Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.     

Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation

The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G1 and G2/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages.     

Red wine polyphenols, in the absence of alcohol, reduce lipid peroxidative stress in smoking subjects

Phenolic compounds in red wine can exert antioxidant effects on in vitro lipoprotein oxidation. This has led to speculation that red wine consumption mediates unique anti-atherosclerotic effects compared to other alcoholic beverages. However, studies assessing the effects of red wine consumption on lipoprotein oxidation ex vivo have not been conclusive. The recent identification of the F2-isoprostanes as oxidative products of arachidonic acid has provided a reliable measure of in vivo lipid peroxidation. This randomized trial investigated changes in plasma and urinary F2-isoprostane concentrations following red wine, white wine, or dealcoholized red wine consumption in humans. Eighteen male smokers consumed, in random order, red wine, white wine, or dealcoholized red wine, for two weeks with one week washout between beverages. Plasma and urinary F2-isoprostane concentrations were measured before and after each beverage. Serum gamma-glutamyl transpeptidase (gamma-GT) and urinary 4-O -methylgallic acid were measured as markers of alcohol consumption and phenolic acid absorption, respectively. Plasma F2-isoprostanes (p < .05) decreased significantly with dealcoholized red wine but not with the alcohol-containing beverages. Urinary excretion of F2-isoprostanes showed a similar trend. gamma-GT decreased significantly with dealcoholized red wine and increased with both alcohol-containing beverages (p < .01). Urinary excretion of 4-O-methylgallic acid increased significantly (p < .001) in the 24 h urine samples following red wine or dealcoholized red wine ingestion, but not with white wine. Serum urate increased and beta-carotene decreased with both alcoholic beverages relative to dealcoholized red wine. There was no change in the antioxidants alpha- and gamma-tocopherol or vitamin C with any of the beverages. The results suggest that polyphenols in dealcoholized red wine can reduce in vivo lipid peroxidation as measured by F2-isoprostanes in smoking subjects. However, no reduction in lipid peroxidation was observed following red or white wine consumption, suggesting that any protective effects of wine drinking on cardiovascular disease are unlikely to be related to inhibition of lipid oxidation.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.     

Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats

Polyphenols from tea and other beverages such as red wine have been regarded with interest as possible chemopreventive agents against cancer. Here we report that red wine polyphenols (50 mg/kg) administered with the diet to F344 rats for 16 weeks inhibited colon carcinogenesis induced by azoxymethane (AOM, 7.4 mg/kg, total dose 74 mg/kg) or dimethylhydrazine (DMH, 30 mg/kg, total dose, 300 mg/kg). Polyphenol-treated animals had a consistently lower tumour yield compared to controls. In polyphenol-treated rats, the main bacterial strains in the faeces at sacrifice were Bacteroides, Lactobacillus and Bifidobacterium spp., whereas microorganisms predominantly identified in control-fed rats were Bacteroides, Clostridium and Propionibacterium spp. Wine polyphenols (57 mg/kg for 10 days, by gavage), administered to rats not treated with carcinogens, produced a significant decrease in the basal level of DNA oxidative damage of the colon mucosa as measured with the comet assay (average pyrimidine oxidation was reduced by 62% and purine oxidation by 57%, p<0.05). To further explore the molecular effects of wine polyphenols we used the microarray technology to study gene expression profiles: rats were treated with 50 mg/kg wine polyphenols for 14 days, mixed in the diet. Global expression analysis of 5707 genes revealed an extensive down-regulation of genes involved in a wide range of physiological functions, such as metabolism, transport, signal transduction and intercellular signalling. By analysing metabolic pathways with the GenMAPP software program we observed that two major regulatory pathways were down-regulated in the colon mucosa of polyphenols-treated rats: inflammatory response and steroid metabolism. We also found a down-regulation of many genes regulating cell surface antigens, metabolic enzymes and cellular response to oxidative stress. In conclusion, reduction of oxidative damage, modulation of colonic flora and variation in gene expression may all concur in the modulation of intestinal function and carcinogenesis by wine polyphenols.     

Importance of the central region of lamprey gonadotropin-releasing hormone III in the inhibition of breast cancer cell growth

Naturally occurring isoforms of the decapeptide gonadotropin-releasing hormone (GnRH) share residues 1-4 and 9-10. lGnRH-III, the third isoform isolated in the sea lamprey has no endocrine effect in mammals but shows a direct antiproliferative effect on human breast, prostate and endometrial cancer cell lines. To investigate these features, residues 5-8 of lGnRH-III were systematically replaced with Ala. The ability of the synthetic analogs to interact with receptors on MDA-MB 231 human breast cancer cells and their effect on the growth of the same cell line were investigated. [Ala6]lGnRH-III and [Ala7]lGnRH-III have neither receptor binding nor antiproliferative activity. Replacement of His5 with Ala resulted in an analog that binds to the receptor but does not have antiproliferative activity. The results are in agreement with previous reports that modifications of Lys at position 8 are well tolerated.     

Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin,the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages

epsilon-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 microM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of epsilon-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 microM vs. 10 to 100 microM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids.     

Dihydro-resveratrol—A potent dietary polyphenol

Dihydro-resveratrol (dihydro-R), a prominent polyphenol component of red wine, has a profound proliferative effect on hormone-sensitive tumor cell lines such as breast cancer cell line MCF7. We found a significant increase in MCF7 tumor cells growth rates in the presence of picomolar concentrations of this compound. The proliferative effect of dihydro-R was not observed in cell lines that do not express hormone receptors (MDA-MB-231, BT-474, and К-562).     

