Lycopene as a guardian of redox signalling

It has been suggested that lycopene, the major carotenoid found in tomato, exhibits health-beneficial effects by virtue of its antioxidant activity. However, recent literature suggests that lycopene can actually "perform" roles independent of such capacity and involving a direct modulation of redox signalling. Reactive oxygen species are known to act as second messengers in the modulation of cellular signalling leading to gene expression changes and pharmacological responses. Lycopene may control redox-sensitive molecular targets, affecting enzyme activities and expressions and modulating the activation of MAPKs and transcription factors, such as NF-κB and AP-1, Nrf2.


INtRoductIoN
Lycopene is present in many fruits and vegetables, with tomatoes and processed tomato products being among the richest sources.Several recent studies suggest that dietary lycopene is able to reduce the risk of chronic diseases such as cancer (Giovannucci, 1999) and cardiovascular diseases (Rao, 2002) Although several mechanisms have been implicated in health-beneficial effects of lycopene, such as modulation of intercellular gap junction communication, hormones, immune system and metabolic pathways, the antioxidant properties of lycopene are thought to be primarily involved in its preventive effects in chronic diseases.Because of its high number of conjugated dienes, lycopene is one of the most potent antioxidants, with a singlet-oxygen-quenching ability twice as high as that of β-carotene and 10 times higher than that of α-tocopherol (Di Mascio et al., 1989).Increased reactive oxygen species (ROS) levels result in oxidative stress that can damage DNA, proteins, and lipids and lead to early cell death.The evidence that lycopene is able to modulate ROS levels is demonstrated by several studies, showing that the carotenoid may chemically interact with ROS and undergo oxidation and may prevent ROS-induced cell damage (Palozza et al., 2010a).
However, recently, a novel mechanism has been hypothesized by which lycopene may act as a beneficial agent in human health.The carotenoid has been reported to directly modulate several redox-sensitive signalling pathways altered in cancer (Palozza et al., 2011a) and cardiovascular diseases (Palozza et al., 2010b), being responsible for cell regulatory functions.The redox molecules regulated by lycopene involve antioxidant response elements (ARE), ROS-producyng enzymes, small-GTPases, mitogen-activated protein kinases (MAPK), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), redox-sensitive proteins involved in cell growth, such as p53 and the Bcl-2 family proteins and the Ku proteins (Fig. 1).This review summarizes the background information about lycopene as a modulator of redox signal.

ModuLAtIoN oF ANtIoxIdANt ResPoNse eLeMeNts ANd Nrf2
Several evidences suggest that lycopene can upregulate the antioxidant electrophile/antioxidant response element (EpRE/ARE) and the nuclear factor E2-related factor 2 (Nrf2), thereby stimulating the production of phase II detoxifying antioxidant enzymes that protect cells from reactive oxygen species and other electrophilic molecules (Ben-Dor et al., 2005).Lycopene has been reported to upregulate the ARE system in HepG2 and MCF-7 cells through the Nrf2 nuclear transcription pathway (Ben-Dor et al., 2005) and the expression of ARE-regulated proteins, including epoxide hydrolase 1 (EPHX1), superoxide dismutase-1 (SOD-1), catalase (CAT), and the metal binding protein transferrin (TF), in the androgensensitive human prostate cell line LNCaP (Goo et al., 2007).Recently, enzymatic metabolites of lycopene have been also reported to be able to induce Nrf2-mediated expression of phase II detoxifying/antioxidant enzymes (Linnewiel et al., 2009).In particular, apo-10′-lycopenoic acid, one of the products of lycopene cleaved by carotene 9′,10′-oxygenase at its 9′,10′ double bond, induced heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligases and increased total intracellular glutathione levels in human bronchial epithelial cells.Such effects were accompanied by a suppression of endogenous ROS generation and H 2 O 2 -induced oxidative damage.Recently, lycopene has been also reported to mitigate the nephrotoxic effect of cisplatin in rat through Nrf2-mediated induction of heme oxygenase-1 (HO-1) (Sahin et al., 2010).However, recently, it has been reported that lycopene supplementation was not able to modulate the oxidant-responsive protein HO-1 in lymphocytes from healthy young men (Markovitch et al., 2009).

ModuLAtIoN oF P450 exPRessIoN
Mammalian cytochromes P450 (P450) are a family of heme-thiolate enzymes involved in the oxidative metabolism of a variety of endogenous and exogenous lipophilic compounds.Poor coupling of the P450 catalytic cycle results in continuous production of ROS, which affects signalling pathways and other cellular functions (Zangar et al., 2004).Several studies have reported the ability of carotenoids to induce enzyme activities associated with P450 family.In particular, Astorg and colleagues proposed that lycopene-induced modulation of the liver metabolizing enzyme, cytochrome P4502E1, was the underlying mechanism of protection against carcinogeninduced preneoplastic lesions in the rat liver (Astorg, et al., 1997).Moreover, the administration of lycopene to rats was shown to induce liver CYP types 1A1/2, 2B1/2 and 3A in a dose-dependent manner [Breinholt et al., 2000).Recently, both lycopene and β-carotene have been shown to activate the cytochrome P450 1A1 gene but only β-carotene was able to induce the retinol dehydrogenase gene in mice (Aung, et al., 2009).The observations that P450 activity was induced at very low lycopene plasma levels suggests that modulation of drug metabolising enzymes by carotenoids may be relevant to humans.

