Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells

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 2′7′-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 μM 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.

Introduction

The peculiar climate of the Mediterranean basin, characterized 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 epidemiological studies, indeed, indicate that these dietetic habits may lower the incidence of several degenerative pathologies, including coronary heart diseases and cancer [4], [5], [6].

A case-control study in Australia suggests the existence of an inverse relationship between the risk of skin cancer and the high intake of antioxidant-containing foods [7]. In this respect, it has been amply reported that natural antioxidants can exert a protective effect against skin inflammation and cancer, induced by ultraviolet (UV) radiation [8]. Medium-wave (UVB)¹-induced carcinogenesis in mouse was suppressed when a green tea polyphenolic fraction was topically applied to the skin or orally administered in the drinking water [9], [10]. It has been shown that quercetin, intraperitoneally administered to rats before long-wave UV (UVA) irradiation, decreases significantly the malondialdehyde concentration and slows down the decrease of enzymatic antioxidant activities [11]. Moreover, a diet rich in lycopene protects the skin against UV-induced erythema formation in humans [12]. Recently, F’guyer et al. [13] demonstrated that topical application of resveratrol to hairless mice results in a significant inhibition of UV-induced skin edema. Recent reports indicate that both orally administered and topically applied vitamin E prevents the UVB-induced skin carcinogenesis in mice [14]. The effect of several antioxidants has been tested in UVA-irradiated human skin fibroblasts, suggesting that vitamin E and vitamin C are potential photoprotectors [15]. Finally, in the recent years it has been reported that olive oil application exerts a protective effect against UVB-induced murine skin tumors [16]. These data encouraged the use of olive oil-based sun lotions, containing high levels of antioxidant compounds.

Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET) is the main ortho-diphenolic compound found in olive oil and responsible for its antioxidant properties [17], [18]. In addition, it should be noted that DOPET, in addition of being an olive oil component, is also a dopamine metabolite [19], [20]. DOPET has been shown to function as an efficient scavenger of peroxyl radicals [21] and contributes toward determining the shelf-life of the oil, preventing its auto-oxidation [17], [18]. Nevertheless the possibility that protection exerted by DOPET may involve actual prevention of radical generation cannot be ruled out. The biological activities of DOPET have been explored by several groups as reviewed by Manna et al. [22]. DOPET in vitro prevents LDL oxidation [23] and platelet aggregation [24] and inhibits 5-and 12-lipoxygenases [25]. It also exerts an inhibitory effect on peroxynitrite-dependent DNA base modifications and tyrosine nitration [26]. Experiments from our laboratory have demonstrated that DOPET, which effectively permeates cell membranes via a passive diffusion mechanism [27], counteracts the cytotoxic effects of reactive oxygen species (ROS) in various human systems. Preincubation of intestinal Caco-2 cells with DOPET prevents the typical damages of oxidative stress [28]. Similarly, DOPET exerts a protective effect against the H₂O₂-induced oxidative hemolysis and malondialdehyde formation in red blood cells [29]. Moreover, this diphenol exerts an antiproliferative effect, inducing apoptosis in HL-60 cells as well as in resting and activated peripheral blood lymphocytes [30]. DOPET bioavailability and metabolism have been widely investigated: it is interesting to note in this respect that the molecule, when intravenously injected to rats, rapidly distributes in all organs and tissues, where it undergoes extensive metabolic transformation [20].

Cultured melanoma cells are a suitable model system for studying the mechanisms by which UVA irradiation might induce structural and functional alterations at the tissue level, as well as the biological implications of such modifications [31], [32], [33]. Nevertheless previous studies suggest that melanoma cells may behave slightly differently from normal melanocytes in their antioxidant response to oxygen [34]; thus suggesting careful extrapolation of results to situations which are operative in vivo. A number of reports, so far, contributed to establish the role of ROS in the mechanism(s) of cell damages caused by UV radiation [16], [35]. Recently, a dramatic increase of both lipid peroxidation products (TBARS) and abnormal isoaspartyl residues have been reported in a human melanoma cell line (M14) exposed to increasing doses of UVA [31]. These abnormal residues, arising from Asn deamidation and/or Asp isomerization, are selectively recognized and methyl-esterified, by protein l-isoaspartate(d-aspartate)-O-methyltransferase (PIMT; EC 2.1.1.77), a ubiquitous housekeeping enzyme identified in both prokaryotes and eukaryotes [36], [37], [38], [39]. PIMT catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to free a-carboxyl groups of l-isoAsp residues, thus activating their conversion into normal l-aspartyl residues [40], [41]. Therefore this repair reaction prevents the accumulation of potentially dysfunctional proteins in cells and tissues [40], [42], [43]. In addition, PIMT-mediated methyl ester formation can be used as a marker reaction to tag these abnormal residues in natural and recombinant proteins [44] and to monitor their increase associated with cell stress conditions [45], [46], [47].

Using this tool, we were able to show that in M14 cells the occurrence of l-isoaspartyl residues is an early event upon UVA-induced cell injury. A potential role of the oxidative stress in the mechanism of this UVA-induced protein damage has been hypothesized [31], [48], [49], [50]. To further assess the existence of a close relationship between oxidation phenomena and protein damage, the possible effects of the natural antioxidant DOPET in this model system have been evaluated. Results indicate that hydroxytyrosol is able to prevent the increase in l-isoaspartyl residues, markers of protein damage, in M14 cells exposed to UVA irradiation. DOPET’s protective effect is paralleled by a decrease of ROS production, thus confirming the role of the oxidative stress in the mechanism of UVA-induced protein damages.