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1.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438649

RESUMO

The transforming growth factor-beta (TGF-ß) plays an important role in pathological fibrosis and cancer transformation. Therefore, the inhibition of the TGF-ß signaling pathway has therapeutic potential in the treatment of cancer. In this study, the binding modes between 47 molecules with a pyrrolotriazine-like backbone structure and transforming growth factor-beta type 1 receptor (TßR1) were simulated by molecular docking using Discovery Studio software, and their structure-activity relationships were analyzed. On the basis of the analysis of the binding modes of ligands in the active site and the structure-activity relationships, 29,254 new compounds were designed for virtual screening. According to the aforementioned analyses and Lipinski's rule of five, five new compounds (CQMU1901-1905) with potential activity were screened through molecular docking. Among them, CQMU1905 is an attractive molecule composed of 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), and 5-azacytosine. Interestingly, 5-FU, 6-MP, and 5-azacytidine are often used as anti-metabolic agents in cancer treatment. Compared with existing compounds, CQMU1901-1905 can interact with target proteins more effectively and have good potential for modification, making them worthy of further study.


Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Citosina/análogos & derivados , Citosina/química , Citosina/farmacologia , Fluoruracila/química , Fluoruracila/farmacologia , Humanos , Mercaptopurina/química , Mercaptopurina/farmacologia , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
2.
Eur J Med Chem ; 180: 15-27, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31299584

RESUMO

Six series of 4-(benzo[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methylpyridin-2-yl)- pyrazoles 18a-d, 19a-d, 22a-d and 3(5)-(6-methylpyridin-2-yl)-4-(thieno[3,2,-c]- pyridin-2-yl)pyrazoles 20a-d, 21a-d, 23c, 23d have been synthesized and evaluated for their activin receptor-like kinase 5 (ALK5) and p38α mitogen activated protein (MAP) kinase inhibitory activities in enzymatic assays. Among these compounds, the most active compound, 22c, inhibited ALK5 phosphorylation with an IC50 value of 0.030 µM in the enzymatic assay. Compound 22c showed four-fold more potent activity against ALK5 kinase than the clinical candidate, compound LY-2157299. The selectivity index of 22c against p38α MAP kinase is 235, which is much higher than that of LY-2157299 (4) and equally selective to that of EW-7197 (218). Compound 22c effectively suppressed protein and mRNA expression of collagen I and α-SMA in TGF-ß-induced LX-2 human hepatic stellate cell (HSC), this result shows that compound 22c has the ability to inhibit the activation of HSC. Compound 22c is expected to be a preclinical candidate for the treatment of hepatic fibrosis.


Assuntos
Desenho de Drogas , Fibrose/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibrose/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Relação Estrutura-Atividade
3.
Int J Mol Sci ; 20(10)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121879

RESUMO

Hepatocyte growth factor (HGF) and transforming growth factor ß1 (TGFß1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFß1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFß1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFß1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFß1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFß-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFß1-RI blockade.


Assuntos
Benzamidas/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Dioxóis/uso terapêutico , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Neoplasias Ósseas/patologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos
4.
Nat Commun ; 10(1): 1665, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971692

RESUMO

Lung cancer is the leading cause of cancer-related deaths worldwide. Tumor suppressor genes remain to be systemically identified for lung cancer. Through the genome-wide screening of tumor-suppressive transcription factors, we demonstrate here that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vivo. Ectopic GATA4 expression results in lung cancer cell senescence. Mechanistically, GATA4 upregulates multiple miRNAs targeting TGFB2 mRNA and causes ensuing WNT7B downregulation and eventually triggers cell senescence. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGF-ß2 level and is significantly associated with poor prognosis. TGFBR1 inhibitors show synergy with existing therapeutics in treating GATA4-deficient lung cancers in genetically engineered mouse model as well as patient-derived xenograft (PDX) mouse models. Collectively, our work demonstrates that GATA4 functions as a tumor suppressor in lung cancer and targeting the TGF-ß signaling provides a potential way for the treatment of GATA4-deficient lung cancer.


