Senescence-associated Β-galactosidase Activity Research Articles

Long-term demyelination and aging-associated changes in mice corpus callosum; evidence for the role of accelerated aging in remyelination failure in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disorder affecting the central nervous system. Evidence suggests that age-related neurodegeneration contributes to disability progression during the chronic stages of MS. Aging is characterized by decreased regeneration potential and impaired myelin repair in the brain. It is hypothesized that accelerated cellular aging contributes to the functional decline associated with neurodegenerative diseases. We assessed the impact of aging on myelin content in the corpus callosum (CC) and compared aging with the long-term demyelination (LTD) consequents induced by 12 weeks of feeding with a cuprizone (CPZ) diet. Initially, evaluating myelin content in 2-, 6-, and 18-month-old mice revealed a reduction in myelin content, particularly at 18 months. Myelin thickness was decreased and the g-ratio increased in aged mice. Although a lower myelin content and higher g-ratio were observed in LTD model mice, compared to the normally aged mice, both aging and LTD exhibited relatively similar myelin ultrastructure. Our findings provide evidence that LTD exhibits the hallmarks of aging such as elevated expression of senescence-associated genes, mitochondrial dysfunction, and high level of oxidative stress as observed following normal aging. We also investigated the senescence-associated β-galactosidase activity in O4+ late oligodendrocyte progenitor cells (OPCs). The senescent O4+/β-galactosidase+ cells were elevated in the CPZ diet. Our data showed that the myelin degeneration in CC occurs throughout the lifespan, and LTD induced by CPZ accelerates the aging process which may explain the impairment of myelin repair in patients with progressive MS.

Investigating the protective effects of Astragalus polysaccharides on cyclophosphamide-induced bone marrow suppression in mice and bone mesenchymal stem cells

BackgroundThis study determines the role and mechanism of APS in cyclophosphamide-induced myelosuppression in mice and bone mesenchymal stem cells (BMSCs) cell model. MethodsCy-induced myelosuppression mice and BMSCs cell model were established. Fifty C57BL/6 mice (weighing 20 ± 2 g) were randomly divided into five groups. Femur and tibia samples, bone marrow samples, and blood samples were collected 3 days after the last injection of Cy. Histopathology changes and cell apoptosis were detected. Cell viability, apoptosis, cycle distribution, reactive oxygen species activity, osteogenesis ability, and protein levels were detected. γ-H2AX and senescence-associated β-galactosidase activity expression was detected by immunofluorescence. Cy-induced senescence and Wnt/β-catenin related protein levels were detected using western blotting. ResultsThe results showed that APS effectively induced Cy-induced histological injury and cell apoptosis rate. After treated with APS, ROS and ALP levels were significantly increased. In BMSCs, cell viability, apoptosis, and cell cycle distribution were also influenced by APS treatment. Compared with the control group, cell viability was significantly increased, the cell apoptosis rate was decreased while the number of cells remained in the G0-G1 phase was increased. Meanwhile, ROS levels were significantly increased in APS group. Cell senescence and Wnt/β-catenin related protein (γ-H2AX, SA-β-gal, p21, p16, p-β-catenin/ β-catenin, c-Myc, and AXIN2) levels were also altered both in vivo and in vitro. Interestingly, the effects of APS were reversed by BML-284. ConclusionOur results indicate that APS protected Cy-induced myelosuppression through the Wnt/β-catenin pathway and APS is a potential therapeutic drug for Cy-induced myelosuppression.

Neem Leaf Extract Exhibits Anti-Aging and Antioxidant Effects from Yeast to Human Cells.

