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1.
Biol Pharm Bull ; 46(2): 177-186, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36724946

RESUMO

Huntington's disease (HD) is a hereditary neurodegenerative disease that involves an expansion of the CAG repeats of the Huntingtin (HTT) gene, but the disease onset and progression do not necessarily correspond to the extent of CAG repeats. Decreased mitochondrial complex II activity has also been reported to be closely associated with disease pathogenesis. Here, we examined the mechanism of cell death induced by 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, using striatal cells (STHdhQ111 cells) derived from HD model mice with mutant HTT carrying the CAG repeat extended. Treatment with 3-NP (5 mM) enhanced cell death in STHdhQ111 compared to STHdhQ7 cells with normal HTT. Ferrostatin-1, an inhibitor of ferroptosis, and deferoxamine, an iron chelator, markedly inhibited 3-NP-induced cell death in both the STHdh cell lines. On the other hands, cell death was not abrogated by a broad-spectrum caspase inhibitor, Z-VAD-FMK, indicating that this cell death was caspase-independent. Cell death caused by 3-NP is suggested to be due to ferroptosis. Furthermore, 3-NP-induced cell death was markedly inhibited by GSK2795039, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) inhibitor, suggesting that cell death is mainly mediated by intracellular superoxide anion (O2-) production through NOX2. Furthermore, a mitochondria-targeted superoxide dismutase mimetic (Mito-TEMPO), partially inhibited 3-NP-induced cell death, suggesting that O2- production in the mitochondria is partially responsible for cell death. These results indicate that 3-NP-induced cell death in the STHdhQ111 cells is caspase-independent, non-apoptotic, and that ferroptotic cell death is mainly induced via NOX2 activation.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas do Tecido Nervoso/metabolismo , Caspases/metabolismo , Doença de Huntington/induzido quimicamente , Doença de Huntington/genética , Doença de Huntington/metabolismo
2.
Hum Exp Toxicol ; 42: 9603271231152831, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36650058

RESUMO

BACKGROUND: We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study. METHODS AND RESULTS: Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-ß-gal (senescence-associated ß-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs. CONCLUSION: CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.


Assuntos
Proteína Rica em Cisteína 61 , Insuficiência Ovariana Primária , Piroptose , Animais , Feminino , Humanos , Ratos , Caspases/metabolismo , Proliferação de Células , Ciclofosfamida/toxicidade , Células da Granulosa/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Insuficiência Ovariana Primária/induzido quimicamente , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo
3.
J Immunol ; 210(4): 459-474, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36602965

RESUMO

Leptospira interrogans are bacteria that can infect all vertebrates and are responsible for leptospirosis, a neglected zoonosis. Some hosts, such as humans, are susceptible to the disease, whereas mice are resistant and get chronically colonized. Although leptospires escape recognition by some immune receptors, they activate the NOD-like receptor pyrin 3-inflammasome and trigger IL-1ß secretion. Classically, IL-1ß secretion is associated with lytic inflammatory cell death called pyroptosis, resulting from cytosolic LPS binding to inflammatory caspases, such as caspase 11. Interestingly, we showed that L. interrogans and Leptospira biflexa do not trigger cell death in either murine, human, hamster, or bovine macrophages, escaping both pyroptosis and apoptosis. We showed, in murine cells, that the mild IL-1ß secretion induced by leptospires occurred through nonlytic caspase 8-dependent gasdermin D pore formation and not through activation of caspase 11/noncanonical inflammasome. Strikingly, we demonstrated a potent antagonistic effect of pathogenic L. interrogans and their atypical LPS on spontaneous and Escherichia coli LPS-induced cell death. Indeed, LPS of L. interrogans efficiently prevents caspase 11 dimerization and subsequent massive gasdermin D cleavage. Finally, we showed that pyroptosis escape by leptospires prevents massive IL-1ß release, and we consistently found no major role of IL-1R in controlling experimental leptospirosis in vivo. Overall, to our knowledge, our findings described a novel mechanism by which leptospires dampen inflammation, thus potentially contributing to their stealthiness.


