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FoxO3 axis in bronchial epithelial cells
Experimental Gerontology 81 (2016) 119–128
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells E. Pace a,⁎, S. Di Vincenzo a, M. Ferraro a, A. Bruno a, P. Dino a, M.R. Bonsignore b, S. Battaglia b, F. Saibene c, L. Lanata c, M. Gjomarkaj a a b c
Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Palermo, Italy Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S), University of Palermo, Palermo, Italy Dompè Medical Affair, Milan, Italy
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Article history: Received 30 March 2016 Received in revised form 18 May 2016 Accepted 25 May 2016 Available online 26 May 2016 Section Editor: Werner Zwerschke Keywords: Cigarette smoke SIRT1 FoxO3 Carbocysteine Bronchial epithelial cells
a b s t r a c t Background: Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aims: Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. Methods: 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. Results: CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. Conclusions: The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Chronic obstructive pulmonary disease (COPD) is a progressive disease arising from an inflammatory response to irritants, among which cigarette smoke is the most frequent (Sethi and Murphy, 2001; Wilson, 2000, 2001). Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence within the lung, an event characterized by loss of replicative potential, increased beta galactosidase activity, and resistance to cell apoptosis (Beltrami et al., 2011). Cell senescence is believed to play an
Abbreviations: CSE, cigarette smoke extracts; COPD, chronic obstructive pulmonary disease; SIRT1, sirtuin1; FoxO3, Forkhead box O3; CARB, carbocysteine. ⁎ Corresponding author at: Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa, 153, 90146 Palermo, Italy. E-mail address: [email protected] (E. Pace).
http://dx.doi.org/10.1016/j.exger.2016.05.013 0531-5565/© 2016 Elsevier Inc. All rights reserved.
active role in COPD pathogenesis. COPD is considered a disease of the lung inflammaging, which is associated with the DNA damage response, transcription activation and chromatin modifications (MacNee, 2011). Anti-aging sirtuin1 (SIRT1), a NAD +-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD (Rajendrasozhan et al., 2008). SIRT1 protects against emphysema through FoxO3-mediated reduction of cellular senescence (Yao et al., 2012). SIRT1 downregulates inflammaging via regulating also p53, p21, nuclear factor kappa B, histones and a variety of proteins involved in DNA damage and repair (Yao et al., 2012; Atkins et al., 2014). Since incidence of COPD increases with age, chronic inflammation and cellular senescence may be intertwined in the pathogenesis of premature aging (MacNee, 2011). The airway epithelium, besides actively contributing to airway defence mechanisms, exerts an important role in coordinating local inflammation and immune responses (Kato and Schleimer, 2007). It is
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conceivable that when epithelial cells are chronically exposed to cigarette smoke they differentiate in a functional senescent phenotype with a reduced reparative potential and higher pro-inflammatory properties. In this context, secretion of IL-8, a hallmark of senescence-associated secretory phenotype (Hara et al., 2012) and a potent chemokine for neutrophils, is increased within the airways of smokers and of COPD patients and upon TLR4 activation and ERK 1/2 activation in cigarette smoke extracts (CSE) stimulated bronchial epithelial cells in vitro (Pace et al., 2008a). Carbocysteine, an anti-oxidant and mucolytic agent, was shown to reduce the severity and the rate of exacerbations in COPD patients (Zheng et al., 2008). In vitro, carbocysteine counteracted the increase in p21 and IL-8 mRNA and protein in bronchial epithelial cells exposed to CSE (Pace et al., 2013a). It is conceivable that carbocysteine could restore SIRT1/FoxO3 axis in bronchial epithelial cells and counteract some pro-senescence molecular events promoted by cigarette smoke exposure. The aim of this study was to investigate the relationship between SIRT1/FoxO3 axis, cellular senescence, regulation of cell cycle progression and apoptosis mechanisms in response to cigarette smoke in bronchial epithelial cells and to assess the effects of carbocysteine on cigarette smoke-induced molecular events. 2. Materials and methods 2.1. Preparation of cigarette smoke extracts (CSE) Commercial cigarettes (Marlboro) were used in this study. Cigarette smoke solution was prepared as described previously (Pace et al., 2008a). Each cigarette was smoked for 5 min and one cigarette was used per 10 ml of PBS to generate a CSE-PBS solution. The CSE solution was filtered through a 0.22 μm-pore filter to remove bacteria and large particles. The smoke solution was then adjusted to pH 7.4 and used within 30 min of preparation. This solution was considered to be 100% CSE and diluted to obtain the desired concentration in each experiments. The concentration of CSE was calculated spectrophotometrically measuring the OD as previously described at the wavelength of 320 nm. The pattern of absorbance, among different batches, showed very little differences and the mean OD of the different batches was 1.37 ± 0.16. The presence of contaminating LPS in undiluted CSE was assessed by a commercially available kit (Cambrex Corporation, East Rutherfort, New Jersey, USA) and was below the detection limit of 0.1 EU/ml. 2.2. Bronchial epithelial cell cultures 16-HBE cells, an immortalized normal bronchial epithelial cell line, were used in this study. Bronchial epithelial cells were maintained in MEM (Gibco, BRL, Germany), supplemented with 10% fetal calf serum (Euroclone, Milan, Italy). Cell cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37 °C. Cells were cultured in the presence and absence of CSE (10% and 20%) and CARB at 10−4 M (Dompè, Italy) for 24 h. No pre-treatment with CARB was performed in CSE exposed cells. CSE and CARB were simultaneously added to bronchial epithelial cells. The used drug concentration was selected in previous published studies (Pace et al., 2013b). At the end of stimulation, cells, cell extracts, and cell culture supernatants were collected for further evaluations. 2.3. Short term cell proliferation The short term cell proliferation of 16HBE cells exposed to the previously described stimuli was measured using carboxyfluorescein succinimidyl ester (CFSE) labeling assay (Pace et al., 2012a). CFSE is used to fluorescently label live cells and is equally partitioned to daughter cells during division. CSE and carbocysteine stimulated 16HBE were incubated with CFSE (Molecular Probes, Inc. Eugene, OR) (at a final concentration of 5 μM) at 37 ° C for 10 min. Cell proliferation was assessed
after 72 h by flow-cytometry and proliferated cells showed reduced CFSE expression. 2.4. Long term cell proliferation The long term proliferation of 16HBE cells exposed to the previously described stimuli was assessed by clonogenic assay as previously described (Bruno et al., 2014). Using 35-mm well of 6-well plate (Falcon, Becton Dickinson, Franklin Lakes, N.J.) a lower layer was prepared using complete MEM medium in 0.5% agarose. The cells were harvested and seeded (5 × 104) on the upper layer with 0.3% agarose prepared with the same medium as the lower layer, and finally incubated for 21 days at 37 °C in an atmosphere containing 5% CO2. At the end of incubation, colonies were counted under an inverted phase-contrast microscope (Leica, Wetzlar, Germany). The experiments were conducted in triplicate. Colonies were defined as cell aggregates with at least 40 cells. The true number of colonies was calculated as the number of aggregates on the positive control subtracted from the number of colonies on the experimental plates. Results are cell colony numbers. 2.5. Beta galactosidase staining 16HBE cells were exposed to the previously described stimuli and βgalactosidase (SA-βGal) staining was assessed as previously described (Itahana and Campisi, 2007). For senescence-activated β-galactosidase (SA-βGal) staining, we used Senescence-βGal Staining Kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, cells were washed twice with PBS, fixed with 2% formaldehyde/0.2% glutaralde-hyde in PBS for 10 min, washed twice with PBS, and incubated with fresh SA-b-gal staining solution containing 1 mg/ m1 X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in 40 mM citric acid/sodium phosphate buffer (pH 6.0) for 12 h at 37 °C in a CO2 free atmosphere. The SA-b-gal-stained cells obtained were photographed under an inverted microscope (Nikon Eclipse TS100, Nikon, Melville, NY, USA 1009). Briefly, cells were washed twice with PBS, fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 10 min, washed twice with PBS, and incubated with fresh SA-β-gal staining solution containing 1 mg/ ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in 40 mM citric acid/sodium phosphate buffer (pH 6.0) at 37 °C in a CO2 free atmosphere overnight. In the assay, senescence-associated β-galactosidase catalyzes the hydrolysis of X-gal, which results in the accumulation of a distinctive blue color in senescent cells. The images were captured by Leica microscope, Wetzlar, Germany. 