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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.

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.

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.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.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.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.

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&ndash, 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 &lt, 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.

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.

OBJECTIVES: Examine the effect of a carotenoid-rich Tomato Nutrient Complex on blood pressure and perform a dose-response analysis and uncover the optimal effective supplementation dose in maintaining blood pressure within normal range among individuals with systolic blood pressure between 130–140 mmHg, otherwise healthy subjects. METHODS: In this double-blind, randomized, placebo-controlled study, different doses of the Tomato Nutrient Complex were examined, corresponding to 5 mg, 15 mg, and 30 mg lycopene. The effect of the tomato-derived treatment was compared with 15 mg of synthetic Lycopene and placebo over 8 weeks of treatment. In addition, we analyzed the bioavailability of the carotenoids following treatment with different doses of the Tomato Nutrient Complex in a group of 25 healthy volunteers. Volunteers were treated for four weeks with Tomato Nutrient Complex providing 2, 5 and 15 mg lycopene. RESULTS: Results indicate that treatment for 8 weeks with Tomato Nutrient Complex standardized to contain 15 mg or 30 mg of lycopene was associated with statistically significant reductions in mean SBP of 9.7 mmHg and 7 mmHg compared to baseline values respectively. Treatment with the lower dose Tomato Nutrient Complex standardized for 5 mg of lycopene, or treatment with 15 mg of synthetic lycopene as a standalone did not show a significant effect. In parallel, we analyzed the bioavailability of the carotenoids in healthy subjects. In a group of 25 healthy volunteers treated for 4 weeks with different doses of the Tomato Nutrient Complex. we identified a dose dependent and significant increase in blood level of lycopene, phytoene, and phytofluene. CONCLUSIONS: Overall, the results suggest that the carotenoids in the Tomato Nutrient Complex are well absorbed in a dose dependent manner and that only the carotenoid levels achieved by the dose corresponding to 15 mg lycopene or more, is correlated to a beneficial effect on systolic blood pressure while lower doses and standalone lycopene are not enough to drive the effect. FUNDING SOURCES: Lycored.

OBJECTIVES: To demonstrate that the combination of carotenoids and polyphenols produce a synergistic photo-protective effect from UV-induced damage to skin cells. Mechanistically, this can be partially explained by modulation of the NFkB and antioxidant response element/Nrf2 (ARE/Nrf2) transcription systems that are important in this process. METHODS: In skin keratinocytes and fibroblasts, we analyzed the effects of carotenoids and polyphenol combinations on ARE/Nrf2 and NFkB transcriptional activity (measured by reporter gene assays), as well as on UVB-induced IL-6 and matrix metalloproteinase (MMP-1) expression (measured by ELISA). RESULTS: A combination of Tomato Nutrient Complex (containing lycopene) with rosemary extract (containing carnosic acid) synergistically inhibited NFkB and activated ARE/Nrf2 transcription systems in epidermal keratinocytes and dermal fibroblasts. This combination of phytonutrients also synergistically inhibited the UVB-induced release of the cytokine IL-6. Inflammatory cytokines such as IL-6 and TNFα induce the expression of MMPs, which leads to collagen breakdown. We found that MMP-1 secretion was induced in human dermal fibroblasts by the pro-inflammatory cytokine TNFα. Pre-treatment of these cells with carnosic acid resulted in reduced MMP-1 protein levels. These 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, we checked the human bioavailability of carotenoids from Tomato Nutrient Complex and found nearly a two-fold increase in their plasma concentrations. CONCLUSIONS: This study demonstrates that combination of dietary carotenoids and polyphenols synergistically inhibit UV-induced skin cell damage. The NFKB and ARE/Nrf2 transcription systems are likely involved in these effects. FUNDING SOURCES: Lycored.

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.

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.

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.

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 IKKβ and the p65 subunit. Direct interaction with IKKβ 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.

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.

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.

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.

