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1.
J Cancer Res Clin Oncol ; 145(9): 2261-2271, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31367836

RESUMO

PURPOSE: To investigate the role of sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) in bladder cancer progression and invasion. METHODS: We cultured three bladder cancer cell lines, muscle-invasive T24 and 5637, and non-muscle-invasive KK47, in the presence of a recombinant-Shh (r-Shh) protein or cyclopamine, a Shh signaling inhibitor, to investigate proliferation and expression of EMT markers. Wound-healing assays and transwell assay were performed to evaluate cell invasion and migration. Mice were then inoculated with bladder cancer cells and treated with cyclopamine. Mouse tumor samples were stained for Shh signaling and EMT markers. RESULTS: R-Shh protein enhanced cell proliferation, whereas cyclopamine significantly suppressed cell proliferation, especially in invasive cancer (5637 and T24) (p < 0.05). R-Shh protein promoted EMT, suppressed E-cadherin and enhanced N-cadherin and vimentin and Gli1, an Shh downstream molecule, while cyclopamine blocked EMT, especially in 5637 and T24. Cyclopamine also inhibited cell invasion and migration in vitro. In the animal study, intraperitoneal injection of cyclopamine significantly suppressed tumor growth in 5637 and T24 in mice (p = 0.01 and p = 0.004, respectively) and slightly suppressing KK47 tumor growth (p = 0.298). Significant cyclopamine-induced suppression of Gli1 in 5637 and T24 mouse tumors (both p = 0.03) was seen, suggesting that muscle-invasive bladder cancer may be more dependent on Shh signaling than non-muscle-invasive bladder cancer. CONCLUSIONS: Shh signaling and EMT were especially enhanced in muscle-invasive bladder cancer progression and invasion, and suppressed by the inhibition of Shh signaling.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Proteínas Hedgehog/fisiologia , Neoplasias Musculares/secundário , Neoplasias da Bexiga Urinária/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Musculares/metabolismo , Invasividade Neoplásica , Transdução de Sinais/fisiologia , Neoplasias da Bexiga Urinária/metabolismo
2.
Nature ; 571(7764): 284-288, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263273

RESUMO

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.


Assuntos
Membrana Celular/química , Proteínas Hedgehog/agonistas , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Esteróis/farmacologia , Animais , Sítios de Ligação , Técnicas Biossensoriais , Domínio Catalítico/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacologia , Proteínas Hedgehog/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Receptor Patched-1/antagonistas & inibidores , Receptor Patched-1/metabolismo , Conformação Proteica , Estabilidade Proteica , Anticorpos de Cadeia Única/imunologia , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/química , Esteróis/química , Esteróis/metabolismo , Proteínas de Xenopus/química
3.
Chem Biol Interact ; 310: 108759, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31326407

RESUMO

Sustained exogenous stimuli induce oxidative stress in granulosa cells and cause cell apoptosis, thereby resulting in follicular atresia. Hyperoside is a natural flavonoid that possesses anti-oxidant activity. The present study aimed to evaluate the effect of hyperoside on hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in granulosa cells. Cell viability was measured using MTT assay. The malondialdehyde (MDA) level and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were detected to reflect the oxidative stress. Flow cytometry was performed to measure the apoptotic rate. Western blot was carried out to determine the expression of Bcl-2, Bax, Sonic hedgehog (SHH), Gli1, and smoothened (SMO). The mRNA levels of SHH, Gli1, and SMO were analyzed using qRT-PCR. We found that hyperoside improved cell viability in H2O2-stimulated granulosa cells. The increased MDA level and decreased activities of SOD, GSH-Px, and CAT caused by H2O2 stimulation were reversed by hyperoside treatment. The apoptotic rate of H2O2-stimulated granulosa cells was reduced after treatment with hyperoside. Hyperoside treatment caused a decrease in Bax expression and an increase in Bcl-2 expression in H2O2-stimulated granulosa cells. The mRNA and protein levels of SHH, Gli1, and SMO in H2O2-stimulated granulosa cells were elevated by hyperoside treatment. Suppression of SHH pathway by cyclopamine attenuated the protective effects of hyperoside on H2O2-induced injury. In short, hyperoside protected granulosa cells from H2O2-induced cell apoptosis and oxidative stress via activation of the SHH signaling pathway.