2.6. Western blot analysis The expression of SIRT1 and FoxO3 in 16HBE cells exposed to the previously described stimuli was evaluated by western blot analysis as previously described (Pace et al., 2008b). To study SIRT1 and FoxO3 nuclear translocation, the protein extracts were treated to separate the cytoplasmic and nuclear protein fractions by using a commercial kit “NEPER Nuclear and Cytoplasmic Extraction Reagents” following the manufacturer's directions (Thermo Scientific; Waltham, MA). An amount of 30 μg of total proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and blotted onto nitrocellulose membranes. Membranes were incubated with a IgG rabbit polyclonal antibody anti-SIRT1 (1:500; overnight) (sc-15404 - Santa Cruz Biotechnology, Dallas, TX-USA) and a IgG goat polyclonal antibody anti-FoxO3 (1:750; overnight) (sc-9812 - Santa Cruz Biotechnology, Dallas, TX-USA). Then membranes were stripped and incubated with rabbit polyclonal Lamin B1 antibody (#9087, Cell Signaling Technology, Danvers, MA-USA) for assessing quality control of nuclear extracts. Revelation was performed with an enhanced chemiluminescence system (GE Healthcare, Chalfont St. Giles, UK) followed by autoradiography. Negative controls were performed without
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primary antibody or including an isotype control antibody. Data are expressed as densitometric arbitrary units by correction with the density of the bands obtained for Lamin B1. To assess acetylation of FoxO3, the total protein extracts were immunoprecipitated for FoxO3 with an IgG goat polyclonal antibody anti-FoxO3 (2 μg; sc-9812 Santa Cruz Biotechnology), which was added to 300 μg of sample proteins in a final volume of 400 μl, and incubated overnight at 4 °C with gentle shaking. Protein-A/G agarose beads (16 μl; sc-2003 Santa Cruz Biotechnology) were added to each sample and kept for 3 h at 4 °C on a rotating rocker. The beads were washed 3 times with lysis buffer and resuspended in reducing sample buffer for western blot analysis. The immunoprecipitated FoxO3 agarose bead suspension was resolved by SDS-PAGE. To assess FoxO3 acetylation, the membranes of immunoprecipitated FoxO3 were blotted against anti-acetylated lysine (#9441 - Cell Signaling). Revelation was performed with an enhanced chemiluminescence system (GE Healthcare, Chalfont St. Giles, UK) followed by autoradiography. 2.7. Immunoprecipitation and SIRT1 activity SIRT1 activity of 16HBE cells exposed to the previously described stimuli was assessed as previously described (Caito et al., 2010a). SIRT1 was immunoprecipitated from nuclear cell lysates (100 μg) by overnight incubation with anti-SIRT1 antibody at 4 °C and then incubated with protein G-agarose beads (sc-2003 - Santa Cruz Biotechnology, Dallas, TX-USA). Beads were extensively washed with lysis buffer. SIRT1 activity was measured using a commercial kit (Enzo Lifesciences, Farmingdale, NY, USA). An acetylated lysine substrate is incubated with samples with SIRT1 activity. Deacetylation sensitizes the substrate such that treatment with the SIRT1 developer in the second step releases a fluorescent product. The assay was performed exactly as recommended by the manufacturer. Fluorescence was measured in a microplate reader using Ex 355 and Em 460. 2.8. Immunofluorescence 16HBE were incubated with anti-SIRT1 and anti-FoxO3 antibodies followed by FITC and PE conjugated secondary antibodies, respectively. The cells were analysed by fluorescence microscope Axioskop 2 Zeiss microscope (Heidelberg, Germany). 2.9. ChiP assay ChiP analysis was performed using the EZ-ChIP kit (UpstateMillipore Corporate- Billerica, MA) as previously described (Pace et al., 2012b) in 16HBE cells exposed to the previously described stimuli. The crosslinked chromatin were sonicated to lengths spanning 200– 1000 bp. The samples were precleared with 60 μl of Protein G Agarose and then incubated with a IgG goat polyclonal antibody anti-FoxO3 [10 μg] (sc-9812 - Santa Cruz Biotechnology, Dallas, TX-USA). Immunocomplexes were precipitated using Protein G Agarose. After washing, DNA fragments were isolated and purified with columns. PCR was performed using primers for the promoter region of survivin gene: forward 5′ – TGA GCT GAG ATC ATG CCA CT – 3′ and reverse 5′ – CTG GTG CCT CCA CTG TCT TT – 3′.
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Fluorescein Isothiocyanate-conjugated anti rabbit immunoglobulin G or with a mouse anti-human p21 antibody (sc-817; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by FITC-conjugated goat antimouse (DAKO) and then evaluated by flow cytometry. Rabbit or mouse negative control immunoglobulins (DAKO) were used for negative controls. Flow cytometry analyses were performed on a Becton Dickinson FACSCalibur System. Analysis was done on 10,000 acquired events for each sample using cellQuest acquisition and data analysis software (Becton Dickinson). Data are expressed as percentage of positive cells.
2.11. Statistics Data are expressed as mean counts ± standard deviation. Kolmorogov-Smirnov Normality test was initially performed to assess whether parametric analyses of data could be performed. Comparison between different experimental conditions was evaluated by ANOVA post-hoc test. p b 0.05 was accepted as statistically significant.
3. Results 3.1. Effects of CSE and carbocysteine (CARB) on short and long term cell proliferation of bronchial epithelial cells Epithelial cell senescence is associated with loss of replicative potential. Firstly, short-term cell proliferation in bronchial epithelial cells exposed to CSE was assessed by flow cytometry using the CFSE method. CSE reduced, in a dose dependent manner, short-term cell proliferation (reduction versus baseline after CSE exposure: 10% = 25 ± 10; p b 0.006; 20% = 41 ± 4; p b 0.0001); incubation with CARB significantly counteracted this phenomenon in cells exposed to CSE 10% (increase versus CSE 10% = 7.4 ± 4; p b 0.03) but not in cells exposed to CSE 20% (Fig. 1A–B). To further explore the effects of both CSE and CARB on cell proliferation, clonogenic assay, a method used for assessing long-term cell proliferation, was performed. CSE reduced, in a dose dependent manner (reduction versus baseline after CSE exposure: 10% = 27.6 ± 12; p b 0.005; CSE 20% = 61 ± 17; p b 0.001) long-term cell proliferation and incubation with CARB significantly counteracted this phenomenon in cells exposed to both CSE 10% and CSE 20% (increase versus CSE 10% = 25 ± 26; p b 0.04 increase versus CSE 20% = 87 ± 58; p b 0.006) (Fig. 1C).
3.2. Effects of CSE and CARB on beta galactosidase staining of bronchial epithelial cells Increased beta galactosidase staining is considered a gold standard to identify senescent cells. CSE increased beta galactosidase staining and incubation with CARB completely reverted this phenomenon (Fig. 2A). CSE increased p21 expression and CARB counteracted this phenomenon (Fig. 2B).
2.10. Expression of p21 and survivin by flow-cytometry
3.3. Effects of CSE and CARB on nuclear SIRT1 expression by bronchial epithelial cells
The expression of p21 and survivin by flow cytometry were assessed as previously described (Pace et al 2013a) (Chiappara et al, 1832). Briefly, 16HBE cells exposed to the previously described stimuli, were fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature. After two washes in permeabilization buffer (PBS containing 1% FCS, 0.3% saponin, and 0.1% sodium azide) for 5 min at 4°C, the cells were stained with a rabbit polyclonal anti-human survivin antibody (NB500-201; Novus Biologicals. Littleton, CO, USA) followed by a
SIRT1 plays a relevant role in anti-aging processes. The effects of both CSE and CARB on nuclear translocation of SIRT1 was assessed by western blot analysis. CSE in a dose dependent manner reduced nuclear expression of SIRT1 (reduction versus baseline after CSE exposure: 10% = 19.8 ± 10; p b 0.03; 20% = 28.6 ± 11; p b 0.01) and incubation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 16.2 ± 11; p b 0.04 increase versus CSE 20% = 20 ± 6; p b 0.007) (Fig. 3).
