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1.
Adv Immunol ; 162: 59-108, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38866439

RESUMEN

Apoptotic pore formation in mitochondria is the pivotal point for cell death during mitochondrial apoptosis. It is regulated by BCL-2 family proteins in response to various cellular stress triggers and mediates mitochondrial outer membrane permeabilization (MOMP). This allows the release of mitochondrial contents into the cytosol, which triggers rapid cell death and clearance through the activation of caspases. However, under conditions of low caspase activity, the mitochondrial contents released into the cytosol through apoptotic pores serve as inflammatory signals and activate various inflammatory responses. In this chapter, we discuss how the formation of the apoptotic pore is regulated by BCL-2 proteins as well as other cellular or mitochondrial proteins and membrane lipids. Moreover, we highlight the importance of sublethal MOMP in the regulation of mitochondrial-activated inflammation and discuss its physiological consequences in the context of pathogen infection and disease and how it can potentially be exploited therapeutically, for example to improve cancer treatment.


Asunto(s)
Apoptosis , Mitocondrias , Membranas Mitocondriales , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Animales , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inflamación/inmunología , Caspasas/metabolismo , Transducción de Señal , Neoplasias/inmunología , Neoplasias/metabolismo
2.
Physiol Plant ; 176(3): e14401, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38899462

RESUMEN

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.


Asunto(s)
Chlamydomonas reinhardtii , Proteolisis , Estrés Salino , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/metabolismo , Proteolisis/efectos de los fármacos , Caspasas/metabolismo , Caspasas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética
3.
Mar Drugs ; 22(6)2024 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-38921562

RESUMEN

Experiments conducted on triple-negative breast cancer have shown that fucoidan from Lessonia trabeculata (FLt) exhibits cytotoxic and antitumor properties. However, further research is necessary to gain a complete understanding of its bioactivity and level of cytotoxicity. The cytotoxic effect of FLt was determined by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was analyzed using annexin V and caspase 3/7 staining kit and DNA fragmentation. In addition, transcriptional expression of antiapoptotic (Bcl-2 and XIAP) and proapoptotic (caspase 8, caspase 9, and AIF) genes were analyzed in TNBC 4T1 cells. After 72 h of culture, the IC50 for FLt was 561 µg/mL, while doxorubicin (Dox) had an IC50 of 0.04 µg/mL. In addition, assays for FLt + Dox were performed. Annexin V and caspase 3/7 revealed that FLt induces early and late-stage apoptosis. DNA fragmentation results support necrotic death of 4T1 cells. Similarly, transcripts that prevent cell death were decreased, while transcripts that promote cell death were increased. This study showed that FLt induces apoptosis by both caspase-dependent and caspase-independent mechanisms. These findings suggest that FLt may have potential applications in breast cancer treatment. Further research will provide more information to elucidate the mechanism of action of FLt.


Asunto(s)
Apoptosis , Caspasas , Polisacáridos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Polisacáridos/farmacología , Animales , Femenino , Caspasas/metabolismo , Ratones , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Humanos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Fragmentación del ADN/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Kelp
4.
Cell Rep ; 43(5): 114251, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38761374

RESUMEN

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.


Asunto(s)
Diferenciación Celular , Daño del ADN , Proteínas de Drosophila , Macrófagos , Fagocitosis , Animales , Macrófagos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Transducción de Señal , Caspasas/metabolismo , Activación Enzimática , Desoxirribonucleasas/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo
5.
Int J Mol Sci ; 25(10)2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38791499

RESUMEN

The activation of caspases is a crucial event and an indicator of programmed cell death, also known as apoptosis. These enzymes play a central role in cancer biology and are considered one promising target for current and future advancements in therapeutic interventions. Traditional methods of measuring caspase activity such as antibody-based methods provide fundamental insights into their biological functions, and are considered essential tools in the fields of cell and cancer biology, pharmacology and toxicology, and drug discovery. However, traditional methods, though extensively used, are now recognized as having various shortcomings. In addition, these methods fall short of providing solutions to and matching the needs of the rapid and expansive progress achieved in studying caspases. For these reasons, there has been a continuous improvement in detection methods for caspases and the network of pathways involved in their activation and downstream signaling. Over the past decade, newer methods based on cutting-edge state-of-the-art technologies have been introduced to the biomedical community. These methods enable both the temporal and spatial monitoring of the activity of caspases and their downstream substrates, and with enhanced accuracy and precision. These include fluorescent-labeled inhibitors (FLIs) for live imaging, single-cell live imaging, fluorescence resonance energy transfer (FRET) sensors, and activatable multifunctional probes for in vivo imaging. Recently, the recruitment of mass spectrometry (MS) techniques in the investigation of these enzymes expanded the repertoire of tools available for the identification and quantification of caspase substrates, cleavage products, and post-translational modifications in addition to unveiling the complex regulatory networks implicated. Collectively, these methods are enabling researchers to unravel much of the complex cellular processes involved in apoptosis, and are helping generate a clearer and comprehensive understanding of caspase-mediated proteolysis during apoptosis. Herein, we provide a comprehensive review of various assays and detection methods as they have evolved over the years, so to encourage further exploration of these enzymes, which should have direct implications for the advancement of therapeutics for cancer and other diseases.


