Expression Of Senescence-associated Secretory Phenotype Factors Research Articles

Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

Hyperinflammatory responses are pivotal in the cardiomyocyte senescence pathophysiology, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted RND3's suppressive effect on IL33 expression. This study aims to explore the role of RND3 in IL33/ST2 signaling activation and in cardiomyocyte senescence. Intramyocardial injection of exogenous IL33 reduces the ejection fraction and fractional shortening of rats, inducing the appearance of senescence-associated secretory phenotype (SASP) in myocardial tissues. Recombinant IL33 treatment of AC16 cardiomyocytes significantly upregulated expression of SASP factors like IL1α, IL6, and MCP1, and increased the p-p65/p65 ratio and proportions of SA-β-gal and γH2AX-positive cells. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) and ST2 antibody astegolimab treatments mitigated above effects. RND3 gene knockout H9C2 cardiomyocytes using CRISPR/Cas9 technology upregulated IL33, ST2L, IL1α, IL6, and MCP1 levels, decreased sST2 levels, and increased SA-β-gal and γH2AX-positive cells. A highly possibility of binding between RND3 and IL33 proteins was showed by molecular docking and co-immunoprecipitation, and loss of RND3 attenuated ubiquitination mediated degradation of IL33; what's more, a panel of ubiquitination regulatory genes closely related to RND3 were screened using qPCR array. In contrast, RND3 overexpression in rats by injection of AAV9-CMV-RND3 particles inhibited IL33, ST2L, IL1α, IL6, and MCP1 expression in cardiac tissues, decreased serum IL33 levels, and increased sST2 levels. These results suggest that RND3 expression in cardiomyocytes modulates cell senescence by inhibiting the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.

GPX4 Over-expression Is Associated with Increased Senescence Burden and Metabolic Dysfunction

Chronic inflammation is a hallmark of obesity, diabetes, and cardiovascular disease (CVD), contributing to morbidity and mortality worldwide. Adipose tissue inflammation is commonly observed in obesity-related diseases and is considered a critical driver of metabolic dysfunction. Cellular senescence, a state of cell cycle arrest, accumulates in adipose tissues in obesity and contributes to tissue inflammation through the production of pro-inflammatory factors known as the SASP (senescence associated secretory phenotype). Novel therapeutics targeting senescent cells have been shown to improve metabolic function in obesity, although the underlying mechanisms of senescent cell accumulation and clearance are not well known. Here, we compared a genetically modified mouse model that overexpresses glutathione peroxidase 4 (GPX4), with wildtype (WT) littermate mice. GPX4 is an antioxidant, selenoenzyme, and a key regulator of ferroptosis, an iron-dependent form of regulated cell death. Previous in vitro studies have shown that activation of ferroptosis through inhibition of GPX4 is associated with a decrease in senescence burden, suggesting that ferroptosis may serve as a novel senolytic pathway. We hypothesized that overexpression of GPX4 in the transgenic (TG) mice will reduce ferroptosis, increase senescence burden, and impair metabolic function compared to WT mice. To determine the effects of ferroptosis on metabolic function, GPX4 WT and TG mice were fed either a 60% high fat diet (HFD) or normal chow (NC) ad libitum for 8-10 weeks. We found higher body, liver, and white adipose tissue (WAT) mass in GPX4 TG compared to WT mice fed NC and HFD (Interaction all p<0.0003, 0.0001, 0.0044, n=8-10/group). After HFD, T cell infiltration in the WAT was higher in GPX4 TG mice compared to WT (Interaction all p<0.0253), and both CD8 and CD4 were significantly different from NC controls, contributing to metabolic dysfunction in these animals (p=0.027,0.207, n=4/group). Mice fed HFD demonstrated increased glucose intolerance compared to NC controls (p=0.029, p=0.0216, n=8-12/group), however, the TG HFD mice were trending towards higher glucose intolerance compared to WT HFD (p=0.17). Compared to NC and HFD fed WT mice, there was increased gene expression for the senescence markers, p16 (all p<0.0041) and p21(all p<0.002), as well as increased expression of SASP factors, CXCL2 (all p<0.0055), TNFa (all p<0.0159), and PAI1 (all p<0.05, n=5-7/group) in the HFD fed TG mice, indicating increased senescence burden in the TG HFD mice. Malondialdehyde (MDA), a byproduct and marker of ferroptosis, was elevated in the HFD fed WT mice (p=0.0002) compared to all other groups, indicating increased ferroptosis. HFD fed TG mice also had increased expression of ferritin (all p<0.0079, n=5/group) compared to all other groups, suggesting resistance to ferroptosis that may contribute to the increased senescence burden in the TG HFD group. Together these data demonstrate that increases in cellular senescence and impairments in metabolic function after HFD are exacerbated by increased GPX4 expression, suggesting that GPX4 may be a novel target to decrease senescence and improve metabolic function through induction of the ferroptosis pathway. Funded in part by awards from National Institute of Health Awards R01 AG 060395, R01 AG048366, T32 HL139451-05, T32 HL007576, R01 AG077751, Nora Eccles Treadwell Foundation (NEFT), and Veteran's Affairs Merit Review Award I01 BX004492. 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.