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Fig. 1. Effects of CARB on cell proliferation. Bronchial epithelial cells (16HBE) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) and short-term cell proliferation was evaluated with CFSE by flow cytometry (A–B) (n = 5) and long-term cell proliferation was evaluated by clonogenic assay (C) (n = 6). A. Data are expressed as percentage of cells with low CFSE expression (means ± SD). *p b 0.05 (ANOVA). B. Representative histograms were shown. C. Data are expressed as numbers of cell colonies (means ± SD). *p b 0.05 (ANOVA).
3.4. Effects of CSE and CARB on SIRT1 activity in bronchial epithelial cells Oxidants, aldehydes, and cigarette smoke induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. We further assessed the effects of both CSE and CARB on SIRT1 deacetylase activity in bronchial epithelial cells. CSE reduced deacetylase activity of SIRT1, in a dose dependent manner (reduction versus baseline after CSE exposure: 10% = 27.6 ± 9; p b 0.006; 20% = 30.6 ± 9; p b 0.003), and co-stimulation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 14.6 ± 13; p b 0.05 increase versus CSE 20% = 18.2 ± 9; p b 0.01) (Fig. 4A). 3.5. Effects of CSE and CARB on SIRT1/FoxO3 intracellular localization in bronchial epithelial cells SIRT1 deacetylases and activates FoxO3 transcription factor that translocates into the nucleus up-regulating anti-aging genes and down regulating aging genes. To assess SIRT1/FoxO3 intracellular localization, an immunofluorescence approach was used. CSE mainly reduced nuclear SIRT1 localization, and CARB counteracts this phenomenon (Fig. 4B). 3.6. Effects of CSE and CARB on nuclear FoxO3 expression by bronchial epithelial cells The amount of active FoxO is constantly replenished by deacetylation enzymes such as the SIRTs. FoxO3 acetylation may result into loss of DNA binding and nuclear export of FoxO3. Since CSE reduced
SIRT1 deacetylase activity, the effects of CSE on nuclear expression and acetylation of FoxO3 were assessed by western blot analysis. CSE reduced nuclear expression of FoxO3, in a dose dependent manner (reduction versus baseline of CSE 10% = 19.4 ± 14; p b 0.02; reduction versus baseline of CSE 20% = 28 ± 17; p b 0.006), and co-stimulation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 9.5 ± 13; p = 0.05 increase versus CSE 20% = 16 ± 13; p b 0.05) (Fig. 5A–B). CSE increased the acetylation of FoxO3 and CARB counteracted this effect (Fig. 5C). 3.7. Effects of SIRT1 and FoxO3 on survivin expression by bronchial epithelial cells Survivin is essential in protecting cells from entering apoptosis and in controlling cell growth. An age-related accumulation of survivin probably contributes to the apoptosis resistance observed in aged as well as in senescent cells. Since SIRT1 controls FoxO3 activation and FoxO3 positioned on survivin gene promoter blocks survivin gene transcription, the role of both SIRT1 and FoxO3 on survivin was explored. As shown in Fig. 6A, CSE or sirtinol, an inhibitor of SIRT1 activity, induced survivin protein expression. Furthermore, by ChiP assay, it was demonstrated that CSE reduced the localization of FoxO3 on survivin promoter (Fig. 6B). 3.8. Effects of CSE and CARB on survivin expression by bronchial epithelial cells Finally, the effects of both CSE and carbocysteine on the expression of survivin protein were explored by flow cytometry. CSE increased, in
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Fig. 2. Effects of CARB on senescence markers. Bronchial epithelial cells (16HBE) (n = 3) were incubated with CSE (20%) and/or CARB (10−4 M) and were assessed for beta galactosidase activity and p21 expression. A. Beta galactosidase activity was evaluated by beta galactosidase staining (n = 3). One representative experiment was shown. B. p21 expression was evaluated by flow cytometry. Representative histograms were shown.
a dose dependent manner, survivin expression (increase versus baseline of CSE 10% = 13 ± 10; p b 0.01; increase versus baseline of CSE 20% = 26 ± 16; p b 0.002) and the co-stimulation with CARB significantly counteracted this phenomenon (reduction versus CSE 10% = 17 ± 13; p b 0.02 reduction versus CSE 20% = 16 ± 11; p b 0.007) (Fig. 7A–B). The total results of the present study are summarized in Fig. 8. 4. Discussion The present study demonstrates for the first time that carbocysteine, a mucolytic drug with potent anti-oxidant activities, is able to counteract several molecular events associated to cellular senescence in bronchial epithelial cells exposed to cigarette smoke. The used model resembles the effects of cigarette smoke in heavy smokers (Su et al., 1998). In particular, the specific effects on cell proliferation, beta galactosidase activity, SIRT1/FoxO3 axis and cell apoptosis inhibitor proteins (survivin) were evaluated. Targeting lung inflammaging, by using pharmacological SIRT1 activators and an anti-oxidant approach, could be a promising therapeutic intervention for COPD/emphysema (Karrasch et al., 2008). Chronic systemic inflammation characterizes aging and has been correlated with many diseases, most of them age-related. Chronic inflammation and cellular senescence (also called inflammaging) are deeply involved in the pathogenesis of premature lung aging, which is considered as an important contributing factor in driving chronic obstructive pulmonary disease (COPD), a disease whose incidence
increases with age (MacNee, 2011; Karrasch et al., 2008). SIRT1, an antiaging protein, is reduced in lungs of mice exposed to cigarette smoke and in patients with COPD (Yao et al., 2014). In this regard, it has been demonstrated that SIRT1, activating FoxO3 transcription factor, reduces cigarette smoke induced oxidative stress and contributes to its protective effects against lung inflammaging and subsequent development of COPD (Yao et al., 2012). Cellular senescence is a specialized form of growth arrest induced by various stressful stimuli (Qi et al., 2014). Here, we demonstrated that CSE reduced short- and long-term cell proliferation, and incubation with CARB significantly counteracted these CSE effects. Previous works from our laboratory showed that smoking and in vitro treatment with cigarette smoke extracts (CSE) increased reactive oxygen species production in bronchial epithelial cells, which was attenuated by carbocysteine treatment (Pace et al., 2013a, 2013b). Environmental stress, including cigarette smoke, hastens the shriveling of the tips of telomeres and alters the phenotype and the metabolism thus shortening cellular life span and accelerating the process of premature senescence (Beltrami et al., 2011). Replicative senescence is the terminal growth arrest that occurs in most normal human cells after a fixed number of divisions in vitro aimed to prevent genomic instability. Cells undergoing stress induced premature senescence share many cellular and molecular features as those undergoing replicative senescence, but they differ in the aspect of the time at which these features exhibit. Increased expression of p21(Cip1/Waf1) (p21), an inhibitor of the cyclin-dependent kinase contributes to reduce the replicative potential of the senescent cells (Jackson and Pereira-Smith, 2006; Yew et al.,
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Fig. 3. Effects of CARB on SIRT1 nuclear expression. Bronchial epithelial cells (16HBE) (n = 5) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, nuclear proteins were collected and analysed for SIRT1 expression by western blot analysis. Membranes were then stripped and incubated with anti-Lamin B1 antibody. A. Densitometric analysis of SIRT1 expression. Signals corresponding to SIRT1 on the various western blots were semiquantified by densitometric scanning, normalized and expressed after correction with the density of the band obtained for Lamin B1. Data are expressed as arbitrary units ± SD. *p b 0.05 (ANOVA). B. Representative western blot performed on nuclear extracts. Lane1 = baseline; lane 2 = CSE 10%; lane 3 = CSE 20%; lane 4 = CARB (10−4 M); lane 5 = CSE 10% + CARB (10−4 M); lane 6 = CSE 20% + CARB (10−4 M).
