Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid.
It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.
El-Masry
Eman M
EM
Department of Pharmacology, Duke University Medical Center, Box 3813, Durham, NC 27710, USA.
Abou-Donia
Mohamed B
MB
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors*; D000970:Antineoplastic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015153:Blotting, Western; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D019450:Genes, MDR / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D037121:Plant Lectins / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D027981:Symporters
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D000975:Antioxidants; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D027981:Symporters; C034981:tomato lectin; C021499:lactose permease; D001205:Ascorbic Acid