Senescence-associated Β-galactosidase Activity Research Articles

Etoposide Triggers Cellular Senescence by Inducing Multiple Centrosomes and Primary Cilia in Adrenocortical Tumor Cells.

Etoposide (ETO) has been used in treating adrenocortical tumor (ACT) cells. Our previous study showed that ETO inhibits ACT cell growth. In the present study, we show that ETO treatment at IC50 (10 μM) inhibited ACT cell growth by inducing cellular senescence rather than apoptosis. Several markers of cellular senescence, including enlarged nuclei, activated senescence-associated β-galactosidase activity, elevated levels of p53 and p21, and down-regulation of Lamin B1, were observed. We further found that ETO induced multiple centrosomes. The inhibition of multiple centrosomes accomplished by treating cells with either roscovitine or centrinone or through the overexpression of NR5A1/SF-1 alleviated ETO-induced senescence, suggesting that ETO triggered senescence via multiple centrosomes. Primary cilia also played a role in ETO-induced senescence. In the mechanism, DNA-PK-Chk2 signaling was activated by ETO treatment; inhibition of this signaling cascade alleviated multiple ETO-induced centrosomes and primary cilia followed by reducing cellular senescence. In addition to DNA damage signaling, autophagy was also triggered by ETO treatment for centrosomal events and senescence. Importantly, the inactivation of DNA-PK-Chk2 signaling reduced ETO-triggered autophagy; however, the inhibition of autophagy did not affect DNA-PK-Chk2 activation. Thus, ETO activated the DNA-PK-Chk2 cascade to facilitate autophagy. The activated autophagy further induced multiple centrosomes and primary cilia followed by triggering senescence.

Inhibition of SIRT1 Limits Self-Renewal and Oncogenesis by Inducing Senescence of Liver Cancer Stem Cells

PurposeCancer stem cells (CSCs) have been considered involving in tumorigenesis, local recurrence, and therapeutic drug resistance of hepatocellular carcinoma (HCC). To investigate novel and effective methods for targeting hepatic CSCs is crucial for a permanent cure of liver cancer.MethodsThe expression level of SIRT1 was detected in CSCs of HCC tissues and cancer cell lines. Expression of CSC markers, the self-renewal and tumorigenic ability of liver CSCs were analyzed with SIRT1 inhibition. Cellular senescence-related markers were used to detect CSCs senescence after inhibition of SIRT1.ResultsSIRT1 was highly expressed in CSCs of HCC cell lines and human HCC tissues. In vitro study revealed that decreasing of SIRT1 level significantly downregulated the stemness-associated genes of liver CSCs and reduced the CSC stemness properties. Also, downregulated SIRT1 suppressed liver CSCs proliferation by decreasing their self-renewal abilities. Furthermore, CSCs with decreased SIRT1 expression showed limited tumorigenicity and formed smaller HCC tumor in vivo. And SIRT1 decreased CSCs became more susceptible to chemotherapeutic drugs. Mechanistically, SIRT1 decreased CSCs became senescence through the activation of p53-p21 and p16 pathway. The data further indicated that the tumor formed from SIRT1-knockdown CSCs exhibited higher senescence-associated β-galactosidase (SA-β-Gal) activity but lower proliferative capacity.ConclusionTaken together, these findings pointed that induction of senescence in liver CSCs is an effective tumor suppression method for HCC, and SIRT1 may be served as a promising target for HCC treatment.

Antiretroviral Tenofovir Induces Senescence-Associated β-Galactosidase Activity in Primary Human Brain Vascular Cells in Multi-Layer Three-Dimensional Co-Culture

