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1.
Cell Mol Life Sci ; 79(10): 510, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36066676

RESUMO

Oncosis (from Greek ónkos, meaning "swelling") is a non-apoptotic cell death process related to energy depletion. In contrast to apoptosis, which is the main form of cell death induced by anticancer drugs, oncosis has been relatively less explored but holds potential to overcome drug resistance phenomena. In this study, we report a novel rationally designed mitochondria-targeted iridium(III) complex (OncoIr3) with advantageous properties as a bioimaging agent. OncoIr3 exhibited potent anticancer activity in vitro against cancer cells and displayed low toxicity to normal dividing cells. Flow cytometry and fluorescence-based assays confirmed an apoptosis-independent mechanism involving energy depletion, mitochondrial dysfunction and cellular swelling that matched with the oncotic process. Furthermore, a Caenorhabditis elegans tumoral model was developed to test this compound in vivo, which allowed us to prove a strong oncosis-derived antitumor activity in animals (with a 41% reduction of tumor area). Indeed, OncoIr3 was non-toxic to the nematodes and extended their mean lifespan by 18%. Altogether, these findings might shed new light on the development of anticancer metallodrugs with non-conventional modes of action such as oncosis, which could be of particular interest for the treatment of apoptosis-resistant cancers.


Assuntos
Antineoplásicos , Neoplasias , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Morte Celular , Linhagem Celular Tumoral , Irídio/farmacologia , Necrose , Neoplasias/tratamento farmacológico
2.
Curr Protoc ; 2(9): e525, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36069669

RESUMO

Unicellular eukaryotic organisms such as yeast and protozoa serve as useful models for studying the impact of chemicals on cell physiology, cellular growth, and genome duplication. The yeast Saccharomyces cerevisiae has been widely used to assess apoptosis induced by chemicals due to its genetic tractability, ease of evaluation, and readily available impact assessment tools. Apoptosis in S. cerevisiae is characterized by many features, including increased cell death, loss of membrane integrity, release of caspases, chromatin condensation, and nuclear fragmentation, which are similar to the ones observed in mammalian cells. Current methods of apoptosis assessment typically require specialized equipment and reagents, which limits wide adoption. Here, we describe a rapid, inexpensive, and easy-to-perform assay in yeast for the analysis of late-stage apoptotic features in cells treated with a chemical. We describe a protocol for assessing loss of cell survival and changes in the nucleus. We demonstrate the approach by using acetic acid and hydrogen peroxide as test chemicals. This assay for the study of late-stage apoptotic features in S. cerevisiae can be performed reliably and rapidly by any laboratory with basic equipment and may be extended for studying apoptosis in similar single-cell organisms after treatment with toxicological agents. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culture of Saccharomyces cerevisiae, treatment with acetic acid or hydrogen peroxide, and semi-quantitative growth assay Basic Protocol 2: DAPI staining and fluorescence microscopy for the assessment of change in nucleus-to-cytoplasm ratio and nuclear integrity.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácido Acético/metabolismo , Animais , Apoptose/fisiologia , Caspases/metabolismo , Peróxido de Hidrogênio/metabolismo , Mamíferos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Oxid Med Cell Longev ; 2022: 9148257, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36062190

RESUMO

Neuronal apoptosis after subarachnoid hemorrhage (SAH) is believed to play an important role in early brain injury after SAH. The energy metabolism of neuron is closely related to its survival. The transient hyperglycemia caused by insulin resistance (IR) after SAH seriously affects the prognosis of patients. However, the specific mechanisms of IR after SAH are still not clear. Studies have shown that α-KG takes part in the regulation of IR and cell apoptosis. In this study, we aim to investigate whether α-KG can reduce IR after SAH, improve the disorder of neuronal glucose metabolism, alleviate neuronal apoptosis, and ultimately play a neuroprotective role in SAH-induced EBI. We first measured α-KG levels in the cerebrospinal fluid (CSF) of patients with SAH. Then, we established a SAH model through hemoglobin (Hb) stimulation with HT22 cells for further mechanism research. Furthermore, an in vivo SAH model in mice was established by endovascular perforation. Our results showed that α-KG levels in CSF significantly increased in SAH patients and could be used as a potential prognostic biomarker. In in vitro model of SAH, we found that α-KG not only inhibited IR-induced reduction of glucose uptake in neurons after SAH but also alleviated SAH-induced neuronal apoptosis. Mechanistically, we found that α-KG inhibits neuronal IR by inhibiting S6K1 activation after SAH. Moreover, neuronal apoptosis significantly increased when glucose uptake was reduced. Furthermore, our results demonstrated that α-KG could also alleviate neuronal apoptosis in vivo SAH model. In conclusion, our study suggests that α-KG alleviates apoptosis by inhibiting IR induced by S6K1 activation after SAH.