Assuntos
Fator de Transcrição GATA4/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Proteínas Wnt/metabolismo , Células A549 , Animais , Senescência Celular/genética , Regulação para Baixo , Feminino , Fator de Transcrição GATA4/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Mol Life Sci ; 76(15): 3005-3018, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31006037

RESUMO

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Longo não Codificante/metabolismo , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Neuroglia/citologia , Neuroglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
6.
Gynecol Oncol ; 153(3): 639-650, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30928020

RESUMO

OBJECTIVE: To test whether platelets could increase invasion potential and initiate EMT in ovarian cancer cells via a TGF-ß signaling pathway. METHODS: Blood samples were collected in 69 patients with ovarian cancer, 16 patients with benign ovarian tumor and 64 healthy donors. SK-OV-3 and OVCAR-3 ovarian cancer cells were treated with platelets. Transwell assays were used to analyze the invasive capacity, and EMT was assessed by microarray analysis, quantitative real-time PCR (qPCR) and Western blotting. Activation of TGF-ß pathway was examined by ELISA and Western blotting. TGF-ß type I receptor (TßR I) inhibitor A83-01 was used to confirm the role of TGF-ß pathway in vitro and in vivo. RESULTS: Clinical data showed ovarian cancer patients with elevated platelet counts had a higher incidence of advanced stages. Treatment with platelets increased the invasive properties of both cell lines. Mesenchymal markers (snail family transcriptional repressor-1, vimentin, neural cadherin, fibronectin-1 and matrix metalloproteinase-2) were up-regulated in platelet-treated cells, while the epithelial marker (epithelial cadherin) was down-regulated. Higher TGF-ß level was observed in patients with elevated platelet counts when compared to the subjects. Higher levels of TGF-ß were also found in culture medium treated with platelets, and cells treated with platelets also showed increased phosphorylation of Smad2. TßR I inhibitor A83-01 reversed the EMT-like alterations and inhibited platelet-induced invasion in vitro and in vivo. CONCLUSION: Platelet increased invasion potential and induced EMT in ovarian cancer cells in a TGF-ß dependent pathway. Platelet-derived TGF-ß may be useful as a new target treatment for ovarian cancer.


Assuntos
Plaquetas , Transição Epitelial-Mesenquimal , Neoplasias Ovarianas/sangue , Fator de Crescimento Transformador beta/metabolismo , Adulto , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Transição Epitelial-Mesenquimal/genética , Feminino , Fibronectinas/genética , Expressão Gênica , Voluntários Saudáveis , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , Contagem de Plaquetas , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/genética , Tiossemicarbazonas/farmacologia , Fator de Crescimento Transformador beta/sangue , Regulação para Cima , Vimentina/genética
7.
Cell Death Dis ; 10(2): 124, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30741930

RESUMO

Skin fibrosis is mainly characterized by excessive collagen deposition. Studies have recently identified a number of flavonoids with variable structures that have the potency of inhibiting collagen synthesis and thus attenuating organ fibrosis. In this study, we found that flavones with 5, 7, 3', 4' hydroxy substitution reduced collagen expression most efficiently. Among those flavones, luteolin, quercetin, and myricetin were selected for follow-up. In vivo, the three compounds ameliorated skin fibrosis and reduced collagen deposition. Further analysis showed the compounds had significant inhibition on the proliferation, activation and contractile ability of dermal fibroblasts in vitro and in vivo. More importantly, we revealed that luteolin, quercetin, and myricetin selectively downregulated the phosphorylation of Smad2/3 in TGF-ß/Smads signaling via binding to activin receptor-like kinase 5 (ALK5) and impairing its catalytic activity. We also found flavones with 5, 7, 3', 4' hydroxy substitution showed stronger affinity with ALK5 compared with other flavonoids. Herein, we identified at least in part the underlying molecular basis as well as the critical structures that contribute to the antifibrotic bioactivity of flavones, which might benefit drug design and modification.


