Resveratrol-treated Cells Research Articles

SIRT1 inhibits apoptosis of degenerative human disc nucleus pulposus cells through activation of Akt pathway

Many studies have demonstrated that SIRT1, an NAD(+)-dependent deacetylase, reduces apoptosis in several different cells. However, the role of SIRT1 in apoptosis of disc nucleus pulposus (NP) cells remains unclear. The present study was performed to determine whether degenerative human NP would express SIRT1, and to investigate the role of SIRT1 in NP cells apoptosis. The expression of SIRT1 in disc NP of patients (>55 years) with lumbar disc degenerative disease (DDD) and the disc NP of patients (<25 years) with lumbar vertebra fracture (LVF) was assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blot analysis. The results showed that SIRT1 mRNA and protein levels were greater in LVF disc NP than those in DDD disc NP. Degenerative human NP cells were treated in culture with activator or inhibitor of SIRT1, resveratrol or nicotinamide, or SIRT1 small interfering RNA (siRNA), and cell apoptosis was quantified via flow cytometry. The rate of apoptosis was far fewer in resveratrol-treated NP cells than in SIRT1 siRNA-transfected or nicotinamide-treated NP cells. After SIRT1 siRNA was transfected, NP cells decreased phosphorylation of Akt, while resveratrol phosphorylated Akt. Treatment with LY294002 or Akt siRNA increased the rate of apoptosis. Our results suggested that SIRT1 plays a critical role in survival of degenerative human NP cells through the Akt anti-apoptotic signaling pathway.

Resveratrol induces nuclear factor-κB activity in human cardiac cells

BackgroundResveratrol is a grape polyphenol that prevents cardiac hypertrophy and protects the heart from ischemic injury, metabolic dysregulation, and inflammatory processes in several murine models. Methods and resultsThe aim of this study was to investigate the effects of resveratrol on the inflammatory processes in human cardiac AC16 cells in order to gain a better understanding of its cardioprotective mechanisms in the human heart. Resveratrol induced the DNA-binding activity of the pro-inflammatory transcription factor NF-κB in AC16 cells, and exacerbated the increase caused by tumor necrosis factor-α (TNF-α). In accordance with this, resveratrol increased the expression of the pro-inflammatory genes ICAM-1 (intercellular adhesion molecule-1) and TNF-α. In contrast, resveratrol decreased the expression of pro-inflammatory genes IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1). Likewise, resveratrol also induced inflammation in rat neonatal cardiomyocytes, and in the heart of mice fed a standard chow diet supplemented with resveratrol (1g/kg diet) for four months. Western-blot analyses revealed that NF-κB p65 subunit levels were upregulated in an IκB-dependent manner in the nuclei of resveratrol-treated human cardiac cells. Finally, resveratrol activated the signal transducer and activator of transcription 3 (STAT3) signaling and induced the expression of its anti-apoptotic downstream effector Bcl-xL, both involved in the cardioprotective survival activating factor enhancement (SAFE) pathway. ConclusionsResveratrol enhanced NF-κB activity in human and murine cardiac cells, in a process that coincided with the activation of STAT3 and anti-apoptotic downstream effectors. Therefore, activation of the SAFE pathway by resveratrol might be involved in the cardioprotective effects of this compound.

Effects of resveratrol on enrichment of adipose-derived stem cells and their differentiation to osteoblasts in two-and three-dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose-derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271(+), CD73(+) and CD45(-)) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11(+) and OCN(+). Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µM) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two-dimensional (2D) cultures as a function of alkaline phosphatase-specific activity and osteocalcin production. In addition, cells were seeded onto three-dimensional (3D) poly-ε-caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro-CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol-treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy.

