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1.
Phytomedicine ; 67: 153163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901891

RESUMO

BACKGROUND: Renal interstitial fibrosis is a common pathway through which chronic kidney disease progresses to end-stage renal disease. There are currently no effective drugs available to treat kidney fibrosis, so traditional medicine is likely to be a candidate. The therapeutic potential of saikosaponin B2 (SSB2), a biologically active ingredient of Radix Bupleuri, on renal fibrosis has not been reported. METHODS: A unilateral ureteral obstruction (UUO) model was conducted to induce renal interstitial fibrosis in mice. SSB2's effect was valuated by histological staining and exploring the changes in expression of relative proteins and mRNAs. A conditional medium containing sonic hedgehog variant protein stimulating normal rat kidney interstitial fibroblast cells (NRK-49F) was used in an in vitro model to determine the possible mechanism. The molecular target of SSB2 was verified using several mutation plasmids. RESULTS: SSB2 administration reduced kidney injury and alleviated interstitial fibrosis by decreasing excessive accumulation of extracellular matrix components in UUO mice. It could also reduce the expression of α-SMA, fibronectin and Gli1, a crucial molecule of the hedgehog (Hh) signaling pathway both in vivo and in vitro. In NIH-3T3 cells simulated by conditional medium containing sonic hedgehog variant protein, SSB2 showed the ability to decrease the expression of Gli1 and Ptch1 mRNA. Using a dual-luciferase reporter assay, SSB2 suppressed the Gli-luciferase reporter activity in NIH-3T3 cells, and the IC50 was 0.49 µM, but had no effect on the TNF-α/NF-κB and Wnt/ß-catenin signaling pathways, indicating the inhibition selectivity on the Hh signaling pathway. Furthermore, SSB2 failed to inhibit the Hh pathway activity evoked by ectopic expression of Gli2ΔN and Smo D473H, suggesting that SSB2 might potentially act on smoothened receptors. CONCLUSION: SSB2 could attenuate renal fibrosis and decrease fibroblast activation by inhibiting the Hh signaling pathway.


Assuntos
Proteínas Hedgehog/metabolismo , Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Animais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Células HEK293 , Humanos , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Células NIH 3T3 , Ácido Oleanólico/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
2.
J Cancer Res Clin Oncol ; 146(2): 297-304, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31960187

RESUMO

PURPOSE: Itraconazole is an antifungal drug that has been clinically used for over 30 years. In recent years, scholars have discovered that it possesses an anticancer effect. Moreover, its mechanism has been clarified to some degree. What deserves to be mentioned is that itraconazole acting on the Hedgehog pathway has made a new progress in the treatment of cancers. While interestingly, studies have demonstrated that the Hedgehog pathway is largely activated in different cancer cells. RESULT: This review tries to highlight the effect of itraconazole on smoothened receptor (SMO) in the Hedgehog pathway, thereby reducing the glioma-associated oncogene homolog (GLI) release and finally exhibiting a range of anticancer effects, promoting apoptosis of cancer cells, and inhibiting proliferation by indirect inhibition of NF-κB pathway and inflammation, moreover, promoting the expression of cyclin-dependent kinase inhibitors, inhibiting the expression of target genes transcribed by GLI such as BCL-2 and Cyclin-D1. Besides, itraconazole increases the number of Bnip3, subsequently, inducing the dissociation of the Beclin-1/BCL-2 binding complex, as a result of ultimately promoting autophagy of cancer cells. CONCLUSION: As a new anticancer drug, whether itraconazole eventually entering clinical application requires the joint eforts of all scholars. In any case, an in-depth study on itraconazole will bring new hope for cancer patients in the near future.


Assuntos
Proteínas Hedgehog/metabolismo , Itraconazol/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Ensaios Clínicos Fase II como Assunto , Humanos , Itraconazol/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo
3.
Mol Pharmacol ; 97(1): 23-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707356

RESUMO

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.