Neem leaves have long been used in traditional medicine for promoting longevity. However, the precise mechanisms underlying their anti-aging effects remain elusive. In this study, we investigated the impact of neem leaf extract (NLE) extracted from a 50% ethanol solution on the chronological lifespan of Saccharomyces cerevisiae, revealing an extension in lifespan, heightened oxidative stress resistance, and a reduction in reactive oxygen species. To discern the active compounds in NLE, LC/MS and the GNPS platform were employed. The majority of identified active compounds were found to be flavonoids. Subsequently, compound-target pharmacological networks were constructed using the STP and STITCH platforms for both S. cerevisiae and Homo sapiens. GOMF and KEGG enrichment analyses of the predicted targets revealed that "oxidoreductase activity" was among the top enriched terms in both yeast and human cells. These suggested a potential regulation of oxidative stress response (OSR) by NLE. RNA-seq analysis of NLE-treated yeast corroborated the anti-oxidative effect, with "oxidoreductase activity" and "oxidation-reduction process" ranking high in enriched GO terms. Notably, CTT1, encoding catalase, emerged as the most significantly up-regulated gene within the "oxidoreductase activity" cluster. In a ctt1 null mutant, the enhanced oxidative stress resistance and extended lifespan induced by NLE were nullified. For human cells, NLE pretreatment demonstrated a decrease in reactive oxygen species levels and senescence-associated β-galactosidase activity in HeLa cells, indicative of anti-aging and anti-oxidative effects. This study unveils the anti-aging and anti-oxidative properties of NLE while delving into their mechanisms, providing novel insights for pharmacological interventions in aging using phytochemicals.

The role of cellular senescence in neurodegenerative diseases.

Increasing evidence has revealed that cellular senescence drives NDs, including Alzheimer's disease (AD) and Parkinson's disease. Different senescent cell populations secrete senescence-associated secretory phenotypes (SASP), including matrix metalloproteinase-3, interleukin (IL)-1α, IL-6, and IL-8, which can harm adjacent microglia. Moreover, these cells possess high expression levels of senescence hallmarks (p16 and p21) and elevated senescence-associated β-galactosidase activity in in vitro and in vivo ND models. These senescence phenotypes contribute to the deposition of β-amyloid and tau-protein tangles. Selective clearance of senescent cells and SASP regulation by inhibiting p38/mitogen-activated protein kinase and nuclear factor kappa B signaling attenuate β-amyloid load and prevent tau-protein tangle deposition, thereby improving cognitive performance in AD mouse models. In addition, telomere shortening, a cellular senescence biomarker, is associated with increased ND risks. Telomere dysfunction causes cellular senescence, stimulating IL-6, tumor necrosis factor-α, and IL-1β secretions. The forced expression of telomerase activators prevents cellular senescence, yielding considerable neuroprotective effects. This review elucidates the mechanism of cellular senescence in ND pathogenesis, suggesting strategies to eliminate or restore senescent cells to a normal phenotype for treating such diseases.

Targeting pathological cells with senolytic drugs reduces seizures in neurodevelopmental mTOR-related epilepsy.

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated β-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.

Allicin regulates Sestrin2 ubiquitination to affect macrophage autophagy and senescence, thus inhibiting the growth of hepatoma cells

BackgroundAllicin regulates macrophage autophagy and senescence, and inhibits hepatoma cell growth. This study investigated the mechanism by which allicin inhibits the growth of hepatoma cells. MethodsHepa1–6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking. ResultsData analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice. ConclusionAllicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.

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.

Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. We assessed cell morphology, senescence-associated β-galactosidase (SA-β-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-β-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

Downregulation of Histone H3.3 Induces p53-Dependent Cellular Senescence in Human Diploid Fibroblasts.

Cellular senescence is an irreversible growth arrest that acts as a barrier to cancer initiation and progression. Histone alteration is one of the major events during replicative senescence. However, little is known about the function of H3.3 in cellular senescence. Here we found that the downregulation of H3.3 induced growth suppression with senescence-like phenotypes such as senescence-associated heterochromatin foci (SAHF) and β-galactosidase (SA-β-gal) activity. Furthermore, H3.3 depletion induced senescence-like phenotypes with the p53/p21-depedent pathway. In addition, we identified miR-22-3p, tumor suppressive miRNA, as an upstream regulator of the H3F3B (H3 histone, family 3B) gene which is the histone variant H3.3 and replaces conventional H3 in active genes. Therefore, our results reveal for the first time the molecular mechanisms for cellular senescence which are regulated by H3.3 abundance. Taken together, our studies suggest that H3.3 exerts functional roles in regulating cellular senescence and is a promising target for cancer therapy.

