stilbenes and thiazolyl-blue

The purpose of this study was to evaluate the effect of resveratrol on cytogenetic damages of iododeoxyuridine (IUdR) and x-ray megavoltage radiation (6 MV) in spheroid model of U87MG glioblastoma cancer cell line using clonogenic and alkaline comet assay. Cells were cultured as spheroids (350 µm) that were treated with 20 μM resveratrol, 1 μM IUdR and 2 Gy of 6 MV x-ray. After treatment, viability of the cells, colony forming ability and the induced DNA damages were examined using trypan blue dye exclusion, colonogenic and alkaline comet assay, respectively. Our results showed that resveratrol could significantly reduce the colony number and induce the DNA damages of the cells treated with IUdR in combination with 6 MV x-ray radiation. That results indicated that resveratrol as an inhibitor of hypoxia inducible factor 1 alpha (HIF-1α) protein in combination with IUdR as a radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Glioblastoma; Humans; Idoxuridine; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Trypan Blue; X-Rays

Resveratrol has been shown to be a potential chemopreventive and anticancer agent, inducing apoptosis in a variety of cancer cells. The present study was performed to evaluate the effect of resveratrol on A549 human lung adenocarcinoma epithelial cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide evaluation demonstrated that the exposure of cells to increasing concentrations of resveratrol (0-175 µM) for 24 h resulted in a decrease in cell viability (IC50 85.5 µM). Annexin V/propidium iodide double stain verified apoptosis in A549 cells, while negligible cell cytotoxity (≥ 0.5 %) was observed in all untreated incubations. Using colorimetric assay kits, induction of caspase-3, but not of caspase-8, activity was detected in response to resveratrol (> 130 µM). Confirmatory evidence of this finding was provided by Western blotting, indicating expression of cleaved caspase-3 levels in a concentration-dependent manner with a minimum resveratrol concentration of 65 µM required for activation of this protease, while that of caspase-8 remained unaffected. The apoptotic process was associated with reactive oxygen species production in a concentration-dependent manner, evidenced by microscopic examination and fluorescence-activated cell sorting analysis using the 2',7'-dichlorofluorescein diacetate assay. In the presence of the mitochondrial electron transport chain inhibitor rotenone, reactive oxygen species production and the concomitant apoptotic cell population were significantly reduced. This finding suggested that the resveratrol-induced apoptosis was mediated via a mitochondrial pathway alignment in human A549 cells. Although effective levels were observed at high concentrations, the outcome may well differ under in vivo conditions. Finally, experiments reaffirmed the chemical instability of trans-resveratrol, suggesting the need for protection of the solutions from extended exposure to light.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Survival; Epithelial Cells; Fluoresceins; Humans; Lung Neoplasms; Mitochondria; Reactive Oxygen Species; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles

Resveratrol interacts with the complex III of the respiratory chain, is a radical scavenger and also suppressor of radical formation in the mitochondria. It reduces the intracellular calcium levels in pre- and postsynaptic neurons and also may inhibit the pro-apoptotic factors in glutamate overflow that occurs, e.g. in excitotoxicity. In cell cultures, glutamate overflow leads to formation of free radicals and results in apoptosis. This increase of radical concentration is enhanced by influx of cations like iron or copper ions into the cell. In present study, the beneficial action of resveratrol was investigated in glutamate-affected dissociated cultures of mice mesencephalic primary cultures. On the 10th day in vitro, 5 mM of glutamate was administered for 15 min and the cultures were further maintained in medium containing 0, 0.01, 0.1 or 1 μM of resveratrol. Resveratrol reduced glutamate-induced damages. The number of dopaminergic neurons was increased and their morphology ameliorated when resveratrol followed glutamate treatment. A significant reduction of glutamate-induced radical formation in cultures treated with resveratrol corresponded with a considerable high antioxidative potential of this stilbene determined using the DPPH assay. In addition, ICP-OES was set up to measure the tissues' copper and iron contents in organotypic cortical cultures of glutamate treated (0 or 30 μM) slices and those in which resveratrol (0, 0.01, 0.1 or 1 μM) was co-administered. Levels of copper were dose-dependently increased, and also the concentration of iron was higher in resveratrol-treated organotypic cultures. The hypothesis that resveratrol has beneficial actions against glutamate damages was verified.

