Enzastaurin, a Protein Kinase Cβ Inhibitor, Suppresses Signaling through the Ribosomal S6 Kinase and Bad Pathways and Induces Apoptosis in Human Gastric Cancer Cells

Abstract

Activation of protein kinase C (PKC) has been implicated in gastric carcinogenesis. Enzastaurin is an oral ATP-competitive inhibitor of the PKCB isozyme. Although enzastaurin was initially advanced to the clinic based on its antiangiogenic activity, it is also known to have a direct effect on a variety of human cancer cells, inducing apoptosis by inhibiting the Akt signal pathway. However, data on enzastaurin for gastric cancer are limited. Therefore, this study was performed to assess the antitumor activity of enzastaurin on gastric cancer cells and to investigate the underlying antitumor mechanisms. Enzastaurin suppressed the proliferation of cultured gastric cancer cells and the growth of gastric carcinoma xenografts. Enzastaurin did not have an effect on gastric cancer cell cycle progression; however, it had a direct apoptosis-inducing effect through the caspase-mediated mitochondrial pathway. Glycogen synthase kinase 3B phosphorylation, a reliable pharmacodynamic marker of enzastaurin activity, and Akt phosphorylation were both decreased after treatment with enzastaurin. Although the p90 ribosomal S6 kinase (Rsk) was also dephosphorylated, Erk phosphorylation was not affected in the enzastaurin-treated gastric cancer cells. Enzastaurin activated Bad, one of the Bcl-2 proapoptotic proteins, through dephosphorylation at Ser 112 , and depletion of Bad activity resulted in resistance to enzastaurin-induced apoptosis and cytotoxicity in gastric cancer cells. These data suggest that enzastaurin induces apoptosis through Rsk-mediated and Badmediated pathways, besides inhibiting the Akt signal cascade. Furthermore, enzastaurin had synergistic or additive effects when combined with 5-fluorouracil, cisplatin, paclitaxel, or irinotecan. These results warrant further clinical investigation of enzastaurin for gastric cancer treatment. [Cancer Res 2008;68(6):1916-26]

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

“…Thus, when combining these two agents, enzastaurin can facilitate pemetrexed-damaged cells to undergo apoptosis. In line with our observations, previous studies showed little effects of enzastaurin alone on cell cycle progression (Lee et al, 2008), but others reported that the non-selective PKC inhibitor UCN-01 was able to inhibit the phosphorylation of Cdc25C and abrogate the G2/M checkpoint, potentiating the cytotoxicity of a variety of anticancer agents (Yu et al, 1998;Graves et al, 2000). However, enzastaurin was also able to influence other proteins involved in cell cycle regulation.…”

“…Enzastaurin was originally evaluated in human tumour xenograft-bearing mice for its antiangiogenic activity upon PKCb inhibition, as it showed reduction of plasma VEGF levels together with a significant decrease in intratumoural vessel density (Keyes et al, 2004). However, several studies have shown that enzastaurin exhibits direct growth inhibiting effects on a wide array of cultured human tumour cells (Graff et al, 2005;Oberschmidt et al, 2005;Querfeld et al, 2006;Rizvi et al, 2006;Podar et al, 2007;Spalding et al, 2007;Lee et al, 2008). Recent studies suggest that the antitumour effects of enzastaurin are mediated through interference with the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Graff et al, 2005;Querfeld et al, 2006;Rizvi et al, 2006;Lee et al, 2008), an important pathway regulating the apoptotic response.…”

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3β

“…However, we did not observe antiproliferative or toxic effects of enzastaurin on IL-2-stimulated NK cells at therapeutic relevant concentrations ranging from 0.625 µM to 2.5 µM. Notably, enzastaurin concentrations higher than 2.5 µM were required to affect cell proliferation and viability of some tumour cell types with highly activated PKCß [38,40]. However, enzastaurin concentrations between 0.625 µM and 2.5 µM inhibited dose-dependently GSK3ß phosphorylation in NK cells indicating an uncoupling of anti-proliferative effects of enzastaurin from effects on GSK3ß signalling.…”

“…Enzastaurin has been reported to induce apoptosis in several tumors by interfering with signaling through the PI3K/AKT pathway [1,37,38]. We investigated by western blot the influence of enzastaurin on AKT activity and its downstream effectors in NK cells.…”

Modulation of 4E-BP1 Function as a Critical Determinant of Enzastaurin-Induced Apoptosis

“…Enzastaurin induces apoptosis in a wide variety of human cancer cell lines including glioblastoma multiforme (1, 28), cutaneous T-cell lymphoma (29), colon (1), gastric (30), multiple myeloma (31,32), and Waldenstrom's macroglobulinemia (33). Our data indicate that enzastaurin treatment blocks 4E-BP1 phosphorylation and increases the association of eIF4E with 4E-BP1.…”