| IntroductionMultidrug resistance (MDR) is a major obstacle in the treatment of malignant carcinoma. Mechanistically, the resistance phenomena may be explained by a number of aspects,. One of the most widely studied aspects of MDR is the reduction of intracellular drug accumulation, in which ATP binding cassette (ABC) transporters expressed in the plasma membrane are notorious mediators of MDR.Multidrug-resistance-associated proteints (MRPs) belong to subfamily C of the ABC transporter superfamily (ABCC). In human, the MRP family comprises several members (MRP1-MRP9), of which, MRP1plays a very important role in MDR.Although MRP1has emerged as an important contributor to chemoresistance, the molecular mechanism for the induction of MRP1has not been clarified. However, there is substantial evidence that indicates the induction of phse II enzymes and MRPs is similar. As the induction of phase II enzymes is mainly mediated through antioxidant response elements (ARE or EpREs), it suggests that the most likely candidate for a concerted regulation of expression of MRPs is also ARE, which has a common5’-(G/A)TGACnnnGC(G./A)-3’motif. Recently, in the mouse Mrp2, Mrp3and Mrp4gene, ARE-like sequences were identified, suggesting the possible role of ARE motif in the regulatory expression of Mrpl.Nrf2was identified as the main transcription factor that mediates ARE-driven transcription. It regulates the antioxidant response by introducing the expression of genes bearing an ARE in their regulatory regions, such as NQO1, GCS, and HO-1. Activation of the Nrf2pathway composes a cellular protective system that promotes cell survival under detrimental environments. However, recent studies have shown that constitutively high level of Nrf2promotes cancer formation and contributes to chemoresistance, which is called dark side of Nrf2pathway. However, how Nrf2plays such a role still remains unknown. For instance, which molecular that is tightly regulated by Nrf2pathway is responsible for chemoresistance? Is MRP1the one of the candidates?In order to confirm above hypothesis, which is Nrf2-ARE pathway regulating expression of MRP1, this study investigated the expressive relationship between Nrf2and Mrpl and their contribution to drug resistance in tumor using two experimental models—two established human small-cell lung cancer cell lines with different sensitivity to antineoplastic drugs, and clinical specimens of human lung carcinoma, breast carcinoma, gastric carcinoma and intestinal carcinoma. Furthermore, with luciferase reporter gene assay and CHIP assay, the direct interaction between Nrf2and promoter of Mrp1gene was determined. This study investigated the potential dark side of Nrf2-ARE pathway in regulation of MRP1expression. It may benefit us in searching for the original key points in the process of multi-drug resistance, and may be used as a potential target for tumor chemotherapy.Part Ⅰ Promotion of multidrug resistance by Nrf2in multidrug resistant H69AR small-cell lung cancer cells.Purpose:To investigate the effect of Nrf2on multidrug resistance of multidrug resistant H69AR small-cell lung cancer cells.Methods:Two lines of small-cell lung cancer cells were chosen in this study. One is drug sensitive H69cell. The other is multidrug resistant H69AR cell. Realtime PCR and Western blot assays were utilized to compare the different expression level of Nrf2, MRPs and phase Ⅱ enzymes between them. MTT assay was used to detect different drug sensitivity between the two cell lines. Nrf2siRNA duplex oligoribonucleotides were transfected into H69AR cells. Thereafter, Western blot assay was used to detect different expression of Nrf2and MRPl before and after Nrf2siRNA transfection. MTT assay was utilized to detect different drug sensitivity of H69AR cells before and after Nrf2siRNA transfection.Results:Compared to H69cells, mRNA and protein expression level of Nrf2and Mrp1was much higher in H69AR cells. Therefore, H69AR cells are more multidrug resistant. Reducing the expression of Nrf2by RNA interference in H69AR cells decreased protein expression of MRP1. Accordingly, H69AR cells after Nrf2siRNA transfection are more drug sensitive.Conclusions:Nrf2promotes multidrug resistance of multidrug resistant H69AR small-cell lung cancer cells via up-regulation of MRP1expression.Part Ⅱ Promotion of Mrpl expression by Nrf2via direct binding to ARE sites in promoter region of MrplPurpose:To explore how Nrf2-ARE pathway regulates Mrpl expression and to make sure that Mrpl is the downstream target gene of Nrf2.Methods:In the5’-flanking promoter region of human Mrp1gene, possible ARE sequences were searched. The two putative ARE sequences and their mutant counterparts with point mutations against the key sites were chemically synthesized. Luciferase reporter gene plasmids carrying wild-type and mutant ARE sequences were constructed and transfected into MDA-MB-231cells. Luciferase reporter gene assay was utilized to determine the different luciferase activity after overexpression of Nrf2. Primers against the ARE sequence in Mrpl promoter were designed. And finally CHIP assay was carried out to make sure that Nrf2directly binds to ARE sites of Mrpl.Results:In the5’-flanking region of the human Mrpl gene, two putative ARE sites were identified, which was named ARE1and ARE2. Luciferase reporter gene assay indicated that over-expression of Nrf2increased ARE1and ARE2mediated induction of promoter activity, while mutation of the ARE1and ARE2sites decreased the induction. Chromatin immunoprecipitation assay in231cells demonstrated that exposure of tBHQ promoted the expression of transcription factor Nrf2, which were recruited to ARE1and ARE2sites of Mrpl promoter. Meanwhile, with comparative CHIP analysis, it showed increased binding of Nrf2to the endogenous ARE1and ARE2elements in H69AR compared with H69cells. Conclusions:In the promoter region of Mrp1, two ARE sites were identified, which was named ARE1and ARE2. Transcription factor Nrf2can directly bind to ARE1and ARE2.Part Ⅲ Expression of Nrf2, NQO1and Mrpl in human cancer tissues and their correlation with one anther.Purpose:To clarify expression of Nrf2, NQO1and Mrp1in small-cell lung cancer, breast cancer, gastric cancer and colon cancer and their correlation with one another.Methods:IHC was performed in40human carcinoma samples (with small-cell lung cancer, breast cancer, gastric cancer and colon cancer10samples each) to investigate Nrf2, NQO1and Mrpl expression. The IHC staining sections were analyzed and measured by software i-Solution to quantitate the expression of Nrf2, Mrp1and NQO1in all cases. To confirm the correlation of their expression by twos, the Pearson correlation test was next performed.Results:Nrf2immunoreactivity was readily detected in the nucleus and occasionally in the cytoplasm. The expression of Mrp1was showed both on cell membrane and in cytoplasm whereas NQO1was in cytoplasm. The staining data and statistical analysis demonstrated all three proteins were expressed much higher in tumor tissue than adjacent non-tumor tissue. And more important, the expression of the two genes--Nrf2and Mrp1was correlative. Pearson’s correlation coefficient between Nrf2and Mrpl was0.899,0,871,0.866and0.800in small-cell lung cancer, breast cancer, gastric cancer and colon cancer tissues (P<0.001).Conclusions:The expression of Nrf2, NQO1and Mrpl were correlative and were expressed higher in malignant tumors than adjacent non-tumor tissue... |
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