RESUMEN
Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1ß and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.
Asunto(s)
Proliferación Celular , Ferroptosis , Inflamación , Células Intersticiales del Testículo , Ferroptosis/efectos de los fármacos , Animales , Masculino , Ratones , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Inflamación/patología , Inflamación/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Ciclohexilaminas , FenilendiaminasRESUMEN
Vascular cognitive impairment (VCI) is the second leading cause of dementia with limited treatment options, characterised by cerebral hypoperfusion-induced white matter rarefaction (WMR). Subcortical VCI is the most common form of VCI, but the underlying reasons for region susceptibility remain elusive. Recent studies employing the bilateral cortical artery stenosis (BCAS) method demonstrate that various inflammasomes regulate white matter injury and blood-brain barrier dysfunction but whether caspase-1 inhibition will be beneficial remains unclear. To address this, we performed BCAS on C57/BL6 mice to study the effects of Ac-YVAD-cmk, a caspase-1 inhibitor, on the subcortical and cortical regions. Cerebral blood flow (CBF), WMR, neuroinflammation and the expression of tight junction-related proteins associated with blood-brain barrier integrity were assessed 15 days post BCAS. We observed that Ac-YVAD-cmk restored CBF, attenuated BCAS-induced WMR and restored subcortical myelin expression. Within the subcortical region, BCAS activated the NLRP3/caspase-1/interleukin-1beta axis only within the subcortical region, which was attenuated by Ac-YVAD-cmk. Although we observed that BCAS induced significant increases in VCAM-1 expression in both brain regions that were attenuated with Ac-YVAD-cmk, only ZO-1 and occludin were observed to be significantly altered in the subcortical region. Here we show that caspase-1 may contribute to subcortical regional susceptibility in a mouse model of VCI. In addition, our results support further investigations into the potential of Ac-YVAD-cmk as a novel treatment strategy against subcortical VCI and other conditions exhibiting cerebral hypoperfusion-induced WMR.
Asunto(s)
Clorometilcetonas de Aminoácidos , Disfunción Cognitiva , Sustancia Blanca , Animales , Ratones , Sustancia Blanca/metabolismo , Encéfalo/metabolismo , Caspasa 1/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.
Asunto(s)
Furina , Peroxidasa , Humanos , Células HL-60 , Furina/metabolismo , Furina/genética , Peroxidasa/metabolismo , Granulocitos/metabolismo , Granulocitos/citología , Diferenciación Celular , Subtilisinas/metabolismo , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/genética , Clorometilcetonas de Aminoácidos/farmacologíaRESUMEN
Organophosphates cause hyperstimulation of the central nervous system, leading to extended seizures, convulsions, and brain damage. Sarin is a highly toxic organophosphate nerve agent that has been employed in several terrorist attacks. The prolonged toxicity of sarin may be enhanced by the neuroinflammatory response initiated by the inflammasome, caspase involvement, and generation/release of proinflammatory cytokines. Since neurodegeneration and neuroinflammation are prevalent in sarin-exposed animals, we were interested in evaluating the capacity of quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), a pan caspase inhibitor to attenuate neuroinflammation following sarin exposure. To test this hypothesis, sarin-exposed C57BL/6 mice were treated with Q-VD-OPh or negative control quinolyl-valyl-O-methylglutamyl-[-2,6-difluorophenoxy]-methyl ketone, sacrificed at 2- and 14-day time points, followed by removal of the amygdala and hippocampus. A Bio-Rad 23-Plex cytokine analysis was completed on each tissue. The results suggest that exposure to sarin induced a dramatic increase in interleukin-1ß and 6 other cytokines and a decrease in 2 of the 23 cytokines at 2 days in the amygdala compared with controls. Q-VD-OPh attenuated these changes at the 2-day time point. At 14 days, six of these cytokines were still significantly different from controls. Hippocampus was less affected at both time points. Diazepam, a neuroprotective drug against nerve agents, caused an increase in several cytokines but did not have a synergistic effect with Q-VD-OPh. Treatment of sarin exposure with apoptosis inhibitors appears to be a worthwhile approach for further testing as a comprehensive counteragent against organophosphate exposure. SIGNIFICANCE STATEMENT: A pan inhibitor of caspases (Q-VD-OPh) was proposed as a potential antidote for sarin-induced neuroinflammation by reducing the level of inflammation via inflammasome caspase inhibition. Q-VD-OPh added at 30 minutes post-sarin exposure attenuated the inflammatory response of a number of cytokines and chemokines in the amygdala and hippocampus, two brain regions sensitive to organophosphate exposure. Apoptotic marker reduction at 2 and 14 days further supports further testing of inhibitors of apoptosis as a means to lessen extended organophosphate toxicity in the brain.
