Molecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells

Erlotinib is commonly used as a second line treatment in non-small cell lung cancer patients with sensitizing EGFR mutations. In EGFR-wild type patients, however the results are limited. Therefore we evaluated whether the combination of the Protein kinase C-β inhibitor enzastaurin with erlotinib could enhance the effect in the A549 and H1650 cell lines. Cytotoxicity of erlotinib, enzastaurin and their 72-h simultaneous combination was assessed with the MTT assay. The pharmacologic interaction was studied using the method of Chou and Talalay, cell cycle perturbations were assessed by flow cytometry and modulation of ERK1/2 and AKT phosphorylation was determined with ELISA. For protein phosphorylation of GSK3β we performed Western Blot analysis and a Pamgene phosphorylation array, while RT-PCR was used to investigate VEGF and VEGFR-2 expression before and after drug treatments. A synergistic interaction was found in both cell lines with mean CI of 0.58 and 0.63 in A549 and H1650 cells, respectively. Enzastaurin alone and in combination with erlotinib increased the percentage of cells in S and G2M phase, mostly in H1650 cells, while AKT, ERK1/2 and GSK3β phosphorylation were reduced in both cell lines. VEGF expression decreased 5.0 and 6.9 fold in A549 cells after enzastaurin alone and with erlotinib, respectively, while in H1650 only enzastaurin caused a relevant reduction in VEGF expression. The array showed differential phosphorylation of EGFR, GSK3β, EphA1 and MK14. In conclusion, enzastaurin is a protein kinase Cβ inhibitor, working on several cellular signaling pathways that are involved in proliferation, apoptosis and angiogenesis. These features make it a good compound for combination therapy. In the present study the combination of enzastaurin and erlotinib gives synergistic results, warranting further investigation