Cell type-dependent modulation of senescence features using Weo electrolyzed water.

Electrolyzed-reduced water has powerful antioxidant properties with constituents that scavenge reactive oxygen species (ROS), which are known to be produced by several intrinsic and extrinsic processes. When there is an imbalance between ROS production and antioxidant defenses, oxidative stress occurs. Persistent oxidative stress leads to cellular senescence, an important hallmark of aging, and is involved in several age-related conditions and illnesses. This study aims to investigate whether Weo electrolyzed water (WEW) could modulate the phenotype of senescent cells. We compared normal human lung fibroblasts (BJ) and breast cancer cells (T47D) treated with hydrogen peroxide (H2O2) to induce senescence. We assessed the molecular impact of WEW on markers of cellular senescence, senescence-associated secretory phenotype (SASP) factors, and stress response genes. Treatment with WEW modulated markers of cellular senescence, such as the senescence-associated ß-galactosidase (SA-ß-gal) activity, EdU incorporation and p21 expression, similarly in both cell types. However, WEW modulated the expression of SASP factors and stress response genes in a cell type-dependent and opposite fashion, significantly decreasing them in BJ cells, while stimulating their expression in T47D cells. Reduction in the expression of SASP factors and stress-related genes in BJ cells suggests that WEW acts as a protective factor, thereby reducing oxidative stress in normal cells, while making cancer cells more sensitive to the effects of cellular stress, thus increasing their elimination and consequently reducing their deleterious effects. These findings suggest that, due to its differential effects as a senomorphic factor, WEW could have a positive impact on longevity and age-related diseases.

Long Non-Coding RNA KCNQ1OT1 Regulates Protein Kinase CK2 Via miR-760 in Senescence and Calorie Restriction.

Long non-coding RNAs (lncRNAs) play important biological roles. Here, the roles of the lncRNA KCNQ1OT1 in cellular senescence and calorie restriction were determined. KCNQ1OT1 knockdown mediated various senescence markers (increased senescence-associated ß-galactosidase staining, the p53-p21Cip1/WAF1 pathway, H3K9 trimethylation, and expression of the senescence-associated secretory phenotype) and reactive oxygen species generation via CK2α downregulation in human cancer HCT116 and MCF-7 cells. Additionally, KCNQ1OT1 was downregulated during replicative senescence, and its silencing induced senescence in human lung fibroblast IMR-90 cells. Additionally, an miR-760 mimic suppressed KCNQ1OT1-mediated CK2α upregulation, indicating that KCNQ1OT1 upregulated CK2α by sponging miR-760. Finally, the KCNQ1OT1-miR-760 axis was involved in both lipopolysaccharide-mediated CK2α reduction and calorie restriction (CR)-mediated CK2α induction in these cells. Therefore, for the first time, this study demonstrates that the KCNQ1OT1-miR-760-CK2α pathway plays essential roles in senescence and CR, thereby suggesting that KCNQ1OT1 is a novel therapeutic target for an alternative treatment that mimics the effects of anti-aging and CR.

Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells.

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA ß-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases.

Senotherapeutic-like effect of Silybum marianum flower extract revealed on human skin cells.

Cellular senescence causes irreversible growth arrest of cells. Prolonged accumulation of senescent cells in tissues leads to increased detrimental effects due to senescence associated secretory phenotype (SASP). Recent findings suggest that elimination of senescent cells has a beneficial effect on organismal aging and lifespan. In this study, using a validated replicative senescent human dermal fibroblasts (HDFs) model, we showed that elimination of senescent cells is possible through the activation of an apoptotic mechanism. We have shown in this replicative senescence model, that cell senescence is associated with DNA damage and cell cycle arrest (p21, p53 markers). We have shown that Silybum marianum flower extract (SMFE) is a safe and selective senolytic agent targeting only senescent cells. The elimination of the cells is induced through the activation of apoptotic pathway confirmed by annexin V/propidium iodide and caspase-3/PARP staining. Moreover, SMFE suppresses the expression of SASP factors such as IL-6 and MMP-1 in senescent HDFs. In a co-culture model of senescent and young fibroblasts, we demonstrated that senescent cells impaired the proliferative capacities of young cells. Interestingly, when the co-culture is treated with SMFE, the cell proliferation rate of young cells is increased due to the decrease of the senescent burden. Moreover, we demonstrated in vitro that senescent fibroblasts trigger senescent process in normal keratinocytes through a paracrine effect. Indeed, the conditioned medium of senescent HDFs treated with SMFE reduced the level of senescence-associated beta-galactosidase (SA-ß-Gal), p16INK4A and SASP factors in keratinocytes compared with CM of senescent HDFs. These results indicate that SMFE can prevent premature aging due to senescence and even reprograms aged skin. Indeed, thanks to its senolytic and senomorphic properties SMFE is a candidate for anti-senescence strategies.

