Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.277
Filtrar
1.
J Immunol ; 207(10): 2411-2416, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34663620

RESUMO

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.


Assuntos
Caspase 3/imunologia , Caspase 8/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas de Ligação a Fosfato/imunologia , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Morte Celular , Citocinas , Vírus da Influenza A/imunologia , Vírus da Influenza A/metabolismo , Camundongos , Infecções por Orthomyxoviridae/metabolismo
2.
Environ Toxicol Pharmacol ; 88: 103750, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34597787

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease with limited therapeutic management approaches. The present study evaluated the potential therapeutic impact of betulin on acetic acid (AA)-induced UC in rats. UC was induced by intracolonic instillation of AA (3% v/v). Rats were treated with betulin (8 mg/kg, I.P., once daily) four days post AA instillation and for 14 consecutive days. Betulin attenuated AA-induced UC as evidenced by retracted macroscopic scores, serum CRP titre and LDH activity, attenuated histopathological hallmarks of UC including mucosal necrosis, haemorrhage, congestion and inflammatory cells infiltration. Moreover, betulin dampened UC-associated colonic inflammatory load with modulation of TLR4/NF-kB axis and reduction in colonic inflammatory cytokines; TNF-α, IL1ß and IL-6. Nevertheless, betulin suppressed colonic apoptosis with reduced colonic caspase-3 and caspase-8 expression. The current findings confirm a beneficial therapeutic impact of betulin against UC. The prospective underlying mechanisms include down-regulation of TLR4/NF-κB and the subsequent downstream signalling pathways.


Assuntos
Anti-Inflamatórios/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Triterpenos/uso terapêutico , Ácido Acético , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Citocinas/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Triterpenos/farmacologia
3.
Chem Biol Interact ; 350: 109704, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655567

RESUMO

Pyroptosis is a novel type of pro-inflammatory programmed cell death that has been strongly reported to be related to inflammation, immune, and cancer. Dihydroartemisinin (DHA) has good anti-tumor properties. However, the exact mechanism by which DHA induces pyroptosis to inhibit esophageal squamous cell carcinoma (ESCC) remains unclear. After applying DHA treatment to ESCC, we found that some dying cells exhibited the characteristic morphology of pyroptosis, such as blowing large bubbles from the cell membrane, accompanied by downregulation of pyruvate kinase isoform M2 (PKM2), activation of caspase-8/3, and production of GSDME-NT. Meanwhile, it was accompanied by an increased release of LDH and inflammatory factors (IL-18 and IL-1ß). Both knockdown of GSDME and application of caspase-8/3 specific inhibitors (z-ITED-FMK/Ac-DEVD-CHO) significantly inhibited DHA-induced pyroptosis. However, the former did not affect the activation of caspase-3. In contrast, overexpression of PKM2 inhibited caspase-8/3 activation as well as GSDME-N production. Furthermore, both si-GSDME and OE-PKM2 inhibited DHA-induced pyroptosis in vivo and in vitro. Therefore, the results suggest that DHA can induce pyroptosis of ESCC cells via the PKM2-caspase-8/3-GSDME pathway. Implication: In this study, we identified new mechanism of DHA in inhibiting ESCC development and progression, and provide a potential therapeutic agent for the treatment of ESCC.


Assuntos
Artemisininas/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Piroptose/efeitos dos fármacos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Piroptose/fisiologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochemistry ; 60(37): 2824-2835, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34472839

RESUMO

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.


Assuntos
Caspase 3/química , Caspase 6/química , Caspase 9/metabolismo , Sequência de Aminoácidos/genética , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 8/metabolismo , Caspase 9/fisiologia , Caspases/metabolismo , Ativação Enzimática , Humanos , Transdução de Sinais/genética
5.
Cell Rep ; 36(8): 109614, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34433041

RESUMO

Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1ß. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora.


