Ganoderic Acid D Protects Human Amniotic Mesenchymal Stem Cells against Oxidative Stress-Induced Senescence through the PERK/NRF2 Signaling Pathway.
ABSTRACT: Aging is an important risk factor in the occurrence of many chronic diseases. Senescence and exhaustion of adult stem cells are considered as a hallmark of aging in organisms. In this study, a senescent human amniotic mesenchymal stem cell (hAMSC) model subjected to oxidative stress was established in vitro using hydrogen peroxide. We investigated the effects of ganoderic acid D (GA-D), a natural triterpenoid compound produced from Ganoderma lucidum, on hAMSC senescence. GA-D significantly inhibited ?-galactosidase (a senescence-associated marker) formation, in a dose-dependent manner, with doses ranging from 0.1??M to 10??M, without inducing cytotoxic side-effects. Furthermore, GA-D markedly inhibited the generation of reactive oxygen species (ROS) and the expression of p21 and p16 proteins, relieved the cell cycle arrest, and enhanced telomerase activity in senescent hAMSCs. Furthermore, GA-D upregulated the expression of phosphorylated protein kinase R- (PKR-) like endoplasmic reticulum kinase (PERK), peroxidase III (PRDX3), and nuclear factor-erythroid 2-related factor (NRF2) and promoted intranuclear transfer of NRF2 in senescent cells. The PERK inhibitor GSK2656157 and/or the NRF2 inhibitor ML385 suppressed the PERK/NRF2 signaling, which was activated by GA-D. They induced a rebound for the generation of ROS and ?-galactosidase-positive cells and attenuated the differentiation capacity. These findings suggest that GA-D retards hAMSC senescence through activation of the PERK/NRF2 signaling pathway and may be a promising candidate for the discovery of antiaging agents.
Project description:BACKGROUND:Mesenchymal stem cells (MSCs) were considered a regenerative therapeutic approach in both acute and chronic diseases. However, whether MSCs regulate the antioxidant metabolism of CD4+ T cells and weaken immunosenescence remains unclear. Here, we reported the protective effects of hPMSCs in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal-induced mouse aging model. METHODS:In vivo study, 40 male C57BL/6 mice (8?weeks) were randomly divided into four groups: control group, D-gal group, hPMSC group, and PBS group. In in vitro experiment, human naive CD4+ T (CD4CD45RA) cells were prepared using a naive CD4+ T cell isolation kit II and pretreated with the Akt inhibitor LY294002 and Nrf2 inhibitor ML385. Then, isolated naive CD4+ T cell were co-cultured with hPMSCs for 72?h in the absence or presence of anti-CD3/CD28 Dynabeads and IL-2 as a mitogenic stimulus. Intracellular ROS changes were detected by flow cytometry. The activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were measured by colorimetric analysis. The senescent T cells were detected SA-?-gal stain. The expression of aging-related proteins was detected by Western blotting, RT-PCR, and confocal microscopy. RESULTS:We found that hPMSC treatment markedly decreased the ROS level, SA-?-gal-positive cells number, senescence-associated secretory phenotype (IL-6 and OPN) expression, and aging-related protein (P16 and P21) expression in senescent CD4+ T cells. Furthermore, hPMSC treatment effectively upregulated Nrf2 nuclear translocation and the expression of downstream target genes (HO-1, CAT, GCLC, and NQO1) in senescent CD4+ T cells. Moreover, in vitro studies revealed that hPMSCs attenuated CD4+ T cell senescence by upregulating the Akt/GSK-3?/Fyn pathway to activate Nrf2 functions. Conversely, the antioxidant effects of hPMSCs were blocked by the Akt inhibitor LY294002 and Nrf2 inhibitor ML385 in senescent CD4+ T cells. CONCLUSIONS:Our results indicate that hPMSCs attenuate D-gal-induced CD4+ T cell senescence by activating Nrf2-mediated antioxidant defenses and that upregulation of Nrf2 by hPMSCs is regulated via the Akt/GSK-3?/Fyn pathway.
Project description:Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-?-galactosidase (?-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased ?-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in ?-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.
Project description:Vascular endothelial cell senescence is a leading cause of age-associated and vascular diseases. Mammalian target of rapamycin complex 2 (mTORC2) is a conserved serine/threonine (Ser/Thr) protein kinase that plays an important regulatory role in various cellular processes. However, its impact on endothelial senescence remains controversial. In this study we investigated the role and molecular mechanisms of mTORC2 in endothelial senescence. A replicative senescence model and H<sub>2</sub>O<sub>2</sub>-induced premature senescence model were established in primary cultured human umbilical vein endothelial cells (HUVECs). In these senescence models, the formation and activation of mTORC2 were significantly increased, evidenced by the increases in binding of Rictor (the essential component of mTORC2) to mTOR, phosphorylation of mTOR at Ser2481 and phosphorylation of Akt (the effector of mTORC2) at Ser473. Knockdown of Rictor or treatment with the Akt inhibitor MK-2206 attenuated senescence-associated ?-galactosidase (?-gal) staining and expression of p53 and p21 proteins in the senescent endothelial cells, suggesting that mTORC2/Akt facilitates endothelial senescence. The effect of mTORC2/Akt on endothelial senescence was due to suppression of nuclear factor erythroid 2-related factor 2 (Nrf2) at the transcriptional level, since knockdown of Rictor reversed the reduction of Nrf2 mRNA expression in endothelial senescence. Furthermore, mTORC2 suppressed the expression of Nrf2 via the Akt/GSK-3?/C/EBP? signaling pathway. These results suggest that the mTORC2/Akt/GSK-3?/C/EBP?/Nrf2 signaling pathway is involved in both replicative and inducible endothelial senescence. The deleterious role of mTORC2 in endothelial cell senescence suggests therapeutic strategies (targeting mTORC2) for aging-associated diseases and vascular diseases.
