Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53

Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependent increase in S phase and a concomitant reduction in the G(1) phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.     

Colorimetric heparinase assay for alternative anti-metastatic activity

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

气象因子和矿质元素对虎杖根茎白藜芦醇含量的影响

对全年各月虎杖材料中白藜芦醇含量,及其Ca、Cu、Fe、K、Mg、Na、Zn 8种矿质元素积累进行检测,结合气象因子动态变化,采用主成分分析法,研究了不同季节虎杖白藜芦醇含量动态变化与其矿质元素、气象因子动态变化的关系．结果表明,Cu与Fe,Mg与K、Zn,Ca与月日照时数,K与Zn相关系数分别为0．812、0．871、0．793、0．602、0．729,呈极显著正相关;Cu、Mn与月平均气温,Fe与K相关系数分别为-0．738、-0．712、-0．766,呈极显著负相关;Cu与白藜芦醇含量呈显著负相关．找出了影响虎杖白藜芦醇含量动态变化的5个主成分,其中“酶促反应促进性矿质营养因子”,“高温、强照射气象因子”的贡献率分别为35．539％、33．358％,合计为68．897％,二者对不同季节虎杖白藜芦醇的变异贡献率最大．同时进行白藜芦醇含量的动态变化与主成分的多元相关、回归分析,建立了回归方程。     

Coordinate induction by UV light of stilbene synthase,phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in leaves of vitaceae

A stilbene synthase catalyzing the formation of resveratrol from 4-hydroxycinnamoyl-CoA and malonyl-CoA was found in the leaves of several Vitaceae. This stilbene synthase and two other enzymes functioning on the route from phenylalanine to stilbenes were induced concurrently upon irradiation of the leaves with UV light. With leaves of Cissus antarctica, an increase of stilbene synthase activity, more than hundred-fold, was observed with a maximum appearing 15 h after the induction with UV light.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - Mes morpholinoethanesulfonic acid     

Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis

Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.     

Ameliorative effects of resveratrol on liver injury in streptozotocin‐induced diabetic rats

The objective of this study was to investigate the ameliorative property and potential mechanism of resveratrol (RVT) in a dose of 10 mg/kg for 15 consecutive days against liver injury in streptozotocin‐induced diabetic rats. Diabetic rats significantly (P < 0.05) exhibited liver injury manifested by increased aspartylaminotransferase, alanine aminotransferase, and bilirubin; disturbed liver weight to body weight; and confirmed by hematoxylin and eosin staining. Liver from diabetic rats exhibited significant increase in malondialdehyde level and significant decrease in reduced glutathione, glutathione‐S‐transferase, quinone reductase, catalase, and superoxide dismutase. Diabetic rats showed significant disturbance in serum lipid profile. Treatment with RVT significantly (P < 0.05) abrogated diabetes‐induced perturbation in these parameters and liver histology. These data suggest that RVT treatment is associated with promising hepatoprotective effect against diabetes‐induced liver damage via reduction of serum glucose level and oxidative damage and improving serum lipid profile. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:384–392, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21432     

Effects of resveratrol on HepG2 cells as revealed by H-NMR based metabolic profiling

Background

Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis.

Methods

We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a ¹H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways.

Results

Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation.

Conclusions

Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation.

General significance

NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.     

Activity of resveratrol triesters against primary acute lymphoblastic leukemia cells

Resveratrol is a common polyphenol of plant origin known for its cancer prevention and other properties. Its wider application is limited due to poor water solubility, low stability, and weak bioavailability. To overcome these limitations, a series of 13 novel resveratrol triesters were synthesized previously. In this paper, we describe the synthesis of 3 additional derivatives and the activity of all 16 against primary acute lymphoblastic leukemia cells. Of these, 3 compounds were more potent than resveratrol (IC₅₀ = 10.5 µM) namely: resveratryl triacetate (IC₅₀ = 3.4 µM), resveratryl triisobutyrate (IC₅₀ = 5.1 µM), and resveratryl triisovalerate (IC₅₀ = 4.9 µM); all other derivatives had IC₅₀ values of >10 µM. Further studies indicated that the active compounds caused G1 phase arrest, increased expression of p53, and induced characteristics of apoptotic cell death. Moreover, the compounds were only effective in cycling cells, with cells arrested in G1 phase being refractory.