Abstract
5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2′-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5′-adenosyl)-l-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,α-tosyl-l-phenylalanine chloromethyl ketone, N,p-tosyl-l-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.
Footnotes
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This study was supported by the Deutsche Forschungsgemeinschaft [Grant KU 1340]; and by Deutsche Forschungsgemeinschaft Research Training Group [Grant IRTG 1331].
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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.108.143560.
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ABBREVIATIONS: 5-aza-CR, 5-azacytidine; 5-aza-dCR, 5-aza-2′-deoxycytidine; TLCK, N,p-tosyl-l-lysine chloromethyl ketone; TPCK, N,α-tosyl-l-phenylalanine chloromethyl ketone; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; DR, death receptor; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ActD, actinomycin D; CHX, cycloheximide; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone; NF, nuclear factor; RG 108, 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)proprionic acid.
- Received July 15, 2008.
- Accepted September 29, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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