Combined Effects of Carotenoids and Polyphenols in Balancing the Response of Skin Cells to UV Irradiation.

Oral carotenoids and polyphenols have been suggested to induce photo-protective effects. The aim of the study was to test whether the combination of carotenoids and polyphenols produce greater protective effects from UV-induced damage to skin cells. Such damage is characterized by inflammation and oxidative stress; thus, the photo-protective effect can be partially explained by modulating the nuclear factor kappa B (NFκB) and antioxidant response element/Nrf2 (ARE/Nrf2) transcription systems, known as important regulators of these two processes. Indeed, it was found in keratinocytes that carotenoids and polyphenols inhibit UVB-induced NFκB activity and release of cytokine IL-6. A combination of tomato extract with rosemary extract inhibited UVB-induced release of IL-6 more than each of the compounds alone. Moreover, this combination synergistically activated ARE/Nrf2 transcription systems. Inflammatory cytokines such as IL-6 and TNFα induce the expression of matrix metalloproteinases (MMPs), which leads to collagen breakdown; thus, it is important to note that carnosic acid reduced TNFα-induced MMP-1 secretion from human dermal fibroblasts. The in vitro results suggest beneficial effects of phytonutrient combinations on skin health. To assure that clinical experiments to prove such effects in humans are feasible, the human bioavailability of carotenoids from tomato extract was tested, and nearly a twofold increase in their plasma concentrations was detected. This study demonstrates that carotenoids and polyphenols cooperate in balancing UV-induced skin cell damage, and suggests that NFκB and ARE/Nrf2 are involved in these effects.

The anti-cancer effects of carotenoids and other phytonutrients resides in their combined activity.

Epidemiological studies have consistently shown that regular consumption of fruits and vegetables is strongly associated with reduced risk of developing chronic diseases, such as cancer. It is now accepted that the actions of any specific phytonutrient alone do not explain the observed health benefits of diets rich in fruits and vegetables as nutrients that were taken alone in clinical trials did not show consistent preventive effects. The considerable cost and complexity of such clinical trials requires prudent selection of combinations of ingredients rather than single compounds. Indeed, synergistic inhibition of prostate and mammary cancer cell growth was evident when using combinations of low concentrations of various carotenoids or carotenoids with retinoic acid and the active metabolite of vitamin-D. In this study we aimed to develop simple and sensitive in vitro methods which provide information on potent combinations suitable for inclusion in clinical studies for cancer prevention. We, thus, used reporter gene assays of the transcriptional activity of the androgen receptor in hormone-dependent prostate cancer cells and of the electrophile/antioxidant response element (EpRE/ARE) transcription system. We found that combinations of several carotenoids (e.g., lycopene, phytoene and phytofluene), or carotenoids and polyphenols (e.g., carnosic acid and curcumin) and/or other compounds (e.g., vitamin E) synergistically inhibit the androgen receptor activity and activate the EpRE/ARE system. The activation of EpRE/ARE was up to four fold higher than the sum of the activities of the single ingredients, a robust hallmark of synergy. Such combinations can further be tested in the more complex in vivo models and human studies.

Polyphenols, isothiocyanates, and carotenoid derivatives enhance estrogenic activity in bone cells but inhibit it in breast cancer cells.

While exposure to estrogens is a major risk factor of breast and endometrial cancer, it well established that estrogens are beneficial for bone health. We have previously shown that carotenoids inhibit estrogen signaling in breast and endometrial cancer cells. The aim of this study was to compare the effects of various phytonutrients, (carotenoid derivatives, polyphenols, isothiocyanates) on estrogenic activity in breast cancer cells and osteoblast-like cells. All the tested phytonutrients inhibited estrogen response element (ERE) transactivation in breast cancer cells. In contrast, these compounds either did not affect or enhanced ERE activity and the expression of several bone-forming genes. These results were obtained using two osteoblast-like cell lines, MG-63 human osteosarcoma cells stably transfected with estrogen receptor-α (ERα) and MC3T3-E1 mouse calvaria-derived cells expressing endogenous ER. Phytonutrients-induced ERE inhibition in breast cancer cells, and its potentiation in osteoblast-like cells were associated both with a decrease and a rise in total and nuclear ERα levels, respectively. Phytonutrients activated the electrophile/antioxidant response element (EpRE/ARE) transcription system to various extents in both cancer and bone cell lines. Overexpression of Nrf2, the major EpRE/ARE activating transcription factor, mimicked the effects of phytonutrients, causing inhibition and enhancement of ERE transactivation in breast cancer cells and in osteoblast-like cells, respectively. Moreover, reduction in Nrf2 levels by RNAi led to a decrease in the phytonutrient potentiation of ERE activity transactivation in osteoblast-like cells. These findings suggest that the enhancement and inhibition of estrogen signaling by phytonutrients in bone-derived cells and breast cancer cells, respectively, is partially mediated by the activation of the Nrf2/ARE system.