Assuntos
Proliferação de Células/efeitos dos fármacos , Flavonas/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Pele/patologia , Animais , Sítios de Ligação , Domínio Catalítico , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno/genética , Colágeno/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Flavonas/química , Flavonas/metabolismo , Humanos , Hidroxilação , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Fosforilação/efeitos dos fármacos , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Pele/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
8.
Biochem Biophys Res Commun ; 510(1): 53-58, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30660362

RESUMO

PURPOSE: The aim of this study was to investigate the mechanism of let-7a-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis (PMOP) mice. METHODS: A mouse model of PMOP was established and osteoporosis model was identified by micro-CT scan. BMSCs in the sham group and PMOP group were cultured and osteogenic differentiation was induced. The expression of let-7a-5p in BMSCs was detected by qRT-PCR, and BMSCs was induced by osteogenic differentiation in sham and PMOP group. The BMSCs treated by let-7a-5p mimics, let-7a-5p inhibitor and negative control were named as let-7a-5p mimics group, mimics NC group, let-7a-5p inhibitor group and inhibitor NC group, respectively. ALP staining and alizarin red staining were used to detect osteogenic differentiation ability, qRT-PCR and western blot were used to detect the expression of Runt-related transcription factor 2 (Runx2) and Osterix. The targeting relationship between let-7a-5p and TGFBR1 were verificated by target scan and luciferase reporter gene assay. RESULTS: The PMOP mouse model was successfully established. The expression of let-7a-5p in BMSCs of PMOP group was significantly higher than that in the sham group (P < 0.05). Let-7a-5p reduced the expression of ALP and the formation of calcified nodules, while also inhibited the expression of Runx2 and Osterix. TGFBR1 is the target gene of let-7a-5p. CONCLUSION: Let-7a-5p might inhibit the osteogenic differentiation of BMSCs in PMOP mice by regulating TGFBR1.


Assuntos
Células-Tronco Mesenquimais/citologia , MicroRNAs/fisiologia , Osteogênese/efeitos dos fármacos , Osteoporose Pós-Menopausa/prevenção & controle , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , MicroRNAs/farmacologia , Fator de Transcrição Sp7/antagonistas & inibidores
9.
Proc Natl Acad Sci U S A ; 116(9): 3883-3892, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30655341

RESUMO

The endometrial lining of the uterine cavity is a highly dynamic tissue that is under the continuous control of the ovarian steroid hormones, estrogen and progesterone. Endometrial adenocarcinoma arises from the uncontrolled growth of the endometrial glands, which is typically associated with unopposed estrogen action and frequently occurs in older postmenopausal women. The incidence of endometrial cancer among younger women has been rising due to increasing rates of obesity, a major risk factor for the disease. The transforming growth factor ß (TGFß) family is a highly conserved group of proteins with roles in cellular differentiation, proliferation, and cancer. Inactivating mutations in the genes encoding the TGFß cell surface receptors (TGFBR1/ALK5 and TGFBR2) have been detected in various human cancers, indicating that a functional TGFß signaling pathway is required for evading tumorigenesis. In this study, we present a mouse model with conditional inactivation of activin receptor-like kinase 5 (ALK5) in the mouse uterus using progesterone receptor cre ("Alk5 cKO") that develops endometrial adenocarcinoma with metastasis to the lungs. The cancer and metastatic lung nodules are estrogen dependent and retain estrogen receptor α (ERα) reactivity, but have decreased levels of progesterone receptor (PR) protein. The endometrial tumors develop only in Alk5 cKO mice that are mated to fertile males, indicating that TGFß-mediated postpartum endometrial repair is critical for endometrial function. Overall, these studies indicate that TGFß signaling through TGFBR1/ALK5 in the endometrium is required for endometrial homeostasis, tumor suppression, and postpartum endometrial regeneration.


Assuntos
Adenocarcinoma/genética , Carcinogênese/genética , Neoplasias do Endométrio/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Útero/metabolismo , Adenocarcinoma/patologia , Animais , Modelos Animais de Doenças , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Útero/patologia
10.
Invest New Drugs ; 37(1): 118-126, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29995286

