| Mucoepidermoid carcinoma (MEC) is the most common malignacy in salivary glands and accounts for about 30% of malignant tumors in salivary glands in Chinese population. Although it sometimes shows a slow growth resembling a benign lesion, this neoplasm can be highly aggressive with a dismal prognosis. A 5-year disease-free survival rate between 0 and 43% has been demonstrated in the patients with high-grade tumors.A major reason of failure in chemotherapy was multidrug resistance (MDR) of tumor. Multidrug resistance is the phenomenon that when a certain kind of tumor is resistant to a certain kind of antineoplastic drug, it can also be resistant to different kinds of drugs with different structures and mechanism of action. This phenomenon can be either inborn or induced after birth. Many different mechanisms cause MDR. Now, based on the common biology , it is assumed that P-gp is over programmed by MDR-1 gene, and it is combined with drugs or remove the drugs directly from cell envelope. Then the toxicity of the drugs is cut down.. We assume that MDR-1 may be involved in the muti drug resistance of MEC. Therefore, in this study, we established and characterized a multi drug resistant cell line Mc3/5-FU derived from highly metastatic human salivary gland mucoepidermoid carcinoma cell line Mc3. We examined the drug resistency and the expression of MDR-1 gene and P-gp protein in the cells. Furthermore, we constructed MDR-1-shRNA expression vector and silenced MDR-1 in Mc3/5-FU cells.The study includs two parts as follows:1. Establishment of a multidrug resistant human salivary gland Mucoepidermoid Carcinoma Mc3/5-FU Cell line and it's biological chaicteristicsMc3 cells were planted into athymic mice and the mice were given 5-FU by intraperitoneal injection. After in vivo induction the tumor cells were in vitro cultured and exposed to 5-FU at a constant concentration The in vivo and in vitro induction was repeted regularly and a cell line resitent to 5-FU was obtained and named Mc3/5-FU. The morphology of the cells was studied by microscopy and electron microscopy, cell proliferation by cellcounting, drug resistence by MTT assay. The expression of MDR-1 gene was examined by RT-PCR, P-gp protein expression by and immunofluorescence technique. The morphology of Mc3 and Mc3/5-FU cells was not obviously different. The population doubling time of Mc3 and Mc3/5-FU cells was 60h and 55h respectively. The resistance index of Mc3/5-FU cells to 5-FU, BLM, VCR, VBL and CDDP was 13.3, 4.2, 6.4, 6.9 and 0.72 respectively. PCR electrophoresis assay showed that expression of MDR-1 gene was higher in Mc3/5-FU cells than in Mc3. Mc3/5-FU was p-gp positive while Mc3 was negative. MRP-1 gene and MRP protein were expressed stronger in Mc3/5-FU cells than in Mc3.2. Establishment of stably transfected cell line by MDR-1-shRNA eukaryotic expression vector .The plasmid expressing shRNA homologous to MDR-1 mRNA was constructed with shRNA expressing vector pRNAT-U-6.1/Neo based on the principle of siRNA design.The combinant plasmid was transfected stably into Mc3/5-FU Cells using Lipofectamine 2000. Finally, the shRNA efficiency was detected by RT-PCR analysis. We found that MDR-1-shRNA could effectively block the expression of MDR-1 in Mc3/5-FU cells .Conclusion:1. Mc3/5-FU is multi drug resistant.2. MDR-1 gene was overexpressed in Mc3/5-FU cells and P-gp protein was expressed in Mc3/5-FU but not in Mc3 cells.. MRP-1 gene and MRP protein were expressed stronger in Mc3/5-FU cells than in Mc3.3. Regular repeted in vivo and in vitro induction with anti cancer drug may be a effective way to establish multi drug resistant cell line .4. Transfection of Mc3/5-FU cells with MDR-1-shRNA may block the expression of MDR-1 gene.
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