| Globally, gastric cancer (GC) is the second mostly frequent cause of cancer-related mortality and the incidence is much higher at Asia than other geographic areas. In china, gastric cancer is the second leading cause of cancer-related mortality and has the third highest incidence. Because gastric cancer is usually diagnosed in the advanced or metastatic stage, the prognosis of gastric cancer is generally rather poor, with a5-year survival rate of less than30%. Surgical operation remains the standard therapy strategy at present, but recurrence appears in approximately50-70%of patients with advanced disease. Therefore, chemotherapy has been widely accepted as palliative treatment for both resectable and advanced gastric cancer, leading to improvements in quality of life for patients and prolonged survival. Although gastric cancer is considered as relatively insensitive to chemotherapeutics and several important chemotherapeutic agents such as cisplatin (DDP) and5-fluorouracil (5-FU) are effective to date, the survival time has been unsatisfactory so far. Multiple lines of evidence suggest that long-term chemotherapy often fails to eliminate all cancer cells due to the development of multidrug resistance (MDR), which can further caused the cancer recurrence.AnxA2(ANXA2), a calcium dependent phospholipid binding protein, is involved in maintaining the plasticity and rearrangement of the actin cytoskeleton. There is overwhelming evidence that ANXA2is a multifunctional protein which is involved in tumor progression, proliferation, apoptosis, and signaling transduction. ANXA2may act as tumor suppressors or tumor promoters depending on the types of tumor cells and tissues. For example, increased ANXA2tissue levels have been observed in malignancies of the liver and breast, however, decreased ANXA2tissue levels have been observed in malignancies of the esophageal and prostate. Therefore, ANXA2may serve as an ideal target for cancer therapy and a biomarker for studying tumor progression, proliferation, apoptosis, and tumor patients’ prognosis. However, the role of ANXA2in gastric cancer is not clear.Objectives To investigate the ANXA2expression in gastric cancer as well as its impact and mechanisms on drug resistance. To clarify the role of ANXA2in the development of gastric cancer occur, as to provide theoretical basis for targeted therapy of gastric cancer.Methods ANXA2expression 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 ANXA2were designed and synthesized and then transfected into resistant gastric cancer SGC7901/DDP cells. ANXA2expression was detected by RT-qPCR and Western blotting. Drug sensitivity of SGC7901/DDP cells to one P-gp-related drug (doxorubicin) and on P-gp-non-related drugs (5-FU and cisplatin) were measured by the SRB assay. In addition, the effect of ANXA2siRNA on the expression of MDR-related genes and activation of some molecular in signaling pathway were also observed.Results are as follows:Part one: The study of ANXA2expression in gastric cancer tissuesIn the first part, we investigated the expression of ANXA2in gastric cancer tissue by quantitative real-time RT-PCR and Western blot. The results are as follows:1Expression differences of ANXA2mRNA between gastric cancer tissue and adjacent normal tissuesThe mRNA level of ANXA2in the clinical samples of gastric cancer and adjacent normal tissues were detected by RT-PCR. The results showed that ANXA2mRNA expression was significantly higher in cancer tissues compared to that in adjacent normal tissues, suggesting that Annexin A3plays an important role in gastric carcinogenesis.2Expression differences of ANXA2protein between gastric cancer tissue and adjacent normal tissuesThe protein level of ANXA2in the clinical samples of gastric cancer and adjacent normal tissues were detected by western blot. The results showed that ANXA2protein expression was significantly higher in cancer tissues compared to that in adjacent normal tissues, suggesting that Annexin A3plays an important role in gastric carcinogenesis.Part two: The study of ANXA2expression in gastric cancer differentiationIn the second part, to investigate the significance of ANXA2in gastric carcinoma differentiation, we detected the expression of Annexin A3in gastric cancer tissue and gastric cancer cell lines with the various differentiat ion by quantitative real-time RT-PCR, and Western blot.1Correlation of Annexin A3expression with gastric cancer tissues with various differentiation gradesGenerally, the poorer GC differentiation, the worse the prognosis was. To assess whether ANXA2were related with GC differentiation, the expression of ANXA2in GC tissues from stage I-Ⅲ GC patients was further detected by RT-QPCR, and western blotting analysis. And the results showed that the higher differentiation stage, the lower expression level of ANXA2was, indicating that the expression level of ANXA2was negative correlated with the differentiation stage of gastric tumor.2Correlation of Annexin A3expression with cultured gastric cancer cell lines with various differentiation gradesTo further verify the correlation of ANXA2expression with GC differentiation and the feasibility of following study for mechanism, ANXA2expression in GC cell lines (MKN28, SGC7901and BGC823) with various differentiation stage was also detected. quantitative real-time RT-QPCR, western blotting analysis, and immunocytochemistry staining. The results of RT-QPCR and western blotting analysis showed that ANXA2expression were MKN-28, SGC-7901and BGC-823in increasing order, which is consistent with ANXA2expression in differentiation stage of gastric cancer tissues. Part three: Effect of ANXA2-siRNA transfection to chemosensitivity of gastric cancerIn the third part, to investigate the significance ANXA2expression of in a gastric cancer cell line SGC7901and a DDP resistant gastric cancer cell line, and gastric cancer cell resistance change after ANXA2-siRNA transfection, to make clear ANXA2mechanism