Overexpression of SIRT6 in porcine fetal fibroblasts attenuates cytotoxicity and premature senescence caused by D-galactose and tert-butylhydroperoxide

SIRT6, a member of the yeast silent information regulator 2 (SIR2) family, possesses both robust ADP-ribosyltransferase activity and protein deacetylase activity depending on NAD(+). It has been shown to maintain genomic stability and telomere integrity, and to prevent age-related disorders and premature ageing. However, the mechanism by which SIRT6 overexpression affects cellular ageing is not well understood. In this study, we investigated the effect of SIRT6 overexpression on cytotoxicity and ageing syndromes. A full-length cDNA of porcine SIRT6 was inserted into pcDNA3.1(-) and subsequently transfected into porcine fetal fibroblasts (PFFs). Overexpression of SIRT6 was identified by quantitative real-time polymerase chain reaction and western blot assay. The cells were incubated with D-galactose and tert-butylhydroperoxide at their cytotoxic concentrations. The damage caused by the stresses was detected with fluorescence microscopy using 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder analysis, and observation under transmission electron microscopy. The results showed that SIRT6 overexpression in cells decreased damage to the nuclei. It also protected against the generation of DNA fragmentation and damage in the ultramicrostructure of the cells, especially damage to mitochondria. Our observations suggested that one function of SIRT6 in PFFs was to dampen cytotoxicity, and, therefore, to decrease the damage that causes premature senescence.     

Cardiolipin induces premature senescence in normal human fibroblasts

Lipids seem to have various roles in cellular senescence. We found that cardiolipin very sensitively inhibits growth of normal human fibroblasts, whereas other phospholipids do not at 100 times higher concentrations. Growth arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. Senescence markers such as the p21(waf1/sdi-1), fibronectin, and collagenase-I genes were significantly upregulated by cardiolipin. In addition, caldiolipin significantly increased in normally senesced human fibroblasts leaving other phospholipids unaltered. These results suggest that accumulation of cardiolipin is one of the causes for replicative senescence.     

Inonotus obliquus protects against oxidative stress-induced apoptosis and premature senescence

In this study, we investigated the cytoprotective effects of Inonotus obliquus against oxidative stress-induced apoptosis and premature senescence. Pretreatment with I. obliquus scavenged intracellular ROS and prevented lipid peroxidation in hydrogen peroxide-treated human fibroblasts. As a result, I. obliquus exerted protective effects against hydrogen peroxide-induced apoptosis and premature senescence in human fibroblasts. In addition, I. obliquus suppressed UV-induced morphologic skin changes, such as skin thickening and wrinkle formation, in hairless mice in vivo and increased collagen synthesis through inhibition of MMP-1 and MMP-9 activities in hydrogen peroxide-treated human fibroblasts. Taken together, these results demonstrate that I. obliquus can prevent the aging process by attenuating oxidative stress in a model of stress-induced premature senescence.     

UVB-induced premature senescence of human diploid skin fibroblasts

In this work, we show that repeated stresses with UVB (290-320 nm) induce stress-induced premature senescence (SIPS) of skin human diploid fibroblasts (HDFs). HDFs at early cumulative population doublings were exposed three or five times to increasing subcytotoxic doses of UVB with one stress per day. After 2 days of recovery, several biomarkers of replicative senescence were established. First, there was an increase in the proportion of cells positive for senescence-associated beta-galactosidase activity. Second, there was a loss of replicative potential as assessed by a very low level of [3H]-thymidine incorporation. Third, the steady-state level of the mRNA of three senescence-associated genes, i.e. fibronectin, osteonectin and SM22, was increased in HDFs at 72 h after three and five exposures to UVB. In conclusion, these results suggest that it is possible to induce SIPS in HDFs after repeated exposures to subcytotoxic doses of UVB. This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in skin, as shown recently with HDFs in H(2)O(2)-induced premature senescence.     

Autophagy impairment induces premature senescence in primary human fibroblasts

Background

Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells.

Methodology/Principal Findings

Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways.

Conclusion

Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts.     

Schisandrin B protects against tert-butylhydroperoxide induced cerebral toxicity by enhancing glutathione antioxidant status in mouse brain

Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from Fructus Schisandrae, has been shown to produce antioxidant effect on rodent liver and heart. A mouse model of tert-butylhydroperoxide (t-BHP) induced cerebral toxicity was adopted for examining the antioxidant potential of Sch B in the brain. Intracerebroventricular injection of t-BHP caused a time-dependent increase in mortality rate in mice. The t-BHP toxicity was associated with an increase in the extent of cerebral lipid peroxidation and an impairment in cerebral glutathione antioxidant status, as evidenced by the abrupt decrease in reduced glutathione (GSH) level and the inhibition of Se-glutathione peroxidase activity at 5 min following t-BHP challenge. Sch B pretreatment (1 or 2 mmol/kg/day × 3) produced a dose-dependent protection against t-BHP induced mortality. The protection was associated with a decrease in the extent of lipid peroxidation and an enhancement in glutathione antioxidant status in brain tissue detectable at 5 min post t-BHP challenge, with the assessed biochemical parameters being returned to normal values at 60 min in Sch B pretreated mice at a dose of 2 mmol/kg. The ensemble of results suggests the antioxidant potential of Sch B pretreatment in protecting against cerebral oxidative stress.     

Stress-induced premature senescence in BJ and hTERT-BJ1 human foreskin fibroblasts

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric DNA-binding complex of the subunits alpha and beta with relevance in O(2) and energy homeostasis. The labile component, HIF-1alpha, is not only activated by hypoxia but also by peptides such as insulin and interleukin-1 (IL-1) in normoxia. We investigated whether inhibitors of mitogen-activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3-kinase (PI3K: LY 294002) do not only lower the hypoxia-induced, but also the insulin- and IL-1-induced HIF-1alpha accumulation and HIF-1 DNA-binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF-1 activation in a dose-dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF-1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF-1alpha levels at non-toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF-1 but it is also crucially involved in the response to insulin and IL-1.     