Skeletal muscle aging, cellular senescence, and senotherapeutics: Current knowledge and future directions.

Cellular senescence is a state of cell cycle arrest induced by several forms of metabolic stress. Senescent cells accumulate with advancing age and have a distinctive phenotype, characterized by profound chromatin alterations and a robust senescence-associated secretory phenotype (SASP) that exerts negative effects on tissue health, both locally and systemically. In preclinical models, pharmacological agents that eliminate senescent cells (senotherapeutics) restore health and youthful properties in multiple tissues. To date, however, very little is understood about the vulnerability of terminally-differentiated skeletal muscle fibers and the resident mononuclear cells that populate the interstitial microenvironment of skeletal muscle to senescence, and their contribution to the onset and progression of skeletal muscle loss and dysfunction with aging. Scientific advances in these areas have the potential to highlight new therapeutic approaches to optimize late-life muscle health. To this end, this review highlights the current evidence and the key questions that need to be addressed to advance the field's understanding of cellular senescence as a mediator of skeletal muscle aging and the potential for emerging senescent cell-targeting therapies to counter age-related deficits in muscle mass, strength, and function. This article is part of the Special Issue - Senolytics - Edited by Joao Passos and Diana Jurk.

Early elimination of uremic toxin ameliorates AKI-to-CKD transition.

Acute kidney injury (AKI)-related fibrosis is emerging as a major driver of chronic kidney disease (CKD) development. Aberrant kidney recovery after AKI is multifactorial and still poorly understood. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a detrimental factor of renal fibrosis. However, the mechanisms underlying IS-related aberrant kidney recovery after AKI is still unknown. The present study aims to elucidate the effects of IS on tubular damage and its involvement in the pathogenesis of AKI-to-CKD transition. Our results showed that serum IS started to accumulate associated with the downregulation of tubular organic anion transporter but not observed in the small-molecule uremic toxins of the unilateral ischemia-reperfusion injury (UIRI) without a contralateral nephrectomy model. Serum IS is positively correlated with renal fibrosis and binding immunoglobulin protein (BiP) and CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression induction in the UIRI with a contralateral nephrectomy model (UIRI+Nx). To evaluate the effects of IS in the AKI-to-CKD transition, we administered indole, a precursor of IS, at the early stage of UIRI. Our results demonstrated IS potentiates renal fibrosis, senescence-associated secretory phenotype (SASP), and activation of endoplasmic reticulum (ER) stress, which is attenuated by synergistic AST-120 administration. Furthermore, we clearly demonstrated that IS exposure potentiated hypoxia-reperfusion (H/R) induced G2/M cell cycle arrest, epithelial-mesenchymal transition (EMT) and aggravated ER stress induction in vitro. Finally, the ER chemical chaperon, 4-phenylbutyric acid (4-PBA), successfully reversed the above-mentioned AKI-to-CKD transition. Taken together, early IS elimination in the early stage of AKI is likely to be a useful strategy in the prevention and/or treatment of the AKI-to-CKD transition.

A guide to senolytic intervention in neurodegenerative disease.

Cellular senescence is a potential tumor-suppressive mechanism that generally results in an irreversible cell cycle arrest. Senescent cells accumulate with age and actively secrete soluble factors, collectively termed the 'senescence-associated secretory phenotype' (SASP), which has both beneficial and detrimental effects. Although the contribution of senescent cells to age-related pathologies has been well-established outside the brain, emerging evidence indicates that brain cells also undergo cellular senescence and contribute to neuronal loss in the context of age-related neurodegenerative diseases. Contribution of senescent cells in the pathogenesis of neurological disorders has led to the possibility of eliminating senescence cells via pharmacological compounds called senolytics. Recently several senolytics have been demonstrated to elicit improved cognitive performance and healthspan in mouse models of neurodegeneration. However, their translation for use in the clinic still holds several potential challenges. This review summarizes available senolytics, their purported mode of action, and possible off-target effects. We also discuss possible alternative strategies that may help minimize potential side-effects associated with the senolytics approach.

