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Lin K, Luo X, Du C, Zuo C, Li Z, Zhang G, Li C, Zhu L. ANRIL modulates endothelial senescence and angiogenesis through SASP-driven miR146a regulation in age-related vascular dysfunction. Mech Ageing Dev 2025; 225:112058. [PMID: 40222710 DOI: 10.1016/j.mad.2025.112058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2025] [Revised: 03/25/2025] [Accepted: 04/08/2025] [Indexed: 04/15/2025]
Abstract
Vascular aging, marked by endothelial cell (EC) dysfunction and compromised angiogenesis, is a central driver of age-related ischemic diseases. Although lncRNAs have emerged as pivotal regulators of endothelial function, their specific roles in endothelial aging remain enigmatic. In this study, we identify the lncRNA ANRIL as a crucial modulator of endothelial dysfunction during aging. By analyzing publicly available lncRNA sequencing datasets comparing young and old ECs, we pinpointed ANRIL and validated its role through a replicative senescence model in human umbilical vein ECs (HUVECs) and FACS sorting of skeletal muscle ECs from aged mice. While ANRIL showed minimal direct effects on angiogenesis, functional assays and transcriptomic analysis revealed its profound impact on the senescence-associated secretory phenotype (SASP). Remarkably, ANRIL regulates the expression of miR146a in ECs, which is transferred to macrophages, where it inhibits VEGF secretion and disrupts endothelial neovascularization. In vivo, ANRIL downregulation in a murine hindlimb ischemia model significantly enhanced neovascularization and restored blood flow, revealing its therapeutic potential for ischemic diseases. These findings position ANRIL as a novel, potent regulator of endothelial senescence, offering new insights into the molecular basis of vascular aging and suggesting ANRIL as a promising therapeutic target to mitigate age-related vascular dysfunction.
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Affiliation(s)
- Kechuan Lin
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China; The Third Xiangya Hospital of Central South University, Changsha, Hunan 410008, China
| | - Xin Luo
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China
| | - Can Du
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China
| | - Chenzhe Zuo
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China
| | - Zhenyu Li
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China
| | - Guogang Zhang
- National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China; The Third Xiangya Hospital of Central South University, Changsha, Hunan 410008, China
| | - Chuanchang Li
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China
| | - Lingping Zhu
- Department of geriatric, Coronary Circulation Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China; National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan 410008, China.
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Prasad K, Kaul SC, Wadhwa R, Guruprasad KP, Satyamoorthy K. Cellular oxidative stress and sirtuins mediate regulation of senescence and neuronal differentiation by withaferin A. Free Radic Biol Med 2025; 233:174-185. [PMID: 40154756 DOI: 10.1016/j.freeradbiomed.2025.03.038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2025] [Revised: 03/21/2025] [Accepted: 03/25/2025] [Indexed: 04/01/2025]
Abstract
Withaferin A (WA) and Withanone (WN), the steroidal lactones are pharmacologically established for anticancer and chemopreventive effects in certain cancers. However, their effects on redox modulations, mechanisms stimulating senescence and neuronal differentiation in neuroblastoma cells are less understood. Here we examined the influence of WA on perturbations in the molecular architecture of growth, differentiation and senescence of human brain cancer cell SH SY5Y in vitro and test its efficacy in mouse tumor models. We found senescence induction amplified by WA as determined by a senescence-associated β-galactosidase assay. This led us to evaluate DNA damage which was enhanced as measured by phospho-γH2AX foci formation, directed by reactive oxygen species (ROS) production as determined by flow cytometry and confocal imaging. Furthermore, we assessed the influence of DNA damage on cell cycle arrest and DNA repair. Neurosphere formation assay was performed to demonstrate the stem cell inhibitory potential of WA. Subcutaneous xenograft of neuroblastoma cells in athymic Balb/c mice was performed followed by treatment with WA and tumor growth inhibition was established. Withania somnifera (WS) extract and WA induced alterations in ROS, triggering DNA damage and concomitantly regulated SIRTs expression leading to activation of senescence in SH SY5Y cells. Upon prolonged incubation, differentiation into neuronal lineages was confirmed by using differentiation markers such as neurofilament medium, nestin, MAP2 and synaptophysin as measured by immunofluorescence and flow cytometry. The results suggest a complex interplay between the induction of senescence and concurrent neuronal differentiation of SH SY5Y cells mediated by early alterations in SIRT1 and SIRT3. Thus, we report the senescence and differentiation potential of WS extracts and WA through ROS that are mediated via modulation of SIRT1, SIRT3 and mitochondria function.
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Affiliation(s)
- Keshava Prasad
- Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, India.
| | - Sunil C Kaul
- AIST-INDIA DAILAB, DBT-AIST International Center for Translational & Environmental Research (DAICENTER), National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, 305-8565, Japan
| | - Renu Wadhwa
- AIST-INDIA DAILAB, DBT-AIST International Center for Translational & Environmental Research (DAICENTER), National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, 305-8565, Japan
| | - Kanive P Guruprasad
- Centre for Ayurvedic Biology, Department of Ageing Research, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, India
| | - Kapaettu Satyamoorthy
- SDM Centre for Cellular and Molecular Sciences, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara (SDM) University, Manjushree Nagar, Sattur, Dharwad, Karnataka, 580009, India.
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Zhang W, Cheung TH. Detection of Cellular Senescence on Murine Muscle Tissue Sections by Senescence-Associated β-Galactosidase Staining. Methods Mol Biol 2025. [PMID: 40172829 DOI: 10.1007/7651_2025_612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/04/2025]
Abstract
Staining for the presence of senescence-associated beta-galactosidase (SA-β-gal) serves as a crucial indicator for cellular senescence. This staining assay is based on the elevated activity of β-galactosidase in senescent cells, which cleaves the X-Gal substrate to produce an insoluble blue product in acidic conditions. This enables X-Gal to be visualized using microscopy. In this study, we describe the identification of SA-β-gal+ cells within murine muscle tissues.
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Affiliation(s)
- Wenxin Zhang
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Daniel and Mayce Yu Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Tom H Cheung
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Daniel and Mayce Yu Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China.
- Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China.
- Guangdong Provincial Key Laboratory of Brain Science, Disease and Drug Development, Shenzhen-Hong Kong Institute of Brain Science, HKUST Shenzhen Research Institute, Shenzhen, China.
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Morgado LAL, Rodrigues LMZ, Silva DCF, da Silva BD, Irigoyen MCC, Takano APC. NF-κB-Specific Suppression in Cardiomyocytes Unveils Aging-Associated Responses in Cardiac Tissue. Biomedicines 2025; 13:224. [PMID: 39857807 PMCID: PMC11762954 DOI: 10.3390/biomedicines13010224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2024] [Revised: 01/10/2025] [Accepted: 01/12/2025] [Indexed: 01/27/2025] Open
Abstract
Background/Objectives: Aging is associated with structural and functional changes in the heart, including hypertrophy, fibrosis, and impaired contractility. Cellular mechanisms such as senescence, telomere shortening, and DNA damage contribute to these processes. Nuclear factor kappa B (NF-κB) has been implicated in mediating cellular responses in aging tissues, and increased NF-κB expression has been observed in the hearts of aging rodents. Therefore, NF-κB is suspected to play an important regulatory role in the cellular and molecular processes occurring in the heart during aging. This study investigates the in vivo role of NF-κB in aging-related cardiac alterations, focusing on senescence and associated cellular events. Methods: Young and old wild-type (WT) and transgenic male mice with cardiomyocyte-specific NF-κB suppression (3M) were used to assess cardiac function, morphology, senescence markers, lipofuscin deposition, DNA damage, and apoptosis. Results: Kaplan-Meier analysis revealed reduced survival in 3M mice compared to WT. Echocardiography showed evidence of eccentric hypertrophy, and both diastolic and systolic dysfunction in 3M mice. Both aged WT and 3M mice exhibited cardiac hypertrophy, with more pronounced hypertrophic changes in cardiomyocytes from 3M mice. Additionally, cardiac fibrosis, senescence-associated β-galactosidase activity, p21 protein expression, and DNA damage (marked by phosphorylated H2A.X) were elevated in aged WT and both young and aged 3M mice. Conclusions: The suppression of NF-κB in cardiomyocytes leads to pronounced cardiac remodeling, dysfunction, and cellular damage associated with the aging process. These findings suggest that NF-κB plays a critical regulatory role in cardiac aging, influencing both cellular senescence and molecular damage pathways. This has important implications for the development of therapeutic strategies aimed at mitigating age-related cardiovascular diseases.
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Affiliation(s)
| | | | | | - Bruno Durante da Silva
- Unidade de Hipertensao, Instituto do Coracao, Hospital das Clinicas, Faculdade de Medicina, Universidade de Sao Paulo (InCor-HCFMUSP), Sao Paulo 05403-000, Brazil
| | - Maria Claudia Costa Irigoyen
- Unidade de Hipertensao, Instituto do Coracao, Hospital das Clinicas, Faculdade de Medicina, Universidade de Sao Paulo (InCor-HCFMUSP), Sao Paulo 05403-000, Brazil
| | - Ana Paula Cremasco Takano
- Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil
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Makalakshmi MK, Banerjee A, Pathak S, Paul S, Sharma NR, Anandan B. A pilot study on the efficacy of a telomerase activator in regulating the proliferation of A375 skin cancer cell line. Mol Biol Rep 2024; 52:69. [PMID: 39704853 DOI: 10.1007/s11033-024-10161-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Accepted: 12/09/2024] [Indexed: 12/21/2024]
Abstract
INTRODUCTION The changes in histone modifications are linked to the progression of benign and normal tissue to malignancy. Thus, numerous findings suggest that targeting epigenetic factors might be a focus for anti-cancer treatment. In this study, we tested the hypothesis that telomerase activator might be a potential epigenetic regulator in combatting skin cancer cell proliferation. METHODS Melanoma cell line A375 cells were treated with telomerase activator TA-65. Cell senescence assay was done to evaluate the senescence induction. Morphological changes and differences in expression of HDACs and hTERT genes were studied. Further, hyaluronidase and anti-oxidant assays were also performed. Additionally, telomerase enzyme and 20S proteasome activity was also studied. RESULTS Morphological changes were observed in treated cells and it is evident that telomerase activator induced cellular senescence in high concentrations. From our results, it is evident that HDAC8 and HDAC10 expression was upregulated, whereas hTERT gene expression was downregulated in treated groups. This suggests that the telomerase activator has a regulatory role in skin cancer cells proliferation by targeting the epigenetic factors. CONCLUSION Targeting HDACs and hTERT in the treatment of melanoma is a prominent concern. In our current study, we highlight the most recent research, although in its initial stage, involving various epigenetic factors involved in melanoma cells proliferation.
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Affiliation(s)
- M K Makalakshmi
- Medical Biotechnology, Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, India
| | - Antara Banerjee
- Medical Biotechnology, Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, India.
- Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, 603103, India.
| | - Surajit Pathak
- Medical Biotechnology, Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, India.
- Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, 603103, India.
| | - Sujay Paul
- Tecnologico de Monterrey, School of Engineering and Sciences, Campus Queretaro, Av. Epigmenio Gonzalez, No. 500 Fracc. San Pablo, Querétaro, CP 76130, Mexico
| | - Neeta Raj Sharma
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
| | - B Anandan
- Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamilnadu, 600113, India.
