One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.

Cellular senescence in testicular cancer. Is there a correlation with the preoperative markers and the extent of the tumor? An experimental study.

PURPOSE: The aim of this experimental study is to investigate the correlation between the presence of senescent cells and the tumor size, the lymphovascular invasion (LVI), the invasion of rete testis (RTI), the preoperative tumor markers or pathological stage in patients who underwent orchiectomy for malignant purposes. METHODS: This experimental study included patients with a history of radical orchiectomy performed from January 2011 to January 2019. The testicular tissue specimens underwent an immunohistopathological process for the detection of the presence of cellular senescence. Besides, the tumor size, the histopathological type, the pathological stage of the tumor and the presence of Lymphovascular (LVI) or rete testis (RTI) invasions were also recorded. Additionally, the preoperative serum levels of alpha-fetoprotein, beta-human chorionic gonadotropin and lactate dehydrogenase were recorded. After the completion of immunohistochemical analysis, the rate of senescent cells in each specimen was also recorded. RESULTS: The mean senescent cell rate was estimated to be 14.11±11.32% and 15.46±10.58% in patients with presence of LVI or absence of LVI, respectively (p=0.46). The mean senescent cell rate was calculated at 18.13±12.26% and 12.56±9.38% (p=0.096) in patients with presence of RTI or absence of RTI, respectively. The mean senescent cell rate in the pT1 group was calculated at 14.58 ± 9.82%, while in T2 and T3 groups the mean senescent cell rate was estimated to be 15.22 ± 12.03% and 15.35 ± 14.21%, respectively (p=0.98). A statistically significant correlation was detected between the senescence rate and the tumor size (Pearson score 0.40, p=0.027) and between the rate of senescent cells and the preoperative level of lactate dehydrogenase (LDH) (Pearson score -0.53, p=0.002). CONCLUSIONS: The presence of cellular senescence was correlated with the extent of the testicular tumor in terms of tumor size as well as the preoperative level of the LDH serum marker.

Pulsed Electromagnetic Fields (PEMFs) Trigger Cell Death and Senescence in Cancer Cells.

The currently available anti-cancer therapies, such as gamma-radiation and chemotherapeutic agents, induce cell death and cellular senescence not only in cancer cells but also in the adjacent normal tissue. New anti-tumor approaches focus on limiting the side effects on normal cells. In this frame, the potential anti-tumor properties of Pulsed Electromagnetic Fields (PEMFs) through the irradiation of breast cancer epithelial cells (MCF-7 and MDA-MB-231) and normal fibroblasts (FF95) were investigated. PEMFs had a frequency of 8 Hz, full-square wave type and magnetic flux density of 0.011 T and were applied twice daily for 5 days. The data collected showcase that PEMF application decreases the proliferation rate and viability of breast cancer cells while having the opposite effect on normal fibroblasts. Moreover, PEMF irradiation induces cell death and cellular senescence only in breast cancer cells without any effect in the non-cancerous cells. These findings suggest PEMF irradiation as a novel, non-invasive anti-cancer strategy that, when combined with senolytic drugs, may eliminate both cancer and the remaining senescent cells, while simultaneously avoiding the side effects of the current treatments.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

Targeting senescence and inflammation in chronic destructive TNF-driven joint pathology.

We had shown that administration of the senolytic Dasatinib abolishes arthritis in the human TNF transgenic mouse model of chronic destructive arthritis when given in combination with a sub-therapeutic dose of the anti-TNF mAb Infliximab (1 mg/kg). Herein, we found that while the number of senescent chondrocytes (GL13+/Ki67-), assessed according to guideline algorithmic approaches, was not affected by either Dasatinib or sub-therapeutic Infliximab monotherapies, their combination reduced senescent chondrocytes by 50 %, which was comparable to levels observed with therapeutic Infliximab monotherapy (10 mg/kg). This combination therapy also reduced the expression of multiple factors of senescence-associated secretory phenotype in arthritic joints. Studies to elucidate the interplay of inflammation and senescence may help in optimizing treatment strategies also for age-related pathologies characterized by chronic low-grade joint inflammation.

A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer.

BACKGROUND: Prostate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed. METHODS: Androgen-dependent (LNCaP) and -independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy. RESULTS: We demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression. CONCLUSIONS: We identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.

The Dual Role of Oxidative-Stress-Induced Autophagy in Cellular Senescence: Comprehension and Therapeutic Approaches.

The contemporary lifestyle of the last decade has undeniably caused a tremendous increase in oxidative-stress-inducing environmental sources. This phenomenon is not only connected with the rise of ROS levels in multiple tissues but is also associated with the induction of senescence in different cell types. Several signaling pathways that are associated with the reduction in ROS levels and the regulation of the cell cycle are being activated, so that the organism can battle deleterious effects. Within this context, autophagy plays a significant role. Through autophagy, cells can maintain their homeostasis, as if it were a self-degradation process, which removes the "wounded" molecules from the cells and uses their materials as a substrate for the creation of new useful cell particles. However, the role of autophagy in senescence has both a "dark" and a "bright" side. This review is an attempt to reveal the mechanistic aspects of this dual role. Nanomedicine can play a significant role, providing materials that are able to act by either preventing ROS generation or controllably inducing it, thus functioning as potential therapeutic agents regulating the activation or inhibition of autophagy.

Increase of HO-1 Expression in Critically Ill COVID-19 Patients Is Associated with Poor Prognosis and Outcome.