Senescence-associated hyper-activation to inflammatory stimuli in vitro.

Aging is associated with an increased susceptibility to adverse inflammatory conditions such as sepsis and cytokine storm. We hypothesized that senescent cells (SnCs) play a central role in this age-associated pathology in part due to their expression of the senescence-associated secretory phenotype (SASP), which may prime SnCs to inflammatory stimulation. To test this hypothesis, we examined the expression of various inflammatory cytokines and chemokines at the levels of gene transcription and protein production in various SnCs in vitro in response to lipopolysaccharide (LPS), interleukin-1ß (IL1ß), and tumor necrosis factor α (TNFα) stimulation. We found that SnCs not only expressed higher basal levels of various inflammatory cytokines and chemokines as a manifestation of the SASP, but more importantly exhibited hyper-activation of the induction of a variety of inflammatory mediators in response to LPS, IL1ß and TNFα stimulation as compared with non-SnCs. This senescence-associated hyper-activation is likely mediated in part via the p38MAPK (p38) and NFκB pathways because LPS stimulation elicited significantly higher levels of p38 phosphorylation and NFκB p65 nuclear translation in SnCs when compared to their non-senescent counterparts and inhibition of these pathways with losmapimod (a p38 specific inhibitor) and BMS-345541 (a selective NFκB inhibitor) attenuated LPS-induced expression of IL6, TNFα, CCL5, and IL1ß mRNA in SnCs. These findings suggest that SnCs may play an important role in the age-related increases in the susceptibility to developing an exacerbated inflammatory response and highlight the potential to use senotherapeutics to ameliorate the severity of various devastating inflammatory conditions in the elderly.

The Cancer SENESCopedia: A delineation of cancer cell senescence.

Cellular senescence is characterized as a stable proliferation arrest that can be triggered by multiple stresses. Most knowledge about senescent cells is obtained from studies in primary cells. However, senescence features may be different in cancer cells, since the pathways that are involved in senescence induction are often deregulated in cancer. We report here a comprehensive analysis of the transcriptome and senolytic responses in a panel of 13 cancer cell lines rendered senescent by two distinct compounds. We show that in cancer cells, the response to senolytic agents and the composition of the senescence-associated secretory phenotype are more influenced by the cell of origin than by the senescence trigger. Using machine learning, we establish the SENCAN gene expression classifier for the detection of senescence in cancer cell samples. The expression profiles and senescence classifier are available as an interactive online Cancer SENESCopedia.

Astrocyte senescence and SASP in neurodegeneration: tau joins the loop.

Tau accumulation is a core component of Alzheimer's disease and other neurodegenerative tauopathies. While tau's impact on neurons is a major area of research, the effect of extracellular tau on astrocytes is largely unknown. This article summarizes our recent studies showing that astrocyte senescence plays a critical role in neurodegenerative diseases and integrates extracellular tau into the regulatory loop of senescent astrocyte-mediated neurotoxicity. Human astrocytes in vitro undergoing senescence were shown to acquire the inflammatory senescence-associated secretory phenotype (SASP) and toxicity to neurons, which may recapitulate aging- and disease-associated neurodegeneration. Here, we show that human astrocytes exposed to extracellular tau in vitro also undergo cellular senescence and acquire a neurotoxic SASP (e.g. IL-6 secretion), with oxidative stress response (indicated by upregulated NRF2 target genes) and a possible activation of inflammasome (indicated by upregulated ASC and IL-1ß). These findings suggest that senescent astrocytes induced by various conditions and insults, including tau exposure, may represent a therapeutic target to inhibit or delay the progression of neurodegenerative diseases. We also discuss the pathological activity of extracellular tau in microglia and astrocytes, the disease relevance and diversity of tau forms, therapeutics targeting senescence in neurodegeneration, and the roles of p53 and its isoforms in astrocyte-mediated neurotoxicity and neuroprotection.