Asunto(s)
Clorometilcetonas de Aminoácidos , Agentes Nerviosos , Quinolinas , Sarín , Ratones , Animales , Sarín/toxicidad , Inhibidores de Caspasas/farmacología , Inhibidores de Caspasas/uso terapéutico , Enfermedades Neuroinflamatorias , Inflamasomas , Ratones Endogámicos C57BL , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Encéfalo , Citocinas , Agentes Nerviosos/farmacología , Caspasas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Organofosfatos/farmacología , Cetonas/efectos adversosRESUMEN
Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by â¼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.
Asunto(s)
Trombosis , Tromboembolia Venosa , Humanos , Ratones , Animales , Anticoagulantes/uso terapéutico , Trombina/uso terapéutico , Tromboembolia Venosa/tratamiento farmacológico , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Clorometilcetonas de Aminoácidos/farmacología , Clorometilcetonas de Aminoácidos/uso terapéutico , IsquemiaRESUMEN
The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Enoxaparina/farmacología , Furina/antagonistas & inhibidores , Espermina/análogos & derivados , Zeaxantinas/farmacología , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , COVID-19/transmisión , COVID-19/virología , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Enoxaparina/química , Enoxaparina/metabolismo , Furina/química , Furina/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Espermina/química , Espermina/metabolismo , Espermina/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Replicación Viral , Zeaxantinas/química , Zeaxantinas/metabolismoRESUMEN
The protozoan parasite Perkinsus marinus causes Dermo disease in eastern oysters, Crassostrea virginica, and can suppress apoptosis of infected hemocytes using incompletely understood mechanisms. This study challenged hemocytes in vitro with P. marinus for 1 h in the presence or absence of caspase inhibitor Z-VAD-FMK or Inhibitor of Apoptosis protein (IAP) inhibitor GDC-0152. Hemocytes exposure to P. marinus significantly reduced granulocyte apoptosis, and pre-incubation with Z-VAD-FMK did not affect P. marinus-induced apoptosis suppression. Hemocyte pre-incubation with GDC-0152 prior to P. marinus challenge further reduced apoptosis of granulocytes with engulfed parasite, but not mitochondrial permeabilization. This suggests P. marinus-induced apoptosis suppression may be caspase-independent, affect an IAP-involved pathway, and occur downstream of mitochondrial permeabilization. P. marinus challenge stimulated hemocyte differential expression of oxidation-reduction, TNFR, and NF-kB pathways. WGCNA analysis of P. marinus expression in response to hemocyte exposure revealed correlated protease, kinase, and hydrolase expression that could contribute to P. marinus-induced apoptosis suppression.
Asunto(s)
Crassostrea/parasitología , Clorometilcetonas de Aminoácidos , Animales , Apicomplexa , Apoptosis , Caspasas , Hemocitos/parasitología , Interacciones Huésped-Parásitos , Proteínas Inhibidoras de la Apoptosis , FN-kappa B , Oxidación-Reducción , Estrés OxidativoRESUMEN
zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.