Objective: Differentiation therapy with the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D3 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 1,25D3 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 1,25D3, in the induction of differentiation (EC50 = 0.059 ± 0.011, 0.275 ± 0.093 and 0.652 ± 0.085 nM, respectively) and growth arrest (IC50 = 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 1,25D3 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 1,25D3 analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) is a powerful differentiation agent, which has potential for treatment of myeloid leukemias and other types of cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that carnosic acid, the major rosemary polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D(3) in human myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D(-) murine myelomonocytic leukemia cells induced by 1 nM 1,25D(3) or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by carnosic acid (10 microM) or the carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0 + G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p. injections of 2 microg Ro25-4020 in Balb/c mice bearing WEHI-3B D(-) tumors produced a significant delay in tumor appearance and reduction in tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 microg Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a vitamin D derivative can cooperate not only in inducing leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for chemoprevention or differentiation therapy of myeloid leukemia.

The possible involvement of several transcription systems in the anticancer activity of carotenoids is the focus of this review. Carotenoids modulate the basic mechanisms of cell proliferation, growth factor signaling, gap junctional intercellular communication, and produce changes in the expression of many proteins participating in these processes. The changes in the expression of multiple proteins suggest that the initial effect of carotenoids involves modulation of transcription. We and others have found evidence for the role of several transcription systems, such as the retinoid receptors, activator protein-1 (AP-1), peroxisome proliferator-activated receptors (PPAR), xenobiotic receptors and the antioxidant response element (ARE), in the anticancer activity of carotenoids. The observed modulation of a network of transcription systems may provide the molecular basis for the synergistic anticancer effects of the combinations of various carotenoids together with other dietary and pharmacologic compounds.

Lycopene, the main carotenoid found in tomatoes and tomato-based products, has been reported to be protective against several types of cancer. Assessment of changes in plasma concentration of carotenoids following ingestion of lycopene-rich food sources does not necessarily predict changes in lycopene concentration or distribution of its isomers in other body tissues. Our aim was to determine the relationship between concentrations of lycopene and other tomato carotenoids in human serum and body tissues after tomato-oleoresin supplementation. Tomato lycopene oleoresin (30 mg/d) or a placebo was administered for 1 to 7 weeks to seventy-five volunteers undergoing elective haemorrhoidectomy or peri-anal fistulotomy. Carotenoid concentration and isomer distribution in blood and in the surgically removed skin and adipose tissues was measured by HPLC. The serum concentration of lycopene increased after supplementation from 0·26 (sd0·12) to 0·52 (sd0·25) μmol/l (n35;Pn40), lycopene serum concentration did not change significantly. Serum lycopene concentration after treatment was 2·2-fold greater in the lycopene group than in the placebo group, a slightly higher ratio than that found in skin and adipose tissue (1·6- and 1·4-fold higher than the placebo, respectively). A significant correlation between serum and tissue concentrations was found for both β-carotene and lycopene in the placebo group, whereas in the lycopene-supplemented group the correlation between serum and tissues remained the same for β-carotene but for lycopene was weak. Lycopene supplementation did not significantly change the proportion of all-trans v. cisisomers in the serum and tissues, despite the fact that more than 90 % of the supplemented lycopene was in the all-transform. These results show that tomato-oleoresin supplementation increases lycopene concentrations in serum and in adipose tissue and skin. The ability to increase lycopene levels in tissues is one of the prerequisites for using it as a food supplement with health benefits.

There is extensive evidence that high intake of fruits and vegetables is associated with decreased risk of many types of cancers. Thus, it is widely accepted that diet changes are a powerful means to prevent cancer. Although there is a growing interest in the role of the tomato carotenoid lycopene in cancer prevention and treatment, we hypothesize that a single micronutrient cannot replace the power of the concerted action of multiple agents derived from a diet rich in fruits and vegetables. Indeed, we found that lycopene can synergize with other phytonutrients in the inhibition of cancer cell growth. The mechanism underlying the inhibitory effects of lycopene and other carotenoids involves interference in several pathways related to cancer cell proliferation and includes changes in the expression of many proteins participating in these processes, such as connexins, cyclins, cyclin-dependent kinases, and their inhibitors. These changes in protein expression suggest that the initial effect involves modulation of transcription by ligand-activated nuclear receptors or by other transcription factors. It is feasible to suggest that carotenoids and their oxidized derivatives interact with a network of transcription systems that are activated by different ligands at low affinity and specificity and that this activation leads to the synergistic inhibition of cell growth.

Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models. In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death. Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl. Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid. Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression. These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.

