Establishment Of A Multidrug Resistant Human Salivary Gland Adenoid Cystic Carcinoma Cell Line And Initial Study Of Its Multidrug Resistance Characteristic

Salivary gland adenoid cystic carcinoma, which comprized 27% of all salivary gland malignant tumors,is one of the most frequently encountered malignance.It is very common in submandibular gland and small-salivary gland and has a high metastatic potential.MDR is the most common impediment to successful chemotherapy for a variety of cancers.MDR characterized by cross resistance to antineoplastic drugs, is mostly caused by over-expression of MDR1 gene encoding P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) transporter superfamily. Althrough P-gp overpression has been associated with multidrug resistance in many tumours and cell lines,there are a number of examples of multidrug resisitant cell lines and tumours where P-gp is not involved.The multidrug resistance-associated proteins (MRPs) are members of ATP-binding cassette superfamily of transporter proteins. The expression of MRPs in normal tissues has been examined using both mRNA and protein ananlysis.MRPs are thought to be like P-gp,having both physiological and a protective role against xenobiotics.As MRP1 is located at the basolateral side of epithelial cells,it tends to pump drugs into the body,rather than away from it via the bile,urine or the gut in the way that P-gp dose. MRP1 has been found overexprssed in multidrug-resisitant cell line derived from many different tissue and tumour types,including small- and large-cell lung cancers,carcinomas of the colon,breast, bladder,prostate,thyroid and cervix,gloma,neuroblastoma,and various forms of leukaemia. Again like P-gp activity,active transport of compounds to ATPase activity,and this has also been shown to be stimulated in isolated membrane vesicles from MRP1-transfected cells,via the addition of the anthracyclines daunomycin and doxorubicin and the Vinca alkaloids vincristine and vinblastine.In this study a multidrug resistant cell line ACC-2/BLM of human salivary gland adenoid cystic carcinoma was established and its biological characteristics was studied.The study included three parts as follows:1. Human salivary gland adenoid cystic carcinoma cells of ACC-2 were treated by repeated 24-hour-exposure to high dose of Bleomycin(20?/ml). Drug sensitivity was evaluated by MTT assay. After 10 month treatment the resistance index (RI) of the cells to BLM, 5-FU,CDDP,CTX and VCR was 7.299, 1.03, 2.15, 1.114 and 5.96 respectively and the cell line was named ACC-2/BLM. The biologicol charecteristics of the cell lines was studied by cell counting, clonogenic assay, electron microscop observation and flow cytometry(FCM). Compared with ACC-2 cells the population doubling time(DT) of ACC-2/BLM was increased,S phase cell number in cell cell cycle decreased , G1 and G2 phase cell number incresed. Clonogenecity of ACC-2/BLM cells increased. Lysosome in ACC-2/BLM cells increased.2. RT-PCR examination revealed that MDR1 mRNA was not expressed in bouth ACC-2 and ACC-2/BLM cells. MRP1 mRNA expression was stronger in ACC-2/BLM cells than in ACC-2. Immunofluorescence assay showed that P-gp was not expressed in bouth ACC-2 and ACC-2/BLM cells. MRP protein expression was stronger in ACC-2/BLM cells than in ACC-23. Two DNA sequences containing a small hairpin structure were designed and synthesized. The complement form was obtained by annealing and cloning into vector pGPU6/GFP/Neo, and the recombinant plasmid was transformed into strain DH5α. The plasmid identified by restriction enzyme analysis was used for sequencing. ACC-2/BLM cells were transfected with plasmid vector Lipofectamine 2000, then RT-PCR was employed to test the expression of MRP1 mRNA in ACC-2/BLM cell lines and selected according to G418. Transfection ratio of The most effective shRNA was selected from the 4 designed shRNAs. The expression of MRP1 mRNA was decreased in ACC-2/BLM cells 72h after trnsfection.4,Comparison between ACC-2 and ACC-2/BLM cells with Gene chip Agilent containning 41000 human gens showed that in ACC-2/BLM cells 514 gens were up-regulated and 469 down- regulated.Conclusion: ACC-2/BLM cell line has the multidrug resistant characteristics. The expression of MRP1 gene may be related to the multidrug resis tance of ACC-2/BLM cells.The recombinant plasmid was cloned, the constructed expression plasmid pGPU6/GFP/Neo-shRNA-D can effectively inhibit the expression of MRP1 mRNA. Multiple genes were involved in the multidrug resistance of ACC-2/BLM cells.