Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide.

Several recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H₂O₂) in the culture medium. The aim of this study was to clarify the role played by H₂O₂ in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H₂O₂-producing properties of 3,4-DHPEA. The proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. It was found that the H₂O₂-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H₂O₂ accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H₂O₂ from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H₂O₂ accumulation, it was found that superoxide dismutase increased the H₂O₂ production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O₂) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H₂O₂-producing activity of 3,4-DHPEA. The pro-oxidant activity of 3,4-DHPEA deeply influences its 'in vitro' chemopreventive activities. The main initiation step in the H₂O₂-producing activity is the auto-oxidation of 3,4-DHPEA by O₂ with the formation of the semiquinone, superoxide ions (O₂(-)) and 2H(+).