Air Pollution Drives Macrophage Senescence through a Phagolysosome-15-Lipoxygenase Pathway.

Urban particulate matter (PM; uPM) poses significant health risks, particularly to the respiratory system. Fine particles, such as PM2.5, can penetrate deep into the lungs and exacerbate a range of health problems, including emphysema, asthma, and lung cancer. PM exposure is also linked to extrapulmonary disorders such as heart and neurodegenerative diseases. Moreover, prolonged exposure to elevated PM levels can reduce overall life expectancy. Senescence is a dysfunctional cell state typically associated with age but can also be precipitated by environmental stressors. This study aimed to determine whether uPM could drive senescence in macrophages, an essential cell type involved in particulate phagocytosis-mediated clearance. Although it is known that uPM exposure impairs immune function, this deficit is multifaceted and incompletely understood, partly because of the use of particulates such as diesel exhaust particles as a surrogate for true uPM. uPM was collected from several locations in the United States, including Baltimore, Houston, and Phoenix. Bone marrow-derived macrophages were stimulated with uPM or reference particulates (e.g., diesel exhaust particles) to assess senescence-related parameters. We report that uPM-exposed bone marrow-derived macrophages adopt a senescent phenotype characterized by increased IL-1α secretion, senescence-associated β-galactosidase activity, and diminished proliferation. Exposure to allergens failed to elicit such a response, supporting a distinction between different types of environmental exposure. uPM-induced senescence was independent of key macrophage activation pathways, specifically inflammasome and scavenger receptors. However, inhibition of the phagolysosome pathway abrogated senescence markers, supporting this phenotype's attribution to uPM phagocytosis. These data suggest that uPM exposure leads to macrophage senescence, which may contribute to immunopathology.

The Combined Anti-Aging Effect of Hydrolyzed Collagen Oligopeptides and Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Human Skin Fibroblasts.

Stem cell-derived exosomes (SC-Exos) are used as a source of regenerative medicine, but certain limitations hinder their uses. The effect of hydrolyzed collagen oligopeptides (HCOPs), a functional ingredient of SC-Exos is not widely known to the general public. We herein evaluated the combined anti-aging effects of HCOPs and exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) using a senescence model established on human skin fibroblasts (HSFs). This study discovered that cells treated with HucMSC-Exos + HCOPs enhanced their proliferative and migratory capabilities; reduced both reactive oxygen species production and senescence-associated β-galactosidase activity; augmented type I and type III collagen expression; attenuated the expression of matrix-degrading metalloproteinases (MMP-1, MMP-3, and MMP-9), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α); and decreased the expression of p16, p21, and p53 as compared with the cells treated with HucMSC-Exos or HCOPs alone. These results suggest a possible strategy for enhancing the skin anti-aging ability of HucMSC-Exos with HCOPs.

Ochratoxin A (OTA) causes intestinal aging damage through the NLRP3 signaling pathway mediated by calcium overload and oxidative stress.

Ochratoxin A (OTA) is a widespread environmental toxin that poses a serious threat to human and animal health. OTA has been shown to cause cellular and tissue damage and is a global public health problem. However, the effects of OTA on gastrointestinal aging have not been reported. The aim of this study was to investigate the effects of OTA on intestinal aging in vitro and in vivo. In vitro experiments showed that OTA induced cellular inflammation through calcium overload and oxidative stress, significantly up-regulated the expression of P16, P21, and P53 proteins, markedly increased senescence-associated β-galactosidase activity (SA-β-gal) positive cells, and obviously decreased the expression of proliferating cell nuclear antigen (PCNA) proteins, which led to intestinal cell senescence. Meanwhile, we found that treatment with β-carotene ameliorated OTA-induced intestinal cell senescence. Consistent with the results of the in vitro experiments, in vivo studies showed that the intestinal aging of mice fed OTA was significantly higher than that of the control group. In conclusion, OTA may induce intestinal aging through calcium overload, oxidative stress and inflammation. This study lays a foundation for further research on the toxicological effects of OTA.