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 β-galactosidase (SA-β-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.

Cellular senescence promotes meibomian gland dysfunction in a chronic graft-versus-host disease mouse model

PurposeAging is a well-established risk factor for meibomian gland dysfunction (MGD). We previously reported an accelerated cellular senescence phenomenon in the lacrimal glands of a murine model of chronic graft-versus-host disease (cGVHD). Herein, we aimed to elucidate the relationship between cellular senescence and MGD in cGVHD mice, utilizing the senolytic agent ABT-263. MethodsA cGVHD mouse model was established through allogeneic bone marrow transplantation (BMT) from B10.D2 to BALB/c mice. Subsequently, cGVHD mice were treated with either ABT-263 or vehicle. The eyelids of recipients were analyzed at 4-week intervals post-BMT in both groups. ResultsMeibomian gland (MG) area was significantly smaller in cGVHD mice than in syngeneic control mice. ABT-263-treated mice retained a significantly larger MG area than their vehicle-treated counterparts. Pathological and immunohistochemical examinations revealed significant reductions in eyelid tissue inflammation and pathological fibrosis in the ABT-263 group compared to that in the vehicle-treated group. Additionally, expression of DNA damage markers, senescent cell markers, and senescence-associated secretory phenotype (SASP) factors was elevated in the eyelids of cGVHD mice compared with that in syngeneic mice. The expression of these cellular senescence-associated molecules was considerably suppressed in ABT-263-treated eyelids compared to that in vehicle-treated ones. ConclusionsCellular senescence, along with expression of SASP factors, exhibited increased activity in the eyelids, particularly in the MGs of cGVHD mice. ABT-263 mitigated the severity of MGD. These findings highlight the potential of targeting cellular senescence as an effective approach for MGD treatment in cGVHD.

Bazi Bushen capsule improves the deterioration of the intestinal barrier function by inhibiting NLRP3 inflammasome-mediated pyroptosis through microbiota-gut-brain axis.