Assuntos
Resistência à Insulina , Hemorragia Subaracnóidea , Animais , Apoptose/fisiologia , Glucose , Ácidos Cetoglutáricos , Camundongos , Fosforilação , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/tratamento farmacológico , Hemorragia Subaracnóidea/metabolismo
4.
Methods Cell Biol ; 172: 17-36, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064223

RESUMO

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.


Assuntos
Apoptose , Fosfatidilserinas , Animais , Anexina A5/metabolismo , Anexina A5/farmacologia , Apoptose/fisiologia , Morte Celular , Citometria de Fluxo/métodos , Humanos , Camundongos
5.
Methods Mol Biol ; 2543: 1-11, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36087254

RESUMO

This chapter describes a simple, nondestructive, annexin V apoptosis detection method that can be employed in real time over a 48-h test exposure. The real-time functionality allows for temporal resolution of apoptotic and cell death responses during the test exposure and obviates the need for onerous sample preparation and time course protocols associated with other annexin V methods. Further, this technique is eminently accessible to a wide range of laboratories because it does not require flow cytometry or other cytometric methods. It was developed for use with a variety of microplate well densities and with standard multimodal plate readers. The central feature of this assay is that it continuously reports the residency status of phosphatidylserine (PS) on the exofacial surface of a cell as it is translocated from the inner membrane leaflet during the apoptotic process. This homogenous, no-wash assay is made possible by two optimized and distinct annexin V fusion proteins which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During an apoptotic event, the luminescent signal arising from an assay well is proportional to the number of cells with PS exposure, and fluorescence intensity correlates with the degree of cell death (secondary necrosis). Conversely, untreated cells contribute negligible luminescent or fluorescent signals throughout the time course. The data collected from these assay measures provide for both standard potency determinations and kinetic characterization of dose- and agent-dependent apoptotic responses, from early through late phases.


Assuntos
Apoptose , Fosfatidilserinas , Anexina A5/metabolismo , Apoptose/fisiologia , Corantes , Citometria de Fluxo/métodos , Humanos , Necrose , Fosfatidilserinas/metabolismo
6.
Methods Mol Biol ; 2543: 45-55, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36087258

RESUMO

Apoptotic cells are cleared from the body principally through recognition and engulfment by neighboring phagocytes, a process known as efferocytosis. During efferocytosis, phagocytes are recruited to the site/activated by "find me" signals released from apoptotic cells, precisely identify apoptotic cells by the recognition of "eat me" signals on the apoptotic cell surface, and engulf the apoptotic cells to prevent secondary necrosis and inflammation. Thus, efferocytosis is critical for tissue homeostasis in normal physiology. However, efferocytosis of apoptotic tumor cells-performed by tumor-associated macrophages-suppresses immunity within the tumor microenvironment and limits the antitumor response. This phenomenon is further exacerbated in tumor residual disease because of the high apoptotic cell burden generated by cytotoxic therapies. Blocking efferocytosis could be a powerful approach to boost tumor immunogenicity, particularly as a combination approach with cytotoxic therapies that produce many apoptotic cells, but little is currently known about the immune response to efferocytosis. Moreover, there is a dearth of in vivo models available to study the immunologic and therapeutic consequences of blocking efferocytosis in tumor residual disease.Here, we describe a model that enables in vivo studies of tumor immunology in the aftermath of cytotoxic therapy with an emphasis on the impact of efferocytosis. Orthotopic HER2+ mammary tumors are established in immune-competent mice, followed by a single administration of lapatinib, a receptor tyrosine kinase inhibitor of HER2, to the mice that induces widespread, transient apoptosis in the tumor microenvironment. In the days following lapatinib treatment, agents that block efferocytosis such as BMS-777607 are administered. Tissue is collected from cohorts of mice at day 2 (after lapatinib treatment only) to assess apoptosis, day 8 (after lapatinib treatment followed by blockade of efferocytosis) to assess the immune response to apoptosis and efferocytosis, and day 28 (after 4 consecutive weeks of treatment) to assess therapeutic efficacy. This model enables mechanistic studies of tumor immunology in residual disease as well as therapeutic efficacy studies of targeted agents that disrupt efferocytosis.