Assuntos
Leptospira interrogans , Leptospirose , Cricetinae , Animais , Bovinos , Camundongos , Humanos , Leptospira interrogans/metabolismo , Lipopolissacarídeos , Inflamassomos/metabolismo , Macrófagos , Leptospirose/metabolismo , Leptospirose/microbiologia , Interleucina-1beta/metabolismo , Caspases/metabolismo , Piroptose , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
4.
Mol Med Rep ; 27(2)2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36601752

RESUMO

The cell­killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat­induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre­treatment. Apoptosis was measured by Annexin V­FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT­induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase­3, caspase­8 and phosphorylated­JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD­FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT­induced apoptosis via a reactive oxygen species mediated mitochondria/caspase­dependent pathway in U937 cells.


Assuntos
Hipertermia Induzida , Leucemia Monocítica Aguda , Humanos , Células U937 , Cálcio/metabolismo , Apoptose , Cumarínicos/farmacologia , Caspases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxirredução , Potencial da Membrana Mitocondrial
5.
Physiol Biochem Zool ; 96(1): 53-61, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36626842

RESUMO

AbstractIn most systems, the caspase cascade is activated during cellular stress and results in inflammation and apoptosis. Hibernators experience stressors such as extremely low body temperatures, bradycardia, possible ischemia and reperfusion, and acidosis. However, widespread inflammation and apoptosis would represent an energetic expense that is incompatible with hibernation. To better understand global caspase regulation during hibernation, we employed a systems-level approach and analyzed 11 caspases in ground squirrel liver that are involved in inflammatory (caspases 1, 4, 5, 11, and 12) and apoptotic (caspases 2, 6, 7, 8, 9, and 10) pathways. Western blots revealed liberation of active forms for two inflammatory (caspases 11 and 12) and two apoptotic (caspases 6 and 9) caspases during hibernation (e.g., p15, the most active fragment of caspase 6, increased 8.26±0.70-fold in interbout-aroused animals). We used specific peptide substrates to interrogate the four seemingly activated caspases and demonstrated no expected increases in proteolytic activity. Specific targets of these four caspases were similarly not cleaved, demonstrating that initiation of caspase activation may occur without concomitant downstream effects. Similarly, we found no evidence for upstream activation for caspase 9 signaling based on permeabilization of the outer mitochondrial membrane. We contend that these caspases are suppressed after seeming activation during hibernation. Incomplete caspase signaling is effectively mitigating the induction of widespread inflammation and apoptosis during hibernation.


Assuntos
Hibernação , Doenças dos Roedores , Animais , Caspases/metabolismo , Sciuridae/fisiologia , Transdução de Sinais , Apoptose , Inflamação , Hibernação/fisiologia
6.
Proc Natl Acad Sci U S A ; 120(2): e2210181120, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36595704

RESUMO

Malaria, caused by Plasmodium parasites is a severe disease affecting millions of people around the world. Plasmodium undergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening blood-stage of malaria. Although the natural immune responses impeding Plasmodium infection and development in the liver are key to controlling clinical malaria and transmission, those remain relatively unknown. Here we demonstrate that the DNA of Plasmodium parasites is sensed by cytosolic AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. Remarkably, Caspase-1 was observed to undergo unconventional proteolytic processing in hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D, but not inflammasome-associated proinflammatory cytokines. Nevertheless, this resulted in the elimination of Plasmodium-infected hepatocytes and the control of malaria infection in the liver. Our study uncovers a pathway of natural immunity critical for the control of malaria in the liver.


