Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells.

Hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth-suppressive and pro-apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro-apoptotic activity of 3,4-DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4-DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V-FITC kit. The concentration of H(2)O(2) in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. The pro-apoptotic effect of 3,4-DHPEA (100 muM) was prevented by N-acetyl-cysteine, ascorbate, and alpha-tocopherol. Catalase enzyme suppressed the 3,4-DHPEA-induced apoptosis, while the Fe(II)-chelating reagent o-phenantroline showed no effect, suggesting the involvement of H(2)O(2 )but not of OH(*). Indeed, 3,4-DHPEA caused accumulation of H(2)O(2) in the culture medium. Tyrosol (p-hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4-DHPEA but not able to generate H(2)O(2), did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4-DHPEA is mediated by the production of H(2)O(2) in the culture medium.