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1.
Clin Exp Immunol ; 216(3): 272-279, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38457368

RESUMO

Macrophage activation syndrome (MAS) is a life-threatening condition, characterized by cytopenia, multi-organ dysfunction, and coagulopathy associated with excessive activation of macrophages. In this study, we investigated the roles of alpha2-antiplasmin (α2AP) in the progression of MAS using fulminant MAS mouse model induced by toll-like receptor-9 agonist (CpG) and D-(+)-galactosamine hydrochloride (DG). α2AP deficiency attenuated macrophage accumulation, liver injury, and fibrin deposition in the MAS model mice. Interferon-γ (IFN-γ) is associated with macrophage activation, including migration, and plays a pivotal role in MAS progression. α2AP enhanced the IFN-γ-induced migration, and tissue factor production. Additionally, we showed that fibrin-induced macrophage activation and tumor necrosis factor-α production. Moreover, the blockade of α2AP by neutralizing antibodies attenuated macrophage accumulation, liver injury, and fibrin deposition in the MAS model mice. These data suggest that α2AP may regulate IFN-γ-induced responses and be associated with macrophage activation and fibrin deposition in the MAS progression.


Assuntos
Modelos Animais de Doenças , Fibrina , Interferon gama , Síndrome de Ativação Macrofágica , Ativação de Macrófagos , Macrófagos , alfa 2-Antiplasmina , Animais , Fibrina/metabolismo , Camundongos , alfa 2-Antiplasmina/metabolismo , Ativação de Macrófagos/imunologia , Síndrome de Ativação Macrofágica/imunologia , Interferon gama/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Knockout , Fator de Necrose Tumoral alfa/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Galactosamina
2.
Mol Med Rep ; 28(4)2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37594053

RESUMO

Previously, using three types of cationic lipids, the effect of phospholipids in liposomal formulations on gene-knockdown efficacy was determined after in vitro and in vivo transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) containing various cationic lipids and phospholipids. In the present study, six other types of cationic lipids, namely N,N-dimethyl-N-tetradecyltetradecan-1-aminium bromide, N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 2-[bis{2-(tetradecanoyloxy)ethyl}amino]-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-6-14), 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP) and 1,2-dioleoyl-3-dimethylammonium-propane were selected, and the effect of phospholipids in liposomal formulations containing each cationic lipid on gene-knockdown was evaluated. A total of 30 types of cationic liposomes composed of each cationic lipid with phosphatidylethanolamine containing unsaturated or saturated diacyl chains (C14, C16 or C18) were prepared. Regardless of the type of cationic lipid, the inclusion of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the liposomal formulations resulted in injectable size of siRNA lipoplexes after mixing of siRNA and cationic liposomes. Transfection of their lipoplexes with luciferase (Luc) siRNA into human breast cancer MCF-7-Luc cells stably expressing Luc led to a strong knockdown of Luc. Furthermore, the systemic injection of siRNA lipoplexes composed of DC-1-16, DC-6-14, DOTMA or DSTAP with DOPE resulted in siRNA accumulation in the lungs. Significant gene-knockdown was observed in the lungs of mice following the systemic injection of siRNA lipoplexes containing DC-1-16 and DOPE. Cationic liposomes composed of DC-1-16 and DOPE serve as potential carriers for in vitro and in vivo siRNA transfection.


Assuntos
Neoplasias Mamárias Animais , Fosfolipídeos , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , Lipossomos , Brometos , Cloretos , Propano , Cátions
3.
Mol Med Rep ; 26(2)2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35686555

RESUMO

Formulation of cationic liposomes is a key factor that determine the gene knockdown efficiency by cationic liposomes/siRNA complexes (siRNA lipoplexes). Here, to determine the optimal combination of cationic lipid and phospholipid in cationic liposomes for in vitro and in vivo gene knockdown using siRNA lipoplexes, three types of cationic lipid were used, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB) and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12). Thereafter, 30 types of cationic liposome composed of each cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (C14, C16, or C18) were prepared. The inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains with DOTAP- or DDAB-based cationic liposomes induced strong luciferase gene knockdown in human breast cancer MCF-7-Luc cells stably expressing luciferase gene. Furthermore, the inclusion of phosphatidylcholine or phosphatidylethanolamine containing saturated and short dialkyl chains or unsaturated and long dialkyl chains into TC-1-12-based cationic liposomes resulted in high gene knockdown efficacy. When cationic liposomes composed of DDAB/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), TC-1-12/DOPE and TC-1-12/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine were used, significant gene knockdown occurred in the lungs of mice following systemic injection of siRNA lipoplexes. Overall, the present findings indicated that optimal phospholipids in cationic liposome for in vitro and in vivo siRNA transfection were affected by the types of cationic lipid used.


