In the article titled “Resveratrol Attenuates Copper-Induced Senescence by Improving Cellular Proteostasis” [
Copper sulfate-induced premature senescence (CuSO4-SIPS) consistently mimics molecular mechanisms of replicative senescence, particularly at the endoplasmic reticulum proteostasis level. In fact, disruption of protein homeostasis has been associated with age-related cell/tissue dysfunction and susceptibility to human disorders. Resveratrol is a polyphenolic compound with proven antiaging properties under certain conditions. In this setting, we aimed to evaluate the ability of resveratrol to attenuate cellular senescence induction and to unravel related molecular mechanisms. Resveratrol attenuated typical senescence-induced alterations to cell morphology, senescence-associated beta-galactosidase activity, and cell proliferation in CuSO4-SIPS WI-38 fibroblasts. The mechanisms implicated in this antisenescence effect seem to be independent of the regulation of senescence-associated genes and proteins but are reliant on cellular proteostasis improvement. In fact, resveratrol supplementation restores copper-induced increases in protein content, attenuates immunoglobulin-binding protein levels, and reduces carbonylated and polyubiquitinated proteins by inducing autophagy. Our data provide compelling evidence for the beneficial effects of resveratrol by mitigating stressful consequences associated with CuSO4-SIPS via modulation of protein quality control systems. These findings highlight the importance of balanced cellular proteostasis and add further knowledge regarding molecular mechanisms mediating the antisenescence effects of resveratrol. Moreover, they contribute to the identification of specific molecular targets whose modulation may prevent age-associated cell dysfunction and improve human healthspan.
Normal somatic dividing cells have been proven to be a valuable in vitro model to study cellular senescence and unravel molecular mechanisms and pathways implicated in the human aging process. The well-known model of replicative senescence (RS) is achieved when human diploid fibroblasts (HDFs) spontaneously stop dividing after an initial active period of population doublings (PDs) and become unresponsive to mitogenic stimuli [1]. In addition to irreversible cell cycle arrest, RS fibroblasts exhibit other typical, morphological, and molecular features, such as increased cellular volume, higher senescence-associated beta-galactosidase (SA beta-gal) activity, and increased expression of senescence-associated genes and proteins [2, 3]. A similar senescent phenotype, termed stress-induced premature senescence (SIPS), can be attained by exposing HDFs to subcytotoxic doses of oxidative stress inducers, such as hydrogen peroxide (H2O2-SIPS) [4], tert-butyl hydroperoxide, ultraviolet B radiation [3], or copper sulfate (CuSO4-SIPS) [5]. Recently, the latter was shown to mimic the RS model better than the most frequently used H2O2-SIPS model [6].
Resveratrol is a natural polyphenolic compound that has been shown to increase the maximum lifespan of several organisms, such as
At the cellular level, resveratrol has been shown to attenuate senescent features in both RS [12] and H2O2-SIPS [13, 14] cellular models. These antiaging effects have long been associated with the ability of resveratrol to activate sirtuin 1 (Sirt1) deacetylase [15]. Further, it has been demonstrated that Sirt1 overexpression attenuates senescence and extends the replicative lifespan of several cultured cell types [16–18], while its inhibition results in increased cellular senescence [16]. Downregulation of Sirt1 has been associated with aging [19] and has been observed in cellular senescence models [20, 21], further demonstrating its preventive role in the features of senescence. Besides the ability of resveratrol to modulate signal transduction pathways via activation of Sirt1 [14, 22], several other biological events have been assigned to be responsible for its positive effects, including its ability to increase stress resistance [12], induce telomerase activity [23], decrease the secretion of senescence-associated proinflammatory proteins [24], and inhibit the mechanistic target of rapamycin (mTOR) [13]. Resveratrol has also been found to modulate protein quality control cellular responses, as it regulates the expression of heat shock molecular chaperones [25] and promotes cellular protein degradation mechanisms, namely, the ubiquitin-proteasome system (UPS) [26, 27] and lysosomal autophagy [28, 29]. Moreover, resveratrol was able to increase the lifespan of
In the present study, we aimed to evaluate the ability of resveratrol to attenuate the establishment of cellular senescence upon CuSO4 induction, unraveling the molecular mechanisms that might be involved. We found that resveratrol supplementation was able to reduce the appearance of some senescence-associated features by improving cellular proteostasis, likely via prevention of oxidative damage to proteins and the induction of protein degradation mechanisms, which prevent the accumulation of damaged proteins.