Assuntos
Macrófagos , Neoplasias , Animais , Apoptose/fisiologia , Lapatinib/farmacologia , Macrófagos/metabolismo , Camundongos , Necrose/patologia , Neoplasias/patologia , Fagocitose , Microambiente Tumoral
7.
Methods Mol Biol ; 2543: 57-69, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36087259

RESUMO

Apoptosis and necrosis are the two sides of the cell death penumbra. Apoptosis is a well-studied model of cell death wherein the cell destroys itself employing a predefined form of active signaling without the release of soluble cytoplasmic contents to the external environment. Compared to apoptosis, necrosis is a nonspecific form of sudden cell death in response to an invasive external stimulus which in turn is devoid of active programmed intracellular signaling leading to the sudden release of the soluble cellular contents consequent to the rupture of the cell membrane. This fundamental difference between apoptosis and necrosis made us believe that the former is the safe form of cell death and the latter is an undesirable one which often elicits an inflammatory response to the adjacent cells. Recent studies have shown that necrosis also involves a few defined cellular and complex biochemical events similar to apoptosis rendering it difficult to distinguish these two events at the single-cell level using the currently used popular assays.Here we provide a newly described detailed methodology encompassing cell system development along with a multiparametric flow cytometry-based approach to discriminate apoptotic cells from necrotic cells using a stable cell line expressing genetically encoded probe for detecting caspase activation and DsRed targeted at the mitochondria.


Assuntos
Apoptose , Mitocôndrias , Apoptose/fisiologia , Morte Celular , Citometria de Fluxo/métodos , Humanos , Mitocôndrias/metabolismo , Necrose/metabolismo
8.
Front Immunol ; 13: 886374, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36110858

RESUMO

Fibrosis is defined as the abnormal and excessive deposition of extracellular matrix (ECM) components, which leads to tissue or organ dysfunction and failure. However, the pathological mechanisms underlying fibrosis remain unclear. The inflammatory response induced by tissue injury is closely associated with tissue fibrosis. Recently, an increasing number of studies have linked necroptosis to inflammation and fibrosis. Necroptosis is a type of preprogrammed death caused by death receptors, interferons, Toll-like receptors, intracellular RNA and DNA sensors, and other mediators. These activate receptor-interacting protein kinase (RIPK) 1, which recruits and phosphorylates RIPK3. RIPK3 then phosphorylates a mixed lineage kinase domain-like protein and causes its oligomerization, leading to rapid plasma membrane permeabilization, the release of cellular contents, and exposure of damage-associated molecular patterns (DAMPs). DAMPs, as inflammatory mediators, are involved in the loss of balance between extensive inflammation and tissue regeneration, leading to remodeling, the hallmark of fibrosis. In this review, we discuss the role of necroptotic DAMPs in tissue fibrosis and highlight the inflammatory responses induced by DAMPs in tissue ECM remodeling. By summarizing the existing literature on this topic, we underscore the gaps in the current research, providing a framework for future investigations into the relationship among necroptosis, DAMPs, and fibrosis, as well as a reference for later transformation into clinical treatment.


Assuntos
Alarminas , Apoptose , Alarminas/metabolismo , Apoptose/fisiologia , DNA , Fibrose , Humanos , Inflamação/patologia , Interferons , Necrose/patologia , RNA , Receptores de Morte Celular
9.
Mediators Inflamm ; 2022: 2356507, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36117589

RESUMO

Ischemic stroke (IS) is a general term for necrosis of brain tissue caused by stenosis, occlusion of arteries supplying blood to the brain (carotid artery and vertebral artery), and insufficient blood supply to the brain. Cerebral ischemia is the main kind of IS causing cell damage. However, the underlying mechanism still needs to be clarified further. In this study, it was demonstrated that FFAR1 was a hub gene in IS. The expression of FFAR1 was increased in PC12 cells with OGD/R treatment. FFAR1 deficiency inhibited cell viability and induced cell apoptosis, which was reversed by FFAR1 overexpression. Moreover, candesartan, as a compound targeting FFAR1, facilitated cell viability and reduced cell apoptosis. The expression of ITGA4 was also high in OGD/R-PC12 cells as FFAR1. Furthermore, FFAR1 deficiency retarded the increasing of cell viability and inhibition of cell apoptosis by downregulation of Bax and Cleaved Caspase-3 in OGD/R-PC12 cells with candesartan treatment. In conclusion, candesartan may regulate neuronal apoptosis through FFAR1/ITGA4 axis.


