Mechanistic aspects of carotenoid health benefits - where are we now?

Dietary intake and tissue levels of carotenoids have been associated with a reduced risk of several chronic diseases, including cardiovascular diseases, type 2 diabetes, obesity, brain-related diseases and some types of cancer. However, intervention trials with isolated carotenoid supplements have mostly failed to confirm the postulated health benefits. It has thereby been speculated that dosing, matrix and synergistic effects, as well as underlying health and the individual nutritional status plus genetic background do play a role. It appears that our knowledge on carotenoid-mediated health benefits may still be incomplete, as the underlying mechanisms of action are poorly understood in relation to human relevance. Antioxidant mechanisms - direct or via transcription factors such as NRF2 and NF-κB - and activation of nuclear hormone receptor pathways such as of RAR, RXR or also PPARs, via carotenoid metabolites, are the basic principles which we try to connect with carotenoid-transmitted health benefits as exemplified with described common diseases including obesity/diabetes and cancer. Depending on the targeted diseases, single or multiple mechanisms of actions may play a role. In this review and position paper, we try to highlight our present knowledge on carotenoid metabolism and mechanisms translatable into health benefits related to several chronic diseases.

Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity.

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

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.

Effect of Lumenato oral supplementation on plasma carotenoid levels and improvement of visual and experiential skin attributes.

BACKGROUND: Cellular metabolism and exposure to solar irradiation result in generation of free radicals which are destructive and can lead to premature aging. Antioxidants and free radical scavengers such as carotenoids successfully protect from these free radicals by quenching and neutralizing them thereby strengthening skin barrier which leads to improved skin moisturization, desquamation, and a more youthful look. This study was designed to evaluate the consumer-perceived efficacy of an oral supplement (Lumenato™) containing a mix of tomato carotenoids and oil-soluble vitamins in improving skin appearance after 12 weeks of supplement use. MATERIALS AND METHODS: Plasma levels of phytoene, phytofluene, zeta-carotene, and lycopene were quantitated before and after 1-, 2-, 3-, and 4-week administration of Lumenato by 24 healthy volunteers. Part II of the study addressed skin visual attributes as assessed by validated tools (questionnaires). A total of 60 females, aged 35 to 55 years, completed part II of the study. The subjects answered questionnaires pertaining to their assessment of skin appearance before and after 12 weeks of taking the supplement. RESULTS: There was a significant increase (p < 0.001) in plasma levels of phytoene, phytofluene, and zeta-carotene after 1- to 4-week treatment with Lumenato. After 12 weeks of using the supplement, the score of different skin parameters was reported to significantly improve (p < 0.001). Improvement was recorded in skin elasticity, firmness, brightness, skin tone, reduction in dark spots and periorbital dark circles, skin hydration, texture and fine lines and wrinkles. A significant (p < 0.001) improvement in overall skin condition after using the supplement was observed. The subjects noticed statistically significant (p < 0.001) improvement in skin elasticity, firmness, brightness, skin tone, reduction in dark spots and periorbital dark circles, skin hydration, texture and fine lines and wrinkles after 12 weeks of using the supplement. The overall skin condition also exhibited a significant improvement (p < 0.001). Self-assessed improvement of the face was identified at the first time point (4 weeks) and improved significantly (p < 0.001) for the 12 weeks of use. Interestingly, these improvements persisted even after treatment was stopped. CONCLUSION: Based on the confines and conditions of this study, the use of oral supplement containing a mix of tomato carotenoids significantly increased plasma levels of phytoene, phytofluene, and zeta-carotene, and continuous use resulted in improved facial skin attributes which were palpable by the consumers and continued even after treatment was stopped.

Effects of golden tomato extract on skin appearance-outlook into gene expression in cultured dermal fibroblasts and on trans-epidermal water loss and skin barrier in human subjects.

SCOPE: Two experiments were performed to test the effects of rich tomato extract (Golden Tomato Extract, GTE) on human skin. In one experiment, the effects of this extract on gene expression in cultured human dermal fibroblasts were examined. In a second experiment, human subjects consumed the extract and trans-epidermal water loss (TEWL), and aspects of skin appearance were monitored. METHODS AND RESULTS: Primary human dermal fibroblasts in culture were treated with the extract. After six hours, RNA was extracted, and gene expression was examined using Affymetrix Human Clariom D array processing. For the clinical study, 65 human subjects consumed a capsule once a day for 16 weeks, and various skin parameters were assessed at predetermined time intervals. Among the genes upregulated by GTE are genes that augment innate immunity, enhance DNA repair, and the ability to detoxify xenobiotics. GTE significantly reduced TEWL in subjects who had high TEWL at baseline, but it had no effect on TEWL in subjects who had lower TEWL at baseline. CONCLUSIONS: Golden tomato extract may provide benefits to the skin by enhancing innate immunity and other defense mechanisms in the dermis and by providing antioxidants to the skin surface to optimize TEWL and the appearance of the skin.

