Abstract:

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.

Key words: Colorectal cancer, Dihydropyrimidine dehydrogenase, Autophagy, Light chain 3, P62