Asunto(s)
Caspasas , Caspasas/metabolismo , Humanos , Animales , Apoptosis , Transferencia Resonante de Energía de Fluorescencia/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Inhibidores de Caspasas/farmacología , Colorantes Fluorescentes/química
6.
Biochem Biophys Res Commun ; 717: 150029, 2024 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-38714015

RESUMEN

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.


Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Dominios Proteicos , Multimerización de Proteína , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteína 10 de la LLC-Linfoma de Células B/química , Proteína 10 de la LLC-Linfoma de Células B/genética , Humanos , Cristalografía por Rayos X , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Caspasas/metabolismo , Caspasas/química
7.
Retrovirology ; 21(1): 8, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38693565

RESUMEN

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.


Asunto(s)
Linfocitos T CD4-Positivos , Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Animales , Ratones , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , VIH-1/efectos de los fármacos , Humanos , Linfocitos T CD4-Positivos/inmunología , Tejido Linfoide/virología , Tejido Linfoide/inmunología , Carga Viral/efectos de los fármacos , Bazo/virología , Bazo/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Caspasas/metabolismo , Inhibidores de Caspasas/farmacología , Antirretrovirales/uso terapéutico
8.
Int J Biol Macromol ; 270(Pt 2): 132476, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38777016

RESUMEN

Gasdermin (GSDM) proteins are executioners of pyroptosis in many species. Gasdermin proteins can be cleaved at their linker region between the amino domain (NT) and carboxyl domain (CT) by enzymes. The released GSDM-NTs bind cell membrane and form pores, thereby leading to the release of cellular components and lytic cell death. GSDM-mediated pyroptosis is considered to play important role in immune responses. However, little is known about the GSDM proteins and GSDM-mediated pyroptosis in birds. In the current study, genes encoding chicken gasdermin A (chGSDMA) and chGSDME were cloned. The cleavage of chGSDMA and chGSDME by chicken caspase-1 (chCASP1), chCASP3 and chCASP7 and the cleavage sites were determined. The chGSDMA-NT obtained form chCASP1-mediated cleavage and chGSDME-NT obtained from chCASP3/chCASP7-mediated cleavage could bind and damage cell membrane and lead to cell death of HEK293 cells. chGSDMA-NT also strongly localized to and formed puncta in nucleus. Besides, both chGSDMA-NT and chGSDME-NT showed growth inhibition and bactericidal activity to bacteria. In chickens challenged with Pasteurella multocida and Salmonella typhimurium, the expression of chGSDMA and chGSDME was upregulated and the activation of chCASP3 and the cleavage of chGSDME were observed. The work provides essential information for expanding our knowledge on pyroptosis in birds.


Asunto(s)
Caspasas , Pollos , Piroptosis , Animales , Humanos , Células HEK293 , Caspasas/metabolismo , Pasteurella multocida , Proteolisis , Proteínas Aviares/metabolismo , Proteínas Aviares/genética , Secuencia de Aminoácidos , Gasderminas
9.
Placenta ; 151: 37-47, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38703713

RESUMEN

Caspases, a family of cysteine proteases, are pivotal regulators of apoptosis, the tightly controlled cell death process crucial for eliminating excessive or unnecessary cells during development, including placental development. Collecting research has unveiled the multifaceted roles of caspases in the placenta, extending beyond apoptosis. Apart from their involvement in placental tissue remodeling via apoptosis, caspases actively participate in essential regulatory processes, such as trophoblast fusion and differentiation, significantly influencing placental growth and functionality. In addition, growing evidence indicates an elevation in caspase activity under pathological conditions like pre-eclampsia (PE) and intrauterine growth restriction (IUGR), leading to excessive cell death as well as inflammation. Drawing from advancements in caspase research and placental development under both normal and abnormal conditions, we examine the significance of caspases in both cell death (apoptosis) and non-cell death-related processes within the placenta. We also discuss potential therapeutics targeting caspase-related pathways for placenta disorders.