The Protective Effect of Carotenoids, Polyphenols, and Estradiol on Dermal Fibroblasts under Oxidative Stress.

Skin ageing is influenced by several factors including environmental exposure and hormonal changes. Reactive oxygen species (ROS), which mediate many of the effects of these factors, induce inflammatory processes in the skin and increase the production of matrix metalloproteinases (MMPs) in dermal fibroblasts, which leads to collagen degradation. Several studies have shown the protective role of estrogens and a diet rich in fruits and vegetables on skin physiology. Previous studies have shown that dietary carotenoids and polyphenols activate the cell's antioxidant defense system by increasing antioxidant response element/Nrf2 (ARE/Nrf2) transcriptional activity and reducing the inflammatory response. The aim of the current study was to examine the protective effect of such dietary-derived compounds and estradiol on dermal fibroblasts under oxidative stress induced by H2O2. Human dermal fibroblasts were used to study the effect of H2O2 on cell number and apoptosis, MMP-1, and pro-collagen secretion as markers of skin damage. Treatment of cells with H2O2 led to cell death, increased secretion of MMP-1, and decreased pro-collagen secretion. Pre-treatment with tomato and rosemary extracts, and with estradiol, reversed the effects of the oxidative stress. This was associated with a reduction in intracellular ROS levels, probably through the measured increased activity of ARE/Nrf2. Conclusions: This study indicates that carotenoids, polyphenols, and estradiol protect dermal fibroblasts from oxidative stress-induced damage through a reduction in ROS levels.

Seaweeds fast EDC bioremediation: Supporting evidence of EE2 and BPA degradation by the red seaweed Gracilaria sp., and a proposed model for the remedy of marine-borne phenol pollutants.

In the last few decades, Endocrine Disrupting Chemicals (EDCs) have taken significant roles in creating harmful effects to aquatic organisms. Many proposed treatment applications are time consuming, expensive and focus mainly on waste water treatment plants (WWTP), which are indeed a major aquatic polluting source. Nonetheless, the marine environment is the ultimate sink of many pollutants, e.g. EDCs, and has been largely neglected mainly due to the challenge in treating such salty and immense open natural ecosystems. In this study we describe the bromination and the yet unpresented degradation process of high concentrations (5 mg/L) of phenolic EDCs, by the marine red macroalgaeGracilaria sp. As shown, 17α-Ethinylestradiol (EE2), a well-known contraceptive drug, and one of the most persistent phenol EDCs in the environment, was eliminated from both the medium and tissues of the macroalga, in addition to the degradation of all metabolites as verified by the nil estrogenic activity recorded in the medium. Validation of the proposed bromination-degradation route was reinforced by identifying Bisphenol A (BPA) brominated degradation products only, following 168H of incubation in the presence of Gracilaria sp. As demonstrated in this assay for EE2, BPA and finally for paracetamol, it is likely that the phenol scavenging activity is nonspecific and, thus, possibly even a wider scope of various other phenol-based pollutants might be treated in coastal waters. As far as we know, Gracilaria sp. is the only marine sessile organism able of degrading various phenol based pollutants. The worldwide distribution of many Gracilaria species and their wide aquaculture knowhow, suggest that bioremediation based on these seaweeds is a possible cost effective progressive solution to the treatment of a wide scope of phenols at the marine environment.

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

Recent Progress in Discovering the Role of Carotenoids and Metabolites in Prostatic Physiology and Pathology-A Review-Part II: Carotenoids in the Human Studies.

Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention related to chronic diseases. One organ that has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives-retinoids and apo-carotenoids) are involved in a plethora of intra- and intercellular signaling, cell growth, and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids, such as lycopene (LYC) and ß-carotene (BC), in prostatic physiology and pathology, the present review aimed to cover the past ten years of research in this regard. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed in this second part. The objective of this compilation was to emphasize the present state of knowledge about the most potent molecular targets of carotenoids, as well as to propose promising carotenoid agents for the prevention and possible treatment of prostatic diseases.

