Irreversible Cell Cycle Arrest Research Articles

Mitochondrial fatty acid oxidation drives senescence.

Cellular senescence is a stress-induced irreversible cell cycle arrest involved in tumor suppression and aging. Many stresses, such as telomere shortening and oncogene activation, induce senescence by damaging nuclear DNA. However, the mechanisms linking DNA damage to senescence remain unclear. Here, we show that DNA damage response (DDR) signaling to mitochondria triggers senescence. A genome-wide small interfering RNA screen implicated the outer mitochondrial transmembrane protein BNIP3 in senescence induction. We found that BNIP3 is phosphorylated by the DDR kinase ataxia telangiectasia mutated (ATM) and contributes to an increase in the number of mitochondrial cristae. Stable isotope labeling metabolomics indicated that the increase in cristae enhances fatty acid oxidation (FAO) to acetyl-coenzyme A (acetyl-CoA). This promotes histone acetylation and expression of the cyclin-dependent kinase inhibitor p16INK4a. Notably, pharmacological activation of FAO alone induced senescence both in vitro and in vivo. Thus, mitochondrial energy metabolism plays a critical role in senescence induction and is a potential intervention target to control senescence.

Combined HDAC8 and checkpoint kinase inhibition induces tumor-selective synthetic lethality in preclinical models.

The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergizes with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.

Topical Platelet Exosomes Reduce Senescence Signaling in Human Skin: An Exploratory Prospective Trial.

Cellular senescence, an irreversible cell cycle arrest with secretory phenotype, is a hallmark of skin aging. Regenerative exosome-based approaches, such as topical human platelet extract (HPE), are emerging to target age-related skin dysfunction. To evaluate the cellular and molecular effects of topical HPE for skin rejuvenation after 12 weeks of twice daily use. Skin biopsies were obtained for histological evaluation of senescence markers, p16INK4a and p21CIP1/WAF1. Telomere-associated foci, coassociation of telomeres, and DNA damage marker, γH2AX, were assessed. RNA sequencing evaluated senescence associated secretory phenotype (SASP) and extracellular matrix pathways. p16INK4a and p21CIP1/WAF1 staining in senescent skin cells revealed low and high expression subgroups that did not correspond to chronological age. Topical HPE significantly reduced high p16INK4a cells in the dermis (p = .02). There was also a decrease in telomere damage after topical HPE (p = .03). In patients with high senescent cells at baseline, there was a 40% reduction in proinflammatory SASP. Extracellular matrix remodeling pathways, including collagen and elastic fibers, were up-regulated. Topical HPE, applied on intact skin, reduced senescence signaling and senescence-associated telomere damage after 12 weeks of twice daily use, targeting a path for skin longevity or healthy skin aging.

Pan-cancer characterization of cellular senescence reveals its inter-tumor heterogeneity associated with the tumor microenvironment and prognosis

Cellular senescence (CS) is characterized by the irreversible cell cycle arrest and plays a key role in aging and diseases, such as cancer. Recent years have witnessed the burgeoning exploration of the intricate relationship between CS and cancer, with CS recognized as either a suppressing or promoting factor and officially acknowledged as one of the 14 cancer hallmarks. However, a comprehensive characterization remains absent from elucidating the divergences of this relationship across different cancer types and its involvement in the multi-facets of tumor development. Here we systematically assessed the cellular senescence of over 10,000 tumor samples from 33 cancer types, starting by defining a set of cancer-associated CS signatures and deriving a quantitative metric representing the CS status, called CS score. We then investigated the CS heterogeneity and its intricate relationship with the prognosis, immune infiltration, and therapeutic responses across different cancers. As a result, cellular senescence demonstrated two distinct prognostic groups: the protective group with eleven cancers, such as LIHC, and the risky group with four cancers, including STAD. Subsequent in-depth investigations between these two groups unveiled the potential molecular and cellular mechanisms underlying the distinct effects of cellular senescence, involving the divergent activation of specific pathways and variances in immune cell infiltrations. These results were further supported by the disparate associations of CS status with the responses to immuno- and chemo-therapies observed between the two groups. Overall, our study offers a deeper understanding of inter-tumor heterogeneity of cellular senescence associated with the tumor microenvironment and cancer prognosis.