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KUSHIDA C, TAMURA N, KASASHIMA Y, SATO K, ARAI K. Characterization of senescent mesenchymal stem/stromal cells derived from equine bone marrow and the effects of NANOG on the senescent phenotypes. J Vet Med Sci 2024; 86:930-937. [PMID: 38972751 PMCID: PMC11422694 DOI: 10.1292/jvms.24-0161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Accepted: 06/27/2024] [Indexed: 07/09/2024] Open
Abstract
In equine regenerative medicine using bone marrow-derived mesenchymal stem/stromal cells (BM-MSC), the importance of the quality management of BM-MSC has been widely recognized. However, there is little information concerning the relationship between cellular senescence and the stemness in equine BM-MSC. In this study, we showed that stemness markers (NANOG, OCT4, SOX2 and telomerase reverse transcriptase) and colony forming unit-fibroblast apparently decreased accompanied with incidence of senescence-associated β-galactosidase-positive cells by repeated passage. Additionally, we suggested that down-regulation of cell proliferation in senescent BM-MSC was related to increased expression of cyclin-dependent kinase inhibitor 2B (CDKN2B). On the other hand, forced expression of NANOG into senescent BM-MSC brought upregulation of several stemness markers and downregulation of CKDN2B accompanied with restoration of proliferation potential and osteogenic ability. These results suggested that expression of NANOG was important for the maintenance of the stemness in equine BM-MSC.
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Affiliation(s)
- Chiho KUSHIDA
- Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo, Japan
- National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo, Japan
| | - Norihisa TAMURA
- Laboratory of Clinical Science and Pathobiology, Equine Research Institute, Japan Racing Association, Tochigi, Japan
| | - Yoshinori KASASHIMA
- Laboratory of Clinical Science and Pathobiology, Equine Research Institute, Japan Racing Association, Tochigi, Japan
| | - Kota SATO
- National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo, Japan
| | - Katsuhiko ARAI
- Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Tokyo, Japan
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Montero-Martin N, Girón MD, Vílchez JD, Salto R. Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells. Int J Mol Sci 2024; 25:9150. [PMID: 39273113 PMCID: PMC11394838 DOI: 10.3390/ijms25179150] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2024] [Revised: 08/13/2024] [Accepted: 08/21/2024] [Indexed: 09/15/2024] Open
Abstract
Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.
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Affiliation(s)
- Nora Montero-Martin
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, E18071 Granada, Spain
| | - María D Girón
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, E18071 Granada, Spain
| | - José D Vílchez
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, E18071 Granada, Spain
| | - Rafael Salto
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, E18071 Granada, Spain
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Godoy MCXD, Monteiro GA, Moraes BHD, Macedo JA, Gonçalves GMS, Gambero A. Addition of Polyphenols to Drugs: The Potential of Controlling "Inflammaging" and Fibrosis in Human Senescent Lung Fibroblasts In Vitro. Int J Mol Sci 2024; 25:7163. [PMID: 39000270 PMCID: PMC11241747 DOI: 10.3390/ijms25137163] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Revised: 06/25/2024] [Accepted: 06/26/2024] [Indexed: 07/16/2024] Open
Abstract
The combination of a polyphenol, quercetin, with dasatinib initiated clinical trials to evaluate the safety and efficacy of senolytics in idiopathic pulmonary fibrosis, a lung disease associated with the presence of senescent cells. Another approach to senotherapeutics consists of controlling inflammation related to cellular senescence or "inflammaging", which participates, among other processes, in establishing pulmonary fibrosis. We evaluate whether polyphenols such as caffeic acid, chlorogenic acid, epicatechin, gallic acid, quercetin, or resveratrol combined with different senotherapeutics such as metformin or rapamycin, and antifibrotic drugs such as nintedanib or pirfenidone, could present beneficial actions in an in vitro model of senescent MRC-5 lung fibroblasts. A senescent-associated secretory phenotype (SASP) was evaluated by the measurement of interleukin (IL)-6, IL-8, and IL-1β. The senescent-associated β-galactosidase (SA-β-gal) activity and cellular proliferation were assessed. Fibrosis was evaluated using a Picrosirius red assay and the gene expression of fibrosis-related genes. Epithelial-mesenchymal transition (EMT) was assayed in the A549 cell line exposed to Transforming Growth Factor (TGF)-β in vitro. The combination that demonstrated the best results was metformin and caffeic acid, by inhibiting IL-6 and IL-8 in senescent MRC-5 cells. Metformin and caffeic acid also restore cellular proliferation and reduce SA-β-gal activity during senescence induction. The collagen production by senescent MRC-5 cells was inhibited by epicatechin alone or combined with drugs. Epicatechin and nintedanib were able to control EMT in A549 cells. In conclusion, caffeic acid and epicatechin can potentially increase the effectiveness of senotherapeutic drugs in controlling lung diseases whose pathophysiological component is the presence of senescent cells and fibrosis.
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Affiliation(s)
- Maria Carolina Ximenes de Godoy
- School for Life Sciences, Pontifical Catholic University of Campinas (PUC-Campinas), Av. John Boyd Dunlop, Campinas 13034-685, SP, Brazil
| | - Gabriela Arruda Monteiro
- School for Life Sciences, Pontifical Catholic University of Campinas (PUC-Campinas), Av. John Boyd Dunlop, Campinas 13034-685, SP, Brazil
| | - Bárbara Hakim de Moraes
- School for Life Sciences, Pontifical Catholic University of Campinas (PUC-Campinas), Av. John Boyd Dunlop, Campinas 13034-685, SP, Brazil
| | - Juliana Alves Macedo
- Department of Food and Nutrition, School of Food Engineering, State University of Campinas, Campinas 13083-862, SP, Brazil
| | - Gisele Mara Silva Gonçalves
- School for Life Sciences, Pontifical Catholic University of Campinas (PUC-Campinas), Av. John Boyd Dunlop, Campinas 13034-685, SP, Brazil
| | - Alessandra Gambero
- School for Life Sciences, Pontifical Catholic University of Campinas (PUC-Campinas), Av. John Boyd Dunlop, Campinas 13034-685, SP, Brazil
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Raviola S, Griffante G, Iannucci A, Chandel S, Lo Cigno I, Lacarbonara D, Caneparo V, Pasquero S, Favero F, Corà D, Trisolini E, Boldorini R, Cantaluppi V, Landolfo S, Gariglio M, De Andrea M. Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells. Commun Biol 2024; 7:292. [PMID: 38459109 PMCID: PMC10924099 DOI: 10.1038/s42003-024-05957-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2023] [Accepted: 02/22/2024] [Indexed: 03/10/2024] Open
Abstract
Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.
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Affiliation(s)
- Stefano Raviola
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Gloria Griffante
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Andrea Iannucci
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
- Department of Biomedicine and Prevention, University of Rome "Tor Vergata", Rome, Italy
| | - Shikha Chandel
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Irene Lo Cigno
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Davide Lacarbonara
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Valeria Caneparo
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
| | - Selina Pasquero
- Viral Pathogenesis Unit, Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy
| | - Francesco Favero
- Bioinformatics Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Bioinformatics Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Davide Corà
- Bioinformatics Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Bioinformatics Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Elena Trisolini
- Pathology Unit, Department of Health Sciences, University of Eastern Piedmont, Novara, Italy
| | - Renzo Boldorini
- Pathology Unit, Department of Health Sciences, University of Eastern Piedmont, Novara, Italy
| | - Vincenzo Cantaluppi
- Nephrology and Kidney Transplantation Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Santo Landolfo
- Viral Pathogenesis Unit, Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy
| | - Marisa Gariglio
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy
- Molecular Virology Unit, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy
| | - Marco De Andrea
- Intrinsic Immunity Unit, CAAD - Center for Translational Research on Autoimmune and Allergic Disease, University of Eastern Piedmont, Novara, Italy.
- Viral Pathogenesis Unit, Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy.
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de Farias JO, da Costa Sousa MG, Martins DCM, de Oliveira MA, Takahashi I, de Sousa LB, da Silva IGM, Corrêa JR, Silva Carvalho AÉ, Saldanha-Araújo F, Rezende TMB. Senescence on Dental Pulp Cells: Effects on Morphology, Migration, Proliferation, and Immune Response. J Endod 2024; 50:362-369. [PMID: 38211820 DOI: 10.1016/j.joen.2023.12.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 12/29/2023] [Accepted: 12/30/2023] [Indexed: 01/13/2024]
Abstract
INTRODUCTION Evidence indicates that senescence can affect essential dental pulp functions, such as defense capacity and repair, consequently affecting the successes of conservative endodontic treatments. This study aims to evaluate the effects of senescence on the morphology, migration, proliferation, and immune response of human dental pulp cells. METHODS Cells were treated with doxorubicin to induce senescence, confirmed by β-galactosidase staining. Morphological changes, cellular proliferation, and migration were evaluated by scanning electron microscopy, trypan blue cells, and the scratch method, respectively. Modifications in the immune response were evaluated by measuring the genes for pro-inflammatory cytokines tumor necrosis factor alpha and interleukin (IL)-6 and anti-inflammatory cytokines transforming growth factor beta 1 and IL-10 using the real time polymerase chain reaction assay. RESULTS An increase in cell size and a decrease in the number of extensions were observed in senescent cells. A reduction in the proliferative and migratory capacity was also found in senescent cells. In addition, there was an increase in the gene expression of inflammatory cytokines tumor necrosis factor alpha and IL-6 and a decrease in the gene expression of IL-10 and transforming growth factor beta-1, suggesting an exacerbated inflammatory situation associated with immunosuppression. CONCLUSIONS Cellular senescence is possibly a condition that affects prognoses of conservative endodontic treatments, as it affects primordial cellular functions related to this treatment.
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Affiliation(s)
- Jade Ormondes de Farias
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil
| | - Maurício Gonçalves da Costa Sousa
- Division of Biomaterials and Biomechanics, Department of Restorative, Dentistry, School of Dentistry, Oregon Health & Science University, Portland Oregon; Knigth Cancer Precision Biofabrication Hub, Knigth Cancer Institute, Oregon, Health and Science University, Portland, Oregon; Cancer Early Detection Advanced Research Center, Oregon Health Science, University, Portland, Oregon
| | - Danilo César Mota Martins
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil
| | - Mayara Alves de Oliveira
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil
| | - Isadora Takahashi
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil
| | - Larissa Barbosa de Sousa
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil
| | | | - José Raimundo Corrêa
- Laboratório de Microscopia e Microanálise, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, Brazil
| | - Amandda Évelin Silva Carvalho
- Laboratório de Hematologia e Células-tronco, Faculdade de Ciências da Saúde, Universidade de Brasília, Brasília, Brazil
| | - Felipe Saldanha-Araújo
- Laboratório de Hematologia e Células-tronco, Faculdade de Ciências da Saúde, Universidade de Brasília, Brasília, Brazil
| | - Taia Maria Berto Rezende
- Pós-graduação em Ciências da Saúde, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil; Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica, de Brasília, Brasília, Brazil; Departamento de Odontologia, Faculdade de Ciências de Saúde, Universidade, Brasília, Brazil; Pós-graduação em Odontologia, Faculdade de Ciências de Saúde, Universidade de Brasília, Brasília, Brazil.
