| Objective:Esophageal cancer is the sixth most common cause of cancer deaths worldwide and has a high incidence in Fujian Province.Chemotherapy is one of the main way for treating esophageal cancer.However,drug resistance often occurs in the later stage of the treatment.It has shown that autophagy plays an important role in tumor drug resistance.In this study,firstly,the m RNA expression of autophagy regulating protein UNC-51-like kinase 1(ULK1)in esophageal cancer was analyzed by RT-q PCR.Then,the over-expression or knockdown cell model was established and the proliferation of these cells were observed and compared.Finally,two ULK1 inhibitors,MRT68921 and SBI-0206965,were used to treat human esophageal cancer cells solely or combined with cisplatin.The proliferation of treated esophageal cancer cells were analyzed to test our hypothesis: ULK1 inhibitor could enhance the cytotoxicity of cisplatin on human esophageal cancer cells.Methods:1.The expression data of ULK1 gene in esophageal cancer tissues were downloaded from Oncomine,a database of tumor-related gene expression.10 pairs of human esophageal cancer tissues and normal esophageal tissues were collected.Total RNA was extracted,and RNA expression levels of autophagy related genes in human esophageal cancer tissues were detected by reverse transcription quantitative real-time PCR(RT-q PCR).2.(1)The ULK1 gene was overexpressed/knocked down in esophageal cancer cells,ECA-109,and the levels of ULK1 and phosphorylated ULK1(p-ULK1)after transfection were detected by Western Blot;(2)By the colony formation assay,the effect of ULK1 overexpress/knock-down on the colony formation ability of esophageal cancer cells was observed in the overexpressed/knocked down model of ULK1 of human esophageal cancer cells.3.(1)The effect of non-selective ULK1 inhibitor,MRT68921,and selective ULK1 inhibitor,SBI-0206965 on the proliferation ability of human esophageal cancer cell line ECA109 cells was evaluated by CCK-8;(2)The human esophageal cancer cells were treated with SBI-0206965 and rapamycin.Cell proteins were collected to detect the expression levels of autophagy-related proteins LC3,P62 and p-ATG14;(3)Cytotoxicity test and cell growth curve mapping were applied to observe the ability of SBI-0206965 on the enhancement of the toxicity of cisplatin on human esophageal cancer cells;(4)Western Blot was used to detect the level of cell proliferation protein Cyclin D1,and the mechanism of SBI-0206965 to enhance the toxicity of cisplatin on human esophageal carcinoma cells was preliminarily explored.Results:1.The analysis of tumor database,Oncomine showed that ULK1 was highly expressed in esophageal squamous cell carcinoma.The results showed that the expression level of ULK1 and ATG14 m RNA in esophageal cancer tissues were higher than that in normal esophageal tissues,while the expression levels of p62 was lower than that in normal tissues,with statistically significant differences(p<0.01).2.(1)Through transfection test,ULK1 protein was successfully overexpressed and knocked down in human esophageal cancer cells;(2)The results of the colony formation assay showed that the cell clone formation ability of the knock-down ULK1 group was significantly decreased compared with the control group and the blank group.3.(1)Both inhibitors of ULK1 can inhibit the proliferation of human esophageal cancer cells in a time-dependent and concentration-dependent manner;(2)SBI-0206965 blocks the phosphorylation of ATG14 further and reduces the expression level of p-ATG14 by blocking the formation of ULK1 complex,thus reducing the formation of autophagosome to inhibit autophagy;(3)Low concentration SBI-0206965 combined with cisplatin can enhance the toxicity of cisplatin to human esophageal cancer cells;(4)SBI-0206965 combined with cisplatin can enhance the toxicity of cisplatin on human esophageal cancer cells by reducing the expression level of Cyclin D1.Conclusion:1.The m RNA expression of ULK1 are up-regulated in human esophageal cancer tissues;2.The expression of knocking down of ULK1 in esophageal cancer cells can reduce the proliferation ability of human esophageal cancer cells;3.The ULK1 inhibitor is able to inhibit the proliferation of human esophageal cancer cells in a time-dependent and concentration-dependent manner;4.The ULK1 inhibitor combined with cisplatin can further inhibit autophagy by inhibiting the activity of ULK1 kinase,thus enhancing the toxicity of the cisplatin to human esophageal cancer cells. |