Assuntos
Malária , Parasitos , Plasmodium , Animais , Humanos , Hepatócitos/metabolismo , Fígado , Malária/parasitologia , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo
7.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36674798

RESUMO

Pyroptosis is a programmed cell death characterized by the rupture of the plasma membranes and release of cellular content leading to inflammatory reaction. Four cellular mechanisms inducing pyroptosis have been reported thus far, including the (i) caspase 1-mediated canonical, (ii) caspase 4/5/11-mediated non-canonical, (iii) caspase 3/8-mediated and (iv) caspase-independent pathways. Although discovered as a defense mechanism protecting cells from infections of intracellular pathogens, pyroptosis plays roles in tumor initiation, progression and metastasis of tumors, as well as in treatment response to antitumor drugs and, consequently, patient outcome. Pyroptosis induction following antitumor therapies has been reported in several tumor types, including lung, colorectal and gastric cancer, hepatocellular carcinoma and melanoma. This review provides an overview of the cellular pathways of pyroptosis and discusses the therapeutic potential of pyroptosis induction in cancer, particularly in melanoma.


Assuntos
Melanoma , Piroptose , Humanos , Apoptose , Caspase 1/metabolismo , Caspases/metabolismo , Melanoma/tratamento farmacológico
8.
Am J Physiol Cell Physiol ; 324(2): C222-C235, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36622073

RESUMO

This study investigates the mechanism by which microRNA (miR)-30e-3p reduces coronary microembolism (CME)-induced cardiomyocyte pyroptosis and inflammation. Cardiac function tests, histological staining, and transmission electron microscopy were performed on CME-model rats injected with adeno-associated viral vectors. Cardiomyocytes were transfected 24 h before a cellular model of pyroptosis was established via treatment with 1 µg/mL lipopolysaccharide (LPS) for 4 h and 5 mM ATP for 30 min. Pyroptosis, inflammation, and Wnt/ß-catenin signaling in cardiomyocytes were detected. Dual-luciferase reporter assays and/or RNA pull-down assays were performed to verify the binding of miR-30e-3p to HDAC2 mRNA or HDAC2 to the SMAD7 promoter. Chromatin immunoprecipitation was used to assess the level of H3K27 acetylation at the SMAD7 promoter. miR-30e-3p and SMAD7 expression levels were downregulated and HDAC2 expression was upregulated with CME. The overexpression of miR-30e-3p restored cardiac functions in CME-model rats and reduced serum cTnI, IL-18, and IL-1ß levels, microinfarcts, inflammatory cell infiltration, apoptosis, collagen content, and GSDMD-N, cleaved caspase-1, and NLRP3 expression in the myocardium, but these effects were reversed by SMAD7 knockdown. The overexpression of miR-30e-3p or knockdown of HDAC2 reduced LDH, IL-18, and IL-1ß secretion, propidium iodide intake, and GSDMD-N, NLRP3, cleaved caspase-1, Wnt3a, Wnt5a, and ß-catenin expression in the cardiomyocyte model. miR-30e-3p inhibited the expression of HDAC2 by binding HDAC2 mRNA. HDAC2 repressed the expression of SMAD7 by catalyzing H3K27 deacetylation at the SMAD7 promoter. miR-30e-3p, by binding HDAC2 to promote SMAD7 expression, reduces CME-induced cardiomyocyte pyroptosis and inflammation.


Assuntos
MicroRNAs , Miócitos Cardíacos , Ratos , Animais , Miócitos Cardíacos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Interleucina-18/metabolismo , beta Catenina/metabolismo , Piroptose/genética , Inflamação , RNA Mensageiro , Caspases/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Histona Desacetilase 2/genética
9.
Elife ; 122023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36645406

RESUMO

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.


Assuntos
Disenteria Bacilar , Shigella , Camundongos , Animais , Disenteria Bacilar/microbiologia , Inflamassomos/metabolismo , Morte Celular , Shigella flexneri/metabolismo , Caspases/genética , Caspases/metabolismo
10.
Cell Rep Med ; 4(1): 100899, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36652908

RESUMO

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.


