Study On Establishment Of The Stably Expressing Human BCRP-COS-7Cell Line

Cancer has become a high incidence of serious harm to human health and life, and itstreatment including chemotherapy, surgery, radiation therapy and biological therapy. Amongthem, drug therapy plays a decisive role in the treatment of neoplastic process. In recent years,the continuous emergence of new anticancer drugs and targeted drug brings new hope forcancer patients. However, drug resistance has become a major bottleneck in the treatment ofcancer. Drug resistance of tumor cell, especially multidrug resistance (MDR) is the mainreason for the failure of chemotherapy, and also an urgent problem need to be solved in thisfield. MDR[1]is that with the devastating complication of cancer cells becomingsimultaneously resistant to many structurally and mechanistically unrelated drugs, the efficacyof chemotherapeutic management of cancer often becomes severely limited. In human tumorcells, several transporter proteins can be involved in MDR. These proteins—P-gp, MRP1,MRP2, MRP3, and BCRP—all belong to the ABC transporter family. They act as effluxpumps, which result in decreased intracellular concentrations of cytotoxic drugs.Breast cancer resistance protein (BCRP) is a principal cause of multidrug resistancetransmembrane protein. BCRP stably expression cell line is the basis of the study associateddrug resistance. In this study, human BCRP CDS was amplified from cDNA of MCF-7/ADRcell, constructed human BCRP expressing plasmid pcDNA3.1(+)-BCRP, transfected COS-7cells and established stable COS-7/BCRP cell lines with600μg/ml of G418. The transportactivity of highly-stably expressing human BCRP was confirmed by efflux of BCRP substratemitoxantrone. The main research contents include the following aspects:.1. The cloning, sequence analysis of human BCRP CDS and construction of thepcDNA3.1(+)-BCRP expressing plasmidTotal RNA from MCF-7/ADR cell line were reverse-transcripted to cDNA. A pair ofprimer was designed to amplify BCRP CDS according to GenBank (BC092408.1). CDSwas amplified with cDNA as templates. CDS was cloned into pMD-19T Simple vectorand sequencing results showed that the amplified CDS was1836bp in length and encoded611amino acids. The DNA sequence and its amino acid sequence showed a completehomology with the reference sequence on GenBank database. Then the CDS wassubcloned into pcDNA3.1(+) vector by Hind III/BamH I,the result vector designatedpcDNA3.1(+)-BCRP.2. pcDNA3.1(+)-BCRP transfection with COS-7and resistant cell clones screeningThe linearized plasmid pcDNA3.1(+)-BCRP were transfected into COS-7cells. After transfection, cells were passaged with a ratio of1:15. The resistant cell colonies were emergedand picked up after G418selection for three weeks.3. BCRP integration and expression identification of G418-resistant cell colonies andbiological activity detectionGenomic PCR, SDS-PAGE were performed to verify the BCRP expressing stably in theG418-resistant cell lines. Mitoxantrone, BCRP efflux substrate, was used to identify theprotein activity. For the study, the incubation time was30min. The lysate samples wereanalysed by LC-MS/MS. Then calculated the concentration of ingredients in cells, comparedthe accumulation per minute by certain protein. The results showed that accumulation ofmitoxantrone was significantly decreased in BCRP highly expressed cell line compared withwild type cell. All data showed that the highly-stably expressing COS-7/BCRP cell line weresuccessfully established. The resulting COS-7/BCRP could be applicated for further study ofBCRP-mediated durg transport.