Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Abstract

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKCb inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription -PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3b and Akt phosphorylation, which was additionally reduced by drug combination (À58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (450% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients.

Enzastaurin shows preclinical antitumor activity against human transitional cell carcinoma and enhances the activity of gemcitabine

“…Additionally, the combination of gemcitabine and enzastaurin was synergistic for inhibiting cell viability and metabolic activity in vitro. Similar synergism has been recently shown preclinically between enzastaurin and pemetrexed against non-small cell lung cancer (26). In vitro migration of both cell-lines was inhibited at an earlier time point by enzastaurin independent of antiviability activity.…”

Sp1 inhibition-mediated upregulation of VEGF165b induced by rh-endostatin enhances antiangiogenic and anticancer effect of rh-endostatin in A549

“…Growth inhibition was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction method [24]. Three thousand cells per well plated into 96-well plates separately were incubated in RPMI 1640 containing 10% newborn bovine serum for 24 h. Next, after treatment, cells were incubated for 2 h at 37°C in 50 μl per well MTT solution (2 mg ml −1 ; Sigma).…”

A phase II study of enzastaurin in combination with erlotinib in patients with previously treated advanced non-small cell lung cancer

“…Combination studies with pemetrexed, gemcitabine, and capecitabine have been conducted without unexpected safety findings [28][29][30].…”