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11
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Lee SK, Han MS, Tung CH. In vivo senescence imaging nanoprobe targets the associated reactive oxygen species. NANOSCALE 2024; 16:1371-1383. [PMID: 38131616 DOI: 10.1039/d3nr04083f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2023]
Abstract
Cellular senescence, a cell-cycle arrest state upon stress or damage, can adversely impact aging and cancers. We have designed a novel near infrared fluorogenic nanoprobe, named D3, which can only be turned on by highly elevated levels of reactive oxygen species (ROS), critical players for the induction and maintenance of senescence, for real-time senescence sensing and imaging. In contrast to glowing senescent cells, non-senescent cells whose ROS levels are too low to activate the D3 signal remain optically silent. Upon systemic injection into senescent tumor-bearing mice, the D3 nanoprobe quickly accumulates in tumors, and its fluorescence signal is turned on specifically by senescence-associated ROS in the senescent tumors. The fluorescence signal at senescent tumors was 3-fold higher than that of non-senescent tumors. This groundbreaking design introduces a novel activation mechanism and a powerful imaging nanoprobe to identify and assess cellular senescence in living organisms.
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Affiliation(s)
- Seung Koo Lee
- Molecular Imaging Innovations Institute, Department of Radiology, Weill Cornell Medicine, 413 East 69th Street, Box 290, New York, NY 10021, USA.
| | - Myung Shin Han
- Molecular Imaging Innovations Institute, Department of Radiology, Weill Cornell Medicine, 413 East 69th Street, Box 290, New York, NY 10021, USA.
| | - Ching-Hsuan Tung
- Molecular Imaging Innovations Institute, Department of Radiology, Weill Cornell Medicine, 413 East 69th Street, Box 290, New York, NY 10021, USA.
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12
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Heo JW, Lee HE, Lee J, Choi LS, Shin J, Mun JY, Park HS, Park SC, Nam CH. Vutiglabridin Alleviates Cellular Senescence with Metabolic Regulation and Circadian Clock in Human Dermal Fibroblasts. Antioxidants (Basel) 2024; 13:109. [PMID: 38247533 PMCID: PMC10812742 DOI: 10.3390/antiox13010109] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 01/10/2024] [Accepted: 01/12/2024] [Indexed: 01/23/2024] Open
Abstract
The process of cellular senescence, which is characterized by stable cell cycle arrest, is strongly associated with dysfunctional cellular metabolism and circadian rhythmicity, both of which are reported to result from and also be causal to cellular senescence. As a result, modifying any of them-senescence, metabolism, or the circadian clock-may affect all three simultaneously. Obesity accelerates aging by disrupting the homeostasis of reactive oxygen species (ROS) via an increased mitochondrial burden of fatty acid oxidation. As a result, if senescence, metabolism, and circadian rhythm are all linked, anti-obesity treatments may improve metabolic regulation while also alleviating senescence and circadian rhythm. Vutiglabridin is a small molecule in clinical trials that improves obesity by enhancing mitochondrial function. We found that chronic treatment of senescent primary human dermal fibroblasts (HDFs) with vutiglabridin alleviates all investigated markers of cellular senescence (SA-β-gal, CDKN1A, CDKN2A) and dysfunctional cellular circadian rhythm (BMAL1) while remarkably preventing the alterations of mitochondrial function and structure that occur during the process of cellular senescence. Our results demonstrate the significant senescence-alleviating effects of vutiglabridin, specifically with the restoration of cellular circadian rhythmicity and metabolic regulation. These data support the potential development of vutiglabridin against aging-associated diseases and corroborate the intricate link between cellular senescence, metabolism, and the circadian clock.
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Affiliation(s)
- Jin-Woong Heo
- School of Undergraduate Studies, Daegu Gyeongbuk Institute of Science and Technology, College of Transdisciplinary Studies, Daegu 42988, Republic of Korea; (J.-W.H.); (J.L.)
- Aging and Immunity Laboratory, Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Republic of Korea
| | - Hye-Eun Lee
- School of Medicine, Kyungpook National University, Daegu 41566, Republic of Korea;
- Neural Circuit Research Group, Korea Brain Research Institute, Daegu 41062, Republic of Korea;
| | - Jimin Lee
- School of Undergraduate Studies, Daegu Gyeongbuk Institute of Science and Technology, College of Transdisciplinary Studies, Daegu 42988, Republic of Korea; (J.-W.H.); (J.L.)
| | - Leo Sungwong Choi
- Glaceum Incorporation, Research Department, Suwon 16675, Republic of Korea; (L.S.C.); (J.S.); (H.-S.P.)
| | - Jaejin Shin
- Glaceum Incorporation, Research Department, Suwon 16675, Republic of Korea; (L.S.C.); (J.S.); (H.-S.P.)
| | - Ji-Young Mun
- Neural Circuit Research Group, Korea Brain Research Institute, Daegu 41062, Republic of Korea;
| | - Hyung-Soon Park
- Glaceum Incorporation, Research Department, Suwon 16675, Republic of Korea; (L.S.C.); (J.S.); (H.-S.P.)
| | - Sang-Chul Park
- Future Life and Society Research Center, Advanced Institute of Aging Science, Chonnam National University, Gwangju 61186, Republic of Korea;
| | - Chang-Hoon Nam
- Aging and Immunity Laboratory, Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Republic of Korea
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13
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Jouabadi SM, Ataabadi EA, Golshiri K, Bos D, Stricker BHC, Danser AHJ, Mattace-Raso F, Roks AJM. Clinical Impact and Mechanisms of Nonatherosclerotic Vascular Aging: The New Kid to Be Blocked. Can J Cardiol 2023; 39:1839-1858. [PMID: 37495207 DOI: 10.1016/j.cjca.2023.07.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Revised: 07/07/2023] [Accepted: 07/20/2023] [Indexed: 07/28/2023] Open
Abstract
Ischemic cardiovascular disease and stroke remain the leading cause of global morbidity and mortality. During aging, protective mechanisms in the body gradually deteriorate, resulting in functional, structural, and morphologic changes that affect the vascular system. Because atherosclerotic plaques are not always present along with these alterations, we refer to this kind of vascular aging as nonatherosclerotic vascular aging (NAVA). To maintain proper vascular function during NAVA, it is important to preserve intracellular signalling, prevent inflammation, and block the development of senescent cells. Pharmacologic interventions targeting these components are potential therapeutic approaches for NAVA, with a particular emphasis on inflammation and senescence. This review provides an overview of the pathophysiology of vascular aging and explores potential pharmacotherapies that can improve the function of aged vasculature, focusing on NAVA.
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Affiliation(s)
- Soroush Mohammadi Jouabadi
- Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands; Department of Epidemiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Ehsan Ataei Ataabadi
- Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Keivan Golshiri
- Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Daniel Bos
- Department of Epidemiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands; Department of Radiology and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Bruno H C Stricker
- Department of Epidemiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - A H Jan Danser
- Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Francesco Mattace-Raso
- Division of Geriatric Medicine, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Anton J M Roks
- Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
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14
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Lo Cigno I, Calati F, Girone C, Borgogna C, Venuti A, Boldorini R, Gariglio M. SIRT1 is an actionable target to restore p53 function in HPV-associated cancer therapy. Br J Cancer 2023; 129:1863-1874. [PMID: 37838812 PMCID: PMC10667542 DOI: 10.1038/s41416-023-02465-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Revised: 09/26/2023] [Accepted: 10/04/2023] [Indexed: 10/16/2023] Open
Abstract
BACKGROUND Our aim was to evaluate the efficacy and anti-cancer action of a precision medicine approach involving a novel SIRT1-dependent pathway that, when disrupted, leads to the restoration of a functional p53 in human papillomavirus (HPV)-transformed cells. METHODS The anticancer potential of inhibiting SIRT1 was evaluated by examining the effects of the specific SIRT1 inhibitor EX527 (also known as Selisistat) or genetic silencing, either individually or in conjunction with standard chemotherapeutic agents, on a range of HPV+ cancer cells and a preclinical mouse model of HPV16-induced cancer. RESULTS We show that SIRT1 inhibition restores a transcriptionally active K382-acetylated p53 in HPV+ but not HPV- cell lines, which in turn promotes G0/G1 cell cycle arrest and inhibits clonogenicity specifically in HPV+ cells. Additionally, EX527 treatment increases the sensitivity of HPV+ cells to sublethal doses of standard genotoxic agents. The enhanced sensitivity to cisplatin as well as p53 restoration were also observed in an in vivo tumorigenicity assay using syngeneic C3.43 cells harbouring an integrated HPV16 genome, injected subcutaneously into C57BL/6J mice. CONCLUSIONS Our findings uncover an essential role of SIRT1 in HPV-driven oncogenesis, which may have direct translational implications for the treatment of this type of cancer.
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Affiliation(s)
- Irene Lo Cigno
- Virology Unit, Department of Translational Medicine, Eastern Piedmont University, Novara, Italy
| | - Federica Calati
- Virology Unit, Department of Translational Medicine, Eastern Piedmont University, Novara, Italy
| | - Carlo Girone
- Virology Unit, Department of Translational Medicine, Eastern Piedmont University, Novara, Italy
| | - Cinzia Borgogna
- Virology Unit, Department of Translational Medicine, Eastern Piedmont University, Novara, Italy
| | - Aldo Venuti
- HPV Unit, UOSD Tumor Immunology and Immunotherapy, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Renzo Boldorini
- Pathology Unit, Department of Health Sciences, Eastern Piedmont University, Novara, Italy
| | - Marisa Gariglio
- Virology Unit, Department of Translational Medicine, Eastern Piedmont University, Novara, Italy.
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15
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Cicardi M, Hallgren J, Mawrie D, Krishnamurthy K, Markandaiah S, Nelson A, Kankate V, Anderson E, Pasinelli P, Pandey U, Eischen C, Trotti D. C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress. iScience 2023; 26:107505. [PMID: 37664610 PMCID: PMC10470315 DOI: 10.1016/j.isci.2023.107505] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 05/10/2023] [Accepted: 07/26/2023] [Indexed: 09/05/2023] Open
Abstract
The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is aberrantly translated in the sense and antisense directions into dipeptide repeat proteins, among which poly proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo. PR partitions to the nucleus when heterologously expressed in neurons and other cell types. We show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR strongly accumulates in the nucleolus, a nuclear structure critical in regulating the cell stress response. We determined that, in neurons, PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels also prevented PR-mediated neurotoxicity both in in-vitro and in-vivo models. We investigated if PR could induce the senescence phenotype in neurons. However, we did not observe any indications of such an effect. Instead, we found evidence for the induction of programmed cell death via caspase-3 activation.
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Affiliation(s)
- M.E. Cicardi
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - J.H. Hallgren
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - D. Mawrie
- Center for Neuroscience, Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
| | - K. Krishnamurthy
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - S.S. Markandaiah
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - A.T. Nelson
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - V. Kankate
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - E.N. Anderson
- Center for Neuroscience, Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
| | - P. Pasinelli
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - U.B. Pandey
- Center for Neuroscience, Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
| | - C.M. Eischen
- Sidney Kimmel Cancer Center, Department of Pharmacology, Physiology, and Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - D. Trotti
- Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
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16
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Iriki T, Iio H, Yasuda S, Masuta S, Kato M, Kosako H, Hirayama S, Endo A, Ohtake F, Kamiya M, Urano Y, Saeki Y, Hamazaki J, Murata S. Senescent cells form nuclear foci that contain the 26S proteasome. Cell Rep 2023; 42:112880. [PMID: 37541257 DOI: 10.1016/j.celrep.2023.112880] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 05/26/2023] [Accepted: 07/13/2023] [Indexed: 08/06/2023] Open
Abstract
The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.