Cellular Senescence: The Driving Force of Musculoskeletal Diseases.

The aging of the world population is closely associated with an increased prevalence of musculoskeletal disorders, such as osteoporosis, sarcopenia, and osteoarthritis, due to common genetic, endocrine, and mechanical risk factors. These conditions are characterized by degeneration of bone, muscle, and cartilage tissue, resulting in an increased risk of fractures and reduced mobility. Importantly, a crucial role in the pathophysiology of these diseases has been proposed for cellular senescence, a state of irreversible cell cycle arrest induced by factors such as DNA damage, telomere shortening, and mitochondrial dysfunction. In addition, senescent cells secrete pro-inflammatory molecules, called senescence-associated secretory phenotype (SASP), which can alter tissue homeostasis and promote disease progression. Undoubtedly, targeting senescent cells and their secretory profiles could promote the development of integrated strategies, including regular exercise and a balanced diet or the use of senolytics and senomorphs, to improve the quality of life of the aging population. Therefore, our review aimed to highlight the role of cellular senescence in age-related musculoskeletal diseases, summarizing the main underlying mechanisms and potential anti-senescence strategies for the treatment of osteoporosis, sarcopenia, and osteoarthritis.

TRPM7 controls skin keratinocyte senescence by targeting intracellular calcium signaling.

Cellular senescence is described as an irreversible cell cycle arrest for proliferating cells and is associated with the secretion of senescence associated secretory phenotype factors. It has been known to accumulate with age and is regarded as a key driver of aging-associated skin pathologies. However, the lack of markers of skin senescence and partially understood skin cellular senescence mechanisms has limited the exploration of skin aging and anti-skin aging strategies. Recently, intracellular calcium signaling has emerged as an important regulator of cellular senescence and aging. However, little is known about the modulation of skin cellular senescence by calcium-associated factors. Here, we found that the expression of calcium channel transient receptor potential melastatin 7 (TRPM7) is elevated during skin keratinocyte senescence and aging. Importantly, TRPM7 promotes skin keratinocyte senescence by triggering intracellular calcium transfer from the endoplasmic reticulum to the mitochondria; accumulation of mitochondrial calcium then induces a drop in mitochondrial membrane potential and reactive oxygen species production, leading to subsequent nuclear enlargement and DNA damage. Altogether, these findings indicate that TRPM7 controls skin keratinocyte senescence through regulating intracellular calcium signaling, and thus, shed light on novel strategies for anti-skin aging therapy.

To target cellular senescence in diabetic kidney disease: the known and the unknown.

Cellular senescence represents a condition of irreversible cell cycle arrest, characterized by heightened senescence-associated beta-galactosidase (SA-β-Gal) activity, senescence-associated secretory phenotype (SASP), and activation of the DNA damage response (DDR). Diabetic kidney disease (DKD) is a significant contributor to end-stage renal disease (ESRD) globally, with ongoing unmet needs in terms of current treatments. The role of senescence in the pathogenesis of DKD has attracted substantial attention with evidence of premature senescence in this condition. The process of cellular senescence in DKD appears to be associated with mitochondrial redox pathways, autophagy, and endoplasmic reticulum (ER) stress. Increasing accumulation of senescent cells in the diabetic kidney not only leads to an impaired capacity for repair of renal injury, but also the secretion of pro-inflammatory and profibrotic cytokines and growth factors causing inflammation and fibrosis. Current treatments for diabetes exhibit varying degrees of renoprotection, potentially via mitigation of senescence in the diabetic kidney. Targeting senescent cell clearance through pharmaceutical interventions could emerge as a promising strategy for preventing and treating DKD. In this paper, we review the current understanding of senescence in DKD and summarize the possible therapeutic interventions relevant to senescence in this field.

Advances in Nanotherapy for Targeting Senescent Cells.

