Establishment Of Cell Line Highly Expressing BCRP And Investigation Of Its Drug Resistant Mechanism

Objects: To establish a cell line expressing BCRP and investigate the mechanism of its drug resistance.Methods: The aimed segments were isolated from the cell line Mcf-7/Adr by the method of reverse transcription polymerase chain reaction and were inserted into a eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/BCRP. The recombinant plasmid, first propagated in Escherichia coli DH5α,then extracted,purified and digested with BamH I and Xho I,was confirmed to contain full length of BCRP cDNA by agarose gel analysis and DNA sequence analysis.Then the PA317 cells were transfected with the plasmid using electroporation,while the cells The rate of transfection was observed by the cells which were transfected with the plasmid pcDNA3.1/EGFP.Then the transfected cells were screened with antibiotic G418. Single clones expressing BCRP were obtained by limited-dilution method. The mRNA expression of BCRP in positive clones were detected by RT-PCR. While,the expression of BCRP protein in the positive clones was evaluated by Western blot and immunohistochemistry. When we got the drag-resistance cell line,we did the cell venenosus trial with MTZ and excretion of Rho123 trial to indicate the BCRP protein function.In order to investigate the mechanism of its drug resistance, we detected the Akt protein of PI3K-Akt pathway.Results: A recombinant eukaryotic expression plasmid PCDNA3.1-BCRP was successfully constructed. Then the mixed PA317 clones expressing BCRP were selected by G418 for two weeks after transfection.Through limited-dilution cloning, three single clones were obtained,and the best one named as B11.The BCRP mRNA transcription was detected by RT-PCR in the positive clone. The BCRP protein was identified from the whole cell protein of PA317/BCRP by Western blot and immunohistochemistry.The cell venenosus trial with MTZ showed that the cell PA317/BCRP had the lower inhibition ratio than the cell PA317 and PA317/pcDNA3.1. The excretion of Rho123 trial showed that the PA317/BCRP had higher excretion. Thus we proved that the cell line was stably established.Further more, we found that the akt protein had higher expression in PA317/BCRP, contrst to the cell PA317 and PA317/pcDNA3.1.It may indicated that PI₃K-Akt pathway activation had relation with the drug resistance.Conclusion: A cell line expressing the BCRP protein stably was established. It provided a useful tool to investgate the role of BCRP in the multidrug resistance of oncotherapy. Further more,we found that the Akt protein was up-regulated in the PA317/BCRP, contrst to the cell PA317 and PA317/pcDNA3. It may indicated that PI3K-Akt pathway activation had relation with the drug resistance of BCRP.It gave us some information which we can use to discover some new drugs to resist its drug resistance.