Assuntos
Sítios de Ligação/genética , Bioensaio/métodos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Compostos de Boro/química , Cinamatos/farmacologia , Descoberta de Drogas/métodos , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Luciferases/química , Morfolinas/farmacologia , Nanoestruturas/química , Purinas/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
4.
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
5.
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
6.
J Neurooncol ; 144(1): 11-20, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31177425

RESUMO

AIMS: Skull base meningiomas represent approximately 25% of all meningiomas, nearly 20% of which are atypical or anaplastic. To date, effective medical treatments for meningiomas are still lacking. Genetic aberrations (TRAF7, KLF4, AKT1, and SMO) and the effects of genetic aberrations on the expression of inhibitory immune checkpoint molecules (PD-L1, IDO, and TDO2) in skull base meningiomas are still unclear. METHODS: Genetic alterations in the four genes were identified in 92 skull base meningiomas by Sanger sequencing. The expression differences in immune checkpoints between mutant and wild-type (WT) tumors were determined by immunohistochemistry (IHC) and Western blot (WB). RESULTS: The four mutations were not concurrently detected in the patients with skull base meningiomas. Among the tumors from the KLF4-mutated group, almost half were petroclival meningiomas. KLF4- and TRAF7-mutated tumors were predominantly secretory meningiomas. SMO-mutated tumors exhibited higher calcification, and half of these tumors were observed in the brain midline. Receiver operating characteristic curve analysis indicated that tumor volume can predict KLF4 and TRAF7 mutation status with high sensitivity and specificity, respectively. The IHC and WB analyses indicated that PD-L1, IDO, and TDO2 levels in tumors with TRAF7 mutations were significantly higher than those in WT tumors. Meanwhile, there was a significant difference in TDO2 between tumors with AKT1 mutations and WT tumors. Specifically, TRAF7 mutations could play a key role in skull base meningiomas by regulating the expression of inhibitory immune checkpoints and thus suppressing immune responses. CONCLUSIONS: Checkpoint inhibitors may be potential strategies for targeted immunotherapies of these mutant meningiomas.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Meníngeas/patologia , Meningioma/patologia , Mutação , Neoplasias da Base do Crânio/patologia , Adolescente , Adulto , Idoso , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Feminino , Seguimentos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/imunologia , Neoplasias Meníngeas/metabolismo , Meningioma/genética , Meningioma/imunologia , Meningioma/metabolismo , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Curva ROC , Neoplasias da Base do Crânio/genética , Neoplasias da Base do Crânio/imunologia , Neoplasias da Base do Crânio/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Adulto Jovem
7.
Anal Bioanal Chem ; 411(22): 5721-5727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31214754

RESUMO

Metastases are the leading causes of death in cancer patients. Due to intimate connection with metastasis, Smoothened (Smo) has become a therapeutic target for antimetastatic drugs and can provide early warning of metastasis in breast cancer. Thus, we have developed an electrochemical method in Smo analysis based on small-molecule drugs. Smo on the metastatic cell surface can be internalized after combination with the small-molecule drug. The surplus small-molecule drug and rolling circle amplification (RCA) primer are competitively binding with capture probe on the electrode surface through the click chemical reaction. After RCA reaction, methylene blue is used to label the RCA product. In this process, the more Smo on the metastatic cell surface, the more RCA primer is bound with peptide on the electrode. Therefore, the obtained signal response is positively correlated to Smo on the cancer cells. Moreover, the RCA provides sufficiently high sensitivity, enabling the limit of detection of Smo to be calculated as 0.1 pM (S/N = 3). Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future. Graphical abstract.


Assuntos
Receptor Smoothened/metabolismo , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas/métodos , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Ligação Proteica
8.
PLoS Genet ; 15(5): e1007711, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120883

RESUMO

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.


Assuntos
Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Proteínas Hedgehog/genética , Receptor Smoothened/genética , Obstrução do Fluxo Ventricular Externo/genética , Septo Interventricular/metabolismo , Animais , Movimento Celular , Proliferação de Células , Embrião de Mamíferos , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica , Haploinsuficiência , Proteínas Hedgehog/metabolismo , Heterozigoto , Humanos , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Receptor Smoothened/metabolismo , Tronco Arterial/anormalidades , Tronco Arterial/metabolismo , Obstrução do Fluxo Ventricular Externo/metabolismo , Obstrução do Fluxo Ventricular Externo/patologia , Septo Interventricular/patologia
9.
Cells ; 8(5)2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117185

RESUMO

7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cetocolesteróis/farmacologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Autofagossomos/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor Smoothened/metabolismo
10.
Dev Biol ; 452(1): 55-65, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31071314