Assuntos
Técnicas de Silenciamento de Genes , Lipossomos , Animais , Cátions , Feminino , Humanos , Luciferases , Pulmão , Células MCF-7 , Camundongos , Fosfatidilcolinas , Fosfatidiletanolaminas , Fosfolipídeos , RNA Interferente Pequeno/genética , Transfecção
4.
Mol Biol Rep ; 49(1): 205-215, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34709571

RESUMO

BACKGROUND: Diabetic nephropathy (DN), is microvascular complication of diabetes causes to kidney dysfunction and renal fibrosis. It is known that hyperglycemia and advanced glycation end products (AGEs) produced by hyperglycemic condition induce myofibroblast differentiation and endothelial-to-mesenchymal transition (EndoMT), and exacerbate fibrosis in DN. Recently, we demonstrated that α2-antiplasmin (α2AP) is associated with inflammatory response and fibrosis progression. METHODS: We investigated the role of α2AP on fibrosis progression in DN using a streptozotocin-induced DN mouse model. RESULTS: α2AP deficiency attenuated EndoMT and fibrosis progression in DN model mice. We also showed that the high glucose condition/AGEs induced α2AP production in fibroblasts (FBs), and the reduction of receptor for AGEs (RAGE) by siRNA attenuated the AGEs-induced α2AP production in FBs. Furthermore, the bloackade of α2AP by the neutralizing antibody attenuated the high glucose condition-induced pro-fibrotic changes in FBs. On the other hand, the hyperglycemic condition/AGEs induced EndoMT in vascular endothelial cells (ECs), the FBs/ECs co-culture promoted the high glucose condition-induced EndoMT compared to ECs mono-culture. Furthermore, α2AP promoted the AGEs-induced EndoMT, and the blockade of α2AP attenuated the FBs/ECs co-culture-promoted EndoMT under the high glucose condition. CONCLUSIONS: The high glucose conditions induced α2AP production, and α2AP is associated with EndoMT and fibrosis progression in DN. These findings provide a basis for clinical strategies to improve DN.


Assuntos
Anticorpos Neutralizantes/farmacologia , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , alfa 2-Antiplasmina/genética , Animais , Linhagem Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Células NIH 3T3
5.
J Basic Clin Physiol Pharmacol ; 33(5): 633-638, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34913624

RESUMO

OBJECTIVES: Depression is a psychiatric disorder that affects about 10% of the world's population and is accompanied by anxiety. Depression and anxiety are often caused by various stresses. However, the etiology of depression and anxiety remains unknown. It has been reported that alpha2-antiplasmin (α2AP) not only inhibits plasmin but also has various functions such as cytokine production and cell growth. This study aimed to determine the roles of α2AP on the stress-induced depression and anxiety. METHODS: We investigated the mild repeated restraint stress-induced depressive and anxiety-like behavior in the α2AP+/+ and α2AP-/- mice using the social interaction test (SIT), sucrose preference test (SPT), and elevated plus maze (EPM). RESULTS: The stresses such as the mild repeated restraint stress suppressed α2AP expression in the hippocampus of mice, and the treatment of fluoxetine (selective serotonin reuptake inhibitor [SSRI]) recovered the stress-caused α2AP suppression. We also showed that α2AP deficiency promoted the mild restraint stress-stimulated depression-like behavior such as social withdrawal and apathy and apoptosis in mice. In contrast, α2AP deficiency attenuated the mild restraint stress induced the anxiety-like behavior in mice. CONCLUSIONS: α2AP affects the pathogenesis of depression and anxiety induced by stress.


