Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40202, USA.
Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
Genes (Basel). 2023 Mar 31;14(4):843. doi: 10.3390/genes14040843.
Breast cancer is one of the leading causes of cancer death. Recent studies found that arylamine -acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer, further suggesting NAT1 could be a potential therapeutic target for breast cancer. Previous publications have established that knockout (KO) in breast cancer cell lines leads to growth reduction both in vitro and in vivo and metabolic changes. These reports suggest that NAT1 contributes to the energy metabolism of breast cancer cells. Proteomic analysis and non-targeted metabolomics suggested that KO may change the fate of glucose as it relates to the TCA/KREB cycle of the mitochondria of breast cancer cells. In this current study, we used [U-C]-glucose stable isotope resolved metabolomics to determine the effect of KO on the metabolic profile of MDA-MB-231 breast cancer cells. We incubated breast cancer cells (MDA-MB-231 cells) and Crispr KO cells (KO#2 and KO#5) with [U-C]-glucose for 24 h. Tracer incubation polar metabolites from the cells were extracted and analyzed by 2DLC-MS, and metabolite differences were compared between the parental and KO cells. Differences consistent between the two KO cells were considered changes due to the loss of NAT1. The data revealed decreases in the C enrichment of TCA/Krebs cycle intermediates in KO cells compared to the MDA-MB-231 cells. Specifically, C-labeled citrate, isocitrate, a-ketoglutarate, fumarate, and malate were all decreased in KO cells. We also detected increased C-labeled -lactate levels in the KO cells and decreased C enrichment in some nucleotides. Pathway analysis showed that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and the TCA cycle were most affected. These data provide additional evidence supporting the impacts of knockout on cellular energy metabolism. The data suggest that NAT1 expression is important for the proper functioning of mitochondria and the flux of glucose through the TCA/Krebs cycle in breast cancer cells. The metabolism changes in the fate of glucose in KO breast cancer cells offer more insight into the role of NAT1 in energy metabolism and the growth of breast cancer cells. These data provide additional evidence that NAT1 may be a useful therapeutic target for breast cancer.
乳腺癌是癌症死亡的主要原因之一。最近的研究发现，芳基胺乙酰转移酶 1（NAT1）在乳腺癌中经常上调，这进一步表明 NAT1 可能是乳腺癌的潜在治疗靶点。先前的出版物已经确定，在乳腺癌细胞系中敲除（KO）会导致体外和体内生长减少和代谢变化。这些报告表明，NAT1 有助于乳腺癌细胞的能量代谢。蛋白质组分析和非靶向代谢组学表明，KO 可能会改变葡萄糖的命运，因为它与乳腺癌细胞线粒体的 TCA/KREB 循环有关。在本研究中，我们使用 [U-C]-葡萄糖稳定同位素分辨代谢组学来确定 KO 对 MDA-MB-231 乳腺癌细胞代谢谱的影响。我们用 [U-C]-葡萄糖孵育乳腺癌细胞（MDA-MB-231 细胞）和 Crispr KO 细胞（KO#2 和 KO#5）24 小时。从细胞中提取示踪孵育的极性代谢物，并通过 2DLC-MS 进行分析，比较亲本细胞和 KO 细胞之间的代谢物差异。在两个 KO 细胞中一致的差异被认为是由于 NAT1 缺失而导致的变化。数据显示，与 MDA-MB-231 细胞相比，KO 细胞中 TCA/Krebs 循环中间产物的 C 富集减少。具体来说，KO 细胞中的 C 标记柠檬酸、异柠檬酸、a-酮戊二酸、富马酸和苹果酸均减少。我们还检测到 KO 细胞中 C 标记的 -乳 酸水平升高和一些核苷酸中 C 富集减少。通路分析表明，精氨酸生物合成、丙氨酸、天冬氨酸和谷氨酸代谢以及 TCA 循环受到的影响最大。这些数据提供了更多证据支持 NAT1 缺失对细胞能量代谢的影响。数据表明，NAT1 表达对于乳腺癌细胞中线粒体的正常功能和葡萄糖通过 TCA/Krebs 循环的通量很重要。KO 乳腺癌细胞中葡萄糖命运的代谢变化提供了更多关于 NAT1 在能量代谢和乳腺癌细胞生长中的作用的见解。这些数据提供了更多证据表明，NAT1 可能是乳腺癌的一个有用的治疗靶点。
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