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1.
J Biochem ; 170(4): 445-451, 2021 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33964134

RESUMO

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.


Assuntos
Adrenomedulina/análogos & derivados , Adrenomedulina/química , Albumina Sérica Humana/química , Adrenomedulina/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia em Gel/métodos , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Masculino , Maleimidas/metabolismo , Peso Molecular , Ratos , Ratos Wistar , Receptores de Adrenomedulina/metabolismo , Albumina Sérica Humana/farmacocinética
2.
Biol Pharm Bull ; 43(11): 1799-1803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33132326

RESUMO

Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8-37) or AM (22-52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.


Assuntos
Adrenomedulina/farmacologia , Proteína 1 Modificadora da Atividade de Receptores/agonistas , Proteína 2 Modificadora da Atividade de Receptores/agonistas , Proteína 3 Modificadora da Atividade de Receptores/agonistas , Adrenomedulina/análogos & derivados , Células HEK293 , Meia-Vida , Humanos , Polietilenoglicóis/química , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Biochem Biophys Res Commun ; 529(3): 778-783, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32736707

RESUMO

Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.


Assuntos
Adrenomedulina/química , Adrenomedulina/metabolismo , Trombina/metabolismo , Adrenomedulina/síntese química , Adrenomedulina/farmacocinética , Animais , Células HEK293 , Humanos , Masculino , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Proteólise , Ratos Wistar
4.
Oncol Rep ; 38(1): 506-514, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28586026

RESUMO

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC. Galectin-9 (Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8 (IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Galectinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Autofagia , Caspase 3/metabolismo , Caspase 9/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Galectinas/genética , Galectinas/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Interleucina-8/metabolismo , Queratina-18/metabolismo , MicroRNAs/isolamento & purificação , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico
5.
Int J Oncol ; 48(3): 1165-74, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26797414

RESUMO

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. There are various histological types of GBC, and the vast majority of GBC cases are adenocarcinomas. Squamous and adenosquamous carcinomas are rare GBC subtypes that are traditionally considered to be more aggressive and to be associated with a poorer prognosis than adenocarcinoma. Galectin-9 (Gal-9), a tandem-repeat-type galectin, has been reported to induce apoptosis-mediated elimination of various cancers, including hepatocellular carcinoma, cholangiocarcinoma, and hematologic malignancies. Therefore, we investigated the antitumor effects of Gal-9 on GBC in vitro and in vivo. In our in vitro experiments, Gal-9 suppressed cell proliferation in various GBC cell lines but not in the OCUG-1 cell line, which represents a poorly differentiated type of adenosquamous carcinoma. Gal-9 induced the apoptosis of Gal-9-sensitive GBC cells by increasing the levels of caspase-cleaved keratin 18 and phosphorylated p53. However, Gal-9 did not affect the expression of various cell cycle-related proteins. In addition, Gal-9 suppressed tumor growth by implanted human GBC cells in a xenograft model. Furthermore, Gal-9 induced the phosphorylation of the Ephrin type-B receptor, and the microRNA (miRNA) expression profile was markedly altered by Gal-9. Based on these results, various miRNAs might contribute to the suppression of tumor growth. Our data reveal that Gal-9 suppresses the growth of GBC, possibly by inducing apoptosis and altering miRNA expression. Thus, Gal-9 might serve as a therapeutic agent for the treatment of GBC.


Assuntos
Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Galectinas/metabolismo , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Queratina-18/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Mutação , Fosforilação , Ensaios Antitumorais Modelo de Xenoenxerto
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