The Mechanism Of How Nuclear Multidrug-resistance Related Protein1Contributes To Multidrug-resistance Of Mucoepidermoid Carcinoma

Mucoepidermoid carcinoma (MEC) is the most common primary oral andmaxillofacial malignant salivary gland tumor in occidental and Chinese population[1,2,3]. Itcomprises about10%of all salivary gland neoplasms and about35%of malignant lesions[4]. In pediatric patients, MEC comprises about16%of all salivary gland neoplasms andabout51%of malignant lesions[5]. MEC is divided into low, intermediate and high gradeon the basis of morphological and cytologic features. The low and intermediate gradeMEC usually have a high survival rate, while the high grade MEC usually has very poorprognosis whose5-year survival is only about30%[6,7]. MEC usually has lowchemosensitivity, so chemotherapy is largely used for palliation of unresectable ormetastatic disease. Complete surgical resection is the standard treatment for MEC patients[8].The multidrug resistance-related protein1(MRP1or ABCC1) is a membrane-bound, energy-dependent efflux transporter belonging to the superfamily of ABC transporters.MRP1was firstly discovered in a multidrug-resistant small-cell lung cancer cell linewithout overexpressing ABCB1(MDR1)[9]. Afterward, increased expression of MRP1hasbeen reported as a predictor of poor response tochemotherapy in a variety ofhematological and solid tumors[10]. In tumor cells, MRP1is predominantly localized onthe cytomembrane, which suggests the transporter role of MRP1in clinicalchemoresistance, nevertheless MRP1can also be detected in the cytoplasm[11,12,13,14,15,16].In most normal tissues, MRP1is localized on the basolateral cellular surface, whichresults in the efflux of its substrates into the blood[17]. Multidrug resistance protein1(MDR1or ABCB1) and Multidrug resistance-related protein1(MRP1or ABCC1), bothbelonging to the ATP-binding cassette superfamily of membrane-bound transporters, aretwo genes found highly related to multidrug-resistance. The human MDR1spans over100kb on human chromosome7q21and the human MRP1spans over194kb on chromosome16p13.1. The amino-acid sequence of the two genes resembles only to a modest extent (～15%). The substrates for ABCB1/MDR1are commonly hydrophobic drugs having neutralor positive charge and MRP1commonly transports neutral and anionic hydrophobicnatural products. Unlike MDR1, MRP1also transports glutathione and other drugconjugates[18]. Despite the comprehensive study of MRP1, the crystal structure and thetransport mechanism of MRP1remains elusive[19], and the broadly existed polymorphismsand mutation of MRP1in tumors also make its function unpredictable[20,21].In this study, we found nuclear MRP1in the MEC cells for the first time and provedthat the nuclear translocation of MRP1was the reason causing multidrug-resistance ofMC3/5FU cells. To explore how MRP1lead to multidrug-resistance in the nuclei of MECcells, we investigated the MDR1expression in the MC3/5FU cells as the MRP1wasdownregulated. We also investigated the expression pattern of MDR1and MRP1in127cases of MEC patients by immunohistochemistry and found the correlation betweenMRP1and MDR1in MEC for the first time. We further ensured the nuclear expression ofMRP1in MEC and checked expression of nuclear MRP1in other normal and tumortissues. From the results above, we thought the new function of the MRP1made it a promising target in MEC clinical treatment.Part1: Downregulation of MRP1leads to the decrease of multidrug resistance inmucoepidermoid carcinoma cell lineAim：This part was to investigate the role of MRP1in mucoepidermoid carcinoma.Subjects and methods: A highly metastatic human mucoepidermoid carcinoma cell lineMC3[22] and its5FU-resistant subline MC3/5FU was established and stored in ourlaboratory. The short hairpin RNA (shRNA) expressing plasmid was constructed andstable gene transfected MC3/5FU cells were selected. Reverse transcription polymerasechain reaction (RT-PCR) was used to check the RNA expression changes. Western blotwas used to analysis the protein expression changes. Cell growth and chemoresistance wasmeasured by methyl thiazolyl tetrazolium assay (MTT assay). Apoptosis of cells wasdetected by TUNEL staining. Student’s t-test and one-way ANOVA (LSD) were used fordetermining the significance of difference in comparisons. Calculations were carried outby SPSS version12. P value of less than0.05was considered statistically significant.Results: Downregulation of MRP1sensitized MC3/5FU cells to chemotherapeutic drugs(5FU, ADM, PADM, BLM-A5, CDDP and TAX), depressed the growth of MC3/5FU cellsand