Hypoxia promotes temozolomide resistance in glioblastoma cells via ROS- mediated up-regulation of TRPM2

Abstract

Abstract Background: Hypoxia, an essential feature of gliomas, is thought to promote chemo-resistance by regulating reactive oxygen species (ROS) levels. Transient receptor potential melastatin 2 (TRPM2) is one of the ion transport proteins and is involved in the regulation of oxidative stress. However, relationship between ROS and TRPM2 expression in hypoxia-induced temozolomide (TMZ) resistance of glioblastoma cells remains unclear. Methods: U87MG cells were cultured with different concentrations of TMZ for the indicated times under normoxia (21% O2) or hypoxia (2.5% O2). Cell viability was detected with WST-1 test and observed by a neurite outgrowth assay. The intracellular ROS scavenging activity was detected according to the H2DCF-DA method. The cells were also treated with the scavenger of ROS NAC and the inhibitor of TRPM2 2-APB. Impaired mitochondrial membrane potential (ΔΨm) and intensity of intracellular Ca2+ were measured under fluorescence microscope. Online database was used to assess the relationship between MGMT and TRPM2 expression level. Western blot was used to analyze the protein levels of TRPM2, MGMT, MSH3 and APNG. Results: Compared with the normoxia group, hypoxia significantly promoted glioma cells survival after treatment by TMZ (200µM) for 24 h or 48 h, accompanied with reduction of mitochondrial dysfunction and intracellular ROS. However, the baseline levels of ROS were mildly increased under hypoxia, which had no impact on mitochondrial function in glioma cells. Additional, TRPM2 expression was obviously increased under hypoxia and inhibited by NAC in glioma cells. We found that the expression levels of TRPM2 were positively correlated with MGMT both in online database (rho=0.165, P < 0.05) and GBM cancer tissues (r=0.9302, P < 0.05). Over-expression of TRPM2 participated in the up-regulation of APNG and MGMT, but down-regulation of MSH3 in glioma cells under hypoxia. Our findings also demonstrated that the treatment group during NAC or 2-APB add-on could significantly attenuate calcium influx, followed by increasing mitochondrial dysfunction and cytotoxicity in glioma cells, in comparison with TMZ alone. Conclusion: The hypoxia-induced up-regulation of baseline ROS levels contributes to the decrease in the sensitivity of glioma cells to TMZ via promoting demethylation and inhibiting DNA mismatch repair. Moreover, TRPM2-mediated Ca2+ influx attenuates mitochondria dysfunction and then protects glioma cells against TMZ damage. TRPM2 may be a potential target in adjuvant treatment with TMZ for glioblastoma multiforme (GBM) patients.