IntroductionIn the current context of early diagnosis of HIV infection, immediate initiation of antiretroviral (ARV) therapy, and lifelong chronic treatment, the potential ARV toxicity is of particular concern. Emtricitabine (FTC) and tenofovir (TFV) are commonly used as backbone drugs in ARV regimens recommended for initial therapy of HIV infection. Here we assessed the effects of FTC and TFV exposure on senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence, in human brain vascular cells.DesignMulti-layer three-dimensional cell co-cultures and in vitro assays.MethodsTo mimic the small vessel wall structure in vivo, three types of primary human brain vascular cells (endothelial cells, smooth muscle cells, and pericytes) were co-cultured on three Alvetex Scaffold disks placed on top of each other in order (three-layer three-dimensional cell co-cultures) and exposed to clinically relevant concentrations of ARV drugs (FTC, TFV, or FTC+TFV combination) or vehicle for eight days (four or five biological replicates per condition, 18 replicates totally). The SA-β-Gal activity was quantitatively assayed in vitro by using the chemiluminescent Galacto-Star System (T1012; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) in 54 protein lysates extracted from individual cell-culture disks. Three-factor analysis of variance (cell type, FTC, TFV) was used to assess differences in the SA-β-Gal activity levels normalized by the corresponding total protein concentrations.ResultsThere was a trend for the FTC by TFV interaction effect on SA-β-Gal activity (P = 0.058). The effects of FTC and TFV were not significantly different among the three cell types. The overall effect of FTC was not significant when controlling for TFV and cell type. The overall effect of TFV was significant when controlling for FTC and cell type (F(1,48) = 30.61, P < 0.001, partial η2 = 0.389). In the absence of FTC, TFV raised SA-β-Gal activity by 0.631 units on average, regardless of cell type (P < 0.001, partial η2 = 0.368). In the presence of FTC, TFV raised SA-β-Gal activity by 0.303 units on average, regardless of cell type (P = 0.015, partial η2 = 0.118).ConclusionOur preliminary findings suggest that primary human brain vascular cells exposed to TFV at clinically relevant concentrations undergo cellular senescence. This potential adverse effect of TFV should be further studied in animal models of HIV infection.

PTH Protects Osteocytes From Oxidative Stress and Cellular Senescence

Age-induced osteoporosis is characterized by a progressive decline in bone formation and increase in bone resorption with uncoupled activities of osteoblasts and osteoclasts. Parathyroid hormone (PTH) is used in the clinic to treat osteoporosis due to its anabolic actions on bone via binding to the PTH receptor (PPR). The receptor is highly expressed in cells of the osteoblastic lineage, including osteocytes. Osteocytes are the most abundant cells in bone and serve as a key regulator of bone remodeling. Despite the significant role of PPR signaling in skeletal homeostasis, its function in osteocytes during aging remains unclear. We have gathered preliminary data demonstrating that mice lacking PPR predominantly in osteocytes (Dmp1-PPRKO) have marked age-induced bone loss due to increased bone resorption and suppressed bone formation. These mice, with aging, develop characteristics of skeletal senescence: a decrease in osteoprogenitors and an increase in bone marrow adiposity and p16Ink4a/Cdkn2a expression in bone. Since senescence of cells in the bone microenvironment has been reported as a cause of age-induced bone loss, we hypothesized that PPR signaling protects osteocytes from senescence. To test this hypothesis, we generated osteocytes (Ocy454-12H), in which the PPR expression was ablated using CRISPR/Cas9 technique. Ocy454-12H-PPRKO and Ocy454-12H-PPRCtrl cells were treated with PTH followed by an exposure to hydrogen peroxide (H2O2). High levels of intracellular reactive oxygen species (ROS), including H2O2, promote protein and DNA oxidation, resulting in cell death and senescence. PTH treatment significantly suppressed the increase in H2O2-induced cell death, measured by resazurin-based assays, in PPRCtrl but not in PPRKO cells. We analyzed intracellular ROS levels using a fluorescent probe and found that PTH treatment significantly suppressed the increase in ROS upon H2O2 exposure, suggesting an antioxidant function of PTH in osteocytes. To further investigate if PTH prevents osteocytes from oxidative stress-induced senescence, we examined senescence-associated β-galactosidase (SA β-gal) activity in cells that were treated with PTH followed by an exposure to low doses of H2O2. Compared to untreated and PPRKO groups, treatment with PTH significantly decreased the number of SA β-gal positive cells, demonstrating that PPR signaling protects osteocytes, and possibly other osteoblastic cells, from H2O2-induced cellular senescence. PTH treatment reduced mRNA expression of p21/Cdkn1a. Taken together these results demonstrate that PPR signaling is important to protect osteocytes from cellular senescence.

Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood CD8+ cells during healthy aging

Abstract Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo, and if it has a role in immunosenesence, remains controversial due to the lack of specific markers to identify these cells. Human CD8+ T cells from younger and older donors were isolated from blood and labeled with a fluorescent substrate for senescence-associated b-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of CD8+ T cells with high SA-βGal activity in the older cohort. SA-βGal high CD8+ T cells displayed increased p21, p16INK4a, and inflammatory cytokine transcripts. They also had greater DNA damage response foci and p16INK4a protein and had deficient proliferation. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed cells with low, medium, and high SA-βGal had distinct profiles. In addition, SA-βGal high CD8+ T cells had larger mitochondrial mass than cells with low SA-βGal as measured by MitoID staining. This may indicate differences in metabolism, which may contribute to senescence. The presence of p16INK4a, DNA damage, and inflammatory cytokines and deficient proliferation demonstrate that SA-βGal is a strong indicator of cellular senescence in CD8+. T cells. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence.

Cellular senescence as a target to inhibit epithelial‐mesenchymal transition in HK‐2 cells

Cellular senescence is a program that induces a stable growth arrest accompanied by metabolic reprogramming, phenotypic changes, increased senescence-associated β-galactosidase (SA-β-gal) activity, and secretion of senescence-associated secretory phenotype. It has been demonstrated that cellular senescence is associated with epithelial-mesenchymal transition (EMT) and chronic kidney disease, and higher SA-β-gal activity were found in proximal and distal tubules of feline chronic kidney disease (CKD). Studies showed that elimination of senescent cells by FOXO4 DRI, a peptide antagonist to block the interaction of forkhead box O 4 (FOXO4) and p53, improved kidney function in aging mice. The represent study tested the hypothesis that inhibition of cellular senescence alleviates TGF-β1-induced EMT in human renal epithelial (HK-2) cells. Cultured HK-2 cells were divided into 3 groups: Ctrl, TGF-β1 and FOXO4 DRI+TGF-β1. Western blot analysis showed that a senescent biomarker, p16, was significantly higher in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups (1±0.15, 2±0.1, 0.96±0.15 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). Another important senescent indicator, SA-β-gal staining, was also significantly increased in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups(1±0.27, 7.2±0.39, 4.6±0.38 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). Meanwhile, Western blot analysis showed that the EMT markers, α-SMA and collagen I/III, were significantly higher in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups (α-SMA：1±0.05, 1.4±0.05, 0.96±0.1 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05; and collagen I/III: 1±0.03, 1.49±0.06, 0.91±0.1 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). These results suggest that targeting cellular senescence might be a potential therapeutic strategy for CKD.

Senescence-associated β-galactosidase reveals the abundance of senescent CD8+ T cells in aging humans.

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.

Edible Bird’s Nest Extract Reduced Expression of Senescence Markers in Bone Marrow Mesenchymal Stem Cells

Edible bird’s nest (EBN) is often consumed as a health food due to its suggested health benefits, including anti-aging effects, however the mechanism is still unknown. This study investigated the effect of EBN extract using long term expansion bone marrow-derived mesenchymal stem cells (BMMSCs) as an aging model. Passage 5 (P5) and passage 8 (P8) BMMSCs were treated with EBN extract, and their proliferation, senescence-associated β-galactosidase (SA-β-Gal) activity, and expression of p16 INK4a were analyzed. Treatment of BMMSCs with EBN extract decreased population doubling time (PDT) in P5 but not in P8 BMMSCs. In P5 BMMSCs, 200 ppm EBN extract increased BMMSCs proliferation, with PDT reduced by 27.6%. However, 200 ppm EBN extracts did not affect P8 BMMSCs proliferation, although it increased BMMSCs viability. Treatment of P5 and P8 BMMSCs with 200 ppm EBN extract decreased SA-β-Gal activity by 54.8% and 47.1% of the control, respectively (P&lt;0.05). Levels of p16 INK4a expression were 5.4-fold lower in P5 BMMSCs treated with 200 ppm EBN extract compared to control (P&lt;0.05). Similarly, treatment of P8 BMMSCs with 200 ppm EBN extract reduced p16 INK4a mRNA level by 7.9-fold compared to the control (P&lt;0.05). In order to investigate the pathway of EBN extract inhibition, we further analyzed IL-6 and NF-κB1 expression. Treatment of P5 and P8 BMMSCs with 200 ppm EBN extract reduced IL-6 mRNA levels by 7.9-fold and 2.1-fold of control, respectively (P&lt;0.05). We found that 200 ppm EBN extract reduced NF-κB1 mRNA level approximately 2.4-fold both in P5 and P8 BMMSCs (P&lt;0.05). Thus, EBN extract reduces markers of senescence, indicated by decreased SA-β-Gal activity and p16 INK4a mRNA level, and this correlated with reduced messenger RNA levels of the pro-inflammatory factor IL-6 and the transcription factor NF-κB1.

