Mechanism Of 5-FU Mediated Chemotherapeutic Insensitivity Mediated By Autophagy In Cholangiocarcinoma

Cholangiocarcinoma(CCA)is a malignant tumor that originate d in the hepatobiliary system.Its histological type is more common in a denocarcinoma.The anatomical site of the intersection of the cystic duct and the common hepatic duct divides and the malignant tumor originatin g from the epidermis of the intrahepatic biliary branch bifurcation above the intersection is called Intrahepatic cholangiocarcinoma(ICCA);it occur s below the point of intersection.The malignant tumor in the common b ile duct epithelial cells is called extrahepatic bile duct cancer(ECCA),th e latter including ampulla cancer.And between the two,the tumor that o ccurs in the common hepatic duct is also called hilar cholangiocarcinoma (HCC).The pathogenesis of bile duct cancer is occult,with slow onset,long duration,such as jaundice,abdominal discomfort or pain,anorexia, weight loss and other clinical symptoms are not typical,not prominent,easily with other system diseases,such as chronic cholecystitis,chronic st omach ten Duodenal inflammation,pleurisy,large blood vessels,or spinal disease are confused and difficult to identify and value.However,chola ngiocarcinoma itself has a high degree of malignancy and a low degree of differentiation.It is easy to invade the blood vessels or lymphatic ves sels to cause local or distant metastasis in the early stage,or infiltrate a nd diffuse along the anatomical pathways of the bile duct,involving the surrounding normal tissue cells,resulting in the final diagnosis to the mi ddle and late stages.Or the basic chance of complete surgical resection of the surgery is lost.Therefore,comprehensive supportive care for intern al medicine is the first choice,especially for cytotoxic chemotherapy.Ma ny previous studies have confirmed that chemotherapy is the main treatm ent method for inoperable cholangiocarcinoma,especially gemcitabine + cisplatin chemotherapy as the first choice,except for his use of single or five-FU alone platinum or platinum-based drugs,5-FU + platinum Or co mprehensive supportive therapy including these drugs.However,currently available epidemiological data show that the 5-year survival rate for clin ical treatment of advanced bile duct cancer is still low.Even if there are indications for surgery,more than half of patients still have higher mort ality after surgery.This indicates that palliative care The poor therapeutic effect of sexual therapy may be related to the insensitivity and drug res istance of tumors to chemotherapeutic drugs.At the same time,the disco mfort caused by chemotherapy also significantly affects the daily life of patients.Some people think that the autophagic behavior of cancer cells i s one of the causes of the above-mentioned consequences.OBJECTIVE: To study the effect of fluorouracil on the proliferation of cholangiocytes (HCCC-9810),observe the changes in cell proliferation and autophagy,a nd block autophagy,further observe the relationship between autophagy a nd cell proliferation,and thus preliminary study of cholangiocarcinoma.T he mechanism of chemotherapy insensitivity.METHODS AND RESULT S: Firstly,different concentrations of fluorouracil(50-200ug/ml)were use d to intervene the CCA cell line HCCC-9810 for 24 h.CCK-8 microplate reader was used to detect the proliferation of cancer cells.The concentr ation of chemotherapeutic drugs(150ug/ml)was the best inhibitory conce ntration.At the same time,the cell morphological changes were observed under a microscope.The results showed that as the concentration of che motherapeutic drugs increased,the proliferation of cells was inhibited gra dually,and the concentration of 0-150ug/ml fluorouracil was gradually inc reased.The linear relationship in the range,but when the maximum conc entration of 200ug/ml is reached,the proliferation of cells is inhibited bu t slightly decreased.The results show that the cholangiocarcinoma cells d o have chemotherapy-insensitive phenomenon.In this study,Western-blot method was used to detect the expression of autophagy marker Beclin-1. The results showed that Beclin-1 levels gradually increased with the inc reasing concentration of chemotherapeutic drugs in a dose-dependent man ner,indicating that chemotherapy can induce autophagy pathway activatio n in cancer cells.,and the performance is positively related.After autop hagy was blocked by 3-MA,an autophagy inhibitor,the above method w as used again to detect cell proliferation and autophagy.Finally,using re al-time fluorescence quantitative PCR to detect the m RNA level of the A MP-dependent protein kinase(AMPK)-activated protein kinase(AMPK)g ene,it was found that m RNA expression of AMPK was positively correl ated with the concentration of chemotherapeutic drugs,and Beclin-1The protein expression was similar,AMPK gene was up-regulated.After the a utophagy inhibitor 3-MA was added to inhibit the autophagic behavior of cancer cells,the AMPK gene was significantly down-regulated and was lower than that of the control group.CONCLUSION: Fluorouracil can i nhibit the proliferation of CCA cell line(HCCC-9810)and induce autoph agy;autophagy activation may be one of the mechanisms of HCCC-9810 cell chemotherapy insensitive and avoiding adverse environment,and thi s process may be related to the upregulation of AMPK gene.