Assuntos
Células da Granulosa/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Ovário/citologia , Quercetina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Feminino , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras , Quercetina/farmacologia , Quercetina/uso terapêutico , Ratos , Transdução de Sinais
4.
Adv Exp Med Biol ; 1143: 95-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338817

RESUMO

Despite the significant progress that has been made in understanding the biology of leukemia stem cells (LSCs), some key questions regarding the concept of LSCs have not as yet been satisfactorily addressed experimentally. As a result, the clinical relevance of LSCs remains less than clear due to controversies caused largely by technical limitations in efficiently identifying LSCs. This has impeded our ability to fully address the features of genetic heterogeneity and metabolic/epigenetic plasticity of pre-LSCs and LSCs. With the development and use of humanized immunocompromised mice, we are able to more precisely analyze LSCs for their functions and interaction with the bone marrow niche. In addition, some promising targets in LSCs have recently been identified, including Sonic Hedgehog (SHH) and BCL-2, which are highly expressed in AML cells. It is hopeful that new anti-LSC compounds will be tested fully in clinical trials for their efficacy in treating human leukemias.


Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Animais , Medula Óssea/fisiopatologia , Progressão da Doença , Proteínas Hedgehog/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/fisiopatologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
5.
Anticancer Res ; 39(7): 3353-3363, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262856

RESUMO

Vitamin D, or more precisely its active metabolite calcitriol (1,25-(OH)2D3), plays a fundamental role in bone metabolism and differentiation as well as in intestinal absorption of calcium and regulation of calcium-phosphate metabolism. Recent decades have brought about the discovery of the role of calcitriol in processes regulating cell differentiation, proliferation, angiogenesis and apoptosis. This creates the potential for numerous therapeutic applications of vitamin D in diseases associated with autoaggressive immune responses or in cancer. This study presents selected issues regarding current knowledge of the anti-cancer mechanisms of vitamin D.


Assuntos
Neoplasias , Vitamina D/fisiologia , Vitaminas/fisiologia , Animais , Apoptose , Ciclo Celular , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Receptores de Calcitriol/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta/efeitos adversos
6.
Cell Mol Life Sci ; 76(20): 3939-3952, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201464

RESUMO

Articular cartilage is formed at the end of epiphyses in the synovial joint cavity and permanently contributes to the smooth movement of synovial joints. Most skeletal elements develop from transient cartilage by a biological process known as endochondral ossification. Accumulating evidence indicates that articular and growth plate cartilage are derived from different cell sources and that different molecules and signaling pathways regulate these two kinds of cartilage. As the first sign of joint development, the interzone emerges at the presumptive joint site within a pre-cartilage tissue. After that, joint cavitation occurs in the center of the interzone, and the cells in the interzone and its surroundings gradually form articular cartilage and the synovial joint. During joint development, the interzone cells continuously migrate out to the epiphyseal cartilage and the surrounding cells influx into the joint region. These complicated phenomena are regulated by various molecules and signaling pathways, including GDF5, Wnt, IHH, PTHrP, BMP, TGF-ß, and FGF. Here, we summarize current literature and discuss the molecular mechanisms underlying joint formation and articular development.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Regulação da Expressão Gênica , Cápsula Articular/metabolismo , Via de Sinalização Wnt , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular , Condrócitos/citologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Cápsula Articular/citologia , Cápsula Articular/crescimento & desenvolvimento , Osteogênese/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
7.
Nat Commun ; 10(1): 2410, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160587

RESUMO

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.


