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1.
Biol Pharm Bull ; 47(9): 1494-1503, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39261049

RESUMO

Cancer chemotherapy increases the risk of thrombosis; however, the mechanisms underlying this thrombosis are not completely understood. Plasminogen activator inhibitor (PAI)-1 is a key molecule in the fibrinolytic system that inhibits tissue plasminogen activator and urokinase, which converts plasminogen into plasmin; therefore, excess PAI-1 increases the risk of thrombosis. In this study, we investigated whether temporary treatment of the human luminal A-type breast cancer cell line MCF-7 with antitumor drugs clinically used for breast cancer therapy promotes PAI-1 production. Treatment of MCF-7 cells with paclitaxel (PTX), a microtubule-stabilizing antitumor drug, at 1 µM for 2 h elevated the PAI-1 concentration of the conditioned medium at 48 h after treatment but not in those treated with tamoxifen and cyclophosphamide. Microtubule assembly inhibitors vinblastine (VBT) and vincristine (VCT) also increased the PAI-1 concentration in the conditioned medium. PAI-1 (SERPINE1) expression was upregulated in MCF-7 cells after PTX, VBT, and VCT treatment; this increase in expression persisted for eight days. In contrast, PAI-1 production in MDA-MB-231 cells treated with PTX, VBT, or VCT did not increase with increasing PAI-1 concentration. This study demonstrated that temporary low-dose treatment with microtubule-associated anticancer drugs increased PAI-1 release from MCF-7 cells but not from MDA-MB-231 cells. These results indicate that chemotherapy against luminal A-type breast cancer using microtubule-associated drugs may cause thrombosis through the inhibition of the fibrinolytic system by PAI-1.


Assuntos
Neoplasias da Mama , Paclitaxel , Inibidor 1 de Ativador de Plasminogênio , Vimblastina , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Paclitaxel/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vimblastina/farmacologia , Células MCF-7 , Feminino , Linhagem Celular Tumoral , Antineoplásicos Fitogênicos/farmacologia , Vincristina/farmacologia
2.
J Med Food ; 26(11): 843-848, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37862040

RESUMO

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine and health supplement in Japan. Ashitaba yellow stem exudate (AYE) contains abundant chalcones and thus has the potential to treat and prevent many pathological states such as cancer, inflammation, obesity, diabetics, thrombosis, and hypertension. Levels of plasminogen activator inhibitor 1 (PAI-1), a key regulator of the fibrinolytic system, increase with age in mouse plasma. Therefore, we aimed to determine the effects of AYE on plasma thrombotic parameters in aging mice. Long-term (52 weeks) AYE supplementation significantly decreased age-induced increases of PAI-1 in mouse plasma. Supplementation with AYE decreased levels of the acute-phase and fibrinolytic protein plasma plasminogen, and significantly decreased those of tumor necrosis factor α. These results suggested that continuous intake of AYE throughout life decreases age-induced systemic inflammation and prevents thrombotic tendencies without affecting body weight gain in aged mice. Our findings showed that supplementing diets with AYE might help to prevent thrombotic diseases in elderly individuals.


Assuntos
Angelica , Trombose , Humanos , Animais , Camundongos , Idoso , Inibidor 1 de Ativador de Plasminogênio , Aumento de Peso , Inflamação/tratamento farmacológico , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Exsudatos e Transudatos , Suplementos Nutricionais
3.
Biol Pharm Bull ; 45(12): 1772-1783, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36450530

RESUMO

Solid tumors habitually harbor regions with insufficient oxygen away from vasculature. Hypoxia is an important factor that confers malignant phenotypes like chemoresistance to tumor cells. We have demonstrated that cathepsin G (CG) stimulates cell aggregation in breast cancer MCF-7 cells by activating insulin-like growth factor-1 signaling. We investigated whether cancer cell aggregates induced by CG acquire hypoxia-dependent chemoresistance. Pimonidazole staining and hypoxia-inducible factor (HIF)-1α and -2α expression indicated that the core of the cell aggregates was hypoxic. Electrophoretic mobility shift and reporter assays showed that the CG-induced cell aggregates displayed transcriptional activity through HIF-responsive elements. Moreover, HIF target genes PGK1 and SLC2A1 demonstrated upregulated expression in CG-induced cell aggregates, indicating that the aggregates expressed functional HIF. Doxorubicin (DXR)-induced cytotoxicity was significantly lower in the cell aggregates induced by CG compared with monolayer cells under normoxia. Unexpectedly, the upregulation of P-glycoprotein expression, which is reported to be a HIF-target gene, and decreasing intracellular accumulation of DXR was not detected in the cell aggregates as opposed to in monolayer cells under normoxia. Additionally, reduction of DXR sensitivity in the aggregates was not suppressed by treatment with the HIF inhibitor, YC-1 and HIF-1α small interfering RNA (siRNA). Therefore, we conclude that cell aggregation induced by CG decreases DXR sensitivity via a HIF-independent mechanism.