Project description:Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age-related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence-associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal ?-galactosidase, and this has been exploited as a marker for senescence (senescence-associated ?-galactosidase activity). Consequently, we hypothesized that galactose-modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose-modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal ?-galactosidase (GLB1)-dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole-body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of ?-catenin-positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose-modified duocarmycin prodrugs to treat senescence-related pathologies.
Project description:Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ?400?000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap 'N' Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.
Project description:Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated ?-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6? signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6? in senescent cells. Moreover, ATF6? drives the increase of senescence associated-?-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6? in the establishment of morphological features of senescence in normal human primary fibroblasts.
Project description:It has been over half a century since cellular senescence was first noted and characterized, and yet no consensus senescent marker has been reliably established. This challenge is compounded by the complexity and heterogenic phenotypes of senescent cells. This necessitates the use of multiple biomarkers to confidently characterise senescent cells. Despite cytochemical staining of senescence associated-beta-galactosidase being a single marker approach, as well as being time and labour-intensive, it remains the most popular detection method. We have developed an alternative flow cytometry-based method that simultaneously quantifies multiple senescence markers at a single-cell resolution. In this study, we applied this assay to the quantification of both replicative and induced senescent primary cells. Using this assay, we were able to quantify the activity level of SA ?-galactosidase, the expression level of p16INK4a and ?H2AX in these cell populations. Our results show this flow cytometric approach to be sensitive, robust, and consistent in discriminating senescent cells in different cell senescence models. A strong positive correlation between these commonly- used senescence markers was demonstrated. The method described in this paper can easily be scaled up to accommodate high-throughput screening of senescent cells in applications such as therapeutic cell preparation, and in therapy-induced senescence following cancer treatment.
Project description:Cellular senescence, a state of irreversible growth arrest and altered cell function, causes aging-related diseases. Hence, treatment modalities that could target aging cells would provide a robust therapeutic avenue. Herein, for the first time, we utilized CD9 receptors (overexpressed in senescent cells) for nanoparticle targeting in addition to the inherent ?-galactosidase activity. In our study, CD9 monoclonal antibody-conjugated lactose-wrapped calcium carbonate nanoparticles loaded with rapamycin (CD9-Lac/CaCO3/Rapa) were prepared for targeted rapamycin delivery to senescent cells. The nanoparticles exhibited an appropriate particle size (~130?nm) with high drug-loading capacity (~20%). In vitro drug release was enhanced in the presence of ?-galactosidase suggesting potential cargo drug delivery to the senescent cells. Furthermore, CD9-Lac/CaCO3/Rapa exhibited high uptake and anti-senescence effects (reduced ?-galactosidase and p53/p21/CD9/cyclin D1 expression, reduced population doubling time, enhanced cell proliferation and migration, and prevention of cell cycle arrest) in old human dermal fibroblasts. Importantly, CD9-Lac/CaCO3/Rapa significantly improved the proliferation capability of old cells as suggested by BrdU staining along with significant reductions in senescence-associated secretory phenotypes (IL-6 and IL-1?) (P?<?0.05). Altogether, our findings suggest the potential applicability of CD9-Lac/CaCO3/Rapa in targeted treatment of senescence.
Project description:Staining mice tissues for ?-galactosidase activity is a fundamental tool to detect age- or disease-associated cellular senescence. However, reported analyses of positivity for senescence-associated ?-galactosidase activity or for other markers of senescence in post-mitotic cells of healthy murine tissues have been fragmentary or inconclusive. Here, we attempted to independently deepen this knowledge using multiple senescence markers within the same cells of wild type mice entering middle age (9 months of age). A histochemistry protocol for the pH-dependent detection of ?-galactosidase activity in several tissues was used. At pH 6, routinely utilized to detect senescence-associated ?-galactosidase activity, only specific cellular populations in the mouse body (including Purkinje cells and choroid plexus in the central nervous system) were detected as strongly positive for ?-galactosidase activity. These post-mitotic cells were also positive for other established markers of senescence (p16, p21 and DPP4), detected by immunofluorescence, confirming a potential senescent phenotype. These data might contribute to understanding the functional relation between the senescence-associated ?-galactosidase activity and senescence markers in post-mitotic cells in absence of disease or advanced aging.
Project description:Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.