WI-38 human fetal lung fibroblasts were purchased from The European Collection of Cell Cultures (ECACC) and were cultivated in complete Basal Medium Eagle (BME) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 humidified atmosphere. WI-38 cells are young, with less than 30 PDs, and enter senescence at 45 PDs or above. For the induction of CuSO4-SIPS, subconfluent young WI-38 fibroblasts were exposed to 350
Cell morphology evaluation was performed 72 h after copper removal via optical inspection with an inverted microscope. To assess the presence of senescent cells, SA beta-gal was detected 72 h after copper removal as previously described [5]. The percentage of SA beta-gal-positive cells in each condition was determined by microscopically counting 400 total cells/well from at least three independent experiments.
To assess the effects of the different treatments on cell proliferation and total protein content, cell numbers were determined and a sulforhodamine B (SRB) assay [30] was performed over time after copper removal. Briefly, 3000 cells/well were seeded in 96-well culture plates, treated for 24 h with CuSO4 (or Na2SO4 for controls), and then analyzed at different time points (0, 24, 48, and 72 h) while recovering in the presence or absence of resveratrol. For cell number determination, cells were trypsinized and stained with trypan blue; viable cells were microscopically counted in a Neubauer chamber. The total number of cells per well for each condition at the different time points was calculated and plotted; at
Protein levels were assessed 72 h after CuSO4 exposure by western blot analysis. WI-38 cells exposed to the different treatments were washed with PBS and scraped on ice in a lysis buffer (10 mM Tris, pH 7.4; 100 mM NaCl; 1 mM EDTA; and 0.1% Triton X-100) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). After the Bradford assay was conducted, 20
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Previously, it was demonstrated that Sirt1 expression decreases with increasing PDs [20] and in H2O2-SIPS cellular models [21]. Here, Sirt1 mRNA and protein levels were evaluated by qPCR and western blot, respectively, in CuSO4-induced senescent WI-38 fibroblasts. Similarly to other RS and SIPS models, gene (Figure
Reduced sirtuin 1 (Sirt1) expression in copper sulfate stress-induced premature senescent (CuSO4-SIPS) fibroblasts is restored by the addition of resveratrol. (a-b) WI-38 fibroblasts were incubated with 350
Senescent cells usually present typical morphological alterations, increased levels of SA beta-gal, and irreversible inhibition of cell proliferation. Therefore, these three features were evaluated to assess the effects of resveratrol in CuSO4-SIPS fibroblasts. Briefly, cell proliferation was assessed by counting viable cells 0, 24, 48, and 72 h after copper removal. Cell morphology was observed at the final time point (72 h), and the percentage of SA beta-gal-positive cells was quantified for each condition. As shown in Figure
Resveratrol attenuates the appearance of typical senescence-associated features induced by CuSO4. (a) Cell morphology was evaluated 72 h after the removal of 350
Similar to previously reported results [6], 34% of cells were positive for SA beta-gal in the CuSO4-SIPS cellular model (Figure
There are several genes and proteins, such as p21, ApoJ, and TGF
Resveratrol supplementation does not affect copper-induced expression of senescence-associated molecules. (a) Transcript-relative levels of cyclin-dependent kinase inhibitor 1A (p21), apolipoprotein J (ApoJ), and transforming growth factor beta 1 (TGF
A proteostasis imbalance is a major hallmark of aging [32] and has been demonstrated at the cellular level by increased intracellular protein content [33]. To measure cellular protein accumulation for each experimental condition, the ratio between total protein content and cell number, here defined as the protein load index (PLI), was calculated 0, 24, 48, and 72 h after CuSO4 removal (sodium sulfate for controls). Assuming that the PLI equals 1 immediately after stress removal, PLI values were 1.7-fold higher in CuSO4-SIPS cells at 48 and 72 h than in the respective control cells (Figure
Imbalance in CuSO4-induced proteostasis is attenuated by resveratrol. The protein load index (PLI), used as a measure of cellular protein accumulation, was calculated as the ratio between total protein content and cell number for each condition at different time points after CuSO4 treatment (0, 24, 48, and 72 h). PLI values were normalized to the initial time point (0 h), and the relative values are plotted for the indicated conditions. Data are presented as means ± SEM of at least three independent experiments.