Aging is an inevitable process in the human body, and cellular senescence refers to irreversible cell cycle arrest caused by external aging-promoting mechanisms. Moreover, as age increases, the accumulation of senescent cells limits both the health of the body and lifespan and even accelerates the occurrence and progression of age-related diseases. Therefore, it is crucial to delay the periodic irreversible arrest and continuous accumulation of senescent cells to address the issue of aging. The fundamental solution is targeted therapy focused on eliminating senescent cells or reducing the senescence-associated secretory phenotype. Over the past few decades, the remarkable development of nanomaterials has revolutionized clinical drug delivery pathways. Their unique optical, magnetic, and electrical properties effectively compensate for the shortcomings of traditional drugs, such as low stability and short half-life, thereby maximizing the bioavailability and minimizing the toxicity of drug delivery. This article provides an overview of how nanomedicine systems control drug release and achieve effective diagnosis. By presenting and analyzing recent advances in nanotherapy for targeting senescent cells, the underlying mechanisms of nanomedicine for senolytic and senomorphic therapy are clarified, providing great potential for targeting senescent cells.

Crosstalk between the DNA damage response and cellular senescence drives aging and age-related diseases.

Cellular senescence is a crucial process of irreversible cell-cycle arrest, in which cells remain alive, but permanently unable to proliferate in response to distinct types of stressors. Accumulating evidence suggests that DNA damage builds over time and triggers DNA damage response signaling, leading to cellular senescence. Cellular senescence serves as a platform for the perpetuation of inflammatory responses and is central to numerous age-related diseases. Defects in DNA repair genes or senescence can cause premature aging disease. Therapeutic approaches limiting DNA damage or senescence contribute to a rescued phenotype of longevity and neuroprotection, thus suggesting a mechanistic interaction between DNA damage and senescence. Here, we offer a unique perspective on the crosstalk between the DNA damage response pathway and senescence as well as their contribution to age-related diseases. We further summarize recent progress on the mechanisms and therapeutics of senescence, address existing challenges, and offering new insights and future directions in the senescence field.

Reduced dynamicity and increased high-order protein assemblies in dense fibrillar component of the nucleolus under cellular senescence

Cellular senescence, which is triggered by various stressors, manifests as irreversible cell cycle arrest, resulting in the disruption of multiple nuclear condensates. One of the affected structures is the nucleolus, whose tripartite layout, separated into distinct liquid phases, allows for the stepwise progression of ribosome biogenesis. The dynamic properties of dense fibrillar components, a sub-nucleolar phase, are crucial for mediating pre-rRNA processing. However, the mechanistic link between the material properties of dense fibrillar components and cellular senescence remains unclear. We established a significant association between cellular senescence and alterations in nucleolar materiality and characteristics, including the number, size, and sphericity of individual subphases of the nucleolus. Senescent cells exhibit reduced fibrillarin dynamics, aberrant accumulation of high-order protein assemblies, such as oligomers and fibrils, and increased dense fibrillar component density. Intriguingly, the addition of RNA-interacting entities mirrored the diminished diffusion of fibrillarin in the nucleolus during cellular senescence. Thus, our findings contribute to a broader understanding of the intricate changes in the materiality of the nucleolus associated with cellular senescence and shed light on nucleolar dynamics in the context of aging and cellular stress.

Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer's brain haemodynamics.

Cholinergic innervation in the brain is involved in modulating neurovascular function including cerebral blood flow haemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer's disease, albeit the aetiology remains to be clarified. In the current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry (LDF) in 3xTgAD Alzheimer's disease model) and wild-type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) was higher in young 3xTgAD versus in wild-type mice (48.35; 95% CI:27.03-69.67 versus 22.70; CI:15.5-29.91, P < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 versus 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3×TgAD mice. This suggests endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within the basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters (occasionaly), were observed at the basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell-cycle arrest in old 3xTgAD versus wild-type mice (P = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within the basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Pearson's correlation coefficient = 0.62) and the loss of cholinergic cells in vulnerable basal forebrain environment, was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF haemodynamics. Our study revealed the early change of CBF haemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age-related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at the basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer's disease.

SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Primary cilia as a targetable node between biliary injury, senescence and regeneration in liver transplantation

Biliary complications are a major cause of morbidity and mortality in liver transplantation. Up to 25% of patients that develop biliary complications require additional surgical procedures, re-transplantation or die in the absence of a suitable regraft. Here, we investigate the role of the primary cilium, a highly specialised sensory organelle, in biliary injury leading to post-transplant biliary complications. Human biopsies were used to study the structure and function of primary cilia in liver transplant recipients that develop biliary complications (n= 7) in comparison with recipients without biliary complications (n= 12). To study the biological effects of the primary cilia during transplantation, we generated murine models that recapitulate liver procurement and cold storage, and assessed the elimination of the primary cilia in biliary epithelial cells in the K19CreERTKif3afl/fl mouse model. To explore the molecular mechanisms responsible for the observed phenotypes we used invitro models of ischemia, cellular senescence and primary cilia ablation. Finally, we used pharmacological and genetic approaches to target cellular senescence and the primary cilia, both in mouse models and discarded human donor livers. Prolonged ischemic periods before transplantation result in ciliary shortening and cellular senescence, an irreversible cell cycle arrest that blocks regeneration. Our results indicate that primary cilia damage results in biliary injury and a loss of regenerative potential. Senescence negatively impacts primary cilia structure and triggers a negative feedback loop that further impairs regeneration. Finally, we explore how targeted interventions for cellular senescence and/or the stabilisation of the primary cilia improve biliary regeneration following ischemic injury. Primary cilia play an essential role in biliary regeneration and we demonstrate that senolytics and cilia-stabilising treatments provide a potential therapeutic opportunity to reduce the rate of biliary complications and improve clinical outcomes in liver transplantation. Up to 25% of liver transplants result in biliary complications, leading to additional surgery, retransplants, or death. We found that the incidence of biliary complications is increased by damage to the primary cilium, an antenna that protrudes from the cell and is key to regeneration. Here, we show that treatments that preserve the primary cilia during the transplant process provide a potential solution to reduce the rates of biliary complications.

Faulty Gap Filling in Nucleotide Excision Repair Leads to Double-Strand Break Formation in Senescent Cells

The change of repair efficiency of UV-induced pyrimidine dimers due to aging was examined in replicatively senesced fibroblasts. The fibroblasts with repeated passages showed the characteristics of cellular senescence including irreversible cell cycle arrest, elevated β-galactosidase activity and senescence-associated secretory phenotype. The incision efficiency of oligonucleotide containing UV lesions was similar regardless of cell doubling levels, but the gap filling process was impaired in replicatively senescent cells. The releases of XPG, PCNA and RPA from damaged sites were delayed, which might have disturbed the DNA polymerase progression. The persistent single stranded DNA (ssDNA) was likely converted to double strand breaks (DSBs), leading to ATM phosphorylation and 53BP1 foci formation. γ-H2AX induction mainly occurred in G1 phase in senescent cells, not in S phase like in normal cells, indicating replication stress-independent DSBs might be formed. Mre11 having nuclease activity accumulated to damaged sites at early time point after UV irradiation but not released in senescent cells. The pharmacological studies using specific inhibitors for the nuclease activity suggested that Mre11 contributed to the enlargement of ssDNA gap, facilitating the DSB formation.

A Platinum-Aluminum Bimetallic Salen Complex for Pro-senescence Cancer Therapy.

Cell senescence is defined as irreversible cell cycle arrest, which can be triggered by telomere shortening or by various types of genotoxic stress. Induction of senescence is emerging as a new strategy for the treatment of cancer, especially when sequentially combined with a second senolytic drug capable of killing the resulting senescent cells, however severely suffering from the undesired off-target side effects from the senolytic drugs. Here, we prepare a bimetalic platinum-aluminum salen complex (Alumiplatin) for cancer therapy-a combination of pro-senesence chemotherapy with in situ senotherapy to avoid the side effects. The aluminum salen moiety, as a G-quadruplex stabilizer, enhances the salen's ability to induce cancer cell senescence and this phenotype is in turn sensitive to the cytotoxic activity of the monofunctional platinum moiety. It exhibits an excellent capability for inducing senescence, a potent cytotoxic activity against cancer cells both in vitro and in vivo, and an improved safety profile compared to cisplatin. Therefore, Alumiplatin may be a good candidate to be further developed into safe and effective anticancer agents. This novel combination of cell senescence inducers with genotoxic drugs revolutionizes the therapy options of designing multi-targeting anticancer agents to improve the efficacy of anticancer therapies.