Nuclear factor-kappaB as a switch in regulation of resveratrol-mediated apoptosis and erythrocytic differentiation in human leukaemia cells

Resveratrol exerts functions relating apoptosis and differentiation; nonetheless, the mechanisms remain unclear. We examined the regulation of resveratrol on apoptosis and erythrocytic (E)-differentiation in leukaemia cells. The results showed that resveratrol treatment for 24h induced apoptosis in human leukaemia NB4, HL-60, and U937 cells. Resveratrol-treated K562 cells underwent erythrocyte (E)-differentiation and cell cycle arrest in S-phase, without apoptotic induction in the early stage, but with marked apoptosis in the later stages. Resveratrol-induced apoptosis could be accompanied by P38 downregulation, and E-differentiation could occur with PI3K/Akt upregulation. Downstream NF-κB was increased by resveratrol treatment for 24h, while luciferase activity was decreased by resveratrol treatment for 72h. Similar changes were discovered for nuclear levels and NF-κB-DNA binding activity. In conclusion, resveratrol induced differential responses through different signalling pathways, including PI3K/Akt and P38/MAPK, leading to opposing expressions of NF-κB. NF-κB could be a switch for regulating E-differentiation and apoptosis in resveratrol-stimulated K562 cells.

Metabolic Patterns and Biotransformation Activities of Resveratrol in Human Glioblastoma Cells: Relevance with Therapeutic Efficacies

Background Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).Methodology/Principal FindingsCiting resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.Conclusions/SignificanceLN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.

MicroRNA-622 functions as a tumor suppressor by targeting K-Ras and enhancing the anticarcinogenic effect of resveratrol

Aberrant expression of microRNA (miRNA) has been previously demonstrated to play an important role in a wide range of cancer types and further elucidation of its role in the mechanisms underlying tumorigenesis, anticarcinogenesis and potential chemotherapeutics is warranted. We chose the anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-transformed human bronchial epithelial cell line 16HBE-T to study miRNAs involved in anticarcinogenesis. In resveratrol-treated cells, we found that miR-622 was upregulated, whereas it was downregulated in 16HBE-T cells, suggesting that miR-622 potentially acts as a tumor suppressor. Increasing the level of miR-622 by transient transfection-induced inhibition of proliferation and G(0) arrest in 16HBE-T cells and the lung cancer cell line H460 as demonstrated by cell viability and cell cycle analysis. MiR-622 dramatically suppressed the ability of 16HBE-T cells to form colonies in vitro and to develop tumors in nude mice. According to bioinformatics analysis, K-Ras messenger RNA was predicted as a putative miR-622 target; this was confirmed by western blot and luciferase reporter assays. Cell growth retardation was inhibited upon knockdown of K-Ras and an increase in the level of miR-622 in 16HBE-T cells. Furthermore, miR-622 inhibitor partially impaired the growth of 16HBE-T cells as demonstrated by luciferase reporter activity and K-Ras protein expression in 16HBE-T cells. In summary, miR-622 functions as a tumor suppressor by targeting K-Ras and impacting the anticancer effect of resveratrol. Therefore, miR-622 is potentially useful as a clinical therapy. MiR-622 impacts the K-Ras signal pathway and the potentially anticarcinogenic or chemotherapeutic properties warrant further investigation.

Effects of resveratrol on HepG2 cells as revealed by 1H-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.

Resveratrol Alters microRNA Expression Profiles in A549 Human Non-Small Cell Lung Cancer Cells

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.

Resveratrol rescues cAMP‐dependent anionic transport in the cystic fibrosis pancreatic cell line CFPAC1

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel in the plasma membrane of epithelia whose mutation is the cause of the genetic disease cystic fibrosis (CF). The most frequent CFTR mutation is deletion of Phe(508) and this mutant protein (delF508CFTR) does not readily translocate to the plasma membrane and is rapidly degraded within the cell. We hypothesized that treating epithelial cells with resveratrol, a natural polyphenolic, phyto-ooestrogenic compound from grapes, could modulate both the expression and localization of CFTR. Cells endogenously expressing CFTR (MDCK1 and CAPAN1 cells) or delF508CFTR (CFPAC1 and airway epithelial cells, deriving from human bronchial biopsies) were treated with resveratrol for 2 or 18 h. The effect of this treatment on CFTR and delF508CFTR expression and localization was evaluated using RT-PCR, Western blot and immunocytochemistry. Halide efflux was measured with a fluorescent dye and with halide-sensitive electrodes. Production of interleukin-8 by these cells was assayed by ELISA. Resveratrol treatment increased CFTR expression or maturation in immunoblotting experiments in MDCK1 cells or in CFPAC1 cells. Indirect immunofluorescence experiments showed a shift of delF508CFTR localization towards the (peri)-membrane area in CFPAC1 cells and in human airway epithelial cells. A cAMP-dependent increase in membrane permeability to halide was detected in resveratrol-treated CFPAC1 cells, and was inhibited by a selective inhibitor of CFTR. These results show that resveratrol modulated CFTR expression and localization and could rescue cAMP-dependent chloride transport in delF508CFTR cells.