increased the apoptosis of MC3/5FU cells under5FU.Conclusion: MRP1plays important role in the multidrug-resistance of mucoedpidermoidcarcinoma.Part2: Nuclear translocation of MRP1contributes to multidrug resistance ofmucoepidermoid carcinomaAim：This part is to test the hypothesis that nuclear translocation of MRP1contributes thechemoresistance of mucoepidermoid carcinoma (MEC).Subjects and methods: Immunofluorescent Confocal Laser-Microscopy was used tocheck the Subcellular localization of MRP1in cells. Immunohistochemical studies wasused to find the location of MRP1in MEC tissues and normal salivary gland tissues. Immunohistochemical studies was used to check the location and expression of MRP1innormal tissues and MEC tissues. Furthermore, IHC study of multiple tumor tissue arraysections was applied to investigate the location and expression of MRP1in other normaltissues and other tumor tissues.Student’s t-test and one-way ANOVA (LSD) were used fordetermining the significance of difference in comparisons. Calculations were carried outby SPSS version12. P value of less than0.05was considered statistically significant.Results: After we compared the drug-resistant MEC cells (MC3/5FU) with its parent cells(MC3), we did not find significant quantitative change of MRP1, but, interestingly, wefound that the location of MRP1changed from the cell membrane to the nuclei of MECcells after its chemoresistance increased. For further study, we decreased the expression ofMRP1in MC3/5FU cells through RNA interference and found that downregulation ofMRP1sensitized MC3/5FU cells to chemotherapeutic drugs and the MRP1lost havecome mainly in the nuclei. We also proved the nuclear MRP1, which has not been foundin other normal or tumor tissues yet, was widely existed in MEC tissues.Conclusion: Nuclear translocation of MRP1could be a new mechanism of MRP1leadingto multidrug-resistance (MDR) and new treatment modalities based on this could bedeveloped.Part3: The downregulation of MRP1leads to the downregulation of MDR1expressionAim: This part was to investigate how MRP1contributes to multidrug resistance ofmucoepidermoid carcinoma.Subjects and method: Human MEC tissues were obtained from127patients who hadreceived no pretreatment before undergoing routine surgery for MEC at the Department ofOral and Maxillofacial Surgery, School of Stomatology, the Fourth Military MedicalUniversity, from July2006to July2011. Immunohistochemistry was used to detect theexpression of MRP1and MDR1in MEC tissues. Staining was evaluated independently bytwo of the authors (BL.Cai, Y.Liu). Results were scored in a qualitative and a quantitativeway. To exclude the possibility that MRP1was expressed on the surface of nuclearmembrane of MEC cells, the location of MRP1was layer scanned by immunofluorescent confocal laser-microscopy. Reverse transcription polymerase chain reaction (RT-PCR) wasused to check the RNA expression changes. Western blot was used to analysis the proteinexpression changes. Student’s t-test and one-way ANOVA (LSD) were used fordetermining the significance of difference in comparisons. The relationship betweenMDR1and MRP1(or nuclear MRP1) was analyzed with Spearman’s rank correlationanalysis. Calculations were carried out by software SPSS version12.0, P<0.05wasconsidered statistically significant.Results: After we downregulated the MRP1expression in MC3/5FU cells, themultidrug-resistance-protein1(MDR1or ABCB1) expression decreased. A positivecorrelation between the nuclear MRP1expression and the MDR1expression was alsofound in the immunohistochemistry study of127MEC specimens.Conclusion: The expression of MRP1is positively correlated with MDR1expression.Part4: Down-regulation of MRP1decreased MDR1expression in MC3/5FU cells bydecreasing the Transcriptional Activity of the MDR1Promoter in vitroAim: This part was to investigate the role of MRP1in the MDR1regulation pathway.Subjects and method: Reverse transcription polymerase chain reaction (RT-PCR) wasused to check the RNA expression changes. Western blot was used to analysis the proteinexpression changes. Transient Transfections and luciferase reporter assays were made tocheck the relationship between MRP1and MDR1.Results: Down-regulation of MRP1decreased MDR1expression in MC3/5FU cells bydecreasing the Transcriptional Activity of the MDR1Promoter in vitro.Conclusion: Down-regulation of MRP1decreased MDR1expression in MC3/5FU cellsby decreasing the Transcriptional Activity of the MDR1Promoter in vitro. MRP1’s newfunction and its possible unique sequences in MEC made it a promising target in thetherapy of MEC.