Abstract PS12-03: p53 loss enables HR+ breast cancer escape from CDK4/6 inhibitor-induced quiescence via CDK2

Abstract Background: Inhibition of CDK4/6 kinases has led to improved clinical outcomes in hormone receptor positive (HR+) breast cancer. While these are highly effective therapies, only a minority of patients experience long-term disease control. We sought to determine the genomic configurations and underlying mechanisms associated with long-term response. Methods: To identify genomic patterns associated with clinical outcomes, we analyzed a cohort of 447 patients with metastatic HR+ breast cancer treated at MSK with first-line CDK4/6 inhibitors (CDK4/6i) for which tumor-normal sequencing and long-term clinical follow up were available. To identify the pattern of genomic features associated with longer, intermediate, and short response, we implemented an elastic net Cox regression on binary pathogenic variant status of each gene as well as select clinical features (prior endocrine therapy, endocrine therapy partner, de novo metastatic status). Our principal aim was variable interpretability over pure predictive accuracy. Human HR+ breast cancer models including human breast cancer organoids and cell lines were utilized for mechanistic studies. For validation in a clinical setting, we analyzed the association between Ki67 score after neoadjuvant ribociclib plus endocrine therapy and pre-treatment gene mutation from the FELINE trial [NCT02712723] using a Fisher exact test. Results: Our model identified a “longer response” group (n = 124, 27.7%) from patients with a median progression free survival (PFS) of 32.5 months, compared with an “intermediate” (n = 224, 50.1%, median PFS = 13.7 months) and “short response” group (n = 99, 22.1%, median PFS = 5.84 months). TP53 and MDM2 pathogenic variant status were the most important variables to stratify between these groups, obtaining variable selection frequencies of 1.0 and 0.93 and mean hazard ratios of 2.02 and 1.38, respectively. To elucidate the mechanisms whereby the p53 pathway supports long term response, we generated isogenic and patient derived models of TP53 loss or MDM2 overexpression. Using immunoblotting and cell cycle assays, we found that drug-treated p53 KO cells and MDM2 overexpressing cells effectively suppressed RB1 phosphorylation and blocked in G1 after 24-48 hours. However, upon drug withdrawal, these cells could reenter the cell cycle and promote long-term tumor outgrowth. These effects we observed both in vitro and in vivo. Measures of long-term CDK4/6i response such as expression of senescence associated secretory phenotype genes was abrogated by TP53 loss. Mechanistically, we found persistent phosphorylation of the p130 RB1-like protein in the p53 KO cells. Phosphorylation of p130 impaired its interaction with E2F4, thereby blocking DREAM complex assembly and promoting cell cycle reentry. Inhibition of phosphorylation of p130 via p21 overexpression or by selective CDK2 inhibitors could restore irreversible cell cycle arrest in p53 KO cells. The combination of CDK2 and CDK4/6 inhibition led to long-term tumor growth suppression in models with mutant TP53. To validate the human relevance of TP53 mediating CDK4/6i response, we analyzed longitudinal samples from the FELINE trial that evaluated efficacy and feasibility of neoadjuvant ribociclib plus endocrine therapy. Of 45 evaluable patients, 13 (28.9%) harbored a pre-treatment TP53 loss of function variant. Of these 13 cases, 7 (53.8%) did not achieve a low ( &amp;lt; 10%) Ki-67 upon surgical resection as compared to TP53 wildtype tumors (n=32), only one (3.2%) of which did not achieve a low Ki-67 [OR 32.1, 95% CI 3.28 – 1660.3, p = 0.00026]. Conclusion: Loss of p53 was strongly associated with lack of long-term response to CDK4/6i in patients. Complete inhibition of both CDK4/6 and CDK2 appears to be necessary in order to convert quiescent HR+ tumors cells into durably inhibited and effectively dormant cancers. Citation Format: Rei Kudo, Anton Safonov, Edaise M. da Silva, Qing Li, Hong Shao, Marie Will, Harikrishna Nakshatri, Jorge Reis-Filho, Shom Goel, Andrew Koff, Britta Weigelt, Qamar Khan, Pedram Razavi, Sarat Chandarlapaty. p53 loss enables HR+ breast cancer escape from CDK4/6 inhibitor-induced quiescence via CDK2 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-03.