Recent Progress in Discovering the Role of Carotenoids and Their Metabolites in Prostatic Physiology and Pathology with a Focus on Prostate Cancer-A Review-Part I: Molecular Mechanisms of Carotenoid Action.

Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention and medicine. One organ which has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives-retinoids and other apo-carotenoids) are involved in intra- and intercellular signaling, cell growth and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids such as lycopene (LC) and ß-carotene (BC) in prostatic physiology and pathology, the present review aims to cover the past ten years of research in this area. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed and discussed in the second part. The objective of this compilation is to emphasize the present state of knowledge regarding the most potent molecular targets of carotenoids and their main metabolites, as well as to propose promising carotenoid agents for the prevention and treatment of prostatic diseases.

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.

Synergistic antileukemic activity of carnosic acid-rich rosemary extract and the 19-nor Gemini vitamin D analogue in a mouse model of systemic acute myeloid leukemia.

OBJECTIVE: Differentiation therapy with the hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D(3) induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D(3) analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML. METHODS: Proliferation and differentiation of WEHI-3B D- (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice. RESULTS: 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D(3), in the induction of differentiation (EC(50) = 0.059 +/- 0.011, 0.275 +/- 0.093 and 0.652 +/- 0.085 nM, respectively) and growth arrest (IC(50) = 0.072 +/- 0.018, 0.165 +/- 0.061 and 0.895 +/- 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 +/- 2.5 days in untreated mice to 66.5 +/- 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D(3) combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures. CONCLUSION: Combined effectiveness of 1,25D(3) analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

Tomato lycopene extract supplementation decreases insulin-like growth factor-I levels in colon cancer patients.

Epidemiological studies have shown that high serum levels of insulin-like growth factor-I are associated with an increased risk of colon and other types of cancer. The aim of this study was to determine whether short intervention with dietary tomato lycopene extract will affect serum levels of the insulin-like growth factor system components in colon cancer patients. The study had a double-blind, randomized, placebo-controlled design. Colon cancer patients (n=56), candidates for colectomy, were recruited from the local community a few days to a few weeks before surgery. Personal and medical data were recorded. Plasma concentrations of insulin-like growth factor-I and II and insulin-like growth factor-I-binding protein-3 were assayed by routine laboratory methods. Lycopene was assayed by high-performance liquid chromatography. Plasma lycopene levels increased by twofold after supplementation with tomato lycopene extract. In the placebo-treated group, there was a small nonsignificant increase in lycopene plasma levels. The plasma concentration of insulin-like growth factor-I decreased significantly by about 25% after tomato lycopene extract supplementation as compared with the placebo-treated group (P<0.05). No significant change was observed in insulin-like growth factor-I-binding protein-3 or insulin-like growth factor-II, whereas the insulin-like growth factor-I/insulin-like growth factor-I-binding protein-3 molar ratio decreased significantly (P<0.05). Given that high plasma levels of insulin-like growth factor-I have been suggested as a risk factor for various types of cancer including colon cancer, the results support our suggestion that tomato lycopene extract has a role in the prevention of colon and possibly other types of cancer.

Carnosic acid potentiates the antioxidant and prodifferentiation effects of 1alpha,25-dihydroxyvitamin D3 in leukemia cells but does not promote elevation of basal levels of intracellular calcium.

