Fig. 6. Decreased GS expression in HCC827 cells is associated with acquired resistance to gefitinib.

a According to the MTT assays, HCC827 GR cells became resistant to gefitinib after chronic and repeated exposure to increasing doses of gefitinib compared to HCC827 cells, which were sensitive to gefitinib. b, c Changes in GLUL mRNA expression levels were quantified by qRT-PCR (b), and GS protein levels were examined by western blotting (c) to compare the levels between HCC827 and HCC827 GR cells after exposure to the gefitinib or control treatment for 48 h. The bars shown are normalized to the GAPDH control and represent the mean ± SD of triplicate samples. d Glutamine levels in HCC827 and HCC827 GR cells were assessed after a 24-h exposure to 5 µM and 5 nM gefitinib, respectively. e–g After exposing HCC827 and HCC827 GR cells to 0, 0.1, 1, and 10 µM gefitinib for 72 h, the intracellular ATP (e), GSH (f), and ROS levels (g) were measured using the ATP determination kit, the GSH-Glo glutathione assay kit, and the DCFH-DA reagent, respectively. The gefitinib treatment significantly reduced the normalized ATP and GSH levels in HCC827 cells in a dose-dependent manner. Compared to the stable ROS level in HCC827 cells, the total ROS level in HCC827 GR cells was reduced, indicating scavenging. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, two-tailed Student’s t-test