Assuntos
Inflamassomos , Nanopartículas , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Caspases/metabolismo , Interleucina-1/metabolismo
11.
Ecotoxicol Environ Saf ; 249: 114429, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36516625

RESUMO

Although programmed cell death (PCD) has been reported in phytoplankton, knowledge of the characterization of the PCD pathway and cascade process in different phytoplankton species is still limited. In this study, PCD progression in cyanobacterium Microcystis aeruginosa and green algae Chlorella luteoviridis by paraquat-induced oxidative stress was monitored. The results showed that paraquat-induced PCD in the two species belonged to the caspase-dependent pathway. Dose- and time-dependent PCD characteristics in the two strains under paraquat included the increase in caspase-like activity, DNA fragmentation, and chromatin condensation. However, the signaling pathway and cascade events of PCD in M. aeruginosa and C. luteoviridis differed. In M. aeruginosa, the free Ca2+ concentration was rapidly increased at 8 h, followed by a significant elevation of the reactive oxygen species (ROS) level at 24 h, and eventual cell death. In C. luteoviridis, the mitochondrial apoptosis pathway, revealed by the depolarization of the mitochondrial membrane potential at 1 h and increase in the ROS level and caspase-like activity at 8 h, might contribute to cell death. In addition, the dynamics of ROS levels and metacaspase activity were synchronized, suggesting that paraquat-triggered PCD was ROS-mediated in both M. aeruginosa and C. luteoviridis. These results provide insights into PCD patterns in prokaryotic cyanobacteria and eukaryotic green algae under similar stress.


Assuntos
Chlorella , Cianobactérias , Microcystis , Microcystis/metabolismo , Paraquat/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Chlorella/metabolismo , Apoptose , Caspases/metabolismo , Cianobactérias/metabolismo
12.
Acta Histochem ; 125(1): 151989, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36529079

RESUMO

Regulating macrophage-hepatic stellate cells (HSCs) crosstalk through SIRT1-TLR2/TLR4 has contributed to the essence of new pharmacologic strategies to improve hepatic fibrosis. We investigated how Luteoloside (LUT), one of the flavonoid monomers isolated from Eclipta prostrata (L.) L., modulates macrophage-HSCs crosstalk during hepatic fibrosis. HSC-T6 or rat peritoneal macrophages were activated by TGF-ß or LPS/ATP, and then treated with LUT or Sirtinol (SIRT1 inhibitor) for 6 h. Further, HSCs were cultured with the conditioned medium from the LPS/ATP activated peritoneal macrophages. In HSC-T6 or peritoneal macrophages, LUT could decrease the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. LUT also significantly increased the expressions of SIRT1 and ERRα. And LUT significantly suppressed the releases of pro-inflammatory cytokines, including NLRP3, ASC, caspase-1, IL-1ß, and regulated signaling TLR2/TLR4-MyD88 activation. The expressions of TLR2, TLR4, NLRP3, caspase-1, IL-1ß, α-SMA were increased and the expression of ERRα was decreased by Sirtinol, indicated that LUT might mediate SIRT1 to regulate TLR4 expression and further alleviate inflammation and fibrosis. LUT could regulate SIRT1-mediated TLR4 and ECM in HSCs was reduced, when HSCs were cultured with conditioned medium. Hence, LUT could decrease the expressions of fibrosis markers, reduce the releases of inflammatory cytokines in activated HSCs or macrophages. In conclusion, LUT might be a promising candidate that regulating SIRT1-TLR2/TLR4 signaling in macrophages interacting with HSCs during hepatic fibrosis.


