目的 探討葫蘆素B通過影響鐵死亡途徑提高結(jié)直腸癌細(xì)胞對(duì)5-氟尿嘧啶（5-fluorouracil，5-FU）的敏感性并研究其潛在機(jī)制。方法 構(gòu)建結(jié)直腸癌耐藥細(xì)胞株HCT116/5-FU，用低、高劑量（5、15μg/mL）的葫蘆素B分別處理HCT116和HCT116/5-FU細(xì)胞不同時(shí)間（12、24、48、72 h），細(xì)胞計(jì)數(shù)試劑盒-8（cell counting kit-8，CCK-8）實(shí)驗(yàn)檢測(cè)細(xì)胞活力。利用生物信息學(xué)數(shù)據(jù)庫探究外核苷酸焦磷酸酶/磷酸二酯酶2（ectonucleotide pyrophosphatase/phosphodiesterase 2，ENPP2）在結(jié)直腸癌中的表達(dá)情況及與預(yù)后的關(guān)系。免疫熒光檢測(cè)葫蘆素B對(duì)HCT116/5-FU細(xì)胞中ENPP2及鐵死亡相關(guān)標(biāo)志物Fe²⁺含量、脂質(zhì)過氧化水平等表達(dá)的影響。在HCT116/5-FU細(xì)胞中過表達(dá)ENPP2，檢測(cè)細(xì)胞活力及鐵死亡相關(guān)分子的表達(dá)。結(jié)果 成功構(gòu)建了HCT-116/5-FU耐藥細(xì)胞株，并發(fā)現(xiàn)葫蘆素B對(duì)該細(xì)胞株的生長(zhǎng)具有較好的抑制作用，表現(xiàn)出明顯的時(shí)間-劑量相關(guān)性，15 μg/mL葫蘆素B作用48 h抑制率顯著升高（P＜0.05）。生物信息學(xué)分析顯示，ENPP2基因在結(jié)直腸癌組織中高表達(dá)，且與患者預(yù)后不良密切相關(guān)。葫蘆素B處理可升高HCT-116/5-FU細(xì)胞內(nèi)的Fe²⁺含量及脂質(zhì)過氧化水平（P＜0.05），并影響鐵死亡標(biāo)志分子谷胱甘肽過氧化物酶4（glutathione peroxidase 4，GPX4）、環(huán)氧化酶2（cyclooxygenase 2，COX2）的表達(dá)（P＜0.01）。ENPP2過表達(dá)可降低葫蘆素B對(duì)Fe²⁺含量、脂質(zhì)過氧化和鐵死亡的上調(diào)作用（P＜0.05、0.01）。結(jié)論 葫蘆素B通過抑制ENPP2的表達(dá)影響鐵死亡提高結(jié)直腸癌細(xì)胞耐藥的敏感性。

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, 15 μg/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 Fe²⁺ 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 15 μg/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 Fe²⁺ 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.

R285.5

河南省科技攻關(guān)項(xiàng)目（242102310142）；河南省中醫(yī)藥科學(xué)研究專項(xiàng)課題（2023ZY2084）；河南省中醫(yī)藥拔尖人才培養(yǎng)項(xiàng)目（2022ZYBJ11）