Ascorbic acid inhibits senescence in mesenchymal stem cells through ROS and AKT/mTOR signaling

Mesenchymal stem cell (MSC) aging seriously affects its function in stem cell transplantation for treatment. Extensive studies have focused on how to inhibit senescence in MSCs. However, the mechanism of senescence in MSC was not clear. In this study, we used d-galactose to induce MSC aging. Then we found that the number of aging cells was increased compared with untreated MSCs. We discovered that ascorbic acid could inhibit the production of reactive oxygen species (ROS) and activation of AKT/mTOR signaling in MSCs caused by d-galactose. Especially, when treated together with a ROS scavenger or AKT inhibitor, the senescent cells were obviously decreased in d-galactose-induced MSCs. Taken together, we identify that ascorbic acid owns the potential to inhibit the senescence of MSCs through ROS and Akt/mTOR signaling. Together, our data supports that ascorbic acid can be used to prevent MSCs from senescence, which can enhance the efficiency of stem cell transplantation in the clinic.     

The cell fate: senescence or quiescence

Senescence and quiescence are frequently used as interchangeable terms in the literature unwittingly. Despite the fact that common molecules play role in decision of cell cycle arrest, senescent and quiescent cells have some distinctive phenotypes at both molecular and morphological levels. Thus, in this review we summarized the features of senescence and quiescence with respect to visual characteristics and prominent key molecules. A PubMed research was conducted for the key words; “senescence”, “quiescence” and “cell cycle arrest”. The results which are related to cell cycle control were selected. The selection criteria of the target articles used for this review included also key cell cycle molecules such as p53, pRB, p21, p16, mTOR, p27, etc. The results were not evaluated statistically. The mechanistic target of rapamycin (mTOR) has been claimed to be key molecule in switching on/off senescence/quiescence. Specifically, although maximal p53 activation blocks mTOR and causes quiescence, partial p53 activation sustains mTOR activity and causes senescence subsequently. In broader perspective, quiescence occurs due to lack of nutrition and growth factors whereas senescence takes place due to aging and serious DNA damages. Contrary to quiescence, senescence is a degenerative process ensuing a certain cell death. We highlighted several differences between senescence and quiescence and their key molecules in this review. Whereas quiescence (cell cycle arrest) is only one half of the senescence, the other half is growth stimulation which causes actual senescence phenotype.     

Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

Elimination of proliferating cells unmasks the shift from senescence to quiescence caused by rapamycin

Background

Depending on cellular context, p53-inducing agents (such as nutlin-3a) cause different outcomes including reversible quiescence and irreversible senescence. Inhibition of mTOR shifts the balance from senescence to quiescence. In cell lines with incomplete responses to p53, this shift may be difficult to document because of a high proportion of proliferating cells contaminating arrested (quiescent and senescent) cells. This problem also complicates the study of senescence caused by minimal levels of p21 that are capable to arrest a few cells.

Methodology

During induction of senescence by low levels of endogenous p53 and ectopic p21, cells were co-treated with nocodazole, which eliminated proliferating cells. As a result, only senescent and quiescent cells remained.

Results and Discussion

This approach revealed that rapamycin efficiently converted nutlin-induced-senescence into quiescence. In the presence of rapamycin, nutlin-arrested MCF-7 cells retained the proliferative potential and small/lean morphology. Using this approach, we also unmasked senescence in cells arrested by low levels of ectopic p21, capable to arrest only a small proportion of HT1080-p21-9 cells. When p21 did cause arrest, mTOR caused senescent phenotype. Rapamycin and high concentrations of nutlin-3a, which inhibit the mTOR pathway in these particular cells, suppressed senescence, ensuring quiescence instead. Thus, p21 causes senescence passively, just by causing arrest, while still active mTOR drives senescent phenotype.     

Proteostasis failure and cellular senescence in long‐term cultured postmitotic rat neurons

Cellular senescence, a stress‐induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age‐related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long‐term cultures show molecular changes indicative of both senescence (senescence‐associated β‐galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell‐autonomous neuroprotective response.     

Hypoxia and gerosuppression: The mTOR saga continues

Growth-promoting and nutrient/mitogen-sensing pathways such as mTOR convert p21- and p16-induced arrest into senescence (geroconversion). We have recently demonstrated that hypoxia, especially near-anoxia, suppresses geroconversion. This gerosuppressive effect of hypoxia correlated with inhibition of the mTOR/S6K pathway but not with modulation of the LKB1/AMPK/eEF2 pathway. Here we further show that mTOR inhibition is required for gerosuppression by hypoxia, at least in some cellular models, because depletion of TSC2 abolished mTOR inhibition and gerosupression by hypoxia. Also, in two cancer cell lines resistant to inhibition of mTOR by both p53 and hypoxia, hypoxia did not suppress geroconversion. Therefore, the effects of hypoxia on the oxygen-sensing mTOR pathway and geroconversion are cell type-specific. We also briefly discuss replicative senescence, organismal aging and free radical theory.     