Topics: Animals; Antioxidants; Biphenyl Compounds; Brain; Cells, Cultured; Coloring Agents; Copper; Ethidium; Excitatory Amino Acid Antagonists; Female; Fluorescent Dyes; Glutamic Acid; Iron; Mesencephalon; Mice; Mice, Inbred C57BL; Neurites; Neurons; Organ Culture Techniques; Picrates; Pregnancy; Propidium; Resveratrol; Spectrophotometry, Atomic; Stilbenes; Tetrazolium Salts; Thiazoles; Tyrosine 3-Monooxygenase

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system.

Topics: 4-Butyrolactone; Animals; Animals, Newborn; Antineoplastic Agents, Phytogenic; Astrocytes; Blotting, Western; Cell Communication; Cells, Cultured; Coloring Agents; Connexin 43; Enzyme Inhibitors; Female; Gap Junctions; Image Processing, Computer-Assisted; Male; Microscopy, Fluorescence; Naphthalenes; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Stilbenes; Tetrazolium Salts; Thiazoles

Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of β-amyloid peptide 25-35 (Aβ(25-35)) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aβ(25-35), and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca(2+) homeostasis and attenuated Aβ(25-35) neurotoxicity. Additionally, Aβ(25-35)-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Calcium; Cell Survival; Flow Cytometry; Gene Expression Regulation; Homeostasis; L-Lactate Dehydrogenase; Neurodegenerative Diseases; Neurons; PC12 Cells; Peptide Fragments; Rats; Resveratrol; rho-Associated Kinases; Signal Transduction; Sirtuin 1; Stilbenes; Tetrazolium Salts; Thiazoles

Although tissue-penetrable light (red and NIR) has great potential for spatiotemporally controlled release of therapeutic agents, it has been hampered because of the lack of chemistry translating the photonic energy to the cleavage of a chemical bond. Recently, we discovered that an aminoacrylate group could be cleaved to release parent drugs after oxidation by SO and have called this "photo-unclick chemistry". We demonstrate its application to far-red-light-activated prodrugs. A prodrug of combretastatin A-4 (CA4) was prepared, CMP-L-CA4, where CMP is dithiaporphyrin, a photosensitizer, and L is an aminoacrylate linker. Upon irradiation with 690 nm diode laser, the aminoacrylate linker of the prodrug was cleaved, rapidly releasing CA4 (>80% in 10 min) in CDCl3. In tissue culture, it showed about a 6-fold increase in its IC50 in MCF-7 after irradiation, most likely because of the released CA4. Most significantly, CMP-L-CA4 had better antitumor efficacy in vivo than its noncleavable (NC) analog, CMP-NCL-CA4. This is the first demonstration of the in vivo efficacy of the novel low-energy-light-activatable prodrug using the photo-unclick chemistry.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Chemistry, Pharmaceutical; Coloring Agents; Cross-Linking Reagents; Darkness; Dermatitis, Phototoxic; Fluorescent Dyes; Light; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Porphyrins; Prodrugs; Stilbenes; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tubulin

Resveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems.. Liposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H2DCF-DA dye.. Particle size varied from 134.2 ± 0.265 nm to 103.3 ± 1.687 nm; PDI ≤ 0.3; zeta potential values were greater than -30 mV and PDE ≥ 80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H2O2 further intensified the antioxidative effects of pure RES and liposomal formulations.. The present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS.

Topics: Amidines; Antioxidants; Area Under Curve; Ascorbic Acid; Cell Survival; Coloring Agents; Drug Carriers; Free Radical Scavengers; Humans; Indicators and Reagents; Leukocytes; Liposomes; Luminescence; Luminol; Oxidative Stress; Particle Size; Reactive Oxygen Species; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Vitamins