Assuntos
Células Estreladas do Fígado , Receptor 4 Toll-Like , Animais , Ratos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/uso terapêutico , Caspases/metabolismo , Caspases/uso terapêutico , Comunicação Celular , Meios de Cultivo Condicionados , Citocinas/metabolismo , Fibrose , Células Estreladas do Fígado/metabolismo , Lipopolissacarídeos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sirtuína 1/metabolismo , Sirtuína 1/uso terapêutico , Receptor 2 Toll-Like , Receptor 4 Toll-Like/metabolismo
13.
Mol Biol Rep ; 50(2): 1627-1637, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36562934

RESUMO

BACKGROUND: Systemic inflammatory response could affect many systems. Cardiac dysfunction develops due to cardiovascular system damage and could be mortal. Selenium is a trace element that can be used as a dietary supplement and has antioxidant, anti-inflammatory, and anti-apoptotic properties. This study aims to evaluate the protective effects of selenium on cardiovascular damage via silenced information regulator 1 (SIRT1)/p53 and cytochrome C (Cyt-c)/ caspase-3 (Cas-3) pathways. METHODS AND RESULTS: Thirty-two rats were randomly divided into 4 groups as control, LPS (0.1 mg/kg, intraperitoneally(i.p.), 2-7 days) and LPS + Selenium (LPS-0.1 mg/kg, i.p., 2-7 days, selenium - 100 µg/kg, i.p., 1-7 days) and selenium (100 µg/kg, i.p., 1-7 days) group. On the 8th day of the experiment, rats were sacrificed. Blood samples and half of the left ventricles were collected for biochemical and genetic analysis. The remaining left ventricles and aorta were taken for histological and immunohistochemical analysis. In the LPS group myocardial hemorrhages, hyperemia, and endothelial cell loss were observed. Also, Cas-3 and vascular endothelial growth factor (VEGF) expressions; creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-alpha (TNF-α), ischemia modified albumin (IMA), total oxidant status (TOS), oxidative stress index (OSI) levels; p53, Cyt-c, Cas-3 mRNA expressions increased while total antioxidant status (TAS) levels, glutathione peroxidase (GPx) activity, SIRT1 mRNA expression decreased. Selenium treatment reversed all these changes. CONCLUSION: Selenium showed protective effects on cardiovascular injury via regulating SIRT1/p53 and Cyt-c/Cas-3 pathways. This study enlightened the possible usage of selenium on cardiotoxicity.


Assuntos
Selênio , Ratos , Animais , Selênio/farmacologia , Selênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Caspases/metabolismo , Biomarcadores/metabolismo , Lipopolissacarídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Albumina Sérica , Coração , Estresse Oxidativo , RNA Mensageiro/genética , Apoptose
14.
Bioelectrochemistry ; 150: 108355, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36549173

RESUMO

Irreversible electroporation (IRE) has been reported to variably cause apoptosis, necrosis, oncosis or pyroptosis. Intracellular ATP is a key substrate for apoptosis which is rapidly depleted during IRE, we sought to understand whether intracellular ATP levels is a determinant of the mode of cell death following IRE. A mouse bladder cancer cell line (MB49) was treated with electric fields while increasing the number of pulses at a fixed electric field strength, and pulse width. Cell proliferation and viability and ATP levels were measured at different timepoints post-treatment. Cell death was quantified with Annexin-V/Propidium Iodide staining. Caspase activity was measure with a fluorometric kit and western blotting. A pan-caspase (Z-VAD-FMK) inhibitor was used to assess the impact of signal inhibition. We found cell death following IRE was insensitive to caspase inhibition and was correlated with ATP loss. These findings were confirmed by cell death assays and measurement of changes in caspase expression on immunoblotting. This effect could not be rescued by ATP supplementation. Rapid and acute ATP loss during IRE interferes with caspase signaling, promoting necrosis. Cell necrosis from IRE is expected to be immunostimulatory and may be effective in cancer cells that carry mutated or defective apoptosis genes.


Assuntos
Apoptose , Eletroporação , Camundongos , Animais , Necrose , Morte Celular , Caspases/metabolismo , Trifosfato de Adenosina , Caspase 3/metabolismo , Caspase 3/farmacologia
15.
J Immunol ; 210(3): 322-334, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36525001

RESUMO

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.


