Figure 6. PUMA mediates the chemosensitization effects of regorafenib in vitro and in vivo.

(A) WT and PUMA-KO HCT116 cells were treated with 20 μmol/L regorafenib, 20 mg/L 5-fluorouracil (5-FU), or their combination. Upper, western blot analysis of PUMA expression in WT cells treated for 24 hours; lower, apoptosis in WT and PUMA-KO cells treated for 48 hours analyzed by nuclear staining with Hoechst 33258. (B) WT and PUMA-KO HCT116 cells were treated with 20 μmol/L regorafenib, 25 μmol/L oxaliplatin, or their combination. Upper, western blot analysis of PUMA expression in WT cells treated for 24 hours; lower, apoptosis in WT and PUMA-KO cells treated for 48 hours analyzed as in (A). (C) WT and PUMA-KO HCT116 cells were treated with 20 μmol/L regorafenib, 6 μg/mL of cetuximab, or their combination. Upper, western blot analysis of PUMA expression in WT cells treated for 24 hours; lower, apoptosis in WT and PUMA-KO cells treated for 48 hours analyzed as in (A). (D) Nude mice were injected s.c. with 4 × 10⁶ WT or PUMA-KO HCT116 cells. After 1 week, mice were treated with 15 mg/kg regorafenib daily by oral gavage, 25 mg/kg 5-FU every other day by i.p. injection, or their combination for 10 consecutive days. Tumor volume at indicated time points after treatment was calculated and plotted with p values for indicated comparisons, n=5 in each group. Arrows indicate regorafenib or 5-FU injection. (E) Mice with WT or PUMA-KO HCT116 xenograft tumors were treated with 15 mg/kg regorafenib daily, 25 mg/kg 5-FU every other day, or their combination as in (D) for 4 consecutive days. Paraffin-embedded sections were analyzed by TUNEL staining. TUNEL-positive cells were counted and plotted. (F) Tissue sections from (E) were analyzed by active caspase 3 staining. Active caspase 3-positive cells were counted and plotted. Results in (E) and (F) were expressed as means ± SD of 3 independent experiments. ***, P <0.001; **, P <0.01; *, P <0.05.