DNA damage is a late event in resveratrol-mediated inhibition of Escherichia coli

Resveratrol is an important phytoalexin notable for a wide variety of beneficial activities. Resveratrol has been reported to be active against various pathogenic bacteria. However, it is not clear at the molecular level how this important activity is manifested. Resveratrol has been reported to bind to cupric ions and reduce it. In the process, it generates copper-peroxide complex and reactive oxygen species (ROS). Due to this ability, resveratrol has been shown to cleave plasmid DNA in several studies. To this end, we envisaged DNA damage to play a role in resveratrol mediated inhibition in Escherichia coli. We employed DNA damage repair deficient mutants from keio collection to demonstrate the hypersensitive phenotype upon resveratrol treatment. Analysis of integrity and PCR efficiency of plasmid DNA from resveratrol-treated cells revealed significant DNA damage after 6?h or more compared to DNA from vehicle-treated cells. RAPD-PCR was performed to demonstrate the damage in genomic DNA from resveratrol-treated cells. In addition, in situ DNA damage was observed under fluorescence microscopy after resveratrol treatment. Further resveratrol treatment resulted in cell cycle arrest of significant fraction of population revealed by flow cytometry. However, a robust induction was not observed in phage induction assay and induction of DNA damage response genes quantified by promoter fused fluorescent tracker protein. These observations along with our previous observation that resveratrol induces membrane damage in E. coli at early time point reveal, DNA damage is a late event, occurring after a few hours of treatment.     

Resveratrol affects in vitro rumen fermentation,methane production and prokaryotic community composition in a time- and diet-specific manner

This study aimed to investigate the effect of resveratrol on methane production, rumen fermentation and microbial composition under high-concentrate (HC) and high-forage (HF) diets using the in vitro fermentation system. A total of 25 mg of resveratrol was supplemented into 300 mg of either HC or HF diet. Methane production, total volatile fatty acid (VFA) concentration, molar proportion of VFA, metabolites of resveratrol and prokaryotic community composition were measured after 12 and 24 h of in vitro fermentation. Resveratrol reduced methane production (ml per mg of dry matter degraded) by 41% and 60% under both HC and HF diets (P < 0.001), respectively, and this result could be associated with the lower abundance of Methanobrevibacter (P < 0.001) in response to resveratrol. The molar proportion of propionate was significantly higher in the resveratrol group only under the HC diet (P = 0.045). The relative abundance of 10 bacterial genera was affected by the three-way interaction of treatment, diet and time (P < 0.05). Resveratrol was partly converted to dihydroresveratrol after 24 h of fermentation, and its degradation could be associated with microbes belonging to the order Coriobacteriales. Our results suggest that multiple factors (e.g. diet and time) should be considered in animal experiments to test the effect of polyphenol or other plant extracts on rumen fermentation, methane emission and microbial composition.     

Resveratrol stimulates phenolic metabolism and PSII efficiency in wheat infected with powdery mildew

The beneficial effects of trans-resveratrol on pathogen-response and its physiological mechanisms were studied in two wheat cultivars differing in the level of resistance against powdery mildew (Blumeria graminis (DC.) Speer). Resveratrol induced phenolics metabolism in a manner that lead to increased phenylalanine ammonia-lyase (PAL) and β-D-glucosidase activity in leaf regions showing no symptoms of pathogen infection. In less-resistant cultivar, similar alterations were additionally observed in regions invaded by the pathogen. Resveratrol also stimulated photosynthetic efficiency during pathogenesis and this effect was more prominent in the resistant cultivar. Regulatory function of resveratrol on wheat growth and development was also observed (stimulation of generative induction). High performance liquid chromatography (HPLC) analysis detected a low amount of compound with retention time corresponding to trans-resveratrol in healthy leaves and increased the content of this compound in infected foliage. The multidirectional properties of resveratrol activity in wheat plants are discussed.     

Production of a major stilbene phytoalexin, resveratrol in peanut (Arachis hypogaea) and peanut products: a mini review

As a major stilbene phytoalexin, resveratrol is produced or elicited in several plant species as a part of defense systems protecting plants against diseases. Resveratrol can be present in both the trans- and cis-isomeric forms, and only the trans-form increases the life expectancy and lowers the risk of cardiovascular diseases as the most bioactive form. In addition to the usages for diet and industry, peanut plant (Arachis hypogaea) and peanuts are getting higher attention due to their containment of resveratrol in the kernels and other parts of peanut plant, such as leaves, roots, and peanut shell. Recently, natural resveratrol derived from peanuts has also become a promising nutraceutical agent, promoting human health. Resveratrol has also been detected in peanut products including peanut butters, roasted peanuts, and boiled peanuts. Although, smaller and immature peanuts contain higher levels of resveratrol than mature peanuts, resveratrol in peanuts can also be preserved by cooking or manufacturing processes. Moreover, the amount of resveratrol in peanut plants and peanuts has been found to increase by external stimuli including microbial infection, wounding, UV light irradiation, ultrasonication, yeast extract treatment and by plant stress hormones. In addition, molecular level analysis has confirmed that four resveratrol synthase (RS) genes (RS1, RS2, RS3 and RS4) which catalyze synthesis of resveratrol have been identified in peanuts, and up-regulation of the genes is positively correlated to the increased contents of resveratrol. In this review, we summarize the natural biosynthesis of resveratrol in peanuts and peanut plants, as well as the occurrence of this natural phytoalexin in various peanut products. A brief knowledge on the biosynthetic pathway of resveratrol synthesis has been described. This review also deals on highlighting the effect of various external stimuli (biotic and abiotic stresses) in order to achieve the maximum induction and/or elicitation of resveratrol in peanuts and peanut plants.     

