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

S.J. Byun, Y. Son, H.-O. Pae

Department of Surgery, Wonkwang University School of Medicine, Iksan, Republic of Korea. hopae@wku.ac.kr

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• Pharmacology

OBJECTIVES: AMP-activated protein kinase (AMPK) is suggested to exert cytoprotective and anti-inflammatory effects in endothelial cells, but the precise mechanisms are not fully understood. It has been reported that pharmacological activation of AMPK induces endothelial heme oxygenase-1 (HO-1) expression. β-Lapachone (BL), a well-known substrate of NAD(P)H: quinone oxidoreductase (NQO1), stimulates AMPK activation via NQO1 activation. Here we examined 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 presence of dicoumarol (DC), compound C (CC), or tin protoporphyrin-IX (SnPP), and then challenged 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 expression. AICAR, an AMPK activator, also induced HO-1 expression. In contrast, CC, an inhibitor 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 endothelial cells via AMPK activation, providing one of possible mechanisms by which BL can exert beneficial effects.

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