2011). Since CSE is able to increase p21 and CARB is able to counteract this phenomenon, it is conceivable that here CARB restored cell proliferation in bronchial epithelial cells exposed to cigarette smoke by downregulating p21 expression. The morphology of senescent cells is characterized by large nucleus, increased cytoplasmic granularity and focal enrichment of lysosome-related β-galactosidase activity (Beltrami et al., 2011). A senescence associated β-galactosidase (SA-β-gal), a biomarker, is detected by histochemical staining of cells using the artificial substrate X-gal. The presence of the SA-β-gal biomarker is present in metabolically active cells and is independent from DNA synthesis thus distinguishing senescent from quiescent cells (Itahana and Campisi, 2007; Dimri et al., 1995). In the present study, CSE exposure increased β-galactosidase staining and incubation with carbocysteine was effective in preventing the expression of β-galactosidase in bronchial epithelial cells. Recent findings suggest a relevant role of SIRT1/FoxO3 pathway in cell senescence. Sirtuins (SIRT1–SIRT7) are NAD+ dependent deacetylases which control a wide number of processes implicated in the regulation of homeostasis (Guarente and Franklin, 2011) and SIRT1 (the best characterized member of sirtuins in mammals) has a key role in governing cellular stress management and, consequently, has a key role in healthy lifespan (Salminen et al., 2013). In particular, SIRT1 regulates inflammation, senescence/aging, stress resistance, and deoxyribonucleic acid (DNA) damage repair via deacetylating intracellular signaling molecules and chromatin histones. SIRT1 overexpression attenuated the increased levels of the senescent markers p21, p16, and p53 in the lung of SIRT1 +/− mice and protected against increase of lung senescence induced by cigarette smoke (Yao et al., 2012). On the other hand, uncontrolled or excessive oxidative stress, alters both the expression and the deacetylase activity of SIRT1 (Caito et al., 2010b). On the basis of these
results we tested the effects of CSE and carbocysteine on SIRT1 expression and activity in bronchial epithelial cells. The results clearly show that CSE reduce the nuclear expression and the deacetylase activity of SIRT1. The incubation of bronchial epithelial cells with carbocysteine is effective in counteracting these CSE mediated effects. Forkhead box class O 3 (FoxO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Deacetylation by SIRT1 has been shown to differentially alter DNA binding affinity and increases deacetylated forms of FoxO3 which in turn favor the expression of antioxidant and cytoprotective genes (Tikhanovich et al., 2013). Targeted disruption of FoxO3 resulted in downregulation of antioxidant genes in mouse lungs in response to cigarette smoke exposure and FoxO3 dys-function greatly contributes to lung emphysema (Hwang et al., 2011). CSE highly reduced the nuclear localization of SIRT1, and co-stimulation with carbocysteine was effective in preventing this CSE-mediated effect in bronchial epithelial cells. Furthermore, since increased FoxO3 acetylation may result in loss of DNA binding and nuclear export of FoxO3 (Tikhanovich et al., 2013), we semi-quantified the nuclear expression of FoxO3 and assessed its acetylation. CSE reduce the nuclear expression and increased the acetylation of FoxO3, and incubation with carbocysteine was effective in counteracting this CSE mediated effect in bronchial epithelial cells. Since senescent cells are characterized by resistance to apoptosis, to get a deeper insight into the molecular consequences of the alteration of SIRT1/FoxO3 pathway in bronchial epithelial cells exposed to cigarette smoke, survivin expression was assessed. Survivin is essential in protecting cells from entering apoptosis (Dohi et al., 2004), in controlling cell growth (Wheatley and McNeish, 2005) and contributes to senescence regulation (Unruhe et al., 2015). FoxO3, deacetylated by
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Fig. 4. Effects of CARB on SIRT1 activity and on SIRT1 and FoxO3 intracellular localization. Bronchial epithelial cells (16HBE) (n = 5) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. A. Nuclear proteins were immuno-precipitated and then assessed for SIRT1 activity. Data are expressed as μM ± SD using as reference a curve of the fluor de Lys deacetylated standard (provided with the kit). *p b 0.05 (ANOVA). B. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%) and/or CARB (10−4 M) for 24 h. SIRT1 and FoxO3 intracellular localization was assessed by immunofluorescence. SIRT1 and FoxO3 expression were identified by red and green fluorescence, respectively. Scale bar = 10 μm; magnification = 630×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
SIRT1, within the nucleus binds to the survivin promoter thus blocking the survivin gene transcription (Obexer et al., 2009). In the present study we showed for the first time that altered activity of the SIRT1/ FoxO3 axis in bronchial epithelial cells leads to increased expression of survivin protein by promoting an increased gene transcription. Accordingly, CSE or an inhibitor of the deacetylase activity of SIRT1 (sirtinol), increased the expression of survivin protein and this CSE mediated effect was associated to reduced localization of FoxO3 on the survivin gene promoter. With regard to the effects promoted by carbocysteine, the incubation with carbocysteine, improving SIRT1 and FoxO3 expression/activity was able to reduce survivin protein expression in bronchial epithelial cells exposed to cigarette smoke. In conclusion, the present study provides compelling data on new effects of carbocysteine in counteracting senescence associated molecular events in bronchial epithelial cells exposed to cigarette smoke. Further studies aimed to assess the effects of carbocysteine on cellular senescence in vivo are therefore necessary. Conflicts of interest Elisabetta Pace - Competing interests: She received institutional research funds from Dompè. This relationship did not influence author's objectivity.
Maria Ferraro - Competing interests: None declared. Di Vincenzo Serena - Competing interests: None declared. Andreina Bruno - Competing interests: None declared. Paola Dino - Competing interests: None declared. Maria Rosaria - Competing interests: None declared. Salvatore Battaglia - Competing interests: None declared. Luigi Lanata - Competing interests: Employed by Dompè who is involved in respiratory research and market drugs for the treatment of COPD, these relationships did not influence author' objectivity. Federico Saibene - Competing interests: Employed by Dompè who is involved in respiratory research and market drugs for the treatment of COPD, these relationships did not influence author' objectivity. Mark Gjomarkaj - Competing interests: None declared.
Acknowledgements This work was supported by the Italian National Research Council and by Dompè. Elisabetta Pace designed the study, performed the statistical analysis of the data, wrote the manuscript and declares that she has had access to and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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Fig. 5. Effects of CARB on nuclear expression and on acetylation of FoxO3. Bronchial epithelial cells (16HBE) (n = 8) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, nuclear proteins were collected and analysed for FoxO3 expression by western blot analysis. Membranes were then stripped and incubated with antiLamin B1 antibody. A. Densitometric analysis of FoxO3 expression. Signals corresponding to FoxO3 on the various western blots were semiquantified by densitometric scanning, normalized and expressed after correction with the density of the band obtained for Lamin B1. Data are expressed as arbitrary units ± SD. *p b 0.05 (ANOVA). B. Representative western blot performed on nuclear extracts. Lane1 = baseline; lane 2 = CSE 10%; lane 3 = CSE 20%; lane 4 = CARB (10−4 M); lane 5 = CSE 10% + CARB (10−4 M); lane 6 = CSE 20% + CARB (10−4 M). C. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, immunoprecipitated (IP) for FoxO3 and then assessed for acetylation (IB). A representative experiment is shown.
Fig. 6. Role of SIRT1 and FoxO3 on Survivin expression. A. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%) or Sirtinol (10 μM) for 24 h. Survivin protein expression was assessed by flow cytometry. Data are expressed as percentage of positive cells. Representative histograms were shown. B. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%). Chip assay using anti FoxO3 antibody and PCR using primers spanning the promoter region of survivin gene (forward 5′ – TGA GCT GAG ATC ATG CCA CT – 3′ and reverse 5′ – CTG GTG CCT CCA CTG TCT TT – 3′). One out of two experiments is shown. Lane 1 = Input (total DNA without immunoprecipitation) baseline; lane 2 = Input CSE 20%; lane 3 = Baseline; lane 4 = CSE 20%.
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Fig. 7. Effects of CARB on survivin expression. Bronchial epithelial cells (16HBE) (n = 8) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Survivin expression was assessed by flow cytometry. A. Data are expressed as percentage of survivin positive cells (means ± SD). *p b 0.05 (ANOVA). B. Representative histograms were shown.
Maria Ferraro, Serena Di Vincenzo, Andreina Bruno performed all the experiments of the study and participated to the interpretation of the data. Paola Dino actively contributed to the experiments and to the activities related to the revision process of the manuscript. Maria Rosaria Bonsignore, Salvatore Battaglia, Federico Saibene and Luigi Lanata contributed to the interpretation of the data. Mark Gjomarkaj contributed to the interpretation of the data and to writing the manuscript.