RESUMO

The majority of oligodendrocytes in the neocortex originate from neural progenitors that reside in the dorsal forebrain. We recently showed that Sonic Hedgehog (Shh) signaling in these dorsal progenitors is required to produce normal numbers of neocortical oligodendrocytes during embryonic development. Conditional deletion of the Shh signaling effector, Smo, in dorsal progenitors caused a dramatic reduction in oligodendrocyte numbers in the embryonic neocortex. In the current study, we show that the depleted oligodendrocyte lineage in Smo conditional mutants is able to recover to control numbers over time. This eventual recovery is achieved in part by expansion of the ventrally-derived wild-type lineage that normally makes up a minority of the total oligodendrocyte population. However, we find that the remaining dorsally-derived mutant cells also increase in numbers over time to contribute equally to the recovery of the total population. Additionally, we found that the ways in which the dorsal and ventral sources cooperate to achieve recovery is different for distinct populations of oligodendrocyte-lineage cells. Oligodendrocyte precursor cells (OPCs) in the neocortical white matter recover completely by expansion of the remaining dorsally-derived Smo mutant cells. On the other hand, mature oligodendrocytes in the white and gray matter recover through an equal contribution from dorsal mutant and ventral wild-type lineages. Interestingly, the only population that did not make a full recovery was OPCs in the gray matter. We find that gray matter OPCs are less proliferative in Smo cKO mutants compared to controls, which may explain their inability to fully recover. Our data indicate that certain populations of the dorsal oligodendrocyte lineage are more affected by loss of Shh signaling than others. Furthermore, these studies shed new light on the complex relationship between dorsal and ventral sources of oligodendrocytes in the developing neocortex.


Assuntos
Linhagem da Célula , Proteínas Hedgehog/metabolismo , Neocórtex/embriologia , Oligodendroglia/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Animais , Proteínas Hedgehog/genética , Camundongos , Camundongos Knockout , Neocórtex/citologia , Oligodendroglia/citologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células-Tronco/citologia
11.
Dev Biol ; 450(1): 47-62, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30914320

RESUMO

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.


Assuntos
Proteínas Hedgehog/metabolismo , Hidrocefalia , Organogênese , Prosencéfalo , Receptores Acoplados a Proteínas-G/deficiência , Transdução de Sinais , Animais , Movimento Celular , Deleção de Genes , Proteínas Hedgehog/genética , Hidrocefalia/embriologia , Hidrocefalia/genética , Hidrocefalia/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/anormalidades , Tubo Neural/embriologia , Células Neuroepiteliais/metabolismo , Células Neuroepiteliais/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Prosencéfalo/anormalidades , Prosencéfalo/embriologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismo
12.
Histopathology ; 75(1): 118-127, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30861166

RESUMO

AIMS: Because the hedgehog signalling pathway plays a major role in many types of cancer and can nowadays be targeted by specific compounds, we aimed to investigate the role of this pathway in squamous cell carcinoma of the head and neck. METHODS AND RESULTS: Ninety-eight treatment-naive head and neck cancer specimens were immunohistologically stained for SMO, GLI-1, p53 and p16 expression and correlated with clinicopathological factors. Immunoreactivity for SMO and GLI-1 was found in 20 (20.4%) and 52 (53.1%) cases of tumours, respectively. SMO expression correlated with GLI-1 expression (ρ = 0.258, P = 0.010) in univariate and multivariate analysis (P = 0.007, t = 2.81). In univariate analysis, high SMO expression was associated with shorter overall survival (HR = 0.56; 95% CI = 0.32-0.98; P = 0.044) and disease-free survival (HR = 0.53; 95% CI = 0.30-0.95; P = 0.034). In multivariate cox regression analysis SMO expression showed a trend towards an independent predictor for shorter overall survival (HR = 0.57; 95% CI = 0.30-1.05; P = 0.072) and disease-free survival (HR = 0.53; 95% CI = 0.28-1.02; P = 0.056). In head and neck cancer patients with low tumour p16 expression, SMO expression was an independent factor for overall survival (HR = 0.49; 95% CI = 0.24-0.98; P = 0.043) and disease-free survival (HR = 0.45; 95% CI = 0.22-0.96; P = 0.037). CONCLUSION: Although it needs to be confirmed in larger cohorts, our results suggest that targeting SMO might be a potentially therapeutic option in patients with head and neck cancer. In line, molecular pathological analyses including mutation analysis in the hedgehog pathway might point to additional therapeutic leads.


Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Receptor Smoothened/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Biomarcadores Tumorais/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Proteínas Hedgehog/metabolismo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Estudos Retrospectivos , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
13.
Future Med Chem ; 11(6): 617-638, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30912670

RESUMO

Since the Hedgehog signaling pathway has been associated with cancer, it has emerged as a therapeutic target for cancer therapy. The main target among the key Hedgehog proteins is the GPCR-like Smo receptor. Therefore, some Smo antagonists that have entered clinical trials, including the US FDA-approved drugs vismodegib and sonidegib, to treat basal cell carcinoma and medulloblastoma. However, early resistance of these drugs has spawned the need to understand the molecular bases of this phenomena. We therefore reviewed details about Smo receptor structures and the best Smo antagonist chemical structures. In addition, we discussed strategies that should be considered to develop new, safer generations of Smo antagonists that avoid current clinical limitations.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Receptor Smoothened/antagonistas & inibidores , Animais , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Conformação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/química , Receptor Smoothened/metabolismo
14.
Proc Natl Acad Sci U S A ; 116(12): 5550-5557, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819883

RESUMO

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.


