1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor

Abstract

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

FIGURE 1.

NH125 inhibits eEF2K in vitro and induces cellular eEF2 phosphorylation. A, NH125 inhibits eEF2K in enzymatic assays in the presence of various calmodulin concentrations. B, H1299 cells were treated with NH125 under serum, serum-free, and HBSS conditions for 6 h and processed for Western blot analysis. Phospho-eEF2 (peEF2) antibody was used to measure the phosphorylation level of eEF2. Total eEF2 and β-actin antibodies were used to detect protein levels and served as loading controls. C, additional cell lines including PC3, HeLa, H460, and C6 were treated and processed the same way as in A. Only β-actin was used as loading control because the total eEF2 level tracked well with β-actin under NH125 conditions. D, typical high content analysis images showed PC3 cells treated with NH125 at the indicated concentrations and processed for immunofluorescence staining. Cell nuclei were stained blue. peEF2 was stained green.

FIGURE 2.

Treatment of H460 and H1299 cells with eEF2K siRNA. Cells were transfected with eEF2K siRNA or mock and grown in serum or serum-free conditions for 24 h. Samples were processed for Western blot analysis. Membranes were probed with anti-eEF2K, peEF2, and β-actin antibodies (A). A parallel set of cells transfected with eEF2K siRNA was allowed to grow for 4 days before CyQuant NF analysis for cell proliferation (B). Percent cell proliferation of siRNA-transfected cells over mock transfected control cells was calculated to measure the effect of eEF2K siRNA under either serum or serum-free conditions. Error bars indicate S.E.

FIGURE 3.

Highly selective eEF2K inhibitor A-484954 inhibits eEF2 phosphorylation but has no inhibitory effect on growth. Chemical structure of A-484954 and selective kinase profiles are shown in A. H1299 cells were treated with A-484954 under serum, serum-free, and HBSS conditions at the indicated concentrations for 6 h and processed for Western blot analysis (B). Phospho-eEF2 (peEF2) antibody was used to measure the phosphorylation level of eEF2. Total eEF2 and β-actin antibodies were used to detect protein levels and served as loading controls. Additional cell lines including PC3, HeLa, H460, and C6 cells were treated and processed the same way (C). Only β-actin was used as loading control because the total eEF2 level tracked well with β-actin under A-484954-treated conditions. D, PC3 cells were treated with NH125 or A-484954 under either serum (+Serum) or serum-free (−Serum) conditions and assayed for proliferation using CyQuant NF reagents.

FIGURE 4.

eEF2 phosphorylation correlates with growth inhibition. PC3 cells were treated with the indicated compounds at various concentrations for 6 h and processed for Western blot analysis (A). The membrane was probed with peEF2 and pACC antibodies (A). Because total eEF2 and ACC tracked well with β-actin, only β-actin was shown as the protein loading control. A parallel set of PC3 cells was treated with the compounds at the indicated concentrations for 3 days and subject to CyQuant NF analysis (B). Fluorescence units (FU) were graphed against compound concentrations. Error bar shows S.E. of the samples.

FIGURE 5.

NH125-induced eEF2 phosphorylation is mediated through multiple pathways. PC3 cells were treated with compounds at the given concentrations for 6 h and processed for Western blot analysis. Cpd A, Abbott AMPK inhibitor. Cpd C, AMPK inhibitor compound C.