抑制DNA-PKcs对放射治疗后食管鳞癌细胞系EC109自噬蛋白表达和增殖的影响
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摘要
目的研究抑制DNA-PKcs对放射照射(X-Ray)后食管鳞癌细胞EC109自噬蛋白表达和增殖的影响。方法用NU7441抑制DNA-PKcs;放射照射处理食管鳞癌细胞EC109;实验共分为4组(对照组、NU7441组、X-Ray组、X-Ray+NU7441组)。蛋白免疫印迹(Western blot)法检测自噬蛋白Beclin-1及LC3B的表达;流式细胞术检测凋亡;四甲基偶氮唑盐(MTT)法检测细胞增殖情况。结果 p-DNA-PKcs在NU7441处理食管鳞癌细胞EC109后表达降低,X-Ray照射后表达升高。与未处理细胞(对照组)相比,X-Ray+NU7441组Beclin-1及LC3B均表达升高,与X-Ray组相比,X-Ray+NU7441组Beclin-1表达升高。与对照组相比,X-Ray组及X-Ray+NU7441组凋亡率明显增加,差异有统计学意义(P<0.05)。与X-Ray组相比,X-Ray+NU7441组细胞凋亡率明显增加。MTT结果显示,与对照组相比,X-Ray组及X-Ray+NU7441组增殖明显被抑制,差异有统计学意义(P<0.05)。结论 NU7441抑制EC109细胞DNA-PKcs蛋白表达后,可促进肿瘤细胞自噬蛋白Beclin-1和LC3B表达,促进凋亡、抑制细胞增殖。
Objective To study the effect of DNA-PKcs blocking on the expression of autophagic proteins,and proliferation of esophageal squamous cell carcinoma cells(EC109)after irradiation(X-Ray).Methods NU7441 was used to inhibit DNA-PKcs and X-Ray radiation treatment were used to treat EC109 cells.Th experiment was divided into 4 groups,including control group,NU7441 group,X-Ray group,X-Ray+NU7441 group.The expressions of autophagy protein Beclin-1 and LC3B were detected by Western blot.Apoptosis was detected by flow cytometry.MTT assay was used to detect cell proliferation.Results The expression of p-DNA-PKcs in EC109 cells was decreased after NU7441 treatment and increased after X-ray irradiation.Compared with untreated cells(control group),the expressions of both Beclin-1 and LC3 Bin X-Ray+NU7441 group were increased.Compared with the XRay group,the expression of Beclin-1 in the X-Ray+NU7441 group was increased.Compared with the control group,the apoptotic rate of EC109 cells in the X-Ray group and X-Ray+NU7441 group was significantly increased,the difference was statistically significant(P<0.05).Compared with the X-Ray group the apoptosis rate in the X-Ray-NU7441 group was significantly increased.The MTT results showed that compared with the control group,the proliferation in the X-Ray group and X-Ray+NU7441 group was significantly inhibited,the difference was statistically significant(P<0.05).Conclusion NU7441 inhibits the expression of DNA-PKcs protein in EC109 cells,which could promote the expressions of autophagy protein Beclin-1 and LC3B,promotes apoptosis and inhibits cell proliferation.
Objective To study the effect of DNA-PKcs blocking on the expression of autophagic proteins,and proliferation of esophageal squamous cell carcinoma cells(EC109)after irradiation(X-Ray).Methods NU7441 was used to inhibit DNA-PKcs and X-Ray radiation treatment were used to treat EC109 cells.Th experiment was divided into 4 groups,including control group,NU7441 group,X-Ray group,X-Ray+NU7441 group.The expressions of autophagy protein Beclin-1 and LC3B were detected by Western blot.Apoptosis was detected by flow cytometry.MTT assay was used to detect cell proliferation.Results The expression of p-DNA-PKcs in EC109 cells was decreased after NU7441 treatment and increased after X-ray irradiation.Compared with untreated cells(control group),the expressions of both Beclin-1 and LC3 Bin X-Ray+NU7441 group were increased.Compared with the XRay group,the expression of Beclin-1 in the X-Ray+NU7441 group was increased.Compared with the control group,the apoptotic rate of EC109 cells in the X-Ray group and X-Ray+NU7441 group was significantly increased,the difference was statistically significant(P<0.05).Compared with the X-Ray group the apoptosis rate in the X-Ray-NU7441 group was significantly increased.The MTT results showed that compared with the control group,the proliferation in the X-Ray group and X-Ray+NU7441 group was significantly inhibited,the difference was statistically significant(P<0.05).Conclusion NU7441 inhibits the expression of DNA-PKcs protein in EC109 cells,which could promote the expressions of autophagy protein Beclin-1 and LC3B,promotes apoptosis and inhibits cell proliferation.