Assuntos
AVC Isquêmico , Animais , Apoptose/fisiologia , Benzimidazóis , Compostos de Bifenilo , Caspase 3/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , Ratos , Tetrazóis , Proteína X Associada a bcl-2
10.
Front Immunol ; 13: 917141, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36090995

RESUMO

COVID-19 caused by SARS-CoV-2 can cause various systemic diseases such as acute pneumonia with cytokine storm. Constituted of necroptosis, pyroptosis, and ferroptosis, regulated necrosis constitutes the cell death patterns under the low apoptosis condition commonly observed in COVID-19. Regulated necrosis is involved in the release of cytokines like TNF-α, IL-1 ß, and IL-6 and cell contents such as alarmins, PAMPs, and DAMPs, leading to more severe inflammation. Uncontrolled regulated necrosis may explain the poor prognosis and cytokine storm observed in COVID-19. In this review, the pathophysiology and mechanism of regulated necrosis with the double-edged sword effect in COVID-19 are thoroughly discussed in detail. Furthermore, this review also focuses on the biomarkers and potential therapeutic targets of the regulated necrosis pathway in COVID-19, providing practical guidance to judge the severity, prognosis, and clinical treatment of COVID-19 and guiding the development of clinical anti-SARS-CoV-2 drugs.


Assuntos
COVID-19 , Apoptose/fisiologia , Síndrome da Liberação de Citocina , Humanos , Necrose , SARS-CoV-2
11.
Int J Mol Sci ; 23(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36077484

RESUMO

Peroxisomes are a class of simple organelles that play an important role in plant reactive oxygen species (ROS) metabolism. Experimental evidence reveals the involvement of ROS in programmed cell death (PCD) in plants. Plant PCD is crucial for the regulation of plant growth, development and environmental stress resistance. However, it is unclear whether the ROS originated from peroxisomes participated in cellular PCD. Enzymes involved in the peroxisomal ROS metabolic pathways are key mediators to figure out the relationship between peroxisome-derived ROS and PCD. Here, we summarize the peroxisomal ROS generation and scavenging pathways and explain how peroxisome-derived ROS participate in PCD based on recent progress in the functional study of enzymes related to peroxisomal ROS generation or scavenging. We aimed to elucidate the role of the peroxisomal ROS regulatory system in cellular PCD to show its potential in terms of accurate PCD regulation, which contribute to environmental stress resistance.


Assuntos
Apoptose , Peroxissomos , Apoptose/fisiologia , Redes e Vias Metabólicas , Peroxissomos/metabolismo , Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Physiol Plant ; 174(4): e13753, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36004735

RESUMO

In Nicotiana tabacum, the degeneration of connective tissue and stomium tissue (the stomium and circular cell cluster [CCC]) is essential for anther dehiscence. Both connective cells and CCC cells are crystal idioblasts, and these cells will undergo degeneration after accumulating calcium oxalate (CaOx) crystals. However, detailed data concerning this process are minimal. Therefore, this study used cellular biological and physiological methods to illustrate this relationship. Results demonstrated that tobacco anther dehiscence is a series of timed programmed cell death (PCD) processes that include the CCC, connective tissue, and stomium. The degenerating crystal idioblasts of the tobacco anther were found to possess two hallmark characteristics that distinguished them from normal PCD cells, namely dynamic changes in CaOx crystals and the appearance of numerous peroxisomes. The accumulation of CaOx and the production of H2 O2 occurred simultaneously or successively before PCD. The peak H2 O2 content was found to appear after the insoluble oxalate. Further, CeCl3 cytochemistry staining was used to detect subcellular H2 O2 , and the precipitate of H2 O2 was primarily present in peroxisomes and around CaOx crystals. These results show that anther dehiscence in N. tabacum is a PCD process in which crystal idioblasts play a vital role in CaOx degradation and H2 O2 production.


