Selective Downregulation of the MDR1 Gene Product in Caco-2 Cells by Stable Transfection To Prove Its Relevance in Secretory Drug Transport

Abstract

Considerable interest is focused on overcoming multidrug resistance (MDR) in cancer chemotherapy. The in vitro experiments to characterize P-glycoprotein's (P-gp) function and to decrease its effects have led to a variety of strategies such as addition of competitors or supplementation of the medium with oligonucleotides complementary to the 5'-end of the MDR1-mRNA. For the Caco-2 cell line, an in vitro model for absorption screening, expressing multiple transporters including P-gp, which pumps substances back into the apical solution, P-gp activity might mask other relevant transport proteins' activity. The objective of the present study was to construct a Caco-2 subline with reduced P-gp expression level. Caco-2 cells were transfected by electroporation with two different mammalian expression vectors, and the obtained subclones were investigated at RNA (Northern blotting, RT-PCR), protein (FACS analysis), and functional (transport studies) levels for reduction in P-gp expression. Northern blotting showed that the levels of transcription of the inserted gene were different among the several clones, but those results did not completely correlate with the FACS analysis for P-gp expression. The clones with the strongest reduction in P-gp expression detected by the FACS analysis also showed the lowest secretory fluxes of the P-gp substrate talinolol in transport studies. Repetition of FACS analysis after 7 and 24 months on 20 to 30 passage older subclones still showed reduction in P-gp expression and indicated that they are stably transfected. The new cell lines constructed in the present study provide the possibility to perform in vitro absorption studies in a cell system composed of differentiated enterocytes growing as a monolayer like the normal Caco-2 cell line but with a lower down to almost lacking expression of P-gp.