Assuntos
Colesterol/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Animais , Rastreamento de Células , Camundongos , Transdução de Sinais , Receptor Smoothened/metabolismo
15.
Proc Natl Acad Sci U S A ; 116(3): 874-879, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598432

RESUMO

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.


Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Receptor Smoothened/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , Transdução de Sinais
16.
Med Res Rev ; 39(3): 1137-1204, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30484872

RESUMO

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure-activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine-based delivery approaches for Hh pathway inhibitors are also discussed concisely.


Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Transdução de Sinais , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Proteínas Hedgehog/metabolismo , Humanos , Receptor Smoothened/metabolismo , Relação Estrutura-Atividade
17.
Mol Cell Biochem ; 454(1-2): 11-23, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30251117

RESUMO

Since PI3K/Akt/mTOR and sonic hedgehog (SHH) signaling pathways are highly activated in glioblastoma-initiating cells (GICs), we examined the effects of inhibiting these pathways on GIC characteristics and tumor growth in mice. NVP-LDE-225 (inhibitor of Smoothened) inhibited the expression of Gli1, Gli2, Smoothened, Patched1, and Patched2, and induced the expression of SuFu, whereas NVP-BEZ-235 (dual inhibitor of PI3K and mTOR) inhibited the expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting the self-renewal capacity of GICs, expression of pluripotency maintaining factors (Nanog, c-Myc, Oct4, and Sox2), Musashi1, cyclin D1, and Bcl-2, and transcription and expression of Gli, and in inducing the expression of cleaved caspase-3, cleaved PARP and Bim. Additionally, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting epithelial-mesenchymal transition. Finally, the combination of NVP-LDE-225 and NVP-BEZ-235 was superior in inhibiting tumor growth, regulating the expression of pluripotency promoting factors, stem cell markers, cell cycle, and cell proliferation, and modulating EMT compared to single agent alone. In conclusion, the combined inhibition of PI3K/Akt/mTOR and SHH pathways was superior to single pathway inhibition in suppressing glioblastoma growth by targeting GICs.


Assuntos
Compostos de Bifenilo/farmacologia , Proliferação de Células , Imidazóis/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Piridinas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/fisiopatologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
18.
Genetics ; 211(1): 151-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30446520

RESUMO

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.


Assuntos
Proteínas Fúngicas/genética , Magnaporthe/genética , Receptor Smoothened/genética , Esporos Fúngicos/crescimento & desenvolvimento , Citoesqueleto de Actina/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Microtúbulos/metabolismo , Morfogênese , Oryza/microbiologia , Septinas/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Esporos Fúngicos/genética , Virulência/genética
19.
Basic Clin Pharmacol Toxicol ; 124(6): 660-669, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30548093

RESUMO

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Proteínas Hedgehog/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Alcaloides de Veratrum/farmacologia , Animais , Isquemia Encefálica/patologia , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Receptores Patched/metabolismo , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Acidente Vascular Cerebral/patologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
20.
Life Sci ; 217: 222-228, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30543826

RESUMO

Itraconazole is an antagonist of the component Smoothened of Hedgehog pathway, which can inhibit the growth of medulloblastoma, basal cell carcinoma, and melanoma, etc. To research the binding mechanism of the Smoothened and triazoles, we used docking and molecular dynamics simulations on the Smoothened crystal structure and six triazoles. Unlike vismodegib, itraconazole can effectively bind into the pocket in the C-terminal domain of the Smoothened crystal structure instead of the N-terminal domain. The binding of itraconazole can change the conformation of the N-terminal domain even although itraconazole only had limited area contacting with N-terminal domain of the Smoothened. Besides, the binding of Itraconazole will not affect the binding of vismodegib. The strong binding affinity could be demonstrated between itraconazole and the Smoothened. Posaconazole and ketoconazole also had the strong binding affinity and the similar binding mode with the Smoothened crystal structure.


Assuntos
Receptor Smoothened/metabolismo , Triazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Itraconazol/química , Itraconazol/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/química , Termodinâmica , Triazóis/química
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