To compensate for the altered proteostasis, CuSO4-SIPS cells present higher levels of p-eIF2 [6], which inhibits general protein translation and allows cells to restore homeostasis. A possible explanation for the diminished PLI obtained for copper-treated cells recovering in the presence of resveratrol could be an increase in the inhibition of overall protein synthesis caused by higher p-eIF2. As expected, p-eIF2 levels were higher in CuSO4-treated cells than in control cells, as determined by western blot (Figure
Resveratrol attenuates copper-induced immunoglobulin-binding protein (BiP) upregulation but has no effect on eukaryotic translation initiation factor 2 (eIF2) phosphorylation or heat shock protein 90 (HSP90) and HSP70 expression. (a) Phosphorylated eIF2 (p-eIF2) and (b) BiP, HSP90, and HSP70 relative protein levels were determined by western blot 72 h after the removal of 350
The altered proteostasis observed in CuSO4-SIPS fibroblasts could be a consequence of a progressive accumulation of oxidatively modified proteins. Protein carbonylation is a type of irreversible protein oxidation that frequently serves as an indicator of increased permanent levels of oxidative stress. Moreover, cellular senescence models [34] and cells treated with oxidative stress inducers [35] were both shown to exhibit increased levels of carbonylated proteins. Herein, carbonyl protein content was evaluated to infer the cellular oxidative status under the different experimental conditions. CuSO4-SIPS cells showed a 13% increase (
CuSO4-induced accumulation of carbonylated and polyubiquitinated proteins is reduced by resveratrol via induction of lysosomal autophagy. (a) Protein carbonyl content and (b) polyubiquitinated (poly-Ub) proteins were evaluated in fibroblasts exposed to the indicated conditions by western blot. Representative blots are depicted and densitometric quantification was normalized by assigning a value of 1 to the control cells in the absence of resveratrol. Ponceau S staining was used to account for differences in protein loading. (c) Lysosomal autophagy was studied by measuring the conversion of LC3-I to LC3-II, a critical step for autophagosome formation, and quantification of P62, a ubiquitin-binding protein that targets Ub substrates to autophagosomes. The LC3-II/LC3-I ratio and relative levels of P62 were evaluated via densitometric quantification and plotted with the assumption that control cells without resveratrol represent a value of 1. Data are presented as means ± SEM of at least three independent experiments.
Depending on the conformation of their polyubiquitin chains, poly-Ub proteins may be degraded either in the proteasome or by lysosomal macroautophagy [36] (termed autophagy from now on for simplicity). Autophagy plays a crucial role in the recycling of dysfunctional organelles and damaged protein aggregates, and it was shown to be induced by resveratrol in order to prevent cellular damage from oxidative stress [28, 29]. In the present study, the induction of autophagy was evaluated by calculating the ratio of LC3-II/LC3-I proteins via western blot, which represents the conversion of LC3-I to LC3-II, an essential step for autophagosome formation. Furthermore, the level of P62 protein, a ubiquitin-binding protein that serves as a link between LC3 and Ub substrates during autophagosome formation, was also evaluated by western blot (Figure
The CuSO4-SIPS cellular model has proven to have major value for studying molecular events that are responsible for the aging process [5, 6, 37]. Furthermore, it provides additional evidence supporting the contribution of copper to age-related functional deterioration and the progression of age-related disorders. The present study shows that CuSO4-induced cell senescence results in reduced Sirt1 expression. As Sirt1 is activated by the polyphenolic compound, resveratrol, the mechanisms and possibility of attenuating this senescent effect via Sirt1 were addressed. We demonstrated that resveratrol supplementation attenuates the copper-induced appearance of some typical features of senescence. In addition, the mechanisms underlying such antisenescence effects of resveratrol involve the modulation of cellular proteostasis, via either protection of proteins from oxidative damage or the induction of protein degradation processes.
The effects of resveratrol on cellular senescence have been investigated; however, the results are contradictory. Specifically, some authors have reported the ability of resveratrol to attenuate cellular aging [12–14], whereas others have shown that it induces senescence [38–41]. In either case, the molecular mechanisms involved in such effects are not fully clear. We believe that these discrepancies can be attributed to the different experimental conditions utilized in these studies. The ability of resveratrol to induce cell senescence is often reported in studies using tumor cell lines [38–40] treated with high concentrations of the compound (above 25
Recently, it was reported that both RS and CuSO4-SIPS models exhibit altered expression of several ER molecular chaperones and enzymes and activated ER UPR pathways [6]. Here, CuSO4-SIPS fibroblasts exhibited greater total protein content, as determined by augmented PLI values; increased expression of BiP, HSP70, and HSP90 molecular chaperones; a rise in the levels of carbonylated proteins; and more poly-Ub proteins, adding further evidence to support the occurrence of proteostasis disruption during senescence. Nevertheless, our hypothesis that increased PLI values reflect impaired proteostasis could be further supported by additional experimental evidence such as the inhibition of protein degradation mechanisms, including autophagy or UPS. At present, the underlying molecular conditions that trigger increases in PLI values are still unknown; however, the typical enlargement of the cell, which is associated with the senescence phenotype, or other mechanisms apart from proteostasis disruption cannot be excluded. CuSO4-SIPS fibroblasts that were allowed to recover in the presence of resveratrol showed improved cellular proteostasis, as their total protein levels were similar to those in controls, BiP chaperone expression was attenuated, and poly-Ub protein levels were reduced. Altogether, these data demonstrate that, in the presence of resveratrol, cells can circumvent copper-induced disruption of cellular proteostasis, which is intimately related to the appearance of the typical senescent phenotype.
