化酶 (PC) 与丙酮酸脱氢酶 (PDH) 的贡献) 衍生克雷布斯循环中间体的活动。我们的数据表明 K562 细胞中 PC 活性确实很高。我们还证明了 K562 细胞的克雷布斯循环的分支入口，其中一个分支使用 PC 衍生的草酰乙酸逆时针运行，另一个分支从 PDH 活性顺时针运行。最后，我们发现 K562 细胞的 PC 活性专门促进 ROS 诱导的草酰乙酸脱羧为丙二酸，以响应 BaP 处理；通过消耗草酰乙酸和丙二酸介导的琥珀酸脱氢酶 (SDH) 抑制，导致进一步的克雷布斯循环破坏，从而导致富马酸双倍减少。结论这项研究将实体癌中 PC 活性的兴趣扩展到包括白血病，并进一步证明了基于示踪剂的核磁共振方法可以更准确地描述克雷布斯循环中碳和代谢物的流动。 此外，我们的研究表明，通过使用 BaP 等提高 ROS 产生的治疗方法，可以利用癌细胞中的 PC 活性作为致命弱点。