Numerous studies have demonstrated the anticancer activity of the tomato carotenoid, lycopene. However, the molecular mechanism of this action remains unknown. Lycopene inhibition of human breast and endometrial cancer cell growth is associated with inhibition of cell cycle progression at the G(1) phase. In this study we determined the lycopene-mediated changes in the cell cycle machinery. Cells synchronized in the G(1) phase by serum deprivation were treated with lycopene or vehicle and restimulated with 5% serum. Lycopene treatment decreased serum-induced phosphorylation of the retinoblastoma protein and related pocket proteins. This effect was associated with reduced cyclin-dependent kinase (cdk4 and cdk2) activities with no alterations in CDK protein levels. Lycopene caused a decrease in cyclin D1 and D3 levels whereas cyclin E levels did not change. The CDK inhibitor p21(Cip1/Waf1) abundance was reduced while p27(Kip1) levels were unaltered in comparison to control cells. Serum stimulation of control cells resulted in reduction in the p27 content in the cyclin E--cdk2 complex and its accumulation in the cyclin D1--cdk4 complex. This change in distribution was largely prevented by lycopene treatment. These results suggest that lycopene inhibits cell cycle progression via reduction of the cyclin D level and retention of p27 in cyclin E--cdk2, thus leading to inhibition of G(1) CDK activities.

It has been suggested that adjunctive growth hormone (GH) therapy improves ovarian response and in vitro fertilization (IVF) outcome in specific groups of patients. The correlation between insulin-like growth factor (IGF) and GH is well established. The aim of this study was to determine whether changes in plasma GH correlate with IGF blood levels in patients during IVF treatment. Thirty-six women undergoing IVF and embryo transfer (ET) were examined. Ovarian stimulation was carried out by gonadotropin-releasing hormone agonists (GnRHa) and gonadotropins. Blood was drawn at the early and late follicular phase, on the day of human chorionic gonadotropin (hCG) injection and at the mid- and the late luteal phases. The samples were assayed for IGF-I, IGF-II, IGF-binding protein-3 (IGF BP-3), GH and estradiol. According to the IGF-I and GH plasma levels, patients were divided into three major groups: Group I consisted of patients in whom peak levels of GH reached more than 4 ng/ml and IGF-I decreased significantly. In this group, estradiol levels were 1863 +/- 149 pg/ml. Group II consisted of patients in whom peak blood GH levels did not exceed 2.5 ng/ml and the IGF-I level remained unchanged. In this group estradiol levels were 630 +/- 57 pg/ml. Group III consisted of patients in whom blood GH levels were low and remained unchanged while estradiol levels were 1600 +/- 420 pg/ml. In this group no significant increase in IGF-levels were observed. There was no significant change in the levels of either IGF-II or IGF BP-3 in any of the groups. We can conclude that (1) there is a negative correlation between GH and IGF-I plasma levels in patients undergoing controlled ovarian hyperstimulation (COH)-IVF, when levels of estradiol and GH are elevated; (2) plasma levels of IGF-I under ovarian hyperstimulation are probably regulated by a multifactorial system; and (3) no correlation was found between the plasma levels of IGF-I and those of IGF-II and IGF BP-3 in all patient groups.

A significant increase in endometrial cancer incidence in tamoxifen-treated breast cancer patients has been reported in many recent studies. The major growth stimulators of endometrial tumors are estrogens, but paradoxically, tamoxifen, a known antiestrogen, also stimulates their growth. The mode of action of estrogen can be partially explained by the modulation of insulin-like growth factor (IGF) autocrine or paracrine action. The purpose of the present study was to examine the involvement of the IGF system in the tamoxifen-stimulated growth of Ishikawa endometrial cancer cells by quantitating the IGF-I receptors and their phosphorylation, as well as membrane associated and secreted IGF-binding proteins (IGFBPs). Tamoxifen did not affect the number or affinity of IGF-I receptors. On the other hand, tamoxifen, similar to estradiol, increased IGF-I-stimulated tyrosine phosphorylation of cellular substrates. In contrast, in MCF-7 mammary cancer cells, tamoxifen reduced IGF-induced tyrosine phosphorylation in the presence of estradiol. The pure antiestrogen LY156758 did not affect Ishikawa basal cell growth but inhibited estradiol- and tamoxifen-induced growth. Growth inhibition by LY156758 of tamoxifen and estradiol-stimulated cells was accompanied by a corresponding inhibition of IGF-stimulated tyrosine phosphorylation. Tamoxifen caused a 3-fold decrease in membrane-associated IGFBPs. Moreover, a reduction in soluble IGFBPs was also observed, making the IGF peptides more available to the receptors. A parallel decrease in IGFBP-3 mRNA was also detected. These experiments suggest that tamoxifen, like estradiol, directly sensitizes endometrial cancer cells to the effects of IGFs that act through the type I receptor. Furthermore, the decrease in IGFBPs and the increase in tyrosine phosphorylation in the presence of tamoxifen provides a molecular mechanism that accounts for the uterotropic effects that are seen with tamoxifen therapy.

