A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

Abstract

Metabolic reprogramming within cancer cells is suggested as a potential barrier to chemotherapy.Additionally, metabolic tumor heterogeneity is one of factor behind discernible hallmarks such as drug resistance, relapse of tumor and the formation of secondary tumors. 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 hypotonic buffer and whole cell lysate was purified by a novel and specifically designed metabolite (100 to 1000 Da) fractionation system as vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique. The authors show the presence of cleaved PARP 1 in MCF-7 cells treated with DOX. Concomitantly, data show 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-Lglutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, the authors report a first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that is potentially linked to the mechanism of cell death in MCF-7 cells. This paper reports on 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.

Hepatitis C Virus Infection Upregulates Plasma Phosphosphingolipids and Endocannabinoids and Downregulates Lysophosphoinositols

“…In contrast, dodecanamide, palmitamide, stearamide, oleamide, and linoleamide were all produced by HepG2 cells treated with extracts of the brown algae Fucus vesiculosus [ 37 ]. Dodecanamide has been reported as an intracellular metabolite in MCF-7 cells after doxorubicin-induced cell death [ 38 ] and with diminished levels in the serum of acute myeloid leukemia patients who responded well to chemotherapy with cytarabine plus anthracycline [ 39 ]. There exist more extensive studies in the literature on palmitamide, palmitoleamide, stearamide, oleamide, and linoleamide [ 37 , 40 , 41 , 42 , 43 , 44 ], but none in relation to HCV and the liver, with the exception of oleamide in our earlier metabolomics report on HCV-infected patients [ 28 ].…”

Free Fatty Acids from Cow Urine DMSO Fraction Induce Cell Death in Breast Cancer Cells without Affecting Normal GMSCs

“…Breast cancer cells were incubated for 72 h at 37 °C. The rest of the procedure was adopted as per the routine protocol [ 39 , 40 ]. The absorbance was recorded using an ELISA reader (Thermo Fisher Scientific, Waltham, WA, USA).…”

“…The rest of the steps were followed as per the manufacturer’s instructions for the use of the annexin V/FITC apoptosis detection kit (ThermoFisher). Then, live and dead cells were recorded with a BD FACSJazz cytometer, and 10,000 events were measured for each sample [ 39 , 40 ].…”

“…After 16–18 h of plating, GMSCs were treated with CUDF and DMSO and incubated for 72 h, as previously described, in the presence of complete DMEM. Next, trypan blue dye exclusion and apoptosis assays were performed as described above to estimate the viability and cell death of GMSCs [ 39 , 40 ]…”

“…To identify the intracellular CUDF-derived FFAs, breast cancer cells were suspended in hypotonic buffer (10 mM KCl, 10 mM NaCl, 20 mM Tris, pH 7.4) [ 39 , 40 ]. Three washing steps with PBS were performed to remove the traces of external medium components.…”