Gastrodiae rhizoma attenuates brain aging via promoting neuritogenesis and neurodifferentiation

BackgroundGastrodiae Rhizoma (Tianma), the dried tuber of Gastrodia elata Bl. (Orchidaceae), is listed as a top-grade herbal medicine in Shen-nong Ben-ts'ao Jing and has been used for treating headaches, dizziness, vertigo and convulsion. It has a neuroprotective effect and extends the lifespan in mouse models of Huntington's disease and Niemann-Pick type C disease. However, its effect on senescence remains unknown. PurposeThis study aimed to investigate the anti-aging effects and the underlying mechanism of Gastrodiae Rhizoma. MethodsD-galactose (D-gal)- and BeSO4-induced cellular senescence and senescence-associated β-galactosidase (SA-β-gal) activity were evaluated in SH-SY5Y and PC12 cells. D-gal–induced aging mice were used as an in vivo model. Animal behaviors including nesting and burrowing and Morris water maze were conducted. Neurogenesis in the hippocampus was assessed by immunohistochemistry and confocal microscopy, and the aging-related proteins were assessed by Western blot analysis. The potential neuritogenesis activity of the partially purified fraction of Gastrodiae Rhizoma (TM-2) and its major ingredients were investigated in PC12 cells. ResultsTM-2 could improve D-gal-induced learning and memory impairement by inhibiting oxidative stress, increasing hippocampal neurogenesis and regulating the SH2B1-Akt pathway. Moreover, N6-(4-hydroxybenzyl)adenine riboside (T1-11) and parishins A and B, three constituents of TM-2, had anti-aging activity, as did T1-11 and parishin A induced neuritogenesis. ConclusionOur data suggested that TM-2 slowed down D-gal-induced cellular and mouse brain aging. These results indicate that Gastrodiae Rhizoma has a beneficial effect on senescence. It may be used for neuroprotection and promoting neurogenesis.

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

POS-385 CXCR2 Plays a Role in Promoting Renal Fibrosis by Accelerating Tubular Cell Senescence

Renal fibrosis is the common pathologic feature for all kinds of chronic kidney diseases (CKD). The premature aging in renal tubular cells plays an important role in the progression of renal fibrosis. However, the underlying mechanisms of tubular senescence have not been elucidated. In this study, we explored the role of CXCR2 in tubular senescence. The expression of CXCR2 in the kidneys of various CKD animal models was assessed. Mitochondrial function, tubular cellular senescence, and renal fibrosis were analyzed in CXCR2-overexpressed or IL-8, a CXCR2 ligand-treated CKD mice and tubular cells. A siRNA strategy or specific inhibitor to CXCR2 was also adopted. CXCR2 was highly upregulated in various CKD models, and colocalized with the senescence-related protein p16INK4A. CXCR2 expression was level intimately correlated with the extent of renal fibrosis, mitochondrial dysfunction, and p16INK4A expression. Furthermore, ectopic expression of CXCR2 in unilateral ischemia-reperfusion injury (UIRI) and unilateral ureteral obstruction (UUO) induced further damage of mitochondrial function and exacerbated renal fibrosis. Activation of CXCR2 via the ligand, IL-8 binding exacerbated tubular senescence, and mitochondrial dysfunction which were evidenced by decrease in mitochondrial biogenesis-related transcription factors peroxisome proliferator-activated receptor-coactivator-1α (PGC-1α) and transcription factor A, mitochondrial (TFAM), increase in p16INK4Aexpression and senescence-associated β-galactosidase (SA-β-gal) activity in vivo and vitro. Conversely, SB225002, a specific inhibitor of CXCR2, repressed UUO-induced renal fibrosis and impeded senescence in cultured tubular epithelial cells. In UIRI mice, CXCR2-accelerated renal fibrosis was inhibited by shRNA-mediated silencing of p16INK4A. Overexpression of CXCR2 remarkably downregulated expressional levels of PGC-1α and TFAM, but upregulated levels of senescence-related p16INK4A and γH2AX, and promoted fibrosis-related morphology changes in tubular cells. Thus, CXCR2 drives renal fibrosis through inducing tubular senescence. The underlying mechanism is related to its regulation on mitochondrial dynamic. Although the more specific signaling mechanisms need to be clarified, our study still provides an important clue showing CXCR2 as a potential target for the prevention and treatment of CKD.