Neuroprotective Effects of Natural Products: Interaction with Intracellular Kinases,Amyloid Peptides and a Possible Role for Transthyretin

Various studies reported on the neuroprotective effects of natural products, particularly polyphenols, widely present in food and beverages. For example, we have shown that resveratrol, a polyphenol contained present in red wine and other foods, activates the phosphorylation of protein kinase C (PKC), this effect being involved in its neuroprotective action against Ass-induced toxicity. Moreover, tea-derived catechin gallate esters inhibit the formation Ass oligomers/fibrils, suggesting that this action likely contributes to their neuroprotective effects. Interestingly, the effects of polyphenols may be attributable, at least in part, to the presence of specific binding sites. Autoradiographic studies revealed that these binding sites are particularly enriched in choroids plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to prevent Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins leading to their neuroprotective actions.     

Evaluation of synthetic isoflavones on cell proliferation, estrogen receptor binding affinity, and apoptosis in human breast cancer cells

Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.     

Flavonoids and steroid hormone-dependent cancers

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

Mechanisms of growth inhibition by antiestrogens and progestins in human breast and endometrial cancer cells.

Marked changes in both growth factor and proto-oncogene expression occur due to treatment of hormonally-responsive human cancers with progestins and antiestrogens. In human endometrial cancer cell lines the antiproliferative effects of progestins and antiestrogens in a particular cell line appear to be associated with similar effects on growth factor and/or proto-oncogene expression. This suggests that although these compounds initially interact with different steroid hormone receptors, the molecular mechanisms of their growth inhibition may be essentially similar. In the case of human breast cancer cell lines, however, the effects of progestins and antiestrogens on gene regulation are often different, suggesting that the molecular mechanisms of progestin and antiestrogen growth inhibition may be essentially dissimilar.     

Isoflavones from black chickpea (Cicer arietinum L) sprouts with antioxidant and antiproliferative activity

Black chickpea is a good source of bioactive compounds, particularly isoflavones. Sprouting improves nutraceutical value in chickpea seeds. This study aimed to explore the role of sprouting of black chickpea seeds on the synthesis of isoflavones and evaluate the impact of the soluble isoflavone on cellular antioxidant activity (CAA) and antiproliferative activity in breast cancer cells. Isoflavones were identified and quantified by HPLC-UV-MS. The CAA and antiproliferative activity were determined in HepG2 cells and MDA-MB-231 cancer cells, correspondingly. In sprouted black chickpea, six isoflavones (formononetin, biochanin-A, and its glycosides) were identified and the total isoflavones content increased (0.31 to 35.72 µgBA/mg of extract). The CAA was increased five times from 137.2 to 788.2 µMEQ/100 g of sample. The bioactive compounds in sprouted chickpea decreased the proliferation of MDA-MB-231 cell line. Also caused morphological changes such as cell shrinkage, rounding and nuclear fragmentation. The results herein suggest that bioactive compounds, as isoflavones, in sprouted black chickpea showed a potential antioxidant and antiproliferative activity. Therefore, it may be considered as a value-added product or ingredient for produce functional foods.     

Antiproliferative effects of melatonin and CGP 52608

The antiproliferative effects of melatonin and CGP 52608, an exogenous ligand for RZR/ROR receptors, are compared in the present paper. Both compounds exerted similar inhibitory effects on the proliferation of neoplastic cells in mouse colonic adenocarcinoma, DU 145 human prostate cancer, MCF-7 human breast carcinoma, and rat diethylstilbestrol-induced prolactinoma. Although it has been suggested that melatonin may influence the proliferation of tumor cells via RZR/ROR receptors, it cannot be excluded that the antiproliferative effects of melatonin and CGP 52608 are unrelated and mediated by different intracellular mechanisms.     

Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway

Several epidemiological studies have indicated that abundant consumption of foods from plant origin is associated with a reduced risk of developing several types of cancers. This chemopreventive effect is related to the high content of these foods in phytochemicals, such as polyphenols, that interfere with several processes involved in cancer progression including tumor cell growth, survival and angiogenesis. In addition to the low intake of plant-based foods, increased body mass and physical inactivity have recently emerged as other important lifestyle factors influencing cancer risk, leading to the generation of low-grade chronic inflammatory conditions which are a key process involved in tumor progression. The objectives of the current study are to investigate the inhibitory effects of these polyphenols on angiogenesis triggered by an inflammatory cytokine (IL-6) and to determine the mechanisms underlying this action. We found that, among the tested polyphenols, apigenin and luteolin were the most potent angiogenesis inhibitors through their inhibitory effect on the inflammatory cytokine IL-6/STAT3 pathway. These effects resulted in modulation of the activation of extracellular signal-regulated kinase-1/2 signaling triggered by IL-6, as well as in a marked reduction in the proliferation, migration and morphogenic differentiation of endothelial cells. Interestingly, these polyphenols also modulated the expression of IL-6 signal transducing receptor (IL-6Rα) and the secretion of the extracellular matrix degrading enzyme MMP-2 as well as the expression of suppressor of cytokine signaling (SOCS3) protein. Overall, these results may provide important new information on the role of diet in cancer prevention.     

Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?

Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. This apparent disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. However, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that are achievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more important in vivo. Many studies of the biological effects of polyphenols in cell culture have been affected by their ability to oxidise in culture media, and awareness of this problem can avoid erroneous claims.     

Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.