The antiproliferative properties of lycopene, the major tomato carotenoid, were compared with those of alpha- and beta-carotene. Lycopene, delivered in cell culture medium from stock solutions in tetrahydrofuran, strongly inhibited proliferation of endometrial (Ishikawa), mammary (MCF-7), and lung (NCI-H226) human cancer cells with half-maximal inhibitory concentration of 1-2 microM; alpha- and beta-carotene were far less effective inhibitors. For example, in Ishikawa cells, a 4-fold higher concentration of alpha-carotene or a 10-fold higher concentration of beta-carotene was needed for the same order of growth suppression. The inhibitory effect of lycopene was detected after 24 hours of incubation, and it was maintained for at least three days. In contrast to cancer cells, human fibroblasts were less sensitive to lycopene, and the cells gradually escaped growth inhibition over time. In addition to its inhibitory effect on basal endometrial cancer cell proliferation, lycopene also suppressed insulin-like growth factor-I-stimulated growth. Insulin-like growth factors are major autocrine/paracrine regulators of mammary and endometrial cancer cell growth. Therefore, lycopene interference in this major autocrine/paracrine system may open new avenues for research on the role of lycopene in the regulation of endometrial cancer and other tumors.

1α,25-dihydroxyvitamin D3 (1,25D) is a powerful differentiation agent, which has potential for treatment of acute myeloid leukemia (AML), but induces severe hypercalcemia at pharmacologically active doses. We have previously shown that carnosic acid (CA), the polyphenolic antioxidant from rosemary plant, markedly potentiates differentiation induced by low concentrations of 1,25D in human AML cell lines. Here, we demonstrated similar enhanced differentiation responses to the 1,25D/CA combination in primary leukemic cells derived from patients with AML, and determined the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in these effects using U937 human monoblastic leukemia cells as the model. CA strongly transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels. Interestingly, 1,25D potentiated the effects of CA on these activities. Stable transfection of wild-type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These opposite effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid X receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed opposing changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the differentiation in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important part in the cooperative induction of myeloid leukemia cell differentiation by 1,25D and a plant polyphenol.

Bombesin-like peptides including neuromedin B have been proposed as autocrine or paracrine growth factors for carcinomas. To examine signal transduction and regulation of cell growth by NMB, transfectants were created with the rat NMB receptor (NMB-R) gene in BALB/3T3 cells which do not express an endogenous bombesin peptide receptor. The resultant cell line, NMB-8, expresses 800,000 NMB binding sites/cell. Addition of NMB has a biphasic effect on [3H]thymidine ([3H]dT) incorporation in confluent and quiescent cells: up to 10 nM of NMB causes a 1.5-3-fold stimulation of [3H]dT incorporation, but at greater than 10 nM there is inhibition of [3H]dT incorporation, and at 100 nM of NMB there is inhibition of cell growth. NMB causes protracted increases in intracellular Ca2+, and pertussis toxin (PT)-insensitive phosphatidylinositol (PI) turnover. NMB-mediated increase in membrane phospholipase-C activity is stimulated by guanosine 5'-O-(3-thiotriphosphate). Arachidonate release is also activated by NMB in a PT-insensitive manner. Brief exposure to 12-tetradecanoylphorbol 13-acetate inhibits NMB-mediated PI turnover but not arachidonate release. Thus, in NMB-8 cells, distinct mechanisms govern NMB-mediated phospholipase-C activation and arachidonate release. Also, neuromedin-B is potentially a bifunctional regulator of cell growth.