Intermittent oral supplementation with fisetin improves arterial function in doxorubicin chemotherapy-treated mice by suppressing cellular senescence

Introduction. Arterial dysfunction (endothelial dysfunction and aortic stiffening) is a key antecedent to the development of cardiovascular diseases. Cellular senescence, a state of nearly irreversible cell cycle arrest, develops in arteries following doxorubicin (Doxo) chemotherapy treatment and induces arterial dysfunction. Fisetin is a natural senolytic (senescent cell clearing compound) that improves arterial function in old mice. However, it is unknown if fisetin supplementation can decrease cellular senescence to reverse Doxo-induced arterial dysfunction. Purpose. To determine if fisetin supplementation can improve arterial function following Doxo treatment by decreasing cellular senescence. Methods &amp; Results. Cell culture. Human aortic endothelial cells were treated with fisetin for 48 hr (0.25, 0.5, 1 uM) following Doxo-induced cellular senescence (200 nm Doxo for 48 hr). 1 uM fisetin lowered senescence burden (senescence-associated β-galactosidase staining) by ~50% ( P = 0.0003). Animals. Young adult (4 mo) male/female p16-3MR mice received Doxo (10 mg/kg) or sham (saline) intraperitoneally. Four weeks later (established time-point of Doxo-induced arterial dysfunction), animals were treated daily with vehicle (V; 10% EtOH, 30% PEG400, 60% Phosal 50 PG) or fisetin (F; 100 mg/kg) via oral gavage using an intermittent 1 week on - 2 weeks off - 1 week on dosing paradigm, yielding 4 groups (Sham-V; Sham-F; Doxo-V; Doxo-F). Data from Sham-V and Sham-F groups were similar for all outcomes. Data are combined due to no sex differences. Endothelial function. Endothelial function (carotid artery endothelium dependent dilation [EDD] to acetylcholine [ACh]) and nitric oxide (NO)-mediated EDD (± L-NAME) were measured following fisetin (or vehicle) supplementation. Doxo-V mice had lower peak EDD relative to Sham-V (73 ± 2 v. 89 ± 2 %; P = 0.003), and this difference was ameliorated in Doxo-F animals (88 ± 3 %). Likewise, Doxo-V mice had lower NO-mediated EDD compared to Sham-V (39 ± 7% v. 55 ± 8%, P = 0.047), and this difference was ameliorated in Doxo-F mice (66 ± 7%). Incubation of carotid arteries ex vivo with ganciclovir (GCV) to clear p16+ senescent cells in p16-3MR mice normalized peak EDD (Sham-V, 93 ± 1; Doxo-V, 93 ± 2; Doxo-F, 91 ± 2%), suggesting that reducing cellular senescence is the mechanism by which fisetin supplementation improved endothelial function. Aortic stiffness. The Doxo-induced increase in aortic stiffness (aortic pulse wave velocity [PWV]) (Doxo-V, 426 ± 20 vs. Sham-V, 332 ± 5 cm/sec; P &lt; 0.0001) was reversed following fisetin supplementation in Doxo-F mice (pre-fisetin: 426 ± 10 v. post-fisetin: 345 ± 5 cm/sec; P = 0.001). The differences in PWV were associated with corresponding differences in ex vivo aortic intrinsic mechanical wall stiffness (Doxo-V, 5991 ± 362 v. Doxo-F, 3996 ± 415 vs. Sham-V, 4813 ± 133 kPa; P = 0.010). Ex vivo incubation of aorta rings with GCV for 48h ameliorated group differences in aortic intrinsic mechanical wall stiffness, suggesting a causal role for suppression of cellular senescence in the response. Conclusion: Intermittent fisetin supplementation may be a novel strategy to favorably modulate cellular senescence, a key cellular signaling pathway mediating arterial dysfunction with Doxo chemotherapy. R21 AG078408 &amp; K99 HL159241. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Causal analysis on DNA methylation reveals key regulators in aging and senescence