RESUMO

Background Galunisertib inhibits type I transforming growth factor-beta receptor serine/threonine kinase. The primary objective of this study was to evaluate the safety and tolerability of galunisertib in combination with sorafenib in Japanese patients with unresectable hepatocellular carcinoma. Patients and methods This open-label, dose-escalation, multicenter, nonrandomized phase 1b study consisted of two dose levels of galunisertib, 160 or 300 mg/day, in combination with sorafenib 800 mg/day. Galunisertib 80 mg or 150 mg was administered orally twice daily for 14 days followed by 14 days of rest plus sorafenib 400 mg administered orally twice daily for 28 days. The dose-limiting toxicity evaluation was 28 days after the first dose. Safety measures, pharmacokinetics, and antitumor activity were assessed. Results Fourteen patients, 7 at each galunisertib dose, were enrolled and treated. Three dose-limiting toxicities were reported for 2 patients. The most common treatment-emergent adverse events (TEAEs) were hypophosphatemia (14 patients [100%]), palmar-plantar erythrodysesthesia syndrome (12 patients [85.7%]), and decreased platelet count (10 patients [71.4%]). The most common grade ≥ 3 TEAEs were hypophosphatemia (10 patients [71.4%]) and palmar-plantar erythrodysesthesia syndrome (7 patients [50.0%]). No grade 5 TEAEs were reported. The pharmacokinetic profile of galunisertib in combination with sorafenib was similar to that previously reported for galunisertib. Eleven patients had a best overall response of stable disease, and 1 patient achieved a partial response by hepatocellular carcinoma-specific modified RECIST. Conclusions These data are consistent with the known safety profile for galunisertib and sorafenib and confirm tolerability of the recommended dose of galunisertib (150 mg twice daily for 14 days) in combination with sorafenib in Japanese patients with unresectable hepatocellular carcinoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Prognóstico , Pirazóis/administração & dosagem , Quinolinas/administração & dosagem , Sorafenibe/administração & dosagem , Distribuição Tecidual
11.
Prostate ; 79(1): 31-43, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30155899

RESUMO

BACKGROUND: Prostate cancer progression is navigated by the androgen receptor (AR) and transforming-growth factor-ß (TGF-ß) signaling. We previously demonstrated that aberrant TGF-ß signaling accelerates prostate tumor progression in a transgenic mouse model of prostate cancer via effects on epithelial-mesenchymal transition (EMT), driving castration-resistant prostate cancer (CRPC). METHODS: This study examined the antitumor effect of the combination of TGF-ß receptor I (TßRI) inhibitor, galunisertib, and FDA-approved antiandrogen enzalutamide, in our pre-clinical model. Age-matched genotypically characterized DNTGFßRII male mice were treated with either galunisertib and enzalutamide, in combination or as single agents in three "mini"-trials and the effects on tumor growth, phenotypic EMT, and actin cytoskeleton were evaluated. RESULTS: Galunisertib in combination with enzalutamide significantly suppressed prostate tumor growth, by increasing apoptosis and decreasing cell proliferation of tumor cell populations compared to the inhibitor as a monotherapy (P < 0.05). The combination treatment dramatically reduced cofilin levels, actin cytoskeleton regulator, compared to single agents. Treatment with galunisertib targeted nuclear Smad4 protein (intracellular TGF-ß effector), but had no effect on nuclear AR. Consequential to TGF-ß inhibition there was an EMT reversion to mesenchymal-epithelial transition (MET) and re-differentiation of prostate tumors. Elevated intratumoral TGF-ß1 ligand, in response to galunisertib, was blocked by enzalutamide. CONCLUSION: Our results provide novel insights into the therapeutic value of targeting TGF-ß signaling to overcome resistance to enzalutamide in prostate cancer by phenotypic reprogramming of EMT towards tumor re-differentiation and cytoskeleton remodeling. This translational work is significant in sequencing TGF-ß blockade and antiandrogens to optimize therapeutic response in CRPC.


Assuntos
Antineoplásicos/administração & dosagem , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Pirazóis/administração & dosagem , Quinolinas/administração & dosagem , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Feniltioidantoína/administração & dosagem , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
12.
Pulm Pharmacol Ther ; 54: 31-38, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448291

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease, and consequently, effective antifibrotic drugs are strongly desired. Although we have previously reported a validated Col1a1-Luc Tg rat model for fibrosis, there are only a few mouse models that enable the evaluation of fibrosis in a short time period and with high sensitivity. Therefore, we generated a Col1a1-internal ribosome entry site (IRES)-Luc knock-in (KI) mouse in which the IRES-luciferase gene construct was inserted into the 3'-UTR of the type I collagen alpha 1 gene (Col1a1). There was a high correlation between luciferase activity and hydroxyproline content in the KI mice, which is similar to the result that we have previously reported for the Col1a1-Luc Tg rat model. In a bleomycin (BLM)-induced lung fibrosis model, luciferase activity in the lung showed a significant increase 3 days after BLM treatment, while only a slight increase was observed in the hydroxyproline content. An ALK-5 inhibitor-R-268712-was effective in inhibiting the luciferase activity in both the in vivo BLM-induced lung fibrosis model and in vitro primary mouse lung fibroblasts. This suggests that fibroblasts are the major collagen-producing cells in lung fibrosis. In human lung fibroblasts, TGF-ß stimulation induced α-smooth muscle actin as observed by immunostaining, suggesting that myofibroblast transdifferentiation (MTD) plays an important role in lung fibrosis. Together, these results indicated that ALK-5 inhibitors might affect lung fibrosis mainly via the inhibition of MTD. Thus, the Col1a1-IRES-Luc KI mouse might be useful for the evaluation of antifibrotic effects and their underlying mechanisms.