The well-documented antioxidant properties of resveratrol are the likely contributors to the cell proteostasis-maintenance effect reported here, as resveratrol is known to protect proteins from becoming oxidized in a concentration- and time-dependent manner. In fact, using in vitro oxidative-stressed erythrocytes, resveratrol prevented protein oxidation, reaching a maximum protective effect between 30 and 60 min after its addition; this phenomenon was slightly reduced over time [42]. In the current study, resveratrol supplementation for 72 h attenuated the amount of carbonylated proteins in copper-treated cells, an effect that was close to reaching statistical significance. A time-course evaluation of protein carbonylation for 72 h would add further information regarding the existence of time-dependent variations in the ability of resveratrol to protect proteins from oxidation.
Another important resveratrol contribution for the modulation of cellular proteostasis is its ability to regulate protein degradation mechanisms, such as UPS [26, 27] or lysosomal autophagy [28, 29]. Both mechanisms have been shown to be intimately related, as autophagy is activated to compensate for UPS inhibition [43]. In brief, autophagy is crucial for degrading dysfunctional organelles and damaged protein aggregates and involves the formation of autophagosomes that are targeted to lysosomes for the degradation of their inner content. Autophagosome formation occurs in successive stages that depend on the concerted action of several proteins [44]. The cytosolic soluble protein, LC3-I, is particularly important in this process because it is lipidated to form LC3-II, which integrates the autophagosome membrane. As such, conversion is essential for elongation and maturation of autophagosomes; the LC3-II/LC3-I ratio is usually used to detect autophagy activation. In addition, the P62 protein is crucial for targeting poly-Ub substrates to autophagosomes via LC3 binding [44], and its detection further indicates such activation. Here, CuSO4-SIPS cells exhibited an increase in LC3-I to LC3-II conversion and P62 protein levels; when they were allowed to recover in the presence of resveratrol, the LC3-II/LC3-I ratio and P62 protein levels were even higher, indicating an enhanced induction of autophagy and targeting of poly-Ub substrates to the autophagosome. These results agree with previous in vitro [45] and in vivo [28] studies demonstrating that oxidative stress promotes increases in the LC3-II/LC3-I ratio, which is further enhanced in the presence of resveratrol. Moreover, it was recently shown that resveratrol promotes the flux of proteins through the autophagosomal-lysosomal pathway, thus attenuating the dysfunctional effects caused by the intracellular accumulation of damaged or defective proteins [27]. These results are concordant with those of the current study, favoring resveratrol antisenescence effects because of improved cellular proteostasis via autophagy induction. However, the present study has some limitations regarding the actual induction of autophagy by resveratrol, and further functional studies monitoring autophagosome number and the autophagic flux [46] in the presence of resveratrol would clarify its effect on such processes. Moreover, given the proven crosstalk between autophagy and proteasomal degradation [47], we cannot exclude the beneficial effects resulting from the ability of resveratrol to modulate UPS.
This study demonstrates that resveratrol attenuates the induction of cell senescence resulting from CuSO4 exposure. Such effects result from the ability of resveratrol to promote cellular adaptive mechanisms, such as autophagy upregulation, which sustain cellular proteostasis and confer cellular resistance to stress. Cellular proteostasis maintenance was found to be crucial to prevent the development of senescent phenotypes. These data also uncover molecular targets, the modulation of which likely prevents age-associated cell and tissue functional deterioration and improves human healthspan.
The authors declare that they have no conflicts of interest.
Liliana Matos was supported by a Ph.D. fellowship SFRH/BD/61820/2009 from Programa Operacional Ciência e Inovação 2010, Fundo Social Europeu, and Fundação para a Ciência e Tecnologia.