Assuntos
Carnívoros/metabolismo , Evolução Molecular , Inflamassomos/metabolismo , Zoonoses/patologia , Animais , Caspase 1/genética , Caspase 1/metabolismo , Caspase 8/metabolismo , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Morte Celular , Linhagem Celular , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas NLR/genética , Proteínas NLR/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhi/patogenicidade , Zoonoses/imunologia , Zoonoses/parasitologia
6.
J Vis Exp ; (174)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34398143

RESUMO

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1ß-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.


Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Receptor fas , Apoptose , Caspase 8/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Transdução de Sinais , Receptor fas/metabolismo
7.
Am J Physiol Cell Physiol ; 321(4): C644-C653, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34432536

RESUMO

Endothelial dysfunction is associated with the initiation of sepsis-associated organ failure. Bacterial quorum-sensing molecules act as pathogen-associated molecular patterns; however, the effects of quorum-sensing molecules on endothelial cells remain less understood. This study investigated the molecular mechanisms of quorum-sensing molecule-induced cell death and their interaction with lipopolysaccharide (LPS) in human umbilical vein endothelial cells. Endothelial cells were treated with N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and LPS derived from Pseudomonas aeruginosa. Treatment with 3OC12-HSL reduced cell viability in a dose-dependent manner, and cotreatment with 3OC12-HSL and LPS enhanced cell death. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay revealed an increase in apoptotic cell death following 3OC12-HSL treatment; furthermore, cotreatment with 3OC12-HSL and LPS enhanced apoptosis. Western blotting revealed that treatment with 3OC12-HSL activated the receptor-interacting protein kinase 1 (RIPK1) pathway, leading to an increase in the levels of cleaved caspase 8 and 3. In addition, we found that treatment with necrostatin-1, an RIPK1 inhibitor, reduced cell death and ameliorated the activation of the RIPK1-dependent apoptotic pathway in 3OC12-HSL-treated cells. In conclusion, 3OC12-HSL induced endothelial cell apoptosis via the activation of the RIPK1 pathway, independent of LPS toxicity. Inhibition of RIPK1 may act as a therapeutic option for preserving endothelial cell integrity in patients with sepsis by disrupting the mechanism by which quorum-sensing molecules mediate their toxicity.


Assuntos
4-Butirolactona/análogos & derivados , Apoptose/efeitos dos fármacos , Homosserina/análogos & derivados , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , 4-Butirolactona/toxicidade , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Ativação Enzimática , Homosserina/toxicidade , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Lipopolissacarídeos/toxicidade , Transdução de Sinais
8.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299199

RESUMO

Continuing our studies on the mechanisms underlying the cytotoxicity of potential drugs, we have described several aspects of the in vitro anticancer activity of ruthenium(II) and platinum(II) complexes with bioactive, synthetic aminoflavone ligands. We examined the mechanism of proapoptotic activity of cis-dichlorobis(3-imino-2-methoxyflavanone)ruthenium(II), cis-dichlorobis(3-imino-2-ethoxyflavanone)ruthenium(II), and trans-dichlorobis(3-aminoflavone)platinum(II). Cisplatin was used as a reference compound. The cytotoxicity was investigated by MTT assay. The mechanism of proapoptotic activity of the tested compounds was investigated by evaluation of caspase-8 activity, cytometric analysis of annexin-V positive cells, and mitochondrial potential loss measurement. The results showed that ruthenium compounds break partially or completely the cisplatin resistance by activating the caspase 8-dependent apoptosis pathway and loss of mitochondrial membrane potential. Platinum compounds also have a cytostatic effect, but their action requires more exposure time. Potential mechanisms underlying drug resistance in the two pairs of cancer cell lines were investigated: total glutathione content, P-glycoprotein activity, and differences in the activity of DNA repair induced by nucleotide excision. Results showed that cisplatin-resistant cells have elevated glutathione levels relative to sensitive cells. Moreover, they indicated the mechanisms enabling cells to avoid apoptosis caused by DNA damage. Pg-P activity has no effect on the development of cisplatin resistance in the cell lines described.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Flavonoides/farmacologia , Neoplasias/tratamento farmacológico , Compostos de Platina/farmacologia , Compostos de Rutênio/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Cisplatino/farmacologia , Complexos de Coordenação/química , Resistencia a Medicamentos Antineoplásicos , Flavonoides/química , Humanos , Ligantes , Neoplasias/metabolismo , Neoplasias/patologia , Compostos de Platina/química , Compostos de Rutênio/química , Células Tumorais Cultivadas
9.
Immunity ; 54(8): 1758-1771.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34256013

