白藜芦醇通过NF—κB依赖途径下调MGMT增强胶质瘤细胞对替莫唑胺药物的敏感性

Resveratrol reverses temozolomide resistance by down-regulation of O （6）-methylguanine- DNAmethyltransferase in T98G glioblastoma cells v/a nuclear factor-KB-dependent pathway

目的研究白藜芦醇增强恶性胶质瘤细胞对替莫唑胺（TMZ）药物敏感性的分子机 制。方法体外常规培养人源性恶性胶质瘤细胞系T98G细胞，MTT检测100、200、400、800 μmol/L 白藜芦醇，50、100、200、400 μmol/L TMZ,TMZ（100 μmol/L）与白藜芦醇（100 μmol/L）联用处 理不同时间后细胞活力的变化；Hoechst 33342染色观察100 μmol/L白藜芦醇、TMZ单独及联合处 理对T98G细胞核形态的影响；流式细胞仪和Western blotting分别检测细胞凋亡、06-甲基鸟嘌呤 -DNA甲基转移酶（MGMT）、核因子KB（NF_KB）信号通路相关蛋白的表达。结果不同浓度白藜 芦醇作用T98G细胞24、72h后活力下降,24、72h半数抑制浓度（IC50）分别为127.5、86.2 μmol/L;不 同浓度TMZ作用T98G细胞72h后活力下降，24、72hIC5Q分别为2.5、3.2mmol/L;联合处理组较 白藜芦醇、TMZ单独处理组细胞活力下降，差异有统计学意义（p〈0.05）。Hoechst 33342染色、流式细 胞仪分别显示白藜芦醇和TMZ处理导致瘤细胞细胞核发生凋亡形态改变，凋亡率增加，二者联合 处理的作用更加显著。与对照组比较，100 （Jimol/L TMZ组细胞MGMT蛋白的表达上升，100 μmol/L 白藜芦醇组、联合处理组MGMT蛋白无表达或弱表达，差异有统计学意义（P〈0.05）。与对照组相比，白藜芦醇组细胞ikB_a的表达上调，NF-kB p65的表达下调；TMZ组IkB-a的表达下调，NF-kB p65 的表达上调；联合处理组NF-kB p65的表达下调，差异均有统计学意义（P〈0.05）。 结论NF-kB 信号依赖通路下调MGMT表达是白藜芦醇增强胶质瘤细胞对TMZ药物敏感性的重要分子机制。

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-KB （NF-KB） -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/xmol/L resveratrol, or 50, 100, 200 and 400 μmol/L TMZ or TMZ （100 Ixmol/L） combined with resveratrol （100 Iμmol/L）; Hoechst33342 staining was employed to observe the effects of 100 μmol/L resveratrol, 100/μmol/L TMZ or TMZ （100/xmol/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 of MGMT, NF-KB 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 ixmol/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 （/＇〈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 Ixmol/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 ]kB-a expression and decreased NF-KB p65 expression in the resveratrol treatment group （P〈0.05）, significantly decreased IkB-ot expression and increased NF-KB p65 expression in the TMZ treatment group （P〈0.05）, and decreased NF-KB 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-KB-dependent pathway is one of the important molecular mechanism of resveratrol reversing temozolomide resistance.