干预自噬影响DPD的表达促进5-FU抗结直肠癌疗效的实验研究

Experimental study on influence of autophagy on DPD expression and its effect on chemotherapy with 5-FU in colorectal cancer

背景与目的:结直肠癌作为常见的消化系统恶性肿瘤之一,其化疗与耐药一直以来备受关注,5-氟尿嘧啶(5-fluorouracil,5-FU)是结直肠癌的一线化疗药物,其疗效常因耐药或不良反应而受到影响。二氢嘧啶脱氢酶(dihydropyrimidine dehydrogenase,DPD)是5-FU代谢的关键限速酶,其表达或降解可能成为影响5-FU疗效的因素。自噬是细胞内蛋白质代谢的重要途径,其在化疗诱导的细胞死亡或增殖抑制中的作用仍存在争议。本研究旨在探讨自噬在结直肠癌化疗过程中的作用以及干预自噬影响5-FU耐药的机制。方法:体外细胞培养人结肠癌HCT-8、COLO205、LOVO和SW480细胞系,观察不同细胞系中DPD的表达对5-FU敏感性的影响,通过药物敏感性实验筛选出5-FU敏感细胞,检测5-FU敏感细胞株及其相对应的耐药细胞株中DPD的表达、自噬水平及自噬关键因子微管相关蛋白轻链3(light chain 3,LC3)、P62的表达,并通过诱导/抑制细胞自噬,观察调控自噬状态对DPD的表达变化以及肿瘤细胞生物学行为和化疗抵抗能力的影响,通过UbiBrowser数据库筛选及免疫共沉淀实验(co-inmunoprecipitation,Co-IP)实验验证DPD降解过程中的E3连接酶,探讨自噬降解DPD逆转5-FU耐药的分子机制。结果:DPD低表达的细胞系对5-FU的敏感性更强,其中COLO205细胞系在4种细胞中DPD表达量最高,并对5-FU的耐药性最强,而HCT-8细胞DPD表达最低并对5-FU最为敏感。与HCT-8细胞相比,HCT-8/FU耐药细胞表达较高水平的DPD,以及较低的基础自噬水平;雷帕霉素(rapamycin,RAPA)介导的自噬激活增强了细胞的自噬水平,降低了DPD表达,同时降低了细胞增殖、侵袭和5-FU耐药性;3甲基腺嘌呤(3-methyladenine,3-MA)及羟氯喹(hydroxychloroquine,HCQ)介导的自噬抑制减弱了细胞的自噬水平,增加了DPD表达,增强了5-FU耐药性。5-FU与自噬激活剂联合应用对细胞的抑效果要远远强于单用5-FU及与自噬抑制剂联合应用的效果;DPD的降解需要完整的自噬流的参与,其中自噬溶酶体的形成是DPD降解的关键步骤之一,而单一的自噬体无法对DPD进行降解。E3连接酶NEDD4在HCT-8/5-FU细胞中与DPD和P62蛋白免疫共沉淀,在DPD的自噬降解中发挥作用。结论:自噬参与DPD降解影响结直肠癌细胞对化疗药物的敏感性,激活自噬可促进DPD降解和抑制5-FU的分解代谢,可能成为逆转结直肠癌5-FU耐药的新途径。

Background and purpose:Colorectal cancer is one of the most common digestive system malignancies,and chemotherapy for colorectal cancer and drug resistance have always been concerns.5-fluorouracil(5-FU)is the first-line chemotherapy drug for colorectal cancer,and its efficacy is often affected by drug resistance or adverse reactions.Dihydropyrimidine dehydrogenase(DPD)is a key rate-limiting enzyme in 5-FU metabolism,and its expression or degradation may be a factor affecting the efficacy of 5-FU.Autophagy is an important pathway of intracellular protein metabolism,and its role in chemotherapy-induced cell death or proliferation inhibition is still controversial.This study aimed to investigate the role of autophagy in colorectal cancer chemotherapy and the mechanism by which autophagy affects 5-FU drug resistance.Methods:Human colon cancer HCT-8,COLO205,LOVO and SW480 cell lines were cultured in vitro to observe the effect of DPD expression on 5-FU sensitivity.5-FU sensitive cells were screened by drug sensitivity test.The expression of DPD,the level of autophagy and the expressions of microtubule-associated protein light chain 3(LC3)and P62 in 5-FU-sensitive cell lines and their corresponding drug-resistant cell lines were detected.The effect of autophagy on the expression of DPD and the biological behavior and chemotherapy resistance of tumor cells were observed.UbiBrowser database screening and co-inmunoprecipitationn(Co-IP)experiment were used to verify the E3 ligase in the process of DPD degradation,and to investigate the molecular mechanism of reversing 5-FU resistance caused by autophagy degradation of DPD.Results:The COLO205 cell line had the highest DPD expression and was the most resistant to 5-FU,while the HCT-8 cell line had the lowest DPD expression and was the most sensitive to 5-FU.Compared with HCT-8/5-FU cells,HCT-8/FU-resistant cells expressed higher levels of DPD and lower levels of basal autophagy.Rapamycin(RAPA)-mediated autophagy activation enhanced the autophagy level,decreased the expression of DPD,and decreased cell proliferation,invasion and 5-FU resistance.The inhibition of autophagy mediated by 3-Methyladenine(3-MA)and hydroxychloroquine(HCQ)attenuated the autophagy level,increased the expression of DPD,and enhanced the drug resistance of 5-FU.The inhibitory effect of 5-FU combined with autophagy activator was much stronger compared with 5-FU alone or combined with autophagy inhibitor.The degradation of DPD required the participation of complete autophagic flow,among which the formation of autophagic lysosomes was one of the key steps of DPD degradation,however a single autophagosome could not degrade DPD.E3 ligase NEDD4 co-immunoprecipitated with DPD and P62 proteins in HCT-8/5-FU cells and played a role in autophagic degradation of DPD.Conclusion:The involvement of autophagy in DPD degradation affects the sensitivity of colorectal cancer cells to chemotherapy drugs.Activation of autophagy can promote the degradation of DPD and inhibit the catabolism of 5-FU,which may be a new way to reverse 5-FU resistance in colorectal cancer.