RESUMO

Apoptosis can potently defend against intracellular pathogens by directly killing microbes and eliminating their replicative niche. However, the reported ability of Mycobacterium tuberculosis to restrict apoptotic pathways in macrophages in vitro has led to apoptosis being dismissed as a host-protective process in tuberculosis despite a lack of in vivo evidence. Here we define crucial in vivo functions of the death receptor-mediated and BCL-2-regulated apoptosis pathways in mediating protection against tuberculosis by eliminating distinct populations of infected macrophages and neutrophils and priming T cell responses. We further show that apoptotic pathways can be targeted therapeutically with clinical-stage compounds that antagonize inhibitor of apoptosis (IAP) proteins to promote clearance of M. tuberculosis in mice. These findings reveal that any inhibition of apoptosis by M. tuberculosis is incomplete in vivo, advancing our understanding of host-protective responses to tuberculosis (TB) and revealing host pathways that may be targetable for treatment of disease.


Assuntos
Apoptose/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Neutrófilos/imunologia , Tuberculose Pulmonar/imunologia , Animais , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Dipeptídeos/uso terapêutico , Humanos , Indóis/uso terapêutico , Ativação Linfocitária/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/imunologia , Tiazóis/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico
10.
Lab Invest ; 101(10): 1363-1370, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34234270

RESUMO

The wound-healing process is a natural response to burn injury. Resveratrol (RES) may have potential as a therapy for wound healing, but how and whether RES regulates skin repair remains poorly understood. Human epidermal keratinocyte (HaCaT) cells were treated with lipopolysaccharide (LPS), and a mouse skin wound-healing model was established. Cell viability and apoptosis were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide or flow cytometry. Cell proliferation was assessed by cell viability and colony-formation analyses. Cell migration was tested by wound-healing analysis. The microRNA-212 (miR-212) and caspase-8 (CASP8) levels were determined by quantitative reverse transcription polymerase chain reaction and western blotting. The correlation between miR-212 and CASP8 was analyzed by dual-luciferase reporter analysis. Skin wound healing in mice was assessed by measuring the wound area and gap after hematoxylin-eosin (HE) staining. RES reduced the LPS-induced reduction in viability and apoptosis in HaCaT cells. miR-212 expression was reduced by LPS and increased by exposure to RES. RES promoted cell proliferation and migration after LPS treatment by increasing miR-212 levels. CASP8 was a target of miR-212. CASP8 silencing promoted cell proliferation and migration, which was reversed by miR-212 knockdown in LPS-treated HaCaT cells. RES promoted skin wound healing in mice, which was reduced by miR-212 knockdown. Thus, RES facilitates cell proliferation and migration in LPS-treated HaCaT cells and promotes skin wound-healing in a mouse model by regulating the miR-212/CASP8 axis.


Assuntos
Caspase 8/metabolismo , MicroRNAs/metabolismo , Resveratrol/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
Molecules ; 26(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071530

RESUMO

Anti-cancer activity of catechin nanoemulsions prepared from Oolong tea leaf waste was studied on prostate cancer cells DU-145 and DU-145-induced tumors in mice. Catechin nanoemulsions composed of lecithin, Tween-80 and water in an appropriate proportion was prepared with high stability, particle size of 11.3 nm, zeta potential of -67.2 mV and encapsulation efficiency of 83.4%. Catechin nanoemulsions were more effective than extracts in inhibiting DU-145 cell growth, with the IC50 being 13.52 and 214.6 µg/mL, respectively, after 48 h incubation. Furthermore, both catechin nanoemulsions and extracts could raise caspase-8, caspase-9 and caspase-3 activities for DU-145 cell apoptosis, arresting the cell cycle at S and G2/M phases. Compared to control, catechin nanoemulsion at 20 µg/mL and paclitaxel at 10 µg/mL were the most effective in reducing tumor volume by 41.3% and 52.5% and tumor weight by 77.5% and 90.6% in mice, respectively, through a decrease in EGF and VEGF levels in serum.


