Mechanistic understanding of β-cryptoxanthin and lycopene in cancer prevention in animal models

To better understand the potential function of carotenoids in the chemoprevention of cancers, mechanistic understanding of carotenoid action on genetic and epigenetic signaling pathways is critically needed for human studies. The use of appropriate animal models is the most justifiable approach to resolve mechanistic issues regarding protective effects of carotenoids at specific organs and tissue sites. While the initial impetus for studying the benefits of carotenoids in cancer prevention was their antioxidant capacity and pro-vitamin A activity, significant advances have been made in the understanding of the action of carotenoids with regards to other mechanisms. This review will focus on two common carotenoids, provitamin A carotenoid β-cryptoxanthin and non-provitamin A carotenoid lycopene, as promising chemopreventive agents or chemotherapeutic compounds against cancer development and progression. We reviewed animal studies demonstrating that β-cryptoxanthin and lycopene effectively prevent the development or progression of various cancers and the potential mechanisms involved. We highlight recent research that the biological functions of β-cryptoxanthin and lycopene are mediated, partially via their oxidative metabolites, through their effects on key molecular targeting events, such as NF-κB signaling pathway, RAR/PPARs signaling, SIRT1 signaling pathway, and p53 tumor suppressor pathways. The molecular targets by β-cryptoxanthin and lycopene, offer new opportunities to further our understanding of common and distinct mechanisms that involve carotenoids in cancer prevention.This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Carotenoids and fatty liver disease: Current knowledge and research gaps

Carotenoids form an important part of the human diet, consumption of which has been associated with many health benefits. With the growing global burden of liver disease, increasing attention has been paid on the possible beneficial role that carotenoids may play in the liver. This review focuses on carotenoid actions in non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD). Indeed, many human studies have suggested an association between decreased circulating levels of carotenoids and increased incidence of NAFLD and ALD. The literature describing supplementation of individual carotenoids in rodent models of NAFLD and ALD is reviewed, with particular attention paid to β-carotene and lycopene, but also including β-cryptoxanthin, lutein, zeaxanthin, and astaxanthin. The effect of beta-carotene oxygenase 1 and 2 knock-out mice on hepatic lipid metabolism is also discussed. In general, there is evidence to suggest that carotenoids have beneficial effects in animal models of both NAFLD and ALD. Mechanistically, these benefits may occur via three possible modes of action: 1) improved hepatic antioxidative status broadly attributed to carotenoids in general, 2) the generation of vitamin A from β-carotene and β-cryptoxanthin, leading to improved hepatic retinoid signaling, and 3) the generation of apocarotenoid metabolites from β-carotene and lycopene, that may regulate hepatic signaling pathways. Gaps in our knowledge regarding carotenoid mechanisms of action in the liver are highlighted throughout, and the review ends by emphasizing the importance of dose effects, mode of delivery, and mechanism of action as important areas for further study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Mammalian Carotenoid-oxygenases: Key players for carotenoid function and homeostasis

Humans depend on a dietary intake of lipids to maintain optimal health. Among various classes of dietary lipids, the physiological importance of carotenoids is still controversially discussed. On one hand, it is well established that carotenoids, such as β,β-carotene, are a major source for vitamin A that plays critical roles for vision and many aspects of cell physiology. On the other hand, large clinical trials have failed to show clear health benefits of carotenoids supplementation and even suggest adverse health effects in individuals at risk of disease. In recent years, key molecular players for carotenoid metabolism have been identified, including an evolutionarily well conserved family of carotenoid-oxygenases. Studies in knockout mouse models for these enzymes revealed that carotenoid metabolism is a highly regulated process and that this regulation already takes place at the level of intestinal absorption. These studies also provided evidence that β,β-carotene conversion can influence retinoid-dependent processes in the mouse embryo and in adult tissues. Moreover, these analyses provide an explanation for adverse health effects of carotenoids by showing that a pathological accumulation of these compounds can induce oxidative stress in mitochondria and cell signaling pathways related to disease. Advancing knowledge about carotenoid metabolism will contribute to a better understanding of the biochemical and physiological roles of these important micronutrients in health and disease. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Carotenoid actions and their relation to health and disease