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Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells E. Pace a,⁎, S. Di Vincenzo a, M. Ferraro a, A. Bruno a, P. Dino a, M.R. Bonsignore b, S. Battaglia b, F. Saibene c, L. Lanata c, M. Gjomarkaj a a b c
Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Palermo, Italy Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S), University of Palermo, Palermo, Italy Dompè Medical Affair, Milan, Italy
a r t i c l e
i n f o
Article history: Received 30 March 2016 Received in revised form 18 May 2016 Accepted 25 May 2016 Available online 26 May 2016 Section Editor: Werner Zwerschke Keywords: Cigarette smoke SIRT1 FoxO3 Carbocysteine Bronchial epithelial cells
a b s t r a c t Background: Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aims: Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. Methods: 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. Results: CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. Conclusions: The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Chronic obstructive pulmonary disease (COPD) is a progressive disease arising from an inflammatory response to irritants, among which cigarette smoke is the most frequent (Sethi and Murphy, 2001; Wilson, 2000, 2001). Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence within the lung, an event characterized by loss of replicative potential, increased beta galactosidase activity, and resistance to cell apoptosis (Beltrami et al., 2011). Cell senescence is believed to play an
Abbreviations: CSE, cigarette smoke extracts; COPD, chronic obstructive pulmonary disease; SIRT1, sirtuin1; FoxO3, Forkhead box O3; CARB, carbocysteine. ⁎ Corresponding author at: Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa, 153, 90146 Palermo, Italy. E-mail address: [email protected] (E. Pace).
http://dx.doi.org/10.1016/j.exger.2016.05.013 0531-5565/© 2016 Elsevier Inc. All rights reserved.
active role in COPD pathogenesis. COPD is considered a disease of the lung inflammaging, which is associated with the DNA damage response, transcription activation and chromatin modifications (MacNee, 2011). Anti-aging sirtuin1 (SIRT1), a NAD +-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD (Rajendrasozhan et al., 2008). SIRT1 protects against emphysema through FoxO3-mediated reduction of cellular senescence (Yao et al., 2012). SIRT1 downregulates inflammaging via regulating also p53, p21, nuclear factor kappa B, histones and a variety of proteins involved in DNA damage and repair (Yao et al., 2012; Atkins et al., 2014). Since incidence of COPD increases with age, chronic inflammation and cellular senescence may be intertwined in the pathogenesis of premature aging (MacNee, 2011). The airway epithelium, besides actively contributing to airway defence mechanisms, exerts an important role in coordinating local inflammation and immune responses (Kato and Schleimer, 2007). It is
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conceivable that when epithelial cells are chronically exposed to cigarette smoke they differentiate in a functional senescent phenotype with a reduced reparative potential and higher pro-inflammatory properties. In this context, secretion of IL-8, a hallmark of senescence-associated secretory phenotype (Hara et al., 2012) and a potent chemokine for neutrophils, is increased within the airways of smokers and of COPD patients and upon TLR4 activation and ERK 1/2 activation in cigarette smoke extracts (CSE) stimulated bronchial epithelial cells in vitro (Pace et al., 2008a). Carbocysteine, an anti-oxidant and mucolytic agent, was shown to reduce the severity and the rate of exacerbations in COPD patients (Zheng et al., 2008). In vitro, carbocysteine counteracted the increase in p21 and IL-8 mRNA and protein in bronchial epithelial cells exposed to CSE (Pace et al., 2013a). It is conceivable that carbocysteine could restore SIRT1/FoxO3 axis in bronchial epithelial cells and counteract some pro-senescence molecular events promoted by cigarette smoke exposure. The aim of this study was to investigate the relationship between SIRT1/FoxO3 axis, cellular senescence, regulation of cell cycle progression and apoptosis mechanisms in response to cigarette smoke in bronchial epithelial cells and to assess the effects of carbocysteine on cigarette smoke-induced molecular events. 2. Materials and methods 2.1. Preparation of cigarette smoke extracts (CSE) Commercial cigarettes (Marlboro) were used in this study. Cigarette smoke solution was prepared as described previously (Pace et al., 2008a). Each cigarette was smoked for 5 min and one cigarette was used per 10 ml of PBS to generate a CSE-PBS solution. The CSE solution was filtered through a 0.22 μm-pore filter to remove bacteria and large particles. The smoke solution was then adjusted to pH 7.4 and used within 30 min of preparation. This solution was considered to be 100% CSE and diluted to obtain the desired concentration in each experiments. The concentration of CSE was calculated spectrophotometrically measuring the OD as previously described at the wavelength of 320 nm. The pattern of absorbance, among different batches, showed very little differences and the mean OD of the different batches was 1.37 ± 0.16. The presence of contaminating LPS in undiluted CSE was assessed by a commercially available kit (Cambrex Corporation, East Rutherfort, New Jersey, USA) and was below the detection limit of 0.1 EU/ml. 2.2. Bronchial epithelial cell cultures 16-HBE cells, an immortalized normal bronchial epithelial cell line, were used in this study. Bronchial epithelial cells were maintained in MEM (Gibco, BRL, Germany), supplemented with 10% fetal calf serum (Euroclone, Milan, Italy). Cell cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37 °C. Cells were cultured in the presence and absence of CSE (10% and 20%) and CARB at 10−4 M (Dompè, Italy) for 24 h. No pre-treatment with CARB was performed in CSE exposed cells. CSE and CARB were simultaneously added to bronchial epithelial cells. The used drug concentration was selected in previous published studies (Pace et al., 2013b). At the end of stimulation, cells, cell extracts, and cell culture supernatants were collected for further evaluations. 2.3. Short term cell proliferation The short term cell proliferation of 16HBE cells exposed to the previously described stimuli was measured using carboxyfluorescein succinimidyl ester (CFSE) labeling assay (Pace et al., 2012a). CFSE is used to fluorescently label live cells and is equally partitioned to daughter cells during division. CSE and carbocysteine stimulated 16HBE were incubated with CFSE (Molecular Probes, Inc. Eugene, OR) (at a final concentration of 5 μM) at 37 ° C for 10 min. Cell proliferation was assessed
after 72 h by flow-cytometry and proliferated cells showed reduced CFSE expression. 2.4. Long term cell proliferation The long term proliferation of 16HBE cells exposed to the previously described stimuli was assessed by clonogenic assay as previously described (Bruno et al., 2014). Using 35-mm well of 6-well plate (Falcon, Becton Dickinson, Franklin Lakes, N.J.) a lower layer was prepared using complete MEM medium in 0.5% agarose. The cells were harvested and seeded (5 × 104) on the upper layer with 0.3% agarose prepared with the same medium as the lower layer, and finally incubated for 21 days at 37 °C in an atmosphere containing 5% CO2. At the end of incubation, colonies were counted under an inverted phase-contrast microscope (Leica, Wetzlar, Germany). The experiments were conducted in triplicate. Colonies were defined as cell aggregates with at least 40 cells. The true number of colonies was calculated as the number of aggregates on the positive control subtracted from the number of colonies on the experimental plates. Results are cell colony numbers. 2.5. Beta galactosidase staining 16HBE cells were exposed to the previously described stimuli and βgalactosidase (SA-βGal) staining was assessed as previously described (Itahana and Campisi, 2007). For senescence-activated β-galactosidase (SA-βGal) staining, we used Senescence-βGal Staining Kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, cells were washed twice with PBS, fixed with 2% formaldehyde/0.2% glutaralde-hyde in PBS for 10 min, washed twice with PBS, and incubated with fresh SA-b-gal staining solution containing 1 mg/ m1 X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in 40 mM citric acid/sodium phosphate buffer (pH 6.0) for 12 h at 37 °C in a CO2 free atmosphere. The SA-b-gal-stained cells obtained were photographed under an inverted microscope (Nikon Eclipse TS100, Nikon, Melville, NY, USA 1009). Briefly, cells were washed twice with PBS, fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 10 min, washed twice with PBS, and incubated with fresh SA-β-gal staining solution containing 1 mg/ ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in 40 mM citric acid/sodium phosphate buffer (pH 6.0) at 37 °C in a CO2 free atmosphere overnight. In the assay, senescence-associated β-galactosidase catalyzes the hydrolysis of X-gal, which results in the accumulation of a distinctive blue color in senescent cells. The images were captured by Leica microscope, Wetzlar, Germany. 