SOCS‐3 antagonizes pro‐apoptotic effects of TRAIL and resveratrol in prostate cancer cells

Therapy for advanced prostate cancer is only palliative and its improvement could be achieved by sensitization to pro-apoptotic agents to which resveratrol belongs. We investigated the interaction between the tumor-selective apoptosis inducer tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and suppressor of cytokine signaling (SOCS-3), an antiapoptotic molecule which is up-regulated in prostate cancer. Expression of SOCS-3 and TRAIL (death) receptors was determined by Western blot after treatment with TRAIL in prostate cancer cell lines. Binding of SOCS-3 to death receptors was investigated by immunoprecipitation. Apoptosis rate was determined by a propidium iodide assay after treatment by TRAIL and resveratrol. SOCS-3, whose expression was differentially regulated by TRAIL in androgen-insensitive prostate cell lines, binds to death receptor 4. Overexpression of SOCS-3 reduced apoptosis in TRAIL- and resveratrol-treated DU145 cells and SOCS-3 siRNA increased apoptosis in TRAIL-treated PC-3 and LNCaP-IL-6+ cells. Our results strongly suggest that SOCS-3 is one of the proteins which influence the ability of TRAIL and resveratrol to cause programmed cell death in prostate cancer.

Resveratrol Inhibits Monocytic Cell Chemotaxis to MCP-1 and Prevents Spontaneous Endothelial Cell Migration Through Rho Kinase-Dependent Mechanism

Inflammatory cell recruitment and intimal neovascularization contribute to atherosclerotic plaque destabilization. The anti-inflammatory red wine polyphenol, resveratrol, has been implicated in cardiovascular protection. In this study, we investigated the effects of resveratrol on endothelial and monocytic cell migration. Human umbilical vein endothelial cell (EC) migration was assessed in a modified barrier assay. Chemotaxis of THP-1 monocytic cells towards monocyte chemoattractant protein (MCP)-1 was determined using a Boyden chamber. Erk phosphorylation downstream of MCP-1 receptor and activation of myosin phosphatase targeting subunit 1 (pMYPT1) downstream of Rho kinase were determined by Western blotting. In resveratrol-treated cells, progressive shape elongation was observed, evident after 6h of treatment. Treatment with resveratrol (1-20 µmol/L) dose-dependently inhibited EC migration. This effect of resveratrol was independent of nuclear factor (NF)-kappaB and sirtuin 1, but was abrogated in the presence of Rho kinase inhibitors. Moreover, resveratrol induced pMYPT1 activation, indicating a novel mechanism of resveratrol activity in EC. In monocytic cells, treatment with resveratrol significantly inhibited chemotaxis towards MCP-1 already at 1 µmol/L. At a resveratrol concentration of 10 µmol/L, chemotaxis was reduced nearly to the negative control (unstimulated with MCP-1) levels. This effect was independent of NF-kappaB and RhoA signaling. In resveratrol treated monocytic cells, MCP-1-induced Erk phosphorylation downstream of CCR2 receptor was dose-dependently inhibited, as observed by Western blot analysis. Resveratrol dose-dependently inhibited endothelial cell migration and MCP-1-induced monocytic cell chemotaxis. This activity may contribute to the cardioprotective effects of resveratrol by inhibition of intimal neovascularization and monocyte recruitment into the artery wall.