Overexpressing fusion proteins of 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS) in tobacco plants leading to resveratrol accumulation and improved stress tolerance

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a phytoalexin produced in plants in response to pathogen attack as a part of plant defense response and it is also a highly bioactive substance of pharmaceutical interest. To obtain transgenic plants with a high level of resveratrol, two enzymes in the last two steps of resveratrol synthesis, 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS), were fused together by a glycine–serine–glycine (GSG) tripeptide linker, and the 4CL-GSG-STS construct driven by a CaMV35S promoter was transformed into tobacco (Nicotiana benthamiana) by Agrobacterium-mediated method. In the transgenic plants, a high resveratrol level was detected (21.05 µg/g fresh weight) by high-pressure liquid chromatography (HPLC), which is higher than previous transgenic plants with only STS gene overexpression. In addition to resistance to pathogen, transgenic plants showed improved tolerance to salt and osmotic stresses, and the lower level of malondialdehyde (MDA) in transgenic plants suggested that resveratrol could protect plant membrane lipid from peroxidation under abiotic stresses.     

Resveratrol enhances the cytotoxic profile of docetaxel and doxorubicin in solid tumour cell lines in vitro

Objectives: Resveratrol, with its robust antioxidant activity, has frequently been suggested as potentially having activity in cancer prevention and some recent reports have indicated that it has cancer treatment potential for several types of neoplasia. It has been found to block p‐glycoprotein and to protect against several chemotherapeutic agents’ side effects. In this study, we assessed interactive characteristics of resveratrol with docetaxel and doxorubicin and further investigated molecular bases of this interaction in cells of three different solid tumour lines (MCF‐7, HeLa and HepG2). Materials and methods and results: Resveratrol per se was found to have anti‐cancer properties, but with relatively low potency in all tested cell lines (IC₅₀ ranged from 35.1 to 83.8 μM). Doxorubicin and docetaxel showed IC₅₀ ranging from 0.48 to 0.72 μM and from 25.9 to 77.8 nM, respectively. Resveratrol in combination with doxorubicin and docetaxel significantly increased potencies of both chemotherapeutic agents showing IC₅₀ ranging from 0.12 to 0.34 μM and from 7.2 to 53.02 nM, respectively. The combination index showed synergistic interaction between resveratrol and doxorubicin or docetaxel on MCF‐7 cells, and additive interactions on HeLa and HepG2 cells. Real time PCR revealed that expression of Bax and Bcl‐2 was simultaneously elevated on combination of resveratrol with doxorubicin or docetaxel in all tested cell lines, whereas p53 exhibited marginal elevation in MCF‐7 and HepG2 cells. In addition, p‐glycoprotein efflux activity was significantly inhibited, with subsequent accumulation of p‐glycoprotein substrate in intracellular compartments. Expression level of mdr1 gene was downregulated after resveratrol combined with doxorubicin or docetaxel in all tested cell lines. Conclusion: Resveratrol potentiates cytotoxic properties of both cancer drugs used in the study through increasing their intracellular level due to p‐glycoprotein inhibition and downregulation of mdr1 gene.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of long-term cultivation on resveratrol accumulation in a high-producing cell culture of Vitis amurensis

Resveratrol is a polyphenol, present in grapes, peanuts, and other plant sources, with a wide range of valuable biological activities. We established a Vitis amurensis cell culture accumulating high levels of resveratrol by introducing the rolB gene of Agrobacterium rhizogenes in the V. amurensis genome, and studied the stability of resveratrol accumulation during 27 months of continuous subculturing. This study demonstrates a decline in the high level of resveratrol production by the rolB transgenic cell line during its long-term cultivation. Elicitation of the rolB transgenic calli with methyl jasmonate and salicylic acid, which are known to stimulate the production of plant secondary metabolites, resulted in a recovery of resveratrol accumulation in the rolB transgenic cell culture, while the empty vector-transformed culture with trace starting content of resveratrol exhibited low inducibility to the treatment.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

A plant pathogen modulates the effects of secondary metabolites on the performance and immune function of an insect herbivore