Assuntos
Vesículas Extracelulares , Inflamassomos , Humanos , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/metabolismo , Macrófagos/metabolismo , Caspases/metabolismo , Vesículas Extracelulares/metabolismo , Escherichia coli/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Crescimento Neural/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/farmacologia
16.
J Mol Biol ; 434(4): 167273, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-34599942

RESUMO

The gasdermin family of pore-forming proteins (PFPs) has recently emerged as key molecular players controlling immune-related cell death in mammals. Characterized mammalian gasdermins are activated through proteolytic cleavage by caspases or serine proteases, which remove an inhibitory carboxy-terminal domain, allowing the pore-formation process. Processed gasdermins form transmembrane pores permeabilizing the plasma membrane, which often results in lytic and inflammatory cell death. While the gasdermin-dependent cell death (pyroptosis) has been predominantly characterized in mammals, it now has become clear that gasdermins also control cell death in early vertebrates (teleost fish) and invertebrate animals such as corals (Cnidaria). Moreover, gasdermins and gasdermin-like proteins have been identified and characterized in taxa outside of animals, notably Fungi and Bacteria. Fungal and bacterial gasdermins share many features with mammalian gasdermins including their mode of activation through proteolysis. It has been shown that in some cases the proteolytic activation is executed by evolutionarily related proteases acting downstream of proteins resembling immune receptors controlling pyroptosis in mammals. Overall, these findings establish gasdermins and gasdermin-regulated cell death as an extremely ancient mechanism of cellular suicide and build towards an understanding of the evolution of regulated cell death in the context of immunology. Here, we review the broader gasdermin family, focusing on recent discoveries in invertebrates, fungi and bacteria.


Assuntos
Bactérias , Caspases , Fungos , Invertebrados , Proteínas Citotóxicas Formadoras de Poros , Piroptose , Animais , Bactérias/metabolismo , Caspases/metabolismo , Fungos/metabolismo , Humanos , Invertebrados/metabolismo , Peptídeo Hidrolases/metabolismo , Porinas , Piroptose/fisiologia
17.
Sci Rep ; 12(1): 22390, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36575196

RESUMO

Selective elimination of tumors has always been the mainstay of oncology research. The on-going research underlying the cellular apoptotic mechanisms reveal caspases activation, especially the key effector caspase-3, as a personalized tumor-selective therapeutic strategy. Our continued research protocol has exploited new optimized Passerini α-acyloxy carboxamides as efficient apoptotic inducers via caspase-3/7 dependent mechanism with highly selective anticancer profiles. The adopted design rationale relied on excluding structural alerts of previous leads, while merging various pharmacophoric motifs of natural and synthetic caspase activators via optimized one-pot Passerini reaction conditions. The prepared compounds resulting from Passerini reaction were screened for their cytotoxic activities against colorectal Caco-2 and liver HepG-2 cancer cells compared to normal fibroblasts utilizing MTT assay. Notably, all compounds exhibited promising low-range submicromolar IC50 against the studied cancer cell lines, with outstanding tumor selectivity (SI values up to 266). Hence, they were superior to 5-fluorouracil. Notably, 7a, 7g, and 7j conferred the highest potencies against Caco-2 and HepG-2 cells and were selected for further mechanistic studies. Caspas-3/7 activation assay of the hit compounds and flow cytometric analysis of the treated apoptotic cancer cells demonstrated their significant caspase activation potential (up to 4.2 folds) and apoptotic induction capacities (up to 58.7%). Further assessment of Bcl2 expression was performed being a physiological caspase-3 substrate. Herein, the three studied Passerini adducts were able to downregulate Bcl2 in the treated Caco-2 cells. Importantly, the mechanistic studies results of the three hits echoed their preliminary MTT antiproliferative potencies data highlighting their caspase-3 dependent apoptotic induction. Finally, the in silico predicted physicochemical and pharmacokinetic profiles, as well as ligand efficiency metrics were drug-like.


