Figure 1.

Cytotoxic effects of APG in GC cell lines. (A,D) Cell viability of dose- (0, 30, 50, and 70 µM, 24 h) and time-dependent (50 µM; 8, 16, and 24 h) APG, in GC cell lines, including AGS, SNU-216, NCI-N87, SNU-638, MKN-7, and MKN-74, and lung normal cell line MRC5 measured using the WST-1 assay on 96-well plates. (B,C) AGS cells (1 × 10⁷) were implanted (sc) into the thigh on the right hind leg of nude mice (n = 10/group). APG (200 or 300 mg/kg) or PBS was administered intraperitoneally (ip) once a day for two days. The longest (L) and shortest (W) axes of the tumors were measured, and the tumor volume (mm³) was calculated as LW2/2. Body weights of the AGS tumor xenograft mice were determined twice a week during the experiment. (E–H) APG was treated in a time-dependent manner (50 µM; 8, 16, and 24 h), and the LDH assay and caspase-3 and caspase-9 activity assays were performed. Western blot analyses of cleaved caspase-3 and -9 for the indicated times in APG-treated AGS and SNU-638 cells; * p < 0.05. β-actin was used as a protein loading control. (I–L) The effect of Z-VAD-FMK (50 μM) and APG treatment. AGS and SNU-638 cells were pre-treated with Z-VAD-FMK for 4 h and were subsequently treated with APG (50 μM, 24 h). Cell viability was determined using the WST-1 assay; cell cytotoxicity was monitored using the LDH assay, and caspase-3 activity was assessed using the caspase-3 activity assay; * p < 0.05, n.s; no signaificant. Sampling of total lysates was performed using the Western blot assay to identify the activation of apoptosis marker cleaved caspase-3. β-actin was used as a protein loading control.