Assuntos
Catequina/química , Emulsões/química , Nanopartículas/química , Folhas de Planta/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Chá/química , Animais , Antineoplásicos/farmacologia , Apoptose , Caspase 8/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Endocitose , Humanos , Concentração Inibidora 50 , Lecitinas/química , Limite de Detecção , Masculino , Camundongos , Camundongos SCID , Nanotecnologia/métodos , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Tamanho da Partícula , Polissorbatos/química , Controle de Qualidade , Solventes , Água/química
12.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071450

RESUMO

Receptor-interacting protein kinase 1 (RIPK1) is a key component of the tumor necrosis factor (TNF) receptor signaling complex that regulates both pro- and anti-apoptotic signaling. The reciprocal functions of RIPK1 in TNF signaling are determined by the state of the posttranslational modifications (PTMs) of RIPK1. However, the underlying mechanisms associated with the PTMs of RIPK1 are unclear. In this study, we found that RING finger protein 4 (RNF4), a RING finger E3 ubiquitin ligase, is required for the RIPK1 autophosphorylation and subsequent cell death. It has been reported that RNF4 negatively regulates TNF-α-induced activation of the nuclear factor-κB (NF-κB) through downregulation of transforming growth factor ß-activated kinase 1 (TAK1) activity, indicating the possibility that RNF4-mediated TAK1 suppression results in enhanced sensitivity to cell death. However, interestingly, RNF4 was needed to induce RIPK1-mediated cell death even in the absence of TAK1, suggesting that RNF4 can promote RIPK1-mediated cell death without suppressing the TAK1 activity. Thus, these observations reveal the existence of a novel mechanism whereby RNF4 promotes the autophosphorylation of RIPK1, which provides a novel insight into the molecular basis for the PTMs of RIPK1.


Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Animais , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Immunoblotting , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Knockout , Fosforilação , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
13.
Cell Death Dis ; 12(7): 647, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168123

RESUMO

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood-brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Lactonas/farmacologia , Inibidores de Proteassoma/farmacologia , Pirróis/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Glioma/metabolismo , Glioma/patologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pirimidinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Esferoides Celulares , Tiofenos/farmacologia , Fatores de Tempo
14.
J Am Soc Nephrol ; 32(8): 1987-2004, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34135081

RESUMO

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.


Assuntos
Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Isquemia/fisiopatologia , Rim/fisiopatologia , Insuficiência Renal Crônica/fisiopatologia , Quinases Ativadas por p21/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Doença Crônica , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Dasatinibe/farmacologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Humanos , Isquemia/etiologia , Rim/irrigação sanguínea , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteopontina/genética , Inibidores de Proteínas Quinases/farmacologia , Obstrução da Artéria Renal/complicações , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/patologia , Análise de Sequência de RNA , Análise de Célula Única , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Regulação para Cima , Quinases Ativadas por p21/genética
15.
Methods Mol Biol ; 2255: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033089

RESUMO

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.


Assuntos
Apoptose , Western Blotting/métodos , Caspase 8/metabolismo , Caspase 9/metabolismo , Neoplasias Ovarianas/patologia , Ativação Enzimática , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
16.
Chem Biol Interact ; 344: 109535, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34051208

