Resveratrol Inhibits Proliferation and Promotes Apoptosis of Neuroblastoma Cells: Role of Sirtuin 1
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- Pizarro, J.G., Verdaguer, E., Ancrenaz, V. et al. Neurochem Res (2011) 36: 187. doi:10.1007/s11064-010-0296-y
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Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-β. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.
KeywordsNeuroblastomaB65ResveratrolSirtinolSilent information regulator (SIRT1)Sirtuin 1Caspase 3Apoptosis
Resveratrol (RESV) is a natural phytoalexin, chemically known as 3,4′,5-trihydroxystilbene, and is a polyphenolic antioxidant present in red wine, grapes, berries, peanuts, etc. . Proposed benefits of RESV include neuroprotection, as well as cancer suppression [2–4]. The cancer chemopreventive potential of RESV is related to activation of the mitochondrial pathway of apoptosis and targeting of the proteins involved in the PI3K/AKT pathway, which are strongly implicated in cancer progression [5–8]. Moreover, RESV can activate silent information regulator 2 (Sirtuin 2) family deacetylase activities [9–11].
Sirtuins belong to a family of histone deacetylase proteins (HDACs), which are divided into three classes. Classes I and II are called the classical HDACs and consist of zinc-dependent hydrolases, while class III, sirtuins, catalyse deacetylation by a different mechanism using NAD+ as a cofactor [12, 13]. Since the discovery of the involvement of SIRT 1 in apoptosis, cell survival, transcription, metabolism and aging, the interests in SIRT1 physiological effects has increased [12, 14–16]. Current research is therefore focused to understand the mechanisms involved in the ability of RESV to increase the activity of SIRT1 and on the intracellular pathways regulated by this protein.
Interestingly, SIRT1 activation or over-expression has a role in apoptosis regulation, through inactivating the tumour suppressor protein p53 by deacetylation and also via FOXO transcriptions factors regulation [17–20]. Likewise, SIRT1 can regulate cellular metabolism through NF-κβ modulation and influences muscle differentiation . Exposure of keratinocytes to UV radiation and cerebellar granule cells to MPP+ causes a SIRT1 down-regulation [22, 23]. To our knowledge, it has still not been demonstrated that if the antiproliferative effects of RESV are mediated by SIRT1 activation. Here we evaluate the involvement of SIRT1 in the control of cell cycle progression and apoptosis induction in a rat dopaminergic neuroblastoma cell line B65 exposed to RESV. To test the involvement of SIRT1, we inhibited its activity with sirtinol and nicotinamide.
Materials and Methods
Pharmacological agents used in this study include resveratrol, sirtinol and nicotinamide (Sigma Chemical Co., St. Louis, MO, USA), cell culture media and fetal calf serum (FCS) (GIBCO, Life Technologies, Paisley, UK), monoclonal antibodies against SIRT1 (Millipore Corp., Bedford, MA), acetylated form of NFkβ-p65, lys379 acetyl-p53, ser473 p-AKT (Cell Signalling Technology, Denvers, MA), cyclin D, cyclin E, cyclin A, cdk2 and actin (Santa Cruz Biotechnology, Santa Cruz, CA), and peroxidase-conjugated IgG secondary antibody (Amersham Corp., Arlington Heights, IL).
Flow cytometry experiments were carried out using an Epics XL flow cytometer. Optical alignment was based on the optimised signal from 10 nm fluorescent beads (Immunocheck, Epics Division). Stained cells were visualized under UV illumination using the 20× objective of a Nikon Eclipse fluomicroscope. Western blot analyses were performed with polyvinylidene fluoride (PVDF) sheets (ImmobilonTM-P, Millipore Corp., Bedford, MA) and a transblot apparatus (BioRad). Immunoreactive protein was visualized using a chemiluminescence-based detection kit following the manufacturer’s protocol (ECL kit; Amersham Corp.).
Cell Culture and Treatment
The dopaminergic neuroblastoma B65 cell line was purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were plated at 200 cells/mm2 and cultured in DMEM media containing 10% FCS and 2 mM glutamine for 24 h prior to addition of chemicals. In toxicity experiments, cells were exposed to RESV at concentrations ranging from 25 to 100 μM, for 15 min to 6 h, in time course experiments. For MTT test and flow cytometry analysis, cells were exposed to resveratrol for 24 h. In the experiments, we observed the effects of RESV in the presence of the SIRT1 inhibitors, sirtinol and nicotinamide.
Assessment of Cell Viability
B65 cells were used after 24 h of in vitro culture. To assess cell viability loss, we used the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] method. MTT was added to the cells at a final concentration of 250 μM and incubated for 1 h, allowing the reduction in MTT to produce a dark blue formazan product. Media were then removed, and cells were dissolved in dimethylsulfoxide. Formazan production was measured by the absorbency change at 570 nm using a microplate reader (BioRad Laboratories, CA, USA). Viability results were expressed as percentages. The absorbency measured from non-treated cells was taken to be 100%.
