Resveratrol reverses temozolomide resistance by down-regulation of O (6)-methylguanine-DNAmethyltransferase in T98G glioblastoma cells via nuclear factor-κB-dependent pathway

Abstract

Objective Glioblastoma,being one of the most malignant forms of adult brain tumors,is usually associated with a dismal prognosis.Given that the protein expression of O (6)-methylguanine-DNA methyltransferase (MGMT) is the most important determinant of temozolomide (TMZ) resistance,great efforts have been made to suppress it by regulating MGMT-related transcription factors.Resveratrol is a terpenoid that exhibits broad pro-apoptotic activity in various types of cancers,including glioblastoma.However,the effects of resveratrol on nuclear factor-κB (NF-κB) -MGMT signaling in glioblastomas have not yet been fully elucidated.In this article,we want to find that the molecular mechanisms of resveratrol reverses temozolomide resistance in glioblastoma cells.Methods Human malignant glioblastomas cell line T98G glioma cells was conventionally cultured in vitro; MTT assay was employed to detect the cell viability after being treated with dimethylsulfoxide (control group),or 100,200,400 and 800 μmol/L resveratrol,or 50,100,200 and 400 μmol/L TMZ or TMZ (100 μmol/L) combined with resveratrol (100 μmol/L); Hoechst33342 staining was employed to observe the effects of 100 μmol/L resveratrol,100 μmol/L TMZ or TMZ (100 μmol/L) combined with resveratrol (100 μmol/L) on T98G nuclear morphology changes; Flow cytometry and Western blotting were used to detect the T98G cell apoptosis and expressions ofMGMT,NF-κB signaling pathway related proteins.Results T98G cells after being treated with different concentrations of resveratrol for 24 and 72 h,the cell vitality decreased with 50％ inhibiting concentration (IC50) reaching 127.5 and 86.2 μmol/L,respectively; T98G cells after being treated with different concentrations of TMZ for 24 and 72 h,the cell vitality decreased with IC50 reaching 2.5 and 3.2 mmol/L,respectively; significant decreased cell vitality in the combination treatment with TMZ and resveratrol group was noted as compared with that in the TMZ or resveratrol treatment groups (P＜0.05).Hoechst 33258 staining and Western blotting revealed apoptotic morphological changes of T98G cell nuclei and increased apoptosis in the TMZ or resveratrol treatment groups,and more obvious changes were noted in the combination treatment with TMZ and resveratrol group.As compared with that in the control group,significantly increased MGMT protein expression in the 100 μmol/LTMZ treatment group was noted (P＜0.05),while no obvious MGMT protein expression was noted in the resveratrol treatment group and combination treatment with TMZ and resveratrol group; as compared with those in the control group,significantly increased IkB-α expression and decreased NF-κB p65 expression in the resveratrol treatment group (P＜0.05),significantly decreased IkB-α expression and increased NF-κB p65 expression in the TMZ treatment group (P＜0.05),and decreased NF-κB p65 expression in the combination treatment with TMZ and resveratrol group (P＜0.05).Conclusion Down-regulation of MGMT in T98G glioblastoma cells by NF-κB-dependent pathway is one of the important molecular mechanism of resveratrol reversing temozolomide resistance. Key words: Glioblastoma; Temozolomide; Resveratrol; O-6-methlguanine-DNA methyltransferase; Nuclear factor-κB