SIRT1 (mammalian ortholog of the yeast silent information regulator 2) is a NAD-dependent histone deacetylase belonging to the multigene family of sirtuins. Anecdotal and epidemiologic observations provide evidence for beneficial effects of the calorie restriction mimetic resveratrol (RES), a SIRT1 activator in preventing cardiovascular diseases and cancer. Although SIRT1 possesses both tumorigenic and antitumorigenic potential, the molecular mechanisms underlying SIRT1-mediated tumor progression or inhibition are poorly understood. In this study, we investigated the role of SIRT1 in multiple human prostate cancer cell lines and prostate-specific PTEN knockout mouse model using resveratrol. Androgen-independent prostate cancer cell lines (C42B, PC3, and DU145) express higher levels of SIRT1 than androgen-responsive (LNCaP) and nontumorigenic prostate cells (RWPE-1). Resveratrol enhanced this expression without any significant effect on SIRT1 enzymatic activity. Inhibition of SIRT1 expression using shRNA enhanced cell proliferation and inhibited autophagy by repressing phosphorylation of S6K and 4E-BP1. These biologic correlates were reversed in the presence of resveratrol. Analysis of prostates from dietary intervention with resveratrol showed a significant reduction in prostate weight and reduction in the incidence of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions by approximately 54% with no significant change in body weight. Consistent with the in vitro findings, resveratrol intervention in the PTEN knockout mouse model was associated with reduction in the prostatic levels of mTOR complex 1 (mTORC1) activity and increased expression of SIRT1. These data suggest that SIRT1/S6K-mediated inhibition of autophagy drives prostate tumorigenesis. Therefore, modulation of SIRT1/S6K signaling represents an effective strategy for prostate cancer prevention.

Topics: Animal Feed; Animals; Cell Line, Tumor; Cell Survival; Diet; Humans; Immunohistochemistry; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Mutation; Phosphorylation; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Resveratrol; Ribosomal Protein S6 Kinases; RNA, Small Interfering; Signal Transduction; Sirtuin 1; Stilbenes; Tetrazolium Salts; Thiazoles; Time Factors; TOR Serine-Threonine Kinases

Resveratrol is an edible polyphenolic phytoalexin present in different plant species and plays important role in inhibiting proliferation and inducing apoptosis of pancreatic cancer cells. In this paper, the mechanism of resveratrol on PANC-1, CFPAC-1 and MIA Paca-2 cells apoptosis was examined.. We first evaluated the effect of resveratrol on viability of PANC-1, CFPAC-1 and MIA Paca-2 cells using MTT assay. Next, we performed real-time PCR to assess the effect of resveratrol on miR-21 expression. We also used Western blot to measure BCL-2 protein levels after down-regulation of miR-21 expression. Finally, we evaluated the effect of miR-21 on resveratrol-induced anti-tumor activity using miR-21 mimic.. Resveratrol induced a significant inhibition of PANC-1, CFPAC-1 and MIA Paca-2 cells viability in a dose-dependent manner. Resveratrol also decreased the expression of miR-21. Besides, down-regulation of miR-21 expression can inhibit BCL-2 expression in PANC-1, CFPAC-1 and MIA Paca-2 cells. Over-expression of miR-21 expression can reverse down-regulation of BCL-2 expression and apoptosis induced by resveratrol.. In this study, we demonstrated that the effect of resveratrol on apoptosis is due to inhibiting miR-21 regulation of BCL-2 expression.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Pancreatic Ducts; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles

Resveratrol promotes differentiation and apoptosis of medulloblastoma cells by suppressing STAT3 signaling and a range of cancer-associated gene expression. However, Bcl-2, a common target of STAT3 and NF-κB signaling, is distinctly up-regulated in resveratrol-treated medulloblastoma cells, indicating potential effects of NF-κB in Bcl-2 expression and anti-medulloblastoma efficiency of resveratrol. To clarify this point, the status of NF-κB signaling and the consequence of NF-κB inhibition in UW228-2 and UW228-3 medulloblastoma cells without and with resveratrol treatment were evaluated by several experimental approaches. The results revealed that resveratrol activated NF-κB signaling in both cell lines at the 4-h treatment point, and the treated cells sequentially exhibited Bcl-2 up-regulation, neuronal-like phenotype with synaptophisin expression, and, eventually, apoptosis. Pyrrolidine dithiocarbamate (PDTC) treatment inhibited NF-κB activation and Bcl-2 expression and committed resveratrol-treated cells to apoptosis at the 8-h time point without the step of neuron-oriented differentiation. On the other hand, a single 50 μg/ml lipopolysaccharide (LPS) treatment activated NF-κB signaling accompanied with sustained proliferation and neuron-like differentiation. Tissue microarray-based immunohistochemical staining showed significantly different (P < 0.001) p65 nuclear translocation between the neurons of tumor-surrounding cerebella (10/10; 100%) and medulloblastoma tissues (20/117; 17.09%). Additionally, synaptophysin production was found in 83.64% of p65-positive and in 40.35% of p65-negative medulloblastoma cases. Our in-vitro and in-vivo results thus demonstrate the dual effects of NF-κB signaling on medulloblastoma cells by delaying resveratrol-induced apoptosis by up-regulating Bcl-2 expression or by involvement in neuronal-like differentiation in the absence of resveratrol. Therefore, appropriate inhibition of NF-κB activation may enhance the anti-medulloblastoma efficacy of resveratrol.