Assuntos
Colágeno Tipo I/genética , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Bleomicina/administração & dosagem , Transdiferenciação Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Fibrose Pulmonar/fisiopatologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
13.
J Cell Biochem ; 120(1): 848-860, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30161276

RESUMO

Expression of the transcriptional regulator, E26 transformation-specific 1 (ETS1), is elevated in human prostate cancers, and this is associated with more aggressive tumor behavior and a rapid progression to castrate-resistant disease. Multiple ETS1 isoforms with distinct biological activities have been characterized and in 44 matched nonmalignant and malignant human prostate specimens, messenger RNAs for two ETS1 isoforms, ETS1p51 and ETS1p42, were detected, with ETS1p51 levels significantly lower in prostate tumor compared to matched nonmalignant prostate tissues. In contrast, ETS1p51 protein, the only ETS1 isoform detected, was expressed at significantly higher levels in malignant prostate. Analysis of epithelial-to-mesenchymal transition (EMT)-associated genes regulated following overexpression of ETS1p51 in the LNCaP prostate cancer cell line predicted promotion of transforming growth factor ß (TGFß) signaling and of EMT. ETS1p51 overexpression upregulated cellular levels of the EMT transcriptional regulators, ZEB1 and SNAIL1, resulted in reduced expression of the mesenchymal marker vimentin with concomitantly elevated levels of claudin 1, an epithelial tight junction protein, and increased prostate cancer cell migration and invasion. ETS1p51-induced activation of the pro-EMT TGFß signaling pathway that was predicted in polymerase chain reaction arrays was verified by demonstration of elevated SMAD2 phosphorylation following ETS1p51 overexpression. Attenuation of ETS1p51 effects on prostate cancer cell migration and invasion by inhibition of TGFß pathway signaling indicated that ETS1p51 effects were in part mediated by induction of TGFß signaling. Thus, overexpression of ETS1p51, the predominant ETS1 isoform expressed in prostate tumors, promotes an EMT program in prostate cancer cells in part via activation of TGFß signaling, potentially accounting for the poor prognosis of ETS1-overexpressing prostate tumors.


Assuntos
Transição Epitelial-Mesenquimal/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dioxóis/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Proteína Smad2/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
14.
J Clin Invest ; 129(2): 569-582, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30511964

RESUMO

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-ß pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-ß signaling, EMT, and cell motility, which is effectively blocked by the TGF-ß receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-ß signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-ß signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.


Assuntos
Fator 3-alfa Nuclear de Hepatócito , Proteínas de Neoplasias , Neoplasias de Próstata Resistentes à Castração , Pirazóis/farmacologia , Quinolinas/farmacologia , Transdução de Sinais , Fator de Crescimento Transformador beta3 , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
PLoS Genet ; 14(12): e1007790, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30557355