Roxithromycin attenuates bleomycin-induced pulmonary fibrosis by targeting senescent cells.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease with a poor prognosis. Emerging evidence has revealed that targeting senescent cells may be a potential treatment for IPF. In this study, we aimed to explore whether roxithromycin (RXM) can improve lung fibrosis by targeting senescent cells. First, we confirmed the ability of RXM to selectively kill senescent cells by inducing apoptosis and inhibiting the expression of senescence-associated secretory phenotype (SASP) factors, suggesting the potential role of RXM as a "senolytic" and "senomorphic" drug. Next, we observed that TGF-ß- and senescent cell-induced lung fibroblast activation was inhibited by RXM treatment, which prompted us to further investigate its effect in vivo. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, RXM was shown to attenuate lung injury, inflammation, and fibrosis. Furthermore, the senescent phenotype of lung tissues induced by BLM was significantly diminished after RXM administration, indicating the potential of RXM as an antifibrotic and antisenescent agent. Interestingly, NADPH oxidase 4 (NOX4), implicated in lung fibrosis and cell senescence, was shown to be inhibited by RXM treatments. The antifibroblast activation and antisenescent effects of RXM were abolished in NOX4 knockdown cells, demonstrating that RXM may ameliorate BLM-induced pulmonary fibrosis by targeting senescent cells mediated by the NOX4 pathway. Collectively, these data demonstrated that RXM may be a potential clinical agent for IPF and further supported the notion that targeting cellular senescence is a promising treatment for progressive age-related disease.

Cellular senescence: Silent operator and therapeutic target in cancer.

The process of carcinogenesis and its progression involves an intricate interplay between a number of signaling networks, metabolic pathways and the microenvironment. These include an alteration in the cellular redox metabolism and deregulation of cell cycle checkpoints. Similar to the dichotomy of redox signaling in cancer cell fate and state determination, a diverging effect of an irreversible cell cycle arrest or senescence on carcinogenesis has been demonstrated. In this regard, while overwhelming oxidative stress has a damaging effect on tissue architecture and organ function and promotes death execution, a mild "pro-oxidant" environment is conducive for cell proliferation, growth and survival. Similarly, cellular senescence has been shown to elicit both a tumor suppressor and an oncogenic effect in a context-dependent manner. Notably, there appears to be a crosstalk between these two critical regulators of cell fate and state, particularly from the standpoint of the divergent effects on processes that promote or abate carcinogenesis. This review aims to provide an overview of these overarching themes and attempts to highlight critical intersection nodes, which are emerging as potential diagnostic and/or therapeutic targets for novel anticancer strategies.

Senolytic agent Quercetin ameliorates intervertebral disc degeneration via the Nrf2/NF-κB axis.

OBJECTIVE: Intervertebral disc degeneration (IDD) represents major cause of low back pain. Quercetin (QUE) is one of the approved senolytic agents. In this study, we evaluated the protective effects of QUE on IDD development and its underlying mechanism. METHODS: Effects of senolytic agent QUE on the viability of nucleus pulposus cells (NPCs) were measured by CCK-8 assays and EdU staining. The senescence associated secreted phenotype (SASP) factors expressions were measured by qPCR, western blot, and ELISA; and NF-κB pathway was detected by immunofluorescence and western blot. Molecular docking was applied to predict the interacting protein of QUE; while Nrf2 was knocked down by siRNAs to confirm its role in QUE regulated senescence phenotype. X-ray, MRI, Hematoxylin-Eosin and Safranin O-Fast green staining were performed to evaluate the therapeutic effects of QUE on IDD in the puncture-induced rat model. RESULTS: In in vitro experiments, QUE inhibited SASP factors expression and senescence phenotype in IL-1ß-treated NPCs. Mechanistically, QUE suppressed IL-1ß induced activation of the NF-κB pathway cascades; it was also demonstrated in molecular docking and knock down studies that QUE might bind to Keap1-Nrf2 complex to suppress NF-κB pathway. In vivo, QUE ameliorated the IDD process in the puncture-induced rat model. CONCLUSIONS: Together the present work suggests that QUE inhibits SASP factors expression and senescence phenotype in NPCs and ameliorates the progression of IDD via the Nrf2/NF-κB axis, which supports senolytic agent QUE as a potential therapeutic agent for the treatment of IDD.