Fig. 2.

Combination of lapatinib and NVP-AEW541 is hyper-additive but not reversing cisplatin resistance in HCC38CisR. a Coincubation with 1.5 μM NVP-AEW541 significantly decreased IC₅₀ of lapatinib in HCC38CisR, whereas this treatment had no effect in HCC38. b Coincubation with 2 μM lapatinib significantly decreased IC₅₀ of NVP-AEW541 in HCC38CisR but had no effect in HCC38. c In HCC38CisR (but not in HCC38), the combination of NVP-AEW541 and lapatinib significantly induced apoptosis in a hyper-additive manner (***p < 0.001). NVP-AEW541 and lapatinib were used at 2 μM. Cells were treated for 48 h and the amount of apoptotic nuclei in the control was subtracted from treated samples. d Effect of NVP-AEW541 or lapatinib (2 μM, respectively) on cell cycle in HCC38CisR. Combination of 2 μM NVP-AEW541 and 2 μM lapatinib significantly (***p < 0.001) increased cell population in G₁ (77.7 ± 1.2% vs. 67.3 ± 1.4%) while reducing cell population in G₂/M phase (14.2 ± 1.5% vs. 25.7 ± 1.6%). Incubation time was 48 h. e Western blot analysis of p-EGFR, p-IGF1R, and p-Akt upon treatment of HCC38CisR with an IC₅₀ of lapatinib or NVP-AEW541 or both compounds for 6 h. f Densitometric analysis of the protein bands for p-AKT, p-EGFR, and p-IGF1R of HCC38CisR were performed using ImageJ software (NIH). Data are means ± SD, n = 3. All values have been normalized to untreated HCC38 CisR. Statistical analysis was performed using one-way ANOVA test (* p < 0.05). g Effect of 1 μM lapatinib and 1.5 μM NVP-AEW541 on cisplatin sensitivity either alone or in combination. Lapatinib and/or NVP-AEW541 were added 48 h prior to cisplatin treatment. IC₅₀ of cisplatin did not significantly differ. All data shown are mean +/− SEM, n = 3, except (e) showing a representative experiment out of 3