引文
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[3]YANG C,WANG Q,LIU X,et al.NU7441enhances the radiosensitivity of liver cancer cells[J].Cell Physiol Biochem,2016,38(5):1897-1905.
[4]ZHAO Y,THOMAS H D,BATEY M A,et al.Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441[J].Cancer Res,2006,66(10):5354-5362.
[5]CISZEWSKI W M,TAVECCHIO M,DASTYCH J,et al.DNA-PK inhibition by NU7441sensitizes breast cancer cells to ionizing radiation and doxorubicin[J].Breast Cancer Res Treat,2014,143(1):47-55.
[6]TANG D,KANG R,LIVESEY K M,et al.Endogenous HMGB1regulates autophagy[J].J Cell Biol,2010,190(5):881-892.
[7]VALENCIA T,KIM J Y,ABU-BAKER S,et al.Metabolic reprogramming of stromal fibroblasts through p62-mTORC1signaling promotes inflammation and tumorigenesis[J].Cancer Cell,2014,26(1):121-135.
[8]KANZAWA T,GERMANO I M,KOMATA T,et al.Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells[J].Cell Death Differ,2004,11(4):448-457.
[9]ZENG X,YAN T,SCHUPP J E,et al.DNA mismatch repair initiates 6-thioguanine--induced autophagy through p53activation in human tumor cells[J].Clin Cancer Res,2007,13(4):1315-1321.
[10]BAEH,GUANJL.Suppression of autophagy by FIP200deletion impairs DNA damage repair and increases cell death upon treatments with anticancer agents[J].Mol Cancer Res,2011,9(9):1232-1241.
[11]赵晓静,马军,袁翎,等.食管鳞癌细胞系TE7放射照射后miRNAs及DNA修复相关因子的表达[J].郑州大学学报(医学版),2017,52(3):247-250.
[12]DERIANO L,GUIPAUD O,MERLE-B?RAL H,et al.Human chronic lymphocytic leukemia B cells can escape DNA damage-induced apoptosis through the nonhomologous end-joining DNA repair pathway[J].Blood,2005,105(12):4776-4783.
[13]XIN Y,JIANG F,YANG C,et al.Role of autophagy in regulating the radiosensitivity of tumor cells[J].J Cancer Res Clin Oncol,2017,143(11):2147-2157.
[14]PALUMBO S,PIRTOLI L,TINI P,et al.Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments[J].J Cell Biochem,2012,113(7):2308-2318.
[15]YU L,SHANG Z F,HSU F M,et al.NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor[J].Oncotarget,2015,6(6):3848-3860.
[16]LIANG B,KONG D,LIU Y,et al.Autophagy inhibition plays the synergetic killing roles with radiation in the multi-drug resistant SKVCR ovarian cancer cells[J].Radiat Oncol,2012,7:213.
[17]HAN M W,LEE J C,CHOI J Y,et al.Autophagy inhibition can overcome radioresistance in breast cancer cells through suppression of TAK1activation[J].Anticancer Res,2014,34(3):1449-1455.
[18]卢小迪,朱小东,赵伟,等.抑制磷酸腺苷激活的蛋白激酶活性增强人鼻咽癌细胞放射敏感性[J].重庆医学,2014(22):2900-2902.
[19]SHAHEEN F S,ZNOJEK P,FISHER A,et al.Targeting the DNA double Strand break repair machinery in prostate cancer[J].PLoS One,2011,6(5):e20311.