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1.
J Oleo Sci ; 69(9): 1087-1093, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32788522

RESUMO

Previously, we reported that the polar lipid fraction from the golden oyster mushroom, Pleurotus citrinopileatus, suppresses colon injuries which result from apoptosis induced by inflammatory stresses in vivo and in vitro (Yamashita et al., J. Oleo Sci., 69, 751-757 (2020)). Here, we investigated the use of lipid classes in mushroom polar lipid fraction in alleviating colon injury using differentiated Caco-2 cells as an intestinal tract model. The mushroom polar lipid fraction was separated into four fractions using silica thin layer chromatography. Each mushroom polar lipid fraction suppressed lipopolysaccharide (LPS)-induced decreases in the viability of intestinal cells, and the effects of sphingolipid fractions were significantly stronger than those of fraction that did not contain sphingolipids. Addition of sphingolipid fractions suppressed the expression of apoptosis-related proteins (e.g., death receptors and caspases) in the LPS-treated cells. Mushroom polar lipids, especially sphingolipids suppress intestinal apoptosis induced by inflammatory stress, and highly polar sphingolipids may exert stronger suppressive effects.


Assuntos
Apoptose/efeitos dos fármacos , Doenças do Colo/tratamento farmacológico , Doenças do Colo/patologia , Fitoterapia , Pleurotus/química , Esfingolipídeos/isolamento & purificação , Esfingolipídeos/farmacologia , Apoptose/genética , Células CACO-2 , Caspases/genética , Caspases/metabolismo , Fracionamento Químico , Doenças do Colo/induzido quimicamente , Expressão Gênica , Humanos , Técnicas In Vitro , Inflamação , Lipopolissacarídeos , Esfingolipídeos/uso terapêutico
2.
Nat Commun ; 11(1): 2249, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382010

RESUMO

Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca2+ for their activation and substrate processing. However, the Ca2+-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Imunidade Vegetal/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Caspases/genética , Caspases/metabolismo , Cristalografia por Raios X , Imunidade Vegetal/genética
3.
Chem Biol Interact ; 320: 109005, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32109484

RESUMO

The mortality rates for acute myeloid leukemia are very high, necessitating the search for novel chemotherapeutic candidates. Herein, we investigated the anticancer potential of a new synthetic compound, 2-ethyl-3-methyliden-1-tosyl-2,3-dihydroquinolin-4-(1H)-one (AJ-374) against myeloid leukemia HL-60 cell line. This analog was selected from the small library of synthetic dihydroquinolinones on the basis of its strong antiproliferative activity against HL-60 cells and 30-fold lower cytotoxicity towards healthy HUVEC cells. AJ-374 promoted the arrest of the cells in the subG0/G1 phase of the cell cycle in the first 24 h. Treatment of HL-60 cells with AJ-374 caused an increase in annexin-V positive cells, activation of caspase-8, -9 and -3, dissipation of the mitochondrial membrane potential and enhancement of FAS protein level. Apoptosis induction triggered by this quinolinone was blocked by the pre-treatment of the cells with caspase-8, -9 and -3 inhibitors. The obtained results indicated that AJ-374-induced apoptosis was executed by both, the extrinsic and intrinsic pathways. The cytotoxic activity of AJ-374 was also associated with down-regulation of the mitogen-activated protein kinase (MAPK) pathway and was independent of reactive oxygen species generation. Taken together, these results suggest that AJ-374 exerts a potent anticancer effect on leukemia cells, with a wide safety margin, which makes this analog an attractive drug candidate for further testing.


Assuntos
Apoptose/efeitos dos fármacos , Quinolonas/farmacologia , Caspases/genética , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Quinolonas/química , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Receptor fas/genética , Receptor fas/metabolismo
4.
PLoS Pathog ; 16(2): e1008297, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32032391