Inducible COX-2-dependent apoptosis in human ovarian cancer cells

Resveratrol is a naturally occurring trihydroxyl-diphenylethylene compound that has been shown experimentally to have beneficial effects in the treatment of cancer and cardiovascular disease. Resveratrol induces programmed cell death (apoptosis) in these cells and activates important signal transducing proteins including extracellular signal-regulated kinases (ERKs) 1 and 2 in cancer cells. Resveratrol also causes nuclear accumulation of the enzyme cyclooxygenase (COX)-2 and of the oncogene suppressor protein, p53. We have studied the molecular basis of the anticancer actions of resveratrol using human ovarian carcinoma (OVCAR-3) cells. Our findings include the following: (i) nuclear accumulation of COX-2 in resveratrol-treated cells is blocked by the ERK1/2 inhibitor, PD98059; (ii) an inhibitor of COX-2 activity, NS398, prevents accumulation of ERK1/2, COX-2, activated p53 and small ubiquitin-like modifier (SUMO-1) in the nucleus; (iii) apoptosis, quantitated by nucleosome enzyme-linked immunosorbent assay and the nuclear abundance of the pro-apoptotic protein, BcL-xs, were inhibited by NS398. This finding implicates nuclear COX-2 in p53-mediated apoptosis induced by resveratrol. Sumoylation is important to stabilization of p53 and a COX-2-SUMO-1 interaction suggests sumoylation of COX-2 in resveratrol-treated cells and (iv) chromatin immunoprecipitation studies showed binding of induced nuclear COX-2 to the promoter region of PIG3 and Bax, pro-apoptotic gene targets of transcriptionally active p53. Nuclear accumulation of activated ERK1/2 and sumolyated COX-2 are essential to resveratrol-induced pSer-15-p53-mediated apoptosis in human ovarian cancer cells.

Inhibition of NF-κB signaling commits resveratrol-treated medulloblastoma cells to apoptosis without neuronal differentiation

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.

Modulation of cancer cell proliferation by cell survival signal Akt and tumor suppressive energy sensor AMP-activated protein kinase in colon cancer cells treated with resveratrol

It has been well known that resveratrol inhibits the proliferation of various cancer cells. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, has emerged as a potent target for cancer prevention and/or treatment. It has been found that the activation of AMPK by resveratrol was crucial for the inhibition of HT-29 colon cancer cells. Resveratrol strongly inhibited phosphorylation of Akt. The possibility whether AMPK activation was essential to the inhibition of p-Akt was investigated in resveratrol-treated cancer cells. The inhibitory effect of resveratrol on Akt was not observed when AMPK activities were blocked by the treatment with AMPK siRNA at a relatively lower level of resveratrol. However, the higher concentrations of resveratrol inhibited Akt without the activation of AMPK. Therefore, it was concluded that resveratrol could modulate Akt AMPK-dependently or AMPK-independently. The inhibition of Akt along with the activation of AMPK may contribute to the unraveling anti-cancer mechanism of resveratrol.

Resveratrol Induces Cell Growth Arrest and Cell Death of Adult T-Cell Leukemia Cells Via Deacetylation of STAT3 by SIRT1