RESUMO

Imatinib, a specific Bcr-Abl tyrosine kinase inhibitor, is the most commonly used drug in the treatment of chronic myeloid leukemia. However, optimal response is not achieved in up to 33% of patients. Therefore, development of novel therapeutic strategies for chronic myeloid leukemia is critical. Betulinic (1) and ursolic (2) acids are natural pentacyclic triterpenes that exhibit antileukemic activities. In this study, we evaluated the effects of pharmacomodulations at the C-3 position of the triterpene moiety of betulinic and ursolic acids on their activity against K562 leukemia cells. Six new derivatives (1a-2c) were synthesized and evaluated for pro-apoptotic and anti-proliferative effects in mammalian and leukemic cells. 2c derivative containing an amine group at the C-3 position of ursolic acid was the most active against leukemia cells with an IC50 value of 5.2 µM after 48 h of treatment. 2c did not exhibit cytotoxic effects against VERO and HepG2 cells and human lymphocytes, showing a good selectivity index for cancer over normal cells. Induced cell death by apoptosis via caspases 3 and 8, and also caused cell cycle arrest as evidenced by accumulation of cells in the G1 phase and decreased cell population in the G2 phase. Furthermore, co-treatment of 2c with imatinib, the chemotherapy drug most commonly used to treat leukemia, resulted in a synergistic effect. Our findings provide a strong rationale for further investigation of combination therapy using the 2c derivative and imatinib in pre-clinical studies.


Assuntos
Antineoplásicos/farmacologia , Mesilato de Imatinib/farmacologia , Triterpenos/farmacologia , Animais , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Triterpenos/síntese química , Células Vero
17.
Int J Cancer ; 149(7): 1408-1420, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34028029

RESUMO

Cysteinyl aspartate specific proteinase (Caspase)-8 has long been considered a promoter of apoptosis and part of the mechanism by which cytotoxic drugs kill cancer cells. With the continuous exploration of the types of programmed cell death, an increasing number of studies have confirmed that caspase-8 plays an important role in cancer. Recently, scholars have proposed the term "PANoptosis," which mainly includes three programmed cell death modes, namely pyroptosis, apoptosis and necroptosis. In addition to mediating endogenous apoptotic pathways, caspase-8 can also participate in the cleavage of gasdermin (GSDM) family proteins to induce pyroptosis. Furthermore, the expression of enzymatically inactive caspase-8 (C362S) can cause embryonic lethality and inflammatory tissue destruction in mice by inducing necroptosis and pyroptosis. Therefore, the activation and deletion of caspase-8 enzyme activity, as well as the knockout of the coding gene, are closely related to "PANoptosis." In addition, caspase-8 can also improve the tumor microenvironment and enhance tumor antiimmunity. Studies have shown that caspase-8 is also associated with tumor growth and invasion, angiogenesis and metastasis, therapeutic resistance and poor clinical outcomes. Therefore, it is very important to measure the cancer-promoting and anticancer effects of caspase-8 and find a balance, and to study its role in the effect of "PANoptosis" in depth. This article reviews the role of caspase-8 in "PANoptosis" in cancer to provide new strategies and targets for cancer.


Assuntos
Apoptose , Caspase 8/metabolismo , Necroptose , Neoplasias/enzimologia , Neoplasias/patologia , Piroptose , Animais , Humanos , Transdução de Sinais
18.
Front Immunol ; 12: 658048, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953720

RESUMO

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm ) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated - together with Linear M1-Ub and cFLIP levels - by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.


Assuntos
Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interleucinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Formação de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Biomarcadores , Caspase 8/metabolismo , Caspase 9 , Comunicação Celular , Sobrevivência Celular , Camundongos , Camundongos Transgênicos , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinas/metabolismo
19.
Cancer Sci ; 112(9): 3507-3519, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34050696

RESUMO

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.


Assuntos
Carcinoma Hepatocelular/metabolismo , Caspase 8/metabolismo , Conexinas/metabolismo , Neoplasias Hepáticas/metabolismo , Necroptose/genética , Coativador 1 de Receptor Nuclear/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Conexinas/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/administração & dosagem , Necroptose/efeitos dos fármacos , Coativador 1 de Receptor Nuclear/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética
20.
Eur J Pharmacol ; 904: 174167, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33974882

RESUMO

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins.


Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Retinopatia Diabética/prevenção & controle , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Resveratrol/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Aldeídos/metabolismo , Animais , Antígenos de Bactérias/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Toxinas Bacterianas/uso terapêutico , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Dasyproctidae , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Retinopatia Diabética/complicações , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Resveratrol/uso terapêutico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/lesões , Estreptozocina , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...