Combination of Enzastaurin and Pemetrexed Inhibits Cell Growth and Induces Apoptosis of Chemoresistant Ovarian Cancer Cells Regulating Extracellular Signal-Regulated Kinase 1/2 Phosphorylation

New strategies in the therapy for malignant diseases depend on a targeted influence on signal transduction pathways that regulate proliferation, cell growth, differentiation, and apoptosis by the activation of serine/threonine kinases. Enzastaurin (LY317615. HCl), a selective inhibitor of protein kinase C beta (PKC beta), is one of these new drugs and causes inhibition of proliferation and induction of apoptosis. Pemetrexed, a multitarget inhibitor of folate pathways, is broadly active in a wide variety of solid tumors. Therefore, the effect of enzastaurin and the combination treatment with pemetrexed was analyzed when applied to the drug-sensitive ovarian cancer cell line HEY and various subclones with drug resistance against cisplatin, etoposide, docetaxel, and paclitaxel, as well as pemetrexed, and gemcitabine. In these novel chemoresistant subclones, the expression of the enzastaurin targets PKC beta II and glycogen synthase kinase 3 beta (GSK3 beta) was analyzed. Exposition to enzastaurin showed various inhibitory effects on phosphorylated forms of GSK3 beta and the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. Cell proliferation experiments identified the cell line-specific half-maximal inhibitory concentration values of enzastaurin and a synergistic inhibitory effect by cotreatment with the antifolate pemetrexed. Induction of apoptosis by enzastaurin treatment was investigated by Cell Death Detection ELISA and immunoblot analyses. Simultaneous treatment with pemetrexed resulted in an enhanced inhibition of proliferation and induction of apoptosis even in partial enzastaurin-resistant cells. Therefore, the combinational effect of enzastaurin and pemetrexed can have promise in clinical application to overcome the fast-growing development of resistance to chemotherapy in ovarian cancer.