RESUMO

Accumulating evidence indicates that miRNAs can be promising diagnostic and/or prognostic markers for various cancers. In this study, we identified a novel miRNA, miR-3607-3p, and its targets in non-small cell lung cancer (NSCLC). The expression of miR-3607-3p was measured and its correlation with patient prognosis was determined. Ectopic expression in NSCLC cells, xenografts, and metastasis models was used to evaluate the effects of miR-3607-3p on proliferation and migration of NSCLC. Luciferase assay and western blotting were performed to validate the potential targets of miR-3607-3p after preliminary screening by microarray analysis and computer-aided algorithms. We demonstrated that miR-3607-3p was downregulated in NSCLC tissues and that miR-3607-3p might act as an independent predictor for overall survival in NSCLC. Moreover, serum miR-3607-3p may be a novel and stable marker for NSCLC. We found that overexpression of miR-3607-3p inhibited cell proliferation, colony formation, migration and invasion, and hampered the cell cycle of NSCLC cell lines in vitro. Our results suggested that miR-3607-3p directly targets TGFBR1 and CCNE2. In accordance with in vitro studies, we confirmed that miR-3607-3p functions as a potent suppressor miRNA of NSCLC. We showed that miR-3607-3p agomir could reduce tumor growth and inhibit TGFBR1 and CCNE2 protein expression. Taken together, our findings indicate that miR-3607-3p can inhibit NSCLC cell growth and metastasis by targeting TGFBR1 and CCNE2 protein expression, and provide new evidence of miR-3607-3p as a potential non-invasive biomarker and therapeutic target for NSCLC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Ciclinas/antagonistas & inibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , RNA Nucleolar Pequeno/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Idoso , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ciclinas/genética , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica , Prognóstico , RNA Nucleolar Pequeno/antagonistas & inibidores , RNA Nucleolar Pequeno/sangue , Receptor do Fator de Crescimento Transformador beta Tipo I/genética
16.
Molecules ; 23(12)2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30572609

RESUMO

The transforming growth factor-ß (TGF-ß), in which overexpression has been associated with various diseases, has become an attractive molecular target for the treatment of cancers. Thirty-two quinoxaline-derivatives of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a⁻d, 15a⁻d, 16a⁻d, 17a⁻d, 18a⁻d, 19a⁻d, 25a, 25b, 25d, 26a, 26b, 26d, 27b, and 27d were synthesized and evaluated for their activin TGF-ß type I receptor kinase and p38α mitogen activated protein (MAP) kinase inhibitory activity in enzymatic assays. Among these compounds, the most active compound 19b inhibited TGF-ß type I receptor kinase phosphorylation with an IC50 value of 0.28 µM, with 98% inhibition at 10 µM. Compound 19b also had good selectivity index of >35 against p38α MAP kinase, with 9.0-fold more selective than clinical candidate, compound 3 (LY-2157299). A molecular docking study was performed to identify the mechanism of action of the synthesized compounds and their good binding interactions were observed. ADMET prediction of good active compounds showed that these ones possess good pharmacokinetics and drug-likeness behavior.


Assuntos
Pirazóis/química , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Quinolinas/química , Relação Estrutura-Atividade
17.
Molecules ; 23(11)2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30384428

RESUMO

As new drugs for the treatment of malignant tumors, transforming growth factor-beta receptor 1 (TGFßR1) antagonists have attracted wide attention. Based on the crystal structure of TGFßR1-BMS22 complex, the pharmacophore model A02 with two hydrogen bond acceptors (HBAs) and four hydrophobic (HYD) properties was constructed. From the common features of active ligands reported in the literature, pharmacophore model B10 was also generated, which has two aromatic ring centers (RAs) and two HYD properties. The two models have high sensitivity and specificity to the training set, and they are highly consistent in spatial structure. Combining the two pharmacophore models, two novel skeleton structures with potential activity were selected by virtual screening from the DruglikeDiverse, MiniMaybridge, and ZINC Drug-Like databases. Four compounds (YXY01⁻YXY04) with potential anti-TGFßR1 activity were designed based on the new skeleton structures. In combination with Lipinski's rules; absorption, distribution, metabolism, excretion, and toxicity (ADMET); and, toxicological properties predicted in the study, YXY01-03 with the novel skeleton, good drug-like properties, and potential activity were finally discovered and may have higher safety relative to BMS22, which may be valuable for further research.


Assuntos
Descoberta de Drogas , Neoplasias/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Interface Usuário-Computador , Cristalografia por Raios X , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Receptor do Fator de Crescimento Transformador beta Tipo I/química , Receptor do Fator de Crescimento Transformador beta Tipo I/genética
18.
Sci Rep ; 8(1): 16065, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375456