RESUMO

Hantaviruses, zoonotic RNA viruses belonging to the order Bunyavirales, cause two severe acute diseases in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantavirus-infected patients show strong cytotoxic lymphocyte responses and hyperinflammation; however, infected cells remain mostly intact. Hantaviruses were recently shown to inhibit apoptosis in infected cells. By inhibiting granzyme B- and TRAIL-mediated apoptosis, hantaviruses specifically and efficiently inhibit cytotoxic lymphocyte-mediated killing of infected cells. Hantaviruses also strongly inhibit apoptosis triggered intrinsically; i.e., initiated through intracellular activation pathways different from those used by cytotoxic lymphocytes. However, insights into the latter mechanisms are currently largely unknown. Here, we dissected the mechanism behind how hantavirus infection, represented by the HFRS-causing Hantaan virus and the HPS-causing Andes virus, results in resistance to staurosporine-induced apoptosis. Less active caspase-8 and caspase-9, and consequently less active caspase-3, was observed in infected compared to uninfected staurosporine-exposed cells. While staurosporine-exposed uninfected cells showed massive release of pro-apoptotic cytochrome C into the cytosol, this was not observed in infected cells. Further, hantaviruses prevented activation of BAX and mitochondrial outer membrane permeabilization (MOMP). In parallel, a significant increase in levels of the pro-survival factor BCL-2 was observed in hantavirus-infected cells. Importantly, direct inhibition of BCL-2 by the inhibitor ABT-737, as well as silencing of BCL-2 by siRNA, resulted in apoptosis in staurosporine-exposed hantavirus-infected cells. Overall, we here provide a tentative mechanism by which hantaviruses protect infected cells from intrinsic apoptosis at the mitochondrial level by inducing an increased expression of the pro-survival factor BCL-2, thereby preventing MOMPs and subsequent activation of caspases. The variety of mechanisms used by hantaviruses to ensure survival of infected cells likely contribute to the persistent infection in natural hosts and may play a role in immunopathogenesis of HFRS and HPS in humans.


Assuntos
Apoptose , Febre Hemorrágica com Síndrome Renal/metabolismo , Potencial da Membrana Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Regulação para Cima , Células A549 , Caspases/genética , Caspases/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Febre Hemorrágica com Síndrome Renal/patologia , Humanos , Membranas Mitocondriais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-32056240

RESUMO

The caspase family proteins are aspartate-specific cysteine proteases that transmit extracellular signals to cells, ultimately cause apoptosis and therefore play a key role in cellular immunity. In this study, we cloned and characterized three caspases from Chinese black sleeper (Bostrychus sinensis), Bscasp-1, Bscasp-8 and Bscasp-9. Real-time PCR analysis showed that Bscasp-1, Bscasp-8 and Bscasp-9 were universally expressed in all tested tissues of B. sinensis. Expression analyses showed that after poly(I:C) stimulation and bacterial (Vibrio parahaemolyticus) infection, the three caspases were significantly upregulated. After poly(I:C) stimulation, the change of Bscasp-1 expression in the head kidney was the most obvious; peak expression was about 80.78-fold more than that of the control. In addition, the expression of Bscasp-8 and Bscasp-9 in the peripheral blood and liver was 167.99- and 17.98-fold higher than that in the control group, respectively. After V. parahaemolyticus infection, the expression peaks of Bscasp-1 and Bscasp-8 in the peripheral blood and spleen were 85.82-fold and 280.83-fold that of the control. However, the expression of Bscasp-9 in the peripheral blood was upregulated only 8.33-fold higher than that in the control group. These results indicate that Bscasp-1, Bscasp-8 and Bscasp-9 are likely involved in response to viral and bacterial infection.


Assuntos
Caspases/genética , Caspases/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caspases/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
6.
Cancer Sci ; 111(4): 1357-1366, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31991041

RESUMO

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (Kd  = 91.4-255 nmol L-1 ); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L-1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nona-arginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L-1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L-1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L-1 r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC16-22 , even at 10 µmol L-1 , compared to untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Proteínas Cromossômicas não Histona/genética , Peptídeos/farmacologia , Survivina/isolamento & purificação , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/química , Caspases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/química , Feminino , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/isolamento & purificação , Imagem Molecular/métodos , Peptídeos/síntese química , Peptídeos/química , Survivina/química , Survivina/genética
7.
Nature ; 577(7792): 706-710, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31942072