Aging is a multifaceted biological phenomenon influenced by genetic, epigenetic, and environmental factors. Cellular senescence, characterized by an irreversible cell cycle arrest, is a pivotal mechanism contributing to aging and age-related diseases. It is also associated with alterations in DNA methylation, a key epigenetic marker. However, the causal link between senescence-associated DNA methylation changes and aging outcomes remains elusive. In our study, we analyzed three distinct methods to induce cellular senescence and systematically evaluated the resultant DNA methylation profiles. Utilizing Mendelian Randomization, we identified that 4.1% of the methylated loci were significantly associated with aging outcomes and accounted for the majority of the observed variance. Further mediation analysis pinpointed key genes, including CISD2, MARS2, and ETFB, that serve as mediators for these epigenetic effects. Experimental validation using Senescence-Associated-β-Galactosidase (SA-β-gal) staining confirmed that knockdown of these genes triggers cellular senescence. Our findings provide compelling evidence for the causal role of DNA methylation in cellular senescence and its impact on aging.

Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via the induction of cellular senescence

ABSTRACT In chronic liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblast-like cells and produce large amounts of extracellular matrix components, e.g. collagen type 1. Cellular senescence is characterized by irreversible cell-cycle arrest, arrested cell proliferation and the acquisition of the senescence-associated secretory phenotype (SASP) and reversal of HSCs activation. Previous studies reported that H2S prevents induction of senescence via its antioxidant activity. We hypothesized that inhibition of endogenous H2S production induces cellular senescence and reduces activation of HSCs. Rat HSCs were isolated and culture-activated for 7 days. After activation, HSCs treated with H2S slow-releasing donor GYY4137 and/or DL-propargylglycine (DL-PAG), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase (CTH), as well as the PI3K inhibitor LY294002. In our result, CTH expression was significantly increased in fully activated HSCs compared to quiescent HSCs and was also observed in activated stellate cells in a in vivo model of cirrhosis. Inhibition of CTH reduced proliferation and expression of fibrotic markers Col1a1 and Acta2 in HSCs. Concomitantly, DL-PAG increased the cell-cycle arrest markers Cdkn1a (p21), p53 and the SASP marker Il6. Additionally, the number of β-galactosidase positive senescent HSCs was increased. GYY4137 partially restored the proliferation of senescent HSCs and attenuated the DL-PAG-induced senescent phenotype. Inhibition of PI3K partially reversed the senescence phenotype of HSCs induced by DL-PAG. Inhibition of endogenous H2S production reduces HSCs activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results show that cell-specific inhibition of H2S could be a novel target for anti-fibrotic therapy via induced cell senescence.

Dihydroartemisinin eliminates senescent cells by promoting autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.

Cellular senescence is an irreversible cell-cycle arrest in response to a variety of cellular stresses, which contribute to the pathogenesis of a variety of age-related degenerative diseases. However, effective antisenescence strategies are still lacking. Drugs that selectively target senescent cells represent an intriguing therapeutic strategy to delay aging and age-related diseases. Thus, we thought to investigate the effects of dihydroartemisinin (DHA) on senescent cells and elucidated its mechanisms underlying aging. Stress-induced premature senescence (SIPS) model was built in NIH3T3 cells using H2O2 and evaluated by β-galactosidasestaining. Cells were exposed to DHA and subjected to cellular activity assays including viability, ferroptosis, and autophagy. The number of microtubule-associated protein light-chain 3puncta was detected by immunofluorescence staining. The iron content was assessed by spectrophotometer and intracellular reactive oxygen species (ROS) was measured by fluorescent probe dichlorodihydrofluorescein diacetate. We found that DHA triggered senescent cell death via ferroptosis. DHA accelerated ferritin degradation via promoting autophagy, increasing the iron contents, promoting ROS accumulation, thus leading to ferroptotic cell death in SIPS cells. In addition, autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. Moreover, Atg5 silencing and autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. We also revealed that the expression of p-AMP-activated protein kinase (AMPK)and p-mammalian target of rapamycin (mTOR) in senescent cells was downregulated. These results suggested that DHA may be a promising drug candidate for clearing senescent cells by inducing autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.