Exercise myokine counteracts chondrocyte senescence and osteoarthritis development through mitochondrial metabolism reprogramming

Purpose: Chondrocyte senescence accelerates apoptosis and extracellular matrix loss, accelerating articular cartilage deterioration in the development of osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5), is an important exercise myokine to slow age-related diseases, like sarcopenia, osteoporosis, obesity, and neural degeneration, etc. Little is known about whether the molecule affect chondrocyte fate or cartilage metabolism in OA joints. This study aimed to investigate what role FNDC5 may play in chondrocyte aging and OA development in human and in mice. Methods: Osteoarthritic cartilage and non-OA cartilage in the leftover articular specimens were biopsied from patients with knee OA who required total knee replacement. FNDC5 transgenic mice were received destabilized medial meniscus (DMM) to induce OA. Articular cartilage damage, osteophyte formation, kinematics, and pain behavior of injured legs were probed using OARSI score, μCT imaging, catwalk, and von Frey filaments test. Senescence-associated β-galactosidase (SA-β-gal) activity, aging makers, and inflammatory cytokines were quantified using biochemical staining and RTPCR. Mitochondrial metabolites and biology were quantified using liquid chromatography-tandem mass spectrometry, seahorse extracellular influx, and laser confocal microscopy. Results: A plenty of SA-β-gal-stained senescent chondrocytes together with weak FNDC5 immunostaining were present in human osteoarthritic cartilage, as compared with the non-OA cartilage. FNDC5 transgenic (FNDC5Tg) mice showed thickened articular cartilage, increased chondrocytes, high subchondral bone mass, and well woven trabecular microstructure. DMM-injured knee joints displayed less OA signs, including articular cartilage erosion, synovitis, and osteophyte formation. FNDC5 overexpression compromised DMM-mediated pain score and irregular walking patterns and repressed gait profiles of injured legs. Likewise, intra-articular injection with Irisin, a soluble form of FNDC5, slowed OA development in DMM-injured legs. In vitro, FNDC5 overexpression reversed IL-1β-inhibited mitochondrial respiration, ATP production, mitochondrial fusion, and mitophagy. FNDC5 improved mitochondrial α-ketoglutarate production, a key TCA cycle metabolite, repressing senescence program (SA-β-gal activity, p16, p21 expression, and IL-6 production) and retaining ECM production in inflamed chondrocytes. Conclusions: FDNC5 loss is correlate with chondrocyte aging in the development of OA in human and in mice. The myokine sustains chondrocytic activity through sustaining mitochondrial TCA cycle metabolism and biology to protect against inflammation-induced senescence. This study shines light onto the mitochondrial mechanism underlying the chondroprotective actions of FNDC5 to articular integrity. Collective investigations also highlight the remedial effects of FNDC5 and Irisin for OA development.

Inhibitory role of ginsenoside Rb2 in endothelial senescence and inflammation mediated by microRNA‑216a.

Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR-216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF-κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR-216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR-216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR-216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre-miR-216a recombinant lentiviruses (Lv-miR-216a) and the number of population-doubling level (PDL) was calculated. Stable overexpression of miR-216a induced a premature senescent-like phenotype, whereas the senescent features and increased activity of senescence-associated β-galactosidase (SA-β-gal) were reversed after Rb2 treatment. The percentage of SA-β-gal-positive cells in senescent PDL25 cells transfected with Lv-miR-216a was decreased 76% by Rb2 treatment compared with the Lv-miR-216a group without Rb2 treatment (P=0.01). Mechanistically, miR-216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF-κB-responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro-inflammatory effects of miR-216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR-216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti-inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR-216a.

Two-Dimensional Design Strategy to Construct Smart Fluorescent Probes for the Precise Tracking of Senescence.