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Affiliation(s)
- Tomohiro Iriki
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Hiroaki Iio
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Shu Yasuda
- Department of Hygienic Chemistry and Medical Research Laboratories, School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 1088641, Japan
| | - Shun Masuta
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Masakazu Kato
- Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo 1648530, Japan
| | - Hidetaka Kosako
- Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Kuramoto-cho, Tokushima 7708503, Japan
| | - Shoshiro Hirayama
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Akinori Endo
- Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 1568506, Japan
| | - Fumiaki Ohtake
- Institute for Advanced Life Sciences, Hoshi University, Shinagawa-ku, Tokyo 1428501, Japan
| | - Mako Kamiya
- Department of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 2268501, Japan
| | - Yasuteru Urano
- Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan; Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Yasushi Saeki
- Division of Protein Metabolism, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 1088639, Japan
| | - Jun Hamazaki
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Shigeo Murata
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan.
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17
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Schmidt DR, Gramatikov IMT, Sheen A, Williams CL, Hurwitz M, Dodge LE, Holupka E, Kiger WS, Cornwall-Brady MR, Huang W, Mak HH, Cormier KS, Condon C, Dane Wittrup K, Yilmaz ÖH, Stevenson MA, Down JD, Floyd SR, Roper J, Vander Heiden MG. Ablative radiotherapy improves survival but does not cure autochthonous mouse models of prostate and colorectal cancer. COMMUNICATIONS MEDICINE 2023; 3:108. [PMID: 37558833 PMCID: PMC10412558 DOI: 10.1038/s43856-023-00336-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Accepted: 07/24/2023] [Indexed: 08/11/2023] Open
Abstract
BACKGROUND Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. METHODS Here we report the development, testing, and validation of a platform to immobilize and target tumors in mice with stereotactic ablative RT (SART). Xenograft and autochthonous tumor models were treated with hypofractionated ablative doses of radiotherapy. RESULTS We demonstrate that hypofractionated regimens used in clinical practice can be effectively delivered in mouse models. SART alters tumor stroma and the immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. Complete pathologic responses were achieved in xenograft models, but not in GEMMs. CONCLUSIONS While SART is capable of fully ablating xenografts, it is unable to completely eradicate disease in GEMMs, arguing that resistance to potentially curative therapy can be modeled in GEMMs.
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Affiliation(s)
- Daniel R Schmidt
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA.
- Harvard Medical School, Boston, MA, USA.
| | - Iva Monique T Gramatikov
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA
| | - Allison Sheen
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Christopher L Williams
- Harvard Medical School, Boston, MA, USA
- Dana-Farber Cancer Institute, Boston, MA, USA
- Department of Radiation Oncology, Brigham and Women's Hospital, Boston, MA, USA
| | - Martina Hurwitz
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Laura E Dodge
- Harvard Medical School, Boston, MA, USA
- Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Boston, MA, USA
- Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Edward Holupka
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - W S Kiger
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Milton R Cornwall-Brady
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Wei Huang
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Howard H Mak
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Kathleen S Cormier
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Charlene Condon
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - K Dane Wittrup
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Ömer H Yilmaz
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Harvard Medical School, Boston, MA, USA
- Department of Pathology, Massachusetts General Hospital, Boston, USA
| | - Mary Ann Stevenson
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Julian D Down
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Scott R Floyd
- Department of Radiation Oncology, Duke University School of Medicine, Durham, NC, USA
| | - Jatin Roper
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Medicine, Division of Gastroenterology, and Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA
| | - Matthew G Vander Heiden
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Dana-Farber Cancer Institute, Boston, MA, USA.
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18
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Salsabila DU, Wardani RK, Hasanah NU, Tafrihani AS, Zulfin UM, Ikawati M, Meiyanto E. Cytoprotective Properties of Citronella Oil (Cymbopogon nardus (L.) Rendl.) and Lemongrass Oil (Cymbopogon citratus (DC.) Stapf) through Attenuation of Senescent-Induced Chemotherapeutic Agent Doxorubicin on Vero and NIH-3T3 Cells. Asian Pac J Cancer Prev 2023; 24:1667-1675. [PMID: 37247287 PMCID: PMC10495914 DOI: 10.31557/apjcp.2023.24.5.1667] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Accepted: 05/22/2023] [Indexed: 05/31/2023] Open
Abstract
OBJECTIVE This study aimed to determine the cytoprotective potentials of citronella (Cymbopogon nardus (L.) Rendl.) essential oil (CO) and lemongrass (Cymbopogon citratus (DC.) Stapf) essential oil (LO). METHODS The essential oils from citronella and lemongrass were obtained by steam-water distillation, then analyzed using Gas Chromatography-Mass Spectrophotometry (GC-MS) to determine the chemical constituents. The antioxidant activity of CO and LO was compared using a total antioxidant capacity kit. The viability of normal kidney epithelial cells Vero and fibroblast NIH-3T3 as the cell models were tested using a trypan blue exclusion assay. The effect of cellular senescence inhibition on both cell models was measured using senescence-associated β-galactosidase (SA-β-gal) staining. The mechanism of action of CO and LO in the protection of cellular damage against doxorubicin was also confirmed through 2',7'-dichlorofluorescin diacetate (DCFDA) staining to discover the ability to decrease reactive oxygen species (ROS) levels and a gelatin zymography assay to observe the activity of matrix metalloproteinases (MMPs). RESULTS The major marker components of CO and LO were citronellal and citral, respectively. Both oils showed low cytotoxic activity against Vero and NIH-3T3 cells, with IC50 values of over 40 µg/mL. LO exhibited higher antioxidant capacity than CO, but there was no effect on the intracellular ROS level of both oils on Vero and NIH-3T3 cells. However, CO and LO decreased cellular senescence induced by doxorubicin exposure on both cells, as well as suppressed MMP-2 expression. Conclusion: Both CO and LO decrease the cellular senescence and MMP-2 expression with less cytotoxic effects on normal cells independently from their antioxidant capacities. The results were expected to support the use of CO and LO as tissue protective and anti-aging agents in maintaining the body's cellular health against chemotherapeutics or cellular damaging agents.
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Affiliation(s)
- Dhiya Ulhaq Salsabila
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Ratih Kurnia Wardani
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Nisa Ul Hasanah
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Ahmad Syauqy Tafrihani
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Ummi Maryam Zulfin
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Muthi’ Ikawati
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
- Laboratory of Macromolecular Engineering, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
| | - Edy Meiyanto
- Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
- Laboratory of Macromolecular Engineering, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia.
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19
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Senescent cardiac fibroblasts: A key role in cardiac fibrosis. Biochim Biophys Acta Mol Basis Dis 2023; 1869:166642. [PMID: 36669578 DOI: 10.1016/j.bbadis.2023.166642] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 01/09/2023] [Accepted: 01/11/2023] [Indexed: 01/19/2023]
Abstract
Cardiac fibroblasts are a cell population that controls the homeostasis of the extracellular matrix and orchestrates a damage response to maintain cardiac architecture and performance. Due to these functions, fibroblasts play a central role in cardiac fibrosis development, and there are large differences in matrix protein secretion profiles between fibroblasts from aged versus young animals. Senescence is a multifactorial and complex process that has been associated with inflammatory and fibrotic responses. After damage, transient cellular senescence is usually beneficial, as these cells promote tissue repair. However, the persistent presence of senescent cells within a tissue is linked with fibrosis development and organ dysfunction, leading to aging-related diseases such as cardiovascular pathologies. In the heart, early cardiac fibroblast senescence after myocardial infarction seems to be protective to avoid excessive fibrosis; however, in non-infarcted models of cardiac fibrosis, cardiac fibroblast senescence has been shown to be deleterious. Today, two new classes of drugs, termed senolytics and senostatics, which eliminate senescent cells or modify senescence-associated secretory phenotype, respectively, arise as novel therapeutical strategies to treat aging-related pathologies. However, further studies will be needed to evaluate the extent of the utility of senotherapeutic drugs in cardiac diseases, in which pathological context and temporality of the intervention must be considered.
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20
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Hallmarks and Biomarkers of Skin Senescence: An Updated Review of Skin Senotherapeutics. Antioxidants (Basel) 2023; 12:antiox12020444. [PMID: 36830002 PMCID: PMC9952625 DOI: 10.3390/antiox12020444] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2023] [Revised: 02/06/2023] [Accepted: 02/07/2023] [Indexed: 02/12/2023] Open
Abstract
Aging is a complex process characterized by an ongoing decline in physiological functions, leading to degenerative diseases and an increased probability of death. Cellular senescence has been typically considered as an anti-proliferative process; however, the chronic accumulation of senescent cells contributes to tissue dysfunction and aging. In this review, we discuss some of the most important hallmarks and biomarkers of cellular senescence with a special focus on skin biomarkers, reactive oxygen species (ROS), and senotherapeutic strategies to eliminate or prevent senescence. Although most of them are not exclusive to senescence, the expression of the senescence-associated beta-galactosidase (SA-β-gal) enzyme seems to be the most reliable biomarker for distinguishing senescent cells from those arrested in the cell cycle. The presence of a stable DNA damage response (DDR) and the accumulation of senescence-associated secretory phenotype (SASP) mediators and ROS are the most representative hallmarks for senescence. Senotherapeutics based on natural compounds such as quercetin, naringenin, and apigenin have shown promising results regarding SASP reduction. These compounds seem to prevent the accumulation of senescent cells, most likely through the inhibition of pro-survival signaling pathways. Although studies are still required to verify their short- and long-term effects, these therapies may be an effective strategy for skin aging.
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21
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Fröhlich J, Rose K, Hecht A. Transcriptional activity mediated by β-CATENIN and TCF/LEF family members is completely dispensable for survival and propagation of multiple human colorectal cancer cell lines. Sci Rep 2023; 13:287. [PMID: 36609428 PMCID: PMC9822887 DOI: 10.1038/s41598-022-27261-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Accepted: 12/29/2022] [Indexed: 01/08/2023] Open
Abstract
Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10% of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Survival of these cells and the ability to propagate them demonstrate their complete β-CATENIN- and TCF/LEF-independence. Nonetheless, one β-CATENIN-deficient cell line eventually became senescent, and absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β-CATENIN and TCF7L2 independently control sizeable fractions of their target genes. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.
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Affiliation(s)
- Janna Fröhlich
- grid.5963.9Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 17, 79104 Freiburg, Germany ,grid.5963.9Faculty of Biology, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
| | - Katja Rose
- grid.5963.9Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 17, 79104 Freiburg, Germany
| | - Andreas Hecht
- Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany. .,Faculty of Biology, Albert-Ludwigs-University Freiburg, 79104, Freiburg, Germany. .,BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, 79104, Freiburg, Germany.