Induction of phase II detoxifying enzymes is a major cellular strategy for reducing the risk of cancer. We previously reported that carotenoids activate the electrophile/antioxidant response element (EpRE/ARE) transcription system and induced the expression of phase II enzymes. Various electrophilic phytonutrients have been shown to induce the EpRE/ARE system by disrupting the inhibitory activity of Keap1 on Nrf2, the major EpRE/ARE activating transcription factor. However, hydrophobic carotenoids such as lycopene lack any electrophilic group and, thus, are unlikely to directly activate Nrf2 and the EpRE/ARE system. Here we demonstrate that carotenoid oxidation products are the active mediators in the stimulation of the EpRE/ARE system by carotenoids. Two lines of evidence support this conclusion. (A) The oxidized derivatives, extracted by ethanol from partially oxidized lycopene, transactivated EpRE/ARE with a potency similar to that of the unextracted lycopene mixture, whereas the intact carotenoid showed a nonsignificant effect. (B) Using a series of characterized mono- and diapocarotenoids that potentially can be derived from in vivo metabolism of carotenoids we defined the following structure-activity rules for activation of EpRE/ARE: (I) aldehydes and not acids are the active molecules; (II) the activity depends on the relative position of the methyl group to the terminal aldehyde which determines the reactivity of the conjugated double bond; (III) the optimal length of a dialdehyde derivative is 12 carbons in the main chain of the molecule. The apocarotenals inhibited breast and prostate cancer cell growth with a similar order of potency to the activation of EpRE/ARE. These results may provide a mechanistic explanation for the cancer preventive activity of carotenoids.

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

1α,25-dihydroxyvitamin D3 (1,25D) is known to regulate various cellular functions, including cell proliferation and differentiation. Both 1,25D and its precursor, 25-hydroxyvitamin D3 [25(OH)D], are catabolized by 24-hydroxylase encoded by the CYP24A1 gene, which is one of the most responsive vitamin D receptor (VDR) target genes, and a is candidate oncogene frequently overexpressed in various malignancies. Therefore, compounds which can inhibit CYP24A1 expression and/or activity, thereby reducing degradation of vitamin D derivatives, may potentiate their anticancer effects. We have previously shown that the plant polyphenols carnosic acid, curcumin and silibinin synergistically enhance differentiation of acute myeloid leukemia (AML) cells induced by near physiologic concentrations of 1,25D. This was associated with the transcription factor Nrf2-assisted elevation of both VDR and the retinoid X receptor alpha (RXRα) protein levels. Here, we demonstrate that despite the latter effect the polyphenols markedly inhibited 1,25D-induced CYP24A1 mRNA expression in AML cell lines but not in osteoblast-like cells. Furthermore, while potentiating 1,25D-induced transactivation of the direct repeat-3 (DR3)-type consensus vitamin D response element (VDRE) in AML cells, as measured by the luciferase (Luc) reporter gene assay, the polyphenols strongly inhibited 1,25D-dependent activation of the CYP24-Luc reporter construct containing an early (-298 bp) promoter region with two critical DR3-type VDREs (VDRE1 and VDRE2). In contrast, the polyphenols failed to inhibit and even enhanced 1,25D-induced transactivation of CYP24-Luc transfected into osteoblastic cells. Inactivating mutations of the VDRE1 or VDRE2 sequence in this construct did not abolish the inhibitory effect of polyphenols. However, overexpression of Nrf2 resulted in reduced CYP24-Luc activation by 1,25D, while dominant-negative Nrf2 had an opposite effect. Importantly, feeding of healthy Balb/c mice with standard rodent chow supplemented with carnosic acid-rich rosemary extract resulted in a marked dose- and time-dependent increase in serum levels of 25(OH)D, as compared to control animals. This was associated with a significant decrease in CYP24A1 mRNA levels in the kidneys of these mice. Collectively, the above data suggest that by downregulating CYP24A1 in a tissue-dependent manner, plant polyphenols may potentiate therapeutic and preventive effects of vitamin D derivatives. These findings may also contribute to our understanding of the beneficial effects of healthy diets. (Supported by the Israel Science Foundation grant 635/11 to M.D. and Y.S. and by the American Institute for Cancer Research grant #10A049 to G.P.S. and M.D.). Citation Format: Ehud Sharony, Ze'ev Barvish, Victoria Novik, Marina Khanin, Merav Cohen-Lahav, Doron Amichay, Yoav Sharoni, George P. Studzinski, Michael Danilenko. Plant polyphenols inhibit cellular 24-hydroxylase (CYP24A1) expression and elevate serum 25-hydroxyvitamin D levels. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2015-4655

