RESUMO
Mutations affecting the Wnt/ßcatenin pathway have been identified in 2640% of hepatocellular carcinoma (HCC) cases. Aberrant activation of this pathway leads to uncontrolled cell proliferation and survival. Thus, identifying Wnt/ßcatenin pathway inhibitors may benefit a subset of patients with HCC. In the present study, the effects of sorafenib and a MEK inhibitor on tumor growth and Wnt/ßcatenin signaling in HCC models were evaluated. A ßcatenin mutant and ßcatenin wildtype HCC models were treated once daily with i) 10 mg/kg sorafenib, ii) 15 mg/kg refametinib (or 25 mg/kg selumetinib), or iii) sorafenib/refametinib. Western blotting was employed to determine changes in biomarkers relevant to Wnt/ßcatenin signaling. Apoptosis, cell proliferation and ßcatenin localization were analyzed by immunohistochemistry. Sorafenib/refametinib markedly inhibited tumor growth and cell proliferation, and caused cell death in naïve and sorafenibresistant HCC models. Despite similar total ßcatenin levels, significant reductions in phosphorylated (p)RanBP3 Ser58, pßcatenin Tyr142, active ßcatenin and ßcatenin target genes were observed in sorafenib/refametinibtreated tumors. Greater levels of ßcatenin in sorafenib/refametinibtreated tumors were accumulated at the membrane, as compared with in the control. In vitro, sorafenib/refametinib inhibited the Wnt/ßcatenin pathway and suppressed Wnt3Ainduced plowdensity lipoprotein receptorrelated protein 6 Ser1490, pRanBP3 Ser58 and pßcatenin Tyr142 in HCC cells. Combination of sorafenib and refametinib inhibits the growth of naïve and sorafenib resistant HCC tumors in association with active suppression of ßcatenin signaling regardless of ßcatenin mutational status. Thus, the sorafenib/MEK inhibitor combination may represent an alternative treatment for patients with HCC whose tumors develop resistance to sorafenib therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Benzimidazóis/administração & dosagem , Difenilamina/administração & dosagem , Difenilamina/análogos & derivados , Humanos , Masculino , Camundongos , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Sorafenibe/administração & dosagem , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.
Assuntos
Bioensaio , Galinhas/imunologia , Interferon gama/análise , Interleucina-18/farmacologia , Animais , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Interferon gama/genética , Interferon gama/metabolismo , Cinética , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
OBJECTIVE: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. DESIGN: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. RESULTS: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo. CONCLUSION: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Difenilamina/análogos & derivados , Neoplasias Hepáticas/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Sulfonamidas/farmacologia , Aloenxertos , Regulação Alostérica , Animais , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Sinergismo Farmacológico , Feminino , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Ratos , Sorafenibe , Sulfonamidas/uso terapêuticoRESUMO
Poxviruses have evolved various strategies to counteract the host immune response, one of which is based on the expression of soluble cytokine receptors. Using various biological assays, we detected a chicken interferon-gamma (chIFN-gamma)-neutralizing activity in supernatants of fowlpox virus (FPV)-infected cells that could be destroyed by trypsin treatment. Secreted viral proteins were purified by affinity chromatography using matrix-immobilized chIFN-gamma, followed by two-dimensional gel electrophoresis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis indicated that the viral IFN-gamma-binding protein in question was encoded by the FPV gene 016. The chicken IFN-gamma binding and neutralizing activity of the recombinant FPV016 protein was confirmed using supernatants of cells infected with a recombinant vaccinia virus that lacked its own IFN-gamma-binding protein but instead expressed the FPV016 gene. The FPV016 gene product also neutralized the activity of duck and human IFN-gamma but failed to neutralize the activity of mouse and rat IFN-gamma. Unlike previously known cellular and poxviral IFN-gamma receptors, which all contain fibronectin type III domains, the IFN-gamma-binding protein of FPV contains an immunoglobulin domain. Remarkably, it exhibits no significant homology to any known viral or cellular protein. Because IFN-gamma receptors of birds have not yet been characterized at the molecular level, the possibility remains that FPV016 represents a hijacked chicken gene and that avian and mammalian IFN-gamma receptors have fundamentally different primary structures.
Assuntos
Proteínas de Transporte/genética , Vírus da Varíola das Aves Domésticas/genética , Interferon gama/metabolismo , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/química , Galinhas , Cromatografia , Eletroforese em Gel Bidimensional , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Focalização Isoelétrica , Camundongos , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Codorniz , RNA/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tripsina/farmacologia , Vaccinia virus/genética , Proteínas Virais/químicaRESUMO
The phylogeny of Th1 and Th2 subsets has not been characterized mainly due to the limited information regarding cytokines in nonmammalian vertebrates. In this study, we characterize a Th1-like regulatory system focusing on the IL-18-regulated IFN-gamma secretion. Stimulation of splenocytes with chicken IL-18 induced high levels of IFN-gamma secretion. Depletion of either macrophages or CD4(+) T cells from the splenocyte cultures caused unresponsiveness to IL-18. In contrast, PBL were unresponsive to IL-18 in the presence or absence of macrophages, but IFN-gamma secretion was stimulated by suboptimal anti-TCR cross-linking combined with IL-18. Splenocytes from five different chicken lines responded equally well to the IL-18 treatment. LSL chicken splenocytes, however, responded only to IL-18 when stimulated either with optimal TCR cross-linking alone or suboptimal TCR cross-linking combined with IL-18. IL-18 not only induced IFN-gamma secretion, but also stimulated splenocyte proliferation. This IL-18-induced proliferation was compared with the effects observed with IL-2. Both cytokines activated the splenocytes as demonstrated by increased size and MHC class II Ag up-regulation in the case of IL-18. Phenotypic analyses following 6 days of culture revealed that IL-2 mainly affected the proliferation of CD8(+) cells, whereas IL-18 had an opposite effect and stimulated the proliferation of CD4(+) cells. Taken together, these results demonstrate the conservation of Th1-like proinflammatory responses in the chicken; they characterize IL-18 as a major growth factor of CD4(+) T cells and identify two distinct mechanisms of IL-18-induced IFN-gamma secretion.