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22
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Abstract
Cellular senescence, once thought an artifact of in vitro culture or passive outcome of aging, has emerged as fundamental to tissue development and function. The senescence mechanism importantly halts cell cycle progression to protect against tumor formation, while transiently present senescent cells produce a complex secretome (or SASP) of inflammatory mediators, proteases, and growth factors that guide developmental remodeling and tissue regeneration. Transiently present senescence is important for skin repair, where it accelerates extracellular matrix formation, limits fibrosis, promotes reepithelialization, and modulates inflammation. Unfortunately, advanced age and diabetes drive pathological accumulation of senescent cells in chronic wounds, which is perpetuated by a proinflammatory SASP, advanced glycation end-products, and oxidative damage. Although the biology of wound senescence remains incompletely understood, drugs that selectively target senescent cells are showing promise in clinical trials for diverse pathological conditions. It may not be long before senescence-targeted therapies will be available for the management, or perhaps even prevention, of chronic wounds.
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Affiliation(s)
- Holly N Wilkinson
- Centre for Atherothrombosis and Metabolic Disease, Hull York Medical School, University of Hull, Hull HU6 7RX, United Kingdom
| | - Matthew J Hardman
- Centre for Atherothrombosis and Metabolic Disease, Hull York Medical School, University of Hull, Hull HU6 7RX, United Kingdom
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23
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Dimri M, Dimri GP. The original colorimetric method to detect cellular senescence. Methods Cell Biol 2022; 181:59-72. [PMID: 38302244 DOI: 10.1016/bs.mcb.2022.09.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Cellular senescence, whereby cells cease to proliferate, is known to contribute to the aging process and age-related pathologies. It is elicited either by cell-intrinsic mechanisms such as progressive telomere shortening or due to the extrinsic stress-related factors, which via p53-p21 and p16-pRB tumor suppressor pathways signal cells to cease proliferation. A proper identification and characterization of senescent cells is necessary to understand the process of aging, age-related pathologies, and the development of therapeutics to treat age-related dysfunctions. The landmark discovery of Senescence-Associated-Beta-Galactosidase (SA-β-Gal) marker, and a simple colorimetric method to detect SA-β-Gal greatly facilitated identification of the senescent cells in human and rodent cells pertaining to age-related diseases (Dimri et al., 1995). Despite the availability of additional senescence biomarkers, the SA-β-Gal marker and histochemical detection method remain the most widely used tool to identify senescent cells in vitro and in vivo. Here, we revisit the original colorimetric method to detect senescent cells that was first published in 1995 (Dimri et al., 1995).
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Affiliation(s)
- Manjari Dimri
- Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, United States
| | - Goberdhan P Dimri
- Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, United States.
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24
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Flor A, Pagacz J, Thompson D, Kron S. Far-red Fluorescent Senescence-associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry. J Vis Exp 2022:10.3791/64176. [PMID: 36190263 PMCID: PMC10657218 DOI: 10.3791/64176] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2023] Open
Abstract
Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-β-Gal). Typically, the SA-β-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-β-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-β-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-β-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling. Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.
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Affiliation(s)
- Amy Flor
- Department of Molecular Genetics and Cell Biology, University of Chicago;
| | - Joanna Pagacz
- Department of Molecular Genetics and Cell Biology, University of Chicago
| | - DeShawn Thompson
- Department of Molecular Genetics and Cell Biology, University of Chicago
| | - Stephen Kron
- Department of Molecular Genetics and Cell Biology, University of Chicago
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25
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Lotti R, Palazzo E, Quadri M, Dumas M, Schnebert S, Biondini D, Bianchini MA, Nizard C, Pincelli C, Marconi A. Isolation of an "Early" Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271. Stem Cells 2022; 40:1149-1161. [PMID: 36037263 PMCID: PMC9806768 DOI: 10.1093/stmcls/sxac060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Accepted: 08/16/2022] [Indexed: 01/12/2023]
Abstract
In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.
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Affiliation(s)
- Roberta Lotti
- DermoLab, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Elisabetta Palazzo
- DermoLab, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Marika Quadri
- DermoLab, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Marc Dumas
- LVMH Recherche, Life Sciences Department, Saint Jean de Braye, France
| | | | - Diego Biondini
- Pediatric Surgery Unit, Department of Pediatric Surgery, University of Modena and Reggio Emilia, Modena, Italy
| | - Maria Anastasia Bianchini
- Pediatric Surgery Unit, Department of Pediatric Surgery, University of Modena and Reggio Emilia, Modena, Italy
| | - Carine Nizard
- LVMH Recherche, Life Sciences Department, Saint Jean de Braye, France
| | - Carlo Pincelli
- DermoLab, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Alessandra Marconi
- Corresponding author: Alessandra Marconi, MSc in Biology, Specialist in Clinical Pathology, DermoLab, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Via Del Pozzo 71, 41124 Modena, Italy. Tel: +39 059 4222812; Fax: +39 059 4224271;
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26
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Kudlova N, De Sanctis JB, Hajduch M. Cellular Senescence: Molecular Targets, Biomarkers, and Senolytic Drugs. Int J Mol Sci 2022; 23:ijms23084168. [PMID: 35456986 PMCID: PMC9028163 DOI: 10.3390/ijms23084168] [Citation(s) in RCA: 74] [Impact Index Per Article: 24.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Revised: 03/31/2022] [Accepted: 04/05/2022] [Indexed: 11/16/2022] Open
Abstract
Cellular senescence is defined as irreversible cell cycle arrest caused by various processes that render viable cells non-functional, hampering normal tissue homeostasis. It has many endogenous and exogenous inducers, and is closely connected with age, age-related pathologies, DNA damage, degenerative disorders, tumor suppression and activation, wound healing, and tissue repair. However, the literature is replete with contradictory findings concerning its triggering mechanisms, specific biomarkers, and detection protocols. This may be partly due to the wide range of cellular and in vivo animal or human models of accelerated aging that have been used to study senescence and test senolytic drugs. This review summarizes recent findings concerning senescence, presents some widely used cellular and animal senescence models, and briefly describes the best-known senolytic agents.
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Affiliation(s)
- Natalie Kudlova
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 77147 Olomouc, Czech Republic; (N.K.); (J.B.D.S.)
| | - Juan Bautista De Sanctis
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 77147 Olomouc, Czech Republic; (N.K.); (J.B.D.S.)
- Institute of Molecular and Translational Medicine Czech Advanced Technologies and Research Institute, Palacky University, 77147 Olomouc, Czech Republic
| | - Marian Hajduch
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 77147 Olomouc, Czech Republic; (N.K.); (J.B.D.S.)
- Institute of Molecular and Translational Medicine Czech Advanced Technologies and Research Institute, Palacky University, 77147 Olomouc, Czech Republic
- Correspondence: ; Tel.: +42-0-585632082
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27
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Das JK, Ren Y, Kumar A, Peng HY, Wang L, Xiong X, Alaniz RC, de Figueiredo P, Ren X, Liu X, Ryazonov AG, Yang JM, Song J. Elongation factor-2 kinase is a critical determinant of the fate and antitumor immunity of CD8 + T cells. SCIENCE ADVANCES 2022; 8:eabl9783. [PMID: 35108044 PMCID: PMC8809536 DOI: 10.1126/sciadv.abl9783] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 12/10/2021] [Indexed: 06/14/2023]
Abstract
eEF-2K has important roles in stress responses and cellular metabolism. We report here a previously unappreciated but critical role of eEF-2K in regulating the fate and cytocidal activity of CD8+ T cells. CD8+ T cells from eEF-2K KO mice were more proliferative but had lower survival than their wild-type counterparts after their activation, followed by occurrence of premature senescence and exhaustion. eEF-2K KO CD8+ T cells were more metabolically active and showed hyperactivation of the Akt-mTOR-S6K pathway. Loss of eEF-2K substantially impaired the activity of CD8+ T cells. Furthermore, the antitumor efficacy and tumor infiltration of the CAR-CD8+ T cells lacking eEF-2K were notably reduced as compared to the control CAR-CD8+ T cells. Thus, eEF-2K is critically required for sustaining the viability and function of cytotoxic CD8+ T cells, and therapeutic augmentation of this kinase may be exploited as a novel approach to reinforcing CAR-T therapy against cancer.
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Affiliation(s)
- Jugal Kishore Das
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Yijie Ren
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Anil Kumar
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Hao-Yun Peng
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Liqing Wang
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Xiaofang Xiong
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Robert C. Alaniz
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
| | - Paul de Figueiredo
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
- Norman Borlaug Center, Texas A&M University, College Station, TX 77843, USA
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA
| | - Xingcong Ren
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA
| | - Xiaoqi Liu
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Alexey G. Ryazonov
- Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Jin-Ming Yang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Jianxun Song
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77843, USA
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28
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Lee SK, Shen Z, Han MS, Tung CH. Developing a far-red fluorogenic beta-galactosidase probe for senescent cell imaging and photoablation. RSC Adv 2022; 12:4543-4549. [PMID: 35425504 PMCID: PMC8981090 DOI: 10.1039/d2ra00377e] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2022] [Accepted: 01/31/2022] [Indexed: 01/10/2023] Open
Abstract
A methylene blue (MB)-based beta-galactosidase (β-gal) activatable molecule, Gal-MB, was developed for senescence imaging and light-triggered senolysis. When in contact with LacZ β-gal or senescence-associated β-gal (SA-β-gal), the photoinsensitive Gal-MB becomes fluorescent. Gal-MB also offered selective phototoxicity toward LacZ β-gal expressing cells and drug-induced senescent cells, which express SA-β-gal, after light illumination at 665 nm. A methylene blue (MB)-based beta-galactosidase (β-gal) activatable molecule, Gal-MB, was developed for senescence imaging and light-triggered senolysis.![]()
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Affiliation(s)
- Seung Koo Lee
- Department of Radiology, Molecular Imaging Innovations Institute, Weill Cornell Medicine New York NY 10021 USA
| | - Zhenhua Shen
- Department of Radiology, Molecular Imaging Innovations Institute, Weill Cornell Medicine New York NY 10021 USA
| | - Myung Shin Han
- Department of Radiology, Molecular Imaging Innovations Institute, Weill Cornell Medicine New York NY 10021 USA
| | - Ching-Hsuan Tung
- Department of Radiology, Molecular Imaging Innovations Institute, Weill Cornell Medicine New York NY 10021 USA
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29
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Yang Y, Ding J, Chen Y, Ma G, Wei X, Zhou R, Hu W. Blockade of ASIC1a inhibits acid-induced rat articular chondrocyte senescence through regulation of autophagy. Hum Cell 2022; 35:665-677. [DOI: 10.1007/s13577-022-00676-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Accepted: 01/15/2022] [Indexed: 01/15/2023]
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30
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Ebrahimi N, Rezanejad H, Asadi MH, Vallian S. LncRNA LOC100507144 acts as a novel regulator of CD44/Nanog/Sox2/miR-302/miR-21 axis in colorectal cancer. Biofactors 2022; 48:164-180. [PMID: 34882869 DOI: 10.1002/biof.1813] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Accepted: 11/12/2021] [Indexed: 12/15/2022]
Abstract
Long noncoding RNAs (lncRNAs) appear as vital regulators and biomarkers in many human cancers. LOC100507144 is a validated lncRNA located in the neighborhood of CD44 in a head-to-head configuration, and its expression and function in cancer cells are still unknown. This research aimed to find out more about the expression and function of this lncRNA in colorectal cancer (CRC). Our expression data represented that the expression of LOC100507144 transcript was substantially higher in tumors with advanced stages, lymph node metastasis, and vascular invasion. Loss-of-function examinations demonstrated that LOC100507144 contributed to CRC cell proliferation by restricting apoptosis, cellular senescence, and promoting cell cycle. Gain-of-function experiments also confirmed these results. Our data illustrated that LOC100507144 enhanced the migration and the epithelial to mesenchymal transition (EMT) of CRC cells, accompanied by the generation of cells with stemness characteristics. Our findings revealed that the knocking-down of LOC100507144 inhibited the expression of crucial stemness factors, including CD44, Nanog, and Sox2, and accordingly resulted in suppressing their targets, miR-302 and miR-21. Overall, the current study's findings for the first time reveal that LOC100507144 could enhance CRC progression and metastasis through regulation of the CD44/Nanog/Sox2/miR-302/miR-21 axis.