The senescence-accelerated prone mouse 8 (SAMP8) is a widely used model for accelerating aging, especially in central aging. Mounting evidence indicates that the microbiota-gut-brain axis may be involved in the pathogenesis and progression of central aging-related diseases. This study aims to investigate whether Bazi Bushen capsule (BZBS) attenuates the deterioration of the intestinal function in the central aging animal model. In our study, the SAMP8 mice were randomly divided into the model group, the BZ-low group (0.5 g/kg/d BZBS), the BZ-high group (1 g/kg/d BZBS) and the RAPA group (2 mg/kg/d rapamycin). Age-matched SAMR1 mice were used as the control group. Next, cognitive function was detected through Nissl staining and two-photon microscopy. The gut microbiota composition of fecal samples was analyzed by 16S rRNA gene sequencing. The Ileum tissue morphology was observed by hematoxylin and eosin staining, and the intestinal barrier function was observed by immunofluorescence. The expression of senescence-associated secretory phenotype (SASP) factors, including P53, TNF-α, NF-κB, IL-4, IL-6, and IL-10 was measured by real-time quantitative PCR. Macrophage infiltration and the proliferation and differentiation of intestinal cells were assessed by immunohistochemistry. We also detected the inflammasome and pyroptosis levels in ileum tissue by western blotting. BZBS improved the cognitive function and neuronal density of SAMP8 mice. BZBS also restored the intestinal villus structure and barrier function, which were damaged in SAMP8 mice. BZBS reduced the expression of SASP factors and the infiltration of macrophages in the ileum tissues, indicating a lower level of inflammation. BZBS enhanced the proliferation and differentiation of intestinal cells, which are essential for maintaining intestinal homeostasis. BZBS modulated the gut microbiota composition, by which BZBS inhibited the activation of inflammasomes and pyroptosis in the intestine. BZBS could restore the dysbiosis of the gut microbiota and prevent the deterioration of intestinal barrier function by inhibiting NLRP3 inflammasome-mediated pyroptosis. These results suggested that BZBS attenuated the cognitive aging of SAMP8 mice, at least partially, by targeting the microbiota-gut-brain axis.

NAD+ associated genes as potential biomarkers for predicting the prognosis of gastric cancer.

Nicotinamide adenine dinucleotide (NAD+) plays an essential role in cellular metabolism, mitochondrial homeostasis, inflammation, and senescence. However, the role of NAD+-regulated genes, including coding and long non-coding genes in cancer development is poorly understood. We constructed a prediction model based on the expression level of NAD+ metabolism-related genes (NMRGs). Furthermore, we validated the expression of NMRGs in gastric cancer (GC) tissues and cell lines; additionally, β-nicotinamide mononucleotide (NMN), a precursor of NAD+, was used to treat the GC cell lines to analyze its effects on the expression level of NMRGs lncRNAs and cellular proliferation, cell cycle, apoptosis, and senescence-associated secretory phenotype (SASP). A total of 13 NMRGs-related lncRNAs were selected to construct prognostic risk signatures, and patients with high-risk scores had a poor prognosis. Some immune checkpoint genes were upregulated in the high-risk group. In addition, cell cycle, epigenetics, and senescence were significantly downregulated in the high-risk group. Notably, we found that the levels of immune cell infiltration, including CD8 T cells, CD4 naïve T cells, CD4 memory-activated T cells, B memory cells, and naïve B cells, were significantly associated with risk scores. Furthermore, the treatment of NMN showed increased proliferation of AGS and MKN45 cells. In addition, the expression of SASP factors (IL6, IL8, IL10, TGF-β, and TNF-α) was significantly decreased after NMN treatment. We conclude that the lncRNAs associated with NAD+ metabolism can potentially be used as biomarkers for predicting clinical outcomes of GC patients.

Atractylenolide III suppresses senescence-associated secretome via inhibiting cGAS/NF-κB pathway in hepatic stellate cells.

Senescence of activated hepatic stellate cells (aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression. Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). Induction of aHSCs senescence by inhibiting SASP may be a potential therapeutic model against hepatic fibrosis. To evaluate the role of atractylenolide III (ATR III) in the development of chemotherapeutic drug-induced SASPs in hepatic stellate cells. Etoposide-induced senescent HSC-LX2 model was established and treated with ATR III at different concentrations (20, 30 and 40 μM). We found that ATR III dose-dependently enhanced senescence in etoposide-induced LX2 cells. ATR III dose-dependently decreased the release and expression of SASP factors (interleukin [IL]-1α, IL-1β, IL6 and IL-8) in senescent cells. ATR III regulated cyclic GMP-AMP synthase (cGAS)/nuclear factor κ (NF-κB) signalling to affect SASP expression in senescent cells. The addition of 2'3' cGAMP counteracted the effect of ATR III. The release of SASP factors in the conditioned medium from senescent cells could affect cell migration, proliferation and contraction through paracrine manner. Our results indicated ATR III could still enter senescence and prevent the production of SASP and its paracrine effects in senescent cells, an effect that may be related to the possible inhibition of cGAS/NF-κB signalling by ATR III. Our study proves that ATR III may be an effective potential drug against liver fibrosis by promoting aHSC senescence, which can provide a new choice for the future clinical treatment of liver fibrosis.

