| Salivary gland adenoid cystic carcinoma(ACC) and mucoepidermoid carcinoma(MEC) are the most frequently encountered malignance. The majour treatment of the tumors is surgery, chemotherapy is used for the advanced caces of those with metastasis. But multidrug resistance (MDR) reduces the effectiveness of chemotherapy. MDR is characterized by cross resistance to antineoplastic drugs, and the members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCB1(MDR1), ABCC1(MRP1), play the most importent role in MDR.In priviouse study multidrug resistance cell lines of ACC-2/BLM and MC3/5-FU were established, high expression of ABCC1(MDR1) gene was found in ACC-2/BLM cells. In this study high expression of ABCC1 was found in MC3/5-FU cells. RNAi method was used to silence the ABCC1 gene in ACC-2/BLM and MC3/5-FU cells, ABCC1 expresse was inhibited and the multidrug resistance of the cells was reversed. Methods and results:1. ACC-2/BLM and MC3/5-FU were repetedly treated by in vitro and in vivo induction of Bleomycin(BLM) and 5-Fluorouracil(5-Fu) respectively for 1 year.. The MDR of ACC-2/BLM to BLM, 5-FU, CDDP, CTX and VCR was increased by 33.3%, 29.7%, 5.3%, 14.7% and 10.0% respectively. The MDR of MC3/5-FU to 5-FU, BLM, VBL, VCR, and CDDP was increased by 171.5%, 73.1%, 41.2%, 20.1% and 634.0% respectively.2?Gene express profile of ACC-2/BLM vs ACC-2 cells, MC3/5-FU vs MC3 was examined with Gene chip Agilent containing 45,015 human gens. The data were analysed with DAVID Bioinformatics Resources 6.7 In ACC-2/BLM cells 514 genes were up-regulated(Ratio>2.5), 466 were down- regulated (Ratio<0.4). In MC/5-FU cells 198 genes were up-regulated, 384 were down- regulated. The differentely expressed genes with the Enrichment Score (ES) >1 were analysed by GO . Up-regulated genes in the cells with MDR includes those related to growth factor, extracellular matrix, regulation of cell adhesion,angiogenesis and so on. Down- regulated in the cells with MDR includes those related to response to endogenous stimulus or drug, apoptosis, and so on.3. RT-PCR revealed that CXCR4, HIF1,CA9A and ABCC1 were Up-regulated in ACC-2/BLM and MC3/5-FU cells,TGM2,AIF and P53 were down- regulated. The results of RT-PCR were partly inconsistent with that of gene chip examination.4. In priviouse study shRNAs targeting to ABCC1 gene were designed and constructed into pGPU6/GFP/Neo plasmide. In this study the plasmide countaining ABCC1 shRNAs were tranfected into ACC-2/BLM and MC3/5-FU cells by Lipofectamine 2000. The transfection effecincy to ACC-2/BLM cells was 40-50%,to MC3/5-FU about 30%?RealTime-PCR showed shRNA4 was the most effective in the inhibition of ABCC1 mRNA expression in the cells. The transfected cells were cloned and cultrued. Cell proliferation , clonogenecity and drug sensitivity were examined. The ABCC1 mRNA and protien expression in the transfected cells were decreased, cell proliferation were slowed. The drug resistence of transfected ACC-2/BLM cells to BLM, 5-FU, CDDP, CTX and VCR was reversed by 92.55%, 63.48%, 50.42%, 11.38% and 81.13% respectively;In the in vivo (in nude mice) study the drug resistence of ACC-2/BLM cells to BLM was reversed by 43.8%.The drug resistence of transfected MC3/5-FU cells to CTX,VCR,BLM,CDDP and 5-FU was reversed by 34.7%, 16.4%, 30.1%, 69.9% and 97.9% respectively. In the in vivo (in nude mice) study the drug resistence of MC3/5-FU cells to 5-FU was reversed by 42%?Conclusion:In vitro and in vivo induction may increase the multidrug resistance potencial of ACC-2/BLM and MC3/5FU cells. The MDR of ACC-2/BLM and MC3/5FU cells is related to multile genes. ABCC1 gene plays important role in the MDR of ACC-2/BLM and MC3/5FU cells. Silence of ABCC1 gene by RNAi may inhibit the expression of ABCC1 mRNA and protien, reverse the MDR of ACC-2/BLM and MC3/5FU cells. |
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