Yan Lin-Hai, Wei Wei-Yuan, Cao Wen-Long, Zhang Xiao-Shi, Xie Yu-Bo, Xiao Qiang
Department of Gastrointestinal Surgery, Affiliated Tumor Hospital of Guangxi Medical University Nanning 530021, Guangxi Zhuang Autonomous Region, China.
Department of Surgery, The First Affiliated Hospital of Guangxi Medical University Nanning 530021, Guangxi Zhuang Autonomous Region, China.
Am J Cancer Res. 2014 Dec 15;5(1):321-32. eCollection 2015.
Modulator of multidrug resistance (MDR) gene is a direct transcriptional target of CDX2. However, we still speculate whether CDX2 affects MDR through other ways. In this study, a cisplatin-resistant (SGC7901/DDP) and a 5-fluoro-2, 4(1h,3h)pyrimidinedione-resistant (BGC823/5-FU) gastric cancer cell line with stable overexpression of CDX2 were established. The influence of overexpression of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP and BGC823/5-FU cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2-overexpression lentiviral vector (LV-CDX2-GFP) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining. Results showed that LV-CDX2-GFP led to up-regulation of CDX2 mRNA and protein expression. It significantly inhibited the sensitivity of SGC7901/DDP and BGC823/5-FU cells to cisplatin, doxorubicin, and 5-fluorouracil. Flow cytometry confirmed that the percentage of apoptotic cells decreased after CDX2 up-regulation. This notion was further supported by the observation that up-regulation of CDX2 blocked entry into the M-phase of the cell cycle. Furthermore, up-regulation of CDX2 significantly decreased intracellular accumulation of doxorubicin. In molecular studies, quantitative reverse-transcriptase real-time polymerase chain reaction and western blotting revealed that CDX2 up-regulation could suppress expression of Caspase-3, Caspase-9 and PTEN, and increased the expression of MDR1, MRP, mTOR, HIF-1α.
多药耐药（MDR）基因调节剂是CDX2的直接转录靶点。然而，我们仍推测CDX2是否通过其他方式影响多药耐药。在本研究中，建立了稳定过表达CDX2的顺铂耐药（SGC7901/DDP）和5-氟-2,4（1H,3H）嘧啶二酮耐药（BGC823/5-FU）胃癌细胞系。通过测量SGC7901/DDP和BGC823/5-FU细胞对顺铂、阿霉素和5-氟尿嘧啶的半数抑制浓度（IC50）、阿霉素外排率、凋亡情况以及通过流式细胞术检测细胞周期进程，评估CDX2过表达对多药耐药的影响。此外，我们确定了CDX2过表达慢病毒载体（LV-CDX2-GFP）对肿瘤大小的体内效应，并通过脱氧核苷酸转移酶介导的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。结果显示，LV-CDX2-GFP导致CDX2 mRNA和蛋白表达上调。它显著抑制了SGC7901/DDP和BGC823/5-FU细胞对顺铂、阿霉素和5-氟尿嘧啶的敏感性。流式细胞术证实，CDX2上调后凋亡细胞百分比降低。CDX2上调阻断细胞周期进入M期的观察结果进一步支持了这一观点。此外，CDX2上调显著降低了阿霉素的细胞内蓄积。在分子研究中，定量逆转录实时聚合酶链反应和蛋白质印迹法显示，CDX2上调可抑制半胱天冬酶-3、半胱天冬酶-9和PTEN的表达，并增加多药耐药蛋白1、多药耐药相关蛋白、雷帕霉素靶蛋白、缺氧诱导因子-1α的表达。
