Effects of mTOR inhibitor, everolimus, on proliferation, autophagy and temozolomide sensitivity of glioma cells

Wei Dong,Shuo Sun,Yanbin Wang,Haipeng Liu,Jialiang Wang

doi:10.4314/tjpr.v19i1.12

Abstract

Purpose: To study the effect of the mTOR inhibitor, everolimus, on glioma cell proliferation, autophagy, and drug sensitivity to temozolomide (TMZ).Methods: Human glioma cell lines were cultured in vitro, and the effects of different concentrations of everolimus on the proliferation of brain glial cells were determined using CCK-8 method. The effect of different concentrations of everolimus on brain glial cell levels of autophagy protein were assayed by western blot method.Results: The results of CCK-8 analysis showed that everolimus inhibited the proliferation of glial cells in a time- and concentration-dependent manner. Western blot results showed that the expression levels of autophagy proteins, LC3-II and LC3-II/I, were gradually and concentration-dependently up-regulated, while p62 protein level was gradually decreased concentration-dependently, when compared with blank control (p < 0.05). Treatment with different concentrations of TMZ alone, and in combination with everolimus for 48 h inhibited the proliferation of brain glial cells in a concentration-dependent manner, but the inhibition due to TMZ-everolimus combination was significantly higher than that of TMZ singletreatment (p < 0.05). After 48 h, the expression level of Beclin-1 increased with the ratio of LC3-II/LC-I in TMZ-everolimus group, while the expression level of p62 decreased, when compared with TMZ alone, or control (p < 0.05).Conclusion: Everolimus significantly inhibits the proliferation of glioma cells and promotes the occurrence of autophagy. Combined use of TMZ and everolimus significantly enhances the sensitivity of TMZ to glioma cells, inhibits cell proliferation, and promotes autophagy better than TMZ alone. Keywords: mTOR inhibitor, Everolimus, Glioma cells, Proliferation, Autophagy

Highlights

Glioma is a high-incidence malignant tumor of the central nervous system due to carcinogenesis in the brain and spinal cord
Effect of everolimus concentration on autophagy protein levels in glial cells Western blot results showed that after treatment of glial cells with different concentrations of everolimus, the expression levels of the autophagy proteins LC3-II, LC3-II /I ratio and Beclin-1 were gradually increased in a concentration-dependent manner, when compared with the blank control group, and the protein level of P62 was gradually decreased in a concentration-dependent manner (p < 0.05; Table 2, Figure 2)
The CCK-8 test results showed that treatment of glial cells with different concentrations of TMZ alone or in combination with everolimus for 48h inhibited the proliferation of glial cells in a concentration-dependent manner, and the inhibition due to TMZ/ everolimus combination was significantly higher than that of TMZ alone (p < 0.05; Figure 3)

Summary

Introduction

Glioma is a high-incidence malignant tumor of the central nervous system due to carcinogenesis in the brain and spinal cord. The incidence of glioma accounts for 1- 3 % of all malignant tumors. Most glioma occur in the cerebral hemisphere. Glioma is caused by the interaction between susceptible congenital genetic factors and carcinogenic environmental factors [1]. The symptoms and signs of glioma are related to the location and pathological grade of the tumor. Patients present with headache, nausea, vomiting and blurring, among cultured in an atmosphere of 5 % CO2 at 37 °C and saturated humidity. The cell culture medium was changed every 2 - 3 days, and cells in good state of logarithmic growth were selected for the experiments

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