Based on extensive epidemiological observation, fruits and vegetables that are a rich source of carotenoids are thought to provide health benefits by decreasing the risk of various diseases, particularly certain cancers and eye diseases. The carotenoids that have been most studied in this regard are β-carotene, lycopene, lutein and zeaxanthin. In part, the beneficial effects of carotenoids are thought to be due to their role as antioxidants. β-Carotene may have added benefits due its ability to be converted to vitamin A. Additionally, lutein and zeaxanthin may be protective in eye disease because they absorb damaging blue light that enters the eye. Food sources of these compounds include a variety of fruits and vegetables, although the primary sources of lycopene are tomato and tomato products. Additionally, egg yolk is a highly bioavailable source of lutein and zeaxanthin. These carotenoids are available in supplement form. However, intervention trials with large doses of β-carotene found an adverse effect on the incidence of lung cancer in smokers and workers exposed to asbestos. Until the efficacy and safety of taking supplements containing these nutrients can be determined, current dietary recommendations of diets high in fruits and vegetables are advised.     

Prooxidant effects of beta-carotene in cultured cells

There is a growing body of interest on the role of β-carotene and other carotenoids in human chronic diseases, including cancer. While epidemiological evidence shows that people who ingest more dietary carotenoids exhibit a reduced risk for cancer, results from intervention trials indicate that supplemental β-carotene enhances lung cancer incidence and mortality among smokers. A possible mechanism which can explain the dual role of β-carotene as both a beneficial and a harmful agent in cancer as well as in other chronic diseases is its ability in modulating intracellular redox status. β-Carotene may serve as an antioxidant or as a prooxidant, depending on its intrinsic properties as well as on the redox potential of the biological environment in which it acts. This review summarizes the available evidence for a prooxidant activity of β-carotene in cultured cells, focusing on biochemical and molecular markers of oxidative stress, which have been reported to be enhanced by the carotenoid.     

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following β-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5–2.0 μM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from β-carotene, can exert protective effects against cigarette smoke condensate.     

Carotenoids of dragonflies,from the perspective of comparative biochemical and chemical ecological studies

Carotenoids of 20 species of dragonflies (including 14 species of Anisoptera and six species of Zygoptera) were investigated from the viewpoints of comparative biochemistry and chemical ecology. In larvae, β-carotene, β-cryptoxanthin, lutein, and fucoxanthin were found to be major carotenoids in both Anisoptera and Zygoptera. These carotenoids were assumed to have originated from aquatic insects, water fleas, tadpoles, and small fish, which dragonfly larvae feed on. Furthermore, β-caroten-2-ol and echinenone were also found in all species of larvae investigated. In adult dragonflies, β-carotene was found to be a major carotenoid along with lutein, zeaxanthin, β-caroten-2-ol, and echinenone in both Anisoptera and Zygoptera. On the other hand, unique carotenoids, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene, were present in both Anisoptera and Zygoptera dragonflies. These carotenoids were not found in larvae. Food chain studies of dragonflies suggested that these carotenoids originated from aphids, and/or possibly from aphidophagous ladybird beetles and spiders, which dragonflies feed on. Lutein and zeaxanthin in adult dragonflies were also assumed to have originated from flying insects they feed on, such as flies, mosquitoes, butterflies, moths, and planthoppers, as well as spiders. β-Caroten-2-ol and echinenone were found in both dragonfly adults and larvae. They were assumed to be metabolites of β-carotene in dragonflies themselves. Carotenoids of dragonflies well reflect the food chain during their lifecycle.     