AbstractAbstract 2844Adult T-cell leukemia (ATL) is an aggressive malignancy of peripheral T cells infected with human T-cell leukemia virus type 1 (HTLV-1). The prognosis of aggressive ATL patients remains poor because of its resistance to conventional chemotherapy. Therefore, new therapeutic targets should be explored. We recently reported that signal transducers, and activators of transcription 3 (STAT3)/survivin pathway is critical for cell proliferation and survival of ATL cells (Ito S. et al. Leuk Res. 2010. 34:352-357). Thus, STAT3 may be one of the therapeutic targets in ATL. Recent report suggested that acetylation in the SH2 domain of STAT3 is an important regulatory modification that influences protein-protein interaction and transcriptional regulation. Although STAT3 has been shown to interact with p300 acetyltransferase, the mechanism of deacetylation of STAT3 remains unknown. It has been recently shown that fasting-activated longevity protein sirtuin 1 (SIRT1) deacetylase/STAT3 pathway in the regulation of gluconeogenesis. We hypothesized that STAT3 deacetylation by SIRT1 may influence the cell growth and survival of ATL cells. In this study, we investigated the effects of resveratrol, an activator of SIRT1 deacetylase, on HTLV-1 infected T-cell lines, KUT1 and MT2 cells. We found that resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner by trypan blue exclusion method (P<0.01, ED50=50mM). Resveratrol also induced apoptosis in these cells by flow cytometric analysis with AnnexinV/propidium iodide staining (P<0.01). We next examined intracellular signaling in resveratrol-treated cells. Immunoblot analysis showed the suppression of constitutive phosphorylation of STAT3 at Tyr705 and IkBα at Ser32 but not JAK3 and STAT5 of both cells 24 hours after the treatment of resveratrol. Interestingly, resveratrol reduced acetylation of STAT3 at Lys685. We also observed the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) in resveratrol-treated cells, indicating that resveratrol induces caspase-dependent apoptosis in these cells. To better understand the mechanism by which resveratrol induces STAT3 deacetylation, we examined the relationship between SIRT1 and STAT3. SIRT1 protein expression level was elevated after the treatment of resveratrol. Immunoprecipitation assay revealed the physical interaction of STAT3 with SIRT1 in MT2 cells, suggesting that the mechanism of action of resveratrol involves the deacetylation of STAT3 through the interaction of STAT3 with SIRT1.Our data indicate that resveratrol presents a potent anti-leukemia effect in part via the deacetylation of STAT3 in HTLV-1 infected cells. These mechanistic findings suggest that resveratrol may have a potential in the treatment of ATL. Disclosures:No relevant conflicts of interest to declare.

Differential regulation of proliferation, cell cycle control and gene expression in cultured human aortic and pulmonary artery endothelial cells by resveratrol

Resveratrol is a grape polyphenol with cardioprotective attributes, supported in part by its demonstrated anti-mitogenic, apoptosis-inducing and gene modulatory activities in various cell types known to play an integral role in atherogenesis. To test whether resveratrol exerts similar effects on systemic and pulmonary vasculature, cells derived from different anatomical sites, cultured human aortic and pulmonary artery endothelial cells, respectively denoted HAECs and HPAECs, were exposed to resveratrol for assessment of effects on proliferation, cell cycle distribution, induction of apoptosis, and specific gene expression. Resveratrol inhibited cell proliferation in a time- and dose-dependent manner in HAECs and HPAECs, accompanied by disruption of cell cycle control and progression as assayed by flow cytometry. Analysis of gene changes in resveratrol-treated endothelial cells by RT-PCR showed suppression of nitric oxide synthase (eNOS) and preproendothelin-1 (ppET-1) expression in both cell types. To discover group gene alterations resulting from exposure to resveratrol, changes in mRNA levels were determined by human signal transduction pathway finder cDNA array analysis. The results showed that resveratrol up-regulated levels of cyclin-dependent kinase inhibitor p57, egr-1, forkhead box A2 and c-jun in HAECs, and elevated expression of cathepsin D, ICAM-1, c-jun and patched 1 in HPAECs. In addition, treatment by resveratrol also resulted in attenuated expression of bcl-xl, fibronectin-1, HIP, mdm2, PIG3 and WSB1/SWIP-1 in HAECs, and CDX1, engrailed homolog 1, FASN, fibronectin-1, forkhead box A2, Hoxa-1, hsp27, PIG3, ELAM-1/E-selectin and WSB1/SWIP-1 in HPAECs. These results suggest that resveratrol acts by distinct and overlapping signaling pathways and mechanisms in HAECs and HPAECs, further supporting the notion that the cardioactive activities and effects of this grape polyphenol are contingent upon or influenced by the vascular bed of origin.

Resveratrol-induced autophagy in human U373 glioma cells

Autophagy is an intracellular protein transport process leading to the degradation of organelles and long-lived proteins in eukaryotes. The down-regulation of autophagy observed in cancer cells has been associated with tumor progression. This study investigated autophagy induced by resveratrol, a natural compound, in human glioma cells. Glioma cells were exposed to resveratrol, and the cell growth and autophagic level were evaluated. Resveratrol inhibited growth and induced cell death in U373 glioma cells. When treated with resveratrol, glioma cells stably expressing GFP fused to LC3, recruited more GFP-LC3-labeled autophagosomes, and the percentage of cells with GFP-LC3-labeled autophagosomes increased. Furthermore, in resveratrol-treated glioma cells, pretreatment with P38 or ERK1/2 inhibitors reduced the autophagic level, suggesting that resveratrol-induced autophagy was positively regulated by P38 and the ERK1/2 pathway. The Akt/mTOR pathway was not involved in resveratrol-induced autophagy. Our results suggest that resveratrol has an anticancer effect on glioma cells by inducing autophagy.

