ACSS2 Contributes To The Formation Of Radioresistance In Esophageal Squamous Carcinoma Cells Under Nutrient Stress Condition

【 Objective 】 To investigate the effect of Acyl-coenzyme A synthetase short-chain family member 2(ACSS2)in the of radiotherapy sensitivity of human esophageal squamous cell carcinoma(ESCC),and explored its potential mechanism of radiotherapy sensitivity in esophageal cancer.【Methods】(1)The expression of ACSS2 in esophageal cancer tissues and esophageal cancer cell lines TE-1,TE-10,ECA-109,KYSE-150 cells and normal esophageal epithelial cell line HET-1A cells under normal condition or nutrient stress treatment were detected by Western blotting;(2)Real time PCR was used to detect the expression of ACSS2 m RNA in esophageal cancer cells treated with different time(0 h,24 h,48 h and 7 day)of nutrient stress treatment.Western blotting was used to detect the expression of ACSS2 in different time(0 day,1day,2 day,7 day,30 day).The experimental groups were divided into control group,nutrient stress group,si-ACSS2 group,nutrient stress plus si-ACSS2 group.The expression of Ki67 in different groups were detected by Western blotting;(3)The experimental groups were divided into control group,nutrient stress group,si-ACSS2 group,nutrient stress plus si-ACSS2 group and flow cytometry was used to detect the cell cycle distribution of different treatment groups.The experimental groups were divided into control group,nutrient stress group,si-ACSS2 group,nutrient stress plus si-ACSS2 group The expression of Akt,p-Akt,m TOR and p-m TOR,in different treatment groups were detected by Western blotting.The experimental group was divided into control group,chloroquine group,starvation group,starvation group,Western blotting was used to detect the expression of LC3B-II,ATG5 and ATG7 in different treatment groups;(4)The experimental groups were divided into control group,nutrient stress group,si-ACSS2 group,nutrient stress plus si-ACSS2 group.After irradiation,flow cytometry was used to detect the apoptotic rate of different treatment groups.TheIV expression of caspase-3,Bcl-2 and Bax in different groups were detected by Western blotting;the survival fraction of four groups at different doses of 2 Gy,4 Gy,6Gy and8 Gy were detected by Cell clonal proliferation assay.(5)In esophageal squamous cell carcinoma tissue sections,Immunohistochemical staining was used to detect the expression and correlation of ACSS2 and p-m TOR in cancerous tissues and adjacent tissues of 60 esophageal carcinoma patients and the relationship between the expression of ACSS2 and clinical factors.【Results】(1)The expression of ACSS2 were upregulated after starvation treatment.The expression of ACSS2 existed in four human esophageal squamous cells under normal nutritional conditions but hardly existed in normal esophageal cells HET-1A.The expression of m RNA of esophageal squamous carcinoma cells were significantly increased under nutrient stress treatment and kept a high level.(2)The change of ACSS2 expression had no effect on the cell proliferation and cell cycle of esophageal squamous carcinoma cells.CCK-8 cell proliferation assay showed no significant difference in cell proliferation between different groups at the same time.Flow cytometry was used to detect the cell cycle distribution in different treatment groups,there were not statistical differences.Western blotting was used to detect the expression of Ki-67,and there were no difference in expression in different treatment groups.(3)Down-regulation of the expression of ACSS2 after nutrient starvation treatment promoted the activation of Akt / m TOR signaling pathway and inhibitd the formation of autophagy.The expressions of p-Akt,p-m TOR,LC3B-II,ATG5 and ATG7 in the nutrient starvation group were significantly upregulated compared with the control group,and the up-regulation of the above-mentioned protein was inhibited after down-regulation of ACSS2.(4)Up-regulation of the expression of ACSS2 inhibited apoptosis.Up-regulation of the expression of ACSS2 induced by nutrient stress promoted the up-regulation of Bcl-2 expression in esophageal squamous cells and inhibited theexpression of cleaved caspase-3,resulting in decrease of apoptosis rate after irradiation.In the control group,the expression of anti-apoptotic protein Bcl-2 was up-regulated and the expression of cleaved caspase-3 was down-regulated after irradiation,down-regulation of ACSS2 reversed the trend.Flow cytometry showed that down-regulation of the expression of ACSS2 resulted in up-regulation of early apoptotic rates under nutrient staravation condition.The assay showed that ACSS2 accumulation under nutritional deficiencies promoted the formation of radioresistance,which down-regulation of ACSS2 could reverse this trend.(5)In 60 cases of esophageal carcinoma,ACSS2 was highly expressed in histopathological sections of esophageal squamous cell carcinoma,especially in cell-dense areas.The expression of ACSS2 in tumor tissues of patients with esophageal cancer was higher than that in adjacent tissues(p<0.01).The expression intensity of ACSS2 was independent of the age,sex,tumor location,tumor size and TMN staging of patients,whereas the area of ACSS2 expression was significantly correlated to five-year survival rate and lymph node metastasis(p <0.05).【Conclusions】Up-regulation of ACSS2 induced by nutrient stress promoted the formation of autophagy in esophageal cancer cells via Akt/m TOR pathway,leading to the generation of radiation resistance.This suggested that ACSS2 played an important role in radioresistance of esophageal cancer through regulating autophagy capacity.