Assuntos
Ansiedade/metabolismo , Depressão/metabolismo , alfa 2-Antiplasmina/metabolismo , Animais , Ansiedade/patologia , Apoptose , Comportamento Animal , Citocinas , Depressão/patologia , Fibrinolisina , Fluoxetina/administração & dosagem , Humanos , Camundongos , Inibidores Seletivos de Recaptação de Serotonina , alfa 2-Antiplasmina/deficiência
6.
Mol Med Rep ; 24(2)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34165169

RESUMO

Cationic liposomes can be intravenously injected to deliver short interfering (si)RNAs into the lungs. The present study investigated the effects of sterol derivatives in systemically injected siRNA/cationic liposome complexes (siRNA lipoplexes) on gene­knockdown in the lungs of mice. Cationic liposomes composed of 1,2­dioleoyl­3­trimethylammonium­propane or dimethyldioctadecylammonium bromide (DDAB) were prepared as a cationic lipid, with sterol derivatives such as cholesterol (Chol), ß­sitosterol, ergosterol (Ergo) or stigmasterol as a neutral helper lipid. Transfected liposomal formulations composed of DDAB/Chol or DDAB/Ergo did not suppress the expression of the luciferase gene in LLC­Luc and Colon 26­Luc cells in vitro, whereas other formulations induced moderate gene­silencing. The systemic injection of siRNA lipoplexes formulated with Chol or Ergo into mice resulted in abundant siRNA accumulation in the lungs. In comparison, systemically injected DDAB/Chol or DDAB/Ergo lipoplexes of Tie2 siRNA effectively increased the suppression of the Tie2 mRNA expression in the lungs of mice. These findings indicated that DDAB/Chol and DDAB/Ergo liposomes could function as vectors for siRNA delivery to the lungs.


Assuntos
Cátions/farmacologia , Técnicas de Silenciamento de Genes/métodos , Lipossomos/farmacologia , Pulmão , RNA Interferente Pequeno/administração & dosagem , Esteróis/química , Esteróis/farmacologia , Distribuição Tecidual/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Colesterol/análogos & derivados , Sistemas de Liberação de Medicamentos , Feminino , Inativação Gênica , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Amônio Quaternário , Transfecção
7.
Mol Biol Rep ; 48(4): 3431-3437, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33913094

RESUMO

Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that α2-antiplasmin (α2AP) is elevated in SSc dermal fibroblasts and SSc model mice, and is associated with fibrosis progression and vascular dysfunction. In the present study, we predicted that α2AP could be a target of microRNA-30c (miR-30c) using TargetScan online database, and investigated the effect of miR-30c on the pathogenesis of SSc using a bleomycin-induced SSc model mice. miR-30c attenuated α2AP expression, and prevented the pro-fibrotic changes (increased dermal thickness, collagen deposition, myofibroblast accmulation) and the vascular dysfunction (the reduction of vascular endothelial cells (ECs) and blood flow) in the skin of SSc model mice. Furthermore, miR-30c suppressed pulmonary fibrosis progression in the SSc model mice. miR-30c exerts the anti-fibrotic and anti-angiopathy effects on SSc model mice, and might provide a basis for clinical strategies for SSc.


Assuntos
Escleroderma Sistêmico/metabolismo , Pele/irrigação sanguínea , alfa 2-Antiplasmina/genética , Animais , Bleomicina/toxicidade , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miofibroblastos , Escleroderma Sistêmico/genética , Pele/efeitos dos fármacos , Pele/metabolismo
8.
Mol Med Rep ; 22(5): 4183-4196, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33000194

RESUMO

Polyethylene glycol (PEG)­modifications (PEGylations) of cationic liposome/small interfering RNA complexes (siRNA lipoplexes) can enhance their systemic stability. The present study determined the effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene­silencing effects and siRNA biodistribution after intravenous injection. Three types of dialkyl or trialkyl cationic lipids were used in the current study for the preparation of cationic liposomes. Additionally, various PEGylated siRNA lipoplexes that contained PEG­1,2­distearoyl­sn­-glycero­-3­phosphoethanolamine (DSPE), PEG­1,2­distearoyl­rac­glycero­3­-methylpolyoxyethylene (DSG), PEG­cholesterol (PEG­Chol) and PEG­chondroitin sulfate conjugate (PEG­CS) were prepared. The results revealed that PEGylation of siRNA lipoplexes with PEG­DSPE strongly decreased gene­silencing effects in cells. In contrast, those with PEG­DSG, PEG­Chol and PEG­CS did not largely decrease gene-silencing effects. However, regardless of the PEG­derivative type, PEGylation of siRNA lipoplexes decreased their agglutination with erythrocytes. Furthermore, intravenous injection of PEGylated siRNA lipoplexes markedly decreased the accumulation of siRNA in the lungs, regardless of the type of PEG­derivative. However, non­PEGylated siRNA lipoplexes accumulated mainly in the lungs regardless of the siRNA lipoplex cationic lipid type. The results indicated that PEGylation of siRNA lipoplexes with PEG­DSG, PEG­Chol and PEG­CS may improve systemic stability without losing transfection activity by PEGylation.