Senescent RAW264.7 cells exhibit increased production of nitric oxide and release inducible nitric oxide synthase in exosomes.

Aging cells not only cease growing, but also secrete various proteins such as inflammatory cytokines. This secretory phenomenon is known as the senescence‑associated secretory phenotype (SASP). The aim of the present study was to elucidate the effects of senescence on the differentiation of osteoclast precursors (OCPs) and corresponding SASP. RAW264.7 cells were used as OCPs and were cultured to passage (P)5, P10 and P20. Cell proliferation assays, senescence‑associated β‑galactosidase staining and telomere length quantification were subsequently performed, and it was revealed that replicative senescence was induced at P20. In addition, the level of tartrate‑resistant acid phosphatase activity in P20 cells treated with receptor activator of nuclear factor‑κB ligand was significantly lower than that in P5 and P10 cells. The SASP factors interleukin‑6, tumour necrosis factor‑α and nitric oxide were significantly increased in P20 culture supernatants compared with those in P5 and P10 supernatants. Furthermore, the number of exosomes at P20 was increased compared with that at P5 and P10, and inducible nitric oxide synthase (iNOS) was expressed in exosomes at P20, but not in exosomes at P5. In conclusion, the present study revealed that senescent RAW264.7 cells exhibit increased expression of SASP factors and release iNOS in exosomes.

Senescence of donor cells impairs fat graft regeneration by suppressing adipogenesis and increasing expression of senescence-associated secretory phenotype factors

BackgroundFat grafting has been regarded as a promising approach for regenerative therapy. Given the rapidly aging population, better understanding of the effect of age on fat graft outcomes and the underlying mechanisms is urgently needed.MethodsC57/BL6 mice [old (O, 18–20-month-old) and young (Y, 4-month-old)] were randomized to four fat graft groups [old-to-old (O-O), young-to-young (Y-Y), old-to-young (O-Y), and young-to-old (Y-O)]. Detailed cellular events before and after grafting were investigated by histological staining, RNA sequencing, and real-time polymerase chain reaction. The adipogenic differentiation potential of adipose-derived mesenchymal stem cells (AD-MSCs) from old or young donors was investigated in vitro. Additionally, adipogenesis of AD-MSCs derived from old recipients was evaluated in the culture supernatant of old or young donor fat tissue.ResultsAfter 12 weeks, the volume of fat grafts did not significantly differ between the O-O and O-Y groups or between the Y-Y and Y-O groups, but was significantly smaller in the O-O group than in the Y-O group and in the O-Y group than in the Y-Y group. Compared with fat tissue from young mice, senescence-associated secretory phenotype (SASP) factors were upregulated in fat tissue from old mice. Compared with the Y-O group, adipogenesis markers were downregulated in the O-O group, while SASP factors including interleukin (IL)-6, tumor necrosis factor-α, and IL-1β were upregulated. In vitro, AD-MSCs from old donors showed impaired adipogenesis compared with AD-MSCs from young donors. Additionally, compared with the culture supernatant of young donor fat tissue, the culture supernatant of old donor fat tissue significantly decreased adipogenesis of AD-MSCs derived from old recipients, which might be attributable to increased levels of SASP factors.ConclusionsAge has detrimental effects on fat graft outcomes by suppressing adipogenesis of AD-MSCs and upregulating expression of SASP factors, and fat graft outcomes are more dependent on donor age than on recipient age. Thus, rejuvenating fat grafts from old donors or banking younger adipose tissue for later use may be potential approaches to improve fat graft outcomes in older adults.