Asunto(s)
Autofagia , Caspasas , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis , Caspasa 3 , Inhibidores de Caspasas/farmacología , Caspasas/genética , Caspasas/metabolismo , HumanosRESUMEN
The present study established a necroptosis model in vitro and investigated the role of HMGB1 in cell necroptosis. A combination of tumor necrosis factor-α and z-VAD-fmk was used to induce necroptosis in L929 cells with necroptosis inhibitor necrostatin-1 applied as an intervention. Flow cytometry and transmission electron microscopy (TEM) were used to measure cell necroptosis. Western blotting assay was applied to detect the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL) and HMGB1. Co-immunoprecipitation (Co-IP) assay was used to confirm the interaction between HMGB1 and RIPK3. Our study demonstrated that HMGB1 migrated from the nucleus to the cytoplasm at the onset of necroptosis and was subsequently released passively to the extracellular matrix. Further experiments determined that the binding of HMGB1 with RIPK3 in the cytoplasm was loose during necroptosis. By contrast, when necroptosis was inhibited, the interaction in the cytoplasm was tight suggesting that this association between HMGB1 and RIPK3 might affect its occurrence. In conclusion, the transfer of HMGB1 from nucleus to cytoplasm, and its interaction with RIPK3 might be potentially involved in necroptosis.
Asunto(s)
Proteína HMGB1 , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis , Línea Celular , Citoplasma/metabolismo , Proteína HMGB1/metabolismo , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Necroptosis is a form of regulated programmed cell death that is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting serine/threonine protein kinase-3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL); however, it is not known whether zinc finger protein 91 (ZFP91) is involved in this process. Here, we investigated ZFP91 as a potential mediator of necroptosis. Our mechanistic study demonstrates that ZFP91 promotes RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. ZFP91 stabilized RIPK1 to promote cell death by inducing RIPK1 de-ubiquitination. ZFP91 also significantly increased production of mitochondrial reactive oxygen species (ROS). Accumulation of ROS promoted RIPK3-independent necroptosis triggered by tumor necrosis factor (TNF). in vivo, ZFP91 knockdown alleviated TNFα-induced systemic inflammatory response syndrome (SIRS). These results provide direct evidence that ZFP91 plays an important role in the initiation of RIPK1/RIPK3-dependent necroptosis in vitro and in vivo. We discussed the potential of ZFP91 as a novel therapeutic target for necroptosis-associated diseases.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas Quinasas/genética , Especies Reactivas de Oxígeno , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Transducción de Señal , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Triazoles/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Ovarian cancer is a common gynecological malignancy among female patients and poses a serious threat to women's health. Although it has been established that Fos-like antigen 2 (FOSL2) is linked to ovarian cancer (OC), its exact role in the development of OC remains unknown. OBJECTIVE: This article aims to investigate the role of FOSL2 in ovarian cancer development. METHODS: FOSL2 expression in ovarian carcinoma and adjacent tissues was assessed using real-time fluorescent quantitative PCR and western blot. We constructed OE/sh-FOSL2 plasmids and Caspase-1 specific inhibitors (Yvad-CMK) and transfected A 2780 cells with them to identify the relevant cell functions. Furthermore, we used western blot assay to determine the changes in expression of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteasezymogen procaspase 1 (pro-caspase-1), cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1ß precursor (pro-IL-1ß), interleukin-1ß (IL-1ß), interleukin-18 precursor (pro-IL-18), and interleukin-18 (IL-18). In addition, we measured the concentration of IL-1ß and IL-18 using an enzyme-linked immunosorbent assay (ELISA). Moreover, Tthe level of lactate dehydrogenase (LDH) in the cell supernatant was measured by LDH release assay kit. RESULTS: The expression of FOSL2 was significantly higher compared with the surrounding tissues. The proliferation, migration, and invasion of A2780 cells were enhanced after transfection with OE-FOSL2 plasmids; however, the cell apoptosis was significantly decreased. When FOSL2 was overexpressed, the inflammasome-associated proteins such as ASC, caspase-1, IL-1ß, and IL-18 were downregulated. Furthermore, FOSL2 induced apoptosis and activated the production of inflammasomes in A2780 cells. Co-therapy with Yvad-CMK and substantially inhibited apoptosis and activation of inflammasomes. CONCLUSIONS: Inhibition of FOSL2 promotes the apoptosis of OC cells by mediating the formation of an inflammasome.
