Construction of a functional transporter analysis system using MDR1 knockdown Caco-2 cells.
OBJECTIVE
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.
Watanabe
Tomoko
T
Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Onuki
Reiko
R
Yamashita
Shinji
S
Taira
Kazunari
K
Sugiyama
Yuichi
Y
D001693:Biological Transport, Active; D015153:Blotting, Western; D018938:Caco-2 Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015723:Gene Library; D019450:Genes, MDR / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007408:Intestinal Absorption; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008954:Models, Biological; D030781:Organisms, Genetically Modified; D034622:RNA Interference / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics; D012366:RNA, Transfer, Val / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012366:RNA, Transfer, Val; D014700:Verapamil