RESUMO

Demineralized bone matrix is a widely used allograft from which not only the inorganic mineral but also embedded growth factors are removed by hydrochloric acid (HCl). The cellular response to the growth factors released during the preparation of demineralized bone matrix, however, has not been studied. Here we investigated the in vitro impact of acid bone lysate (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Proteomic analysis of ABL revealed a large spectrum of bone-derived proteins including TGF-ß1. Whole genome microarrays and RT-PCR together with the pharmacologic blocking of TGF-ß receptor type I kinase with SB431542 showed that ABL activates the TGF-ß target genes interleukin 11, proteoglycan 4, and NADPH oxidase 4. Interleukin 11 expression was confirmed at the protein level by ELISA. Immunofluorescence and Western blot showed the nuclear localization of Smad2/3 and increased phosphorylation of Smad3 with ABL, respectively. This effect was independent of whether ABL was prepared from mandible, calvaria or tibia. These results demonstrate that TGF-ß is a major growth factor that is removed upon the preparation of demineralized bone matrix.


Assuntos
Osso Cortical/química , Dentina/química , Proteômica , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Animais , Benzamidas/farmacologia , Técnica de Desmineralização Óssea , Osso Cortical/efeitos dos fármacos , Dentina/efeitos dos fármacos , Dioxóis/farmacologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genoma/efeitos dos fármacos , Humanos , Ácido Clorídrico/farmacologia , Interleucina-11/genética , Análise em Microsséries , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/crescimento & desenvolvimento , Mucosa Bucal/metabolismo , Proteoglicanas/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais , Proteína Smad3/genética , Suínos , Fator de Crescimento Transformador beta/genética
19.
Int J Parasitol ; 48(13): 1023-1033, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30266591

RESUMO

Here we investigated the gene of a transforming growth factor (TGF)-ß type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-ß family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-ß type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-ß type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.


Assuntos
Haemonchus/crescimento & desenvolvimento , Receptor do Fator de Crescimento Transformador beta Tipo I/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Clonagem Molecular , Biologia Computacional , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Feminino , Regulação da Expressão Gênica , Genes Reporter/fisiologia , Teste de Complementação Genética , Cabras , Haemonchus/genética , Haemonchus/fisiologia , Masculino , Conformação Molecular , Filogenia , Pirazóis/farmacologia , Quinolinas/farmacologia , RNA de Helmintos/isolamento & purificação , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/química , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transdução de Sinais , Organismos Livres de Patógenos Específicos
20.
Surgery ; 164(5): 1100-1108, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30172565

RESUMO

BACKGROUND: EW-7197 is an oral transforming growth factor ß type I receptor kinase inhibitor currently undergoing phase I clinical trials for cancer treatment in the United States. This study evaluates whether EW-7197 prevents peritoneal adhesion formation in a rat model. METHODS: Forty-eight female Wistar rats underwent peritoneal adhesion induction by the creation of peritoneal ischemic buttons and were randomly divided into 4 groups of 12 each. The control group received 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction. The 10 mg and 20 mg groups received 10 or 20 mg/kg EW-7197 phosphate dissolved in 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction. The rebound group received 20 mg/kg EW-7197 phosphate dissolved in 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction followed by 0.3 mL vehicle only by gavage once daily for an additional 21 days. After the respective treatments were completed, the animals were euthanized. RESULTS: All rats survived until the end of the study without complications. EW-7197 reduced the incidence, quality, and tenacity of peritoneal adhesions in a dose-dependent manner. Fibrosis and collagen production were reduced in EW-7197-treated peritoneal ischemic buttons. Transforming growth factor ß/Smad2/3 signaling and mesothelial-to-mesenchymal transition were inhibited in EW-7197-treated peritoneal ischemic buttons. Discontinuation of EW-7197 was not associated with rebound effects. CONCLUSION: EW-7197 prevented peritoneal adhesion formation potentially via inhibition of transforming growth factor ß1/Smad2/3-induced mesothelial-to-mesenchymal transition in a rat model.


Assuntos
Compostos de Anilina/farmacologia , Doenças Peritoneais/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Aderências Teciduais/prevenção & controle , Triazóis/farmacologia , Administração Oral , Compostos de Anilina/uso terapêutico , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fibrose , Humanos , Doenças Peritoneais/etiologia , Doenças Peritoneais/patologia , Peritônio/efeitos dos fármacos , Peritônio/patologia , Peritônio/cirurgia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Ratos , Ratos Wistar , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Aderências Teciduais/etiologia , Aderências Teciduais/patologia , Resultado do Tratamento , Triazóis/uso terapêutico
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