Metabolomic analysis of human MCF7 breast carcinoma cells by 1H-NMR identify phospholipid metabolism as an important target of kahalalide F and myoinositol as a drug-response biomarker

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PURPOSE Previous reports have demonstrated a value of HRMAS-proton magnetic resonance spectroscopy (HRMAS-1H-MRS) for metabolomic analysis of cancer cells and identification of drug response pathways and biomarkers (1,2). In this study, HRMAS-1H-MRS was applied to human MCF7 breast carcinoma cells treated by the marine-derived depsipeptide Kahalalide F (KF). KF promotes oncosis cell death and, antitumor activity independent of multidrug resistance (MDR) and of p53 status. The hepatocellular carcinoma, NSCLC and melanoma phase II clinical trials finished in 2006. Because it modulate lysosomal function, we suspected KF to strongly affect membranar phospholipid remodeling pathways. METHODS To test this hypothesis, exponentially growing human breast MCF7 carcinoma cells were treated by KF (5 µM) in kinetic mode. The cell survival was determined with the resazurin reduction test that determines the amount of fluorescent resorufin produced by living cells. Treated and control cells were submitted to proton NMR spectroscopy with a “Brucker DX500” equipped with a high resolution magic angle spinning (HRMAS) probe. Sequences were used after water signal suppression. Spectra were recorded in 1D mode for qualitative response profiles and in 2D mode ([H,H]-TOCSY] for quantification of cell metabolites. RESULTS. 31 metabolites were identified and quantified. As expected, strong alterations of phospholipid metabolism leading to rapid blockade of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PdtEth) biosynthesis and degradation pathways, were detected as early as 6h after MCF7 treatment. These alterations were time-dependent and correlated with Krebs cycle and glycolysis disturbance. Significant changes in the content of osmolytes devoted to protection of cell homeostasis, most notably the sodium transporter myoinositol, and irreversible accumulation of phosphoethanolamine were further observed. CONCLUSION The present data demonstrate that in vitro, human breast carcinoma response to KF involves early and profound alterations of phospholipid metabolism leading to progressive krebs-cycle and glycolysis blockade and to myoinositol accumulation. In MCF7 cultures, myoinositol is a biomarker of KF response. Further studies are needed to determine its value as biomarker of clinical response.