The Effect And Mechanism Of Camptothecin Inhibiting Neddylation To Activate The Protective Autophagy In Esophageal Cancer Cells

Background: Esophageal cancer is the eighth most common cancer in the world,with an increase of 604,100 cases and 544,000 deaths every year,seriously endangering human health and life safety.Despite continuous advances in diagnosis and treatment,the prognosis for esophageal cancer remains poor,with a 5-year survival rate of about15-25%.The treatment esophageal cancer is still confined to surgery,endoscopic resection,radiation and chemotherapy.However,most patients are already at an advanced stage when they are diagnosed.They have lost the opportunity for surgery and require large doses of radiotherapy and chemotherapy,which will have great toxic side effects on patients.Therefore,there is an urgent need to better clarify the molecular mechanism of esophageal cancer and develop new molecular targeted anti-esophageal cancer treatment strategies.Camptothecin(CPT)is an anti-tumor active ingredient extracted from the Asian tree Camptotheca acuminate in my country.Studies have revealed that CPT is a better anti-tumor drug.Protein Neddylation modification is overactivated in tumor tissues,and blocking this pathway has a significant anti-tumor effect,suggesting that Neddylation modification is an important anti-tumor molecular target.Objectives:Studies have shown that Neddylation modification is over-activated in many tumors,and inhibiting Neddylation modification can induce tumor cell apoptosis and autophagy.CPT can induce tumor cell apoptosis and autophagy.However,whether CPT affects the Nedylation modification and the molecular mechanism by which CPT induces autophagy remains unclear in esophageal cancer cells.Design: Evaluate the anti-tumour effect of CPT on esophageal cancer cells in vitro and in vivo;Elucidate that CPT induces autophagy in esophageal cancer cells;Clarify the effect of CPT on Neddylation modification;Explore that CPT promotes protective autophagy of esophageal cancer cells depends on ROS/AMPK/mTOR/ ULK1 pathway.Materials and Methods:1.ATPLite and clone formation assay were used to evaluate the effect of CPT on cell proliferation in esophageal cancer cell lines EC1 and EC109;2.Annexin V-FITC/PI double staining and Western Blot were used to detect the apoptosis of esophageal cancer cells induced by CPT;3.The production of ROS after CPT treatment was determined by H2-DCFDA staining;4.The expression of NF-κB/AMPK/mTOR/ULK1 pathway related proteins was confirmed by Western Blot analysis after inhibition of Neddylation modification,and these proteins were downregulated by si RNA silencing to determine their recovery;5.Use EC109 cells to construct a mouse subcutaneous xenograft model to evaluate the anti-esophageal cancer effect of CPT in vivo.Results: We found that CPT induced apoptosis and autophagy,and CPT-induced autophagy played a protective role in esophageal cancer cells.Mechanically,CPT Induced ROS generation to promote autophagy via AMPK/mTOR/ULK1 pathway.Recovery experiments showed that inhibiting the generation of ROS blocked the autophagy induced by AMPK/mTOR/ULK1 pathway.In addition,blocking the AMPK pathway significantly enhanced CPT-induced apoptosis and proliferation inhibition.Further studies have shown that CPT induces p-κBα accumulation by inhibiting Neddylation,Thereby inhibiting the NF-κB pathway and activating ROS-mediated autophagy.In vivo experiments further confirmed that CPT induces autophagy in esophageal cancer cells by inhibiting Nedylation modification.Conclusions: In our study,we elucidate a novel mechanism of NF-κB/AMPK/mTOR/ULK1 pathway in CPT-induced protective autophagy in esophageal cancer cells,which provides a sound rationale for combinational anti-ESCC therapy with CPT and inhibition AMPK/mTOR/ULK1 pathway.