Prevention of lipopolysaccharide-induced apoptosis by (2S,3S,4R)-N″-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2- dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine, a benzopyran analog, in endothelial cells

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 downregulation of Bax protein and suppression of cytochrome c 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.