Assuntos
Pulmão/química , Polietilenoglicóis/química , RNA Interferente Pequeno/farmacocinética , Administração Intravenosa , Animais , Colesterol/análogos & derivados , Colesterol/química , Sulfatos de Condroitina/química , Feminino , Inativação Gênica , Humanos , Lipossomos , Células MCF-7 , Camundongos , Fosfatidiletanolaminas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Distribuição Tecidual
9.
Pharmaceutics ; 11(4)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991703

RESUMO

In this study, we examined the effect of cationic lipid type in folate (FA)-polyethylene glycol (PEG)-modified cationic liposomes on gene-silencing effects in tumor cells using cationic liposomes/siRNA complexes (siRNA lipoplexes). We used three types of cationic cholesterol derivatives, cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), and cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol), and we prepared three types of FA-PEG-modified siRNA lipoplexes. The modification of cationic liposomes with 1-2 mol % PEG-lipid abolished the gene-silencing effect in human nasopharyngeal tumor KB cells, which overexpress the FA receptor (FR). In contrast, FA-PEG-modification of cationic liposomes restored gene-silencing activity regardless of the cationic lipid type in cationic liposomes. However, the optimal amount of PEG-lipid and FA-PEG-lipid in cationic liposomes for selective gene silencing and cellular uptake were different among the three types of cationic liposomes. Furthermore, in vitro transfection of polo-like kinase 1 (PLK1) siRNA by FA-PEG-modified liposomes exhibited strong cytotoxicity in KB cells, compared with PEG-modified liposomes; however, in in vivo therapy, intratumoral injection of PEG-modified PLK1 siRNA lipoplexes inhibited tumor growth of KB xenografts, as well as that of FA-PEG-modified PLK1 siRNA lipoplexes. From these results, the optimal formulation of PEG- and FA-PEG-modified liposomes for FR-selective gene silencing might be different between in vitro and in vivo transfection.

10.
Curr Pharm Biotechnol ; 20(1): 47-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30727885

RESUMO

BACKGROUND: The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. METHOD: Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. RESULTS AND CONCLUSION: Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.


Assuntos
Interleucina-18/metabolismo , Linfócitos Intraepiteliais/metabolismo , Leucócitos Mononucleares/metabolismo , Sequência de Aminoácidos , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células HEK293 , Humanos , Interleucina-18/genética , Interleucina-18/imunologia , Linfócitos Intraepiteliais/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
11.
J Drug Target ; 27(2): 217-227, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30024300

RESUMO

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.


Assuntos
Lipídeos/química , Pulmão/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Animais , Cátions , Sistemas de Liberação de Medicamentos , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Lipossomos , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , RNA Mensageiro , Receptor TIE-2/genética , Receptor TIE-2/metabolismo
12.
Curr Pharm Biotechnol ; 19(3): 224-231, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29667554

RESUMO

BACKGROUND: Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. METHODS: We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells. RESULTS: The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library. CONCLUSION: We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.


Assuntos
Fosfatase Alcalina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fosfatase Alcalina/genética , Produtos Biológicos/farmacologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon beta/farmacologia , Lipopolissacarídeos/farmacologia , NF-kappa B/genética , NF-kappa B/metabolismo
13.
Oncol Lett ; 14(5): 5157-5166, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29098022

RESUMO

It has been reported that suppression of protein kinase N3 (PKN3) expression in vascular and lymphatic endothelial cells results in the inhibition of tumor progression and lymph node metastasis formation. The present study investigated whether combination therapy of small interfering RNA (siRNA) against PKN3 and doxorubicin (DXR) could increase therapeutic efficacy against liver and lung metastases. In vitro transfection of PKN3 siRNA into PKN3-positive MDA-MB-231, LLC, and Colon 26 cells and PKN3-negative MCF-7 cells did not inhibit cell growth and did not increase sensitivity to DXR. However, following in vivo treatment, PKN3 siRNA suppressed the growth of liver MDA-MB-231 and lung LLC and MCF-7 metastases, although combination therapy with DXR did not increase the therapeutic efficacy. By contrast, in liver MCF-7 metastases, PKN3 siRNA or DXR alone did not exhibit significant inhibition of tumor growth, but their combination significantly improved therapeutic efficacy. Treatment of liver MDA-MB-231 metastases with PKN3 siRNA induced a change in vasculature structure via suppression of PKN3 mRNA expression. PKN3 siRNA may induce antitumor effects in lung and liver metastases by suppression of PKN3 expression in stroma cells, such as endothelial cells. From these findings, PKN3 siRNA alone or in combination with DXR may reduce the tumor growth of liver and lung metastases regardless of PKN3 expression in tumor cells.

