title: Inhibition of expression of the multidrug resistance-associated P-glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate antisense oligonucleotides.
abstract: Multiple drug resistance (MDR) as a result of overexpression of the P-glycoprotein drug transporter, a product of the MDR1 gene, is a significant problem in cancer therapeutics. We demonstrate that phosphorothioate antisense oligonucleotides can reduce levels of MDR1 message, inhibit expression of P-glyco protein, and affect drug uptake in MDR mouse 3T3 fibroblasts. An obligonucleotide (5995) directed against a sequence overlapping the AUG start codon was effective in reduction MDR1 transcript and protein levels when used at submicromolar concentrations in conjunction with cationic liposomes, whereas a scrambled control oligonucleotide (10221) was ineffective. Substantial and specific antisense effects could also be attained with a 5' cholesterol conjugate of the 5995 sequence. In this case, use of cationic liposomes was unnecessary. The 5' cholesterol 5995, but the not 5' cholesterol 10221, reduced MDR1 message and P-glycoprotein levels by 50-60% when used at low micromolar concentrations. In parallel, treatment with 5' cholesterol 5995 also enhanced cellular accumulation of rhodamine 123, a well-known substrate of the P-glycoprotein transporter. The effectiveness of the cholesterol-conjugated 5995 may be due to its rapid and extensive cell uptake, as indicated in flow cytometry and confocal microscopy studies. These observations suggest that cholesterol-conjugated anti-sense oligonucleotides may offer a novel approach to inhibition of P-glycoprotein-mediated MDR and to the modulation of other tumor cell genes whose overexpression contributes to the neoplastic state or to resistance to therapy.
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Alahari
authors.forename: N M
authors.initials: NM
authors.lastname: Dean
authors.forename: M H
authors.initials: MH
authors.lastname: Fisher
authors.forename: R
authors.initials: R
authors.lastname: Delong
authors.forename: M
authors.initials: M
authors.lastname: Manoharan
authors.forename: K L
authors.initials: KL
authors.lastname: Tivel
authors.forename: R L
authors.initials: RL
authors.lastname: Juliano
mesh_terms: D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000818:Animals; D001483:Base Sequence; D002784:Cholesterol / Q000493:pharmacokinetics / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology*; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics; D016914:Ribonuclease H / Q000502:physiology; D013873:Thionucleotides / Q000493:pharmacokinetics / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012235:Rhodamines; D013873:Thionucleotides; D020112:Rhodamine 123; D002784:Cholesterol; D016914:Ribonuclease H