The redox-sensitive GSK3β is a key regulator of glomerular podocyte injury in type 2 diabetic kidney disease

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Involvement of retinoic acid‑inducible gene‑I in radiation‑induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated β-galactosidase (SA-β-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-β-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

PM2.5-induced premature senescence in HUVECs through the SIRT1/PGC-1α/SIRT3 pathway

Vascular endothelial cell senescence plays a pivotal role in the development of atherosclerosis. Recent studies have demonstrated that ambient fine particulate matter (PM2.5) induces stress-induced premature senescence (SIPS) in vascular endothelial cells. However, the precise mechanisms underlying this process remain to be fully elucidated. Cellular senescence is closely associated with reactive oxygen species (ROS), and emerging research has established a strong connection between the SIRT1/PGC-1α/SIRT3 signaling pathway and the antioxidant system in vascular endothelial cells. In this study, we aimed to investigate the impact of PM2.5 on vascular endothelial cell senescence and to elucidate the underlying mechanisms. Our findings revealed that PM2.5 exposure led to an increase in senescence-associated β-galactosidase (SA-β-gal) activity and the expression of the cell cycle-blocking proteins P53/P21 and P16 in human umbilical vein endothelial cells (HUVECs). Flow cytometry analysis demonstrated an elevated proportion of cells arrested in the G0/G1 phase after PM2.5 exposure. In addition, PM2.5-induced cellular senescence was attributed to the disruption of the cellular antioxidative defense system through the SIRT1/PGC-1α/SIRT3 signaling pathway. The expression of cellular senescence markers was reduced after targeted scavenging of mitochondrial ROS using MitoQ. Moreover, treatment with SRT1720, a SIRT1-specific activator, upregulated the SIRT1/PGC-1α/SIRT3 signaling pathway, restored the antioxidant system, and attenuated the expression of cellular senescence markers. Taken together, our results suggest that PM2.5 downregulates the SIRT1/PGC-1α/SIRT3 signaling pathway, resulting in impaired antioxidant defenses in HUVECs. This, in turn, allows for the accumulation of ROS, leading to inhibition of endothelial cell cycle progression and the onset of stress-induced senescence in HUVECs.

Medium-Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease but effective drugs for treatment of AAA are still lacking. Recently, erythropoietin (EPO) is reported to induce AAA formation in apolipoprotein-E knock out (ApoE-/-) mice but an effective antagonistis unknown. In this study, formoterol, a β2 adrenergic receptor (β2AR) agonist, is found to be a promising agent for inhibiting AAA. To test this hypothesis, ApoE-/- mice are treated with vehicle, EPO, and EPO plus low-, medium-, and high-dose formoterol, respectively. The incidence of AAA is 0, 55%, 35%,10%, and 55% in these 5groups, respectively. Mechanistically, senescence of vascular smooth muscle cell (VSMC) is increased by EPO while decreased by medium-dose formoterol both in vivo and in vitro, manifested by the altered expression of senescence biomarkers including phosphorylation of H2AXserine139, senescence-associated β-galactosidase activity, and P21 protein level. In addition, expression of sirtuin 1 (SIRT1) in aorta is decreased in EPO-induced AAA but remarkably elevatedby medium-dose formoterol. Knockdown of β2AR and blockage of cyclic adenosine monophosphate (cAMP) attenuate the inhibitory role of formoterol in EPO-induced VSMC senescence. In summary, medium-dose formoterol attenuates EPO-induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes.

Recently, we reported that oral-epithelial cells (OE) are unique in their response to Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) in that cell cycle arrest (G2/M) occurs without leading to apoptosis. We now demonstrate that Cdt-induced cell cycle arrest in OE has a duration of at least 7 days with no change in viability. Moreover, toxin-treated OE develops a new phenotype consistent with cellular senescence; this includes increased senescence-associated β-galactosidase (SA-β-gal) activity and accumulation of the lipopigment, lipofuscin. Moreover, the cells exhibit a secretory profile associated with cellular senescence known as the senescence-associated secretory phenotype (SASP), which includes IL-6, IL-8 and RANKL. Another unique feature of Cdt-induced OE senescence is disruption of barrier function, as shown by loss of transepithelial electrical resistance and confocal microscopic assessment of primary gingival keratinocyte structure. Finally, we demonstrate that Cdt-induced senescence is dependent upon the host cell protein cellugyrin, a homologue of the synaptic vesicle protein synaptogyrin. Collectively, these observations point to a novel pathogenic outcome in oral epithelium that we propose contributes to both A. actinomycetemcomitans infection and periodontal disease progression.