Assuntos
Neoplasias Cerebelares/imunologia , Macrófagos/imunologia , Meduloblastoma/imunologia , Microambiente Tumoral/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Humanos , Macrófagos/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Microglia/imunologia , Células Mieloides/imunologia , Receptores CCR2/genética , Regulação para Cima
8.
J Biol Regul Homeost Agents ; 33(3): 731-743, 2019 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31232011

RESUMO

Arsenic acts as a human carcinogen and contributes to skin cancer via mechanisms that remain largely unknown. Recent evidence implicates the perturbation of Wnt, Shh and BMP signals as a potential mechanism. We initiated studies to examine gene expression changes in these signaling pathways. Meanwhile, the antagonistic effect of retinoic acid was explored. In this study, HaCaT and NHEK cells were treated with arsenic trioxide (As2O3) alone or in combination with arotinoid trometamol (retinoic acid receptor agonist). Flow cytometric analysis, PCR array and Western blot were used to determine the potential mechanism and signaling pathways associated with arsenic carcinogenesis. The results showed that low concentration As2O3 could stimulate keratinocyte proliferation, and arotinoid trometamol inhibited the process via regulating the expression of about 20 genes. These genes included components of Wnt signaling (CSNK1A1L, CTNNB1, SFRP1, Wnt10B, Wnt11, Wnt16, Wnt5A, Wnt8A), Shh signaling (C6orf138, HHIP, PTCHD1) and BMP signaling pathway (BMP2, BMP7). The changes of some differentially expressed genes of these signaling pathways in As2O3 treatment group were counteracted by the subsequent arotinoid trometamol treatment. Our data suggest that dysregulation and cross-talk of Wnt, Shh and BMP signals play great roles in the process of arsenic-induced carcinogenesis, which could be antagonized by arotinoid trometamol.


Assuntos
Trióxido de Arsênio/farmacologia , Carcinogênese , Queratinócitos/efeitos dos fármacos , Transdução de Sinais , Trometamina/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Linhagem Celular , Proliferação de Células , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias Cutâneas/induzido quimicamente , Via de Sinalização Wnt
9.
Toxicol Lett ; 313: 30-41, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31181250

RESUMO

The hedgehog (HH) signaling pathway plays an important role in lung development, but its significance in silicosis is unclear. We showed that in human coal pneumoconiosis autopsy specimens, Sonic Hedgehog (SHH) and the Glioma-associated oncogene homolog transcription factors family (GLI) 1 proteins were up-regulated, whereas Patch-1 (PTC) was down-regulated. The protein levels of SHH, smoothened (SMO), GLI1, GLI2, α-smooth muscle actin (α-SMA) and collagen type Ⅰ (Col Ⅰ) were also elevated gradually in the bronchoalveolar lavage fluid (BALF) of different stages of coal pneumoconiosis patients, dynamic silica-inhalation rat lung tissue and MRC-5 cells induced by Ang II at different time points, whereas the PTC and GLI3 levels were diminished gradually. Ac-SDKP, an active peptide of renin-angiotensin system (RAS), is an anti-fibrotic tetrapeptide. Targeting RAS axis also has anti-silicotic fibrosis effects. However, their roles on the HH pathway are still unknown. Here, we reported that Ac-SDKP + Captopril, Ac-SDKP, Captopril, or Ang (1-7) could alleviate silicotic fibrosis and collagen deposition, as well as improve the lung functions of silicotic rat. These treatments decreased the expression of SHH, SMO, GLI1, GLI2, α-SMA, and Col Ⅰ and increased the expression of PTC and GLI3 on both the silicotic rat lung tissue and MRC-5 cells induced by Ang II. We also reported that Ang II may promote myofibroblast differentiation via the GLI1 transcription factor and independently of the SMO receptor.


Assuntos
Angiotensina II/farmacologia , Angiotensina I/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Diferenciação Celular/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fibrose Pulmonar/prevenção & controle , Sistema Renina-Angiotensina/efeitos dos fármacos , Silicose/prevenção & controle , Adulto , Idoso , Animais , Antracose/metabolismo , Antracose/patologia , Linhagem Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Hedgehog/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Silicose/metabolismo , Silicose/patologia
10.
Neoplasma ; 66(5): 839-846, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31167533

