| Objective: Gastric cancer is one of the most common tumors in China, and surgical resection is still the primary treatment for gastric cancer. Because of the lack of cancer screening, many patients are diagnosed at an advanced stage and have no chance of surgery. Thus the chemotherapy seems particularly important, and it can not only improve patients outcome, but also improve the quality of life of patients. Cisplatin (cisplatin, DDP) is a basic anticancer drug of chemotherapy, and NCCN treatment guideline suggests the DDP-based combination chemotherapy is a preferable option of gastric cancer. However, the prognosis of gastric cancer is still poor, and the response rate of chemotherapy ranged from 5% to 50%. Due to the development of tumor primary or aqcuired drug resistance, the decreased sensitivity of tumor to chemotherapy eventually leads to treatment failure and tumor progression. Since numerous researches have revealed a lot of mechanisms in the cisplatin resistance, researches on mechanisms of DDP resistance in gastric cancer cells gradually begun after the establishment of gastric cancer cisplatin resistant SGC7901 (SGC7901/DDP) cell line. The development of drug resistance is a complicated process including changes of several genes and multiple factors. With the development of acquiring resistance, the tumor may often become cross-resistance to a range of other type of anti-tumor drugs showing multi-drug resistance phenotype. So finding and screening of genes involved in DDP resistance will help to further study the mechanisms of acquired drug resistance. Most researches of multidrug resistance in gastric cancer are focused on P-glycoprotein (P-gp), lung resistance protein (LRP), multidrug resistance protein 1 (MRP1), and they are reported to act in multidrug resistance mainly by excreting the chemotherapy drugs or changing the intracellular drugs distribution of tumor cells. Furhtermore, excision repair cross-complementing gene 1 (ERCC1), an important part in NER pathway, can quickly repair the damaged DNA, which causes the resistance of tumor cells to DDP. Meanwhile, the recent study found that one of Ras family proteins RhoE is also involved in drug resistance formation of tumor. This study will test the sensitivity changes of cells to other anticancer drugs during the cisplatin-resistant formation in gastric cancer SGC7901/DDP and its parental SGC7901 cell. And expressions of MDR1, LRP, MRP1, RhoE, and ERCC1 mRNA and proteins in both cell lines are also detected. It will help to provide a theoretical basis for the reversal of cisplatin resistance in gastric cancer if the resistance characteristics and possible mechanisms of SGC7901/DDP cells would be ivestigated.Methods: The Sensitivity of the SGC7901/DDP cell line and parental cells to gastric cancer chemotherapeutic drugs cisplatin(DDP), paclitaxel (PTX), 5-fluorouracil(5-FU), doxorubicin (ADR) were determined by MTT assay. RT-PCR and Western blot were used to evaluate the expression of MDR1/P-gp, LRP, MRP1, RhoE and ERCC1 in human gastric cancer cell lines SGC7901 and SGC7901/DDP, initial discussing gastric carcinoma resistance mechanisms.Results:1 The sensitivity of the DDP, PTX, 5-FU, ADR in the SGC7901/DDP and SGC7901 cellsThe MTT results showed that the IC50 of DDP , PTX, 5-FU, ADR in the SGC7901/DDP cells were 11.245±0.272 mg/ L , 3.277±0.426mg/L, 12.154±2.036mg/L, 0.763±0.043mg/L, and in the parental cells were 2.206±0.256mg/L,1.528±0.097 mg/L,4.481±0.544mg/ L,0.229±0.020mg/L , It had different degrees of tolerance to DDP, PTX, 5-FU and ADR, with resistance index 5.08, 2.14, 2.71, 3.33,suggested that the sensitivity of the four drugs in SGC7901/DDP cells were significantly lower than SGC7901 cells(P<0.05).2 Extraction of RNA quality testingThe concentration of RNA extracted from gastric cancer cells of SGC79 01/ DDP and SGC7901 were 936.12mg/L and 875.29mg/L, A260/A280 values was between 1.9 and 2, meanwhile formaldehyde denaturing agarose gel elect -rophoresis showed that 18S and 28S bands were clear,the brightness ratio was about 1:2, indicating that the purity of total RNA meets the experimental requirements and no significant degradation, therefore,the following test can be carried out.3 RT-PCR detected the mRNA expression of MDR1, LRP, MRP1, RhoE and ERCC1The relative expression of MDR1, LRP, MRP1, RhoE and ERCC1 mRNA in SGC7901 cells were 0.050±0.017, 0.689±0.038, 0.595±0.064, 0.616±0.050 , 0.410±0.061, and in SGC7901/DDP cells were 0.128±0.039, 0.938±0.041, 0.655±0.056, 0.591±0.076, 0.462±0.042. Statistical analysis showed that the mRNA expression of MDR1 and LRP in SGC7901/DDP cells were significantly higher than SGC7901 cells(P<0.05), and the mRNA expression of MRP1, RhoE, ERCC1 in both cell lines were no significant difference (P>0.05).4 Western-blot detected the protein expression of MDR1, LRP, MRP1, RhoE and ERCC1The relative expression of MDR1, LRP, MRP1, RhoE and ERCC1 protein in SGC7901 cells were 0.032±0.096,0.409±0.043,0.144±0.037,0.366±0.056,0.268±0.037,and in SGC7901/DDP cells were 0.118±0.020, 0.792±0.075, 0.344±0.037, 0.651±0.062, 0.495±0.053. There were significant differences of the five proteins expression in both cell lines (P<0.05), the protein expression of MDR1 , LRP, MRP1, RhoE, ERCC1 in SGC7901/DDP cells were significantly higher than SGC7901 cells.Conclusions:1 SGC7901/DDP cells showed the characteristic of multidrug resistance, it had cross-resistance to PTX, 5-FU and ADR.2 The mRNA and its encoded protein expression of MDR1 and LRP in SGC7901/DDP cells were higher than SGC7901 cells, and the expression changes of MDR1/P-gp and LRP may be an important mechanism of multidrug resistance in SGC7901/DDP cells.3 The protein expression of MRP1, RhoE and ERCC1 in SGC7901/DDP cells were significantly higher than SGC7901 cells. However, the mRNA expression of MRP1, RhoE and ERCC1 were no significant difference in both cell lines. The protein expression changes of MRP1, RhoE, ERCC1 may be closely related to multidrug resistance, but there is no significant correlation between mRNA expression levels and drug resistant characterastic of gastric cancer cells. Meanwhile, the regulatory mechanisms of MRP1, RhoE, ERCC1 genes in the process of protein translation should be further discussed. |
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