Topics: Anticarcinogenic Agents; Apoptosis; Cell Line, Tumor; Cerebellum; Flow Cytometry; Humans; Lipopolysaccharides; Medulloblastoma; Neurons; NF-kappa B; Proline; Protein Array Analysis; Resveratrol; Signal Transduction; STAT3 Transcription Factor; Stilbenes; Tetrazolium Salts; Thiazoles; Thiocarbamates; Time Factors

Resveratrol, a stilbene type compound identified in wine and fruit juice, has been found to exhibit various pharmacological activities such as anti-oxidative, anti-cancerous, anti-inflammatory and anti-aging effects. Although numerous papers have explored the pharmacology of resveratrol in one particular cellular action, how this compound can have multiple effects simultaneously has not been fully addressed. In this study, therefore, we explored its broad-spectrum inhibitory mechanism using lipopolysaccharide (LPS)-mediated inflammatory responses and reporter gene assays involving overexpression of toll like receptor (TLR) adaptor molecules. Co-transfection of adaptor molecules such as (1) myeloid differentiation primary response gene 88 (MyD88), (2) Toll/4ll-1 Receptor-domain-containing adapter-inducing interferon-beta (TRIF), (3) TRIF-related adaptor molecule (TRAM), or (4) TANK-binding kinase (TBK) 1 strongly enhanced luciferase activity mediated by transcription factors including nuclear factor (NF)-KB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3. Of the adaptor proteins, TRIF and TBK1 but not MyD88 and IKK enhanced luciferase activity mediated by these transcription factors. Resveratrol dose-dependently suppressed LPS-induced NO production in macrophages. It also blocked the increases in levels of mRNA for IFN-1, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) that were induced by LPS. Resveratrol diminished the translocation or activation of IRF-3 at 90min, c-Jun, a subunit of AP-1, and STAT-1 at 120 min, and p50, a subunit of NF-KB, at 60 and 90 min. Resveratrol strongly suppressed the up-regulation of luciferase activity induced by these adaptor molecules with IC50 values of 5 to 65 microM. In particular, higher inhibitory effects of resveratrol were when TRIF or TBK1 were overexpressed following cotransfection of luciferase constructs with IRF-3 binding sequences. Taken together, our data suggest that the suppression of TRIF and TBK1, which mediates transcriptional activation of NF-kappaB, AP-1, and IRF-3, contributes to resveratrol's broad-spectrum inhibitory activity, and that this compound can be further developed as a lead anti-inflammatory compound.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Nucleus; Cells, Cultured; Coloring Agents; Genes, Reporter; Inflammation; Interferon Regulatory Factor-3; Lipopolysaccharides; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Plasmids; Protein Serine-Threonine Kinases; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Stilbenes; Tetrazolium Salts; Thiazoles

Resveratrol has recently been widely reported to be an age-delaying and neuroprotective compound, and it appears to produce these benefits by activating silent mating type information regulation 2 homolog 1 (SIRT1). However, the role that SIRT1 activation plays in the pathogenesis of amyotrophic lateral sclerosis (ALS) remains unclear. In the present study, SIRT1 expression was found to be much lower in the mutant hSOD1G93A-bearing VSC4.1 cells compared to hSOD1wt cells when both were cultured in low-serum medium, indicating the involvement of SIRT1 activation defects in the pathogenesis of ALS under energetic stress. Further investigation revealed that a 24-h treatment with 0.5-20μM resveratrol had a dose-dependent protective effect on this ALS cell model, and the effects of resveratrol on increasing cell viability, preventing cell apoptosis and elevating cellular ATP levels through promoting mitochondria biogenesis were blocked by SIRT1 inhibition. This further demonstrated a role for SIRT1 activation in the protection of neuronal cells from degeneration. These findings suggest that resveratrol can protect the ALS cell model from mutant SOD1-mediated toxicity through up-regulating the expression of SIRT1, which represents a potential therapeutic target for preventing the motor neuron degeneration in ALS patients.

