Abstract
This study describes the antiapoptotic action of (2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31378), a novel benzopyran analog, in human umbilical vein endothelial cells (HUVECs) in comparison with its acetylated metabolite, (2S,3S,4R)-N"-cyano-N-(6-acetylamino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31612), and with α-tocopherol. Exposure of HUVECs to lipopolysaccharide (LPS) (1 μg/ml) induced time- and concentration-dependent cytotoxicity and oligonucleosomal DNA fragmentation. KR-31378, KR-31612, and α-tocopherol potently suppressed LPS-induced cell death in association with significant reduction in the intracellular reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) that are stimulated by LPS. KR-31378 more effectively protected HUVECs from LPS-induced DNA fragmentation and was more effective in peroxyl radical scavenging than α-tocopherol. Incubation with LPS markedly decreased the Bcl-2 level, which was totally reversed by KR-31378 and to a lesser degree by KR-31612 and by α-tocopherol. In contrast, the greatly increased Bax protein and cytochrome c release stimulated by LPS were markedly suppressed by KR-31378 and by KR-31612, and to a lesser degree by α-tocopherol. Taken together, KR-31378 strongly inhibited cell death in HUVECs in association with antiapoptotic effects, which were accompanied by up-regulation of Bcl-2 protein expression and down-regulation of Bax protein and suppression of cytochromec release. KR-31378 also showed the properties to scavenge the intracellular ROS and peroxyl radicals, and to reduce the TNF-α production induced by LPS.
Footnotes
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This study was supported with funding from the Center for Bioactive Substances, Korea Research Institute of Chemical Technology, Daejon, and from the Korea Science and Engineering Foundation, Korea.
- Abbreviations:
- LPS
- lipopolysaccharide
- TNF-α
- tumor necrosis factor-α
- ROS
- reactive oxygen species
- HUVEC
- human umbilical vein endothelial cell
- MTT
- mitochondrial tetrazolium
- DCFH-DA
- 2′,7′-dichlorofluororescine diacetate
- DCFH
- 2′,7′-dichlorofluororescine
- PRAC
- peroxyl radical absorbing capacity
- AAPH
- 2,2′-azobis(2-amidino-propane) dihydrochloride
- β-PE
- β-phycoerythrin
- Received August 9, 2001.
- Accepted November 2, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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