Suppression of thioredoxin-1 induces premature senescence in normal human fibroblasts

Thioredoxin (TRX) is a ubiquitous multifunctional thiol protein that is critically involved in maintaining cellular redox homeostasis. Levels of thioredoxin-1 (TRX1), the major isoform of TRX, have been shown to correlate with organismal lifespan and age-associated tissue deterioration. Accordingly, we investigated the direct functional effects of suppressing TRX1 levels on cellular senescence, a phenomenon intimately linked with tissue degeneration and aging. Here we find that suppression of TRX1 expression via shRNA rapidly induces premature senescence in young human skin fibroblasts through upregulation of the p53/p21^Cip1/Waf1 and p16^INK4a tumor suppressor pathways. Moreover, inhibition of these pathways by introduction of SV40 Large T Antigen prevents TRX1 suppression-induced premature senescence but not susceptibility to oxidative stressors. Thus our results suggest that TRX1 has a role in suppressing senescence in normal cells in addition to its function as a redox-protective protein.     

Zinc protects HeLa-tat cells against free radical cytotoxicity induced by glucose

In the present study, we investigated the protective effect of zinc on the glucose-induced cytotoxicity in HeLa wild and HeLa-tat cells (30 and 20 mmol/l glucose, respectively). HeLa cells transfected with the protein Tat exhibit a lower antioxidant defense system. Incubation of HeLa wild and HeLa-tat cells with high glucose levels led to a rapid increase in generation of reactive oxygen species (ROS). As expected in the presence of high glucose concentrations, the viability was reduced for both cell lines. The redox status essentially regulated by thiol groups may play an important role in the apoptotic process. Thus, we developed a new method using the p-nitrophenyl disulfide to measure cytosolic thiol groups in intact cells. Cellular zinc was measured using inductively coupled plasma mass spectrometry. Intracellular thiol groups and intracellular zinc concentrations were significantly lower in HeLa cells cultured in hyperglycemic conditions, and their concentrations were significantly lower in HeLa-tat cells than in HeLa wild cells. However, the generation of ROS and the induction of apoptosis by a glucose specific mechanism were prevented by zinc (50 micromol/l) and the intracellular thiol groups and zinc concentrations significantly increased in both cell lines to become similar to the initial values. These results suggest that the glucose oxidation and its subsequent effects on the cells can be prevented by a biological antioxidant such as zinc.     

Nuclear swelling occurs during premature senescence mediated by MAP kinases in normal human fibroblasts

Excess thymidine induced premature senescence in normal human fibroblasts (TIG-7), with induction of typical senescence markers. Nuclear swelling, as well as cell swelling, was clearly observed in these senescent cells. Simultaneous addition of MAP kinase inhibitors, U0126, SB203580, and SP60025, effectively suppressed induction of premature senescence and senescence markers.     

Molecular basis for premature senescence induced by surfactants in normal human cells

Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.     

High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts

Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence.     

Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.     

Spontaneous cytotoxicity mediated by human monocyte-macrophages against human fibroblasts infected with herpes simplex virsu--augmentation by interferon

Human monocyte-macrophages (MΦ) displayed spontaneous cytotoxicity for human fibroblasts infected with herpes simples virus (HSV). The HSV-immune status of the MΦ did not affect the spontaneous cytotoxicity. Significant cytotoxicity required an effector cell to target cell ratio of at least 50: 1, and was first observed 12 hr after incubation of MΦ with HSV-infected cells. A 12- to 18-hr preincubation of MΦ with interferon enhanced the cytotoxic activity. Cytotoxicity activity was also observed with cytomegalovirus-infected HF implying that the cytotoxic activity of the MΦ was nonspecific for viral-infected cells.     

Sulforaphane protects human chondrocytes against cell death induced by various stimuli

Chondrocyte cell death can contribute to cartilage degeneration in articular diseases, such as osteoarthritis (OA). Sulforaphane (SFN), a natural compound derived from cruciferous aliment, is well known as an anti-carcinogen, but according to recent evidence it also shows cytoprotective effects on a variety of non-tumoral cells. Therefore we have tested the ability of SFN to protect chondrocytes from cell death in vitro. Treatment of growing monolayer cultures of human C-28/I2 chondrocytes with SFN in the low micro-molecular range for a few days, reduced cell growth without affecting cell survival or inducing apoptosis. However it decreased cell death in C-28/I2 chondrocytes exposed to stimuli previously reported to promptly trigger apoptosis, that is, the cytokine tumor necrosis factor-α (TNF) plus cycloheximide (CHX) or the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) plus CHX. In particular pre-treatment with SFN reduced effector and initiator caspase activities and the associated activation of JNK kinases. SFN exerted a cytoprotective action even versus H(2)O(2) , which differently from the previous stimuli induced cell death without producing an evident caspase activation. SFN pre-treatment also prevented caspase activation in three-dimensional micromass cultures of OA chondrocytes stimulated with growth-related oncogene α (GROα), a pro-apoptotic chemokine. The suppression of caspase activation in micromasses appeared to be related to the inhibition of p38 MAPK phosphorylation. In conclusion, the present work shows that low micro-molecular SFN concentrations exert pro-survival and anti-apoptotic actions and influence signaling pathways in a variety of experimental conditions employing chondrocyte cell lines and OA chondrocytes treated with a range of death stimuli.     

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment

Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated β-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.