Metabonomic study on the antitumor effect of flavonoid derivative 3d in HepG2 cells and its action mechanism

A novel flavonid derivate, 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chlorophenyl)urea (3d) synthesized in our lab possesses potent antitumor activity against HepG2 cells. Our previous studies on pharmacological mechanism of 3d mostly focused on cell and gene levels, little is about its metabolomics study. Herein, an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of 3d on HepG2 cells and its action mechanism. Q-TOF MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. Comparing 3d-treated HepG2 cells with vehicle control (dimethyl sulfoxide), 32 distinct metabolites involved in glutathione metabolism, glycerophospholipid metabolism, cysteine and methionine metabolism, fatty acid metabolism, and phenylalanine metabolism. The reduced level of glutathione (GSH) and decreased ratio of reduced/oxidized glutathione (GSH/GSSG) in 3d-treated cells indicated the increased oxidative stress after 3d treatment. The significant decrease of phosphatidylcholine (PC) levels and increase of lysophosphatidylcholine (LPC) levels suggested alterations in lipid composition which were causally related to decline in mitochondrial function. Depletion of carnitine and increase of long chain carnitines and fatty acids reflected decline in fatty acid metabolism. The further biological experiments including ROS and MMP measurements confirmed the above probabilities presumed from metabolomic results. Our findings suggested that 3d caused the perturbation of multiple cellular pathways. The increased oxidative stress and the resulting mitochondrial dysfunction resulted in the antiproliferative effect of 3d. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of new compounds that distinct from traditional biological studies.