RESUMO

Protein structure prediction can be used to determine the three-dimensional shape of a protein from its amino acid sequence1. This problem is of fundamental importance as the structure of a protein largely determines its function2; however, protein structures can be difficult to determine experimentally. Considerable progress has recently been made by leveraging genetic information. It is possible to infer which amino acid residues are in contact by analysing covariation in homologous sequences, which aids in the prediction of protein structures3. Here we show that we can train a neural network to make accurate predictions of the distances between pairs of residues, which convey more information about the structure than contact predictions. Using this information, we construct a potential of mean force4 that can accurately describe the shape of a protein. We find that the resulting potential can be optimized by a simple gradient descent algorithm to generate structures without complex sampling procedures. The resulting system, named AlphaFold, achieves high accuracy, even for sequences with fewer homologous sequences. In the recent Critical Assessment of Protein Structure Prediction5 (CASP13)-a blind assessment of the state of the field-AlphaFold created high-accuracy structures (with template modelling (TM) scores6 of 0.7 or higher) for 24 out of 43 free modelling domains, whereas the next best method, which used sampling and contact information, achieved such accuracy for only 14 out of 43 domains. AlphaFold represents a considerable advance in protein-structure prediction. We expect this increased accuracy to enable insights into the function and malfunction of proteins, especially in cases for which no structures for homologous proteins have been experimentally determined7.


Assuntos
Aprendizado Profundo , Modelos Moleculares , Conformação Proteica , Proteínas/química , Software , Sequência de Aminoácidos , Caspases/química , Caspases/genética , Conjuntos de Dados como Assunto , Dobramento de Proteína , Proteínas/genética
8.
Nucleic Acids Res ; 48(3): 1494-1507, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31799626

RESUMO

During viral infection, viral nucleic acids are detected by virus sensor proteins including toll-like receptor 3 or retinoic acid-inducible gene I-like receptors (RLRs) in mammalian cells. Activation of these virus sensor proteins induces type-I interferon production and represses viral replication. Recently, we reported that an RLR family member, laboratory of genetics and physiology 2 (LGP2), modulates RNA silencing by interacting with an RNA silencing enhancer, TAR-RNA binding protein (TRBP). However, the biological implications remained unclear. Here, we show that LGP2 enhances apoptosis by upregulating apoptosis regulatory genes during viral infection. Sendai virus (SeV) infection increased LGP2 expression approximately 900 times compared to that in non-virus-infected cells. Then, the induced LGP2 interacted with TRBP, resulting in the inhibition of maturation of the TRBP-bound microRNA (miRNA) and its subsequent RNA silencing activity. Gene expression profiling revealed that apoptosis regulatory genes were upregulated during SeV infection: caspases-2, -8, -3 and -7, four cysteine proteases with key roles in apoptosis, were upregulated directly or indirectly through the repression of a typical TRBP-bound miRNA, miR-106b. Our findings may shed light on the mechanism of apoptosis, induced by the TRBP-bound miRNAs through the interaction of TRBP with LGP2, as an antiviral defense system in mammalian cells.


Assuntos
MicroRNAs/genética , Coativadores de Receptor Nuclear/genética , RNA Helicases/genética , Viroses/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Caspases/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Interferência de RNA , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Viroses/virologia , Replicação Viral/genética
9.
J BUON ; 24(5): 2141-2146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31786887

RESUMO

PURPOSE: Glioblastoma is one of the prevalent types of brain tumors and is responsible for significant number of deaths world over. Glioblastoma is often diagnosed at advanced stages and there are frequent relapses following chemotherapy. Herein, we examined the anticancer effects of a secoiridoid glycoside Sweroside, against a panel of glioblastoma cells. METHODS: CCK8 assay was used to examine the anti-proliferative effects of this molecule. Αcridine orange (AO)/ethidium bromide (EB) and annexin V/propidium iodide (PI) staining assays were used to examine apoptotic cell death. Cell cycle analysis was performed by flow cytometry. The protein expression was examined by western blotting. RESULTS: Sweroside inhibited the growth of the glioblastoma U251 cell with IC50 of 10 µM. However, Sweroside had low cytotoxic effects on the normal astrocytes cells with an IC50 of 100 µM. Sweroside exerted antiproliferative effects on the U251 glioblastoma cells by apoptotic cell death. This was concomitant with upregulation of apoptotic proteins such as caspase 3 and 9, and Bax expressions. Sweroside also induced arrest of the U251 cells at the G0/G1 phase of the cell cycle. Finally, Sweroside also blocked the JNK/p38 MAPK signal pathway concentration-dependently in U251 glioblastoma cells. CONCLUSIONS: Taken together, these results suggest that Sweroside exerts potent anticancer effects on glioblastoma cells and may prove essential in the management of glioblastoma.


