Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells.

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Curcumin induces multiple signaling pathways leading to vascular smooth muscle cell senescence.

Curcumin, a phytochemical present in the spice named turmeric, and one of the promising anti-aging factors, is itself able to induce cellular senescence. We have recently shown that cells building the vasculature senesced as a result of curcumin treatment. Curcumin-induced senescence was DNA damage-independent; however, activation of ATM was observed. Moreover, neither increased ROS production, nor even ATM were indispensable for senescence progression. In this paper we tried to elucidate the mechanism of curcumin-induced senescence. We analyzed the time-dependence of the level and activity of numerous proteins involved in senescence progression in vascular smooth muscle cells and how inhibition p38 or p38 together with ATM, two proteins involved in canonical signaling pathways, influenced cell senescence. We showed that curcumin was able to influence many signaling pathways of which probably none was dominant and sufficient to induce senescence by itself. However, we cannot exclude that the switch between initiation and progression of senescence is the result of the impact of curcumin on signaling pathways engaging AMPK, ATM, sirtuin 1 and p300 and on their reciprocal interplay. Cytostatic concentration of curcumin induced cellular stress, which exceeded the adaptive response and, in consequence, led to cellular senescence, which is triggered by time dependent activation of several signaling pathways playing diverse roles in different phases of senescence progression. We also showed that activity of ß-glucuronidase, the enzyme involved in deconjugation of the main metabolites of curcumin, glucuronides, increased in senescent cells. It suggests a possible local elevation of curcumin concentration in the organism.

The Role of Curcumin in the Modulation of Ageing.

It is believed that postponing ageing is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-ageing role of curcumin and discuss its senolytic potential.

What is and what is not cell senescence.

Cell senescence is a process that occurs due to telomere erosion or can be induced by various stresses. Senescent cells cease to divide but remain alive, metabolically active and able to secrete many molecules. They also show many hallmarks of senescence, such as enlarged size, increased granularity, increased activity of SA-ß-galactosidase, increased level of cyclin-dependent kinase inhibitors, p16 and p21, and DNA damage foci. Originally, cell senescence was attributed to proliferating normal cells, in contrast to cancer cells, which were considered as those endowed with indefinite growth ability. Recently, it has become evident that anticancer treatment induces senescence in cancer cells. Moreover, certain hallmarks of senescence were detected in non-proliferating post-mitotic cells. There are many signalling pathways involved in cell senescence, but the most prevalent is the DNA damage response pathway. In this review we have summarized our long lasting input in the global study of the mechanisms of senescence of normal and cancer cells and discussed the diversity of the concept of cell senescence.

A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.

[The role of NADPH oxidase NOX4 in regulation of proliferation, senescence and differentiation of the cells]. / Rola oksydazy NADPH NOX4 w regulacji procesów proliferacji, starzenia i róznicowania komórek.

NADPH oxidase NOX4 is a source of reactive oxygen species in many tissue of human body. NOX4 products of activity are connected with various processes that take on the cellular and tissue level. One of them is cellular senescence. The role of this oxidase in the regulation of replicative and oncogene-induced senescence was shown in both normal and cancer cells. On the other hand NOX4 also stimulates to proliferation various types of cancer and primary cells, what promotes pathologies. NOX4 participates in epithelial-mesenchymal transition, important for tumor cells invasion and metastasis. Many research concern the role of NOX4 in the physiology and pathology of the cardiovascular system. It was shown that NOX4 has an impact on vasoconstriction, atherosclerosis development, vascular cells hypertrophy, apoptosis and differentiation. NOX4 plays both positive and negative role in the organism. Better understanding of NOX4 regulation and its involvement in signaling pathways give a hope to control the development of many diseases.

[The role of cellular senescence in carcinogenesis and antitumor therapy]. / Rola starzenia komórkowego w kancerogenezie i terapii przeciwnowotworowej.

Cellular senescence is the process that lead to terminal growth arrest induced by unrepairable double strand DNA damage (DSB). Moreover, activation of the oncogenes as well as inhibition of the tumor suppressor genes were shown to contribute to senescence induction and the senescent cells were identified in the premalignant lesions. Thus senescence is considered as an natural antitumor barrier that act at the early stages of cancerogenesis to stop the proliferation of transformed cells. Interestingly, the premalignant cells that escaped senescence and progress into full blown tumor cells still remain sensitive to induction of senescence, for example during chemio- or radiotherapy. Thus, induction of cancer cell senescence, similarly to apoptosis, is considered to restrain tumor growth and thus contribute to effectiveness of anticancer therapy. The senescent cells, although do not proliferate, remain viable and metabolically active. They secret a lot of cytokines, mitogens as well as enzymes degrading extracellular matrix. These factors can have opposing effect on neighboring cells, leading to senescence induction or stimulation of proliferation. Thus, senescence can act as an double edge sword that inhibit the propagation of potentially dangerous, transformed cells on one hand or induce cell division of the same cell on the other. Presently a lot of work is focused on finding new therapeutic strategies that would involve the tumor targeted senescence induction in both early late stages of cancer development. Nevertheless, the unwanted influence of the senescent cells on the microenvironment, requires careful monitoring the effects of pro-senescent therapies in each case.

Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets.

Extracellular vesicles (EVs) enable communication between cells and tissues and are implicated in modulation of tumor immunosuppression. Here, we present a protocol for isolating tumor-derived EVs and assessing their functional influence in cultures with different subsets of human T cells. We describe steps for differential ultracentrifugation, size exclusion chromatography, EVs quantification, and fluorescence-activated cell sorting of human T cells. We then detail procedures for culturing T cells with EVs and using high-resolution spectral flow cytometry phenotyping for the analysis thereof. For complete details on the use and execution of this protocol, please refer to Swatler et al.1 and Swatler et al.2.

Extracellular Vesicles as Next-Generation Biomarkers in Lung Cancer Patients: A Case Report on Adenocarcinoma and Squamous Cell Carcinoma.

Extracellular vesicles (EVs) released from primary cell lines, originating from resected tissues during biopsies in patients with non-small cell lung cancer (NSCLC) revealing adenocarcinoma and squamous cell carcinoma subtypes, were examined for membrane proteomic fingerprints using a proximity barcoding assay. All the collected EVs expressed canonical tetraspanins (CD9, CD63, and CD81) highly coexpressed with molecules such as lysosome-associated membrane protein-1 (LAMP1-CD107a), sialomucin core protein 24 (CD164), Raph blood group (CD151), and integrins (ITGB1 and ITGA2). This representation of the protein molecules on the EV surface may provide valuable information on NSCLC subtypes and offer new diagnostic opportunities as next-generation biomarkers in personalized oncology.

DNA damage-independent apoptosis induced by curcumin in normal resting human T cells and leukaemic Jurkat cells.

Curcumin, a phytochemical derived from the rhizome of Curcuma longa, is a very potent inducer of cancer cell death. It is believed that cancer cells are more sensitive to curcumin treatment than normal cells. Curcumin has been shown to act as a prooxidant and induce DNA lesions in normal cells. We were interested in whether curcumin induces DNA damage and the DNA damage response (DDR) signalling pathway leading to apoptosis in normal resting human T cells. To this end, we analysed DNA damage after curcumin treatment of resting human T cells (CD3(+)) and of proliferating leukaemic Jurkat cells by the fluorimetric detection of alkaline DNA unwinding (FADU) assay and immunocytochemical detection of γ-H2AX foci. We showed that curcumin-treated Jurkat cells and resting T cells showed neither DNA lesions nor did they activate key proteins in the DDR signalling pathway, such as phospho-ATM and phospho-p53. However, both types of cell were equally sensitive to curcumin-induced apoptosis and displayed activation of caspase-8 but not of DNA damage-dependent caspase-2. Altogether, our results revealed that curcumin can induce apoptosis of normal resting human T cells that is not connected with DNA damage.

Fiji-Based Tool for Rapid and Unbiased Analysis of SA-ß-Gal Activity in Cultured Cells.

Normal cells under stressful conditions such as DNA damage or excessive mitogenic signaling may undergo senescence, which is associated with cell cycle arrest and induction of a proinflammatory phenotype. Accumulation of senescent cells may contribute to the shortening of the life span by accelerating aging and promoting chronic diseases. Cytochemical detection of the senescence-associated ß-galactosidase (SA-ß-gal) activity with 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal) is a widely recognised marker of cell senescence. However, its simplicity and cost effectiveness lead to limitations in quantification, which is usually limited to manual counting of the positive cells. In order to address those limitations, we developed a Fiji-based macro extension that performs automatic and unbiased analysis of the integrated density of SA-ß-gal specific signal. Our tool is not only faster than manual counting but also provides extra resolution compared to the manual methods. Our macro extension could be a valuable tool in any senescence research laboratory.

Lack of activity of the mitochondrial large-conductance calcium-regulated potassium channels in senescent vascular smooth muscle cells.

A limited number of studies have shown functional changes in mitochondrial ion channels in aging and senescent cells. We have identified, for the first time, mitochondrial large-conductance calcium-regulated potassium channels in human smooth muscle mitochondria. This channel, with a conductance of 273 pS, was regulated by calcium ions and membrane potential. Additionally, it was activated by the potassium channel opener NS11021 and blocked by paxilline. Importantly, we have shown that senescence of these cells induced by hydrogen peroxide treatment leads to the disappearance of potassium channel protein levels and channel activity measured by the single channel patch-clamp technique. Our data suggest that disturbances in the expression of mitochondrial large conductance calcium-regulated potassium channels may be hallmarks of cellular senescence and contribute to the misregulation of mitochondrial function in senescent cells.

Exosomes derived from lung cancer cells: Isolation, characterization, and stability studies.

Recent advances in nanomedicine have paved the way for developing targeted drug delivery systems. Nanoscale exosomes are present in almost every body fluid and represent a novel mechanism of intercellular communication. Because of their membrane origin, they easily fuse with cells, acting as a natural delivery system and maintaining the bioactivity and immunotolerance of cells. To develop a reconstitutable exosome-based drug candidate for clinical applications, quality assurance by preserving its physical and biological properties during storage is necessary. Therefore, this study aimed to determine the best storage conditions for exosomes derived from lung cancer cells (A549). This study established that the phosphate-buffered saline buffer enriched with 25 mM trehalose is an optimal cryoprotectant for A549-derived exosomes stored at -80°C. Under these conditions, the concentration, size distribution, zeta potential, and total cargo protein levels of the preserved exosomes remained constant.