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Affiliation(s)
- Nasim Ebrahimi
- Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
| | - Hajar Rezanejad
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | - Malek Hossein Asadi
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | - Sadeq Vallian
- Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
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Zhou R, Xie X, Qin Z, Li X, Liu J, Li H, Zheng Q, Luo Y. Cytosolic dsDNA is a novel senescence marker associated with pyroptosis activation. Tissue Cell 2021; 72:101554. [PMID: 33991763 DOI: 10.1016/j.tice.2021.101554] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 03/31/2021] [Accepted: 04/27/2021] [Indexed: 01/07/2023]
Abstract
Cellular senescence has become a research focus because of its dual roles in ageing and tumorigenesis. The biomarkers of senescence are essential for detecting senescent cells and understanding the ageing process and its regulation. Here, we identify cytosolic double-stranded DNA (dsDNA) as a novel sensitive biomarker for cellular senescence of mouse embryonic fibroblasts (MEFs) in response to common types of stimuli, including replicative stress, genetic modification and oxidative stress. We found that the accumulation of cytosolic dsDNA was positively correlated with the senescence process in MEFs and was detectable earlier than senescence-associated β-galactosidase (SA-β-Gal) staining, which is the current gold standard for senescence detection. Due to the immunogenicity of dsDNA, we further investigated the stimulation of two dsDNA sensors, cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS) and absent in melanoma-2 (AIM2). The results showed that the cGAS protein level did not significantly change upon senescence stimulation, while AIM2 expression was significantly upregulated in senescent cells. Surprisingly, we found that ageing-related cytosolic dsDNA induced significant pyroptosis activation in the senescent MEFs. These data reveal novel easy-to-detect biomarker for cellular senescence. The activation of downstream immunological response pathways might add new experimental evidence for inflammatory ageing.
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Affiliation(s)
- Ruoyu Zhou
- Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China; Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China.
| | - Xiaoli Xie
- Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
| | - Ziyi Qin
- Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
| | - Xinbo Li
- Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
| | - Jing Liu
- Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
| | - Haili Li
- Institute of Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shangdong, 250000, China
| | - Quan Zheng
- Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
| | - Ying Luo
- Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China; Laboratory of Molecular Genetics of Ageing & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China; Guizhou Provincial Key Laboratory of Pathogenesis & Drug Development on Common Chronic Diseases, School of Basic Medicine, Guizhou Medical University, Guiyang, Guizhou, 550000, China.
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32
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Sharma SK, Poudel Sharma S, Leblanc RM. Methods of detection of β-galactosidase enzyme in living cells. Enzyme Microb Technol 2021; 150:109885. [PMID: 34489038 DOI: 10.1016/j.enzmictec.2021.109885] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Revised: 07/26/2021] [Accepted: 07/27/2021] [Indexed: 10/20/2022]
Abstract
The application of β-galactosidase enzyme ranges from industrial use as probiotics to medically important application such as cancer detection. The irregular activities of β-galactosidase enzyme are directly related to the development of cancers. Identifying the location and expression levels of enzymes in cancer cells have considerable importance in early-stage cancer diagnosis and monitoring the efficacy of therapies. Most importantly, the knowledge of the efficient method of detection of β-galactosidase enzyme will help in the early-stage treatment of the disease. In this review paper, we provide an overview of recent advances in the detection methods of β-galactosidase enzyme in the living cells, including the detection strategies, and approaches in human beings, plants, and microorganisms such as bacteria. Further, we emphasized on the challenges and opportunities in this rapidly developing field of development of different biomarkers and fluorescent probes based on β-galactosidase enzyme. We found that previously used chromo-fluorogenic methods have been mostly replaced by the new molecular probes, although they have certain drawbacks. Upon comparing the different methods, it was found that near-infrared fluorescent probes are dominating the other detection methods.
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Affiliation(s)
- Shiv K Sharma
- Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, United States; Thomas More University, 333 Thomas More Pkwy, Crestview Hills, KY 41017
| | - Sijan Poudel Sharma
- Department of Biology, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, United States
| | - Roger M Leblanc
- Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, United States.
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33
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Rahmani F, Hashemzehi M, Avan A, Barneh F, Asgharzadeh F, Moradi Marjaneh R, Soleimani A, Parizadeh M, Ferns GA, Ghayour Mobarhan M, Ryzhikov M, Afshari AR, Ahmadian MR, Giovannetti E, Jafari M, Khazaei M, Hassanian SM. Rigosertib elicits potent anti-tumor responses in colorectal cancer by inhibiting Ras signaling pathway. Cell Signal 2021; 85:110069. [PMID: 34214591 DOI: 10.1016/j.cellsig.2021.110069] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Revised: 06/02/2021] [Accepted: 06/25/2021] [Indexed: 02/06/2023]
Abstract
BACKGROUND The therapeutic potency of Rigosertib (RGS) in the treatment of the myelodysplastic syndrome has been investigated previously, but little is known about its mechanisms of action. METHODS The present study integrates systems and molecular biology approaches to investigate the mechanisms of the anti-tumor effects of RGS, either alone or in combination with 5-FU in cellular and animal models of colorectal cancer (CRC). RESULTS The effects of RGS were more pronounced in dedifferentiated CRC cell types, compared to cell types that were epithelial-like. RGS inhibited cell proliferation and cell cycle progression in a cell-type specific manner, and that was dependent on the presence of mutations in KRAS, or its down-stream effectors. RGS increased both early and late apoptosis, by regulating the expression of p53, BAX and MDM2 in tumor model. We also found that RGS induced cell senescence in tumor tissues by increasing ROS generation, and impairing oxidant/anti-oxidant balance. RGS also inhibited angiogenesis and metastatic behavior of CRC cells, by regulating the expression of CD31, E-cadherin, and matrix metalloproteinases-2 and 9. CONCLUSION Our findings support the therapeutic potential of this potent RAS signaling inhibitor either alone or in combination with standard regimens for the management of patients with CRC.
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Affiliation(s)
- Farzad Rahmani
- Iranshahr University of Medical Sciences, Iranshahr, Iran; Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Milad Hashemzehi
- Tropical and Communicable Diseases Research Centre, Iranshahr University of Medical Sciences, Iranshahr, Iran; Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Amir Avan
- Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran; Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad, Iran
| | - Farnaz Barneh
- Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Fereshteh Asgharzadeh
- Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Reyhaneh Moradi Marjaneh
- Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atena Soleimani
- Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohammadreza Parizadeh
- Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad, Iran
| | - Gordon A Ferns
- Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex BN1 9PH, UK
| | - Majid Ghayour Mobarhan
- Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad, Iran
| | - Mikhail Ryzhikov
- Division of Pulmonary and Critical Care Medicine, Washington University, School of Medicine, Saint Louis, MO, USA
| | - Amir Reza Afshari
- Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohammad Reza Ahmadian
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Elisa Giovannetti
- Cancer Pharmacology Lab, AIRC Start-up, University Hospital of Pisa, Pisa, Italy; Department of Medical Oncology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, the Netherlands
| | - Mohieddin Jafari
- Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of Helsinki, Finland
| | - Majid Khazaei
- Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad, Iran.
| | - Seyed Mahdi Hassanian
- Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad, Iran.
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Kerschbaum S, Wegrostek C, Riegel E, Czerny T. Senescence in a cell culture model for burn wounds. Exp Mol Pathol 2021; 122:104674. [PMID: 34437877 DOI: 10.1016/j.yexmp.2021.104674] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 06/25/2021] [Accepted: 08/15/2021] [Indexed: 11/29/2022]
Abstract
Thermal injuries cause severe damage on the cellular and tissue level and are considered especially challenging in the clinical routine. Complex interactions of different cell types and pathways dictate the formation of burn wounds. Thus, complications like burn wound progression, where so far viable tissue becomes necrotic and the size and depth of the wound increases, are difficult to explain, mainly due to the lack of simple model systems. We tested the behavior of human fibroblasts after heat treatment. A prominent response of the cells is to activate the heat shock response (HSR), which is one of the primary emergency mechanisms of the cell to proteotoxic stress factors such as heat. However, after a powerful but not lethal heat shock we observed a delayed activation of the HSR. Extending this model system, we further investigated these static cells and observed the emergence of senescent cells. In particular, the cells became β-galactosidase positive, increased p16 levels and developed a senescence-associated secretory phenotype (SASP). The secretion of cytokines like IL-6 is reminiscent of burn wounds and generates a bystander effect in so far non-senescent cells. In agreement with burn wounds, a wave of cytokine secretion enhanced by invading immune cells could explain complications like burn wound progression. A simple cell culture model can thus be applied for the analysis of highly complex conditions in human tissues.
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Affiliation(s)
- Sarah Kerschbaum
- Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria
| | - Christina Wegrostek
- Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria
| | - Elisabeth Riegel
- Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria
| | - Thomas Czerny
- Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria.
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35
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The Emergence of Senescent Surface Biomarkers as Senotherapeutic Targets. Cells 2021; 10:cells10071740. [PMID: 34359910 PMCID: PMC8305747 DOI: 10.3390/cells10071740] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 07/01/2021] [Accepted: 07/06/2021] [Indexed: 02/08/2023] Open
Abstract
Senescence is linked to a wide range of age-associated diseases and physiological declines. Thus, senotherapeutics are emerging to suppress the detrimental effects of senescence either by senomorphics or senolytics. Senomorphics suppress the traits associated with senescence phenotypes, while senolytics aim to clear senescent cells by suppressing their survival and enhancing the apoptotic pathways. The main goal of these approaches is to suppress the proinflammatory senescence-associated secretory phenotype (SASP) and to promote the immune recognition and elimination of senescent cells. One increasingly attractive approach is the targeting of molecules or proteins specifically present on the surface of senescent cells. These proteins may play roles in the maintenance and survival of senescent cells and hence can be targeted for senolysis. In this review, we summarize the recent knowledge regarding senolysis with a focus on novel surface biomarkers of cellular senescence and discuss their emergence as senotherapeutic targets.
