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 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 extracellular production of H(2)O(2).

Abstract:In the present investigation the ability of different phenolic compounds, either present or not in olive oil, to induce both apoptosis on tumour cells and H 2 O 2 accumulation in cell culture medium was assesed. Among the phenols studied we found that tyrosol (p-HPEA), homovanillic alcohol and protocatechuic, o-coumaric, vanillic, homovanillic, ferulic and syringic acids did not induce either apoptosis on HL60 cells or H 2 O 2 accumulation, while hydroxytyrosol (3,4-DHPEA), 3,4-dihydroxyphenylacetic acid (3,4-DHPA), 3,4-dihydroxy-hydrocinnamic acid (3,4-DHHC) and gallic acid induced both apoptosis and accumulation of H 2 O 2 in the culture medium which were significantly reduced by catalase. In contrast, the dialdehydic form of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) and to tyrosol (p-DHPEA-EDA) induced high level of apoptosis not reduced by catalase. Finally, oleuropein exerted a weak pro-apoptotic effect not mediated by H 2 O 2 release. From these results it is evident that: (i) the cathecol moiety of phenols is necessary but not sufficient to induce apoptosis and H 2 O 2 accumulation; (ii) the 3,4-DHPEA metabolism may partially reduce its pro-apoptotic potential; (iii) the pro-apoptotic activity of 3,4-DHPEA-EDA and p-DHPEA-EDA is not mediated by H 2 O 2 releasing activity.