ModuLAtIoN oF Ros-PRoducINg eNzyMes
Activated macrophages and neutrophils can produce large amounts of superoxide anion and its derivatives via the phagocytic isoform of NAD(P)H oxidase.Increasing evidence suggests that lycopene is able to reduce the levels of ROS through changes in NAD(P)H oxidase expression.Lycopene and β-carotene depletion for 3 weeks increased PMN-H 2 O 2 content after PMA activation, able to stimulate NADPH activation, while supplementation for 5 weeks restored basal H 2 O 2 generation (Walrand et al., 2205).Moreover, lycopene was able to decrease oxysterol-induced inflammation (Palozza et al., 2011b) and ROS production (Palozza et al., 2010c) in THP-1 cells through a decreased expression of NOX-4, one of the homologues of NADPH oxidase.
Lycopene has been also reported to counteract the effects of iNOS by inhibiting nitric oxide (NO) production and/or by decreasing iNOS at protein and mRNA levels.In particular, it has been shown that treatment with lycopene inhibited lipopolysaccharide (LPS)-stimulated NO production in RAW 264.7 macrophage cells (Rafi et al., 2007) and decreased LPS-induced iNOS.Moreover, it has been recently reported that dietary supplementation of lycopene-rich foods conferred a strong in vivo protection of human lymphocytes, against NO 2 • radical (by electron transfer) and 1 O 2 (by energy transfer) (Böhm et al., 2001).
The effect of lycopene and other natural antioxidants, such as quercetin and tyrosol, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression have been studied in RAW 264.7 macrophages stimulated by gliadin in association with interferon-γ (IFN-γ) (De Stefano et al., 2007).The IFN-γ plus gliadin combination treatment was capable of enhancing iNOS and COX-2 gene expression and NF-κB, interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1-α (STAT-1-α) activation induced by ROS generation.Lycopene, quercetin and tyrosol inhibited all these effects, suggesting that they may decrease iNOS and COX-2 gene expression, acting as non toxic agents for the control of pro-inflammatory genes.

ModuLAtIoN oF sMALL-gtPAses
Numerous observations have suggested a role for the Ras superfamily of small GTPase in redox regulation and ROS have been reported as important downstream effectors for Ras protein.We recently demonstrated that lycopene was able to modify Ras activation by decreasing its pharnesylation and by inducing its translocation from the membrane to the cytoplasm in cancer cells (Palozza et al., 2010d) as well as in stimulated macrophages (Palozza et al., 2011c).The changes in Ras activation were strictly related to an inhibition of the expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by the carotenoid and were accompanied by decreased ROS production and MAPK/NF-κB activation.

ModuLAtIoN oF MAPK cAscAdes
Numerous studies show that MAPKs are strongly activated by ROS and that lycopene, alone or in combination with other natural products, modulate MAPK phosphorylation (Sarkar et al., 2009).Lycopene was reported to attenuate the phenotypic and functional maturation of murine bone marrow-dendritic cells, especially in lipopolysaccharide (LPS)-induced DC maturation, by down-regulating the expression of costimulatory molecules (CD80 and CD86) and major histocompatibility complex type II molecules and by inhibiting the activation of MAPK and NF-κB (Kim et al., 2004).The carotenoid was also able to inhibit platelet derived growth factor (PDGF)-BB-induced signalling and migration in human fibroblasts by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2, p38, and jun N-terminal kinase (JNK) (Chan et al., 2009).In recent studies from our laboratory, lycopene suppressed both MAPK phosphorylation and NF-κB activation in oxysterol-stimulated macrophages as well as in prostatic cancer cells (Palozza et al., 2010c).

