葫芦素B通过ENPP2影响铁死亡在结直肠癌耐药中的机制研究  

Study on mechanism of cucurbitacin B affecting ferroptosis via ENPP2 in colorectal cancer drug resistance

作　　者：王凯 高宗跃 左振魁 杨娅娟 周琼阁 WANG Kai;GAO Zongyue;ZUO Zhenkui;YANG Yajuan;ZHOU Qiongge(Department of Anorectal,Henan Province Hospital of Traditional Chinese Medicine(The Second Affiliated Hospital of Henan University of Chinese Medicine),Zhengzhou 450003,China)

机构地区：[1]河南省中医院(河南中医药大学第二附属医院)肛肠科,河南郑州450003

出　　处：《中草药》2025年第6期2009-2016,共8页Chinese Traditional and Herbal Drugs

基　　金：河南省科技攻关项目(242102310142);河南省中医药科学研究专项课题(2023ZY2084);河南省中医药拔尖人才培养项目(2022ZYBJ11)。

摘　　要：目的探讨葫芦素B通过影响铁死亡途径提高结直肠癌细胞对5-氟尿嘧啶(5-fluorouracil,5-FU)的敏感性并研究其潜在机制。方法构建结直肠癌耐药细胞株HCT116/5-FU,用低、高剂量(5、15μg/mL)的葫芦素B分别处理HCT116和HCT116/5-FU细胞不同时间(12、24、48、72 h),细胞计数试剂盒-8(cell counting kit-8,CCK-8)实验检测细胞活力。利用生物信息学数据库探究外核苷酸焦磷酸酶/磷酸二酯酶2(ectonucleotide pyrophosphatase/phosphodiesterase 2,ENPP2)在结直肠癌中的表达情况及与预后的关系。免疫荧光检测葫芦素B对HCT116/5-FU细胞中ENPP2及铁死亡相关标志物Fe^(2+)含量、脂质过氧化水平等表达的影响。在HCT116/5-FU细胞中过表达ENPP2,检测细胞活力及铁死亡相关分子的表达。结果成功构建了HCT-116/5-FU耐药细胞株,并发现葫芦素B对该细胞株的生长具有较好的抑制作用,表现出明显的时间-剂量相关性,15μg/mL葫芦素B作用48 h抑制率显著升高(P<0.05)。生物信息学分析显示,ENPP2基因在结直肠癌组织中高表达,且与患者预后不良密切相关。葫芦素B处理可升高HCT-116/5-FU细胞内的Fe^(2+)含量及脂质过氧化水平(P<0.05),并影响铁死亡标志分子谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、环氧化酶2(cyclooxygenase 2,COX2)的表达(P<0.01)。ENPP2过表达可降低葫芦素B对Fe^(2+)含量、脂质过氧化和铁死亡的上调作用(P<0.05、0.01)。结论葫芦素B通过抑制ENPP2的表达影响铁死亡提高结直肠癌细胞耐药的敏感性。Objective To investigate whether cucurbitacin B(CuB)enhances the sensitivity of colorectal cancer cells to 5-fluorouracil(5-FU)by influencing the ferroptosis pathway and explore its potential mechanisms.Methods A colorectal cancer drug-resistant cell line HCT116/5-FU was established.HCT116 and HCT116/5-FU cells were treated with different concentrations of CuB(5,15μg/mL)for various durations(12,24,48,72 h)and cell viability was assessed using the cell counting kit-8(CCK-8)assay.Public bioinformatics databases were utilized to investigate the expression of ectonucleotide pyrophosphatase/phosphodiesterase(ENPP2)in colorectal cancer and its correlation with prognosis.Immunofluorescence techniques were employed to detect the effects of CuB on the ENPP2 expression and ferroptosis-related markers Fe^(2+)concentration and lipid peroxidation levels in HCT116/5-FU cells.ENPP2 was overexpressed in HCT116/5-FU cells,and then cell viability and the expression of ferroptosis-related molecules were evaluated.Results The HCT116/5-FU drug-resistant cell line was successfully established,and CuB was found to significantly inhibit the growth of this cell line with a clear time-dose dependency(P<0.05).Notably,the inhibition rate of CuB significantly increased at a concentration of 15μg/mL for 48h(P<0.05).Bioinformatics analysis revealed that ENPP2 gene was highly expressed in colorectal cancer tissues and closely associated with poor patient prognosis.The Fe^(2+)concentration and lipid peroxidation levels in HCT116/5-FU cells were increased by CuB treatment(P<0.05),with the expression of ferroptosis-related markers glutathione peroxidase 4(GPX4)and cyclooxygenase 2(COX2)was influenced(P<0.01).Furthermore,overexpression of ENPP2 significantly reduced the up-regulation effects of CuB on intracellular Fe2+concentration,lipid peroxidation levels and ferroptosis(P<0.05,0.01).Conclusion CuB enhances the sensitivity of colorectal cancer cells to drug resistance by influencing ferroptosis through the inhibition of ENPP2 expression.

关 键 词：结直肠癌 葫芦素B 耐药 ENPP2 铁死亡 

分 类 号：R285.5[医药卫生—中药学]