Whole-body senescent cell clearance alleviates age-related brain inflammation and cognitive impairment in mice.

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation.

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

N-Acetyltransferase 10 Promotes Micronuclei Formation to Activate the Senescence-Associated Secretory Phenotype Machinery in Colorectal Cancer Cells

The formation of micronuclei (MN) is prevalent in human cancer cells and its role in activating the senescence-associated secretory phenotype (SASP) machinery has been identified recently. However, the role of MN in regulation of SASP signaling still needs to define in practical cancers. Here, we reported that in colorectal cancer cells the expression of NAT10 (N-acetyltransferase 10) could mediate MN formation through DNA replication and NAT10-positive MN could activate SASP by binding to cGAS. The chemical inhibition of NAT10 by Remodelin or genomic depletion could markedly reduce MN formation, SASP activation, and senescence in colorectal cancer cells. Cell stress such as oxidative or hypoxia could upregulate NAT10 and its associated MN formation senescence and expression of SASP factors. Statistical analysis of clinical specimens revealed correlations between NAT10 expression, MN formation, SASP signaling, and the clinicopathological features of colorectal cancer. Our data suggest that NAT10 increasing MN formation and SASP pathway activation, promoting colorectal cancer progression.

The role of lamin B receptor in the regulation of senescence-associated secretory phenotype (SASP)

Cellular senescence is a phenomenon of irreversible growth arrest of mammalian somatic cells. Senescent cells increase the production of secretory proteins such as inflammatory cytokines, a phenomenon termed senescence-associated secretory phenotype (SASP). SASP is known to have profound effects on organismal health and aging; however, the molecular mechanisms of SASP are not precisely understood. In our previous studies, we have shown that senescent cells show decreased function of lamin B receptor (LBR), a nuclear membrane protein that regulates heterochromatin organization. Here we examined the implication of LBR in the regulation of SASP because senescent cells show altered heterochromatin organization, which would affect gene expression. We found that knock-down of LBR up-regulated the expression of the SASP factors such as IL-6, IL-8, and MMP1 in HeLa cells, even though cellular senescence was not induced by LBR knock-down. Conversely, enforced expression of LBR suppressed their up-regulated expression in senescent cells induced by excess thymidine. Further, our gene expression profile analysis also showed that many secretory proteins were up-regulated by LBR knock-down. We then analyzed the regulatory mechanisms of the expression of SASP factors by LBR, and found that the promoters of these SASP factors associated with LBR in normally growing cells, but dissociated from it in senescent cells. Additionally, we found that enforced expression of LBR decreased the generation of cytoplasmic DNA, which could be involved in SASP, in senescent cells. These findings suggested that LBR would play crucial roles in the regulation of SASP.

Abstract 895: The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts

Abstract Background: Secreted factors from cancer associated fibroblast (CAFs) is likely to be implicated in cancer progression, but the precise mechanism is not fully understood. It is reported that senescent cells survive and secrete some cytokines, so called senescence-associated secretory phenotype (SASP). The purpose of current study is to identify the molecular mechanism underlying the progression of gastric cancer (GC) peritoneal dissemination by CAFs showing SASP. Methods: We examined the senescent state of CAFs activated NFkB signaling by inflammatory cytokines (iCAFs) and evaluated the expression of SASP factors. We investigated the growth ability of GC cell lines treated with iCAFs derived condition medium (CM). We transplanted GC cell lines with intraperitoneal administration to generate peritoneal metastasis mouse model and evaluated the influence of SASP factors on cancer progression. Results: We found that the growth ability of iCAFs decreased and their expression of p16 and p15, which was senescent markers increased. And the comprehensive genomic analysis revealed that iCAFs was highly enriched the senescent related genes and SASP factors related genes. Next, we also found that iCAFs derived CM significantly increased the growth ability of GC cells. Moreover, also in peritoneal metastasis mouse model, iCAFs derived CM remarkably enhanced intraperitoneal tumors. Conclusion: These findings suggest that SASP factors from senescent CAFs, which activated by inflammatory cytokines can promote peritoneal dissemination of GC cells. Citation Format: Tadahito Yasuda, Mayu Koiwa, Tomoyuki Uchihara, Atsuko Yonemura. The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 895.

Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro

Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.

Cytoplasmic [rad]OH scavenger TA293 attenuates cellular senescence and fibrosis by activating macrophages through oxidized phospholipids/TLR4

AimsElucidation of the biological roles of the mitochondrial and cytoplasmic hydroxyl radical (cyto OH) is hampered by the absence of site-specific OH scavengers. Earlier findings using cyto OH scavenger, TA293, indicated that cyto OH causes cellular senescence, and senescence-associated secretory phenotype (SASP) factors secreted from cells cause macrophage infiltration, inflammation, and apoptosis. However, we found that macrophage infiltration occurs before senescent cells appear. We therefore aimed to elucidate how cyto OH-induces macrophage activation and investigate the mechanism by which activated macrophages cause oxidative stress, inflammation, and apoptosis. Main methodsIn vivo imaging of pyocyanin- and TA293-treated, macrophage-depleted Toll-like receptor 4-knockout (TLR4−/−) OKD48- and IDOL-Tg mouse models were used to visualize oxidative stress and inflammation. SA-β-gal and TUNEL staining were used to detect cellular senescence and apoptosis. The mRNA expression of SASP factors were quantified by qRT-PCR. Activation mechanism of cyto OH-mediated macrophages was studied by an ex vivo analysis that created macrophage-activated oxidized phospholipids (OxPLs) using TLR4−/− mice. Key findingsCyto OH produced OxPLs that acted as TLR4 ligands, resulting in macrophage activation. Macrophages were not involved in oxidative stress in tissues or with oxidative damage caused by cyto OH, but significantly exacerbated cellular senescence, inflammation, apoptosis, and fibrosis. SignificanceWe present a novel mechanism by which cyto OH-induced macrophage activation exacerbates cellular senescence, inflammation, apoptosis, and fibrosis independently from the known cyto OH-induced cellular senescence pathway. Notably, through suppression of this pathway, TA293 shows promise as a therapeutic agent to prevent fibrosis caused by cyto OH-induced oxidative stress.

L-Carnitine inhibits the senescence-associated secretory phenotype of aging adipose tissue by JNK/p53 pathway.

Senescence-associated secretory phenotype (SASP) plays a role in aging adipose tissue dysfunction by directly promoting chronic inflammation. The JNK/p53 pathway was reported as a potential mechanism that mediates SASP. In this study, we investigated the effects of L-carnitine, an inhibitor of the JNK/p53 pathway in adipose tissue SASP and dysfunction. Young and aging rat were given L-carnitine by gavage. Next, we detected the senescence, cytokines expression, chronic inflammation and insulin resistance of adipose tissue. Additionally, JNK/p53 pathway was estimated. Our results show a significant increase expression of SASP components in the adipose tissue of aging rats compared to young rats. Further, we found that infiltration of immune cells and the expression of pro-inflammatory cytokines were enhanced in aging adipose tissue while insulin signaling activity was reduced in aging adipose tissue. Interestingly, L-carnitine markedly reduced the expression of SASP factors. L-Carnitine could significantly reduce chronic inflammation, improving insulin resistance. Further, L-carnitine inhibited SASP by inhibiting JNK/p53 pathway. L-Carnitine inhibited SASP by JNK/p53 pathway and attenuated adipose tissue dysfunction of aging.