Assuntos
Glioblastoma/tratamento farmacológico , Glucosídeos Iridoides/farmacologia , MAP Quinase Quinase 4/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Apoptose/efeitos dos fármacos , Caspases/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
10.
Molecules ; 24(22)2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31766230

RESUMO

Numerous studies have indicated that tumor necrosis factor-alpha (TNF-α) could induce cancer cell survival and metastasis via activation of transcriptional activity of NF-κB and AP-1. Therefore, the inhibition of TNF-α-induced NF-κB and AP-1 activity has been considered in the search for drugs that could effectively treat cancer. Dicentrine, an aporphinic alkaloid, exerts anti-inflammatory and anticancer activities. Therefore, we investigated the effects of dicentrine on TNF-α-induced tumor progression in A549 lung adenocarcinoma cells. Our results demonstrated that dicentrine effectively sensitizes TNF-α-induced apoptosis in A549 cells when compared with dicentrine alone. In addition, dicentrine increases caspase-8, -9, -3, and poly (ADP-ribose) polymerase (PARP) activities by upregulating the death-inducing signaling complex and by inhibiting the expression of antiapoptotic proteins including cIAP2, cFLIP, and Bcl-XL. Furthermore, dicentrine inhibits the TNF-α-induced A549 cells invasion and migration. This inhibition is correlated with the suppression of invasive proteins in the presence of dicentrine. Moreover, dicentrine significantly blockes TNF-α-activated TAK1, p38, JNK, and Akt, leading to reduced levels of the transcriptional activity of NF-κB and AP-1. Taken together, our results suggest that dicentrine could enhance TNF-α-induced A549 cell death by inducing apoptosis and reducing cell invasion due to, at least in part, the suppression of TAK-1, MAPK, Akt, AP-1, and NF-κB signaling pathways.


Assuntos
Apoptose , Aporfinas/farmacologia , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aporfinas/química , Biomarcadores , Caspases/genética , Caspases/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
11.
Glycoconj J ; 36(6): 473-485, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31758295

RESUMO

The emergence of multi drug resistance in non-small cell lung cancer (NSCLC) patients is a major challenge towards the efficacy of chemotherapy. Thus, there is an urgent need for the newer, better clinically targeted strategies to treat this disease. Earlier studies from our laboratory revealed the apoptotic activity of Maackia amurensis agglutinin (MAA) in human NSCLC cells. In this study, the effect of MAA on drug resistant NSCLC cells was investigated. Two Paclitaxel-resistant NSCLC sub-lines (A549/PTX100 and NCI-H460/PTX100) were developed from A549 & NCI-H460 cell lines respectively. The generation of drug resistance phenotype was confirmed by the expression of cell surface MDR-1. Both the drug resistant sub-lines showed distinct morphological alterations. MAA interacted with the cell-surface protein(s) of apparent Mr ~66 kDa and induced apoptosis in both the sub-lines through intrinsic/mitochondrial pathway, involving reduction in mitochondrial trans-membrane potential, up-regulation of Bax, unaltered/decreased expression of Bcl-XL, release of mitochondrial cytochrome c into the cytosol and activation of pro-caspases (-9&-3). Our findings highlighted the potential of this plant agglutinin to serve as an apoptosis inducing agent in drug resistant NSCLC cells.


Assuntos
Aglutininas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Maackia/química , Células A549 , Aglutininas/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspases/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína bcl-X/genética
12.
Int J Mol Sci ; 20(22)2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31752432

