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. 2014 Apr 24;5(9):2807–2819. doi: 10.18632/oncotarget.1921

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Copyright: © 2014 Maitra et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Figure 5a — Combination index of reovirus and irinotecan administration in KRAS mutant HCT116 as compared to KRAS wild type isogenic Hke3 cells at 50% and 75% growth inhibition. While synergy is observed in both cell lines (CI<1), a significant difference is detected in the CI between the two cell lines at the respective effective doses (ED; p=0.002 at ED50 and p=0.01at ED75 respectively).b. FACS analysis for quantitative assessment of apoptosis. The combination group showed a greater degree of apoptosis than single agent reovirus (p=0.01) in HCT116, while in the KRAS WT Hke3 cells there was no improvement as compared to single agent reovirus (p=0.11; Figure 5b). Moreover, the apoptosis in the combination was significantly higher in the KRAS mutant cells at 18.44 + 1.07 (mean + SEM) than the KRAS WT cells, at 11.14 + 0.16 (mean + SEM), with a p value of 0.02. c. Western blot assay to determine the expression of p21 and p53 proteins. HCT116 and Hke3 cells were treated with 5MOI reovirus and 2 uM irinotecan as single agent and in combination for 24 hours. Cells were harvested and 50 ugm were loaded per lane. The blot was probed with β-actin to confirm the equal protein load per lane. The adjoining graphs represents the relative densitometry of p53 and p21 proteins normalized to ß-Actin in control, irinotecan, reovirus and combination treatment of HCT116 and Hke3 cells at 48 hours. The expression of p53 was not significant in either of the cell lines but p21 showed a significant upregulation in irinotecan treated groups in both the cell lines where as significant downregulation only in reovirus treated KRAS mutant HCT 116 cells. The graph shows the mean protein densities from two independent experiments and a two tailed t test is employed to generate the p value. d. Scanning electron micrograph of HCT116 and Hke3 cells upon combination treatment at 5K magnification (upper panel) and 10K magnification in the lower panel. More prominent perturbation is observed in KRAS mutant HCT116 when compared to KRAS wildtype Hke3 cells.