Effects Of Chicken Breast Cancer Resistant Protein On Drug Transmembrane Transport And Its Transcription Regulatory Mechanism

Studies in humans and rodents have shown that drug transport mediated by P-gp and BCRP may affect the drug pharmacokinetics and mediate drug-drug interactions,therefore,P-gp and BCRP draw a significant interest in the drug development.However,no detailed information is as yet available on chicken P-gp and BCRP.In this study,BCRP single-and BCRP/P-gp double transfected MDCK cell lines(named MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1,respectively)were successfully constructed using the lentiviral vector system.Then,use the constructed cell lines to screen the substrates of chicken BCRP and P-gp,and further to study the effects of the transports in the transmembrane transport of their substrates.Finally,we discussed the transcriptional regulation mechanism of chicken Abcg2 gene and screened compounds that could regulate the chicken Abcg2 gene promoter.The main contents and results are involved as follows.1.Establishment of MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1 cell linesIn this study,BCRP single and BCRP/P-gp double-transfected MDCK cell lines were generated to develop reliable systems for screening the substrates of these two transporters and to study the interplay between them.The Abcb1 and Abcg2 complementary DNAs(cDNAs)were amplified from ileum of AA broilers first and then were inserted into the lentiviral expression vector.The generated recombinant lentiviral expression vector and lentiviral packaging plasmid were transferred to 293T cells by Lentifectin,generating the viruses containing full-length chicken Abcb1 cDNA and Abcg2 cDNA that named lentiviral-Abcb1 and lentiviral-Abcg2,separately.Lentiviral-Abcb1 and lentiviral-Abcg2 were used to infect MDCK cells and the stable transfectants were selected with 1 μg/mL puromycin and 1200 μg/mL neomycin,separately.Finally,BCRP single and BCRP/P-gp double-transfected MDCK cell lines were selected by limited dilution method.The expression levels of chicken Abcg2 and Abcb1 genes and proteins in MDCK cells were detected through RT-PCR,western blot and fluorescent label protein analysis,and further,the functions of overexpressed proteins were identified by corresponding substrate accumulation and toxicity assays.Results showed that MDCK cells overexpressing BCRP named MDCK-chAbcg2 cell line could overexpressed chicken BCRP compared with MDCK cells,and the proportions of positive cells in 10-and 50-cell passages were 98.7%and 97%,respectively.Compared with MDCK cells,the fluorescence intensity of accumulated mitoxantrone in MDCK-chAbcg2 cells decreased significantly(P<0.01),however,significantly increased in the presence of Ko143,GF120918 and gefitinib,respectively(P<0.01).MDCK-chAbcg2 cells were more resistant to mitoxantrone-induced cell death compared to the MDCK cells with IC50 increased from 1.4 to 5.6,and no significant alterations of BCRP-mediated mitoxantrone transport activity were observed over a period of 50 culture passages.The above results show that the MDCK-chAbcg2 cell line were successfully constructed.MDCK cells overexpressing both BCRP and P-gp named MDCK-chAbcg2/Abcb1 cell line could overexpressed chicken Abcg2 mRNA,Abcb1 mRNA,BCRP and P-gp compared with MDCK cells,and the proportions of positive cells in 10-and 50-cell passages were 95.3%and 94.6%,respectively.Compared with MDCK cells,the fluorescence intensity of accumulated mitoxantrone in MDCK-chAbcg2/Abcb1 cells decreased significantly(P<0.01),however,significantly increased in the presence of Ko143(P<0.01).Similarly,the fluorescence intensity of accumulated Rhol23 in MDCK-chAbcg2/Abcb1 cells decreased significantly(P<0.01),however,significantly increased(P<0.01)in the presence of verapamil.MDCK-chAbcg2/Abcb1 cells were resistant to mitoxantrone-and doxorubicin-induced cell death compared to the MDCK cells with IC50 increased from 1.3 to 6.9 and 0.5 to 1.9,respectively(P<0.01).No significant alterations of BCRP-and P-gp-mediated mitoxantrone and doxorubicin transport activities were observed over a period of 10 and 50 culture passages(P>0.05).In conclusion,BCRP single-and BCRP/P-gp double transfected MDCK cell lines were successfully constructed.These constructed cell models provide useful systems for high-throughput screening of the potential substrates of chicken BCRP and P-gp as well as the drug-drug interaction mediated via chicken BCRP and P-gp,which could provide scientific guidance for clinical medication.2.Screen the substrates of chicken BCRP and P-gp and study the transmembrane transport of the substratesThe purpose of this study is to screen the substrates of chicken BCRP and P-gp,and further to study the roles of the transports in the transmembrane transport of their substrates,which could provide scientific guidance for clinical medication.In this study,The MDCK-chAbcg2 and MDCK-chAbcb1 cells were used to screen the substrates of chicken BCRP and P-gp based on net efflux ratios(NERs)firstly,and then MDCK-chAbcg2/Abcb1 cells were used to study the transmembrane transport of the cosubstrates of P-gp and BCRP by setting up different drug concentration groups and different inhibitor groups.Results showed that enrofloxacin,ciprofloxacin,tilmicosin,sulfadiazine,ampicillin and clindamycin were classified as the substrates of chicken P-gp as their NERs were greater than two.Similarly,enrofloxacin,ciprofloxacin,tilmicosin,florfenicol,ampicillin and clindamycin were classified as the substrates of BCRP.However,ceftiofur,amoxicillin and doxycycline were not substrates of either chicken BCRP or the substrates of chicken P-gp as their NERs were smaller than two.It was also found that enrofloxacin,ciprofloxacin,tilmicosin,ampicillin,and clindamycin were the cosubstrates of chicken P-gp and