Topics: Amyotrophic Lateral Sclerosis; Annexin A5; Blotting, Western; Cell Death; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Antibody Technique; Humans; Mitochondria; Motor Neurons; Mutation; Neuroprotective Agents; Niacinamide; Propidium; Real-Time Polymerase Chain Reaction; Resveratrol; Sirtuin 1; Stilbenes; Superoxide Dismutase; Superoxide Dismutase-1; Tetrazolium Salts; Thiazoles; Up-Regulation

The mechanisms by which resveratrol (3,5,4'-trihydroxy-stilbene) imparts neural effects is not well understood. We previously demonstrated that, depending upon the concentration of resveratrol and the cell type, this compound exerts anti-or pro-oxidant effects. In the present study, we investigated the effects of resveratrol on H(2)O(2)-mediated genotoxicity in C6 astroglial cells (I - 1mM H(2)O(2)/30 min or II - 0.1mM H(2)O(2)/6h), evaluated by micronucleus assay, lipid peroxidation (TBARS) and membrane integrity. H(2)O(2) increased micronuclei to 1.5 (I) and 1.7-fold (II), compared to control cells. This DNA damage was prevented (I) or partially prevented (II) by resveratrol. Oxidative insult also increased TBARS, 52% in I and 38% in II, P<0.05. These effects were prevented by resveratrol in I and increased in II (70% of increase). Present data contribute to the understanding of resveratrol effects under oxidative stress damage.

Topics: Animals; Antioxidants; Ascorbic Acid; Astrocytes; Cell Line; Cell Survival; Coloring Agents; DNA Damage; Hydrogen Peroxide; L-Lactate Dehydrogenase; Membrane Lipids; Micronucleus Tests; Oxidants; Oxidation-Reduction; Rats; Resveratrol; Stilbenes; Tars; Tetrazolium Salts; Thiazoles

Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells.. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (DeltaPsim) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms.. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol.. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coloring Agents; Glioma; Humans; Membrane Potentials; Microscopy, Fluorescence; Mitochondrial Membranes; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles

Beta-amyloid (Abeta) aggregation has been strongly associated with the neurodegenerative pathology and a cascade of harmful event rated to Alzheimer's disease (AD). Inhibition of Abeta assembly, destabilization of preformed Abeta aggregates and attenuation of the cytotoxicity of Abeta oligomers and fibrils could be valuable therapeutics of patients with AD. Recent studies suggested that moderate consumption of red wine and intake of dietary polyphenols, such as resveratrol, may benefit AD phenotypes in animal models and reduce the relative risk for AD clinical dementia. To understand the mechanism of this neuroprotection, we studied the effects of resveratrol, an active ingredient of polyphenols in wine and many plants, on the polymerization of Abeta42 monomer, the destabilization of Abeta42 fibril and the cell toxicity of Abeta42 in vitro using fluorescence spectroscopic analysis with thioflavin T (ThT), transmission electron microscope (TEM), circular dichroism (CD) and MTT assay. The results showed that resveratrol could dose-dependently inhibit Abeta42 fibril formation and cytotoxicity but could not prevent Abeta42 oligomerization. The studies by Western-blot, dot-blot and ELISA confirmed that the addition of resveratrol resulted in numerous Abeta42 oligomer formation. In conjunction with the concept that Abeta oligomers are linked to Abeta toxicity, we speculate that aside from potential antioxidant activities, resveratrol may directly bind to Abeta42, interfere in Abeta42 aggregation, change the Abeta42 oligomer conformation and attenuate Abeta42 oligomeric cytotoxicity.

