Dipartimento di Specialità Medico-Chirurgiche e Sanità Pubblica, Sezione di Epidemiologia Molecolare ed Igiene Ambientale, Università degli Studi di Perugia, Perugia, Italy.
Eur J Nutr. 2012 Jun;51(4):455-64. doi: 10.1007/s00394-011-0230-3. Epub 2011 Jul 30.
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(+).
最近发表的一些数据表明，羟基酪醇[3,4-二羟基苯乙醇（3,4-DHPEA）]对 HL60 细胞的抗增殖和促凋亡作用可能是通过培养基中过氧化氢（H₂O₂）的积累介导的。本研究的目的是阐明 H₂O₂在 3,4-DHPEA 对乳腺癌（MDA 和 MCF-7）、前列腺癌（LNCap 和 PC3）和结肠癌（SW480 和 HCT116）癌细胞系的化学预防活性中的作用，并研究细胞培养基成分的影响和 3,4-DHPEA 产生 H₂O₂特性的基础上可能的机制。
通过 MTT 测定法测量增殖，通过荧光显微镜和流式细胞术测量凋亡。通过亚铁离子氧化-xylenol 橙法测量培养基中 H₂O₂的浓度。
发现 3,4-DHPEA 的诱导 H₂O₂能力完全被丙酮酸所阻止，并且将细胞暴露于不支持 H₂O₂积累的条件下（向培养基中添加过氧化氢酶或丙酮酸）抑制了 3,4-DHPEA 的抗增殖作用。因此，不同细胞系对 3,4-DHPEA 的抗增殖作用的敏感性与它们从培养基中去除 H₂O₂的能力成反比。关于 3,4-DHPEA 导致 H₂O₂积累的机制，发现超氧化物歧化酶增加了 H₂O₂的产生，而酪氨酸酶、微酸性 pH（6.8）和缺乏氧气（O₂）完全阻止了这种活性。此外，不同的过渡金属螯合物化合物不会改变 3,4-DHPEA 的产 H₂O₂活性。
3,4-DHPEA 的促氧化剂活性深刻影响其“体外”化学预防活性。H₂O₂产生活性的主要起始步骤是 O₂对 3,4-DHPEA 的自动氧化，形成半醌、超氧阴离子（O₂(-)）和 2H(+)。
