Yan Lin-Hai, Wei Wei-Yuan, Cao Wen-Long, Zhang Xiao-Shi, Xie Yu-Bo, Xiao Qiang
Department of Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, China.
BMC Cancer. 2014 Dec 3;14:904. doi: 10.1186/1471-2407-14-904.
Routine chemotherapy often cannot achieve good therapeutic effects because of multidrug resistance (MDR). MDR is frequently caused by the elevated expression of the MDR1 gene encoding P-glycoprotein (P-gp). E2F1 is a frequently overexpressed protein in human tumor cells that increases the activity of the MDR1 promoter, resulting in higher P-gp levels. The upregulation of P-gp might contribute to the survival of tumor cells during chemotherapy. E2F1 confers anticancer drug resistance; however, we speculate whether E2F1 affects MDR through other pathways. This study investigated the possible involvement of E2F1 in anticancer drug resistance of gastric carcinoma in vitro and in vivo.
A cisplatin-resistant SGC7901/DDP gastric cancer cell line with stable overexpression of E2F1 was established. Protein expression levels of E2F1, MDR1, MRP, TAp73, GAX, ZEB1, and ZEB2 were detected by western blotting. The influence of overexpression of E2F1 on anticancer drug resistance was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, as well as the rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. We determined the in vivo effects of E2F1-overexpression on tumor size in nude mice, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining.
The SGC7901/DDP gastric cancer cell line stably overexpressing E2F1 exhibited significantly inhibited sensitivity to cisplatin, doxorubicin, and 5-fluorouracil. Flow cytometry confirmed that the percentage of apoptotic cells decreased after E2F1 upregulation, and that upregulation of E2F1 potentiated S phase arrest of the cell cycle. Furthermore, upregulation of E2F1 significantly decreased intracellular accumulation of doxorubicin. Western blot revealed that E2F1 upregulation suppressed expression of GAX, and increased the expression of MDR1, MRP, ZEB1, TAp73, and ZEB2.
Overexpression of E2F1 promotes the development of MDR in gastric carcinoma, suggesting that E2F1 may represent an efficacious target for gastric cancer therapy.
由于多药耐药性（MDR），常规化疗往往无法取得良好的治疗效果。MDR通常是由编码P-糖蛋白（P-gp）的MDR1基因表达升高引起的。E2F1是人类肿瘤细胞中经常过度表达的一种蛋白质，它会增加MDR1启动子的活性，导致P-gp水平升高。P-gp的上调可能有助于肿瘤细胞在化疗期间存活。E2F1赋予抗癌药物抗性；然而，我们推测E2F1是否通过其他途径影响MDR。本研究调查了E2F1在体外和体内对胃癌抗癌药物抗性的可能影响。
建立了稳定过表达E2F1的顺铂耐药SGC7901/DDP胃癌细胞系。通过蛋白质印迹法检测E2F1、MDR1、多药耐药相关蛋白（MRP）、TAp73、GAX、锌指E盒结合蛋白1（ZEB1）和锌指E盒结合蛋白2（ZEB2）的蛋白表达水平。通过测量SGC7901/DDP细胞对顺铂、阿霉素和5-氟尿嘧啶的半数抑制浓度（IC50），以及通过流式细胞术检测阿霉素外排率、细胞凋亡和细胞周期进程，评估E2F1过表达对抗癌药物抗性的影响。我们确定了E2F1过表达对裸鼠肿瘤大小的体内影响，并通过脱氧核苷酸末端转移酶介导的dUTP-生物素缺口末端标记法以及苏木精和伊红染色检测肿瘤组织中的凋亡细胞。
稳定过表达E2F1的SGC7901/DDP胃癌细胞系对顺铂、阿霉素和5-氟尿嘧啶的敏感性显著降低。流式细胞术证实，E2F1上调后凋亡细胞百分比降低，且E2F1上调增强了细胞周期的S期阻滞。此外，E2F1上调显著降低了阿霉素的细胞内蓄积。蛋白质印迹显示，E2F1上调抑制了GAX的表达，并增加了MDR1、MRP、ZEB1、TAp73和ZEB2的表达。
E2F1过表达促进胃癌多药耐药的发展，提示E2F1可能是胃癌治疗的有效靶点。