Analysis of DNA Fragmentation and Cell Cycle Progression by Flow Cytometry
We studied changes in the cell cycle and apoptosis after 24 h of either RESV exposure or pharmacological inhibition of SIRT1 with nicotinamide (0.250–1 mM) and sirtinol (10–50 μM). The inhibitors were dissolved in culture media and added to the cultures 2 h before addition of RESV. In brief, the culture medium was removed, cells were washed in PBS and flow cytometry experiments were carried out adding propidium iodide (PI, 75 μg/ml) 1 h before analysis. The instrument was set up with a standard configuration: excitation of the sample was performed using a 488 nm air-cooled argon-ion laser at 15 mW power. Forward scatter (FSC), side scatters (SSC) and red (620 nm) fluorescence for PI were acquired. Optical alignment was based on optimised signal from 10 nm fluorescent beads. Time was used as an instrument stability control; red fluorescence was projected onto a 1,024 nonparametric histogram. Aggregates were excluded, gating single cells by their area versus peak fluorescence signal.
Assay of Caspase 3 Enzymatic Activity
We used the colorimetric substrate Ac-DEVD-p-nitroaniline for the determination of caspase 3 according to the following method: B65 cells were collected in a lysis buffer (50 mM Hepes, 100 mM NaCl, 0.1% CHAPS, 0.1 mM EDTA, pH 7.4), 24 h after treatment, and 50 μg/ml of protein was incubated with 200 mM of colorimetric substrate in assay buffer (50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 0.1 mM EDTA, pH 7.4) in 96-well plates at 37°C for 24 h. Absorbance of the cleaved product was measured at 405 nm. Results were expressed as a percentage of sample absorbance over control values.
Aliquots of cell homogenate containing 25 μg of protein per sample were analyzed by Western blot. Briefly, samples were placed in sample buffer (0.5 M Tris–HCl pH 6.8, 10% glycerol, 2% (w/v) SDS, 5% (v/v) 2-β-mercaptoethanol, 0.05% bromophenol blue) and denatured by boiling at 95–100°C for 5 min. Samples were then separated by electrophoresis in 10% acrylamide gels, with proteins subsequently transferred to polyvinylidene fluoride sheets (ImmobilonTM-P, Millipore Corp., Bedford, MA, USA) using a transblot apparatus (BioRad). The membranes were blocked for 1 h with 5% non-fat milk dissolved in TBS-T buffer (Tris 50 mM; NaCl 1.5%; Tween 20, 0.05%, pH 7.5). They were then incubated with primary monoclonal antibodies against SIRT1, lys379 acetyl-p53, ser473 p-AKT, total p53, cyclin D, cyclin E, cyclin A, cdk2 and actin. After 4 h or overnight, blots were washed thoroughly in TBS-T buffer and incubated for 1 h with a peroxidase-conjugated IgG antibody (Amersham Corp., Arlington Heights, IL, USA). Immunoreactive protein was visualized using a chemiluminescence-based detection kit following the manufacturer’s instructions (ECL kit; Amersham Corp.).
Digital images were taken with a Chemidoc XRS (Biorad), which enables semi-quantitation of band intensity. The protein load was monitored via immunodetection of actin.
Data are presented as mean ± SEM of at least three or four experiments. In all experiments, data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s tests. P-values lower than 0.05 were considered significant.
Resveratrol Impairs the Proliferation of B65 Cells and Promotes Apoptosis
Resveratrol Impairs the Activity of AKT and SIRT1 Protein
Time-course experiments were performed to study the effects of RESV on SIRT1 and the acetylated forms of p53 and NF-κβ (both substrates of SIRT1). Western blot results show that exposure to RESV decreased SIRT1 content significantly after 15 min and concomitantly, both acetylated forms of p53 and NF-κβ increased significantly at 15 and 30 min, respectively.
SIRT1 Inhibitors Nicotinamide and Sirtinol Do Not Prevent Apoptosis
Likewise, after 24 h treatment of B65 cells with RESV a decrease in the levels of expression of the SIRT1 band was detected. On the other hand, treatment of cells with sirtinol or nicotinamide was unable to reverse the effects of RESV on the expression of SIRT1.
Finally we evaluated if apoptosis induced by RESV was mediated by the activation of caspases, specifically caspase 3 the main executioner caspase. We found a significant increase (* P < 0.05) in caspase 3 activity after 24 h of after 24 h treatment of B65 cells with RESV. This data suggest that apoptosis induced by RESV is caspase dependent.