Asunto(s)
Apoptosis , Caspasas , Placenta , Humanos , Embarazo , Femenino , Caspasas/metabolismo , Placenta/patología , Placenta/metabolismo , Apoptosis/fisiología , Placentación/fisiología , Animales , Enfermedades Placentarias/patología , Enfermedades Placentarias/metabolismo , Preeclampsia/patología , Preeclampsia/metabolismo , Trofoblastos/fisiología , Trofoblastos/patología
10.
J Theor Biol ; 590: 111857, 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-38797470

RESUMEN

Resisting apoptosis is a hallmark of cancer. For this reason, it may be possible to force cancer cells to die by targeting components along the apoptotic signaling pathway. However, apoptosis signaling is challenging to understand due to dynamic and complex behaviors of ligands, receptors, and intracellular signaling components in response to cancer therapy. In this work, we forecast the apoptotic response based on the combined impact of these features. We expanded a previously established mathematical model of caspase-mediated apoptosis to include extracellular activation and receptor dynamics. In addition, three potential threshold values of caspase-3 necessary for the activation of apoptosis were selected to forecast which cells become apoptotic over time. We first vary ligand and receptor levels with the number of intracellular signaling proteins remaining consistent. Then, we vary the intracellular protein molecules in each simulated tumor cell to forecast the response of a heterogeneous population. By leveraging the benefits of computational modeling, we investigate the combined effect of several factors on the onset of apoptosis. This work provides quantitative insights for how the apoptotic signaling response can be forecasted, and precisely triggered, amongst heterogeneous cells via extracellular activation.


Asunto(s)
Apoptosis , Modelos Biológicos , Neoplasias , Transducción de Señal , Humanos , Neoplasias/patología , Neoplasias/metabolismo , Caspasas/metabolismo , Caspasa 3/metabolismo
11.
Nat Commun ; 15(1): 4025, 2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38740804

RESUMEN

Intracellular membranes composing organelles of eukaryotes include membrane proteins playing crucial roles in physiological functions. However, a comprehensive understanding of the cellular responses triggered by intracellular membrane-focused oxidative stress remains elusive. Herein, we report an amphiphilic photocatalyst localised in intracellular membranes to damage membrane proteins oxidatively, resulting in non-canonical pyroptosis. Our developed photocatalysis generates hydroxyl radicals and hydrogen peroxides via water oxidation, which is accelerated under hypoxia. Single-molecule magnetic tweezers reveal that photocatalysis-induced oxidation markedly destabilised membrane protein folding. In cell environment, label-free quantification reveals that oxidative damage occurs primarily in membrane proteins related to protein quality control, thereby aggravating mitochondrial and endoplasmic reticulum stress and inducing lytic cell death. Notably, the photocatalysis activates non-canonical inflammasome caspases, resulting in gasdermin D cleavage to its pore-forming fragment and subsequent pyroptosis. These findings suggest that the oxidation of intracellular membrane proteins triggers non-canonical pyroptosis.


Asunto(s)
Inflamasomas , Proteínas de la Membrana , Oxidación-Reducción , Piroptosis , Humanos , Inflamasomas/metabolismo , Proteínas de la Membrana/metabolismo , Estrés Oxidativo , Catálisis , Estrés del Retículo Endoplásmico , Peróxido de Hidrógeno/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Radical Hidroxilo/metabolismo , Mitocondrias/metabolismo , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Animales , Procesos Fotoquímicos , Pliegue de Proteína , Caspasas/metabolismo , Gasderminas
12.
J Oleo Sci ; 73(6): 911-920, 2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38797691

RESUMEN

Sakuranin is a flavanone which is a class of flavonoids found abundantly in Prunus species. Flavonoids have been long known for their anticancer properties against a range of human cancers. However, there are no previous reports on the anticancer effects of sakuranin flavanone molecule. This study was designed to study the anticancer effects of sakuranin against human oropharyngeal carcinoma cells along with investigating its effects on caspase-mediated apoptosis, mitochondrial membrane potential (MMP) loss, cell migration and invasion and m-TOR/PI3K/AKT signalling pathway. MTT assay was used to study effects on cell viability. The apoptotic studies were carried out through AO/EB staining, annexin V/FITC staining, comet assay and western blotting assay. Transwell chambers assay was used to study effects on cell migration and invasion. Flow cytometry was used to study effects of Sakuranin on mitochondrial membrane potential loss (MMP). Finally, western blotting was used to investigate m-TOR/PI3K/AKT signalling pathway. Results indicated that Sakuranin led to potent cell proliferation inhibition in a dose-dependent manner. Sakuranin also induced apoptotic cell death as indicated by fluorescence microscopy and annexin V/FITC staining assays. The apoptotic induction was mediated via activation of caspase-3, caspase-9, and Bax while as it led to downregulation of Bcl-2. Sakuranin also caused inhibition of cell migration and cell invasion along with causing significant decrease in MMP. Sakuranin also caused inhibition of expressions of proteins related with m-TOR/PI3K/AKT signalling pathway. In conclusion, the current findings clearly indicate anticancer effects of Sakuranin flavanone in human oropharyngeal cancer cells and are mediated via caspase activated apoptosis, inhibition of cell migration and invasion, loss of mitochondrial membrane potential and targeting m-TOR/PI3K/AKT signalling pathway.


