摘要
目的探讨葡萄糖-6-磷酸脱氢酶(G6PD)在食管癌细胞中表达水平及其对细胞化疗耐药的影响和分子机制。方法选取2021年1月至2022年3月河南省人民医院收集的65例食管癌标本和对应癌旁组织作为研究对象,采用蛋白质印迹法(Western blot)分析食管癌和癌旁组织G6PD表达水平;在人食管癌细胞株EC109细胞采用浓度梯度递增法建立紫杉醇耐药细胞株EC109/DOX,分别采用对照短发卡RNA(shRNA)和G6PD shRNA慢病毒感染EC109/DOX细胞系,建立对照组和G6PD KD组;采用G6PD抑制剂处理24 h后。采用细胞计数试剂盒(CCK-8)分析不同处理的细胞增殖能力;采用流式细胞术分析不同处理细胞的耐药能力。采用Western blot分析不同处理对谷胱甘肽S-转移酶P1(GSTP1)表达水平的影响。组间比较采用t检验。结果癌旁组织中G6PD表达水平(0.97±0.16)明显低于食管癌组织中G6PD蛋白表达水平(2.34±0.28),差异有统计学意义(t=32.670,P<0.05)。紫杉醇耐药组患者G6PD蛋白表达水平(2.14±0.16)明显高于紫杉醇化疗敏感组患者G6PD蛋白表达水平(2.56±0.22),差异有统计学意义(t=8.280,P<0.05)。EC109细胞G6PD蛋白表达水平(0.95±0.07)明显低于EC109/DOX细胞G6PD蛋白表达水平(2.21±0.23),差异有统计学意义(t=11.870,P<0.05)。对照组细胞紫杉醇处理24 h和48 h后吸光度值(1.25±0.03、1.98±0.08)明显高于G6PD KD组细胞24 h和48 h吸光度值(0.87±0.02、1.42±0.07),差异有统计学意义(t=21.631、12.571,P<0.05)。对照组细胞紫杉醇处理48 h后凋亡比例[(12.94±1.78)%]明显低于G6PD KD组细胞凋亡比例[(34.56±4.03)%],差异有统计学意义(t=10.961,P<0.05)。紫杉醇处理的EC109/DOX细胞凋亡水平[(12.83±2.25)%]明显低于6-AN和紫杉醇共处理的EC109/DOX细胞凋亡水平[(43.36±4.53)%],差异有统计学意义(t=13.500,P<0.05)。紫杉醇处理对照组和G6PD KD组细胞48 h后,对照组细胞GSTP1蛋白表达水平(12.94±1.78)明显高于G6PD KD组细胞(34.56±4.03),差异有统计学意义(t=6.490,P<0.05)。紫杉醇处理的EC109/DOX细胞GSTP1蛋白表达水平(1.97±0.05)明显高于6-AN和紫杉醇共处理的EC109/DOX细胞GSTP1蛋白表达水平(1.17±0.14),差异有统计学意义(t=12.260,P<0.05)。结论葡萄糖-6-磷酸脱氢酶通过调节GSTP1蛋白表达水平,进而调控着食管癌细胞对紫杉醇耐药性。
Objective To investigate the expression level of glucose 6-phosphate dehydrogenase(G6PD)in esophageal cancer cells and its effect on chemoresistance and molecular mechanism.Methods Totally,65 esophageal cancer specimens and corresponding adjacent tissues collected in our hospital from January 2021 to March 2022 were selected as the research objects.The expression levels of G6PD in esophageal cancer and adjacent tissues were analyzed by Western blotting.Paclitaxel resistant cell line Ec109/DOX was established by concentration gradient method in human esophageal cancer cell line EC109.EC109/DOX cell lines were infected with control short hairpin RNA(shRNA)and G6PD shRNA lentivirus respectively,and the control group and G6PD KD group were established.After treatment with G6PD inhibitor for 24 h,cell counting kit-8(CCK-8)assay was used to analyze the cell proliferation ability of different treatments.The drug resistance of different treated cells was analyzed by flow cytometry.The expression of TPSP1 was analyzed by Western blotting.The measurement data between groups were compared by t-test.Results The expression level of G6PD in adjacent tissues(0.97±0.16)was significantly lower than that in esophageal cancer tissues(2.34±0.28,t=32.670,P<0.05).The expression level of G6PD protein in paclitaxel resistant group(2.14±0.16)was significantly higher than that in paclitaxel sensitive group(2.56±0.22,t=8.280,P<0.05).The expression level of G6PD protein in EC109 cells(0.95±0.07)was significantly lower than that in EC109/DOX cells(2.21±0.23,t=11.870,P<0.05).The light absorption value of cells in the control group(1.25±0.03,1.98±0.08)after 24 and 48 h of paclitaxel treatment was significantly higher than that in G6PD KD group(0.87±0.02,1.42±0.07,t=21.631,12.571,P<0.05].After 48 h of paclitaxel treatment,the apoptosis ratio[(12.94±1.78)%]in the control group was significantly lower than that in the G6PD KD group[(34.56±4.03)%,t=10.961,P<0.05].The apoptosis level of EC109/DOX cells treated with paclitaxel[(12.83±2.25)%]was significantly lower than that of EC109/DOX cells treated with 6-AN and paclitaxel[(43.36±4.53)%,t=13.500,P<0.05].After 48 h of paclitaxel treatment,the expression level of glutathione S-transferase P1(GSTP1)protein in the control group(12.94±1.78)was significantly lower than that in the G6PD KD group(34.56±4.03,t=6.490,P<0.05).The expression level of GSTP1 protein of EC109/DOX cells treated with paclitaxel(1.97±0.05)was significantly higher than that of EC109/DOX cells treated with 6-AN and paclitaxel(1.17±0.14,t=12.260,P<0.05).Conclusion G6PD regulates the resistance of esophageal cancer cells to paclitaxel by regulating the expression of GSTP1 protein.
作者
曹志坤
乔通
高飞
姚文健
Cao Zhikun;Qiao Tong;Gao Fei;Yao Wenjian(Department of Thoracic Surgery,Henan Provincial People’s Hospital,People’s Hospital of Zhengzhou University,School of Clinical Medicine,Henan University,Zhengzhou 450003,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第7期1321-1324,共4页
Chinese Journal of Experimental Surgery
基金
2022年度国家卫生健康委科学研究基金河南省医学科技攻关计划省部共建项目基金(SBGJ202102022)。
关键词
食管癌
紫杉醇
耐药
葡萄糖-6-磷酸脱氢酶
Esophageal cancer
Paclitaxel
Drug resistance
Glucose-6-phosphate dehydrogenase