2.6. Western blot analysis The expression of SIRT1 and FoxO3 in 16HBE cells exposed to the previously described stimuli was evaluated by western blot analysis as previously described (Pace et al., 2008b). To study SIRT1 and FoxO3 nuclear translocation, the protein extracts were treated to separate the cytoplasmic and nuclear protein fractions by using a commercial kit “NEPER Nuclear and Cytoplasmic Extraction Reagents” following the manufacturer's directions (Thermo Scientific; Waltham, MA). An amount of 30 μg of total proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and blotted onto nitrocellulose membranes. Membranes were incubated with a IgG rabbit polyclonal antibody anti-SIRT1 (1:500; overnight) (sc-15404 - Santa Cruz Biotechnology, Dallas, TX-USA) and a IgG goat polyclonal antibody anti-FoxO3 (1:750; overnight) (sc-9812 - Santa Cruz Biotechnology, Dallas, TX-USA). Then membranes were stripped and incubated with rabbit polyclonal Lamin B1 antibody (#9087, Cell Signaling Technology, Danvers, MA-USA) for assessing quality control of nuclear extracts. Revelation was performed with an enhanced chemiluminescence system (GE Healthcare, Chalfont St. Giles, UK) followed by autoradiography. Negative controls were performed without
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primary antibody or including an isotype control antibody. Data are expressed as densitometric arbitrary units by correction with the density of the bands obtained for Lamin B1. To assess acetylation of FoxO3, the total protein extracts were immunoprecipitated for FoxO3 with an IgG goat polyclonal antibody anti-FoxO3 (2 μg; sc-9812 Santa Cruz Biotechnology), which was added to 300 μg of sample proteins in a final volume of 400 μl, and incubated overnight at 4 °C with gentle shaking. Protein-A/G agarose beads (16 μl; sc-2003 Santa Cruz Biotechnology) were added to each sample and kept for 3 h at 4 °C on a rotating rocker. The beads were washed 3 times with lysis buffer and resuspended in reducing sample buffer for western blot analysis. The immunoprecipitated FoxO3 agarose bead suspension was resolved by SDS-PAGE. To assess FoxO3 acetylation, the membranes of immunoprecipitated FoxO3 were blotted against anti-acetylated lysine (#9441 - Cell Signaling). Revelation was performed with an enhanced chemiluminescence system (GE Healthcare, Chalfont St. Giles, UK) followed by autoradiography. 2.7. Immunoprecipitation and SIRT1 activity SIRT1 activity of 16HBE cells exposed to the previously described stimuli was assessed as previously described (Caito et al., 2010a). SIRT1 was immunoprecipitated from nuclear cell lysates (100 μg) by overnight incubation with anti-SIRT1 antibody at 4 °C and then incubated with protein G-agarose beads (sc-2003 - Santa Cruz Biotechnology, Dallas, TX-USA). Beads were extensively washed with lysis buffer. SIRT1 activity was measured using a commercial kit (Enzo Lifesciences, Farmingdale, NY, USA). An acetylated lysine substrate is incubated with samples with SIRT1 activity. Deacetylation sensitizes the substrate such that treatment with the SIRT1 developer in the second step releases a fluorescent product. The assay was performed exactly as recommended by the manufacturer. Fluorescence was measured in a microplate reader using Ex 355 and Em 460. 2.8. Immunofluorescence 16HBE were incubated with anti-SIRT1 and anti-FoxO3 antibodies followed by FITC and PE conjugated secondary antibodies, respectively. The cells were analysed by fluorescence microscope Axioskop 2 Zeiss microscope (Heidelberg, Germany). 2.9. ChiP assay ChiP analysis was performed using the EZ-ChIP kit (UpstateMillipore Corporate- Billerica, MA) as previously described (Pace et al., 2012b) in 16HBE cells exposed to the previously described stimuli. The crosslinked chromatin were sonicated to lengths spanning 200– 1000 bp. The samples were precleared with 60 μl of Protein G Agarose and then incubated with a IgG goat polyclonal antibody anti-FoxO3 [10 μg] (sc-9812 - Santa Cruz Biotechnology, Dallas, TX-USA). Immunocomplexes were precipitated using Protein G Agarose. After washing, DNA fragments were isolated and purified with columns. PCR was performed using primers for the promoter region of survivin gene: forward 5′ – TGA GCT GAG ATC ATG CCA CT – 3′ and reverse 5′ – CTG GTG CCT CCA CTG TCT TT – 3′.
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Fluorescein Isothiocyanate-conjugated anti rabbit immunoglobulin G or with a mouse anti-human p21 antibody (sc-817; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by FITC-conjugated goat antimouse (DAKO) and then evaluated by flow cytometry. Rabbit or mouse negative control immunoglobulins (DAKO) were used for negative controls. Flow cytometry analyses were performed on a Becton Dickinson FACSCalibur System. Analysis was done on 10,000 acquired events for each sample using cellQuest acquisition and data analysis software (Becton Dickinson). Data are expressed as percentage of positive cells.
2.11. Statistics Data are expressed as mean counts ± standard deviation. Kolmorogov-Smirnov Normality test was initially performed to assess whether parametric analyses of data could be performed. Comparison between different experimental conditions was evaluated by ANOVA post-hoc test. p b 0.05 was accepted as statistically significant.
3. Results 3.1. Effects of CSE and carbocysteine (CARB) on short and long term cell proliferation of bronchial epithelial cells Epithelial cell senescence is associated with loss of replicative potential. Firstly, short-term cell proliferation in bronchial epithelial cells exposed to CSE was assessed by flow cytometry using the CFSE method. CSE reduced, in a dose dependent manner, short-term cell proliferation (reduction versus baseline after CSE exposure: 10% = 25 ± 10; p b 0.006; 20% = 41 ± 4; p b 0.0001); incubation with CARB significantly counteracted this phenomenon in cells exposed to CSE 10% (increase versus CSE 10% = 7.4 ± 4; p b 0.03) but not in cells exposed to CSE 20% (Fig. 1A–B). To further explore the effects of both CSE and CARB on cell proliferation, clonogenic assay, a method used for assessing long-term cell proliferation, was performed. CSE reduced, in a dose dependent manner (reduction versus baseline after CSE exposure: 10% = 27.6 ± 12; p b 0.005; CSE 20% = 61 ± 17; p b 0.001) long-term cell proliferation and incubation with CARB significantly counteracted this phenomenon in cells exposed to both CSE 10% and CSE 20% (increase versus CSE 10% = 25 ± 26; p b 0.04 increase versus CSE 20% = 87 ± 58; p b 0.006) (Fig. 1C).
3.2. Effects of CSE and CARB on beta galactosidase staining of bronchial epithelial cells Increased beta galactosidase staining is considered a gold standard to identify senescent cells. CSE increased beta galactosidase staining and incubation with CARB completely reverted this phenomenon (Fig. 2A). CSE increased p21 expression and CARB counteracted this phenomenon (Fig. 2B).
2.10. Expression of p21 and survivin by flow-cytometry
3.3. Effects of CSE and CARB on nuclear SIRT1 expression by bronchial epithelial cells
The expression of p21 and survivin by flow cytometry were assessed as previously described (Pace et al 2013a) (Chiappara et al, 1832). Briefly, 16HBE cells exposed to the previously described stimuli, were fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature. After two washes in permeabilization buffer (PBS containing 1% FCS, 0.3% saponin, and 0.1% sodium azide) for 5 min at 4°C, the cells were stained with a rabbit polyclonal anti-human survivin antibody (NB500-201; Novus Biologicals. Littleton, CO, USA) followed by a
SIRT1 plays a relevant role in anti-aging processes. The effects of both CSE and CARB on nuclear translocation of SIRT1 was assessed by western blot analysis. CSE in a dose dependent manner reduced nuclear expression of SIRT1 (reduction versus baseline after CSE exposure: 10% = 19.8 ± 10; p b 0.03; 20% = 28.6 ± 11; p b 0.01) and incubation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 16.2 ± 11; p b 0.04 increase versus CSE 20% = 20 ± 6; p b 0.007) (Fig. 3).