Inhibition of Osteoclast Differentiation by Carotenoid Derivatives through Inhibition of the NF-ƙB Pathway.

The bone protective effects of carotenoids have been demonstrated in several studies, and the inhibition of RANKL-induced osteoclast differentiation by lycopene has also been demonstrated. We previously reported that carotenoid oxidation products are the active mediators in the activation of the transcription factor Nrf2 and the inhibition of the NF-ƙB transcription system by carotenoids. Here, we demonstrate that lycopene oxidation products are more potent than intact lycopene in inhibiting osteoclast differentiation. We analyzed the structure-activity relationship of a series of dialdehyde carotenoid derivatives (diapocarotene-dials) in inhibiting osteoclastogenesis. We found that the degree of inhibition depends on the electron density of the carbon atom that determines the reactivity of the conjugated double bond in reactions such as Michael addition to thiol groups in proteins. Moreover, the carotenoid derivatives attenuated the NF-ƙB signal through inhibition of IƙB phosphorylation and NF-ƙB translocation to the nucleus. In addition, we show a synergistic inhibition of osteoclast differentiation by combinations of an active carotenoid derivative with the polyphenols curcumin and carnosic acid with combination index (CI) values < 1. Our findings suggest that carotenoid derivatives inhibit osteoclast differentiation, partially by inhibiting the NF-ƙB pathway. In addition, carotenoid derivatives can synergistically inhibit osteoclast differentiation with curcumin and carnosic acid.

Effect of Tomato Nutrient Complex on Blood Pressure: A Double Blind, Randomized Dose⁻Response Study.

Oxidative stress is implicated in the pathogenesis of essential hypertension, a risk factor for cardiovascular morbidity and mortality. Tomato carotenoids such as lycopene and the colorless carotenoids phytoene and phytofluene induce the antioxidant defense mechanism. This double-blind, randomized, placebo-controlled study aimed to find effective doses of Tomato Nutrient Complex (TNC) to maintain normal blood pressure in untreated hypertensive individuals. The effect of TNC treatment (5, 15 and 30 mg lycopene) was compared with 15 mg of synthetic lycopene and a placebo over eight weeks. Results indicate that only TNC treatment standardized for 15 or 30 mg of lycopene was associated with significant reductions in mean systolic blood pressure (SBP). Treatment with the lower dose standardized for 5 mg of lycopene or treatment with 15 mg of synthetic lycopene as a standalone had no significant effect. To test carotenoid bioavailability, volunteers were treated for four weeks with TNC providing 2, 5 or 15 mg lycopene. The increase in blood levels of lycopene, phytoene, and phytofluene was dose dependent. Results suggest that only carotenoid levels achieved by the TNC dose of 15 mg lycopene or higher correlate to a beneficial effect on SBP in hypertensive subjects while lower doses and lycopene alone do not.

Carotenoid derivatives inhibit nuclear factor kappa B activity in bone and cancer cells by targeting key thiol groups.