The tracking of cellular senescence usually depends on the detection of senescence-associated β-galactosidase (SA-β-gal). Previous probes for SA-β-gal with this purpose only cover a single dimension: the accumulation of this enzyme in lysosomes. However, this is insufficient to determine the destiny of senescence because endogenous β-gal enriched in lysosomes is not only related to senescence, but also to some other physiological processes. To address this issue, we introduce our fluorescent probes including a second dimension: lysosomal pH, since de-acidification is a unique feature of the lysosomes in senescent cells. With this novel design, our probes achieved excellent discrimination of SA-β-gal from cancer-associated β-gal, which enables them to track cellular senescence as well as tissue aging more precisely. Our crystal structures of a model enzyme E. coli β-gal mutant (E537Q) complexed with each probe further revealed the structural basis for probe recognition.

Rapamycin Treatment of Tendon Stem/Progenitor Cells Reduces Cellular Senescence by Upregulating Autophagy.

The elderly population is prone to tendinopathy due to aging-related tendon changes such as cellular senescence and a decreased ability to modulate inflammation. Aging can render tendon stem/progenitor cells (TSCs) into premature senescence. We investigated the effects of rapamycin, a specific mTOR inhibitor, on the senescence of TSCs. We first showed that after treatment with bleomycin in vitro, rat patellar TSCs (PTSCs) underwent senescence, characterized by morphological alterations, induction of senescence-associated β-galactosidase (SA-β-gal) activity, and an increase in p53, p21, and p62 protein expression. Senescence of PTSCs was also characterized by the elevated expression of MMP-13 and TNF-α genes, both of which are molecular hallmarks of chronic tendinopathy. We then showed that rapamycin treatment was able to reverse the above senescent phenotypes and increase autophagy in the senescent PTSCs. The activation of autophagy and senescence rescue was, at least partly, due to the translocation of HMGB1 from the nucleus to the cytosol that functions as an autophagy promoter. By reducing TSC senescence, rapamycin may be used as a therapeutic to inhibit tendinopathy development in the aging population by promoting autophagy.

Sirtuin 1 ameliorates defenestration in hepatic sinusoidal endothelial cells during liver fibrosis via inhibiting stress-induced premature senescence.

ObjectivePremature senescence is related to progerin and involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the mechanisms of premature senescence in defenestration of hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects HSECs fenestrae remain elusive.MethodsWe employed the CCl4‐induced liver fibrogenesis rat models and cultured primary HSECs in vitro, administered with the SIRT1‐adenovirus vector, the activator of SIRT1 and knockdown NOX2. We measured the activity of senescence‐associated β‐galactosidase (SA‐β‐gal) in HSECs. Meanwhile, the protein expression of SIRT1, NOX2, progerin, Lamin A/C, Ac p53 K381 and total p53 was detected by Western blot, co‐immunoprecipitation and immunofluorescence.ResultsIn vivo, premature senescence was triggered by oxidative stress during CCl4‐induced HSECs defenestration and liver fibrogenesis, whereas overexpressing SIRT1 with adenovirus vector lessened premature senescence to relieve CCl4‐induced HSECs defenestration and liver fibrosis. In vitro, HSECs fenestrae disappeared, with emerging progerin‐associated premature senescence; these effects were aggravated by H2O2. Nevertheless, knockdown of NOX2, activation of SIRT1 with resveratrol and SIRT1‐adenovirus vector inhibited progerin‐associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co‐localized with the abnormal accumulation of actin filament (F‐actin) in the nuclear envelope of H2O2‐treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1.ConclusionSIRT1‐mediated deacetylation maintains HSECs fenestrae and attenuates liver fibrogenesis through inhibiting oxidative stress‐induced premature senescence.

Senolytic Targeting of Bcl-2 Anti-Apoptotic Family Increases Cell Death in Irradiated Sarcoma Cells.