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36
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Li S, Cheng Y, Chen S, Qin M, Li P, Yang L. In-situ SERS readout strategy to improve the reliability of beta-galactosidase activity assay based on X-gal staining in shortening incubation times. Talanta 2021; 234:122689. [PMID: 34364487 DOI: 10.1016/j.talanta.2021.122689] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 06/22/2021] [Accepted: 07/03/2021] [Indexed: 01/04/2023]
Abstract
Beta-galactosidase (β-gal) activity is closed related with senescence cells and aging-associated diseases, however, the traditional readout of β-gal activity based on X-gal staining was limited to low sensitivity in short incubation times and false positives in long incubation times. Here, we expose the potential role of insoluble X-gal hydrolysates in causing false positives by diffusion pollution depending on organic medium and then propose the in-situ Surface-enhanced Raman spectroscopy (SERS) readout strategy to identify and locate β-gal positive cells. By building the blue-white screening model and fabricating SERS-active needle sensor, the sensitive detection of β-gal has been realized with the detection limit of less than 1 nmol L-1. The in-situ SERS readout strategy is proved to be necessary and feasible to improve the reliability of X-gal staining assay through shortening the time to a few hours. Moreover, its application was also preliminarily evaluated to analyse individual cells and tissues, which showed the well consistency for judgement of β-gal activity cells at different times. Consequently, by improving reliability and reducing time consumption, this SERS readout strategy may be of great significance to promote the application of X-gal staining assay in biology and biomedicine.
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Affiliation(s)
- Shaofei Li
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China; Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, 230026, China; School of Life Science, Anhui University Hefei, Anhui, 230601, China
| | - Yizhuang Cheng
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China; Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, 230026, China
| | - Siyu Chen
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China; Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, 230026, China
| | - Miao Qin
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China; Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, 230026, China
| | - Pan Li
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China.
| | - Liangbao Yang
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, China; Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, 230026, China.
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37
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Koo S, Won M, Li H, Kim WY, Li M, Yan C, Sharma A, Guo Z, Zhu WH, Sessler JL, Lee JY, Kim JS. Harnessing α-l-fucosidase for in vivo cellular senescence imaging. Chem Sci 2021; 12:10054-10062. [PMID: 34377399 PMCID: PMC8317655 DOI: 10.1039/d1sc02259h] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2021] [Accepted: 06/24/2021] [Indexed: 01/10/2023] Open
Abstract
Precise detection of cellular senescence may allow its role in biological systems to be evaluated more effectively, while supporting studies of therapeutic candidates designed to evade its detrimental effect on physical function. We report here studies of α-l-fucosidase (α-fuc) as a biomarker for cellular senescence and the development of an α-fuc-responsive aggregation induced emission (AIE) probe, termed QM-NHαfuc designed to complement more conventional probes based on β-galactosidase (β-gal). Using QM-NHαfuc, the onset of replicative-, reactive oxygen species (ROS)-, ultraviolet A (UVA)-, and drug-induced senescence could be probed effectively. QM-NHαfuc also proved capable of identifying senescent cells lacking β-gal expression. The non-invasive real-time senescence tracking provided by QM-NHαfuc was validated in an in vivo senescence model. The results presented in this study lead us to suggest that the QM-NHαfuc could emerge as a useful tool for investigating senescence processes in biological systems. Evidence of close association of α-fuc with senescence induction highlights the potential of α-fuc as a novel biomarker for cellular senescence. Here, an α-fuc-responsive AIE probe (QM-NHαfuc) allows for the identification of senescent cell in vivo.![]()
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Affiliation(s)
- Seyoung Koo
- Department of Chemistry, Korea University Seoul 02841 Korea
| | - Miae Won
- Department of Chemistry, Korea University Seoul 02841 Korea
| | - Hao Li
- Department of Chemistry, Sungkyunkwan University Suwon 16419 Korea
| | - Won Young Kim
- Department of Chemistry, Korea University Seoul 02841 Korea
| | - Mingle Li
- Department of Chemistry, Korea University Seoul 02841 Korea
| | - Chenxu Yan
- Institute of Fine Chemicals, East China University of Science and Technology Shanghai 200237 China
| | - Amit Sharma
- CSIR-Central Scientific Instruments Organisation Sector-30C Chandigarh 160030 India
| | - Zhiqian Guo
- Institute of Fine Chemicals, East China University of Science and Technology Shanghai 200237 China
| | - Wei-Hong Zhu
- Institute of Fine Chemicals, East China University of Science and Technology Shanghai 200237 China
| | - Jonathan L Sessler
- Department of Chemistry, University of Texas at Austin Austin Texas 78712-1224 USA
| | - Jin Yong Lee
- Department of Chemistry, Sungkyunkwan University Suwon 16419 Korea
| | - Jong Seung Kim
- Department of Chemistry, Korea University Seoul 02841 Korea
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Scaffa AM, Peterson AL, Carr JF, Garcia D, Yao H, Dennery PA. Hyperoxia causes senescence and increases glycolysis in cultured lung epithelial cells. Physiol Rep 2021; 9:e14839. [PMID: 34042288 PMCID: PMC8157762 DOI: 10.14814/phy2.14839] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Revised: 12/08/2020] [Accepted: 12/15/2020] [Indexed: 02/07/2023] Open
Abstract
Supplemental oxygen and mechanical ventilation commonly used in premature infants may lead to chronic lung disease of prematurity, which is characterized by arrested alveolar development and dysmorphic vascular development. Hyperoxia is also known to dysregulate p53, senescence, and metabolism. However, whether these changes in p53, senescence, and metabolism are intertwined in response to hyperoxia is still unknown. Given that the lung epithelium is the first cell to encounter ambient oxygen during a hyperoxic exposure, we used mouse lung epithelial cells (MLE‐12), surfactant protein expressing type II cells, to explore whether hyperoxic exposure alters senescence and glycolysis. We measured glycolytic rate using a Seahorse Bioanalyzer assay and senescence using a senescence‐associated β galactosidase activity assay with X‐gal and C12FDG as substrates. We found that hyperoxic exposure caused senescence and increased glycolysis as well as reduced proliferation. This was associated with increased double stranded DNA damage, p53 phosphorylation and nuclear localization. Furthermore, hyperoxia‐induced senescence was p53‐dependent, but not pRB‐dependent, as shown in p53KO and pRBKO cell lines. Despite the inhibitory effects of p53 on glycolysis, we observed that glycolysis was upregulated in hyperoxia‐exposed MLE‐12 cells. This was attributable to a subpopulation of highly glycolytic senescent cells detected by C12FDG sorting. Nevertheless, inhibition of glycolysis did not prevent hyperoxia‐induced senescence. Therapeutic strategies modulating p53 and glycolysis may be useful to mitigate the detrimental consequences of hyperoxia in the neonatal lung.
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Affiliation(s)
- Alejandro M Scaffa
- Department of Molecular Pharmacology and Physiology, Brown University, Providence, Rhode Island, USA.,Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
| | - Abigail L Peterson
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
| | - Jennifer F Carr
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
| | - David Garcia
- Department of Chemistry, Brown University, Providence, Rhode Island, USA
| | - Hongwei Yao
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
| | - Phyllis A Dennery
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA.,Department of Pediatrics, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA
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Li S, Cheng Y, Qin M, Chen S, Li P, Yang L. Exploring the utility of Au@PVP-polyamide-Triton X-114 for SERS tracking of extracellular senescence associated-beta-galactosidase activity. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2021; 13:2087-2091. [PMID: 33912876 DOI: 10.1039/d1ay00470k] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
A compound with enrichment and SERS enhancement was successfully developed, which could rapidly adsorb X-gal hydrolysates from a liquid matrix in 5 minutes and further be used for SERS analysis with a detection limit of less than 1 × 10-9 mol L-1. This novel strategy will facilitate the development of an analytical approach for cellular senescence.
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Affiliation(s)
- Shaofei Li
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and University of Science and Technology of China, Hefei, Anhui 230026, China and School of Life Science, Anhui University, Hefei, Anhui 230601, China
| | - Yizhuang Cheng
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Miao Qin
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Siyu Chen
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Pan Li
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and Cancer Hospital, Chinese Academy of Sciences, Hefei, Anhui 230031, China
| | - Liangbao Yang
- Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China. and University of Science and Technology of China, Hefei, Anhui 230026, China and Cancer Hospital, Chinese Academy of Sciences, Hefei, Anhui 230031, China
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Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response. Cancers (Basel) 2021; 13:cancers13092115. [PMID: 33925586 PMCID: PMC8123910 DOI: 10.3390/cancers13092115] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 04/18/2021] [Accepted: 04/22/2021] [Indexed: 12/12/2022] Open
Abstract
Simple Summary Chemotherapy is a commonly applied anticancer treatment, however therapy-induced senescent growth arrest has been associated with aggressive disease recurrence. The p21 protein, encoded by CDKN1A, plays a vital role in the induction of senescence. Its transcriptional control by p53 is well-established. However, in many cancers where TP53 is mutated, p21 expression must be triggered by p53-independent mechanisms. We here used a chromatin-directed proteomic approach and identified ZNF84 as a regulator of CDKN1A gene expression in various p53-deficient cell lines. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin and facilitated senescence bypass. Intriguingly, ZNF84 depletion diminished genotoxic burden evoked by doxorubicin. Clinical data association studies indicated the relevance of ZNF84 expression for patient survival. Collectively, we identified ZNF84 as a critical regulator of senescence-proliferation outcome of chemotherapy, opening possibilities for its targeting in novel anti-cancer therapies of p53-mutated tumours. Abstract The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.
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Zia A, Farkhondeh T, Pourbagher-Shahri AM, Samarghandian S. The Roles of mitochondrial dysfunction and Reactive Oxygen Species in Aging and Senescence. Curr Mol Med 2021; 22:37-49. [PMID: 33602082 DOI: 10.2174/1566524021666210218112616] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 12/16/2020] [Accepted: 12/23/2020] [Indexed: 11/22/2022]
Abstract
The aging process deteriorates organs' function at different levels, causing its progressive decline to resist stress, damage, and disease. In addition to alterations in metabolic control and gene expression, the rate of aging has been connected with the generation of high amounts of Reactive Oxygen Species (ROS). The essential perspective in free radical biology is that reactive oxygen species (ROS) and free radicals are toxic, mostly cause direct biological damage to targets, and are thus a major cause of oxidative stress. Different enzymatic and non-enzymatic compounds in the cells have roles in neutralizing this toxicity. Oxidative damage in aging is mostly high in particular molecular targets, such as mitochondrial DNA and aconitase, and oxidative stress in mitochondria can cause tissue aging across intrinsic apoptosis. Mitochondria's function and morphology are impaired through aging, following a decrease in the membrane potential by an increase in peroxide generation and size of the organelles. Telomeres may be the significant trigger of replicative senescence. Oxidative stress accelerates telomere loss, whereas antioxidants slow it down. Oxidative stress is a crucial modulator of telomere shortening, and that telomere-driven replicative senescence is mainly a stress response. The age-linked mitochondrial DNA mutation and protein dysfunction aggregate in some organs like the brain and skeletal muscle, thus contributing considerably to these post-mitotic tissues' aging. The aging process is mostly due to accumulated damage done by harmful species in some macromolecules such proteins, DNA, and lipids. The degradation of non-functional, oxidized proteins is a crucial part of the antioxidant defenses of cells, in which the clearance of these proteins occurs through autophagy in the cells, which is known as mitophagy for mitochondria.