Topics: Amyloid; Amyloid beta-Peptides; Animals; Antioxidants; Benzothiazoles; Cell Proliferation; Circular Dichroism; Dose-Response Relationship, Drug; Humans; Mice; Microscopy, Electron, Transmission; Molecular Conformation; Molecular Structure; Neuroblastoma; Peptide Fragments; Protein Structure, Quaternary; Resveratrol; Spectrometry, Fluorescence; Stilbenes; Tetrazolium Salts; Thiazoles

The present work has evaluated the role of lipopolysaccharide (LPS) stimulation and of TLR4 receptors on the cytotoxicity of resveratrol (RES), a polyphenolic stilbene component of red wine, in TLR4-bearing (i.e., RAW 264.7) and TLR4-deficient (i.e., 10ScNCr/23) macrophages. Based on the results of the MTT assay, DNA fragmentation analysis, and scanning electron microscopic examination, cell stimulation with LPS was found to maintain the viability of, attenuate DNA fragmentation in, and preserve normal morphology of TLR4-proficient macrophages exposed to RES. In contrast, LPS failed to spare TLR4-deficient macrophages from the deleterious effects of RES. Moreover, while LPS treatment conferred protection from RES toxicity in TLR4-bearing macrophages, this stilbene inhibited the production of nitric oxide by stimulated cells in a concentration-dependent fashion. It is therefore likely that the cytotoxic effects of RES towards non-stimulated macrophages and the propensity of RES to inhibit nitric oxide production by activated macrophages are both contributing, at least in part, to the anti-inflammatory activity of this natural product.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line; Cell Survival; Culture Media; DNA Fragmentation; Lipopolysaccharides; Macrophages; Mice; Microscopy, Electron, Scanning; Nitrites; Receptors, Cell Surface; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Toll-Like Receptor 4

Six new stilbenoids, a (bibenzyldihydrophenanthrene) ether designated phoyunnanin D (1), a bis(dihydrophenanthrene) ether designated phoyunnanin E (2), and four stilbenes designated phoyunbene A-D (3-6), were isolated from the air-dried whole plant of Pholidota yunnanensis ROLFE. The new compounds were identified as 7-[2-(3-hydroxyphenethyl)-4-hydroxy-6-methoxyphenoxy]-4-hydroxy-2-methoxy-9,10-dihydrophenanthrene (1), 1-[(9,10-dihydro-4-hydroxy-2-methoxy-7-phenanthrenyl)oxy]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (2), trans-3,3'-dihydroxy-2',4',5-trimethoxystilbene (3), trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene (4), trans-3,3'-dihydroxy-2',5-dimethoxystilbene (5), and trans-3-hydroxy-2',3',5-trimethoxystilbene (6) based on spectroscopic evidence. Furthermore, the inhibitory effects of compounds 1-6 on nitric oxide production in a murine macrophage-like cell line (RAW 264.7) activated by lipopolysaccharide and interferon-gamma were examined.

Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Liquid; Macrophages; Magnetic Resonance Spectroscopy; Mice; Nitric Oxide; Orchidaceae; Phenanthrenes; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Stilbenes; Tetrazolium Salts; Thiazoles

One new stilbene glucoside (6), along with five known compounds (1-5), were isolated from the roots of Polygonum multiflorum Thumb., and their chemical structures established based on physicochemical and spectroscopic data. Of the compounds, compound 3 showed DNA topoisomerase I and II inhibitory activities.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Chemical Phenomena; Chemistry, Physical; Chromatography, Thin Layer; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Glucosides; Humans; Magnetic Resonance Spectroscopy; Molecular Conformation; Plant Roots; Polygonum; Spectrophotometry, Ultraviolet; Stilbenes; Tetrazolium Salts; Thiazoles; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors

Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity.. NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg x kg(-1), and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method.. The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 microg x h x ml(-1) for CA4, and 10.4 and 13.1 microg x h x ml(-1) for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4.. Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Area Under Curve; Bibenzyls; Calibration; Cell Line, Tumor; Chromatography; Chromatography, High Pressure Liquid; Coloring Agents; Female; Humans; Inhibitory Concentration 50; Mass Spectrometry; Mice; Models, Chemical; Phosphorylation; Prodrugs; Stilbenes; Tetrazolium Salts; Thiazoles; Time Factors

Resveratrol, a polyphenol compound found in grapes and red wines, is a prominent anti-cancer agent. In this study, we demonstrate that resveratrol enhanced TNF-alpha, IL-12 and IL-1beta production from LPS activated phorbol myristate acetate (PMA) differentiated THP-1 human macrophages. Expression of CD86 on macrophages was enhanced by resveratrol alone and with LPS. When macrophages were primed with IFN-gamma, resveratrol suppressed the expression of HLA-ABC, HLA-DR, CD80, CD86 and inhibited production of TNF-alpha, IL-12, IL-6 and IL-1beta induced by LPS. The differential impact of resveratrol on expression of CD14 might be correlated with differential response of macrophages to LPS with or without IFN-gamma priming.