Asunto(s)
Apoptosis/genética , Antígeno 2 Relacionado con Fos/genética , Regulación Neoplásica de la Expresión Génica , Inflamasomas/genética , Neoplasias Ováricas/genética , Interferencia de ARN , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 1/genética , Caspasa 1/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Antígeno 2 Relacionado con Fos/metabolismo , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus-host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Muerte Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida/métodos , SARS-CoV-2/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Furanos/farmacología , Humanos , Hidroxicloroquina/farmacología , Imidazoles/farmacología , Indenos/farmacología , Indoles/farmacología , Necroptosis/efectos de los fármacos , Fitoquímicos/farmacología , Piroptosis/efectos de los fármacos , SARS-CoV-2/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Proteínas Virales/antagonistas & inhibidoresRESUMEN
DON is commonly found in foods and feeds; it presents health risks, especially an increase of growth inhibition in humans, particularly infants and young children. However, there are relatively few research studies devoted to the mechanism of DON-mediated growth retardation. Interestingly, our results showed that DON does not cause any significant production of ROS but results in a persistent and significant release of NO with iNOS increasing activity, mitochondrial ultrastructural changes and decreasing ΔΨm. Moreover, the significant decreases in GH production and secretion induced by DON were dose-dependent, accompanied by an increase of caspase 3, 8 and 9, IL-11, IL-lß and GHRH. NO scavenging agent (haemoglobin) and free radical scavenging agent (N-acetylcysteine) partially reversed mitochondrial damage, and Z-VAD-FMK increased the levels of GH and decreased the levels of caspase 3, 8 and 9, while haemoglobin decreased the levels of caspase 3, 8 and 9, indicating that NO is the primary target of DON-mediated inhibition. Present research study firstly demonstrated that NO is a key mediator of DON-induced growth inhibition and plays critical roles in the interference of GH transcription and synthesis. The current research is conducive to future research on the molecular mechanisms of DON-induced growth inhibition in humans, especially children.
Asunto(s)
Caspasas/metabolismo , Contaminación de Alimentos , Trastornos del Crecimiento/metabolismo , Mitocondrias/efectos de los fármacos , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Tricotecenos/toxicidad , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Niño , Preescolar , Exposición a Riesgos Ambientales/efectos adversos , Trastornos del Crecimiento/inducido químicamente , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hemoglobinas/farmacología , Humanos , Lactante , Interleucinas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2'3'-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2'3'-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2'3'-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2'3'-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2'3'-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.
Asunto(s)
Apoptosis , Proteínas de la Membrana/metabolismo , Necroptosis , Oligopéptidos/farmacología , Neoplasias Pancreáticas/patología , Clorometilcetonas de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunomodulación , Factor 1 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , FN-kappa B/metabolismo , Necroptosis/efectos de los fármacos , Nucleótidos Cíclicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias PancreáticasRESUMEN
OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Lesión Renal Aguda/metabolismo , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Nitrógeno de la Urea Sanguínea , Creatinina , Citocinas/metabolismo , Interleucina-18/biosíntesis , Interleucina-1beta/biosíntesis , Interleucina-6/biosíntesis , Riñón/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Sepsis/metabolismoRESUMEN
The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Azoles/farmacología , Chlorocebus aethiops , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/aislamiento & purificación , Proteasas 3C de Coronavirus/metabolismo , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Isoindoles , Leupeptinas/farmacología , Compuestos de Organoselenio/farmacología , Peptidomiméticos , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Proteínas no Estructurales Virales/metabolismoRESUMEN
FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.
