Abstract
Depsipeptide FK228 [(E)-(1S,4S,10S,21R)-7[(Z)-ethylideno]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone], a novel histone deacetylase (HDAC) inhibitor, previously was reported to be a P-glycoprotein (Pgp) substrate. We now expand the investigation to demonstrate that FK228 is a substrate for Pgp and multidrug resistance-associated protein 1 (MRP1). Transport of FK228 across the Caco-2 cell monolayer in apical to basolateral (AP→BL) and basolateral to apical (BL→AP) directions in the absence and presence of Pgp and MRP inhibitors were investigated. An in vitro uptake study in human red blood cells (RBCs) and a cytotoxicity assay in MRP1(-) HL60 and MRP1(+) HL60Adr cells were conducted to show that FK228 is an MRP1 substrate. An FK228-resistant cell line (HCT15R) was developed from HCT15 colon carcinoma and characterized using a 70-oligomer cDNA microarray, reverse transcription-polymerase chain reaction, Western blot analysis, histone acetyltransferase (HAT) and HDAC activity assays, and cytotoxicity assays. FK228 showed a nearly unidirectional flux across the Caco-2 cell monolayer, with the BL→AP apparent permeability coefficient (Papp) 32 times that of AP→BL without apparent saturation. Pgp inhibition decreased the BL→AP Papp and increased the AP→BL Papp. RBC showed a concentration-dependent uptake and saturable efflux of FK228. HL60Adr cells were 4-fold more resistant to FK228 than HL60 cells, and the resistance was reversed by MRP inhibition. Up-regulation of Pgp, but not changes of MRPs or HAT/HDAC enzymatic activities, was the major mechanism for the acquired FK228 resistance. These studies demonstrate that FK228 is a substrate for Pgp and MRP1, and reversible Pgp up-regulation is predominantly involved in FK228 resistance in vitro.
Footnotes
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This work was supported by National Institutes of Health Grant 1R21CA 96323 and by BioMedical Mass Spectrometry Laboratory at The Ohio State University.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.072033.
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ABBREVIATIONS: FK228, (E)-1S,4S,10S,21R)-7[(Z)-ethylideno]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone; HDAC, histone deacetylase; Pgp, P-glycoprotein; MRP, multidrug resistance-associated protein; RBC, red blood cell; RT-PCR, reverse transcription-polymerase chain reaction; HAT, histone acetyl transferase; MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; CsA, cyclosporin A; Ver, (±)-verapamil hydrochloride; HPLC, high-performance liquid chromatography; HBSS, Hanks' balanced salt solution; DPBS, Dulbecco's phosphate-buffered saline; FBS, fetal bovine serum; AP, apical; BL, basolateral; ABC, ATP-binding cassette; PCR, polymerase chain reaction.
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↵1 Current address: Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD.
- Received May 28, 2004.
- Accepted January 4, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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