Topics: Antigen-Presenting Cells; Antigens, CD; B7-2 Antigen; Cell Death; Cell Differentiation; Cell Survival; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-1; Interleukin-12; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Membrane Glycoproteins; Phenotype; Recombinant Proteins; Resveratrol; Stilbenes; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Thymidine; Tumor Necrosis Factor-alpha

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Differentiation; Cell Division; Cells, Cultured; CHO Cells; Chromans; Coloring Agents; Cricetinae; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; G2 Phase; Glutathione; Glutathione Disulfide; Mitosis; Oxidation-Reduction; Reactive Oxygen Species; Resting Phase, Cell Cycle; Resveratrol; S Phase; Stilbenes; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue

Beta-amyloid peptide is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease. There has been compelling evidence supporting the idea that beta-amyloid-induced cytotoxicity is mediated through the generation of reactive oxygen intermediates (ROIs). Considerable attention has been focused on identifying phytochemicals that are able to scavenge excess ROIs, thereby protecting against oxidative stress and cell death. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a phytoalexin found in the skin of grapes, has strong antioxidative properties that have been associated with the protective effects of red wine consumption against coronary heart disease ("the French paradox"). In this study, we have investigated the effects of resveratrol on beta-amyloid-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with beta-amyloid exhibited increased accumulation of intracellular ROI and underwent apoptotic death as determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL staining). Beta-amyloid treatment also led to the decreased mitochondrial membrane potential, the cleavage of poly(ADP-ribose)polymerase, an increase in the Bax/Bcl-X(L) ratio, and activation of c-Jun N-terminal kinase. Resveratrol attenuated beta-amyloid-induced cytotoxicity, apoptotic features, and intracellular ROI accumulation. Beta-amyloid transiently induced activation of NF-kappaB in PC12 cells, which was suppressed by resveratrol pretreatment.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Death; Cell Nucleus; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; In Situ Nick-End Labeling; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; NF-kappa B; Oxidative Stress; Oxygen; PC12 Cells; Peroxides; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Resveratrol; Signal Transduction; Stilbenes; Tetrazolium Salts; Thiazoles

Inhibition of proliferation by resveratrol of CEM-C7H2 lymphocytic leukemia cells was paradoxically associated with an enhanced cellular 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-reducing activity. This phenomenon was most pronounced at the sub-apoptotic concentration range of 5-20 microM resveratrol. The results of our study show that the MTT-reducing activity can be increased by the polyphenolic antioxidant resveratrol without a corresponding increase in the number of living cells and that this occurs at a concentration range of the antioxidant which is not sufficient to induce apoptosis but suffices to slow down cell growth. This phenomenon appears to be restricted to proliferation inhibitors with antioxidant properties and is cell type-specific. Thus, in determining the effects of flavonoids and polyphenols on proliferation, in certain cell types this might represent a pitfall in the MTT proliferation assay.

Topics: Antioxidants; Apoptosis; Artifacts; Cell Division; Colorimetry; Dose-Response Relationship, Drug; Electron Transport; Formazans; Growth Inhibitors; Histocytochemistry; Humans; Indicators and Reagents; Leukemia, T-Cell; Mitochondria; NAD; NADP; Organ Specificity; Oxidation-Reduction; Resveratrol; Stilbenes; Subcellular Fractions; T-Lymphocytes; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

