BackgroundThe majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.ResultsUsing a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.ConclusionThis study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.

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MDA-MB-468乳腺癌细胞与蛋氨酸应激敏感性相关的代谢变化

背景大多数癌细胞对甲硫氨酸具有独特的代谢需求，这在正常的非致瘤细胞中没有观察到。这种表型被描述为“蛋氨酸依赖性”或“蛋氨酸应激敏感性”，其中当蛋氨酸在细胞培养基中被其代谢前体同型半胱氨酸取代时，癌细胞无法增殖。我们专注于三阴性乳腺癌细胞系 MDA-MB-468 及其对蛋氨酸不敏感的衍生细胞系 MDA-MB-468res-R8 对蛋氨酸应激的代谢反应。 结果使用多种技术，包括荧光寿命成像显微镜 (FLIM)和细胞外通量测定，我们发现当在同型半胱氨酸培养基中培养时，MDA-MB-468 和 MDA-MB-468res-R8 细胞类型中氧化磷酸化的代谢下调。通过气相色谱/飞行时间质谱法对在同型半胱氨酸培养基中培养 2 至 24 小时的两种细胞类型进行非靶向代谢组学。我们确定了两种细胞系在特定途径中的独特代谢反应，包括甲硫氨酸补救、嘌呤/嘧啶合成和三羧酸循环。使用氘标记的同型半胱氨酸的稳定同位素示踪剂研究表明同型半胱氨酸代谢转向转硫途径和谷胱甘肽合成。该数据证实了伴随着氧化型谷胱甘肽水平增加的谷胱甘肽水平增加。同型半胱氨酸流量的重定向导致来自同型半胱氨酸的甲硫氨酸生成减少，尤其是在 MDA-MB-468 细胞中。最后，重要的单碳供体 S-腺苷甲硫氨酸 (SAM) 的合成减少，扰乱了 MDA-MB-468 细胞中 SAM 与 S-腺苷高半胱氨酸的比例，这是细胞甲基化潜力的指标。结论本研究表明代谢反应不同甲硫氨酸敏感的 MDA-MB-468 细胞和甲硫氨酸不敏感的衍生细胞系 MDA-MB-468res-R8 之间的差异。当甲硫氨酸被其代谢前体同型半胱氨酸取代时，两种细胞系似乎都经历了氧化应激，迫使细胞将同型半胱氨酸代谢转向转硫途径以增加谷胱甘肽的合成。抗甲硫氨酸应激的 MDA-MB-468res-R8 细胞比对甲硫氨酸应激敏感的 MDA-MB468 细胞更早地对这种细胞应激做出反应，并且能更好地应对代谢需求。此外，