BCRP.MDCK-Abcg2/Abcb1 cells were further used to study the roles of BCRP and P-gp in the transmembrane transport of their cosubstrates.Results showed that BCRP was the major determinant for cellular efflux transport of enrofloxacin at low concentration(2 μM)with the NER of 2.65,while P-gp is responsible for the enrofloxacin efflux at 10μM with the NER of 1.87.However,when the concentration of enrofloxacin was increased to 50 μM,the NERs were close to 1,indicating that both transporters were fully saturated.Both transporters contribute about equally to the active transport for ciprofloxacin and ampicillin over a large concentration range.For tilmicosin,BCRP and P-gp contribute almost equally to the active transport at low concentration(10 μM)with corresponding NERs of 4.26 and 4.54.Upon increasing concentration of tilmicosin to 50μM,P-gp-mediated transport was significantly higher that of BCRP-mediated with NER of 3.99.Clindamycin showed high affinity with P-gp than BCRP at all tested concentrations.In conclusion,some of the commonly used antimicrobial agents licensed in veterinary medicine are also substrates of chicken P-gp or BCRP,and their transmembrane transport process is affected by P-gp or BCRP.Therefore,the effect of these transporters on the disposition of drug in the body should be considered in clinical medication.3.Characterization analysis and regulators identification of chicken abcg2 gene promoterAbcg2 gene encodes an ABC half transporter named breast cancer resistance protein(BCRP)that influence the pharmacokinetics and toxicity of substrate drugs during clinical therapy.In order to clarify the regulate transcription mechanisms of chicken Abcg2 gene,we cloned and characterized its promoter region,and then the core promoter region and the positive and negative regulatory regions of chicken Abcg2 gene were screened by a series of promoter truncation and mutant fragment plasmids.Then through luciferase assay combined with RT-PCR and substrate accumulation assays,we screened the exogenous compounds which could affect the expression of chicken BCRP gene and protein function by regulating the promoter activity of chicken Abcg2 gene.Finally,we also try to identify the interaction sites with Abcg2 gene promoter of these four selected regulators by progressive deletions and mutations assay.Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Spl sites,which are upstream from two putative CpG islands.Luciferase reporter assay conducted both in LMH cells and chicken primary hepatocytes mapped a basal promoter to nucleotides from-110 bp to+30 bp,that responsible for the constitutive expression of Abcg2.CXR,ER,GR and SP1 transcription factor binding sites positively regulate the activity of chicken Abcg2 promoter,while NF-κB transcription factor binding sites negatively regulate the activity of chicken Abcg2 promoter.The results of luciferase reporter gene experiment showed that ivermectin,daidzein,berberine,rifampicin,clotrimazole,enrofloxacin,tilmicosin,sulfadiazine,doxycycline and ceftiofur activated the promoter activity of chicken Abcg2 gene.The activation effects in LMH cells were 1.4-3.2 times that in control group(P<0.05),while 1.5-4.9 times in chicken primary hepatocytes(P<0.05).However,LPS significantly inhibited the activity of promoter of chicken Abcg2 gene,and the inhibitory effects of LPS on LMH cells and chicken primary hepatocytes were 0.56 and 0.6 times of those in control group respectively(P<0.05).RT-PCR results showed that berberine,tilmicosin and LPS significantly reduced the expression of Abcg2 gene in LMH cells by 0.59,0.66 and 0.56 times(P<0.05),while 0.70,0.73 and 0.25 times(P<0.05)in chicken primary hepatocytes.However,daidzein,clotrimazole,enrofloxacin,sulfadiazine,ceftiofur,doxycycline and ivermectin significantly increased the expression level of chicken Abcg2 gene in LMH cells by 1.40-5.57 times as compared with the control group(P<0.05),and 1.25-1.92 times as compared with the control group in chicken primary hepatocytes(P<0.05).Mitoxantrone accumulation test showed that the fluorescence intensity of mitoxantrone in berberine and LPS treated cells increased significantly to 157%and 121%of the control group(P<0.05),while that in daidzein,clotrimazole and ivermectin treated cells decreased significantly to 71%,75%and 69%of the control group(P<0.01).In conclusion,luciferase reporter assay combined with PCR and mitoxantrone accumulation assay showed that daidzein,clotrimazole,ivermectin and LPS could significantly affect the expression and protein function of chicken BCRP gene by regulating the promoter activity of chicken Abcg2 gene.Therefore,we further screened the sites where these four compounds interact with the chicken Abcg2 gene promoter.The results showed that daidzein and clotrimazole may regulate the promoter activity of chicken Abcg2 gene through CXR transcription factor binding site,Ivermectin and LPS may participate in the transcription regulation of chicken Abcg2 gene through Spl and NF-κB transcription factor binding sites respectively.These above results provide a theoretical basis for further study of transcriptional regulation of chicken BCRP.In conclusion,BCRP single and BCRP/P-gp double-transfected MDCK cell lines named MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1 were successfully constructed in this study.The MDCK-chAbcg2 and MDCK-chAbcb1 cell lines were used to successfully screen the substrates of chicken BCRP and P-gp.The MDCK-chAbcg2/Abcb1 cells was used to study the effects of the transports in transmembrane transport of their substrates The transcriptional regulation mechanism of chicken Abcg2 gene was also reported in this paper.The results of this study enrich the basic theory and database of chicken transporter research field,and provide theoretical basis for rational drug use in chicken clinic.