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

Abstract

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 Cbeta (PKCbeta), 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 PKCbetaII and glycogen synthase kinase 3beta (GSK3beta) was analyzed. Exposition to enzastaurin showed various inhibitory effects on phosphorylated forms of GSK3beta 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.

Prolonged Mechanical Ventilation Alters the Expression Pattern of Angio-neogenetic Factors in a Pre-Clinical Rat Model

“…Images were taken and densitometrically analyzed using ImageJ (v1.46k; National Institute of Health, Bethesda, MA, USA). HEY cells (ovarian cancer cell line, kind gift from Prof Meinhold – Heerlein, Aachen, [17]), which had been incubated in an hypoxic chamber for 96 h were used as positive controls for occurrence of hypoxia related proteins (HIF-1α, 2α and GLUT 4) [17]. For Western blot pictures indicating proper antibody binding (see Figure S2).…”

Combined treatment with TRAIL and PPARγ ligands overcomes chemoresistance of ovarian cancer cell lines

“…The parental ovarian cancer cell line HEY emanated from ascites cells of a moderately diVerentiated serous-papillary ovarian carcinoma and served as origin for cultivation of the cytostatic drug-resistant subpopulations (previously described (Brautigam et al 2009)). For the TRAIL-resistant control cell line, parental HEY cells were treated with increasing concentrations of TRAIL twice per week for 2 month until the cells exhibited signiWcant resistance.…”

“…The appropriate cytostatic drug for each of the resistant subclones was additionally supplied (2 g/mL cisplatin; 5 g/mL etoposide; 15 ng/mL docetaxel; 40 ng/mL paclitaxel; 3 M pemetrexed; 5 M gemcitabine; 10 ng/mL TRAIL). Resistance was proven by MTS Assay (CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay from Promega, Mannheim, Germany) as shown previously (Brautigam et al 2009). Stimulation experiments for immunoblot analyses were performed under serum-starved conditions.…”

Combinatorial inhibition of Plk1 and PKCβ in cancer cells with different p53 status

“…The activity of the serine/threonine kinase AKT, which promotes cell proliferation by inhibiting cell cycle inhibitors [3], is reduced under these circumstances via the inhibition of PKCβ [8]. Enzastaurin is used as a single agent or in combination with chemotherapeutic agents, targeted therapies or irradiation (combination with Pemetrexed [9], with Gemcitabine or Cisplatin [10] or with irradiation [4,11,12]). There are also successful trials in combination with other targeted GSK3β inhibitors [13] or with anti-neoplastic agents [14].…”