Purpose: The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption.
Methods: The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA(val)-shRNA expression vector.
Results: In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells.
Conclusions: MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.