Inhibition Of EEF-2Kinase Augments The Antitumor Activity Of Temozolomide Against Glioma

Objective: In this study, we investigate that whether targeting eEF-2kinase couldimprove the sensitivity of glioma cells to treatment with temozolomide and increase thetreatment effects of this chemotherapeutic drug.Methods: Combined treatment with inhibition of eEF-2kinase by siRNA orNH125and temozolomide in cell culture and in an intracranial xenograft model, weundertake a comprehensive in vitro and in vivo studies by using cellular, molecuarbiology and animal experiment methods. In vitro experiments, to verify the changeof the effect of combination therapy treatment of malignant tumor, human glioma celllines LN229and U251was divided into different treatment groups for24h: TMZ group,eEF-2kinase siRNA or NH125group, TMZ combined with the eEF-2kinase inhibitorgroup and vehicle control group. After different treatment, glioma cell proliferationviability was measured by CCK-8assay; cell ability to migrate was measured by woundmigration assay and invasion was detected by transwell invasion assay. In vivoexperiments, to assess the therapeutic effect of the combination therapy on the originalglioma transplantation, the human glioma cell LN229was first inoculated into brains ofnude mice, and then were given NS, TMZ, NH125drug alone and TMZ+NH125combined treatment. Animal body weight and physical signs were monitored dailyduring the experiments; at day7,14,21after inoculation, the animals were sacrificedand the brains were fixed, embedded, and then stained with hematoxylin eosin (HE) orimmunohistochemical stain for eEF-2K. The tumor slices were examined with lightmicroscope; Kaplan-Meier survival curves was drawn and rate of animals lifeprolongation were compared in different drug groups. In addition, to study exploreits possible molecular mechanism, human glioma cell lines LN229and U251wastreated with different drug as described above for24, after different treatment, theactivity of eEF-2kinase, autophagy-associated proteins expression, apoptosis-associatedproteins was measured by Western blot analysis; apoptotic cells were assessed byAnnexin V-FITC staining assay.Results: In vitro studies, our results showedthat combined treatment with eEF-2kinase inhibitors and TMZ could strongly inhibits cell proliferation, reduces the growth of human glioma cells，suppresses glioma cell migration and invasion. That is to say,the inhibition of eEF-2kinase can enhance the sensitivity of glioma cells to thechemotherapy drug TMZ. In vivo studies, our results shown that the orthotopictransplantation tumor model was stable and repeated; the symptoms of thetumor-bearing nude mice are similar to glioma patients the prevalence of early onsetand advanced clinical manifestations. The HE staining photograph of glioma specimensslice shown that our drug treatment may not cure tumor-bearing animals, but delay thegrowth of tumor. And, combined treatment with TMZ and NH125strongly reduced thegrowth of LN229glioma cell-derived tumor xenograft to a much higher extent than thetreatment with TMZ or NH125alone. The survival curves have shown that combinedtreatment with TMZ and NH125greatly prolonged the tumor-bearing mice survivaltimes, as compared to the treatment with TMZ alone. In addition, combinedtreatment with eEF-2kinase inbhibitors and TMZ decreased cell survival by inhibitingautophagy, as evidenced by the decreases in the amount of LC3II and increases in theamount of p62were indicated by western blot results. And, combined treatment with eEF-2kinase inbhibitors and TMZ decreased cell survival by inducing apoptosis, as evidencedby the increases in the amounts of Cleaved caspase3and PARP were indicated by westernblot results. Further studies on apoptosis by Annexin V/PI staining and flow cytometricanalysis shown the percentage of apoptosis cells and apoptotic index augmentedcompared TMZ or eEF-2kinase inhibitors alone and combined treatment with TMZ andeEF-2kinase inhibitors.Conclusion: Inhibition of eEF-2kinase by siRNA or NH125augments theantitumor activity of temozolomide in vitro and in vivo against GBM. The possiblemechanism is that the combination therapy may cure malignant tumor, via inhibiting theautophagic survival and inducing the apoptosis, to promote more tumor cells into thepermanent death pathway.