Aging: A cell source limiting factor in tissue engineering

Tissue engineering has yet to reach its ideal goal, i.e. creating profitable off-the-shelf tissues and organs, designing scaffolds and three-dimensional tissue architectures that can maintain the blood supply, proper biomaterial selection, and identifying the most efficient cell source for use in cell therapy and tissue engineering. These are still the major challenges in this field. Regarding the identification of the most appropriate cell source, aging as a factor that affects both somatic and stem cells and limits their function and applications is a preventable and, at least to some extents, a reversible phenomenon. Here, we reviewed different stem cell types, namely embryonic stem cells, adult stem cells, induced pluripotent stem cells, and genetically modified stem cells, as well as their sources, i.e. autologous, allogeneic, and xenogeneic sources. Afterward, we approached aging by discussing the functional decline of aged stem cells and different intrinsic and extrinsic factors that are involved in stem cell aging including replicative senescence and Hayflick limit, autophagy, epigenetic changes, miRNAs, mTOR and AMPK pathways, and the role of mitochondria in stem cell senescence. Finally, various interventions for rejuvenation and geroprotection of stem cells are discussed. These interventions can be applied in cell therapy and tissue engineering methods to conquer aging as a limiting factor, both in original cell source and in the in vitro proliferated cells.     

Hypoxia and gerosuppression: The mTOR saga continues

Growth-promoting and nutrient/mitogen-sensing pathways such as mTOR convert p21- and p16-induced arrest into senescence (geroconversion). We have recently demonstrated that hypoxia, especially near-anoxia, suppresses geroconversion. This gerosuppressive effect of hypoxia correlated with inhibition of the mTOR/S6K pathway but not with modulation of the LKB1/AMPK/eEF2 pathway. Here we further show that mTOR inhibition is required for gerosuppression by hypoxia, at least in some cellular models, because depletion of TSC2 abolished mTOR inhibition and gerosupression by hypoxia. Also, in two cancer cell lines resistant to inhibition of mTOR by both p53 and hypoxia, hypoxia did not suppress geroconversion. Therefore, the effects of hypoxia on the oxygen-sensing mTOR pathway and geroconversion are cell type-specific. We also briefly discuss replicative senescence, organismal aging and free radical theory.     

A critical role for TORC1 in cellular senescence

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401 and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.Cellular senescence is a process initiated either when cells proliferate past their potential (replicative senescence) or by activation of an oncogenic stress (oncogene-induced senescence). Both of these events are characterized by the activation of a DNA damage response, which is initiated by eroded telomeres in the case of replicative senescence, and aberrant products of DNA replication in the case of oncogene induced senescence.¹ Senescence plays a critical tumor-suppression role in vivo, and alterations in the senescence program are a hallmark of cancer cells. Bypass of senescence is critical for tumor progression and involves the p53 and pRB tumor-suppressor pathways.² Indeed, expression of DNA tumor virus oncoproteins that target p53 and pRB can bypass senescence in cultured cells,³ and concomitant loss of pRB and p53 bypasses senescence in human diploid fibroblasts.⁴ In addition to being an obligatory step for tumor progression, bypass of senescence creates a favorable environment in which additional tumor-promoting mutations can be acquired. For example, inactivation of p53 in the context of telomere erosion promotes rampant genomic instability mediated by cycles of aberrant DNA damage/DNA repair events.⁵In a new study, Kolesnichenko et al. describe a critical role for the mTOR pathway in senescence induction.⁶ This work demonstrates that inhibition of mTOR is sufficient to delay RAS-induced senescence as well as replicative senescence. Using a combination of inhibitory molecules, shRNA-mediated knockdown and expression of inhibitory proteins, the authors demonstrate that inhibition of the TORC1 complex is sufficient to delay senescence induction. These findings are further corroborated by the independent work of Pospelova and colleagues showing that rapamycin treatment delays senescence induction in murine fibroblasts.⁷ These intriguing findings raise the question of why mTOR inhibition inhibits senescence induction. The work of Kolesnchenko and colleagues provides two clues to explain this phenotype. First, mTOR inhibition results in the activation of the pro-survival factor AKT, a factor that could explain how cells can proliferate in the face of an ongoing senescence-inducing signal. In addition, the authors find reduced levels of p53 and its target gene p21 upon mTOR inhibition. These findings are particularly significant considering the critical role for both p53 activation and p21 induction in senescence induction.In conclusion, the finding that inhibition of the TORC1 complex has a profound effect on the onset of senescence might explain why rapamycin treatment had limited success in the treatment of cancer.⁸ On the other hand, rapamycin slows aging and thus delays cancer in mice.⁹     

Aging-suppressants: Cellular senescence (hyperactivation) and its pharmacologic deceleration

Here I overview the accompanying three reports on suppression of cellular senescence with inhibitors of mTOR, PI-3K and MEK. How can growth inhibitors suppress senescence? May these aging-suppressants decelerate organismal aging? To answer these questions, we need to reconsider the meaning of aging.     

