Yi Wang1, Jiang Cao, Su Zeng. 1. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, Zhejiang Province, China.
Abstract
AIM: To establish a high P-glycoprotein (P-gp) expressing cell line as a model for studying drug absorption and distribution, and to explore the preliminary application of this screening model. METHODS: A full-length MDR1 cDNA fragment in plasmid pMDRA1 was first subcloned into plasmid pET28a(+), then MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/MDR1 was transfected into breast cancer cell line Bcap37 using the Superfect transfection reagent. Several stably transfected clones were obtained after selection with G418. Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the cellular location of the expressed protein was determined by immunohistochemical staining. Drug sensitivity assay was used to evaluate the biological function of expressed P-gp. Concentration of quercetin in cells was determined by high-performance liquid chromatography (HPLC). RESULTS: The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. Real-time fluorescent quantitative RT-PCR showed a higher level of P-gp mRNA in transfected cells compared to that in the control cells, and the Western blot result also indicated that P-gp expression in transfected cells was higher than that in control cells. The immunohistochemical staining showed that the expressed P-gp was localized on cell membranes. Drug sensitivity assay showed that the IC50 for adriamycin and colchicine of the transfected cells was higher than that of the control cells. The concentration of quercetin in model cells was lower than that in control cells by HPLC. After P-gp inhibitor verapamil was administered, the concentration of quercetin in model cells was increased. CONCLUSION: A high P-gp expressing cell line can be established, which could provide a suitable in vitro model system for studying drug intestinal absorption mechanism, predicting the drug permeability characteristics and screening new multi-drug resistance reversing agents. With this model, quercetin can be found to be transported by P-gp, and it is a P-gp substrate.
AIM: To establish a high P-glycoprotein (P-gp) expressing cell line as a model for studying drug absorption and distribution, and to explore the preliminary application of this screening model. METHODS: A full-length MDR1 cDNA fragment in plasmid pMDRA1 was first subcloned into plasmid pET28a(+), then MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/MDR1 was transfected into breast cancer cell line Bcap37 using the Superfect transfection reagent. Several stably transfected clones were obtained after selection with G418. Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the cellular location of the expressed protein was determined by immunohistochemical staining. Drug sensitivity assay was used to evaluate the biological function of expressed P-gp. Concentration of quercetin in cells was determined by high-performance liquid chromatography (HPLC). RESULTS: The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. Real-time fluorescent quantitative RT-PCR showed a higher level of P-gp mRNA in transfected cells compared to that in the control cells, and the Western blot result also indicated that P-gp expression in transfected cells was higher than that in control cells. The immunohistochemical staining showed that the expressed P-gp was localized on cell membranes. Drug sensitivity assay showed that the IC50 for adriamycin and colchicine of the transfected cells was higher than that of the control cells. The concentration of quercetin in model cells was lower than that in control cells by HPLC. After P-gp inhibitor verapamil was administered, the concentration of quercetin in model cells was increased. CONCLUSION: A high P-gp expressing cell line can be established, which could provide a suitable in vitro model system for studying drug intestinal absorption mechanism, predicting the drug permeability characteristics and screening new multi-drug resistance reversing agents. With this model, quercetin can be found to be transported by P-gp, and it is a P-gp substrate.
Authors: K S Lown; R R Mayo; A B Leichtman; H L Hsiao; D K Turgeon; P Schmiedlin-Ren; M B Brown; W Guo; S J Rossi; L Z Benet; P B Watkins Journal: Clin Pharmacol Ther Date: 1997-09 Impact factor: 6.875