Background

Metabolic reprogramming within cancer cells has been recognized as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is the one of factors behind discernible hallmarks such as drug resistance, relapse of the tumor and the formation of secondary tumors.

Methods

In this paper, cell-based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in a hypotonic buffer and the whole cell lysate was purified by a novel and specifically designed metabolite (~ 100 to 1000 Da) fractionation system called vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique.

Results

Cleaved PARP 1 in MCF-7 cells treated with DOX was observed in the present study. Concomitantly, data showed the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, we reported the first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that are potentially linked to the mechanism of cell death in MCF-7 cells.

Conclusion

This paper reported novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells. Finally, these findings validate a proof of concept on the applications of novel VTGE assisted purification and analysis of intracellular metabolites from various cell culture models.

中文翻译：

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一种通过阿霉素诱导细胞死亡检测 MCF-7 细胞内代谢物改变的新方法

背景

癌细胞内的代谢重编程已被认为是化疗的潜在障碍。此外，代谢性肿瘤异质性是耐药性、肿瘤复发和继发性肿瘤形成等明显标志背后的因素之一。

方法

在本文中，进行了基于细胞的检测，包括 PI/膜联蛋白 V 染色和免疫印迹检测，以显示用 DOX 处理的 MCF-7 细胞的凋亡细胞死亡。此外，MCF-7 细胞在低渗缓冲液中裂解，整个细胞裂解物通过称为垂直管凝胶电泳 (VTGE) 的新型和专门设计的代谢物（~100 至 1000 Da）分级系统进行纯化。此外，纯化的细胞内代谢物通过 LC-HRMS 技术进行鉴定。

结果

在本研究中观察到用 DOX 处理的 MCF-7 细胞中裂解的 PARP 1。同时，数据显示 MCF-7 细胞中不存在活性半胱天冬酶 3。新发现是确定 VTGE 系统辅助的关键细胞内代谢物，包括脂质（CDP-DG、植物鞘氨醇、十二酰胺）、非脂质（N-乙酰-D-葡萄糖胺、N1-乙酰亚精胺和γ-L-谷氨酰-L-半胱氨酸) 和 DOX 处理的 MCF-7 细胞中的三肽代谢物。有趣的是，我们报告了阿霉素的第一个证据，阿霉素是 MCF-7 细胞中 DOX 的一种苷元形式，可能与 MCF-7 细胞中的细胞死亡机制有关。

结论

本文报道了新的方法和过程，这些方法和过程涉及基于 VTGE 系统纯化 DOX 处理的 MCF-7 细胞的低渗裂解的新型细胞内代谢物。在这里，这些鉴定的细胞内代谢物证实了独立于半胱天冬酶 3 和线粒体诱导的 MCF-7 细胞凋亡细胞死亡。最后，这些发现验证了新型 VTGE 辅助纯化和分析来自各种细胞培养模型的细胞内代谢物的应用的概念证明。