The metabolic response of HepG2 cells to extracellular vesicles derived from Raphanus sativus L. var. caudatus Alef microgreens probed by chemometrics-assisted LC-MS/MS analysis

Extracellular vesicles (EVs) derived from Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef) microgreens were previously reported as novel bioactive bioparticles against cancer. This study aimed to investigate the metabolic disruption associated with the antiproliferative effect against HepG2 liver cancer cells, a representative of metabolizing cells and tissue. In this study, the neutral red uptake assay was performed to screen for the antiproliferative effect and determine the cytotoxic concentrations of EVs against HepG2 cells. An untargeted approach to cellular metabolomics was conducted using liquid chromatography coupled with the high-resolution mass spectrometry system with multivariate and univariate analyses to determine the metabolic changes of HepG2 liver cancer cells after EV treatment. EVs showed an antiproliferative effect in HepG2 cells with a half-maximal inhibitory concentration (IC₅₀) of 685.5 ± 26.4 and 139.7 ± 4.2 μg/ml at 24 and 48 h, respectively. In the metabolomics study, 163 metabolites were annotated, with 61 significantly altered metabolites. Among these significant metabolites, 18 were related to glycerophospholipid metabolism. Phosphatidylcholine-the important lipid building blocks for cell membranes, lipid mediators for cell proliferation, and immunosuppressive signaling-was mainly decreased by EV treatment. The alteration of cellular phospholipids in cancer was discussed. This finding suggested the possible mechanism of anticancer action of EVs by disrupting phospholipid metabolism and survival signaling in cancer cells. Further studies should be made to confirm EVs' potential as single and combination therapy in vivo to reduce cancer resistance. This may close the gap between in vitro study and clinical setting.