Host plant chemical composition critically shapes the performance of insect herbivores feeding on them. Some insects have become specialized on plant secondary metabolites, and even use them to their own advantage such as defense against predators. However, infection by plant pathogens can seriously alter the interaction between herbivores and their host plants. We tested whether the effects of the plant secondary metabolites, iridoid glycosides (IGs), on the performance and immune response of an insect herbivore are modulated by a plant pathogen. We used the IG‐specialized Glanville fritillary butterfly Melitaea cinxia, its host plant Plantago lanceolata, and the naturally occurring plant pathogen, powdery mildew Podosphaera plantaginis, as model system. Pre‐diapause larvae were fed on P. lanceolata host plants selected to contain either high or low IGs, in the presence or absence of powdery mildew. Larval performance was measured by growth rate, survival until diapause, and by investment in immunity. We assessed immunity after a bacterial challenge in terms of phenoloxidase (PO) activity and the expression of seven pre‐selected insect immune genes (qPCR). We found that the beneficial effects of constitutive leaf IGs, that improved larval growth, were significantly reduced by mildew infection. Moreover, mildew presence downregulated one component of larval immune response (PO activity), suggesting a physiological cost of investment in immunity under suboptimal conditions. Yet, feeding on mildew‐infected leaves caused an upregulation of two immune genes, lysozyme and prophenoloxidase. Our findings indicate that a plant pathogen can significantly modulate the effects of secondary metabolites on the growth of an insect herbivore. Furthermore, we show that a plant pathogen can induce contrasting effects on insect immune function. We suspect that the activation of the immune system toward a plant pathogen infection may be maladaptive, but the actual infectivity on the larvae should be tested.     

Induced expression of the Fragaria × ananassa Rapid alkalinization factor-33-like gene decreases anthracnose ontogenic resistance of unripe strawberry fruit stages

Rapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin-like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF-33-like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea, or Penicillium expansum after 24 and 48 h post-infection. To investigate the role of FaRALF-33-like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF-33-like expression in C. acutatum-inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild-type fruits. In contrast, C. acutatum-inoculated white unripe fruits overexpressing the FaRALF-33-like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF-33-like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum.     

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t‐resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β‐cyclodextrins

The growing demand for t‐resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large‐scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β‐cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t‐resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t‐resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non‐bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae was investigated in this study. Resveratrol is widely used in medicine, food, and cosmetic because of its pharmacological properties. However, it has a much lower content in plants compared with its glucoside piceid, which has a much lower bioavailability. Traditionally, the aglycone is acquired by acid or enzymatic hydrolysis of its glucoside, but the violent condition and the acid pollution in hydrolytic reaction and the high cost of the enzyme limit their industrial development. In this paper, fermentation of P. cuspidatum by A. oryzae was successfully performed, during which, piceid was converted to resveratrol with the highest yield of trans-resveratrol 1.35%, 3.6 times higher than that obtained from raw herb by microwave-assisted extraction. Scale-up production was also performed and the yield of trans-resveratrol was 3.1 times higher after 24 h incubation. Therefore, biotransformation is a better method to increase the yield of resveratrol because of its high yield and mild conditions.     

Resveratrol content and expression patterns of stilbene synthase genes in <Emphasis Type="Italic">Vitis amurensis</Emphasis> cells treated with 5-azacytidine

DNA methylation is known to be involved in the regulation of plant development and defense mechanisms. However, there is a general lack of data on the role of methylation in plant secondary metabolism. We have investigated the effect of a cytidine analog, 5-azacytidine (azaC), which is known to block DNA methylation, on resveratrol biosynthesis and stilbene synthase (STS) gene expression in Vitis amurensis cultured cells. Resveratrol is a naturally occurring polyphenol that has been reported to exhibit a wide range of important biological and pharmacological properties. We previously obtained a control cell line of V. amurensis (VV) as well as a rolB-transgenic cell line of V. amurensis (VB2) that has a higher level of resveratrol accumulation. In our experimental setup, the azaC-treated VV and VB2 calli produced 0.092% and 0.455% dry weight (DW) resveratrol, respectively. We found that treatment with 200 μM of azaC resulted in 1.9- and 2.0-fold increases in resveratrol production in VV and VB2 calli, respectively. A quantitative real-time PCR assay for STS gene expression in the azaC-treated VV and VB2 cells revealed that there were statistically increased expression levels of VaSTS10 in VV calli and of VaSTS5, VaSTS6, and VaSTS10 in VB2 calli. These results demonstrate that azaC is able to increase resveratrol production in V. amurensis calli through a mechanism that involves the induction of STS gene expression.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.     

Resveratrol Has No Effect on Lipoprotein Profile and Does Not Prevent Peroxidation of Serum Lipids in Normal Rats

Trans-resveratrol, one of the antioxidants found in red wine, has been the subject of controversial reports regarding its protective role against cardiovascular diseases. In this study we synthesized trans-resveratrol and injected it to rats (20 and 40 mg/kg body weight, once a day for 21 days, i.p.) to determine its effect on the serum lipid profile. Synthetic trans-resveratrol was an effective antioxidant in vitro against hydroxyl radical (I₅₀ = 33 μM). Resveratrol treatment, however, did not have any effect on either the lipid profile or on Cu⁺²-dependent formation of thiobarbi-turic-acid-reactive substances (TBARS) from protein-associated lipids. Since the amount of resveratrol used in these experiments was orders of magnitude higher than the amounts found in wine, these results suggest that if resveratrol has any effect against coronary heart diseases, it is not related to its antioxidant role on lipids or to changes in lipoprotein profile.