Asunto(s)
Apoptosis , Carcinoma de Células Escamosas , Movimiento Celular , Flavanonas , Potencial de la Membrana Mitocondrial , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Humanos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Flavanonas/farmacología , Flavanonas/aislamiento & purificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Serina-Treonina Quinasas TOR/metabolismo , Caspasas/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos
13.
Cells ; 13(9)2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38727308

RESUMEN

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.


Asunto(s)
Apoptosis , Proliferación Celular , Alcaloides Indólicos , Proteína Quinasa 14 Activada por Mitógenos , Simulación del Acoplamiento Molecular , Humanos , Células A549 , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Alcaloides Indólicos/farmacología , Alcaloides Indólicos/química , Simulación de Dinámica Molecular , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo
14.
Dis Model Mech ; 17(5)2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38616733

RESUMEN

Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin-myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.


Asunto(s)
Caspasas , Linfoma de Células B , Proteínas Proto-Oncogénicas c-myc , Quinasas Asociadas a rho , Animales , Quinasas Asociadas a rho/metabolismo , Linfoma de Células B/patología , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Caspasas/metabolismo , Macrófagos/metabolismo , Apoptosis , Ratones , Análisis de Supervivencia , Ratones Transgénicos , Células de la Médula Ósea/metabolismo , Ratones Endogámicos C57BL , Ciclo Celular
15.
Nanoscale ; 16(16): 7976-7987, 2024 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-38567463

RESUMEN

Collective functionalization of the phytochemicals of medicinal herbs on nanoparticles is emerging as a potential cancer therapeutic strategy. This study presents the facile synthesis of surface-functionalized gold nanoparticles using Bacopa monnieri (Brahmi; Bm) phytochemicals and their therapeutically relevant mechanism of action in the colorectal cancer cell line, HT29. The nanoparticles were characterized using UV-visible spectroscopy, TEM-EDAX, zeta potential analysis, TGA, FTIR and 1H NMR spectroscopy, and HR-LC-MS. The particles (Bm-GNPs) were of polygonal shape and were stable against aggregation. They entered the target cells and inhibited the viability and clonogenicity of the cells with eight times more antiproliferative efficacy (25 ± 1.5 µg mL-1) than Bm extract (Bm-EX). In vitro studies revealed that Bm-GNPs bind tubulin (a protein crucial in cell division and a target of anticancer drugs) and disrupt its helical structure without grossly altering its tertiary conformation. Like other antitubulin agents, Bm-GNPs induced G2/M arrest and ultimately killed the cells, as confirmed using flow cytometry analyses. ZVAD-FMK-mediated global pan-caspase inhibition and the apparent absence of cleaved caspase-3 in treated cells indicated that the death did not involve the classic apoptosis pathway. Cellular ultrastructure analyses, western immunoblots, and in situ immunofluorescence visualization of cellular microtubules revealed microtubule-acetylation-independent induction of autophagy as the facilitator of cell death. Together, the data indicate strong antiproliferative efficacy and a possible mechanism of action for these designer nanoparticles. Bm-GNPs, therefore, merit further investigations, including preclinical evaluations, for their therapeutic potential as inducers of non-apoptotic cell death.


Asunto(s)
Autofagia , Neoplasias Colorrectales , Oro , Nanopartículas del Metal , Humanos , Oro/química , Oro/farmacología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Nanopartículas del Metal/química , Autofagia/efectos de los fármacos , Acetilación , Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/tratamiento farmacológico , Células HT29 , Caspasas/metabolismo , Fitoquímicos/farmacología , Fitoquímicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química
16.
Mol Biol Rep ; 51(1): 594, 2024 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-38683374

