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 H₂O₂ in the culture medium was measured by the ferrous ion oxidation‐xylenol orange method. The pro‐apoptotic effect of 3,4‐DHPEA (100 μM) was prevented by N‐acetyl‐cysteine, ascorbate, and α‐tocopherol. Catalase suppressed the 3,4‐DHPEA‐induced apoptosis, while the Fe(II)‐chelating reagent o‐phenantroline showed no effect, suggesting the involvement of H₂O₂ but not of OH^?. Indeed, 3,4‐DHPEA caused accumulation of H₂O₂ in the culture medium. Tyrosol (p‐hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4‐DHPEA but not able to generate H₂O₂, did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4‐DHPEA is mediated by the extracellular production of H₂O₂.