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Fig. 1. Effects of CARB on cell proliferation. Bronchial epithelial cells (16HBE) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) and short-term cell proliferation was evaluated with CFSE by flow cytometry (A–B) (n = 5) and long-term cell proliferation was evaluated by clonogenic assay (C) (n = 6). A. Data are expressed as percentage of cells with low CFSE expression (means ± SD). *p b 0.05 (ANOVA). B. Representative histograms were shown. C. Data are expressed as numbers of cell colonies (means ± SD). *p b 0.05 (ANOVA).
3.4. Effects of CSE and CARB on SIRT1 activity in bronchial epithelial cells Oxidants, aldehydes, and cigarette smoke induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. We further assessed the effects of both CSE and CARB on SIRT1 deacetylase activity in bronchial epithelial cells. CSE reduced deacetylase activity of SIRT1, in a dose dependent manner (reduction versus baseline after CSE exposure: 10% = 27.6 ± 9; p b 0.006; 20% = 30.6 ± 9; p b 0.003), and co-stimulation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 14.6 ± 13; p b 0.05 increase versus CSE 20% = 18.2 ± 9; p b 0.01) (Fig. 4A). 3.5. Effects of CSE and CARB on SIRT1/FoxO3 intracellular localization in bronchial epithelial cells SIRT1 deacetylases and activates FoxO3 transcription factor that translocates into the nucleus up-regulating anti-aging genes and down regulating aging genes. To assess SIRT1/FoxO3 intracellular localization, an immunofluorescence approach was used. CSE mainly reduced nuclear SIRT1 localization, and CARB counteracts this phenomenon (Fig. 4B). 3.6. Effects of CSE and CARB on nuclear FoxO3 expression by bronchial epithelial cells The amount of active FoxO is constantly replenished by deacetylation enzymes such as the SIRTs. FoxO3 acetylation may result into loss of DNA binding and nuclear export of FoxO3. Since CSE reduced
SIRT1 deacetylase activity, the effects of CSE on nuclear expression and acetylation of FoxO3 were assessed by western blot analysis. CSE reduced nuclear expression of FoxO3, in a dose dependent manner (reduction versus baseline of CSE 10% = 19.4 ± 14; p b 0.02; reduction versus baseline of CSE 20% = 28 ± 17; p b 0.006), and co-stimulation with CARB significantly counteracted this phenomenon (increase versus CSE 10% = 9.5 ± 13; p = 0.05 increase versus CSE 20% = 16 ± 13; p b 0.05) (Fig. 5A–B). CSE increased the acetylation of FoxO3 and CARB counteracted this effect (Fig. 5C). 3.7. Effects of SIRT1 and FoxO3 on survivin expression by bronchial epithelial cells Survivin is essential in protecting cells from entering apoptosis and in controlling cell growth. An age-related accumulation of survivin probably contributes to the apoptosis resistance observed in aged as well as in senescent cells. Since SIRT1 controls FoxO3 activation and FoxO3 positioned on survivin gene promoter blocks survivin gene transcription, the role of both SIRT1 and FoxO3 on survivin was explored. As shown in Fig. 6A, CSE or sirtinol, an inhibitor of SIRT1 activity, induced survivin protein expression. Furthermore, by ChiP assay, it was demonstrated that CSE reduced the localization of FoxO3 on survivin promoter (Fig. 6B). 3.8. Effects of CSE and CARB on survivin expression by bronchial epithelial cells Finally, the effects of both CSE and carbocysteine on the expression of survivin protein were explored by flow cytometry. CSE increased, in
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Fig. 2. Effects of CARB on senescence markers. Bronchial epithelial cells (16HBE) (n = 3) were incubated with CSE (20%) and/or CARB (10−4 M) and were assessed for beta galactosidase activity and p21 expression. A. Beta galactosidase activity was evaluated by beta galactosidase staining (n = 3). One representative experiment was shown. B. p21 expression was evaluated by flow cytometry. Representative histograms were shown.
a dose dependent manner, survivin expression (increase versus baseline of CSE 10% = 13 ± 10; p b 0.01; increase versus baseline of CSE 20% = 26 ± 16; p b 0.002) and the co-stimulation with CARB significantly counteracted this phenomenon (reduction versus CSE 10% = 17 ± 13; p b 0.02 reduction versus CSE 20% = 16 ± 11; p b 0.007) (Fig. 7A–B). The total results of the present study are summarized in Fig. 8. 4. Discussion The present study demonstrates for the first time that carbocysteine, a mucolytic drug with potent anti-oxidant activities, is able to counteract several molecular events associated to cellular senescence in bronchial epithelial cells exposed to cigarette smoke. The used model resembles the effects of cigarette smoke in heavy smokers (Su et al., 1998). In particular, the specific effects on cell proliferation, beta galactosidase activity, SIRT1/FoxO3 axis and cell apoptosis inhibitor proteins (survivin) were evaluated. Targeting lung inflammaging, by using pharmacological SIRT1 activators and an anti-oxidant approach, could be a promising therapeutic intervention for COPD/emphysema (Karrasch et al., 2008). Chronic systemic inflammation characterizes aging and has been correlated with many diseases, most of them age-related. Chronic inflammation and cellular senescence (also called inflammaging) are deeply involved in the pathogenesis of premature lung aging, which is considered as an important contributing factor in driving chronic obstructive pulmonary disease (COPD), a disease whose incidence
increases with age (MacNee, 2011; Karrasch et al., 2008). SIRT1, an antiaging protein, is reduced in lungs of mice exposed to cigarette smoke and in patients with COPD (Yao et al., 2014). In this regard, it has been demonstrated that SIRT1, activating FoxO3 transcription factor, reduces cigarette smoke induced oxidative stress and contributes to its protective effects against lung inflammaging and subsequent development of COPD (Yao et al., 2012). Cellular senescence is a specialized form of growth arrest induced by various stressful stimuli (Qi et al., 2014). Here, we demonstrated that CSE reduced short- and long-term cell proliferation, and incubation with CARB significantly counteracted these CSE effects. Previous works from our laboratory showed that smoking and in vitro treatment with cigarette smoke extracts (CSE) increased reactive oxygen species production in bronchial epithelial cells, which was attenuated by carbocysteine treatment (Pace et al., 2013a, 2013b). Environmental stress, including cigarette smoke, hastens the shriveling of the tips of telomeres and alters the phenotype and the metabolism thus shortening cellular life span and accelerating the process of premature senescence (Beltrami et al., 2011). Replicative senescence is the terminal growth arrest that occurs in most normal human cells after a fixed number of divisions in vitro aimed to prevent genomic instability. Cells undergoing stress induced premature senescence share many cellular and molecular features as those undergoing replicative senescence, but they differ in the aspect of the time at which these features exhibit. Increased expression of p21(Cip1/Waf1) (p21), an inhibitor of the cyclin-dependent kinase contributes to reduce the replicative potential of the senescent cells (Jackson and Pereira-Smith, 2006; Yew et al.,
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Fig. 3. Effects of CARB on SIRT1 nuclear expression. Bronchial epithelial cells (16HBE) (n = 5) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, nuclear proteins were collected and analysed for SIRT1 expression by western blot analysis. Membranes were then stripped and incubated with anti-Lamin B1 antibody. A. Densitometric analysis of SIRT1 expression. Signals corresponding to SIRT1 on the various western blots were semiquantified by densitometric scanning, normalized and expressed after correction with the density of the band obtained for Lamin B1. Data are expressed as arbitrary units ± SD. *p b 0.05 (ANOVA). B. Representative western blot performed on nuclear extracts. Lane1 = baseline; lane 2 = CSE 10%; lane 3 = CSE 20%; lane 4 = CARB (10−4 M); lane 5 = CSE 10% + CARB (10−4 M); lane 6 = CSE 20% + CARB (10−4 M).