Differentiation therapy of cancer remains an only partially attained goal. Agents currently under active investigation include derivatives of vitamin D, modeled on its physiological hormone form, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), but the calcemic effects of these compounds preclude their use in the clinic. An approach that may obviate this problem is to combine 1,25D(3) or its derivatives with other agents that increase the antineoplastic effects of low, nontoxic concentrations of vitamin D compounds. We have recently used the plant-derived polyphenolic antioxidant, carnosic acid (CA), to demonstrate an increase in the differentiating action of 1,25D(3) on human leukemia cells under these conditions (M. Danilenko et al., JNCI, 93: 1224-1233, 2001). We now show that treatment of HL60-G cells with either CA or 1,25D(3) alone resulted in a decrease in the intracellular levels of reactive oxygen species. Furthermore, the combination of 10 micro M CA and a low concentration of 1,25D(3) (1 nM) produced an enhanced antioxidant effect, which correlated with the potentiation of monocytic differentiation. Other plant antioxidants tested (curcumin, silibinin, and the organoselenium antioxidant ebselen) also potentiated differentiation induced by 1,25D(3), although alone, they had only minor differentiating effects. Differentiation induced by CA/1,25D(3) combinations was associated with increased intracellular glutathione content, whereas buthionine sulfoxime decreased both differentiation and the cellular glutathione content. This combination also enhanced the activation of the Raf-mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase mitogen-activated protein kinase module and increased the binding of the activator protein-1 (AP-1) transcription factor to its cognate DNA element in the promoter regions of vitamin D receptor gene, suggesting that the mechanism of potentiation is at least in part attributable to induction and activation of components of this mitogen-activated protein kinase pathway. Cell treatment with a high concentration of 1,25D(3) (100 nM) resulted in a substantial elevation of basal intracellular calcium concentration. In contrast, importantly for an eventual clinical application of these studies, the potentiating action of CA on differentiation induced by a low concentration of 1,25D(3) (1 nM) was not accompanied by an elevation of basal intracellular calcium concentration. These findings suggest that combinations of CA with derivatives of vitamin D should be evaluated for use in differentiation therapy of myeloid leukemias.

Carotenoids activate the antioxidant response element transcription system.

Epidemiologic studies have found an inverse association between consumption of tomato products and the risk of certain types of cancers. However, the mechanisms underlying this relationship are not completely understood. One mechanism that has been suggested is induction of phase II detoxification enzymes. Expression of phase II enzymes is regulated by the antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E2-related factor 2). In this study, we determined the role of this transcription system in the induction of phase II enzymes by carotenoids. We found that in transiently transfected cancer cells, lycopene transactivated the expression of reporter genes fused with ARE sequences. Other carotenoids such as phytoene, phytofluene, beta-carotene, and astaxanthin had a much smaller effect. An increase in protein as well as mRNA levels of the phase II enzymes NAD(P)H:quinone oxidoreductase and gamma-glutamylcysteine synthetase was observed in nontransfected cells after carotenoid treatment. Ethanolic extract of lycopene containing unidentified hydrophilic derivatives of the carotenoid activated ARE with similar potency to lycopene. The potency of the carotenoids in ARE activation did not correlate with their effect on intracellular reactive oxygen species and reduced glutathione level, which may indicate that ARE activation is not solely related to their antioxidant activity. Nrf2, which is found predominantly in the cytoplasm of control cells, translocated to the nucleus after carotenoid treatment. Interestingly, part of the translocated Nrf2 colocalized with the promyelocytic leukemia protein in the promyelocytic leukemia nuclear bodies. The increase in phase II enzymes was abolished by a dominant-negative Nrf2, suggesting that carotenoid induction of these proteins depends on a functional Nrf2 and the ARE transcription system.

Cancer-selective cytotoxic Ca2+ overload in acute myeloid leukemia cells and attenuation of disease progression in mice by synergistically acting polyphenols curcumin and carnosic acid.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by extremely heterogeneous molecular and biologic abnormalities that hamper the development of effective targeted treatment modalities. While AML cells are highly sensitive to cytotoxic Ca2+ overload, the feasibility of Ca2+- targeted therapy of this disease remains unclear. Here, we show that apoptotic response of AML cells to the synergistically acting polyphenols curcumin (CUR) and carnosic acid (CA), combined at low, non-cytotoxic doses of each compound was mediated solely by disruption of cellular Ca2+ homeostasis. Specifically, activation of caspase cascade in CUR+CA-treated AML cells resulted from sustained elevation of cytosolic Ca2+ (Ca2+cyt) and was not preceded by endoplasmic reticulum stress or mitochondrial damage. The CUR+CA-induced Ca2+cyt rise did not involve excessive influx of extracellular Ca2+ but, rather, occurred due to massive Ca2+ release from intracellular stores concomitant with inhibition of Ca2+cyt extrusion through the plasma membrane. Notably, the CUR+CA combination did not alter Ca2+ homeostasis and viability in non-neoplastic hematopoietic cells, suggesting its cancer-selective action. Most importantly, co-administration of CUR and CA to AML-bearing mice markedly attenuated disease progression in two animal models. Collectively, our results provide the mechanistic and translational basis for further characterization of this combination as a prototype of novel Ca2+-targeted pharmacological tools for the treatment of AML.