Figure 4

PIM3 inhibition with AZD1208 promotes cisplatin-induced apoptosis. (A) Kinetic phosphorylation curves for peptides identified as potential PIM3 targets were overlaid for both cisplatin-naïve and cisplatin-resistant tumors. Phosphorylation of the pro-apoptotic protein BAD at phosphorylation sites that inhibit apoptosis was increased in resistant compared to naïve tumors in both HuH6 and COA67 xenografts, indicating decreased apoptosis in resistant cells. (B–E) Cisplatin-induced apoptosis was assessed by flow cytometric analysis of Annexin V/PI dual staining. (B) HuH6 and (C) COA67 cisplatin-resistant cells with or without treatment with 1 µM AZD1208 and/or 10 µM cisplatin for 72 h (for HuH6) and 24 h (for COA67) were stained and analyzed. Values expressed as mean percentage ± SEM. PIM inhibition with AZD1208 significantly promoted early (Annexin V + PI- cells, lower right quadrant (D,E)) as well as late (Annexin V + PI + cells, upper right quadrant (D,E)) apoptosis in cisplatin-resistant cells, indicating that the addition of PIM inhibition enhanced cisplatin-mediated apoptosis. Representative contour plots shown for both (D) HuH6 and (E) COA67 cisplatin-resistant cells along with appropriate staining controls (top panels).