14.
Exp Ther Med ; 14(5): 4300-4306, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29067111

RESUMO

Mitotic progression is regulated by the phosphorylation of heat shock transcription factor 1 (HSF1) by polo-like kinase 1 (PLK1); however, this interaction is often deregulated in tumors. High expression levels of PLK1 and HSF1 have been observed in various types of human cancer. In the present study, it was investigated whether small interfering (si)RNA against PLK1 or HSF1 could suppress tumor growth in vitro and in vivo. In vitro transfection of PLK1 and HSF1 siRNA into PKL1- and HSF1-positive human breast tumor MDA-MB-231 and human cervical carcinoma HeLa cells inhibited cell growth via suppression of PLK1 and HSF1 mRNA expression, respectively. However, the transfection of PLK1 or HSF1 siRNA did not significantly affect the cytotoxicity of doxorubicin in HeLa cells. Furthermore, injection of PKL1 or HSF1 siRNA into mice with liver HeLa metastasis suppressed tumor growth. From these findings, PLK1 and HSF1 may be considered to be promising targets for antitumor therapy.

15.
J Cell Physiol ; 231(12): 2761-7, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26989816

RESUMO

The intracellular cysteine protease caspase-1 is critically involved in obesity-induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase-1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD-like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase-1 itself. However, it is not fully understood how caspase-1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF-α-induced activation of caspase-1 is accompanied by robust induction of NLRP3 in 3T3-L1 adipocytes but that caspase-1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3-L1 cells with TNF-α induced mRNA expression and activation of caspase-1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF-α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase-1 induced by TNF-α, suggesting that NLRP3 is dispensable for TNF-α-induced caspase-1 activation. Moreover, we did not detect the basal and TNF-α-induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase-1 activation. These results suggest that TNF-α induces caspase-1 activation in an inflammasome-independent manner in 3T3-L1 cells and that the ERK-dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761-2767, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Caspase 1/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3-L1 , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Am J Physiol Endocrinol Metab ; 310(8): E643-E651, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26860984

RESUMO

Extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance associated with obesity and type 2 diabetes mellitus. We have now examined the potential of pharmacological targeting of the ERK pathway with MEK (ERK kinase) inhibitors (PD184352 and PD0325901) for the treatment of obesity-associated insulin resistance. The effects of PD184352 and PD0325901 on the expression of adipocytokines and lipolysis activity were thus examined in 3T3-L1 adipocytes maintained in long-term culture as a model of adipocyte hypertrophy. Leptin receptor-deficient (db/db) mice and high-fat diet-fed KKAy mice, both of which are models of type 2 diabetes, were also treated orally with PD184352 to examine its effects on the diabetic condition. ERK activity was increased in hypertrophic 3T3-L1 adipocytes as well as in adipose tissue of db/db mice and high-fat diet-fed KKAy mice, and this enhanced ERK signaling was associated with dysregulation of adipocytokine expression and increased lipolysis activity. Specific blockade of the ERK pathway in hypertrophic 3T3-L1 adipocytes by MEK inhibitors ameliorated the dysregulation of adipocytokine expression and suppressed the enhanced lipolysis activity. Furthermore, repeated oral administration of PD184352 normalized hyperglycemia and hyperlipidemia and improved insulin sensitivity and glucose tolerance in the diabetic mice. These results suggest that sustained activation of the ERK pathway in adipocytes is associated with the pathogenesis of type 2 diabetes and that selective blockade of this pathway with MEK inhibitors warrants further study as a promising approach to the treatment of insulin resistance and type 2 diabetes.


