Expression And Significance Of Nrf2 And Its Target Genes In Pulmonary Adenocarcinoma A549 Cells Which Are Resistant To Cisplatin

BACKGROUND:As a kind of severe diseases doing harm to human health, pulmonary cancer ranks the first place in aspects of morbidity and mortality. As the existence of heterogeneity, there is difference biologic activity in different individuals and different type of tumor cells. Different lung cancer cells have not the same sensitivity to anti-cancer drugs. Chemotherapy is the main method for treating lung cancer. Drug resistance is one of clinical major causes for low survival rate of the patients. Cis-dichlorodiamine platinum (CDDP) is effective and widely-used drug, but the result is unsatisfactory because of drug resistance. In order to decrease the harm which chemotherapeutics do to the organism and increase the sensitivity and effectiveness, we must do further research to explore the mechanism of drug resistance to tumor. As a key transcription factor in anti-oxidant reaction NF-E2-related factor 2 combines together with ARE, then regulates the expression of detoxifying enzymes, antioxidase and drug transport pumps. Both domestic and overseas researches proved that chemo-therapeutics is an obvious oxidative stress to cells, so in this condition whether transcription factor can protect lung cancer against DDP, and mediate drug resistance is a problem needing urgent management.AIM:Our Object is to study the expression and significance of transcription factor Nrf2 and its target genes in A549 cells which are resistance to cisplatin and reveal the mechanism behind it.Moreover, this provides a new direction to reverse drug resistance and have significance to avoid and overcome drug resistance of tumor.METHODS:1 Human pulmonary adenocarcinoma A549 and Cis-dichlorodiamine platinum-resistant cell lines were obtained from Tianjin Lung cancer Institutes. The cells were cultured in RPMI-1640, supplemented with 10% fetal bovine serum (FBS), 100μg/ml penicillin and 100μg/ml streptomycin. The cells were maintained at 37℃in a humidified atmosphere with 5% CO2.2 MTT was used to detect the drug resistance index of A549/DDP cells.3 Real time PCR:There are two groups:A549 and A549/DDP cell line. Total RNA was extracted using TRIzol reagents according to the manufacturer's instructions. Isolated RNA was electrophoresed through 1.2% agarose for-maldehydegels to verify the quality of the RNA. The first strand cDNA was generated by reverse transcription. After a sufficient amount of cDNA was obtained, we performed PCR amplification using a real-time PCR cycler.RESULTS:1 We observe the morphous of A549 and A549/DDP cells through the inverted microscope. The former is smaller and spherical, while the later is bigger and spindle-shaped, minority show irregular triangle.2 A549/DDP and A549 cells in our study are of high homology. A549/DDP has stable quality of drug resistance. We perform three independent and repeated MTT essays, and it proves that the growth suppression of the two cell lines have positive correlation with DDP drug concentration(r=0.984, r=0.896).Inhibitory Concentration 50 of A549/DDP and A549 is (8.36±0.44)ug/ml, (0.69±0.09)ug/ml, (t=29.701, p<0.001). Resistance index of A549/DDP is 12.12.3 Real-time PCR shows that the mRNA expression level of transcription factor Nrf2 in A549/DDP cell line is increased compared with A549(p<0.01).At the same time there is more mRNA expression of Keap1 and the target genes of Nrf2, such as NQO1,GSTP1,GCL, HO-1,MRP4 in A549/DDP than the cells which is sensitive to DDP.CONCLUSION:It proves that the transcription factor Nrf2 and it's target genes HO-1, GCL, MRP4, GSTP1, NQO1 are closely related with Cisplatin resistance of pulmonary adenocarcinoma. They mediate Cisplatin-resistance through decreasing apoptosis, accelerating Cisplatin metabolism and changing the distribution of Cisplatin in pulmonary adenocarcinoma cells. Our study reveals that Nrf2-ARE signal passway may be relative with the Cisplatin-resistance to pulmonary adenocarcinoma. Moreover, this provides a new direction to reverse drug resistance and have significance to avoid and overcome drug resistance of tumor.