Overview of mechanisms of action of lycopene

Dietary intakes of tomatoes and tomato products containing lycopene have been shown to be associated with decreased risk of chronic diseases such as cancer and cardiovascular diseases in numerous studies. Serum and tissue lycopene levels have also been inversely related to the risk of lung and prostate cancers. Lycopene functions as a very potent antioxidant, and this is clearly a major important mechanism of lycopene action. In this regard, lycopene can trap singlet oxygen and reduce mutagenesis in the Ames test. However, evidence is accumulating for other mechanisms as well. Lycopene at physiological concentrations can inhibit human cancer cell growth by interfering with growth factor receptor signaling and cell cycle progression specifically in prostate cancer cells without evidence of toxic effects or apoptosis of cells. Studies using human and animal cells have identified a gene, connexin 43, whose expression is upregulated by lycopene and which allows direct intercellular gap junctional communication (GJC). GJC is deficient in many human tumors and its restoration or upregulation is associated with decreased proliferation. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibits a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression in the HL-60 promyelocytic leukemia cell line, suggesting some interaction at a nuclear or subcellular level. The combination of lycopene and lutein synergistically interact as antioxidants, and this may relate to specific positioning of different carotenoids in membranes. This review will focus on the growing body of evidence that carotenoids have unexpected biologic effects in experimental systems, some of which may contribute to their cancer preventive properties in models of carcinogenesis. Consideration of solubility in vitro, comparison with doses achieved in humans by dietary means, interactions with other phytochemicals, and other potential mechanisms such as stimulation of xenobiotic metabolism, inhibition of cholesterogenesis, modulation of cyclooxygenase pathways, and inhibition of inflammation will be considered. This review will point out areas for future research where more evidence is needed on the effects of lycopene on the etiology of chronic disease.     

Carotenoids and genomic stability

Epidemiological evidence abounds for a link between intake of carotenoids from fruit and vegetable foods and relatively low incidence of various cancers. However, intervention trials have shown, in some cases, a significant increase in occurrence of lung cancer in those volunteers taking supplements of beta-carotene. More information is clearly needed about the mechanism of action of carotenoids. Effects of carotenoids on cells in culture include inhibition of DNA synthesis and proliferation, changes in gene expression, decreased micronucleus frequency, and inhibition of transformation via synthesis of gap-junction proteins. Experiments with animal models are unsatisfactory because of the very poor uptake of carotenoids in rodents compared with man. In humans, oxidative damage to lymphocytes correlates negatively with plasma carotenoid concentrations, and the level of DNA damage is susceptible to reduction by carotenoid-rich foods. It seems clear that the carotenoids act as antioxidants in vivo, and yet this activity may not result in cancer prevention.     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

Carotenoids Induce Apoptosis in the T-lymphoblast Cell Line Jurkat E6.1

Epidemiologically, a high-carotenoid intake via a fruit- and vegetable-rich diet is associated with a decreased risk of various forms of cancer. The mechanisms by which carotenoids exert this protective effect are controversial. In this study, we examined the potency of a range of carotenoids commonly found in human plasma to induce apoptosis in Jurkat E6.1 malignant T-lymphoblast cells. At a concentration of 20 &#119 M, the order of potency to induce apoptosis after 24 h was: &#103 -carotene > lycopene > lutein> &#103 -cryptoxanthin=zeaxanthin. Canthaxanthin failed to induce apoptosis under these conditions. &#103 -Carotene induced apoptosis in a time- and concentration-dependent manner with a lowest effective concentration of about 3 &#119 M. Pre-conditioning of &#103 -carotene for 72 h destroyed its pro-apoptotic activity almost completely, whereas degradation for 6 h or less did not, indicating that either &#103 -carotene itself and/or an early degradation product of &#103 -carotene are the death-inducing compounds. Apoptosis induced by &#103 -carotene was characterized by chromatin condensation and nuclear fragmentation, DNA degradation, PARP cleavage and caspase-3 activation. The antioxidant BO-653 inhibited the degradation of &#103 -carotene in vitro and significantly increased its cytotoxicity, indicating that a pro-oxidant effect of &#103 -carotene is unlikely to cause its pro-apoptotic activity. The induction of apoptosis in transformed cells by carotenoids may explain their protective effect against cancer formation in humans. Possible pathways for induction of apoptosis by carotenoids are discussed.     