RESUMO

Overexpression of Sonic hedgehog (Shh) is associated with progression of several cancers. The expression of Shh in non-small cell lung cancer (NSCLC) has been reported with inconsistent results. Lung adenocarcinoma (LAC) and lung squamous cell carcinoma (LSCC) are two major subtypes of NSCLC, which have different genetic genotypes and clinical therapeutic options. The expression of Shh in specimen of patients with NSCLC has yet to be comprehensively determined according to histological subtypes. Shh expression level was determined in 167 NSCLC patients (56 LAC patients and 111 LSCC patients) by immunohistochemical assay (IHC) and disease-free survival and overall survival of patients were analyzed using the Kaplan-Meier method. Shh protein level in pleural effusion from patients with pneumonia or pleural empyema, tuberculosis, LAC and LSCC was measured with enzyme-linked immunoassay (ELISA). We found that Shh expression is increased in tumor tissues from both LAC and LSCC patients compared with the paired adjacent tissues, while Shh level is negatively correlated with tumor differentiation only in LSCC, LSCC patients containing higher-Shh expression have a poorer prognosis. Furthermore, Shh level is elevated in pleural effusion from LSCC patients compared with that of parapneumonic and LAC pleural effusion. Shh expression in tumor tissues or pleural effusion may represent a potential diagnostic and prognostic marker of LSCC patients, pleural effusion Shh may assist to distinguish between LAC and LSCC.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Prognóstico
11.
Nat Commun ; 10(1): 2863, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253779

RESUMO

Cancer stem cells (CSCs) represent a major source of treatment resistance and tumor progression. However, regulation of CSCs stemness is not entirely understood. Here, we report that TSPAN8 expression is upregulated in breast CSCs, promotes the expression of the stemness gene NANOG, OCT4, and ALDHA1, and correlates with therapeutic resistance. Mechanistically, TSPAN8 interacts with PTCH1 and inhibits the degradation of the SHH/PTCH1 complex through recruitment of deubiquitinating enzyme ATXN3. This results in the translocation of SMO to cilia, downstream gene expression, resistance of CSCs to chemotherapeutic agents, and enhances tumor formation in mice. Accordingly, expression levels of TSPAN8, PTCH1, SHH, and ATXN3 are positively correlated in human breast cancer specimens, and high TSPAN8 and ATXN3 expression levels correlate with poor prognosis. These findings reveal a molecular basis of TSPAN8-enhanced Sonic Hedgehog signaling and highlight a role for TSPAN8 in promoting cancer stemness.


Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/fisiologia , Tetraspaninas/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Regulação para Cima
12.
Nature ; 571(7764): 279-283, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168089

RESUMO

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Oxisteróis/química , Receptor Smoothened/química , Receptor Smoothened/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Ligantes , Modelos Moleculares , Oxisteróis/metabolismo , Receptor Patched-1/metabolismo , Conformação Proteica , Transdução de Sinais , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
13.
Cell Prolif ; 52(4): e12637, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31168899

RESUMO

OBJECTIVES: Chondrocyte proliferation and differentiation are crucial for endochondral ossification, but their regulatory mechanism remains unclear. The present study aimed to determine the physiological function of TGFß1 signalling in the proliferation and differentiation of antler chondrocytes and explore its relationship with Notch, Shh signalling and Foxa. MATERIALS AND METHODS: Immunofluorescence, Western blot, MTS assay, flow cytometry, RNA interference and real-time PCR were used to analyse the function and regulatory mechanisms of TGFß1 signalling in antler chondrocyte proliferation and differentiation. RESULTS: TGFß1, TGFBR1 and TGFBR2 were highly expressed in antler cartilage. TGFß1 promoted chondrocyte proliferation, increased the proportion of S-phase cells and induced the expression of hypertrophic chondrocyte markers Col X, Runx2 and Alpl. However, this induction was weakened by TGFß receptor inhibitor SB431542 and Smad3 inhibitor SIS3. Simultaneously, TGFß1 activated Notch and Shh signalling whose blockage attenuated the above effects of rTGFß1, whereas addition of rShh rescued the defects in chondrocyte proliferation and differentiation elicited by SB431542 and SIS3. Further analysis revealed that inhibition of Notch signalling impeded TGFß1 activation of the Shh pathway. Knockdown of Foxa1, Foxa2 and Foxa3 abrogated the effects of TGFß1 on chondrocyte differentiation. Notch and Shh signalling mediated the regulation of Foxa transcription factors by TGFß1. CONCLUSIONS: TGFß1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch-Shh-Foxa pathway.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Chifres de Veado , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Dioxóis/farmacologia , Proteínas Hedgehog/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Isoquinolinas/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptores Notch/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Transdução de Sinais/efeitos dos fármacos
14.
Cancer Treat Rev ; 76: 41-50, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31125907