RESUMEN

BACKGROUND: Metacaspases comprise a family of cysteine proteases implicated in both cell death and cell differentiation of protists that has been considered a potential drug target for protozoan parasites. However, the biology of metacaspases in Plasmodium vivax - the second most prevalent and most widespread human malaria parasite worldwide, whose occurrence of chemoresistance has been reported in many endemic countries, remains largely unexplored. Therefore, the present study aimed to address, for the first time, the expression pattern of metacaspases in P. vivax parasites. METHODS AND RESULTS: P. vivax blood-stage parasites were obtained from malaria patients in the Brazilian Amazon and the expression of the three putative P. vivax metacaspases (PvMCA1-3) was detected in all isolates by quantitative PCR assay. Of note, the expression levels of each PvMCA varied noticeably across isolates, which presented different frequencies of parasite forms, supporting that PvMCAs may be expressed in a stage-specific manner as previously shown in P. falciparum. CONCLUSION: The detection of metacaspases in P. vivax blood-stage parasites reported herein, allows the inclusion of these proteases as a potential candidate drug target for vivax malaria, while further investigations are still required to evaluate the activity, role and essentiality of metacaspases in P. vivax biology.


Asunto(s)
Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Brasil , Humanos , Malaria Vivax/parasitología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Caspasas/genética , Caspasas/metabolismo , Expresión Génica/genética
17.
Burns ; 50(6): 1562-1577, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38570249

RESUMEN

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.


Asunto(s)
Apoptosis , Quemaduras , Fibroblastos , Calor , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , Fibroblastos/patología , Fibroblastos/metabolismo , Quemaduras/patología , Calor/efectos adversos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Parthanatos , Necrosis , Células Cultivadas , Muerte Celular , Piel/patología , Piel/citología , Piel/lesiones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Caspasas/metabolismo , Roturas del ADN de Doble Cadena , Adenosina Trifosfato/metabolismo
18.
Int J Mol Sci ; 25(8)2024 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-38673741

RESUMEN

A widely used organophosphate flame retardant (OPFR), triphenyl phosphate (TPP), is frequently detected in various environmental media and humans. However, there is little known on the human corneal epithelium of health risk when exposed to TPP. In this study, human normal corneal epithelial cells (HCECs) were used to investigate the cell viability, morphology, apoptosis, and mitochondrial membrane potential after they were exposed to TPP, as well as their underlying molecular mechanisms. We found that TPP decreased cell viability in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 220 µM. Furthermore, TPP significantly induced HCEC apoptosis, decreased mitochondrial membrane potential in a dose-dependent manner, and changed the mRNA levels of the apoptosis biomarker genes (Cyt c, Caspase-9, Caspase-3, Bcl-2, and Bax). The results showed that TPP induced cytotoxicity in HCECs, eventually leading to apoptosis and changes in mitochondrial membrane potential. In addition, the caspase-dependent mitochondrial pathways may be involved in TPP-induced HCEC apoptosis. This study provides a reference for the human corneal toxicity of TPP, indicating that the risks of OPFR to human health cannot be ignored.


Asunto(s)
Apoptosis , Supervivencia Celular , Epitelio Corneal , Retardadores de Llama , Potencial de la Membrana Mitocondrial , Mitocondrias , Humanos , Apoptosis/efectos de los fármacos , Retardadores de Llama/toxicidad , Retardadores de Llama/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/citología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Caspasas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Organofosfatos/farmacología , Organofosfatos/toxicidad , Células Cultivadas
19.
Nat Cell Biol ; 26(5): 757-769, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38538834

RESUMEN

Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis.


Asunto(s)
Aciltransferasas , Péptidos y Proteínas de Señalización Intracelular , Lipoilación , Proteínas de Unión a Fosfato , Piroptosis , Animales , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/genética , Aciltransferasas/metabolismo , Aciltransferasas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Humanos , Ratones , Células HEK293 , Ratones Endogámicos C57BL , Lipopolisacáridos/farmacología , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Caspasas/metabolismo , Choque Séptico/metabolismo , Choque Séptico/patología , Choque Séptico/genética , Membrana Celular/metabolismo , Gasderminas , Acetiltransferasas
20.
Chem Biol Drug Des ; 103(3): e14481, 2024 03.
Artículo en Inglés | MEDLINE | ID: mdl-38458969

RESUMEN

Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 µM) had no toxicity to astrocytes. Besides, SSD (4 µM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1ß, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.


Asunto(s)
Bencimidazoles , Carbocianinas , Enfermedades Mitocondriales , Proteína con Dominio Pirina 3 de la Familia NLR , Ácido Oleanólico/análogos & derivados , Saponinas , Ratas , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Pilocarpina/toxicidad , Factor de Necrosis Tumoral alfa/genética , Caspasas/metabolismo , Interleucina-6 , Transducción de Señal , Inflamación/metabolismo
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