RESUMO
Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Ferroptose , Microbioma Gastrointestinal , Glutamina , NADPH Oxidase 1 , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/microbiologia , Colangiocarcinoma/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Humanos , Glutamina/metabolismo , NADPH Oxidase 1/metabolismo , NADPH Oxidase 1/genética , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/microbiologia , Camundongos , Masculino , Linhagem Celular Tumoral , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/genética , Camundongos Nus , Feminino , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptor do Fator de Crescimento Transformador beta Tipo IRESUMO
BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proteínas Inibidoras de Diferenciação , Neoplasias Pulmonares , Proteínas de Neoplasias , Silibina , Silibina/farmacologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Humanos , Animais , Linhagem Celular Tumoral , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/genética , Silimarina/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Morfogenética Óssea 6 , Silybum marianum/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , FemininoRESUMO
Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.
Assuntos
Receptores de Ativinas Tipo I/genética , Antineoplásicos/farmacologia , Neoplasias do Tronco Encefálico/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Administração Oral , Animais , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/mortalidade , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Proliferação de Células , Criança , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/mortalidade , Glioma Pontino Intrínseco Difuso/patologia , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos SCID , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/farmacocinética , Piridinas/farmacocinética , Pirimidinas/farmacocinética , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Structure-activity relationship and crystallographic data revealed that quinazolinone-containing fragments flip between two distinct modes of binding to activin receptor-like kinase-2 (ALK2). We explored both binding modes to discover potent inhibitors and characterized the chemical modifications that triggered the flip in binding mode. We report kinase selectivity and demonstrate that compounds of this series modulate ALK2 in cancer cells. These inhibitors are attractive starting points for the discovery of more advanced ALK2 inhibitors.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinonas/química , Relação Estrutura-Atividade , Receptores de Ativinas Tipo I/química , Receptores de Ativinas Tipo I/metabolismo , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , HumanosRESUMO
Essentials Antiangiogenic drugs are indicated as therapies for hereditary hemorrhagic telangiectasia. We interrogated the response to four antiangiogenic drugs for anemia and intestinal bleeding. Sorafenib and a pazopanib analog significantly improved while erlotinib worsened anemia. Some oral antiangiogenic drugs were effective in reducing intestinal bleeding. SUMMARY: Background Epistaxis and gastrointestinal (GI) tract hemorrhages are common symptoms of aged hereditary hemorrhagic telangiectasia (HHT) patients that result in anemia. Clinical as well as animal studies have suggested that vascular endothelial growth factor (VEGF) neutralizing antibodies lessen hemorrhage associated with adult-onset arteriovenous malformations (AVMs). Objectives The goal of this study is to evaluate potential therapeutic effects of oral delivery of four antiangiogenic tyrosine-kinase inhibitors (TKIs) in the development of adult-onset AVMs in a murine model of HHT. Methods An adult activin receptor-like kinase 1 (Alk1)-inducible knockout (iKO) model was utilized to evaluate the effect of oral administration of sorafenib, sunitinib, erlotinib and a pazopanib analog (GW771806) on hemoglobin level, GI hemorrhages and formation of wound-induced skin AVMs. Results and Conclusions Sorafenib and GW771806 significantly improved, yet erlotinib worsened, anemia and GI-bleeding in the Alk1-iKO model. However, none of these TKIs appeared to be effective for inhibiting the development of wound-induced skin AVMs. Taken together, these results suggest that oral delivery of antiangiogenic TKIs is selectively more effective for GI bleeding than mucocutaneous AVMs, and it may provide an experimental basis for selective therapeutic options depending on the symptoms of HHT.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Telangiectasia Hemorrágica Hereditária/tratamento farmacológico , Telangiectasia Hemorrágica Hereditária/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Administração Oral , Administração Tópica , Anemia/tratamento farmacológico , Anemia/genética , Animais , Malformações Arteriovenosas , Modelos Animais de Doenças , Cloridrato de Erlotinib/administração & dosagem , Hemorragia Gastrointestinal , Hemoglobinas/análise , Processamento de Imagem Assistida por Computador , Indazóis/administração & dosagem , Camundongos , Camundongos Knockout , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Pirimidinas/administração & dosagem , Pele/irrigação sanguínea , Sorafenibe , Sulfonamidas/administração & dosagem , Sulfonas/administração & dosagem , Tirosina/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , CicatrizaçãoRESUMO
Autophosphorylation of kinases influences their conformational state and can also regulate enzymatic activity. Recently, this has become an area of interest for drug discovery. Using Alk2 as an example, we present two protocols - one based on phosphate-binding Alphascreen beads, the other on coupled luminescence measurements of ADP formation - that can be used to screen for inhibitors of autophosphorylation.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Medições Luminescentes/métodos , Fosforilação/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Recombinantes/metabolismoRESUMO
Transforming growth factor ß1 (TGF-ß1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-ß1 exerts stimulatory and inhibitory effect depending on the type I receptor involved in the signaling pathway. The aim of the present work was to study the signaling mechanisms and the intermediates involved in the action of TGF-ß1 on TM3 Leydig cell proliferation in the presence or absence of progesterone. The MTT assay showed that the presence of progesterone in the culture media lead to a proliferative effect that was blocked by Ru 486, an inhibitor of progesterone receptor; and ALK-5 did not participate in this effect. TGF-ß1 (1 ng/ml) increased the expression of p15 (an inhibitor of cell cycle) in TM3 Leydig cells, and this effect was blocked by progesterone (1µM). The expression of PCNA presented a higher increase in the cell cultured with TGF-ß1 plus progesterone than in cells cultured only with TGF-ß1. Progesterone induced the gene expression of endoglin, a cofactor of TGF-ß1 receptor that leads to a stimulatory signaling pathway, despite of the absence of progesterone response element in endoglin gene. In addition, the presence of progesterone induced the gene expression of egr-1 and also KLF14, indicating that this steroid channels the signaling pathway into a non-canonical mechanism. In conclusion, these findings suggest that the proliferative action of TGF-ß1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , DNA Complementar/genética , Endoglina , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Progesterona/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic ossification. FOP is caused by a gain-of-function mutation in ACVR1 encoding the bone morphogenetic protein type II receptor, ACVR1/ALK2. The mutant receptor causes upregulation of a transcriptional factor, Id1. No therapy is available to prevent the progressive heterotopic ossification in FOP. In an effort to search for clinically applicable drugs for FOP, we screened 1,040 FDA-approved drugs for suppression of the Id1 promoter activated by the mutant ACVR1/ALK2 in C2C12 cells. We found that that two antianginal agents, fendiline hydrochloride and perhexiline maleate, suppressed the Id1 promoter in a dose-dependent manner. The drugs also suppressed the expression of native Id1 mRNA and alkaline phosphatase in a dose-dependent manner. Perhexiline but not fendiline downregulated phosphorylation of Smad 1/5/8 driven by bone morphogenetic protein (BMP)-2. We implanted crude BMPs in muscles of ddY mice and fed them fendiline or perhexiline for 30 days. Mice taking perhexiline showed a 38.0 % reduction in the volume of heterotopic ossification compared to controls, whereas mice taking fendiline showed a slight reduction of heterotopic ossification. Fendiline, perhexiline, and their possible derivatives are potentially applicable to clinical practice to prevent devastating heterotopic ossification in FOP.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Fendilina/farmacologia , Células Musculares/metabolismo , Miosite Ossificante/tratamento farmacológico , Ossificação Heterotópica/tratamento farmacológico , Osteoblastos/metabolismo , Perexilina/análogos & derivados , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 1 Inibidora de Diferenciação/genética , Camundongos , Camundongos Mutantes , Células Musculares/patologia , Mutação , Miosite Ossificante/genética , Miosite Ossificante/metabolismo , Miosite Ossificante/patologia , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Osteoblastos/patologia , Perexilina/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.
Assuntos
Receptores de Ativinas Tipo I/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Miosite Ossificante/patologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Mutação de Sentido Incorreto , Miosite Ossificante/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Pele/patologia , TranscriptomaRESUMO
OBJECTIVES: Transforming growth factor-beta(1) (TGF-beta(1)) and its receptors, type 1 (TR-1) and type 2 (TR-2) play important roles in chronic cyclosporine (CsA)-induced nephropathy. Lotensin is known as an angiotensin-converting enzyme inhibitor and may reduce chronic CsA-induced nephropathy. Recently it is reported that Salviae (a Chinese medicine), which can improve microcirculation and decrease the expression of TGF-beta(1) has the same effect as that of lotensin. Therefore, in this study we assessed the effects of Lotensin or Salviae on the chronic CsA-induced upregulation of TGF-beta(1), TR-1, and TR-2 in a rat model. MATERIALS AND METHODS: Sodium-depleted rats were administered CsA by gastric gavage and a new rat model of chronic CsA-induced nephropathy was established. Rats with chronic CsA-induced nephropathy were treated by lotensin or Salviae. The proteins of TGF-beta(1), TR-1, and TR-2, and the mRNA of TR-1 and TR-2 in the kidneys of CsA-treated rats, were measured by immunohistochemistry (IHC) and in situ hybridization (ISH). The results were investigated semiquantitatively by image analysis. RESULTS: Lotensin or Salviae individually attenuated CsA-induced nephropathy in the rat models, and downregulated the protein expressions of TGF-beta(1), TR-1, and TR-2, and the mRNA transcripts of TR-1 and TR-2 in the rat model. CONCLUSION: Our studies show that treatment with lotensin or Salviae is useful in preventing chronic CsA-induced nephropothy.