Assuntos
Doxorrubicina , Neoplasias , Humanos , Catepsina G , Células MCF-7 , Doxorrubicina/farmacologia , Agregação Celular , RNA Interferente Pequeno , Hipóxia
4.
Artigo em Inglês | MEDLINE | ID: mdl-35462067

RESUMO

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase , Neoplasias da Mama , Catepsina G , Proteínas Associadas aos Microtúbulos , Fator de Ativação de Plaquetas , 1-Alquil-2-acetilglicerofosfocolina Esterase/biossíntese , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Feminino , Humanos , Células MCF-7 , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Neutrófilos/metabolismo , Neutrófilos/patologia , Fator de Ativação de Plaquetas/metabolismo
5.
Biol Pharm Bull ; 43(11): 1678-1686, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33132312

RESUMO

Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells. It has been suggested that tumor cell aggregates are associated with tumor embolism, thus CG-induced cell aggregation may promote tumor metastasis. We have revealed that cell aggregation is caused by elevated free insulin-like growth factor (IGF)-1 in the medium, followed by activation of IGF-1 receptor (IGF-1R). However, the molecular mechanism underlying IGF-1 elevation induced by CG remains unclear. Here, we aimed to elucidate the mechanism by examining the degradative effects of CG on IGF-1, and the IGF binding proteins (IGFBPs), which interfere with the binding of IGF-1 to its receptor. CG specifically evoked MCF-7 cell aggregation at less than 1 nM in a dose-dependent manner, however, neutrophil elastase (NE), chymotrypsin, and trypsin did not. Free IGF-1 concentration was continuously elevated in the medium of cells treated with CG, whereas treatments with other serine proteases resulted in only a transient or slight increase. IGFBP-2, the predominant IGFBP in MCF-7 cells, was gradually digested by CG. CG did not cleave IGF-1 for at least 48 h, whereas other proteases completely digested it. Moreover, CG induced continuous phosphorylation of IGF-1R and Akt, whereas NE-induced phosphorylation was transient, possibly due to insulin receptor substrate (IRS)-1 digestion. These results indicated that CG-specific IGF-1 elevation in the medium is caused by digestion of IGFBP-2, not IGF-1. Hence, this study clarifies the molecular mechanism of CG-specific cell aggregation.


Assuntos
Catepsina G/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias/patologia , Agregação Celular , Meios de Cultura/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células MCF-7 , Proteólise , Transdução de Sinais
6.
Cancer Sci ; 108(8): 1574-1583, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28544544

RESUMO

Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells in a process that is dependent on E-cadherin and CG enzymatic activity. While these tumor cell aggregates can cause tumor emboli that could represent intravascular growth and extravasation into the surrounding tissues, resulting in metastasis, the molecular mechanism underlying this process remains poorly characterized. In this study, we aimed to identify the signaling pathway that is triggered during CG-mediated stimulation of cell aggregation. Screening of a library of compounds containing approximately 90 molecular-targeting drugs revealed that this process was suppressed by the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R)-specific kinase inhibitor OSI-906, as well as the multikinase inhibitors axitinib and sunitinib. Antibody array analysis, which is capable of detecting tyrosine phosphorylation of 49 distinct receptor tyrosine kinases, and the results of immunoprecipitation studies indicated that IGF-1R is phosphorylated in response to CG treatment. Notably, IGF-1R neutralization via treatment with a specific antibody or silencing of IGF-1R expression through siRNA transfection suppressed cell aggregation. Furthermore, CG treatment of MCF-7 cells resulted in increased release of IGF-1 into the medium for 24 h, while antibody-mediated IGF-1 neutralization partially prevented CG-induced cell aggregation. These results demonstrate that autocrine IGF-1 signaling is partly responsible for the cell aggregation induced by CG.


Assuntos
Comunicação Autócrina , Neoplasias da Mama/metabolismo , Catepsina G/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptores de Somatomedina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Axitinibe , Agregação Celular/efeitos dos fármacos , Feminino , Humanos , Imidazóis/farmacologia , Indazóis/farmacologia , Indóis/farmacologia , Células MCF-7 , Fosforilação , Pirazinas/farmacologia , Pirróis/farmacologia , Receptor IGF Tipo 1 , Transdução de Sinais/efeitos dos fármacos , Sunitinibe
7.
Mediators Inflamm ; 2014: 971409, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24803743

RESUMO

We previously found that a neutrophil serine protease, cathepsin G, weakens adherence to culture substrates and induces E-cadherin-dependent aggregation of MCF-7 human breast cancer cells through its protease activity. In this study, we examined whether aggregation is caused by degradation of adhesion molecules on the culture substrates or through an unidentified mechanism. We compared the effect of treatment with cathepsin G and other proteases, including neutrophil elastase against fibronectin- (FN-) coated substrates. Cathepsin G and elastase potently degraded FN on the substrates and induced aggregation of MCF-7 cells that had been subsequently seeded onto the substrate. However, substrate-bound cathepsin G and elastase may have caused cell aggregation. After inhibiting the proteases on the culture substrates using the irreversible inhibitor phenylmethylsulfonyl fluoride (PMSF), we examined whether aggregation of MCF-7 cells was suppressed. PMSF attenuated cell aggregation on cathepsin G-treated substrates, but the effect was weak in cells pretreated with high concentrations of cathepsin G. In contrast, PMSF did not suppress cell aggregation on elastase-treated FN. Moreover, cathepsin G, but not elastase, induced aggregation on poly-L-lysine substrates which are not decomposed by these enzymes, and the action of cathepsin G was nearly completely attenuated by PMSF. These results suggest that cathepsin G induces MCF-7 aggregation through a cell-oriented mechanism.


Assuntos
Catepsina G/farmacologia , Agregação Celular/efeitos dos fármacos , Elastase Pancreática/farmacologia , Movimento Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Fluoreto de Fenilmetilsulfonil/farmacologia
8.
Mediators Inflamm ; 2012: 456462, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22919124

RESUMO

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.


Assuntos
Catepsina G/metabolismo , Catepsina G/farmacologia , Agregação Celular/efeitos dos fármacos , Domínio Catalítico , Catepsina G/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Oligopeptídeos/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Ligação Proteica
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