RESUMO

Vaspin, a visceral adipose tissue-derived serine protease inhibitor, is expressed in the porcine ovary; it induces the activation of various kinases and steroidogenesis. The aim of this study was to examine the effect of vaspin on granulosa (Gc) proliferation, cell cycle regulation, and apoptosis. Porcine Gc was incubated with vaspin (0.01-10 ng/mL) for 24 to 72 h, proliferation was measured using alamarBlue assay, cell cycle progression was assessed using flow cytometry, and cyclin (D, E, and A) protein expression was measured using immunoblotting. Apoptosis was assessed by measuring caspase activity using Caspase-glo 3/7 assay. Furthermore, histone-associated DNA fragments levels were measured using a cell-death detection ELISA; BAX (bcl-2-like protein 4), BCL2 (B-cell lymphoma 2), caspases (-3, -8, and -9), p53 mRNA, and protein expression were assessed using real time PCR and immunoblotting. We found that vaspin significantly enhanced Gc proliferation and cell cycle progression into the S and G2/M phases and decreased apoptosis. We observed that siRNA silencing of the glucose-regulated protein (GRP78) receptor and pharmacological inhibitors of mitogen-activated kinase (MAP3/1/ERK1/2), Janus kinase (STAT3) and protein kinase B (AKT) blocked the ability of vaspin cell proliferation and enhanced caspase-3/7 activities. These results suggest that vaspin via mitogenic effect on porcine Gc acts as a new regulator of ovarian growth, development, or folliculogenesis.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células da Granulosa/fisiologia , Serpinas/genética , Transdução de Sinais/genética , Animais , Caspases/genética , Feminino , Proteínas de Choque Térmico/genética , Humanos , MAP Quinase Quinase Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Transcrição STAT3/genética , Suínos
13.
Int J Med Sci ; 16(11): 1412-1423, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31673231

RESUMO

Resistance against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death of cancer cells is a major obstacle in clinical application of TRAIL. Variable response to TRAIL of gastric cancer cells, synergy of TRAIL with bortezomib and potential mechanisms behind the phenomena were investigated in this study. The response to TRAIL varied among six gastric cancer cell lines, which correlated with the expression of apoptotic TRAIL receptors. Analysis of TCGA gene expression data showed that DR4 expression correlated with DR5 in gastric cancer. Although higher expression of DR4 was significantly associated with lower T, N and TNM stages, neither DR4 nor DR5 expression meaningfully influenced overall survival rate. Combined treatment of TRAIL with bortezomib resulted in strong synergistic response with enhanced activation of caspases-8, -9 and -3, and increased Annexin V-binding cell fractions in TRAIL-resistant SNU-216 cells. Bortezomib increased the expression of p21cip1/waf1, but p21cip1/waf1 silencing did not restore cell viability significantly. Bortezomib also increased DR5 expression and knockdown of DR5 expression significantly recovered cell viability reduced by the combination treatment. Bortezomib decreased phosphorylation of ERK1/2, but increased that of JNK. Treatment with either an ERK1/2 inhibitor U0126 or a JNK inhibitor SP600125 rescued SNU-216 from dying of bortezomib or combined treatment. However, upregulation of DR5 by bortezomib was knocked down only by inhibition of ERK1/2 activation significantly, but not by JNK activity inhibition. In summary, upregulation of DR5 by bortezomib is of critical significance in the synergy of bortezomib with TRAIL in apoptosis of TRAIL-resistant SNU-216 and that activity of ERK1/2 is required in the bortezomib-induced DR5 overexpression.


Assuntos
Bortezomib/administração & dosagem , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Neoplasias Gástricas/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/genética , Idoso , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Butadienos/farmacologia , Caspases/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Nitrilos/farmacologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Quinases Ativadas por p21/genética
14.
J BUON ; 24(4): 1532-1537, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31646804

RESUMO

PURPOSE: In this study, the anticancer effects of a natural flavonoid-Tangeretin, were examined against the drug-resistant MDA-MB-231 breast cancer (BC) cell line and the normal breast cell line Hs 841.T. METHODS: The MTT assay was employed for cell viability determination. Apoptosis was demonstrated by DAPI and Annexin V/propidium iodide (PI) staining. Flow cytometric analyses were performed to gain insights about cell cycle distribution. Western blot assay was used for protein expression determination. RESULTS: Tangeretin inhibited the growth of the drug-resistant MDA-MB-231 cells concentration-dependently and its IC50 was 9 µM, whereas the IC50 was >100 µM against the normal cells. The anti-proliferative effects were due to induction of apoptotic cell death. The apoptotic cell percentage was increased from 5.7% to around 69% as the concentration of Tangeretin was increased. Tangeretin also caused an increase in the Bax/Bcl-2 ratio and activation of the Caspase 3, 8 and 9. In addition, Tangeretin led to arrest of the cells at G2/M phase which was accompanied by depletion of cyclin B1 and D. Transwell assay showed that Tangeretin also reduced the invasion of the MDA-MB-231 cells. CONCLUSION: The findings of this study suggest that Tangeretin exerts potent anticancer effects on the MDA-MB-231 cells and may therefore prove a beneficial lead molecule in BC research.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Flavonas/farmacologia , Invasividade Neoplásica/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/genética , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética
15.
Biomed Res Int ; 2019: 9071297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31637258

RESUMO

TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax-members of the Bcl-2 family-are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK-/- cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak-/- cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.