Epidemiological evidence suggests that carotenoids prevent several types of cancer, including mammary and endometrial cancers. On the other hand, such studies have also shown that estrogens are the most important risk factors for these cancer types. Genistein, the phytoestrogen mainly found in soy, also shows significant estrogenic activity when tested at concentrations found in human blood. The aim of this study was to determine whether carotenoids inhibit signaling of steroidal estrogen and phytoestrogen which could explain their cancer preventive activity. Similar to the known effect of 17beta-estradiol (E(2)), treatment of breast (T47D and MCF-7) and endometrial (ECC-1) cancer cells with phytoestrogens induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element (ERE). However, each of the tested carotenoids (lycopene, phytoene, phytofluene, and beta-carotene) inhibited cancer cell proliferation induced by either E(2) or genistein. The inhibition of cell growth by lycopene was accompanied by slow down of cell-cycle progression from G1 to S phase. Moreover, the carotenoids inhibited estrogen-induced transactivation of ERE that was mediated by both estrogen receptors (ERs) ERalpha and ERbeta. The possibility that this inhibition results from competition of carotenoid-activated transcription systems on a limited pool of shared coactivators with the ERE transcription system was tested. Although cotransfection of breast and endometrial cancer cells with four different coactivators (SRC-1, SRC-2, SRC-3, and DRIP) strongly stimulated ERE reporter gene activity, it did not oppose the inhibitory effect of carotenoids. These results suggest that dietary carotenoids inhibit estrogen signaling of both 17beta-estradiol and genistein, and attenuate their deleterious effect in hormone-dependent malignancies.

Insulin-like growth factors (IGFs) play an important role in normal and cancerous cell proliferation. Moreover, in recent studies IGF-I has been implicated as a major cancer risk factor. The tomato carotenoid lycopene and all-trans retinoic acid (atRA) have been shown to inhibit growth factor-induced proliferation of different types of cancer cells. This action is associated with inhibition of cell cycle progression in G0/G1 phase. Cyclin D1 acts as a growth factor sensor in G1 phase and is overexpressed in many breast cancer tumors. We have previously demonstrated that slowdown of serum-stimulated cell cycle progression from G1 to S phase by lycopene correlates with reduction in cyclin D1 levels, suggesting that the expression of this protein is a main target for lycopene's action.To determine whether the reported reduction in cyclin D1 level is the key mechanism for lycopene and atRA inhibitory action on IGF-I-induced cell cycle progression.Human breast (MCF-7) and endometrial (ECC-1) cancer cells were synchronized in G0/G1 phase by serum deprivation followed by stimulation with IGF-I. Cell treatment with lycopene and atRA inhibited IGF-I-stimulated cell cycle progression from G1 to S phase and decreased retinoblastoma protein (pRb) phosphorylation. These events were associated with a reduction in cyclin D1 and p21(CIP1/WAF1) level, but not that of p27(KIP1). To test the hypothesis that the decrease in cyclin D1 has a major role in the inhibitory effects of lycopene and atRA, we examined the ability of these two agents to suppress cell cycle progression in MCF-7.7D1.13 cells which are capable of expressing cyclin D1 under the control of the Zn-inducible metallothionein promoter. Our results showed that ectopic expression of cyclin D1 can overcome cell cycle inhibition caused by lycopene and atRA.Our findings suggest that attenuation of cyclin Dl levels by lycopene and atRA is an important mechanism for the reduction of the mitogenic action of IGF-I.

It is widely accepted that diet changes are a powerful means to prevent cancer. The possible involvement of transcriptional activity in the anticancer activity of carotenoids will be the focus of this review. Carotenoids function as potent antioxidants, and this is clearly a major mechanism of their action. In addition carotenoids action involves interference in several pathways related to cancer cell proliferation and includes changes in the expression of many proteins participating in these processes such as connexins, phase II enzymes, cyclins, cyclin-dependent kinases and their inhibitors. These changes in protein expression suggest that the initial effect involves modulation of transcription by ligand-activated nuclear receptors or by other transcription factors. It is feasible to suggest that carotenoids and their oxidized derivatives interact with a network of transcription systems that are activated by different ligands at low affinity and specificity and that this activation leads to the synergistic inhibition of cell growth.

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.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.