Assuntos
Oxalato de Cálcio , Tabaco , Apoptose/fisiologia , Oxalato de Cálcio/metabolismo , Tabaco/metabolismo
13.
Int J Mol Sci ; 23(16)2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-36012521

RESUMO

Gliomas are highly aggressive cancer types that are in urgent need of novel drugs and targeted therapies. Treatment protocols have not improved in over a decade, and glioma patient survival remains among the worst of all cancer types. As a result, cancer metabolism research has served as an innovative approach to identifying novel glioma targets and improving our understanding of brain tumors. Recent research has uncovered a unique metabolic vulnerability in the sphingolipid pathways of gliomas that possess the IDH1 mutation. Sphingolipids are a family of lipid signaling molecules that play a variety of second messenger functions in cellular regulation. The two primary metabolites, sphingosine-1-phosphate (S1P) and ceramide, maintain a rheostat balance and play opposing roles in cell survival and proliferation. Altering the rheostat such that the pro-apoptotic signaling of the ceramides outweighs the pro-survival S1P signaling in glioma cells diminishes the hallmarks of cancer and enhances tumor cell death. Throughout this review, we discuss the sphingolipid pathway and identify the enzymes that can be most effectively targeted to alter the sphingolipid rheostat and enhance apoptosis in gliomas. We discuss each pathway's steps based on their site of occurrence in the organelles and postulate novel targets that can effectively exploit this vulnerability.


Assuntos
Glioma , Esfingolipídeos , Apoptose/fisiologia , Morte Celular , Ceramidas/metabolismo , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Lisofosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/metabolismo
14.
Commun Biol ; 5(1): 797, 2022 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941180

RESUMO

While major changes in cellular morphology during apoptosis have been well described, the subcellular changes in nuclear architecture involved in this process remain poorly understood. Imaging of nucleosomes in cortical neurons in vitro before and during apoptosis revealed that chromatin compaction precedes the activation of caspase-3 and nucleus shrinkage. While this early chromatin compaction remained unaffected by pharmacological blockade of the final execution of apoptosis through caspase-3 inhibition, interfering with the chromatin dynamics by modulation of actomyosin activity prevented apoptosis, but resulted in necrotic-like cell death instead. With super-resolution imaging at different phases of apoptosis in vitro and in vivo, we demonstrate that chromatin compaction occurs progressively and can be classified into five stages. In conclusion, we show that compaction of chromatin in the neuronal nucleus precedes apoptosis execution. These early changes in chromatin structure critically affect apoptotic cell death and are not part of the final execution of the apoptotic process in developing cortical neurons.


Assuntos
Caspases , Cromatina , Apoptose/fisiologia , Caspase 3 , Caspases/metabolismo , Neurônios/fisiologia
15.
Proc Natl Acad Sci U S A ; 119(33): e2208522119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939714

RESUMO

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.


Assuntos
Apoptose , Linfócitos T CD8-Positivos , Citotoxicidade Imunológica , Proteína Ligante Fas , Linfócitos T Citotóxicos , Fatores de Transcrição , Animais , Apoptose/fisiologia , Cromatina/metabolismo , Citotoxicidade Imunológica/genética , Proteína Ligante Fas/metabolismo , Granzimas/genética , Humanos , Camundongos , Perforina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Curr Biol ; 32(16): R891-R894, 2022 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-35998601

RESUMO

Mitochondria are central to apoptosis, an immunologically silent form of cell death. The mitochondrial, or 'intrinsic', apoptotic pathway is activated when the permeabilized mitochondrial membrane of stressed cells releases apoptotic effectors. A new study now characterizes how mitochondria are involved in the switch from pyroptotic to necroptotic cell death.


Assuntos
Apoptose , Mitocôndrias , Apoptose/fisiologia , Morte Celular , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo
17.
Int J Mol Sci ; 23(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35955748

RESUMO

Repeated and prolonged stress causes hypothalamic-pituitary-adrenal (HPA) dysregulation. Excessive hypothalamic-pituitary-adrenal axis activity has been linked to inadequate activation of the hypothalamus-pituitary-ovarian axis, which controls the growth and development of ovarian follicles and oocytes. Therefore, we assessed the ovarian reserve under high-housing-density-induced prolonged stress, and investigated the mechanisms underlying diminished ovarian reserve in this study. Eight-week-old female C57BL/6 mice were housed for 10 weeks under different housing densities. We then assessed hormone levels, performed histology and immunohistochemistry analyses of ovarian follicles, evaluated ovarian mRNA expression, and measured angiotensin II-mediated apoptosis in vitro. More densely housed mice presented increased corticosterone levels and decreased follicle-stimulating and luteinizing hormone levels. Moreover, mice exposed to prolonged ordinary stress showed a reduced level of serum anti-Müllerian hormone and an increased number of atretic ovarian follicles. Stressed mice showed increased levels of angiotensinogen and angiotensin II in the ovaries and serum. Furthermore, our in vitro study confirmed that high-housing-density-related stress induced granulosa cell apoptosis, resulting in diminished ovarian reserves. Collectively, our findings highlight the importance of women managing everyday stress to maintain their reproductive health.