RESUMO

BACKGROUND: The hedgehog signaling pathway is normally tightly regulated. Mutations in hedgehog pathway components may lead to abnormal activation. Aberrantly activated hedgehog signaling plays a major role in the development of solid and hematological cancer. In recent years, inhibitors have been developed that attenuate hedgehog signaling; 2 have been approved for use in basal cell carcinoma (BCC), while others are under development or in clinical trials. The aim of this review is to provide an overview of known hedgehog inhibitors (HHIs) and their potential for the treatment of hematological cancers and solid tumors beyond BCC. DESIGN: Published literature was searched to identify articles relating to HHIs in noncutaneous cancer. Both preclinical and clinical research articles were included. In addition, relevant clinical trial results were identified from www.clinicaltrials.gov. Information on the pharmacology of HHIs is also included. RESULTS: HHIs show activity in a variety of solid and hematological cancers. In preclinical studies, HHIs demonstrated efficacy in pancreatic cancer, rhabdomyosarcoma, breast cancer, and acute myeloid leukemia (AML). In clinical studies, HHIs showed activity in medulloblastoma, as well as prostate, pancreatic, and hematological cancers. Current clinical trials testing the efficacy of HHIs are underway for prostate, pancreatic, and breast cancers, as well as multiple myeloma and AML. CONCLUSIONS: As clinical trial results become available, it will be possible to discern which additional tumor types are suited to HHI mono- or combination therapy with other anticancer agents. The latter strategy may be useful for delaying or overcoming drug resistance.


Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Ensaios Clínicos como Assunto , Proteínas Hedgehog/metabolismo , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto
15.
Nat Commun ; 10(1): 2320, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127104

RESUMO

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.


Assuntos
Proteínas Hedgehog/ultraestrutura , Receptor Patched-1/ultraestrutura , Domínios Proteicos , Colesterol/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/isolamento & purificação , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Lipoilação , Modelos Moleculares , Receptor Patched-1/isolamento & purificação , Receptor Patched-1/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
16.
Nat Commun ; 10(1): 1929, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028265

RESUMO

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.


Assuntos
Células Endoteliais/patologia , Proteínas Hedgehog/genética , Prolapso da Valva Mitral/patologia , Valva Mitral/patologia , Células-Tronco Pluripotentes/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Endocárdio/metabolismo , Endocárdio/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Valva Mitral/metabolismo , Prolapso da Valva Mitral/genética , Prolapso da Valva Mitral/metabolismo , Prolapso da Valva Mitral/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína Wnt3A/farmacologia
17.
Cell Mol Life Sci ; 76(19): 3875-3889, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30980110

RESUMO

Re-directing mesenchymal stromal cell (MSC) chondrogenesis towards a non-hypertrophic articular chondrocyte-(AC)-like phenotype is important for improving articular cartilage neogenesis to enhance clinical cartilage repair strategies. This study is the first to demonstrate that high levels of non-canonical WNT5A followed by WNT11 and LEF1 discriminated MSC chondrogenesis from AC re-differentiation. Moreover, ß-catenin seemed incompletely silenced in differentiating MSCs, which altogether suggested a role for WNT signaling in hypertrophic MSC differentiation. WNT inhibition with the small molecule IWP-2 supported MSC chondrogenesis according to elevated proteoglycan deposition and reduced the characteristic upregulation of BMP4, BMP7 and their target ID1, as well as IHH and its target GLI1 observed during endochondral differentiation. Along with the pro-hypertrophic transcription factor MEF2C, multiple hypertrophic downstream targets including IBSP and alkaline phosphatase activity were reduced by IWP-2, demonstrating that WNT activity drives BMP and hedgehog upregulation, and MSC hypertrophy. WNT inhibition almost matched the strong anti-hypertrophic capacity of pulsed parathyroid hormone-related protein application, and both outperformed suppression of BMP signaling with dorsomorphin, which also reduced cartilage matrix deposition. Yet, hypertrophic marker expression under IWP-2 remained above AC level, and in vivo mineralization and ectopic bone formation were reduced but not eliminated. Overall, the strong anti-hypertrophic effects of IWP-2 involved inhibition but not silencing of pro-hypertrophic BMP and IHH pathways, and more advanced silencing of WNT activity as well as combined application of IHH or BMP antagonists should next be considered to install articular cartilage neogenesis from human MSCs.