of drug resistance in gastric cancer cell by quantitative real-time RT-PCR and Western blot.1Expression of ANXA2in MDR gastric cancer cellsTo address the role of ANXA2in the multidrug resistance of GC, the IC50values of DDP in the SGC7901/DDP and parent SGC7901cells were determined to establish the multidrug resistance cell line firstly. The results of SRB showed that the IC50values of DDP is (6.08±1.52) μg/ml and (0.26±0.02) μg/ml in the SGC7901/DDP and SGC7901cells, respectively, which indicated that the SGC7901/DDP cells were23.4times more resistant to DDP than SGC7901cells.Further, we compared the expression of ANXA2in DDP-resistant SGC7901/DDP cells and parent SGC7901cells. quantitative real-time RT-QPCR and western blotting analysis results revealed that the expression of ANXA2was higher in SGC7901/DDP cells than that in parent SGC7901cells at both mRNA and protein levels. These data suggest that ANXA2is associated with the development of MDR in gastric cancer cells.2Effect of siRNA on ANXA2expression in SGC7901/DDP cellsTo further investigate the effects of ANXA2in the multidrug resistance of GC, siRNA techniques was employed in this study. We first investigated the ability of an ANXA2siRNA to inhibit the expression of ANXA2in SGC7901/DDP cells. The results of quantitative real-time RT-QPCR and western blotting showed that20,40, and80nM ANXA2siRNA downregulated the endogenous ANXA2mRNA and protein expression efficiently, and that its optimal concentration is80nM, with an inhibition of approximately90%compared to controls, suggesting that ANXA2siRNAs efficiently inhibited the transcription and translation of ANXA2in SGC7901/DDP cells.We also detected ANXA2expression in SGC7901/DDP cells after80nM ANXA2siRNA transfection for24,48, and72h, the results of quantitative real-time RT-QPCR and western blotting showed that endogenous ANXA2mRNA and protein expression were decreased in a time-dependent manner. After80nM ANXA2siRNA transfection for24,48, and72h, more than90%of the ANXA2expression in SGC7901/DDP cells was inhibited compared to controls, suggesting that ANXA2siRNAs efficiently inhibited the transcription and translation of ANXA2in SGC7901/DDP cells.3Effect of ANXA2-siRNA on MDR of SGC7901/DDP cellsAlthough SGC7901/DDP cells were selected with the chemotherapeutic drug cisplatin, they also displayed multiple resistances to other chemotherapeutic drugs. Therefore, we futher investigate the effects of ANXA2siRNA on the drug sensitivity of SGC7901/DDP to one P-gp-related drug (doxorubicin) and on P-gp-non-related drugs (5-FU and cisplatin). The IC50values for doxorubicin,5-fluorouracil, and cisplatin were all higher in SGC7901/DDP cells than that in parent SGC7901cells. The results showed that drug resistant drug resistant SGC7901cells displayed cross resistance to these chemotherapeutic drugs. In addition, after transfection of80nM ANXA2siRNA, the IC50values for doxorubicin,5-fluorouracil, and cisplatin was decreased significantly compared with non-targeted siRNA and Lipofectamine2000TM group, indicating that ANXA2siRNA could partially reverse resistance to DDP in multi-drug resistant SGC7901cells.Part four: Effect of ANXA2on the MDR-related genes expression in SGC7901/DDP cells after ANXA2-siRNA transfectionIn the fourth part, the expression of MDR-related genes including P-gp, MRP1, Bcl-2, and Bax were detected in SGC7901/DDP cells after ANXA2-siRNA transfection by quantitative real-time RT-QPCR and western blotting.1Effect of ANXA2on the mRNA levels of MDR-related genes in SGC7901/DDP cells To further explore the potential mechanism of ANXA2on development of MDR, the MDR-related genes including P-gp, MRP1, Bcl-2, and Bax were detected by quantitative real-time RT-QPCR. We observed that the expression of P-gp, MRP1, Bcl-2was significantly downregulated, while the expression of Bax was markedly upregulated by80nM ANXA2siRNA in SGC7901/DDP cells. However, the expression of GST-π,TOPO-I, and TOPO-II were not changed.2Effect of ANXA2on the protein levels of MDR-related genes in SGC7901/DDP cellsThe protein level of the MDR-related genes were also detected by western blotting. The results showed that the protein expression of P-gp, MRP1, Bcl-2was significantly downregulated, while the expression of Bax was markedly upregulated by80nM ANXA2siRNA in SGC7901/DDP cells. However, the expression of GST-π,TOPO-I, and TOPO-II were not changed.Part five: Effect of ANXA2on the phosphorylation of MAPKs and PI3K/Akt signaling pathway in SGC7901/DDP Cells after ANXA2-siRNA transfectionIn the last part, the phosphorylation of MAPKs and PI3K/Akt signaling molecular were detected in SGC7901/DDP cells after ANXA2-siRNA transfection by western blotting.Western blotting analysis of MAP kinases and AKT revealed that phosphorylation of p38-MAPK and AKT, but not ERK1/2and JNKs was specifically decreased in SGC7901/DDP after transfection of80nM ANXA2siRNA.Conclusions:1Overexpression of ANXA2was found in gastric cancer tissues, and significant relationship between expression of ANXA2and degree of tumor differentiation was also found in present research. All results suggested that ANXA2may play an important role in carcinogenesis and progression of gastric cancer.2ANXA2was nvolved in multidrug resistance of gastric cancer. After endogenous ANXA2was inhibited by siRNA, the chemosensitivity in vitro of SGC7901/DDP became more sensitive, which was related to the ability of ANXA2to regulate multidrug resistance genes such as P-gp, MRP1, Bcl-2, etc.3Activation of p38-MAPK and AKT signaling pathway was inhibited by the ANXA2siRNA transfection in resistant gastric cancer cells. Our results suggested that drug resistance in SGC7901/DDP cells is attributed at least partially to the activation of p38-MAPK and AKT signal pathway and inhibition of these pathways could increase the sensitivity of SGC7901/DDP to P-gp-unrelated drugs as well as P-gp-related drug. |
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