Assuntos
Reserva Ovariana , Angiotensina II , Animais , Apoptose/fisiologia , Feminino , Células da Granulosa , Habitação , Humanos , Sistema Hipotálamo-Hipofisário , Camundongos , Camundongos Endogâmicos C57BL , Sistema Hipófise-Suprarrenal
18.
Int J Mol Sci ; 23(15)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35955839

RESUMO

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway.


Assuntos
Antígenos de Diferenciação de Linfócitos T , Lipossomos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/fisiologia , Humanos , Células Jurkat , Nanopartículas , Isoformas de Proteínas
19.
Biomolecules ; 12(8)2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-36008968

RESUMO

Intervertebral disc degeneration (IVDD) is a common musculoskeletal degenerative disease worldwide, of which the main clinical manifestation is low back pain (LBP); approximately, 80% of people suffer from it in their lifetime. Currently, the pathogenesis of IVDD is unclear, and modern treatments can only alleviate its symptoms but cannot inhibit or reverse its progression. However, in recent years, targeted therapy has led to new therapeutic strategies. Cysteine-containing aspartate proteolytic enzymes (caspases) are a family of proteases present in the cytoplasm. They are evolutionarily conserved and are involved in cell growth, differentiation, and apoptotic death of eukaryotic cells. In recent years, it has been confirmed to be involved in the pathogenesis of various diseases, mainly by regulating cell apoptosis and inflammatory response. With continuous research on the pathogenesis and pathological process of IVDD, an increasing number of studies have shown that caspases are closely related to the IVDD process, especially in the intervertebral disc (IVD) cell apoptosis and inflammatory response. Therefore, herein we study the role of caspases in IVDD with respect to the structure of caspases and the related signaling pathways involved. This would help explore the strategy of regulating the activity of the caspases involved and develop caspase inhibitors to prevent and treat IVDD. The aim of this review was to identify the caspases involved in IVDD which could be potential targets for the treatment of IVDD.


Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Apoptose/fisiologia , Inibidores de Caspase , Caspases/metabolismo , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia
20.
Contrast Media Mol Imaging ; 2022: 7614497, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35992546

RESUMO

Osteoarthritis (OA) is a rheumatic disease and its pathogenesis involves the dysregulation of noncoding RNAs. Therefore, the regulatory mechanism of circular RNA MELK (circMELK) was specified in this work. OA human cartilage tissue was collected, and circMELK, miR-497-5p, and myeloid differentiation factor 88 (MYD88) expression were examined. Human chondrocytes were stimulated with interleukin- (IL-) 1ß and interfered with vectors altering circMELK, miR-497-5p, and MyD88 expression to observe their effects on cell viability, cell cycle and apoptosis, autophagy, and inflammation. The binding relationship between RNAs was verified. The data presented that OA cartilage tissues presented raised circMELK and MYD88 and inhibited miR-497-5p expression. IL-1ß suppressed cell viability, prevented cell cycle, and induced apoptosis, autophagy, and inflammation of chondrocytes. Functionally, IL-1ß-induced changes of chondrocytes could be attenuated by suppressing circMELK or overexpressing miR-497-5p. circMELK acted as a sponge of miR-497-5p while miR-497-5p was a regulator of MYD88. MYD88 restricted the effect of overexpressing miR-497-5p on IL-1ß-stimulated chondrocytes. MYD88 triggered nuclear factor-kappaB (NF-κB) pathway activation. Shortly, CircMELK promotes chondrocyte apoptosis and inhibits autophagy in OA by regulating MYD88/NF-κB signaling axis through miR-497-5p. Our study proposes a new molecular mechanism for the development of OA.


Assuntos
MicroRNAs , Fator 88 de Diferenciação Mieloide , NF-kappa B , Osteoartrite , Proteínas Serina-Treonina Quinases , RNA Circular , Apoptose/genética , Apoptose/fisiologia , Autofagia/genética , Autofagia/fisiologia , Condrócitos/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Circular/genética , RNA Circular/metabolismo
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