beta-Amyloid peptide (beta-AP) elicits a toxic effect on neurons in vitro and in vivo. Many environmental factors including antioxidants, metal ions and proteoglycans modify beta-AP toxicity. We have investigated on PC12 cells, the protective effect from beta-AP (1-41) of two plant polyphenols, resveratrol and catechin. PC12 cells treated with 10(-6)M beta-AP (1-41) for 16h decrease the cell viability at 24% of the control with an IC(50) value of 1.1+/-0.14 x 10(-8)M. Twenty-five micromolar resveratrol and 50 microM catechin protect PC12 cells from beta-AP (1-41) toxicity with the IC(50) value increased at 2.2+/-0.19 x 10(-7)M and at 8.9+/-0.7 x 10(-8)M, respectively. While the protective effect is concentration dependent for catechin, resveratrol shows a concentration-dependent biphasic effect. Up to 50 microM concentration, resveratrol protects PC12 cells from beta-AP (1-41) toxicity. At concentration higher than 50 microM, an inhibitory activity on cell proliferation appears. This antiproliferative effect is shown also in the absence of beta-AP (1-41). Resveratrol and catechin have a synergistic protective action. In the presence of 50 microM catechin and 10 microM resveratrol or 25 microM resveratrol and 10 microM catechin, the toxicity determined by 10(-7)M beta-AP (1-41) is almost completely abolished. Catechin is more effective than resveratrol in protecting PC12 cells from the toxicity of hydrogen peroxide. The protection from Oxygen Reactive Species (ROS) toxicity is concentration dependent for both resveratrol and catechin. In this case the protection is merely additive and the synergistic effect is not observed. These results demonstrate that resveratrol and catechin protect PC12 cells from beta-AP (1-41) toxicity and that their protective effect is synergistic. Such a protective effect probably is not due only to their antioxidant activity. The different chemical and biological activity shown by these compounds on several cell types and the complexity of the beta-AP (1-41) toxicity may explain the synergistic protective effect and suggest that the utilization of different compounds with synergistic activity may protect more effectively from complex mechanisms of toxicity.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Catechin; Cell Count; Cell Death; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Lipid Peroxidation; PC12 Cells; Peptide Fragments; Rats; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Thiobarbiturates

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely used screening method to measure cell viability and proliferation. When testing the effects of kaempferol on breast cancer cell number (crystal violet staining) and viability (MTT tetrazolium assay) conflicting results were obtained. Cell number decreased but MTT formazan formation increased, suggesting a direct interaction of kaempferol with the MTT tetrazolium reduction. Direct reductive potential was observed in a cell-free system for the presumptive phytoestrogens kaempferol and resveratrol, and extracts of Hypericum perforatum L. and Cimicifuga racemosa L. All agents led to instantaneous dark blue formazan formation in the absence of cells. Additionally, antioxidants such as ascorbic acid, vitamin E and N-acetylcysteine interfered with the MTT tetrazolium assay. When MCF7 and HS578 cells treated with kaempferol were washed before addition of MTT tetrazolium, the direct reduction of dye was reduced significantly. These results indicate that the MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential.

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Breast Neoplasms; Cell Division; Cell Survival; Drug Interactions; Estrogens, Non-Steroidal; Flavonoids; Humans; Isoflavones; Kaempferols; Phytoestrogens; Plant Extracts; Plant Preparations; Plants; Quercetin; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vitamin E

We have shown that liver myofibroblasts stimulate in vitro invasion of hepatocellular carcinoma cell lines through a hepatocyte growth factor/urokinase-dependent mechanism. Resveratrol, a grapevine-derived polyphenol, has been shown to inhibit cellular events associated with tumor initiation, promotion and progression. The aim of this study was to evaluate the effects of trans-resveratrol on invasion of the human hepatoma cell line HepG2. Cell invasion was assessed using a Boyden chamber assay. Activation of the HGF signal transduction pathways was evaluated by Western blot with phospho-specific antibodies. Urokinase expression was measured by RT-PCR and zymography. Trans-resveratrol decreased hepatocyte growth factor-induced cell scattering and invasion. It also decreased cell proliferation without evidence for cytotoxicity or apoptosis. Trans-resveratrol did not decrease the level of the hepatocyte growth factor receptor c-met and did not impede the hepatocyte growth factor-induced increase in c-met precursor synthesis. Moreover, trans-resveratrol did not decrease hepatocyte growth factor-induced c-met autophosphorylation, or Akt-1 or extracellular-regulated kinases-1 and -2 activation. Finally, it did not decrease urokinase expression and did not block the catalytic activity of urokinase. In conclusion, our results demonstrate that trans-resveratrol decreases hepatocyte growth factor-induced HepG2 cell invasion by an as yet unidentified post-receptor mechanism.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Division; Cell Survival; Flavonoids; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Phenols; Phosphorylation; Poly(ADP-ribose) Polymerases; Polymers; Polyphenols; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Resveratrol; RNA, Messenger; Stilbenes; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator