乙醇通过激活TGF-β/Smad/P21信号轴促进结直肠癌奥沙利铂的耐药性OA
Ethanol promotes oxaliplatin resistance in colorectal cancer by activating the TGF-β/Smad/P21 signaling axis
目的 探究乙醇促进结直肠癌(CRC)奥沙利铂耐药的作用及其潜在分子机制.方法 整合 PLCO 队列终生饮酒分层数据分析饮酒水平与CRC发生风险的关联;基于 GEO 数据库比较酒精代谢相关基因在 CRC 与正常组织中的表达差异,并在含化疗信息的队列中评估其与复发及生存结局的关系.同时采集无饮酒史健康人群及 CRC 患者粪便样本,采用气相色谱-质谱联用法(GC-MS)检测乙醇水平,以评估肠道基础乙醇水平.体内采用 Lieber-DeCarli 乙醇液体饲料建立持续乙醇暴露小鼠皮下移植瘤模型,在奥沙利铂处理条件下分为对照组、乙醇组与乙醇脱氢酶抑制剂 4(4-MP)组,评估肿瘤生长.体外以SW480与 HCT116细胞为模型,在奥沙利铂处理条件下分为对照组、100乙醇(EtOH)组与200 EtOH组;采用CCK-8评估药物敏感性,并通过 EdU与流式细胞术检测细胞凋亡及细胞周期变化.吡非尼酮(PFD)干预实验分为 对照组、200 EtOH、200 EtOH+PFD与 PFD组,在体内外分别结合小鼠皮下移植瘤模型、EdU实验、流式细胞术及Western blotting评估PFD干预后耐药相关表型及TGF-β/Smad/P21/Rb轴相关蛋白变化趋势.结果 与轻度饮酒者相比,高频率饮酒者的CRC发生风险更高(HR>1,P<0.05).与正常组织相比,CRC组织中乙醇氧化代谢关键酶ADH1B与ADH1C表达下调(P<0.001).在无饮酒史健康人群及CRC患者粪便样本中均可检测到乙醇.在接受化疗的患者中,与高表达组相比,ADH1B/ADH1C低表达组复发率更高且总体生存更差(P<0.05).在奥沙利铂治疗条件下,与对照组相比,Alcohol组终点肿瘤体积更大(P<0.05);4-MP组亦呈增大趋势.与对照组相比,乙醇处理的SW480与HCT116细胞奥沙利铂IC50升高且凋亡比例降低(P<0.05),以及G0/G1期比例增加及S期比例减少(P<0.05).Western blotting显示,乙醇处理细胞中TGF-β1水平升高、p-Smad2增强、P21上调且p-Rb(Ser780/Ser795)降低.与200EtOH组相比,200EtOH+PFD组凋亡比例升高、G0/G1期比例降低且S期比例升高(P<0.05),并伴随p-Smad2与P21下调及p-Rb(Ser780/Ser795)上调.结论 乙醇蓄积激活 TGF-β/Smad/P21 轴诱导CRC细胞周期阻滞,促进奥沙利铂耐药;吡非尼酮阻断可部分逆转上述效应.
Objective To investigate the effect of ethanol for promoting oxaliplatin resistance in colorectal cancer(CRC)and the underlying molecular mechanisms.Methods Lifetime alcohol consumption data from the PLCO cohort were analyzed to assess the association between alcohol drinking and CRC risk.The differential expressions of alcohol metabolism-related genes between CRC and normal tissues and their associations with recurrence and survival in chemotherapy-treated patients were analyzed based on GEO datasets.Fecal samples from healthy non-drinkers and CRC patients were collected for detecting ethanol levels using gas chromatography-mass spectrometry(GC-MS).In a mouse model bearing subcutaneous CRC xenograft treated with oxaliplatin,the effects of continuous ethanol exposure using the Lieber-DeCarli liquid diet and 4-methylpyrazole(4-MP)treatment on tumor growth were evaluated.In cultured SW480 and HCT116 cells,the effect of 100 and 200 mg/dL ethanol on oxaliplatin sensitivity,apoptosis,cell cycle distribution,and TGF-β/Smad/P21/Rb axis proteins were analyzed,and the results were further validated with pirfenidone intervention experiment.Results High-frequency drinkers had a significantly increased risk of CRC.ADH1B and ADH1C were significantly downregulated in CRC tissues,and their low expressions were associated with a higher recurrence rate and poorer overall survival in chemotherapy-treated patients.Ethanol was detected in fecal samples from both healthy individuals and CRC patients.In oxaliplatin-treated tumor-bearing mice,alcohol exposure resulted in a greater tumor volume.In SW480 and HCT116 cells,ethanol significantly increased oxaliplatin IC₅₀,reduced cell apoptosis,induced G0/G1 arrest,decreased S-phase fraction,and upregulated TGF-β1,p-Smad2,and P21 while downregulating p-Rb expression.Pirfenidone partially reversed these changes and attenuated drug resistance of the cells.Conclusion Ethanol accumulation activates the TGF-β/Smad/P21 axis to induce cell cycle arrest and promote oxaliplatin resistance in CRC cells,which can be partially reversed by pirfenidone inhibition.
谭彬;翁诺舟;曾文涛;古家宇;翁炼基;温慧琳;肖浩成;郑克鸿
南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280南方医科大学珠江医院普通外科,广东 广州 510280
乙醇结直肠癌奥沙利铂耐药TGF-β/Smad/P21吡非尼酮
ethanolcolorectal canceroxaliplatin resistanceTGF-β/Smad/P21pirfenidone
《南方医科大学学报》 2026 (5)
1066-1074,9
国家自然科学基金青年项目(82103399)广州市基础研究计划项目(2024A04J9992)Supported by Natural Science Foundation for the Youth(NSFY)of China(82103399).
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