Whole Fruit Phytochemicals Combating Skin Damage and Carcinogenesis

Skin is arguably the largest organ of the body and is continuously subjected to intrinsic, extrinsic, and environmental stresses. Therefore, skin developed elaborate mechanisms to maintain homeostasis, including antioxidant, antiinflammatory, and DNA damage repair capabilities. However, repeated and excessive stresses can overwhelm these systems, causing serious cutaneous damages, including skin carcinogenesis. Phytonutrients present in the diet possess a myriad of health-promoting effects by protecting skin from damaging free radicals as well as by other mechanisms. Although many chemoprotective phytonutrients have been shown to be efficacious individually, a combination of multiple agents could have synergistic response in curtailing or preventing cutaneous damages. Here, we discuss the benefits of natural amalgamation of phytonutrients in select fruits against skin damage including carcinogenesis. However, a majority of these studies have been done in preclinical models. Therefore, clinical studies are needed to determine the human relevance of the available preclinical data, especially in the human population who are at higher risk for skin cancers (e.g., organ transplant patients). In addition, detailed well-structured preclinical animal studies in the models of high-risk skin carcinogenesis could also be useful toward informing the design for human trials.     

Effects of ascorbic acid and ��-carotene on HepG2 human hepatocellular carcinoma cell line

Recent studies have demonstrated that vegetable rich diets have protective effects on the occurrence and prognosis of various cancers. In addition to dietary intakes, ascorbic acid and β-carotene are also taken as supplements. The aim of this study was to assess effects of ascorbic acid, β-carotene and their combinations on human hepatocellular carcinoma cell line HepG2. Ascorbic acid and β-carotene were applied to cells as plasma peak concentrations (70 and 8 μM, respectively) and their half concentrations (35 and 4 μM, respectively) for 24 and 48 h. Genotoxic and cytotoxic effects of ascorbic acid and β-carotene were evaluated by alkali single cell gel electrophoresis (SCGE), acridine orange/ethidium bromide staining patterns of cells (apoptosis and necrosis) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Results of the SCGE demonstrated that both ascorbic acid and β-carotene caused DNA damage on HepG2 which were also concordant to increased apoptosis and necrosis of cells. Increased TBARS values also demonstrated increased lipid peroxidation in these cells. Results of the present study demonstrates that when dietary intakes of ascorbic acid and β-carotene and their relevant achievable plasma level concentrations were considered, both ascorbic acid and β-carotene induce genotoxic and cytotoxic damage on HepG2 together with increased oxidative damage in contrast to their protective effect on healthy cells. This may be correlated to oxidative status and balance of ROS in hepatocellular carcinoma cells.     

Flavonoids and steroid hormone-dependent cancers

Steroid-hormone dependent cancers, including those of the breast, prostate and colon, are leading causes of morbidity and mortality in western countries. In rural Asian areas, these diseases are relatively uncommon. Dietary factors, including low consumption of fruit, vegetables and soy in the west have been shown in various epidemiologic studies as reasons for these differences. This review discusses flavonoids, one component of these plant foods that is being investigated for their role in chemoprevention. Epidemiological, in vitro, animal and human studies shall be explored to look at mechanisms involved, including steroid hormone activity, effects on cell growth, antioxidant activities, inhibition of chemical carcinogenesis and influences on modulators of cancer risk. Although the in vitro and animal models point to several pathways by which flavonoids may reduce incidence of these cancers, the clinical data are still relatively lacking. More research is needed to determine how best to use foods containing these compounds to reduce steroid hormone-dependent cancer risk.     