Simple SummaryLimited volumetric change after pre-operative radiotherapy (RT) suggests that sarcomas generally do not undergo cell death. Senolytic drugs represent a highly promising field as a new therapy approach to drive senescent cancer cells towards cell death to enhance treatment response. Here, we demonstrate that the Bcl-2 family of anti-apoptotic proteins in irradiated senescent sarcoma cells represents a senotherapeutic target to improve the cell death response in RT. This study paves the way for new treatment options in soft tissue sarcoma management.Radiotherapy (RT) is a key component of cancer treatment. Most of the time, radiation is given after surgery but for soft-tissue sarcomas (STS), pre-surgical radiation is commonly utilized. However, despite improvements in RT accuracy, the rate of local recurrence remains high and is the major cause of death for patients with STS. A better understanding of cell fates in response to RT could provide new therapeutic options to enhance tumour cell killing by RT and facilitate surgical resection. Here, we showed that irradiated STS cell cultures do not die but instead undergo therapy-induced senescence (TIS), which is characterized by proliferation arrest, senescence-associated β-galactosidase activity, secretion of inflammatory cytokines and persistent DNA damage. STS-TIS was also associated with increased levels of the anti-apoptotic Bcl-2 family of proteins which rendered cells targetable using senolytic Bcl-2 inhibitors. As oppose to radiation alone, the addition of senolytic agents Venetoclax (ABT-199) or Navitoclax (ABT-263) after irradiation induced a rapid apoptotic cell death in STS monolayer cultures and in a more complex three-dimensional culture model. Together, these data suggest a new promising therapeutic approach for sarcoma patients who receive neoadjuvant RT. The addition of senolytic agents to radiation treatments may significantly reduce tumour volume prior to surgery and thereby improve the clinical outcome of patients.

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence—as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

Anti-senescence and anti-wrinkle activities of 3—bromo—4,5—dihydroxybenzaldehyde from Polysiphonia morrowii Harvey in human dermal fibroblasts

Objective: To investigate the anti-senescence effect of 3-bromo-4,5- dihydroxybenzaldehyde (BDB) from Polysiphonia morrowii Harvey in human dermal fibroblasts (HDF). Methods: HDF were subjected to treatment of BDB and then treated with hydrogen peroxide (H2O2) to induce premature senescence. Senescence-associated β-galactosidase (SA-β-gal) activity in HDF was determined using the SA-β-gal staining method. Intracellular reactive oxygen species (ROS) production was measured using the 2’,7’-dichlorodihydrofluorescein diacetate assay. Western blotting assay was performed to assess the level of antioxidant enzyme glutathione peroxidase 1 (GPX1). In addition, intracellular collagen and collagenase contents were analyzed using the respective ELISA kits. Elastase activity in HDF supernatants was measured from p-nitroaniline release and normalized using total protein content. Results: Treatment of HDF with H2O2 increased the activity of SAP-gal, but BDB pre-treatment resulted in the reduction of SA-β- gal activity. Moreover, BDB significantly reduced H2O2-induced intracellular ROS production. BDB also markedly increased the level of GPX1, which was inhibited by 400 µM of H2O2. Furthermore, in in vitro study, BDB significantly increased intracellular collagen content and decreased matrix metalloproteinase-1 and elastase activities in HDF. Conclusions: Our results demonstrate that BDB shows anti- senescence and anti-wrinkle activities in vitro.

Detection of Protein Aggregation and Proteotoxicity Induced by Angiotensin II in Vascular Smooth Muscle Cells.

Disruption of protein quality control occurs with aging and cardiovascular pathologies including arterial stiffness and hypertension. Angiotensin II (Ang II) is believed to induce endoplasmic reticulum stress in vascular smooth muscle cells (VSMCs), thus contributing to vascular remodeling and dysfunction. However, whether Ang II increases formation of protein aggregates and mediates proteotoxicity in VSMCs remain obscure. Accordingly, this study aimed to establish a quantitative method of protein aggregate detection induced by Ang II and to investigate their potential involvement in inflammatory and senescence responses. Proteostat staining showed increased aggregate numbers per cell on Ang II exposure. Immunoblot analysis further showed an increase in preamyloid oligomer presence in a detergent insoluble protein fraction purified from VSMCs stimulated with Ang II. Moreover, these responses were attenuated by treatment with chemical chaperone, 4-phenylbutyrate. 4-phenylbutyrate further blocked Ang II-induced senescence associated β-galactosidase activity and THP-1 monocyte adhesion in VSMCs. These data suggest that Ang II induces proteotoxicity in VSMCs which likely contributes to aging and inflammation in the vasculature.