Assuntos
Adipócitos/efeitos dos fármacos , Benzamidas/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Difenilamina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Resistência à Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipocinas/metabolismo , Adiponectina/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Dieta Hiperlipídica , Difenilamina/farmacologia , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Teste de Tolerância a Glucose , Hiperlipidemias/metabolismo , Immunoblotting , Técnicas In Vitro , Insulina/metabolismo , Interleucina-6/metabolismo , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores para Leptina/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
17.
J Drug Target ; 24(4): 309-317, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26234899

RESUMO

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/siRNA complex (cationic lipoplex). In this study, we investigated whether siRNA delivered by this sequential injection could significantly suppress mRNA expression of the targeted gene in liver metastasis and inhibit tumor growth. When cationic lipoplex was intravenously injected into mice bearing liver metastasis of human breast tumor MCF-7 at 1 min after intravenous injection of chondroitin sulfate C (CS) or poly-l-glutamic acid (PGA), siRNA was accumulated in tumor-metastasized liver. In terms of a gene silencing effect, sequential injections of CS or PGA plus cationic lipoplex of luciferase siRNA could reduce luciferase activity in liver MCF-7-Luc metastasis. Regarding the side effects, sequential injections of CS plus cationic lipoplex did not exhibit hepatic damage or induction of inflammatory cytokines in serum after repeated injections, but sequential injections of PGA plus cationic lipoplex did. Finally, sequential injections of CS plus cationic lipoplex of protein kinase N3 siRNA could suppress tumor growth in the mice bearing liver metastasis. From these findings, sequential injection of CS and cationic lipoplex of siRNA might be a novel systemic method of delivering siRNA to liver metastasis.


Assuntos
Portadores de Fármacos/química , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/secundário , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Animais , Ânions , Cátions , Colesterol/química , Sulfatos de Condroitina/química , Ácidos Graxos Monoinsaturados/química , Inativação Gênica , Humanos , Injeções Intravenosas , Lipossomos , Neoplasias Hepáticas Experimentais/genética , Luciferases de Vaga-Lume/genética , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Ácido Poliglutâmico/química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Int J Oncol ; 47(1): 211-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25955490

RESUMO

Previously, we found that the injection of zoledronic acid (ZOL) into mice bearing tumor induced changes of the vascular structure in the tumor. In this study, we examined whether ZOL treatment could decrease interstitial fluid pressure (IFP) via change of tumor vasculature, and enhance the antitumor efficacy of liposomal doxorubicin (Doxil®). When ZOL solution was injected at 40 µg/mouse per day for three consecutive days into mice bearing murine Lewis lung carcinoma LLC tumor, depletion of macrophages in tumor tissue and decreased density of tumor vasculature were observed. Furthermore, ZOL treatments induced inflammatory cytokines such as interleukin (IL)-10 and -12, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α in serum of LLC tumor-bearing mice, but not in normal mice, indicating that ZOL treatments might induce an inflammatory response in tumor tissue. Furthermore, ZOL treatments increased antitumor activity by Doxil in mice bearing a subcutaneous LLC tumor, although they did not significantly increase the tumor accumulation of doxorubicin (DXR). These results suggest that ZOL treatments might increase the therapeutic efficacy of Doxil via improvement of DXR distribution in a tumor by changing the tumor vasculature. ZOL treatment can be an alternative approach to increase the antitumor effect of liposomal drugs.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Difosfonatos/administração & dosagem , Doxorrubicina/análogos & derivados , Líquido Extracelular/efeitos dos fármacos , Imidazóis/administração & dosagem , Animais , Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/genética , Difosfonatos/farmacologia , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Ácido Zoledrônico
19.
Biochem Biophys Res Commun ; 433(4): 456-62, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23501104

RESUMO

The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.


Assuntos
Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Sistema de Sinalização das MAP Quinases , Piridinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Benzimidazóis/farmacologia , Sinergismo Farmacológico , Feminino , Células HT29 , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Estresse Oxidativo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Int J Mol Sci ; 14(3): 4596-612, 2013 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-23443160

RESUMO

It has generally been considered that protein phosphatases have more diverse catalytic domain structures and mechanisms than protein kinases; however, gene annotation efforts following the human genome project appeared to have completed the whole array of protein phosphatases. Ser/Thr phosphatases are divided into three subfamilies that have different structures from each other, whereas Tyr phosphatases and dual-specificity phosphatases targeting Tyr, Ser and Thr belong to a single large family based on their common structural features. Several years of research have revealed, however, the existence of unexpected proteins, designated here as "atypical protein phosphatases", that have structural and enzymatic features different from those of the known protein phosphatases and are involved in important biological processes. In this review, we focus on the identification and functional characterization of atypical protein phosphatases, represented by eyes absent (EYA), suppressor of T-cell receptor signaling (Sts) and phosphoglycerate mutase family member 5 (PGAM5) and discuss their biological significance in cellular signaling.

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