Pancreatic cancer (PaCa) is one of the most aggressive cancers and currently incurable with less than 2% five-years survival rate. Among the ever-increasing list of naturally occurring anticancer agents, a Kmala Tree-derived anticancer compound, Rottlerin (ROT), a PKC-delta inhibitor, appears to have anti-proliferative activity due to its effects on several pathways and cell machineries involved in cell survival, apoptosis, autophagy and invasion. Recent studies suggest that ROT is not a specific PKC delta inhibitor and mediates its effects through other mechanisms. We have previously reported that inhibition of eukaryotic elongation factor-2 kinase (eEF-2K), one of the major kinases appears to be activated in rapidly proliferating malignant cells, leads to down-regulation of signaling pathways affecting growth, survival and chemotherapeutic resistance (Tekedereli et al, 2012). Thus, we hypothesized that ROT targets eEF-2K, and inhibition of eEF-2K signaling is responsible of mediating its effects in PaCa cells. We found that ROT treatment (4-10 μM) inhibits the expression of eEF-2K that was associated with apoptosis induction in PaCa cells in a dose and time-dependent manner as detected by Western blot analysis. We also found that ROT treatment inhibits PaCa cell proliferation and mitochondrial activity and modulates G1/S phase progression in these cells. Treating PaCa cells with ROT markedly induced expression of active caspase-9, caspase-3 and cleaved PARP. To demonstrate a direct link between eEF-2K inhibition and ROT-induced effects, we knocked-down eEF-2K by a specific siRNA. Such knockdown was associated with inhibition of cell growth, resulted in modulation of caspase-related events with induction of similar degree of apoptosis, increased the percentage of Annexin V positive apoptotic cells and resulted in induction of cellular shrinkage and blebbing. Moreover, we found that eEF-2K down-regulation is involved in ROT-induced stimulation of extrinsic apoptotic pathway in PaCa cells with concomitant induction of TNF related apoptosis inducing ligand (TRAIL) receptor DR4 and caspase-8 cleavage. In addition, our findings suggest that eEF-2K contributes to the regulation of G1/S phase progression, as in vitro down-modulation eEF-2K resulted in an increase in the expression of p27Kip1 cyclin-dependent kinase inhibitor (CDKI). Collectively, the results of our study demonstrate, for the first time, that down-regulation of eEF-2K contributes to rottlerin-induced effects including, growth inhibition and apoptosis in PaCa cells and clearly demonstrate novel mechanisms of ROT-induced potent antitumor effects. These findings show that the multi-targeted kinase inhibitor, ROT, represents a promising novel agent for PaCa treatment and eEF-2K could represent an attractive target for the future anticancer agents.

Citation Format: Ahmed A. Ashour, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Sultan N. Alpay, Kevin Dalby, Bulent Ozpolat. Inhibition of eukaryotic elongation factor-2 kinase mediates Rottlerin induced effects in apoptosis and cell proliferation inhibition in human pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2013-848