Assuntos
Condrogênese , Células-Tronco Mesenquimais/fisiologia , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomineralização/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Regulação da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Hipertrofia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos SCID , Pessoa de Meia-Idade , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo , Adulto Jovem
18.
Nat Biotechnol ; 37(4): 436-444, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936566

RESUMO

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.


Assuntos
Proteínas Hedgehog/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Animais , Biotecnologia , Padronização Corporal , Diferenciação Celular , Colesterol/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Neurogênese , Organoides/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Transdução de Sinais
19.
Anticancer Res ; 39(4): 2129-2138, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30952759

RESUMO

BACKGROUND/AIM: Early-stage colorectal cancer (CRC) carries a wide range of survival probabilities. Novel biomarkers in this setting are eagerly awaited. Cancer stem cells (CSCs) are considered one of the reasons for treatment failure. This study sought to determine whether activation of pathways governing the function of CSC's could correlate with treatment outcomes. MATERIALS AND METHODS: Tumor specimens from 325 patients were analyzed with immunohistochemistry (IHC) for Hedgehog and Notch pathway activation and results were correlated with prognosis. RESULTS: Positive Notch3 protein expression was an unfavorable prognostic factor for disease-free survival (DFS) and overall survival (OS) (HR=2.43, p=0.024 and HR=2.56, p=0.028, respectively). Activation of the Shh pathway showed univariately longer DFS (HR=0.49, p=0.032). Possible crosstalk between the two pathways was indicated. No further associations between pathway activation and outcome were evident. CONCLUSION: Apart from Notch 3, activation of the pathways, as indicated by IHC expression of their components, did not result in differences in terms of DFS or OS.


Assuntos
Neoplasias Colorretais/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Notch3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Proteína Jagged-1/metabolismo , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor Notch2/metabolismo , Transdução de Sinais , Adulto Jovem
20.
Cell Prolif ; 52(3): e12608, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30932251

RESUMO

OBJECTIVES: We performed histological, cellular and behavioural analyses of the effects of cyclophosphamide (CTX), a chemotherapeutic drug, in the developing cerebellum and aimed to provide valuable insights into clinical application of CTX in children. MATERIALS AND METHODS: C57BL/6 mice and Math1-dependent GFP expression transgenic mice were used in the research. H&E staining was performed to analyse histological effects of CTX in the cerebellum. Staining for EdU and TUNEL was used to estimate the cell proliferation and apoptosis. Rotarod test and hanging wire test were used to evaluate the behavioural functions. Immunofluorescent staining was used to identify the cell types. The differentiation markers and genes related to Sonic Hedgehog (SHH) signalling were measured via quantitative real-time PCR or immunoblotting. RESULTS: We found that while CTX induced a significant reduction in cell proliferation and increased apoptosis in the EGL in 48 hours, the behavioural functions and the multilayer laminar structure of cerebella were largely restored when the mice grew to adults. Mechanistically, granule neuron progenitors, driven by the SHH signalling, enhanced the capability of proliferation quickly after CTX administration was stopped, which allowed the developing cerebellum to catch up and to gradually replenish the injury. CONCLUSION: The chemotherapeutic agent CTX induces an immediate damage to the developing cerebellum, but the cerebellar multilayer laminar structure and motor function can be largely restored if the agent is stopped shortly after use.


Assuntos
Antineoplásicos Alquilantes/efeitos adversos , Comportamento Animal/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Ciclofosfamida/efeitos adversos , Animais , Animais Recém-Nascidos , Antineoplásicos Alquilantes/administração & dosagem , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cerebelo/crescimento & desenvolvimento , Cerebelo/patologia , Criança , Ciclofosfamida/administração & dosagem , Proteínas Hedgehog/metabolismo , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
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