2011). Since CSE is able to increase p21 and CARB is able to counteract this phenomenon, it is conceivable that here CARB restored cell proliferation in bronchial epithelial cells exposed to cigarette smoke by downregulating p21 expression. The morphology of senescent cells is characterized by large nucleus, increased cytoplasmic granularity and focal enrichment of lysosome-related β-galactosidase activity (Beltrami et al., 2011). A senescence associated β-galactosidase (SA-β-gal), a biomarker, is detected by histochemical staining of cells using the artificial substrate X-gal. The presence of the SA-β-gal biomarker is present in metabolically active cells and is independent from DNA synthesis thus distinguishing senescent from quiescent cells (Itahana and Campisi, 2007; Dimri et al., 1995). In the present study, CSE exposure increased β-galactosidase staining and incubation with carbocysteine was effective in preventing the expression of β-galactosidase in bronchial epithelial cells. Recent findings suggest a relevant role of SIRT1/FoxO3 pathway in cell senescence. Sirtuins (SIRT1–SIRT7) are NAD+ dependent deacetylases which control a wide number of processes implicated in the regulation of homeostasis (Guarente and Franklin, 2011) and SIRT1 (the best characterized member of sirtuins in mammals) has a key role in governing cellular stress management and, consequently, has a key role in healthy lifespan (Salminen et al., 2013). In particular, SIRT1 regulates inflammation, senescence/aging, stress resistance, and deoxyribonucleic acid (DNA) damage repair via deacetylating intracellular signaling molecules and chromatin histones. SIRT1 overexpression attenuated the increased levels of the senescent markers p21, p16, and p53 in the lung of SIRT1 +/− mice and protected against increase of lung senescence induced by cigarette smoke (Yao et al., 2012). On the other hand, uncontrolled or excessive oxidative stress, alters both the expression and the deacetylase activity of SIRT1 (Caito et al., 2010b). On the basis of these
results we tested the effects of CSE and carbocysteine on SIRT1 expression and activity in bronchial epithelial cells. The results clearly show that CSE reduce the nuclear expression and the deacetylase activity of SIRT1. The incubation of bronchial epithelial cells with carbocysteine is effective in counteracting these CSE mediated effects. Forkhead box class O 3 (FoxO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Deacetylation by SIRT1 has been shown to differentially alter DNA binding affinity and increases deacetylated forms of FoxO3 which in turn favor the expression of antioxidant and cytoprotective genes (Tikhanovich et al., 2013). Targeted disruption of FoxO3 resulted in downregulation of antioxidant genes in mouse lungs in response to cigarette smoke exposure and FoxO3 dys-function greatly contributes to lung emphysema (Hwang et al., 2011). CSE highly reduced the nuclear localization of SIRT1, and co-stimulation with carbocysteine was effective in preventing this CSE-mediated effect in bronchial epithelial cells. Furthermore, since increased FoxO3 acetylation may result in loss of DNA binding and nuclear export of FoxO3 (Tikhanovich et al., 2013), we semi-quantified the nuclear expression of FoxO3 and assessed its acetylation. CSE reduce the nuclear expression and increased the acetylation of FoxO3, and incubation with carbocysteine was effective in counteracting this CSE mediated effect in bronchial epithelial cells. Since senescent cells are characterized by resistance to apoptosis, to get a deeper insight into the molecular consequences of the alteration of SIRT1/FoxO3 pathway in bronchial epithelial cells exposed to cigarette smoke, survivin expression was assessed. Survivin is essential in protecting cells from entering apoptosis (Dohi et al., 2004), in controlling cell growth (Wheatley and McNeish, 2005) and contributes to senescence regulation (Unruhe et al., 2015). FoxO3, deacetylated by
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Fig. 4. Effects of CARB on SIRT1 activity and on SIRT1 and FoxO3 intracellular localization. Bronchial epithelial cells (16HBE) (n = 5) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. A. Nuclear proteins were immuno-precipitated and then assessed for SIRT1 activity. Data are expressed as μM ± SD using as reference a curve of the fluor de Lys deacetylated standard (provided with the kit). *p b 0.05 (ANOVA). B. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%) and/or CARB (10−4 M) for 24 h. SIRT1 and FoxO3 intracellular localization was assessed by immunofluorescence. SIRT1 and FoxO3 expression were identified by red and green fluorescence, respectively. Scale bar = 10 μm; magnification = 630×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
SIRT1, within the nucleus binds to the survivin promoter thus blocking the survivin gene transcription (Obexer et al., 2009). In the present study we showed for the first time that altered activity of the SIRT1/ FoxO3 axis in bronchial epithelial cells leads to increased expression of survivin protein by promoting an increased gene transcription. Accordingly, CSE or an inhibitor of the deacetylase activity of SIRT1 (sirtinol), increased the expression of survivin protein and this CSE mediated effect was associated to reduced localization of FoxO3 on the survivin gene promoter. With regard to the effects promoted by carbocysteine, the incubation with carbocysteine, improving SIRT1 and FoxO3 expression/activity was able to reduce survivin protein expression in bronchial epithelial cells exposed to cigarette smoke. In conclusion, the present study provides compelling data on new effects of carbocysteine in counteracting senescence associated molecular events in bronchial epithelial cells exposed to cigarette smoke. Further studies aimed to assess the effects of carbocysteine on cellular senescence in vivo are therefore necessary. Conflicts of interest Elisabetta Pace - Competing interests: She received institutional research funds from Dompè. This relationship did not influence author's objectivity.
Maria Ferraro - Competing interests: None declared. Di Vincenzo Serena - Competing interests: None declared. Andreina Bruno - Competing interests: None declared. Paola Dino - Competing interests: None declared. Maria Rosaria - Competing interests: None declared. Salvatore Battaglia - Competing interests: None declared. Luigi Lanata - Competing interests: Employed by Dompè who is involved in respiratory research and market drugs for the treatment of COPD, these relationships did not influence author' objectivity. Federico Saibene - Competing interests: Employed by Dompè who is involved in respiratory research and market drugs for the treatment of COPD, these relationships did not influence author' objectivity. Mark Gjomarkaj - Competing interests: None declared.
Acknowledgements This work was supported by the Italian National Research Council and by Dompè. Elisabetta Pace designed the study, performed the statistical analysis of the data, wrote the manuscript and declares that she has had access to and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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Fig. 5. Effects of CARB on nuclear expression and on acetylation of FoxO3. Bronchial epithelial cells (16HBE) (n = 8) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, nuclear proteins were collected and analysed for FoxO3 expression by western blot analysis. Membranes were then stripped and incubated with antiLamin B1 antibody. A. Densitometric analysis of FoxO3 expression. Signals corresponding to FoxO3 on the various western blots were semiquantified by densitometric scanning, normalized and expressed after correction with the density of the band obtained for Lamin B1. Data are expressed as arbitrary units ± SD. *p b 0.05 (ANOVA). B. Representative western blot performed on nuclear extracts. Lane1 = baseline; lane 2 = CSE 10%; lane 3 = CSE 20%; lane 4 = CARB (10−4 M); lane 5 = CSE 10% + CARB (10−4 M); lane 6 = CSE 20% + CARB (10−4 M). C. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Total proteins were extracted, immunoprecipitated (IP) for FoxO3 and then assessed for acetylation (IB). A representative experiment is shown.
Fig. 6. Role of SIRT1 and FoxO3 on Survivin expression. A. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%) or Sirtinol (10 μM) for 24 h. Survivin protein expression was assessed by flow cytometry. Data are expressed as percentage of positive cells. Representative histograms were shown. B. Bronchial epithelial cells (16HBE) (n = 2) were incubated with CSE (20%). Chip assay using anti FoxO3 antibody and PCR using primers spanning the promoter region of survivin gene (forward 5′ – TGA GCT GAG ATC ATG CCA CT – 3′ and reverse 5′ – CTG GTG CCT CCA CTG TCT TT – 3′). One out of two experiments is shown. Lane 1 = Input (total DNA without immunoprecipitation) baseline; lane 2 = Input CSE 20%; lane 3 = Baseline; lane 4 = CSE 20%.
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Fig. 7. Effects of CARB on survivin expression. Bronchial epithelial cells (16HBE) (n = 8) were incubated with CSE (10% and 20%) and/or CARB (10−4 M) for 24 h. Survivin expression was assessed by flow cytometry. A. Data are expressed as percentage of survivin positive cells (means ± SD). *p b 0.05 (ANOVA). B. Representative histograms were shown.
Maria Ferraro, Serena Di Vincenzo, Andreina Bruno performed all the experiments of the study and participated to the interpretation of the data. Paola Dino actively contributed to the experiments and to the activities related to the revision process of the manuscript. Maria Rosaria Bonsignore, Salvatore Battaglia, Federico Saibene and Luigi Lanata contributed to the interpretation of the data. Mark Gjomarkaj contributed to the interpretation of the data and to writing the manuscript.
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