Zhang Z D, Li Y, Fan Q, Zhao B, Tan B, Zhao X F
Department of General Surgery, the Fourth Affilated Hospital, Hebei Medical University, China.
Neoplasma. 2014;61(6):627-37. doi: 10.4149/neo_2014_078.
Studies have shown that Annexin A2 (ANXA2) is related with tumor proliferation, apoptosis, differentiation, invasion, migration, and drug resistance. The purpose of this study was to investigate the role and its mechanisms of ANXA2 in multi-drug-resistance (MDR) in gastric cancer. ANXA2 expression in both gastric cancer tissues and cell lines were detected by quantitative real-time PCR (RT-qPCR) and Western blotting. The cell proliferation was measured by SRB assay. The pool of siRNA against ANXA2 was designed and synthesized and then transfected into resistant gastric cancer SGC7901/DDP cells. ANXA2 expression was detected by RT-qPCR and Western blotting. Drug sensitivities of SGC7901/DDP cells to P-gp-related drug (doxorubicin) and P-gp-non-related drugs (5-FU and cisplatin) were measured by SRB assay. Expression of MDR-related genes and phosphorylation of AKT and MAPKs were also detected by RT-qPCR and Western blotting. Results showed that ANXA2 expression was significantly higher in gastric specimens than that in normal tissues, and negatively correlated with the differentiation level of gastric cancer. In addition, ANXA2 expression level was higher in SGC7901/DDP cells than that in parent SGC7901 cells. After knock-down ANXA2 expression using ANXA2 small interfering RNA, the drug sensitivity of SGC7901/DDP cells to doxorubicin, 5-FU and DDP increased. Delivery of ANXA2 siRNA significantly downregulated the expression of P-gp, MRP1 and Bcl-2, while markedly upregulated Bax in SGC7901/DDP cells. However, several other MDR factors such as GST-π, TOPO-I and TOPO-II had no obvious changes. Additionally, phosphorylation of P38MAPK and AKT, but not ERK1/2 or JNKs was specifically decreased in SGC7901/DDP cells after ANXA2 siRNA delivery. Importantly, P38MAPK and AKT inhibitor increased the drug sensitivity of SGC701/DDP cells in a similar way as ANXA2 siRNAs does. ANXA2 is involved in gastric cancer MDR through regulating p38MAPK and AKT pathways as well as certain MDR factors.
研究表明，膜联蛋白A2（ANXA2）与肿瘤的增殖、凋亡、分化、侵袭、迁移及耐药性有关。本研究旨在探讨ANXA2在胃癌多药耐药（MDR）中的作用及其机制。采用定量实时PCR（RT-qPCR）和蛋白质免疫印迹法检测胃癌组织及细胞系中ANXA2的表达。采用磺酰罗丹明B（SRB）法检测细胞增殖情况。设计并合成针对ANXA2的小干扰RNA（siRNA）池，然后转染至耐药胃癌SGC7901/DDP细胞中。采用RT-qPCR和蛋白质免疫印迹法检测ANXA2的表达。采用SRB法检测SGC7901/DDP细胞对P-糖蛋白（P-gp）相关药物（阿霉素）和P-gp非相关药物（5-氟尿嘧啶和顺铂）的药物敏感性。采用RT-qPCR和蛋白质免疫印迹法检测多药耐药相关基因的表达以及AKT和丝裂原活化蛋白激酶（MAPKs）的磷酸化情况。结果显示，胃癌标本中ANXA2的表达明显高于正常组织，且与胃癌的分化程度呈负相关。此外，SGC7901/DDP细胞中ANXA2的表达水平高于亲本SGC7901细胞。使用ANXA2小干扰RNA敲低ANXA2表达后，SGC7901/DDP细胞对阿霉素、5-氟尿嘧啶和顺铂的药物敏感性增加。在SGC7901/DDP细胞中，导入ANXA2 siRNA可显著下调P-gp、多药耐药相关蛋白1（MRP1）和Bcl-2的表达，同时显著上调Bax的表达。然而，其他一些多药耐药因子如谷胱甘肽S转移酶π（GST-π）、拓扑异构酶I（TOPO-I）和拓扑异构酶II（TOPO-II）则无明显变化。此外，导入ANXA2 siRNA后，SGC7901/DDP细胞中P38MAPK和AKT的磷酸化水平特异性降低，而细胞外信号调节激酶1/2（ERK1/2）或c-Jun氨基末端激酶（JNKs）的磷酸化水平未降低。重要的是，P38MAPK和AKT抑制剂与ANXA2 siRNA一样，可增加SGC701/DDP细胞的药物敏感性。ANXA2通过调节p38MAPK和AKT信号通路以及某些多药耐药因子参与胃癌多药耐药的发生。
