The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

November 17, 2015

1Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal 2Departamento de Biologia, Laboratório de Biotecnologia e Citómica, CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal

Andreia Ascenso, 1 , 2 , * Tiago Pedrosa, 2 Sónia Pinho, 2 Francisco Pinho, 2 José Miguel P. Ferreira de Oliveira, 2 Helena Cabral Marques, Helena Oliveira, Sandra Simões, and Conceição Santos

reserarchLycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.

Introduction

Human skin is constantly exposed to the UV irradiation that may induce a number of pathobiological cellular changes. Through lipid peroxidation, protein cross-linking, and DNA damage, UV-A and UV-B radiation (UVR) can cause photoaging and photocarcinogenesis [1–3]. Skin has a variety of enzymatic and small molecular antioxidants that can inhibit oxidative damage. However, the excessive ROS production often exceeds the skin antioxidant ability [4]. In this regard, emphasis on developing novel preventive and therapeutic strategies based on phytocompounds capable of ameliorating the adverse effects of ROS has become an important area of research. Moreover, primary prevention approaches of skin cancer proved to be inadequate in lowering the incidence of this type of cancer, emphasizing the need to develop novel skin cancer chemopreventive agents. Among the vast number of photochemoprotective agents, botanical antioxidants have given promising results [4]. Two types of chemopreventive agents could be useful for the management of skin cancer. Primarily, the agents that could inhibit the damage caused by UVR may prevent the formation of initiated cells (cells with cancerous potential). Secondly, the agents that could eliminate the initiated cells may reduce the risk of skin cancer [5].

Lycopene is a powerful antioxidant both in vitro and in vivo against the oxidation of proteins, lipids, and DNA, and it has been identified as one of the most potent scavengers of singlet species of oxygen free radicals—the highest among the carotenoids [6, 7]. At low oxygen tension, it can also scavenge peroxyl radicals, inhibiting the process of lipid peroxidation [8]. Lycopene was reported as the most quickly depleted antioxidant in skin upon exposure to solar radiation [9] and might play a role of protection against UVR. Recent research has been developed to assess if lycopene has potential for prevention of skin cancer. In fact, lycopene has been shown to inhibit proliferation of several types of cancer cells through different mechanisms in in vitro systems [10, 11]. Chemopreventive antioxidants are mostly studied for their role as radical scavengers, but this preventive role can be complemented by a corrective activity as selective inducers of apoptosis in transformed cells [12]. Moreover, Ribaya-Mercado et al. [9] suggested a role of lycopene in mitigating photooxidative damage in tissues.

Keratinocytes are the predominant cell type (95%) in the epidermis, the outermost layer of the skin [13]. Considering that the principal site of action of UV-B is the epidermis layer [14], keratinocytes might be more susceptible to UV-B-induced apoptosis than fibroblasts which are located in dermis layer (reached by UV-A) [15]. However, keratinocytes may be more UV-B resistant in terms of their proliferative ability as measured by colony survival assays and have greater ability for UV-DNA repair [15].

To date, most of the studies on the therapeutic potential of lycopene have been performed in vivo [16, 17]. These studies may be obscured by the complexity of biological system models. In vitro conditions may circumvent some of these contingencies and complement in vivo data within the 3Rs perspective (Refine, Replace, and Reduce). Despite the lower complexity of in vitro systems, the study of cellular photoprotection by antioxidants could be challenging because of the high chemical instability (especially to air and light) and strong lipophilicity of many antioxidant molecules such as lycopene. According to Zefferino et al. [11] in vitro experiments may occasionally produce inconsistent results due to lycopene’s poor solubility in cell culture media [18]. In fact, lycopene is very hydrophobic (log P ≈ 15) and is usually solubilized in organic solvents such as tetrahydrofuran (THF). However, an uncontrolled precipitation process may occur upon addition to aqueous media, besides the high toxicity associated with these solvents. The solubility and uptake of these large crystals in the cells are quite limited and there is almost no protection against chemical degradation [19]. Alternative ways of delivering lipid-soluble compounds include micelles, microemulsions, nanoparticles, water-dispersible beadlets, artificial liposomes, enriched bovine serum, or other formulations, each of which has an influence on the cellular uptake and compounds stability [18,20–23]. According to Palozza et al., niosomes provide a suitable, safe, and low-cost vehicle for β-carotene in cell culture [24]. Lipid-based delivery systems also show UV-blocking effects dependent on lipid composition and the particle size (the smaller the particle size, the higher the sunscreen activity). Lipid matrices can act as sunscreen carriers and increase the sun protection factor obtained after topical application of UV absorbers (BaSO4, SrCO3, and TiO2) incorporated within these carriers because they provide a fixation medium for these pigments [25, 26]. However, the UV-blocking effect of the vehicle is not desired in this case, besides the difficulties of using these hydrophobic systems for cell culture studies.

The main limitations of different vehicles used for lycopene cell delivery are summarized on Table 1. Each vehicle provides specific advantages but also offers some limitations such as cytotoxicity, poor solubility, and crystallization in the cell medium [27].

Table 1

Table 1
Limitations of different vehicles used for lycopene cell delivery (adapted from Lin et al. [27]).
In addition, the half-life of free lycopene in solution at 37°C is less than few hours. Thus, until an efficient method of solubilizing lycopene in aqueous buffers and cell culture media is developed, in vitro studies on the effects of lycopene on living cells will continue to show considerable variation between laboratories and cell lines and should be interpreted with caution [39].

Pfitzner et al. [18] have demonstrated that methyl-β-CD (M-β-CD) was an improved vehicle for the investigation of carotenoids and other lipophilic compounds in in vitro test systems, compared to organic solvents. Carotenoids-M-β-CD complex was superior concerning biological availability, missing cytotoxicity and presenting excellent stability when compared to other application forms such as organic solvents, mixed micelles, liposomes, or beadlets. At least, the solubilization with M-β-CD was easily and reproducibly achievable under routine laboratory conditions.

According to these literature references [18, 27] and preformulations studies, we decided to use another similar CD derivative, dimethyl-β-CD (DM-β-CD), to solubilize and stabilize lycopene for cell exposure experiments. Depending on the formulation and exposure conditions, lycopene has been shown to prevent cellular damage or otherwise to sensitize damaged cells leading to increased cell death. We aimed to study the effects of lycopene as sensitizer and inducer of cell death in UV-B damaged keratinocytes. Our hypothesis was that cells preexposed to lycopene would be more sensitized to death, in case of subsequent irreversible damage by UV-B. For this, the nontumorigenic keratinocyte cell line HaCaT [40] was used. HaCaT cells were preexposed to lycopene for sensitization and subsequently exposed to damaging UV-B irradiation. The effect of lycopene preexposure was analyzed by assays focused on cytotoxicity, genotoxicity, oxidative stress, and apoptosis.

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