Original Contribution Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells Stefania D’Angelo a , Diego Ingrosso a, * , Valentina Migliardi a , Alvara Sorrentino a , Giovanna Donnarumma b , Adone Baroni c , Lucia Masella a , Maria Antonietta Tufano b , Marcello Zappia a , Patrizia Galletti a a Department of Biochemistry and Biophysics bF. Cedrangolo,Q Medical School, Second University of Naples, Naples, Italy b Department of Experimental Medicine, Microbiology and Clinical Microbiology, Medical School, Second University of Naples, Naples, Italy c Department of Dermatology, Medical School, Second University of Naples, Naples, Italy Received 2 July 2004; revised 9 November 2004; accepted 10 December 2004 Available online 13 January 2005 Abstract Previous studies showed that long-wave ultraviolet (UVA) radiation induces severe skin damage through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. Recent results from our laboratory indicate a dramatic increase of both lipid peroxidation products (TBARS) and abnormal l-isoaspartyl residues, marker of protein damage, in UVA-irradiated human melanoma cells. In this study, the effects of hydroxytyrosol (DOPET), the major antioxidant compound present in olive oil, on UVA-induced cell damages, have been investigated, using a human melanoma cell line (M14) as a model system. In UVA-irradiated M14 cells, a protective effect of DOPET in preventing the uprise of typical markers of oxidative stress, such as TBARS and 2V 7V -dichlorofluorescein (DCF) fluorescence intensity, was observed. In addition, DOPET prevents the increase of altered l-isoAsp residues induced by UVA irradiation. These protective effects are dose dependent, reaching the maximum at 400 AM DOPET. At higher concentrations, DOPET causes an arrest of M14 cell proliferation and acts as a proapoptotic stimulus by activating caspase-3 activity. In the investigated model system, DOPET is quantitatively converted into its methylated derivative, endowed with a radical scavenging ability comparable to that of its parent compound. These findings are in line with the hypothesis that the oxidative stress plays a major role in mediating the UVA-induced protein damage. Results suggest that DOPET may exerts differential effects on melanoma cells according to the dose employed and this must always be taken into account when olive oil-derived large consumer products, including cosmetics and functional foods, are employed. D 2004 Elsevier Inc. All rights reserved. Keywords: Hydroxytyrosol; Melanoma cells; Protein damage; Protein deamidation; Protein l-isoaspartyl methyltransferase; UVA radiation; Free radicals Introduction The peculiar climate of the Mediterranean basin, charac- terized by warm and prolonged sunlight irradiation, favors development of plants, such as olive trees and grape, whose fruits require a high proportion of antioxidant molecules [1]. The synthesis of pigments such as flavonoids, anthocyanins, and polyphenols, activated by sun irradiation [2,3], results in dark-colored fruits that, by this way, protect themselves from the noxious effects of prolonged exposure to sunlight. A Mediterranean diet, rich in fruits and vegetables, grants an elevated intake of these antioxidants that may contribute to its beneficial effects on human health. A number of epidemio- 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.12.015 Free Radical Biology & Medicine 38 (2005) 908 – 919 www.elsevier.com/locate/freeradbiomed Abbreviations: AdoHcy, S-adenosylhomocysteine; AdoMet, S-adeno- sylmethionine; COMT, catechol-O-methyltransferase; DCF, 2V 7V -dichloro- fluorescein; DCFH-DA, 2V -7V -dichlorofluorescein diacetate; DOPET, 3, 4-dihydroxyphenylethanol; FCS, fetal calf serum; HPLC, high-perform- ance liquid chromatography; MOPET, 4-hydroxy-3-methoxyphenyl-etha- nol; HVA, homovanillic alcohol; PBS, phosphate-buffered saline; PIMT, protein l-isoaspartate(d-aspartate)-O-methyltransferase (EC 2.1.1.77); ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive lipid peroxidation end products; SE, standard error; UVA, long-wave ultraviolet; UVB, medium-wave ultraviolet. * Corresponding author. Fax: +0390815667608. E-mail address: diego.ingrosso@unina2.it (D. Ingrosso).