雷公藤红素促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性的研究

Celastrol Enhances the TNF-α-induced Apoptosis by Promoting the Deubiquitination of RIP1 in Colon Cancer

关键词:  雷公藤红素  TNF-α  结肠癌  RIP1  CYLD  去泛素化  凋亡

Key words:  celastrol  TNF-α  colon cancer  RIP1  CYLD  deubiquitination  apoptosis

摘要:

目的 探讨中药活性成分雷公藤红素对TNF-α体外抗结肠癌活性的影响并研究其机制。方法 用雷公藤红素联合TNF-α体外治疗结肠癌细胞系SW480，MTT法检测SW480细胞的细胞活力。SW480细胞用雷公藤红素联合TNF-α治疗后，Annexin V/PI染色法检测细胞的凋亡，Western blot法检测细胞caspase-8、caspase-9和caspase-3的活化和对CYLD蛋白表达的影响，并用免疫共沉淀法检测SW480细胞RIP1蛋白的泛素化水平。结果 雷公藤红素联合TNF-α对结肠癌细胞系SW480的细胞活力抑制率和凋亡诱导活性均显著高于雷公藤红素及TNF-α单治疗组。雷公藤红素联合TNF-α对SW480细胞caspase-8、caspase-9和caspase-3的活化显著高于雷公藤红素及TNF-α单治疗组，且两者联合治疗后，caspase-8的活化时间显著早于caspase-9和caspase-3。SW480细胞用雷公藤红素联合TNF-α治疗后，其RIP1蛋白的泛素化水平显著低于雷公藤红素及TNF-α单治疗组。进一步研究发现，雷公藤红素能显著诱导SW480细胞CYLD蛋白的表达，而TNF-α对CYLD的表达水平无影响，当用小干扰RNA沉默CYLD的表达后，雷公藤红素对TNF-α的协同效应丧失。结论 雷公藤红素通过促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性。

Abstract:

OBJECTIVE To investigate the role of celastrol in TNF-α-induced cell death in colon cancer. METHODS SW480 cells were treated with TNF-α combined with celastrol. After that, MTT assay was performed to measure the cell death; Annexin Ⅴ/PI staining was performed to detect the cell apoptosis; Western blot analysis was performed to evaluate the cleavage of caspase-8, caspase-9, caspase-3, and the expression of CYLD; Co-immunoprecipitation was performed to detect the deubiquitination of RIP1 protein. RESULTS Combination with celastrol significantly promoted the cell death and apoptosis induced by TNF-α treatment in SW480. Combination with celastrol significantly promoted the cleavage of caspase-8, caspase-9, and caspase-3 induced by TNF-α treatment. Meanwhile, the activation of caspase-8 is earlier than the activation of caspase-9 and caspase-3. The results of co-immunoprecipitation and western blot indicated that the deubiquitination of RIP1 in TNF-α-treated SW480 cells were significantly enhanced due to the combination of celastrol. In addition, the results of western blot assay demonstrated that the expression of CYLD could be significantly up-regulated due to the celastrol treatment. Moreover, the transfection of CYLD siRNA abolished the synergistic effect on TNF-α-induced cell death in SW480 cells. CONCLUSION Celastrol enhances the TNF-α-induced apoptosis by promoting the deubiquitination of RIP1 in colon cancer.