Figure 3

Cisplatin-resistant hepatoblastoma cells have higher activity and expression of PIM3 kinase and PIM inhibition increases their sensitivity to cisplatin. (A) Kinomic profiling of HuH6 and COA67 cisplatin-naïve and resistant cells was performed using a PamStation12 microarray. The heatmap displays kinomic signature data with kinomic peptides across all conditions (colored by log difference from mean per cell type, per peptide). Clustering is unsupervised, via a geometric means-distance method, with peptides clustered by row, and samples clustered by column. PIM family kinase targets (boxed region shown) were significantly activated in cisplatin-resistant compared to cisplatin-naïve HuH6 and COA67 tumors. (B) Immunoblotting was performed on lysates from both HuH6 and COA67 cisplatin-naïve and cisplatin-resistant tumors from various passages (P). PIM3 expression was higher in cisplatin-resistant compared to cisplatin-naïve tumors and increased as cells became more insensitive to cisplatin. GAPDH was used to confirm equal protein loading. (C) HuH6 and (D) COA67 cisplatin-naïve and resistant cells were treated with 1 µM of AZD1208, 10 µM of cisplatin, or both drugs for 72 h and proliferation evaluated with CellTiter 96 assay. The addition of AZD1208 to cisplatin reduced proliferation of resistant cells to levels comparable to those of naïve cells treated with cisplatin alone, indicating the ability of AZD1208 to sensitize resistant cells to cisplatin. NS not significant.