Our results show that RESV, a polyphenol compound contained in grape skin and its dietary products such as red wine, inhibits cell cycle proliferation in rat dopaminergic B65 cells and induces apoptosis through caspase 3 activation. Although it is well known that RESV has an antiproliferative mechanism, the implication of SIRT1 in RESV inhibition of cell cycle proliferation is poorly understood. In this study, we investigated whether RESV has a direct apoptotic effect on B65 neuroblastoma cells through the up-expression of SIRT1 in vitro. Results from our experiments show that RESV decreased cell viability and induced apoptosis. However, we also provide evidence that the action on B65 cellular proliferation is not due to interaction with SIRT1. Our hypothesis is based in the evidence that when B65 cells are exposed for 24 h to the potent SIRT1 enzyme inhibitors 1 mM nicotinamide and sirtinol 60 μM, in the presence of RESV, neither compound reversed the effects of RESV. Consequently, these results indicate that an alternative mechanism must be adduced to explain the effects of RESV on cell-cycle proliferation. Furthermore, our results demonstrate that expression of SIRT1 significantly decreases following treatment with RESV. Therefore, all these data confirm that SIRT1 is not involved in RESV antiproliferative effects. Similarly, we also studied downstream targets regulated by SIRT1. As commented above, SIRT1 is a deacetylase enzyme, so the loss of SIRT1 protein expression was accompanied by an increase in the expression of acetylated proteins such as p53 and NFκβ. Hence, these data confirm that RESV influences the decrease in B65 SIRT1 expression.
On the other hand, recent results have demonstrated that SIRT1 has a role in cellular proliferation, favouring rather than disabling it [24–29]. Therefore, loss of SIRT1 expression could be responsible for, or at least contribute to, the inhibition of cell proliferation. The inhibitory effects of RESV on cell cycle have been previously demonstrated, as has its ability to mediate tumour cell death selectively through necrosis, apoptosis, autophagy and other mechanisms, although evidence is emerging that certain normal cells such as endothelial cells, lymphocytes, chondrocytes and adipocytes are vulnerable to RESV [27–30]. Likewise, RESV inhibits proliferation and promotes apoptosis of several tumour lines, such as osteosarcoma, epidermoid carcinoma, macroglobulinemia malignant cells or breast cancer cells [15, 16, 18, 20]. Furthermore, in the majority of studies, RESV inhibits cell proliferation and induces apoptosis via different mechanisms [29, 30]. More specifically, for all these cellular models it has been shown that RESV controls cell survival through modulation of key pathways such as phosphoinositide 3-kinase (PI3K), cell cycle changes and JAK/STAT. Phosphoprotein kinase B (PKB/AKT) activity acts downstream of PI3 K and, in turn, modulates glycogen synthase kinase 3 (GSK3), which is involved in cell cycle control and promotes apoptosis [26–31]. Results from our experiments show that, in agreement with the literature, the AKT content of B65 cells exposed to RESV decreases significantly. Moreover, we also found that cell cycle changes could be mediated by multiple targets such as AKT, FOXO or NFκβ [28–31].
Although the list of activities assigned to RESV is long and includes being an activator of sirtuins, it is considered a non specific compound [3, 4, 11]. Currently, SIRT1 activation by RESV is an area of interest, since it leads to improved metabolic control and contributes to protecting cells from several insults such as reactive oxygen species generation and oxidative injury, or from exposure to the neurotoxin 6-hydroxydopamine [32–37]. In neuronal cells, SIRT1 induction by RESV contributes to survival in the presence of MPP+ [38, 39]. RESV also has a neuroprotective effect in dopaminergic neurons in organotypic midbrain slice cultures . However, it remains to be clarified whether these neuroprotective effects in neurons are SIRT1 dependent, since SIRT1 levels decrease during aging and in neurodegenerative disorders such as Alzheimer’s disease [40–42]. Therefore, the neuroprotective effects of RESV may be due to its antioxidant properties .
Whereas in differentiated cells RESV induces the activity of sirtuins, results from our experiments show that in tumour cells, RESV inhibited SIRT1 expression. Unfortunately, this is the first time this effect has been observed, and thus at present we are unable to compare our results with others in vitro models.
In summary, RESV inhibits cell proliferation and induces apoptosis in dopaminergic B65 cells, but this effect is not directly related to SIRT1 activity. We conclude that RESV seems to reduce the proliferation of B65 cells by modulation of distinct pathways: through activation of p53 protein and its downstream effects, and additionally, through negative regulation of oncogenic signal transduction.
We thank the Language Advisory Service of the University of Barcelona for revising this manuscript. This study was supported by grants SAF-2009-13093 from the Ministerio de Educación y Ciencia, PI080400 and PS09/01789 from the Instituto de Salud Carlos III, 2009/SGR00853 from the Generalitat de Catalunya and (063230) from the Fundació La Marató of TV3. 610RT0405 from programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED).