Mel Aggravates Pyroptosis Induced by Temozolomide Through Inhibiting NRF2-are Pathway in Glioblastoma

Abstract Background:Glioblastoma(GBM) is a common malignant tumor of the brain. It has been verified that melatonin(MEL) can inhibit glioma proliferation. But its mechanism has not been fully elucidated. We intend to examine the mechanism of MEL on glioma cells from the perspective of pyroptosis and Nrf2. Methods:Expression of MEL receptor in glioma was detected by western blot. GBM cell viability treated with temozolomide(TMZ) plus MEL was detected by CCK-8. Pyroptosis rate was determinate by flow cytometric analysis. Western blot was used to detect the Nrf2 and pyroptosis related protein level after MEL treatment. Orthotopic tumor growth inhibition study was performed to further investigate the tumor inhibition effect of TMZ plus MEL.Results:We first confirmed MEL receptor was abundant in glioma tissue and cell lines. After combined treatment of TMZ and MEL, cell viability decreased significantly as compared to those of TMZ treatment alone. Also, the ratio of pyroptosis and ROS level increased, followed by elevated expression of pyroptosis related protein. Furthermore, MEL can induce a diminution of Nrf2 expression in glioma in dose- and time-dependent manner. TMZ can increase Nrf2-ARE pathway expression, which also can be deprived by MEL. Its inhibition of Nrf2 depends on dephosphorylation of IGF-1/AKT/mTOR pathway. More importantly, after overexpression of Nrf2 in glioma cells, the level of pyroptosis-related protein elevated by MEL decreased, suggesting that the effect of MEL on promoting pyroptosis is dependent on its inhibition of Nrf2. In vivo results further confirmed that MEL plus TMZ induced significantly decreased tumor size and increased pyroptosis rate, but had no significant effect on mouse body weight, ALT, AST.Conclusion:MEL can inhibit the phosphorylation of IGF-1/AKT/mTOR pathway at millimol level, which further reduces the expression of Nrf2 and promotes pyroptosis of glioma cells. Considering the modest efficacy of TMZ chemotherapy, MEL can be considered as a potential chemotherapy sensitizer to improve the chemotherapy effect of glioma.