Aberrant activation of the nuclear factor kappa B (NFkB) transcription system contributes to cancer progression, and has a harmful effect on bone health. Several major components of the NFkB pathway such as IkB Kinase (IKK) and the NFkB subunits contain cysteine residues that are critical for their activity. The interaction of electrophiles with these cysteine residues results in NFkB inhibition. Carotenoids, hydrophobic plant pigments, are devoid of electrophilic groups, and we have previously demonstrated that carotenoid derivatives, but not the native compounds activate the Nrf2 transcription system. The aim of the current study was to examine whether carotenoid derivatives inhibit NFkB, and, if so, to determine the molecular mechanism underpinning the inhibitory action. We report in the present study that a mixture of oxidized derivatives, prepared by ethanol extraction from partially oxidized lycopene preparation, inhibited NFkB reporter gene activity. In contrast, the intact carotenoid was inactive. A series of synthetic dialdehyde carotenoid derivatives inhibited reporter activity as well as several stages of the NFkB pathway in both cancer and bone cells. The activity of the carotenoid derivatives depended on the reactivity of the electrophilic groups in reactions such as Michael addition to sulfhydryl groups of proteins. Specifically, carotenoid derivatives directly interacted with two key proteins of the NFkB pathway: the IKKß and the p65 subunit. Direct interaction with IKKß was found in an in vitro kinase assay with a recombinant enzyme. The inhibition by carotenoid derivatives of p65 transcriptional activity was observed in a reporter gene assay performed in the presence of excess p65. This inhibition action resulted, at least in part, from direct interaction of the carotenoid derivative with p65 leading to reduced binding of the protein to DNA as evidenced by electrophoretic mobility shift assay (EMSA) experiments. Importantly, we found by using mutation in key cysteine residues of both p65 and IKK that specific thiol groups are essential for NFkB inhibition by carotenoid derivatives. In conclusion, we propose that electrophilic carotenoid derivatives contribute to cancer prevention as well as bone health maintenance via the inhibition of the NFkB transcription system. Pivotal thiol groups of both IKK and p65 play a key role in this process.

The role of lycopene and its derivatives in the regulation of transcription systems: implications for cancer prevention.

Evidence from epidemiologic studies has suggested that carotenoids, and lycopene in particular, decrease the risk of cancer: however, not all studies support this view. To gain insight into the molecular mechanisms whereby lycopene and other carotenoids may exert their chemoprotective effects, we and others performed a series of studies that used a large panel of cancer cell lines of different lineages and animal models of human cancer. In this review we address some of the mechanisms proposed for the cancer-preventive activity of tomato lycopene, focusing on the induction of the antioxidant response element transcription system and the inhibition of the transcriptional activity of sex hormones, such as estrogens and androgens, and the activity of growth factors, such as insulin-like growth factor. We also considered the modulation by lycopene of the transcription factors peroxisome proliferator-activated receptor, retinoid X receptor, liver X receptor, and activating protein-1. The ligands and the phytonutrient regulators of these transcription systems contain electrophilic active groups, whereas lycopene and nonxanthophylic carotenoids are devoid of them. Thus, we suggest that at least some of the cellular effects of carotenoids are mediated through their derivatives formed either by chemical oxidation or by enzymatic cleavage inside the cells. This review highlights findings that pertain to this exciting avenue of research, which is currently under intense scrutiny in several laboratories worldwide.

Carotenoids and apocarotenoids in cellular signaling related to cancer: a review.

The basis for the vivid color of carotenoids and their antioxidant activity is the multiple conjugated double bonds, which are characteristic for these phytonutrients. Moreover, the cleavage of these oxidation-prone double bonds leads to the formation of apocarotenoids. A large number of carbonyl-containing oxidation products are expected to be produced as a result of carotenoid oxidation and these can be further metabolized into the corresponding acids and alcohols. As discussed in this review, many, but not all, of these potential products have been detected and identified in plants as well as in human and animal plasma and tissues. Some of these compounds were found to be biologically active as anticancer agents. In addition to the inhibition of cancer cell proliferation, several carotenoid metabolites were shown to modulate the activity of various transcription systems. These include ligand-activated nuclear receptors, such as the retinoic acid receptor, retinoid X receptor, peroxisome proliferator-activated receptor and estrogen receptor, as well as other transcription systems that have an important role in cancer, such as the electrophile/antioxidant response element pathway and nuclear factor-κB. Therefore, apocarotenoids can be considered as natural compounds with multifunctional, rather than monofunctional, activity and, thus, can be useful in the prevention of cancer and other degenerative diseases.