Mechanism Of The Chloroquine Regulating Autophagy In Esophageal Cancer Cell Line EC109

Esophageal cancer is one of the most invasive human malignant tumors,which constitutes a major health burden around the world.Esophageal squamous cell carcinoma(ESCC)is the main histological subtype of esophageal cancer,and the 5-year survival rate is less than 20%.Surgery is the main treatment for early esophageal cancer,but 50%of patients with esophageal cancer have lost the opportunity of operation for the middle and advanced stage when they are diagnosed,so they can only use non-operative treatment.At present,the benefit of molecular targeted therapy for esophageal cancer is not clear,advanced patients are usually treated by radiotherapy alone or combined with radiotherapy and chemotherapy,in which tolerance often leads to chemotherapy failure,the prognosis is often very poor.The death factors of ESCC patients mainly include late diagnosis,frequent metastasis and chemotherapy resistance.In view of this,while emphasizing the early screening of esophageal cancer,how to effectively increase the sensitivity of chemotherapeutic drugs and inhibit the drug resistance of tumor cells is a major clinical challenge to be solved.Many studies support autophagy as a tumor promoting factor in ESCC.Autophagy inhibition enhances radiation-induced apoptosis and cell cycle arrest in vitro,and supports the protective effect of autophagy.Various studies related to ESCC models in vitro and in vivo also support autophagy inhibition to enhance the efficacy of anticancer therapy and improve the therapeutic response of ESCC patients.As an autophagy inhibitor,studying the effects of chloroquine on autophagy-related proteins in esophageal cancer cells may provide new ideas for anti-tumor research on improving tumor drug resistance and promoting cancer cell apoptosis through targeted regulation of autophagy.Objective:In order to observe the effect of Chloroquine on autophagy flow in EC 109 cell line and to explore the mechanism of Chloroquine inhibiting the binding of autophagosomes and lysosome to lay a laboratory foundation for clinical application of Chloroquine in anti-tumor therapy.Methods:1.GFP-mRFP-LC3 double fluorescent expression EC 109 cells line was constructed.The cells treated with different concentrations(50μmol/L、100μmol/L、200μmol/L)of chloroquine were collected,and the formation of autophagosomes and autophagy lysosomes were observed by confocal microscope.2.EC 109 cells treated with chloroquine were found to have changed the expression of 866 proteins,including 679 up-regulated proteins and 187 down-regulated proteins.Visualization analysis of KEGG metabolic pathway suggests that LAPTM family(including LAPTM4A,LAPTM4B and LAPTM5)and LITAF are closely related to autophagy.3.The cells were treated with different concentrations(50μmol/L、100μmol/L、200μmol/L)of chloroquine,and the total RNA extracted from the cells was collected and reverse transcribed into c-DNA.The changes of mRNA levels of LAPTM(including LAPTM4A,LAPTM4B and LAPTM5)and LITAF were detected by q-PCR,and the total proteins were collected,and the changes of LAPTM(including LAPTM4A,LAPTM4B and LAPTM5)and LITAF protein levels were detected by Western blot.Results:1.The aggregation of autophagic lysosomes in EC 109 cells was significantly reduced after 200μmol/L chloroquine treatment,and the reduction of autophagic flow was more obvious under high concentration than under low concentration.2.In EC 109 cells treated with chloroquine,it was found that the expression of 866 proteins changed,of which 679 proteins were up-regulated and 187 proteins were down-regulated.Visual analysis of KEGG metabolic pathway suggests that the LAPTM family(including LAPTM4A,LAPTM4B and LAPTM5)and LITAF are closely related to autophagy and down-regulated.3.The results of q-PCR and Western Blot showed that the mRNA and protein levels of LAPTM family and LITAF were significantly down-regulated when chloroquine concentration reached a 200μmol/L level,and the expression level of LAPTM5 protein decreased with the increase of CQ dose.Conclusion:Chloroquine can inhibit autophagy by affecting the formation of autophagic lysosomes in EC 109 cells during autophagy.The molecular mechanism may be that chloroquine down-regulates lysosomal associated transmembrane protein(LAPTM)and lipopolysaccharide induced tumor necrosis factor(LITAF),thus blocking the fusion of autophagosomes and lysosomes into autophagolysosomes.This provides a new idea for us to further study how chloroquine affects the mechanism of autophagy flow in tumor cells and to use LAPTM family and LITAF as autophagy regulatory targets.