ModuLAtIoN oF NF-κB ANd AP-1 ActIVAtIoNs
NF-κB is the first eukaryotic transcription factor shown to respond directly to oxidative stress (Schrek et al., 1991).At least two mechanisms contribute to this effect.The first one involves ROS-mediated enhancement of IkB degradation and the second one the oxidative enhancement of upstream signal cascades.It has been found that lycopene inhibited the binding activity of NF-κB and the expression of NF-κB target gene MMP-9, leading to the inhibition of the invasion of human hepatoma cells (Huang, et al., 2007).Such inhibition was mediated by the downregulation of IκB phosphorylation, NF-κB expression, and NF-κB p65 subunit translocation from cytosol to nucleus (Hung et al., 2008).LPS stimulation has been known to activate the MAPK and NF-κB signal pathways.Pre-treatment with lycopene markedly inhibited the LPS-induced upregulation of p-ERK, p-p38, p-JNK, and NF-κB (Kim et al., 2004).Moreover, tomato lycopene extract prevented LPS-induced pro-inflammatory gene expression by blocking NF-κB signalling (Joo et al., 2009).
Many different oxidative stress-inducing stimuli, including smoking exposure, lead to AP-1 activation, with consequent alteration in cell proliferation, differentiation and apoptosis.It has been reported that lycopene is able to inhibit AP-1 signalling in mammary cancer cells (Karas et al., 2000).
The cancer-preventive effect of lycopene mediated by its ability to induce apoptosis has been comprehensively studied (Palozza et al., 2004).Zhang et al. (2003) have found that not the lycopene itself, but its auto-oxidant product induces apoptosis in HL-60 cells.Although many mechanisms have been suggested by which lycopene may control apoptosis, one of the most interesting is the carotenoid ability to modulate Bcl-2, a protein whose antiapoptotic effects has been, at least partially, explained by its antioxidant properties (Palozza et al., 2004).
The Ku proteins are involved in multiple cellular pathways, including DNA repair, telomere maintenance and Bax-mediated apoptosis.Recently, lycopene has been reported to reduce the levels of H 2 O 2 , to arrest cell cycle progression and to inhibit Ku-DNA binding activity, and Ku70 levels in Pancreatic Acinar AR42J cells (Seo et al., 2009).
FRoM exPeRIMeNtAL to HuMAN studIes: eVIdeNce FoR A Redox ActIVIty oF LycoPeNe Some human studies are being conducted to evaluate the influence of lycopene and/or tomato products on the modulation of markers of oxidative stress and on changes in redox signalling.Devaraj et al. (2007) examined the antioxidant potential of 8 weeks of lycopene supplementation (6.5, 15, 30 mg/d) following a 2 week washout period in healthy subjects.None of the lycopene doses had an effect on LDL oxidation rate, plasma lipid peroxidation markers, and urinary F2-isoprostanes.However, the 30 mg/d dose did decrease lymphocyte DNA damage and urinary 8-OHdG concentrations compared to baseline.A similarly designed study was undertaken in diabetics supplemented with 10 mg/d of lycopene.Supplementation did not alter total antioxidant capacity or oxidized-LDL antibody levels, but did decrease serum MDA levels compared to baseline (Neyestani et al., 2007).Zhao et al. (2006) also used a 2 week washout followed by 56 days of supplementation of 12 mg/d of lycopene to healthy, nonsmoking, postmenopausal women.Lycopene supplementation decreased lymphocyte DNA damage, but did not prevent hydrogen peroxideinduced DNA damage.On the other hand, a significant prevention of the H 2 O 2 -induced DNA damage was observed in lycopene-enriched lymphocytes from healthy volunteers supplemented with 15 mg/day lycopene for 1 week (Torbergsen et al., 2000).Moreover, in patients with localized prostate adenocarcinoma, consumption of lycopene-rich tomato sauce-based diet lead to a decrease in the oxidative DNA damage (Chen et al., 2001).An increase in serum lycopene after supplementation has been also reported to reduce lymphocyte DNA comet tail length in healthy men (Kim et al., 2010).Tomato paste rich in lycopene protects against cutaneous photodamage in humans, by reducing mitochondrial DNA (mtDNA) damage (Rizwan et al., 2011).In addition, the consumption of commercial tomato juice increased plasma lycopene levels and the intrinsic resistance of LDL to oxidation in patients with diabetes (Upritchard et al., 2000).
A recent study shows a significant up-regulation of IGFBP-3 and Bax/Bcl-2 ratio and down-regulation of cyclin-D1, p53, and Nrf-2 in cells incubated with sera from men who consumed red tomato paste (Talvas et al., 2010).Moreover, cell incubation with sera from men who consumed purified lycopene led to significant upregulation of IGFBP-3, c-fos, and uPAR (Talvas et al., 2010).
It is widely recognized that most of the inflammatory cytokines are produced by a mechanism involving activation of redox-sensitive molecules, including MAPKs and NF-κB.In a recent review, we reported (Palozza et al., 2010a) that lycopene decreases pro-inflammatory cytokine levels in human subjects (Riso et al., 2006;Upritchard et al., 2000;Watzl et al., 2000).
Although these promising results, some other studies did not show an in vivo antioxidant role of lycopene and/or tomato products (for a review, see Erdman et al., 2009).Therefore, when considering the data as a whole, there is an overall shortage of supportive evidence and more well designed studies are needed to further clarify this potential mechanism in humans.

coNcLusIoNs
This review summarizes the available evidence for a redox role of lycopene in biological environments.The carotenoid can modulate redox signalling, by controlling antioxidant response elements and Nrf2, ROS-producing enzymes, MAPKs, transcription factors, such as NF-κB and AP-1, and redox proteins involved in cell growth.Despite these promising reports, it is difficult at the moment to directly relate available experimental data to human pathophysiology.More well designed human studies and controlled clinical intervention trials are needed to further clarify the redox role of lycopene in vivo.Such studies should take into consideration subject selection, specific markers of analysis, metabolism and isomerization of lycopene, interaction with other antioxidants.