Heme-oxygenase (HO)-1 is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties and previous reports have also emphasized the antiviral properties of HO-1, either directly or via induction of interferons. To investigate the potential role of HO-1 in patients with coronavirus disease 2019 (COVID-19), the present study assessed changes in HO-1 expression in whole blood and tissue samples. Upregulation of HO-1 protein was observed in lung, liver, and skin tissue independently of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presence. A significant increase of blood HO-1 mRNA levels was observed in critically ill COVID-19 patients compared to those in severe COVID-19 patients and healthy controls. This increase was accompanied by significantly elevated levels of serum ferritin and bilirubin in critically ill compared to patients with severe disease. Further grouping of patients in survivors and non-survivors revealed a significant increase of blood HO-1 mRNA levels in the later. Receiver operating characteristic (ROC) analysis for prediction of ICU admission and mortality yielded an AUC of 0.705 (p = 0.016) and 0.789 (p = 0.007) respectively indicating that HO-1 increase is associated with poor COVID-19 progression and outcome. The increase in HO-1 expression observed in critically ill COVID-19 patients could serve as a mechanism to counteract increased heme levels driving coagulation and thrombosis or as an induced protective mechanism.

Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory system can progress to a multisystemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named senescence-associated secretory phenotype (SASP). We investigated whether coronavirus disease 2019 (COVID-19) is associated with cellular senescence and SASP. METHODS: Autopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID-19 controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients. RESULTS: SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed angiotensin-converting enzyme 2 and exhibited increased senescence (p16INK4A and SenTraGor positivity) and interleukin (IL)-1ß and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGor), DNA damage (γ-H2AX) and increased cytokine (IL-1ß, IL-6, CXCL8) and apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extrapulmonary sites (kidney and liver) of a COVID-19 patient. CONCLUSIONS: We demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.

Evaluation of senescent cells in intervertebral discs by lipofuscin staining.

Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-ß-gal staining or p16Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-ß-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.

Biological Effect of Silver-modified Nanostructured Titanium Dioxide in Cancer.

BACKGROUND/AIM: Nanomedicine is a promising scientific field that exploits the unique properties of innovative nanomaterials, providing alternative solutions in diagnostics, prevention and therapeutics. Titanium dioxide nanoparticles (TiO2 NPs) have a great spectrum of photocatalytic antibacterial and anticancer applications. The chemical modification of TiO2 optimizes its bioactive performance. The aim of this study was the development of silver modified NPs (Ag/TiO2 NPs) with anticancer potential. MATERIALS AND METHODS: Ag/TiO2 NPs were prepared through the sol-gel method, were fully characterized and were tested on cultured breast cancer epithelial cells (MCF-7 and MDA-MB-231). The MTT colorimetric assay was used to estimate cellular viability. Western blot analysis of protein expression along with a DNA-laddering assay were employed for apoptosis detection. RESULTS AND CONCLUSION: We show that photo-activated Ag/TiO2 NPs exhibited significant cytotoxicity on the highly malignant MDA-MB-231 cancer cells, inducing apoptosis, while MCF-7 cells that are characterized by low invasive properties were unaffected under the same conditions.

Implication of Dietary Iron-Chelating Bioactive Compounds in Molecular Mechanisms of Oxidative Stress-Induced Cell Ageing.

One of the prevailing perceptions regarding the ageing of cells and organisms is the intracellular gradual accumulation of oxidatively damaged macromolecules, leading to the decline of cell and organ function (free radical theory of ageing). This chemically undefined material known as "lipofuscin," "ceroid," or "age pigment" is mainly formed through unregulated and nonspecific oxidative modifications of cellular macromolecules that are induced by highly reactive free radicals. A necessary precondition for reactive free radical generation and lipofuscin formation is the intracellular availability of ferrous iron (Fe2+) ("labile iron"), catalyzing the conversion of weak oxidants such as peroxides, to extremely reactive ones like hydroxyl (HO•) or alcoxyl (RO•) radicals. If the oxidized materials remain unrepaired for extended periods of time, they can be further oxidized to generate ultimate over-oxidized products that are unable to be repaired, degraded, or exocytosed by the relevant cellular systems. Additionally, over-oxidized materials might inactivate cellular protection and repair mechanisms, thus allowing for futile cycles of increasingly rapid lipofuscin accumulation. In this review paper, we present evidence that the modulation of the labile iron pool distribution by nutritional or pharmacological means represents a hitherto unappreciated target for hampering lipofuscin accumulation and cellular ageing.

Carotid Disease and Ageing: A Literature Review on the Pathogenesis of Vascular Senescence in Older Subjects.

Aging is a natural process that affects all systems of the human organism, leading to its inability to adapt to environmental changes. Advancing age has been correlated with various pathological conditions, especially cardiovascular and cerebrovascular diseases. Carotid artery (CA) is mainly affected by age-induced functional and morphological alterations causing atheromatous disease. The evolvement of biomedical sciences has allowed the elucidation of many aspects of this condition. Symptomatic carotid disease (CD) derives from critical luminar stenosis or eruption of an atheromatous plaque due to structural modifications of the vessels, such as carotid intima-media thickening. At a histologic level, the aforementioned changes are mediated by elastin fragmentation, collagen deposition, immune cell infiltration, and accumulation of cytokines and vasoconstrictors. Underlying mechanisms include chronic inflammation and oxidative stress, dysregulation of cellular homeostatic systems, and senescence. Thus, there is an imbalance in components of the vessel wall, which fails to counteract exterior stress stimuli. Consequently, arterial relaxation is impaired and atherosclerotic lesions progress. This is a review of current evidence regarding the relationship of aging with vascular senescence and CD. A deeper understanding of these mechanisms can contribute to the production of efficient prevention methods and targeted therapeutic strategies.