Assuntos
Apoptose/genética , Caspases/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Citosol/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia
16.
Bioengineered ; 10(1): 501-512, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31633448

RESUMO

The extract of Phyllodium (P.) elegans was investigated for its anti-cancer properties on brain astroglioma cells (U251-MG), colorectal carcinoma cells (HCT116), and malignant melanoma cells (A375). P. elegans methanolic extract (PeME) showed cytotoxicity on all three cancer cell lines tested. The cell viability assay revealed that PeME significantly reduced the viability of these cells. Clear apoptotic features such as cellular morphology, cell shrinkage, and augmentation of dead cells were observed. Flow cytometry and fluorescence staining techniques confirmed the apoptotic property of PeME. In vitro scratch invasion assay showed that cell migration rate was significantly reduced. Fluorescence microscopic studies using 4',6-diamidino-2-phenylindole staining showed early and late signs of apoptosis after PeME treatment. Upon PeME stimulation, activation of caspase-3/-9 and Mu-2-related death-inducing gene (MUDENG, MuD) was observed by western blot analysis. JC-1 staining analysis by flow cytometry showed that PeME depolarized the mitochondria membrane potential (MMP). Collectively, these findings, for the first time, point to the fact that PeME has anti-cancer properties against brain, colon, and skin cancer cell lines by depolarizing the MMP and activating apoptotic signaling through the activation of caspase-3/-9 as well as MuD. This is the first report reporting the anticancer activity of this specific plant extract.[Figure: see text].


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fabaceae/química , Extratos Vegetais/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487808

RESUMO

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.


Assuntos
Bombyx/genética , Resistência à Doença/genética , Proteínas de Insetos/genética , Animais , Apoptose , Bombyx/imunologia , Bombyx/virologia , Caspases/genética , Caspases/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Insetos/metabolismo , Nucleopoliedrovírus/patogenicidade
18.
Nat Commun ; 10(1): 4044, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492850

RESUMO

Acute graft-versus-host disease (GVHD) remains a major obstacle for the wider usage of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is an effective therapy for hematopoietic malignancy. Here we show that caspase-11, the cytosolic receptor for bacterial endotoxin (lipopolysaccharide: LPS), enhances GVHD severity. Allo-HSCT markedly increases the LPS-caspase-11 interaction, leading to the cleavage of gasdermin D (GSDMD). Caspase-11 and GSDMD mediate the release of interleukin-1α (IL-1α) in allo-HSCT. Deletion of Caspase-11 or Gsdmd, inhibition of LPS-caspase-11 interaction, or neutralizing IL-1α uniformly reduces intestinal inflammation, tissue damage, donor T cell expansion and mortality in allo-HSCT. Importantly, Caspase-11 deficiency does not decrease the graft-versus-leukemia (GVL) activity, which is essential to prevent cancer relapse. These findings have major implications for allo-HSCT, as pharmacological interference with the caspase-11 signaling might reduce GVHD while preserving GVL activity.


Assuntos
Caspases/genética , Doença Enxerto-Hospedeiro/genética , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Transdução de Sinais/genética , Animais , Caspases/metabolismo , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/metabolismo , Humanos , Interleucina-1alfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , Análise de Sobrevida , Transplante Homólogo
19.
Proc Natl Acad Sci U S A ; 116(41): 20539-20544, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548372

RESUMO

Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). In this study, we examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. We found that the Drosophila executioner caspases Dcp-1 and Decay, but not Drice, promoted wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, we found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, our results likely highlight the original function of caspase-not cell death, but basal proteolytic cleavages for cell vigor.


Assuntos
Caspases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Discos Imaginais/crescimento & desenvolvimento , Asas de Animais/crescimento & desenvolvimento , Animais , Apoptose , Caspases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Discos Imaginais/metabolismo , Asas de Animais/metabolismo
20.
Molecules ; 24(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505816

RESUMO

Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA's increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA's apoptotic effects on human osteosarcoma cells.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Proliferação de Células/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Piperidonas/farmacologia , Caspases/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
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