Abstract 466: Cellular senescence drives skin carcinogenesis

Abstract Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer, and has a high rate of recurrence and metastasis. Systemic genotoxic chemotherapeutic drugs such as doxorubicin (Doxo) can be used to treat SCC, but these agents can have deleterious long-term side effects, including fueling the development of more aggressive cancers. Some of the adverse effects of genotoxic chemotherapies might be due to their ability to induce cellular senescence. Cellular senescence is a tumor suppressive mechanism that entails a permanent cell growth arrest accompanied by several metabolic changes. Senescent cells increase during aging, and accumulating evidence suggests they can contribute to a variety of age related pathologies, including cancer, by secreting numerous pro-inflammatory molecules, growth factors and proteases, a feature termed the senescence-associated secretory phenotype (SASP). We recently showed that genotoxic and cytotoxic chemotherapeutic agents induce senescence and a SASP in vivo. However, a direct role for senescent cells in skin carcinogenesis in vivo remains unexplored. To investigate the role of senescent cells and the SASP in squamous cell skin carcinogenesis, we first induced senescence with Doxo in p16-3MR transgenic mice. These mice are designed to permit the selective elimination of senescent cells expressing the established senescence marker p16INK4a (p16) by administrating ganciclovir (GCV) which has a high affinity for the herpes simplex viral thymidine kinase (HSV-TK). Phosphorylated GCV is a DNA chain terminator that kills HSV-TK expressing cells by apoptosis. After Doxo treatment, we initiated skin SCC in p16-3MR mice with 7, 12-dimethylbenz[a]anthracene (DMBA), followed by treatment with 12-O-Tetradecanoylphorbol-13-acetate (TPA) to promote skin carcinogenesis. 25 weeks after Doxo treatment, we observed a significant increase in p16 mRNA levels as well as an increase in expression of the SASP factors IL-1α, MMP-9, and IL-17 in the skin of Doxo- compared to PBS- treated mice. Further, the presence of senescent cells resulted in significantly larger and more proliferative tumors compared to PBS-treated mice. Importantly, systemic elimination of Doxo-induced senescent cells using GCV reduced the expression of SASP factors and significantly reduced tumor size, suggesting that senescent cells fuel skin tumor progression. These findings will help our understanding of the role of chemotherapy-induced cellular senescence in skin carcinogenesis and will pave the way for developing novel therapeutics against squamous cell carcinoma and the deleterious side effects of genotoxic chemotherapies. Citation Format: Fatouma Alimirah, Alexis Valdovinos, Emily Chang, Sena Alptekin, Tanya Pulido, Elijah Jones, Chandani Limbad, Michael Velarde, Marco Demaria, Albert Davalos, Judith Campisi. Cellular senescence drives skin carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 466.

Gut Microbiota Promotes Obesity-Associated Liver Cancer through PGE2-Mediated Suppression of Antitumor Immunity.

Obesity increases the risk of cancers, including hepatocellular carcinomas (HCC). However, the precise molecular mechanisms through which obesity promotes HCC development are still unclear. Recent studies have shown that gut microbiota may influence liver diseases by transferring its metabolites and components. Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA), a Gram-positive gut microbial component, promotes HCC development by creating a tumor-promoting microenvironment. LTA enhances the senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) collaboratively with an obesity-induced gut microbial metabolite, deoxycholic acid, to upregulate the expression of SASP factors and COX2 through Toll-like receptor 2. Interestingly, COX2-mediated prostaglandin E2 (PGE2) production suppresses the antitumor immunity through a PTGER4 receptor, thereby contributing to HCC progression. Moreover, COX2 overexpression and excess PGE2 production were detected in HSCs in human HCCs with noncirrhotic, nonalcoholic steatohepatitis (NASH), indicating that a similar mechanism could function in humans.Significance: We showed the importance of the gut-liver axis in obesity-associated HCC. The gut microbiota-driven COX2 pathway produced the lipid mediator PGE2 in senescent HSCs in the tumor microenvironment, which plays a pivotal role in suppressing antitumor immunity, suggesting that PGE2 and its receptor may be novel therapeutic targets for noncirrhotic NASH-associated HCC. Cancer Discov; 7(5); 522-38. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 443.