Asunto(s)
Fusión Celular , Furina/metabolismo , Placentación , Trofoblastos/enzimología , Clorometilcetonas de Aminoácidos/farmacología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Gonadotropina Coriónica/metabolismo , Colforsina/farmacología , Femenino , Furina/antagonistas & inhibidores , Furina/genética , Humanos , Placentación/efectos de los fármacos , Embarazo , Primer Trimestre del Embarazo , Inhibidores de Serina Proteinasa/farmacología , Nacimiento a Término , Trofoblastos/efectos de los fármacosRESUMEN
Neferine, liensinine, and isoliensinine are bisbenzylisoquinoline alkaloids extracted from seed-embryos of Nelumbo nucifera Gaertn. In this study, we evaluated the anticancer activities and mechanism of action of these natural products in prostate cancer cells by MTT, wound healing, ELISA and Western blotting. Neferine, liensinine, and isoliensinine showed growth inhibition and displayed a significant anti-migration activity in prostate cancer cells. They induced apoptosis and autophagy by activating cleaved caspase-9, cleaved PAPR, Bax, LC3B-II, but decreased Bcl-2 and PARP protein expression in LNCaP cells 24 h after treatments. The apoptotic and cytotoxic effects of neferine, liensinine, and isoliensinine were significantly attenuated in the presence of the caspase inhibitor, Z-VAD-FMK. However, the effects were enhanced in the presence of Akt inhibitor (MK2206) and PI3K inhibitor (LY294002). Moreover, neferine, liensinine, and isoliensinine also downregulated the protein expression of androgen receptor, prostate-specific antigen, and type II 5-α-reductase. These results demonstrated that these bisbenzylisoquinoline alkaloids have the potential as promising therapeutics agents. They induced apoptosis via inactivation with the PI3K/AKT signal pathway.
Asunto(s)
Inhibidores de 5-alfa-Reductasa/farmacología , Antagonistas de Receptores Androgénicos/farmacología , Bencilisoquinolinas/farmacología , Isoquinolinas/farmacología , Fenoles/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Clorometilcetonas de Aminoácidos/farmacología , Antagonistas de Andrógenos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Bencilisoquinolinas/química , Productos Biológicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cromonas/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Isoquinolinas/química , Masculino , Morfolinas/farmacología , Nelumbo/química , Fenoles/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.
Asunto(s)
Apoptosis/efectos de los fármacos , Xenoinjertos/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Adolescente , Adulto , Clorometilcetonas de Aminoácidos/uso terapéutico , Animales , Criopreservación/métodos , Femenino , Congelación , Humanos , Lisofosfolípidos/metabolismo , Ratones , Ratones SCID , Folículo Ovárico/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Trasplante Heterólogo/métodos , Adulto JovenRESUMEN
Our previous studies have implicated Caspase-1 signaling in driving the proinflammatory state of acute graft versus host disease (aGVHD). Therefore, we aimed to elucidate the mechanism of Caspase-1 in in murine models of aGVHD through specific inhibition of its activity with the decoy peptide Ac-YVAD-CMK. We transplanted bone marrow from donor C57BL/6 (H-2b) mice into recipient BALB/c (H-2Kd) mice and randomized the recipients into the following treatment cohorts: (1) allogeneic hematopoietic stem cell transplantation and splenic cell infusion control (PBS group); (2) low dose Ac-YVAD-CMK (AC low group); (3) and high dose Ac-YVAD-CMK (AC high group). Indeed, we observed that Caspase-1 inhibition by Ac-YVAD-CMK ameliorated pathological damage and inflammation in the liver, lungs, and colon elicited by aGVHD. This was associated with reduced mortality secondary to aGVHD. Mechanistically, we found that Caspase-1 inhibition modulated donor T cell expansion, restored the balance of Th1/Th17/Treg subsets, and markedly decreased serum levels and aGVHD target organ mRNA expression of IL-1ß, IL-18, and HMGB1. Thus, we demonstrate that inhibition of Caspase-1 by Ac-YVAD-CMK mitigates murine aGVHD by regulating Th1/Th17/Treg balance and attenuating its characteristic proinflammatory state.