Components of the Mediterranean Diet with chemopreventive activity toward colorectal cancer

Colorectal cancer is one of the leading causes of cancer incidence and death worldwide. For the past 30 years researchers have tried to find drugs suitable for the chemoprevention of the diseases. Epidemiological studies have demonstrated that adherence to the Mediterranean Diet (MD) is protective toward colon cancer development. Key components of this diet are olive oil (OO), fruits and vegetables, legumes and fish. Importantly, many bioactives present in these foods have shown anti-cancer activity in in vitro as well as in animal models of colon carcinogenesis, indicating a possible use as preventive agents. However, when translated to human trials, single bioactives have yielded conflicting results mostly due to irreproducibility of pre-clinical data. Since our preclinical models are usually designed to test single molecules at high concentrations and for very short time in order to provide significant effects, future studies should employ multiple bioactives that would resemble a more natural approach with possible significant synergisms that might be critical for cancer prevention. In this review we will discuss data obtained with components of the MD with a specific focus on OO, omega-3 polyunsaturated fatty acids and apples.     

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.     

Maternal-fetal transfer and metabolism of vitamin A and its precursor β-carotene in the developing tissues

The requirement of the developing mammalian embryo for retinoic acid is well established. Retinoic acid, the active form of vitamin A, can be generated from retinol and retinyl ester obtained from food of animal origin, and from carotenoids, mainly β-carotene, from vegetables and fruits. The mammalian embryo relies on retinol, retinyl ester and β-carotene circulating in the maternal bloodstream for its supply of vitamin A. The maternal-fetal transfer of retinoids and carotenoids, as well as the metabolism of these compounds in the developing tissues are still poorly understood. The existing knowledge in this field has been summarized in this review in reference to our basic understanding of the transport and metabolism of retinoids and carotenoids in adult tissues. The need for future research on the metabolism of these essential lipophilic nutrients during development is highlighted. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

The prevention of lung cancer induced by a tobacco-specific carcinogen in rodents by green and black Tea

A growing body of evidence from studies in laboratory animals indicates that green tea protects against cancer development at various organ sites. We have previously shown that green tea, administered as drinking water, inhibits lung tumor development in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone (NNK), a potent nicotine-derived lung carcinogen found in tobacco. The inhibitory effect of green tea has been attributed to its major polyphenolic compound, epigallocatechin gallate (EGCG), and, to a lesser extent, to caffeine. We have also demonstrated that while levels of O6-methylguanine, a critical lesion in NNK lung tumorigenesis, were not affected in lung DNA. However, the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, were significantly suppressed in mice treated with green tea or EGCG. These studies underscore the importance of the antioxidant activity of green tea and EGCG for their inhibitory activity against lung tumorigenesis. Unlike green tea, the effect of black tea on carcinogenesis has been scarcely studied, even though the worldwide production and consumption of black tea far exceeds that of green tea. The oxidation products found in black tea, thearubigins and theaflavins, also possess antioxidant activity, suggesting that black tea may also inhibit NNK-induced lung tumorigenesis. Indeed, bioassays in A/J mice have shown that black tea given as drinking water retarded the development of lung cancer caused by NNK. However, data on the relationship of black tea consumption with the lung cancer risk in humans are limited and inconclusive. There is a need for additional tumor bioassays in animal models to better examine the protective role of black tea against lung cancer. The development of adenocarcinomas and adenosquamous carcinomas in F344 rats upon chronic administration of NNK provides an important and relevant model for lung carcinogenesis in smokers. Thus far, no information was previously available regarding the effects of tea on this model. We conducted a 2-year lifetime bioassay in F344 rats to determine whether black tea and caffeine are protective against lung tumorigenesis induced by NNK. Our studies in both mice and rats have generated important new data that support green and black tea and caffeine as potential preventive agents against lung cancer, suggesting that a closer examination of the roles of tea and caffeine on lung cancer in smokers may be warranted.