FIGURE 5.

ASCT2 knockdown induced apoptosis by suppressing GSH synthesis, thus increasing ROS levels in OSCC. Immunofluorescence staining (A) and Western blotting (B) were used to determine the downregulation of ASCT2 in SCC15 and UM1 cells transfected with ASCT2 siRNA. Moreover, the decrease in ASCT2 was accompanied by increased apoptosis in ASCT2‐knockdown cells (B). CCK8 assays (C) were performed to determine cell growth in SCC15 and UM1 cells under different glutamine concentrations. The proliferation of ASCT2‐knockdown OSCC cells was significantly inhibited compared with the nontargeted siRNA controls and cannot be reversed by adding extra glutamine (6 mmol/L) to the culture medium. Intracellular ROS were stained with a commercial ROS detection kit, and higher ROS levels were observed in OSCC cells transfected with ASCT2 siRNA (D, bar: 50 µm). The intracellular levels of GSH were significantly decreased in SCC15 and UM1 cells transfected with ASCT2 siRNA compared with the control groups (E). NAC, a ROS inhibitor, effectively inhibited apoptosis ROS induced in ASCT2‐knockdown cells (F). *P < 0.05; NS: no statistical significance. ASCT2, alanine‐serine‐cysteine transporter 2; Gln, glutamine; GSH, glutathione; NAC, N‐acetylcysteine; PARP, poly‐ADP‐ribose polymerase