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Affiliation(s)
- Aliabbas Zia
- Department of Biochemistry, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran. Iran
| | - Tahereh Farkhondeh
- Medical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand. Iran
| | | | - Saeed Samarghandian
- Noncommunicable Diseases Research Center, Neyshabur University of Medical Sciences, Neyshabur. Iran
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Verzola D, Saio M, Picciotto D, Viazzi F, Russo E, Cipriani L, Carta A, Costigliolo F, Gaggero G, Salvidio G, Esposito P, Garibotto G, Poggi L. Cellular Senescence Is Associated with Faster Progression of Focal Segmental Glomerulosclerosis. Am J Nephrol 2021; 51:950-958. [PMID: 33440379 DOI: 10.1159/000511560] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Accepted: 09/12/2020] [Indexed: 02/05/2023]
Abstract
BACKGROUND A current, albeit unproven, hypothesis is that an acceleration of cellular senescence is involved in impaired renal repair and progression of glomerular diseases. Focal segmental glomerulosclerosis (FSGS) is a glomerular disease with a substantial risk for progression to ESRD. However, if and to what extent cell senescence predicts a negative outcome in FSGS is still unknown. METHODS The hypothesis that cell senescence represents a proximate mechanism by which the kidney is damaged in FSGS (NOS phenotype) was investigated in 26 consecutive kidney biopsies from adult FSGS cases (eGFR 72 ± 4 mL/min, proteinuria 2.3 ± 0.6 g/day) who were incident for 2 years in a Northern Italian nephrology center and had a 6-year clinical follow-up. RESULTS Cell senescence (p16INK4A, SA-β-galactosidase [SA-β-Gal]) was upregulated by ∼3- to 4-fold in both glomerular and tubular cells in kidney biopsies of FSGS as compared to age-matched controls (p < 0.05-0.01). Tubular SA-β-Gal correlated with proteinuria and glomerulosclerosis, while only as a trend, tubular p16INK4A was directly associated with interstitial fibrosis. At univariate analysis, basal eGFR, proteinuria, and tubular expression of SA-β-Gal and p16INK4A were significantly directly related to the annual loss of eGFR. No correlation was observed between glomerular p16INK4A and eGFR loss. However, at multivariate analysis, eGFR, proteinuria, and tubular p16INK4A, but not SA-β-Gal, contributed significantly to the prediction of eGFR loss. CONCLUSIONS The results indicate that an elevated cell senescence rate, expressed by an upregulation of p16INK4A in tubules at the time of initial biopsy, represents an independent predictor of progression to ESRD in adult patients with FSGS.
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Affiliation(s)
- Daniela Verzola
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Michela Saio
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Daniela Picciotto
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Francesca Viazzi
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Elisa Russo
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Leda Cipriani
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Annalisa Carta
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Francesca Costigliolo
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Gabriele Gaggero
- Division of Pathology, IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Gennaro Salvidio
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Pasquale Esposito
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
| | - Giacomo Garibotto
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy,
| | - Laura Poggi
- Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine and IRCCS Ospedale Policlinico San Martino, University of Genova, Genova, Italy
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Abstract
Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated β-galactosidase activity (SA-β-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.
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Tsai C, Chang C, Lin B, Wu Y, Wu M, Lin L, Huang W, Holz JD, Sheu T, Lee J, Kitsis RN, Tai P, Lee Y. Up-regulation of cofilin-1 in cell senescence associates with morphological change and p27 kip1 -mediated growth delay. Aging Cell 2021; 20:e13288. [PMID: 33336885 PMCID: PMC7811848 DOI: 10.1111/acel.13288] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Revised: 11/05/2020] [Accepted: 11/27/2020] [Indexed: 01/10/2023] Open
Abstract
Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27Kip1 , but not on the p53 and p16INK4 expressions. The knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27Kip1 gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.
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Affiliation(s)
- Cheng‐Han Tsai
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Chun‐Yuan Chang
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Bing‐Ze Lin
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Yu‐Lou Wu
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Meng‐Hsiu Wu
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Liang‐Tin Lin
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Wen‐Chien Huang
- Department of Surgery Division of Thoracic Surgery MacKay Memorial Hospital Taipei Taiwan
| | - Jonathan D. Holz
- Department of Biology University of Rochester Rochester NY14642USA
| | - Tzong‐Jen Sheu
- Department of Orthopaedics Center for Musculoskeletal Research University of Rochester School of Medicine Rochester NY14642USA
| | - Jhih‐Shian Lee
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
| | - Richard N. Kitsis
- Departments of Medicine (Cardiology) and Cell Biology and Wilf Family Cardiovascular Research Institute Albert Einstein College of Medicine Bronx, New York NY USA
| | - Pei‐Han Tai
- Graduate Institute of Oral Biology School of Dentistry National Taiwan University Taipei Taiwan
| | - Yi‐Jang Lee
- Department of Biomedical Imaging and Radiological Sciences National Yang‐Ming University Taipei Taiwan
- Cancer Progression Research Center National Yang‐Ming University Taipei11221Taiwan
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Huang X, Lan M, Wang J, Guo L, Lin Z, Sun N, Wu C, Qiu B. A fluorescence signal amplification and specific energy transfer strategy for sensitive detection of β-galactosidase based on the effects of AIE and host-guest recognition. Biosens Bioelectron 2020; 169:112655. [DOI: 10.1016/j.bios.2020.112655] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2020] [Revised: 09/18/2020] [Accepted: 09/24/2020] [Indexed: 12/16/2022]
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Santin Y, Lluel P, Rischmann P, Gamé X, Mialet-Perez J, Parini A. Cellular Senescence in Renal and Urinary Tract Disorders. Cells 2020; 9:cells9112420. [PMID: 33167349 PMCID: PMC7694377 DOI: 10.3390/cells9112420] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 11/02/2020] [Accepted: 11/04/2020] [Indexed: 02/06/2023] Open
Abstract
Cellular senescence is a state of cell cycle arrest induced by repetitive cell mitoses or different stresses, which is implicated in various physiological or pathological processes. The beneficial or adverse effects of senescent cells depend on their transitory or persistent state. Transient senescence has major beneficial roles promoting successful post-injury repair and inhibiting malignant transformation. On the other hand, persistent accumulation of senescent cells has been associated with chronic diseases and age-related illnesses like renal/urinary tract disorders. The deleterious effects of persistent senescent cells have been related, in part, to their senescence-associated secretory phenotype (SASP) characterized by the release of a variety of factors responsible for chronic inflammation, extracellular matrix adverse remodeling, and fibrosis. Recently, an increase in senescent cell burden has been reported in renal, prostate, and bladder disorders. In this review, we will summarize the molecular mechanisms of senescence and their implication in renal and urinary tract diseases. We will also discuss the differential impacts of transient versus persistent status of cellular senescence, as well as the therapeutic potential of senescent cell targeting in these diseases.
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Affiliation(s)
- Yohan Santin
- Institut des Maladies Métaboliques et Cardiovasculaires, Inserm, Université Paul Sabatier, UMR 1048—I2MC, 31432 Toulouse, France; (Y.S.); (J.M.-P.)
| | - Philippe Lluel
- Urosphere SAS, Rue des Satellites, 31400 Toulouse, France;
| | - Pascal Rischmann
- Department of Urology, Kidney Transplantation and Andrology, Toulouse Rangueil University Hospital, 31432 Toulouse, France; (P.R.); (X.G.)
| | - Xavier Gamé
- Department of Urology, Kidney Transplantation and Andrology, Toulouse Rangueil University Hospital, 31432 Toulouse, France; (P.R.); (X.G.)
| | - Jeanne Mialet-Perez
- Institut des Maladies Métaboliques et Cardiovasculaires, Inserm, Université Paul Sabatier, UMR 1048—I2MC, 31432 Toulouse, France; (Y.S.); (J.M.-P.)
| | - Angelo Parini
- Institut des Maladies Métaboliques et Cardiovasculaires, Inserm, Université Paul Sabatier, UMR 1048—I2MC, 31432 Toulouse, France; (Y.S.); (J.M.-P.)
- Correspondence: ; Tel.: +33-561325601
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Qiu W, Li X, Shi D, Li X, Gao Y, Li J, Mao F, Guo Y, Li J. A rapid-response near-infrared fluorescent probe with a large Stokes shift for senescence-associated β-galactosidase activity detection and imaging of senescent cells. DYES AND PIGMENTS 2020; 182:108657. [DOI: 10.1016/j.dyepig.2020.108657] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage. Int J Mol Sci 2020; 21:ijms21207556. [PMID: 33066270 PMCID: PMC7593953 DOI: 10.3390/ijms21207556] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Revised: 10/06/2020] [Accepted: 10/08/2020] [Indexed: 02/06/2023] Open
Abstract
Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.
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Biomarkers of senescence in non-human primate adipose depots relate to aging. GeroScience 2020; 43:343-352. [PMID: 32705409 DOI: 10.1007/s11357-020-00230-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Accepted: 07/07/2020] [Indexed: 10/23/2022] Open
Abstract
Forty-three female African green monkeys (Chlorocebus aethiops sabaeus) were selected to represent young adult to advanced geriatric ages (7-24 years) to exhibit a wide range of obesity status (8-53% body fat) and diverse metabolic syndrome criteria such as diabetes, dyslipidemia, and hypertension. Subcutaneous and visceral adipose tissues were collected and evaluated for the presence of senescence cells in both whole tissue and single-cell isolates from subcutaneous sources, utilizing senescence-associated β-galactosidase (SAβ-gal) staining. Plasma samples were analyzed for selected metabolic and inflammatory biomarkers related to the senescence-associated secretory profile. Our results indicated that tissue staining scores did not differ between subcutaneous and intra-abdominal visceral depots and were highly related within individuals. Tissue staining was significantly associated with chronological age; however, no associations with fatness or metabolic syndrome criteria were observed. Associations with age were unchanged when obesity status was included in regression models. Isolated cell staining did positively relate to age but not tissue staining, suggesting some of the SAβ-gal-positive cells were stromal vascular cells or small adipocytes, but that mature large adipocytes, filtered out in the cell isolation process, are also likely to exhibit positive SAβ-gal staining. Plasminogen activator inhibitor-1 (PAI-1) concentration in circulation was the sole inflammation-related biomarker that positively associated with age and is considered to be a marker of senescent cell burden. Our study is the largest, most comprehensive assessment of adipose SAβ-gal staining in a relevant animal model of human aging, and confirms that this senescence-associated biomarker specifically indicates an age-related process.
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Mohamad Kamal NS, Safuan S, Shamsuddin S, Foroozandeh P. Aging of the cells: Insight into cellular senescence and detection Methods. Eur J Cell Biol 2020; 99:151108. [PMID: 32800277 DOI: 10.1016/j.ejcb.2020.151108] [Citation(s) in RCA: 141] [Impact Index Per Article: 28.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 07/10/2020] [Indexed: 01/10/2023] Open
Abstract
Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means "growing old," is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.
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Affiliation(s)
- Nor Shaheera Mohamad Kamal
- School of Health Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
| | - Sabreena Safuan
- School of Health Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
| | - Shaharum Shamsuddin
- School of Health Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; USM-RIKEN International Centre for Ageing Science (URICAS), Universiti Sains Malaysia, 11800 Georgetown, Penang, Malaysia
| | - Parisa Foroozandeh
- USM-RIKEN International Centre for Ageing Science (URICAS), Universiti Sains Malaysia, 11800 Georgetown, Penang, Malaysia.
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