Cytoprotective effect of β-lapachone by inducing heme oxygenase-1 expression and AMP-activated protein kinase activation in human endothelial cells.

OBJECTIVES : AMP-activated pro - tein kinase (AMPK) is suggested to exert cytopro - tective and anti-inflammatory effects in endothe - lial cells, but the precise mechanisms are not fully understood. It has been reported that pharmaco - logical activation of AMPK induces endothelial heme oxygenase-1 (HO-1) expression. β-Lapa - chone (BL), a well-known substrate of NAD(P)H: quinone oxidoreductase (NQO1), stimulates AMPK activation via NQO1 activation. Here we ex - amined whether AMPK activation by BL would be linked to HO-1 expression in ECV304 endothelial cells and whether HO-1 expression could mediate the cytoprotective effect of BL. MATERIALS AND METHODS: Endothelial cells were pre-incubated for 6 h with BL or 5- aminoimidazole-4-carboxamide-1- β-D- ribofuranoside (AICAR) in the absence or pres - ence of dicoumarol (DC), compound C (CC), or tin protoporphyrin-IX (SnPP), and then chal - lenged with tumor necrosis factor- α (TNF- α) for 24 h. Cell viability was evaluated by 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. AMPK phosphorylation and HO-1 expression were detected by Western blot analysis. RESULTS: At non-cytotoxic concentrations, BL induced AMPK phosphorylation and HO-1 ex - pression. AICAR, an AMPK activator, also in - duced HO-1 expression. In contrast, CC, an in - hibitor of AMPK activation, and DC, an inhibitor of NQO1, prevented the increase in BL-induced HO-1 expression. Pretreatment with BL or AICAR reduced TNF- α-induced endothelial cell death. Cytoprotection by BL was almost completely abolished by CC and DC and partly by SnPP, a competitive inhibitor of HO-1. CONCLUSIONS: Our results suggest that BL induces cytoprotective HO-1 expression in en - dothelial cells via AMPK activation, providing one of possible mechanisms by which BL can exert beneficial effects.