Combination of dasatinib and quercetin improves cognitive abilities in aged male Wistar rats, alleviates inflammation and changes hippocampal synaptic plasticity and histone H3 methylation profile.

Aging is associated with cognitive decline and accumulation of senescent cells in various tissues and organs. Senolytic agents such as dasatinib and quercetin (D+Q) in combination have been shown to target senescent cells and ameliorate symptoms of aging-related disorders in mouse models. However, the mechanisms by which senolytics improve cognitive impairments have not been fully elucidated particularly in species other than mice. To study the effect of senolytics on aging-related multifactorial cognitive dysfunctions we tested the spatial memory of male Wistar rats in an active allothetic place avoidance task. Here we report that 8 weeks treatment with D+Q alleviated learning deficits and memory impairment observed in aged animals. Furthermore, treatment with D+Q resulted in a reduction of the peripheral inflammation measured by the levels of serum inflammatory mediators (including members of senescent cell secretome) in aged rats. Significant improvements in cognitive abilities observed in aged rats upon treatment with D+Q were associated with changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons and changes in the level of histone H3 trimethylation at lysine 9 and 27 in the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 5 weeks after the cessation of the drugs administration. Our results expand and provide new insights to the existing knowledge associated with effects of senolytics on alleviating age-related associated cognitive dysfunctions.

Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions.

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

A common signature of cellular senescence; does it exist?

Cellular senescence is a stress response, which can be evoked in all type of somatic cells by different stimuli. Senescent cells accumulate in the body and participate in aging and aging-related diseases mainly by their secretory activity, commonly known as senescence-associated secretory phenotype-SASP. Senescence is typically described as cell cycle arrest. This definition stems from the original observation concerning limited cell division potential of human fibroblasts in vitro. At present, the process of cell senescence is attributed also to cancer cells and to non-proliferating post-mitotic cells. Many cellular signaling pathways and specific and unspecific markers contribute to the complex, dynamic and heterogeneous phenotype of senescent cells. Considering the diversity of cells that can undergo senescence upon different inducers and variety of mechanisms involved in the execution of this process, we ask if there is a common signature of cell senescence. It seems that cell cycle arrest in G0, G1 or G2 is indispensable for cell senescence; however, to ensure irreversibility of divisions, the exit from the cell cycle to the state, which we call a GS (Gero Stage), is necessary. The DNA damage, changes in nuclear architecture and chromatin rearrangement are involved in signaling pathways leading to altered gene transcription and secretion of SASP components. Thus, nuclear changes and SASP are vital features of cell senescence that, together with temporal arrest in the cell cycle (G1 or/and G2), which may be followed by polyploidisation/depolyploidisation or exit from the cell cycle leading to permanent proliferation arrest (GS), define the signature of cellular senescence.

Cellular Senescence in Brain Aging.

Aging of the brain can manifest itself as a memory and cognitive decline, which has been shown to frequently coincide with changes in the structural plasticity of dendritic spines. Decreased number and maturity of spines in aged animals and humans, together with changes in synaptic transmission, may reflect aberrant neuronal plasticity directly associated with impaired brain functions. In extreme, a neurodegenerative disease, which completely devastates the basic functions of the brain, may develop. While cellular senescence in peripheral tissues has recently been linked to aging and a number of aging-related disorders, its involvement in brain aging is just beginning to be explored. However, accumulated evidence suggests that cell senescence may play a role in the aging of the brain, as it has been documented in other organs. Senescent cells stop dividing and shift their activity to strengthen the secretory function, which leads to the acquisition of the so called senescence-associated secretory phenotype (SASP). Senescent cells have also other characteristics, such as altered morphology and proteostasis, decreased propensity to undergo apoptosis, autophagy impairment, accumulation of lipid droplets, increased activity of senescence-associated-ß-galactosidase (SA-ß-gal), and epigenetic alterations, including DNA methylation, chromatin remodeling, and histone post-translational modifications that, in consequence, result in altered gene expression. Proliferation-competent glial cells can undergo senescence both in vitro and in vivo, and they likely participate in neuroinflammation, which is characteristic for the aging brain. However, apart from proliferation-competent glial cells, the brain consists of post-mitotic neurons. Interestingly, it has emerged recently, that non-proliferating neuronal cells present in the brain or cultivated in vitro can also have some hallmarks, including SASP, typical for senescent cells that ceased to divide. It has been documented that so called senolytics, which by definition, eliminate senescent cells, can improve cognitive ability in mice models. In this review, we ask questions about the role of senescent brain cells in brain plasticity and cognitive functions impairments and how senolytics can improve them. We will discuss whether neuronal plasticity, defined as morphological and functional changes at the level of neurons and dendritic spines, can be the hallmark of neuronal senescence susceptible to the effects of senolytics.

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response.

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.