Assuntos
Antineoplásicos , Apoptose , Humanos , Caspase 3/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Células CACO-2 , Antineoplásicos/farmacologia , Antineoplásicos/química , Caspases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral
18.
Int J Mol Sci ; 23(23)2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36498874

RESUMO

The present study aims to continue the study of corchorusoside C (1), a cardenolide isolated from Streptocaulon juventas, as a potential anticancer agent. A mechanistic study was pursued in a zebrafish model and in DU-145 prostate cancer cells to investigate the selectivity of 1 towards NF-κB and PARP-1 pathway elements. Compound 1 was found to inhibit the expression of IKKα and NF-κB p65 in TNF-α induced zebrafish and inhibit the expression of NIK in vitro. The protein expression levels of XRCC-1 were increased and p53 decreased in DU-145 cells. XIAP protein expression was initially decreased after treatment with 1, followed by an increase in expression at doses higher than the IC50 value. The activity of caspase-1 and the protein expression levels of IL-18 were both decreased following treatment of 1. The binding interactions for 1 to NIK, XRCC-1, p53, XIAP, and caspase-1 proteins were explored in molecular docking studies. Additionally, the toxicity profile of 1 in zebrafish was favorable in comparison to its analog digoxin and other anticancer drugs at the same MTD in zebrafish. Overall, 1 targets the noncanconical NF-κB pathway in vivo and in vitro, and is well tolerated in zebrafish supporting its potential in the treatment of prostate cancer.


Assuntos
NF-kappa B , Poli(ADP-Ribose) Polimerase-1 , Neoplasias da Próstata , Animais , Humanos , Masculino , Caspases/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/metabolismo , Linhagem Celular Tumoral , Poli(ADP-Ribose) Polimerase-1/metabolismo
19.
J Mol Neurosci ; 72(12): 2464-2472, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36508141

RESUMO

This study was designed to determine the effects and underlying mechanism of honokiol (HNK) on traumatic brain injury (TBI). A rat TBI model was constructed using the modified Feeney free-fall percussion method and treatment with HNK via intraperitoneal injection. The brain tissues of the rats in each group were assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect the level of neuronal apoptosis. Western blots were used to detect the expression levels of apoptosis-related proteins (Bcl-2 and Bax), and ELISAs were used to measure the levels of pro-inflammatory cytokines (IL-18 and IL-1ß) and the activity of caspase-1. In addition, the mitochondrial membrane potential, reactive oxygen species (ROS), and adenosine 5'-triphosphate (ATP) were also measured. Western blots and qRT-PCRs were used to determine the relative expression levels of the mitochondrial unfolded protein response (UPRmt)-related proteins and mRNAs. Based on the experimental results, treatment with HNK was associated with a decrease in the number of TUNEL-positive cells, downregulated Bax expression levels, elevated Bcl-2 expression levels, and inhibition of neuronal apoptosis in the brain tissue of TBI rats. HNK also suppressed neuroinflammation by decreasing IL-1ß and IL-18 levels and caspase-1 activity. Additionally, HNK lowered the mitochondrial membrane potential and ROS levels, increased ATP levels, and improved mitochondrial dysfunction in neural cells. Furthermore, in the investigation of the mechanism of HNK on TBI, we observed that HNK could activate UPRmt by upregulating the mRNA and protein expression levels of HSPA9, CLPP, and HSP60 in the brain tissues of TBI rats. Collectively, HNK reduced mitochondrial dysfunction, inhibited the apoptosis of nerve cells, and attenuated inflammation in the brains of TBI rats. The protective effect of HNK may be achieved through the activation of UPRmt.


Assuntos
Lesões Encefálicas Traumáticas , Interleucina-18 , Ratos , Animais , Interleucina-18/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neurônios/metabolismo , Resposta a Proteínas não Dobradas , Mitocôndrias/metabolismo , Caspases/metabolismo , Caspases/farmacologia
20.
Int J Mol Sci ; 23(24)2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36555405

RESUMO

Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2ß, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.


Assuntos
Caseína Quinase II , Proteínas de Saccharomyces cerevisiae , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/metabolismo , Caspases/genética , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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