The Role Of Upregulation Of The Multidrug Resistance Gene In Drug-Resistant Gastric Carcinoma Cell

Gastric carcinoma is one of the most common malignant tumor in the world. At present, the early detection of the Gastric carcinoma is very difficult, most patients of Gastric carcinoma were diagnosed as advanced gastric carcinoma, therefore, chemotherapy is one of the most important treatment of gastric carcinoma. However, because of the drug-resistant of Gastric carcinoma tumor cells, the chemotherapy result is not satisfactory in a considerable number of cases. Therefore, the study of multidrug resistance mechanism in gastric carcinoma may play an important role in enhancing the effectiveness of chemotherapy and prolonging survival time of patients of gastric carcinoma .The notion of Multidrug resistance(MDR) was proposed to describe the phenomenon that while the tumor cells resist a kind of anticancer drug, it can also resisit other anticancer drugs which may have completely different structure and drug mechanism. In vitro and in vivo study show that the molecular mechanisms of tumor multi-drug resistance is very complex, which involved many factors such as gene mutations, related protein expression, enzyme-mediated, deficiencies of stem cell fuction, et al. The most important multidrug resistance mechanisms involve the over-expression of mdr1 gene. The mdr1 gene encodes a membrane P-glycoprotein of 170KD which has a function of ATP-dependent drug efflux pump. P-gp is a transmembrane protein which was constituted by 1280 amino acid. By activating ATP pump, P-gp can hinder the drug's intracellular passive diffusion and transport the intracellular poison to the outside. The over-expression of the mdr1 gene on the chromosome 7 which encode the P-gP is the most important mechanism of MDR. Many studies showed that, mdr1/pgp was closely related to the patients'chemotherapy effect. Patients with over-expression of mdr1 ofen has limited survival, low rate of remission, or high rate of relapse. Therefore, the detection of mdr1/pgp expression not only can be used as a predictor of tumor treatment result, but also can helps clinicians to formulate the chemotherapy.As one of the most important regulation pathway of cell signaling system, mitogen-activated protein kinase (MAPK) play an important role in regulation of cell growth, proliferation, differentiation, apoptosis, adhesion and migration, influencing the occurrence invasion and metastasis as well as the drug resistance of tumor. So, MAPK may be one of the targets of the anticancer drug.P38 signaling pathway, also known as the emergency of MAPK signaling pathway, is an important composition of MAPK family. P38 plays an important role in systemic inflammatory response, apoptosis, shock, tumor metastasis, drug resistance. Meanwhile, studies in tumor cells show that P-gp expression is correlated with some activated signaling pathway. And researchers have found that in the multi-drug resistance cell lines, some pathways regulate the expression of P-gp, and when the pathway was blocked, P-gp expression significantly decreased. For example, inhibiting P-glycoprotein function and expression can suppress the P-glycoprotein-mediated MDR phenotype as well as improve the effectiveness of chemotherapy. Therefore, we suppose that the signal pathways may played a very important role in the regulation of MDR. In our subject, we attempt to find out the way P38-MAPK pathway to influence the multidrug resistance of tumor arising from the process. And our research may have an significance of the cancer chemotherapy for clinical program and reversal of drug resistance.一,The expression of the multidrug resistance gene in SGC7901 and SGC7901/VCR cellTo determine the characteristic of chemotherapeutic drug resistance of the two cell lines, we treated SGC7901 and SGC7901/VCR cells with different chemotherapeutic drugs(5-fluorouracil,epirubicin,cisplatin). Then determine the cell activity using MTT method, the result show that the survival rate of SGC7901/VCR cell was significantly higher than that of SGC7901, suggesting that SGC7901/VCR probably belongs to multi-drug resistant cell. In order to identify whether the SGC7901/VCR belongs to a specific kind of multi-drug resistant cell line, we detect the multidrug resistance gene expression of the two cell lines using Western blot and RT-PCR method. The results showed that the expression of multidrug resistance gene in SGC7901/VCR cell was significantly higher than SGC7901, identifying the characteristics of multi-drug resistance in SGC7901/VCR cell.二,P38-MAPK pathway in SGC7901 cells and SGC7901/VCRStudies show that the region of human MDR1 promoter contains the transcription factor AP-1 binding site, which was indirectly involved in regulating the expression of P-gp. The activation of AP-1 can resist many kinds of anti-tumor drugs. Therefore, We adopted Luciferase reporter gene system to detect the AP-1 activity of the two types of cells. The results show that the AP-1 activity of SGC7901/VCR was significantly higher than that of SGC7901. As the downstream target gene of the MAPK pathway, AP-1 could significantly change the expression levels of MAPK in some drug-resistant cells. Therefore, we used Western Blot and flow cytometry to determine the expression of MAPK pathway of the two types of cells, we found that the p38 activity of SGC7901/VCR cells was significantly higher than that of SGC7901. Furthermore, to determine the characteristics of SGC7901/VCR cells, we inhibited the expression of p38 by using SB202190 which is the specific inhibitor of the DN-p38 and p38, the result show that the P38 expression of the SGC7901/VCR cells was significantly decreased compared with the control, suggesting that the p38-MAPK/AP-1 way in SGC7901/VCR cells has been activated, which is closely related to the multidrug resistance of the tumor cells. Meanwhile, we also have detected the activity of P38 in SGC7901 which was treated by cisplatin(2μg/ml) consecutive 24-hours, suggesting that p38-MAPK pathway associated with chemotherapy resistance.In order to verify whether the p38-MAPK pathway was related to chemotherapy resistance, we detected the expression of MDR1 and P-gp function in SGC7901/VCR cells which was treated with the specific inhibitor of p38. The experimental results show that, the expression of the multidrug resistance was significantly down in both protein and mRNA level after using the p38 inhibitors. At the same time, we use the flow cytometry to analysis the accumulation and efflux velocity of Rh123 in cell to find out the impact of SB202190 to P-gp function. It was found that the accumulation and efflux of the Rh123 has been increased significantly after using the p38 inhibitors in SGC7901/VCR cells. This experiment suggested that the inhibition of p38 pathway can reduce the expression of MDR1 and P-gp function. Furthermore, we treated the SGC7901/VCR cells in which the p38-MAPK pathway was inhibited separately using 5-fluorouracil, epirubicin, cisplatin. After observe the cell morphology and analysis the cell apoptosis, we found that the inhibition of p38-MAPK pathway can enhance the sensitivity of drug-resistant cells to chemotherapeutic drug.Conclusion:1. In SGC7901/VCR cell, the multidrug resistance is closely related with the activation of p38-MAPK pathway, and there is no significant relationship between multidrug resistance and JNKs or ERK.2. Inhibition of the p38-MAPK pathway using SB202190 can enhance the sensitivity of drug-resistant tumor cells to chemotherapeutic drug.