Geroconversion: irreversible step to cellular senescence

Cellular senescence happens in 2 steps: cell cycle arrest followed, or sometimes preceded, by gerogenic conversion (geroconversion). Geroconvesrion is a form of growth, a futile growth during cell cycle arrest. It converts reversible arrest to irreversible senescence. Geroconversion is driven by growth-promoting, mitogen-/nutrient-sensing pathways such as mTOR. Geroconversion leads to hyper-secretory, hypertrophic and pro-inflammatory cellular phenotypes, hyperfunctions and malfunctions. On organismal level, geroconversion leads to age-related diseases and death. Rapamycin, a gerosuppressant, extends life span in diverse species from yeast to mammals. Stress–and oncogene-induced accelerated senescence, replicative senescence in vitro and life-long cellular aging in vivo all can be described by 2-step model.     

Bradykinin protects cardiac c-kit positive cells from high-glucose-induced senescence through B2 receptor signaling pathway

Cardiac c-kit positive cells are cardiac-derived cells that exist within the heart and have a great many protective effects. The senescence of cardiac c-kit positive cells probably leads to cell dysfunction. Bradykinin plays a key role in cell protection. However, whether bradykinin prevents cardiac c-kit positive cells from high-glucose-induced senescence is unknown. Here, we found that glucose treatment causes the premature senescence of cardiac c-kit positive cells. Bradykinin B2 receptor (B2R) expression was declined by glucose-induced senescence. Bradykinin treatment inhibited senescence and reduced intracellular oxygen radicals according to senescence-associated β-galactosidase staining and 2′,7′-dichlorodihydrofluorescein diacetate staining. Moreover, the mitochondrial membrane potential was damaged, as measured by JC-1 staining. The mitochondrial membrane potential was preserved under bradykinin treatment. The concentration of superoxide was decreased, and the concentration of intracellular adenosine triphosphate was increased after bradykinin treatment. Western blot showed that bradykinin leads to AKT and mammalian target of rapamycin (mTOR) phosphorylation and decreased levels of P53 and P16 when compared with glucose treatment alone. Antagonists of B2R, phosphoinositide 3-kinase (PI3K), mTOR, and B2R small interfering RNA prevented the protective effect of bradykinin. P53 antagonist also inhibited the glucose-induced senescence of cardiac c-kit positive cells. In conclusion, bradykinin prevents the glucose-induced premature senescence of cardiac c-kit positive cells through the B2R/PI3K/AKT/mTOR/P53 signal pathways.     

Telomere, DNA damage, and oxidative stress in stem cell aging

"Stem cell aging" is a novel concept that developed together with the advances of stem cell biology, especially the sophisticated prospectively isolation and characterization of multipotent somatic tissue stem cells. Although being immortal in principle, stem cells can also undergo aging processes and potentially contribute to organismal aging. The impact of an age-dependent decline of stem cell function weighs differently in organs with high or low rates of cell turnover. Nonetheless, most of the organ systems undergo age-dependent loss of homeostasis and functionality, and emerging evidence showed that this has to do with the aging of resident stem cells in the organ systems. The mechanisms of stem cell aging and its real contribution to human aging remain to be defined. Many antitumor mechanisms protect potential malignant transformation of stem cell by inducing apoptosis or senescence but simultaneously provoke stem cell aging. In this review, we try to discuss several concept of stem cell aging and summarize recent progression on the molecular mechanisms of stem cell aging.     

Suppression of replicative senescence by rapamycin in rodent embryonic cells

The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-beta-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC50 around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12–14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-beta-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.     

miRNAs与衰老相关信号通路的研究进展

miRNAs是一类负调控基因表达的内源性非编码小分子RNA,在细胞衰老过程中发挥重要作用. 细胞衰老是指可增殖细胞在各种应激下出现细胞周期阻滞,并且丧失增殖能力,进入一种不可逆的、相对稳定的状态. p53、p21、p16、SIRT1、胰岛素/IGF-1及mTOR等蛋白是衰老相关信号通路中的重要分子,参与细胞衰老过程. 研究表明,miRNAs可以通过调控这些衰老相关蛋白所在的信号通路,促进或延缓细胞衰老. 本文综述细胞衰老相关的miRNAs,以及它们对衰老相关信号通路的影响,为深化认识衰老和衰老相关疾病的分子机制奠定基础.     

Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence

Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21^WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.     

mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: A roadmap from energy metabolism to stem cell renewal and aging

Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced Pluripotent Stem Cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of mitochondrial function and in the accelerated onset of the glycolytic metabolism that is required to fuel reprogramming. By critically exploring how mTOR-regulated senescence, bioenergetic infrastructure and autophagy can actively drive the reprogramming of somatic cells to pluripotency, we define a metabolic roadmap that may be helpful for designing pharmacological and behavioral interventions to prevent or retard the dysfunction/exhaustion of aging stem cell populations.     

Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence

Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21^WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.     

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.