MDM4 was associated with poor prognosis and tumor-immune infiltration of cancers

MDM4 is one of the MDM protein family and is generally recognized as the key negative regulator of p53. As a cancer-promoting factor, it plays a non-negligible role in tumorigenesis and development. In this article, we analyzed the expression levels of MDM4 in pan-cancer through multiple databases. We also investigated the correlations between MDM4 expression and prognostic value, immune features, genetic mutation, and tumor-related pathways. We found that MDM4 overexpression is often accompanied by adverse clinical features, poor prognosis, oncogenic mutations, tumor-immune infiltration and aberrant activation of oncogenic signaling pathways. We also conducted transcriptomic sequencing to investigate the effect of MDM4 on transcript levels in colon cancer and performed qPCR to verify this. Finally, we carried out some in vitro experiments including colony formation assay, chemoresistance and senescence-associated β-galactosidase activity assay to study the anti-tumor treatment effect of small molecule MDM4 inhibitor, NSC146109. Our research confirmed that MDM4 is a prognostic biomarker and potential therapeutic target for a variety of malignancies.

DOP84 Colonic CD4+ T cell senescence is implicated in the progression of experimental colitis in aged mice

Abstract Background Evidence from recent epidemiological data suggests that the burden of IBD in the elderly is increasing globally. However, the mechanisms underlying the age-related IBD susceptibility remain elusive. Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to autoimmune disease. Herein, we sought to determine the role of immunosenescence in age-dependent colitis and to explore the underlying mechanisms. Methods Young (2-3 months), middle-aged (10-12 months), and aged (20-24 months) mice were established models of colitis by 2% dextran sulfate sodium salt (DSS) treatment. Weight loss, colon length, disease activity indexes (DAI) and histological scores were evaluated to assess the severity of colitis. Using a second-generation fluorogenic substrate for β-galactosidase and multi-parameter flow cytometry, we detected the senescence-associated β-galactosidase (SA-βGal) activity in lymphocytes isolated from colon, including CD4+ T cells, CD8+ T cells, B cells, natural killer cells, microphages, neutrophils and dendritic cells. Based on the immunocytes that exhibit greatest age-related increases in SA-βGal activity, inflammatory cytokines such as TNF-α、IFN-γ、IL-17 were evaluated in senescent immunocytes through flow cytometry. Single-cell and bulk RNA-seq of colonic immune cells in mice with different ages were performed to further investigate the precise molecular mechanisms. Results Ageing increased the severity of DSS-induced colitis wherein aged mice exhibited worsened weight loss, shortened colon length, higher DAI and histological scores compared to young mice. The greatest age-associated increases of SA-βGal activity were observed in colonic CD4+ T cell populations. Additionally, aging resulted in systemic activation of CD4+ T cells, exhibiting an expansion of CD4+ effector memory T (TEM) cells in colon, as well as in spleen. Aged colonic CD4+ T cells generate higher levels of IFN-γ and IL-17 comparing to young colonic CD4+ T cells, especially in status of colitis. Moreover, the accumulated CD4+ TEM cells in the aged colon were the main source of IL-17 production. Single-cell analysis revealed that aging increased colonic CD4+ TEM cells, which were associated with T cell-mediated cytotoxicity and cytokine-mediated signaling pathway. Splenic CD4+ TEM cells isolated from aged colitis mice were confirmed to involve in cellular senescence, Th17 differentiation and inflammatory signaling pathway through bulk RNA-seq. Conclusion These results provide a significant insight into the contribution of senescent CD4+ T cells to age-dependent colitis and provide an attractive possibility that targeting T cell specifically might be a potential strategy to treat elderly IBD patients.