Abstract 4138: Resveratrol induces COX-2-dependent apoptosis in human ovarian cancer cells

Abstract Resveratrol is a naturally-occurring trihydroyxl-diphenylethylene compound that has been shown experimentally to have beneficial effects in the treatment of cancer and cardiovascular disease. We have studied the molecular basis of the anti-cancer actions of resveratrol, using human ovarian carcinoma (OVCAR-3) cells. Resveratrol induces programmed cell death (apoptosis) in these cells and activates important signal transducing proteins including extracellular signal-regulated kinases 1 and 2 (ERK1/2) in cancer cells. Resveratrol also causes nuclear accumulation of the enzyme cyclooxygenase-2 (COX-2) and of the oncogene suppressor protein, p53. Expressed constitutively, cytoplasmic COX-2 is a marker of tumor cell aggressiveness, but there is a resveratrol-inducible pool of nuclear COX-2 that is important to activation (Ser-15 phosphorylation) of the oncogene suppressor protein, p53. Our findings include the following: (1) Nuclear accumulation of COX-2 in resveratrol-treated cells is blocked by the ERK1/2 inhibitor, PD98059. (2) An inhibitor of COX-2 activity, NS398, prevents accumulation in the nucleus of ERK1/2, COX-2, activated p53 and SUMO-1. (3) Apoptosis, quantitated by nucleosome ELISA and the nuclear abundance of the pro-apoptotic protein, Bcl-xs, were inhibited by NS398. This finding implicates nuclear COX-2 in p53-mediated apoptosis induced by resveratrol. Sumoylation is important to stabilization of p53 and a COX-2/SUMO-1 interaction suggests sumoylation of COX-2 in resveratrol-treated cells. (4) Chromatin immunoprecipitation (ChIP) studies showed binding of induced nuclear COX-2 to the promoter region of p21, a pro-apoptotic gene target of transcriptionally active p53. In summary, nuclear accumulation of activated ERK1/2 and COX-2 are essential to resveratrol-induced, pSer-15-p53-mediated apoptosis in human ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4138.

The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates

The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome- and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2Delta cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

Beneficial effects of PTP1B deficiency on brown adipocyte differentiation and protection against apoptosis induced by pro- and anti-inflammatory stimuli

Insulin is an inducer of brown fat adipogenesis through the activation of a signalling network that involves positive/negative modulators. Given the importance of brown adipose tissue (BAT) for basal thermogenic energy expenditure, we investigated the role of PTP1B in the acquisition of terminal differentiated phenotype and in the apoptotic responses of brown adipocytes. Immortalized brown preadipocytes lacking (PTP1B -/-) or expressing (PTP1B +/+) PTP1B have been generated. PTP1B deficiency accelerated a full program of brown adipogenesis including induction of transcription factors, coactivators, adipogenic markers and signalling molecules. Fully differentiated PTP1B -/- brown adipocytes were resistant to tumor necrosis factor (TNFα)–induced apoptosis as these cells were protected against caspase-8 activation, FLIP degradation, Bid cleavage and caspase-3 activation compared to wild-type controls. These events were recovered by PTP1B rescue. Survival signalling including phosphorylation of IRS-1 and Akt/PKB and BclxL expression were decreased in TNFα-treated PTP1B -/- cells but not in the wild-type. Similarly, PTP1B -/- brown adipocytes were protected against resveratrol-induced apoptosis. Phosphorylation of Akt/PKB and Foxo1 phosphorylation/acetylation decreased exclusively in resveratrol-treated wild-type cells, leading to nuclear localization of Foxo1 and up-regulation of Bim. Thus, PTP1B inhibition could be of benefit against obesity by counteracting TNFα-induced brown fat atrophy, and combined with resveratrol might improve low-grade inflammation.