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Cancer Feb 2014During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone....
BACKGROUND
During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone. In this study, the authors investigated the components of dysregulated Hedgehog signaling as potential therapeutic targets for osteosarcoma.
METHODS
Small-molecule agonists and antagonists that modulate the Hedgehog pathway at different levels were used to investigate the mechanisms of dysregulation and the efficacy of Hedgehog blockade in osteosarcoma cell lines. The inhibitory effect of a small-molecule Smoothened (SMO) antagonist, IPI-926 (saridegib), also was examined in patient-derived xenograft models.
RESULTS
An inverse correlation was identified in osteosarcoma cell lines between endogenous glioma-associated oncogene 2 (GLI2) levels and Hedgehog pathway induction levels. Cells with high levels of GLI2 were sensitive to GLI inhibition, but not SMO inhibition, suggesting that GLI2 overexpression may be a mechanism of ligand-independent activation. In contrast, cells that expressed high levels of the Hedgehog ligand gene Indian hedgehog (IHH) and the target genes patched 1 (PTCH1) and GLI1 were sensitive to modulation of both SMO and GLI, suggesting ligand-dependent activation. In 2 xenograft models, active autocrine and paracrine, ligand-dependent Hedgehog signaling was identified. IPI-926 inhibited the Hedgehog signaling interactions between the tumor and the stroma and demonstrated antitumor efficacy in 1 of 2 ligand-dependent models.
CONCLUSIONS
The current results indicate that both ligand-dependent and ligand-independent Hedgehog dysregulation may be involved in osteosarcoma. It is the first report to demonstrate Hedgehog signaling crosstalk between the tumor and the stroma in osteosarcoma. The inhibitory effect of IPI-926 warrants additional research and raises the possibility of using Hedgehog pathway inhibitors as targeted therapeutics to improve treatment for osteosarcoma.
Topics: Adolescent; Adult; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Female; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Male; Molecular Targeted Therapy; Nuclear Proteins; Osteosarcoma; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2
PubMed: 24151134
DOI: 10.1002/cncr.28439 -
Oncotarget Mar 2016The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates...
The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.
Topics: Cadherins; Cell Line, Tumor; Cell Movement; Chromones; Enzyme Activation; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Morpholines; Neoplasm Invasiveness; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pyridines; Pyrimidines; Signal Transduction; Snail Family Transcription Factors; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Veratrum Alkaloids; Vimentin; Zinc Finger Protein GLI1
PubMed: 26859575
DOI: 10.18632/oncotarget.7228 -
Molecules (Basel, Switzerland) Mar 2020Peimine (also known as verticine) is the major bioactive and characterized compound of , a traditional Chinese medicine that is most frequently used to relieve a cough....
Peimine (also known as verticine) is the major bioactive and characterized compound of , a traditional Chinese medicine that is most frequently used to relieve a cough. Nevertheless, its molecular targets and mechanisms of action for cough are still not clear. In the present study, potential targets of peimine for cough were identified using computational target fishing combined with manual database mining. In addition, protein-protein interaction (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using, GeneMANIA and Database for Annotation, Visualization and Integrated Discovery (DAVID) databases respectively. Finally, an interaction network of drug-targets-pathways was constructed using Cytoscape. The results identified 23 potential targets of peimine associated with cough, and suggested that MAPK1, AKT1 and PPKCB may be important targets of pemine for the treatment of cough. The functional annotations of protein targets were related to the regulation of immunological and neurological function through specific biological processes and related pathways. A visual representation of the multiple targets and pathways that form a network underlying the systematic actions of peimine was generated. In summary, peimine is predicted to exert its systemic pharmacological effects on cough by targeting a network composed of multiple proteins and pathways.
Topics: Cevanes; Computational Biology; Cough; Drugs, Chinese Herbal; Gene Expression Profiling; Humans; Medicine, Chinese Traditional; Models, Biological; Protein Interaction Maps; Signal Transduction
PubMed: 32131410
DOI: 10.3390/molecules25051105 -
California Medicine Oct 1953The hypotensive action of veratrum viride given intravenously was studied in 24 patients, 22 of them hypertensive and 2 normotensive. Vasodepression of considerable but...
The hypotensive action of veratrum viride given intravenously was studied in 24 patients, 22 of them hypertensive and 2 normotensive. Vasodepression of considerable but variable degree was obtained in all patients. Maximum hypotension occurred 8 to 15 minutes after injection and relative hypotension usually lasted at least two hours. In four patients subnormal hypotension occurred but there were no clinical manifestations of shock. The blood pressure rose promptly when pressor drugs were administered.A dose of 0.3 to 0.5 mg. brought about a satisfactory decrease in blood pressure. The degree of decrease was affected by the speed of administration and in a few patients by idiosyncratic sensitivity to the drug. Veratrum has an extravagal action on the pulse rate, and in that and other respects resembles digitalis. Veratrum should be given with caution to digitalized patients. Atropine reduced but did not abolish the hypotensive effect of veratrum, and was more effective when given before veratrum. This indicates that the parasympathomimetic action of veratrum is important in the mechanism of blood pressure reduction.
Topics: Blood Pressure; Cardiovascular System; Heart Rate; Hematinics; Hypertension; Hypotension; Injections, Intravenous; Parasympathomimetics; Pharmaceutical Preparations; Plant Extracts; Vasoconstrictor Agents; Veratrum; Veratrum Alkaloids; Vitamin B 12
PubMed: 13094542
DOI: No ID Found -
PloS One 2012The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon...
The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.
Topics: Apoptosis; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Butyrates; Cell Survival; Chemokine CCL2; Colonic Neoplasms; Cytokines; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; HT29 Cells; Hedgehog Proteins; Humans; Interleukin-8; Kruppel-Like Transcription Factors; Oncogene Proteins; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; beta Catenin
PubMed: 23028941
DOI: 10.1371/journal.pone.0045332 -
Discovery Medicine Nov 2016Dermal fibrosis is characterized by the activation of the matrix-producing 'positive' myofibroblasts, and the relentless production and deposition of extracellular...
Dermal fibrosis is characterized by the activation of the matrix-producing 'positive' myofibroblasts, and the relentless production and deposition of extracellular matrix. The hedgehog pathway has recently been demonstrated to work in a pro-fibrotic manner in systemic sclerosis (SSc). A negative regulator of the hedgehog pathway (Hh), the suppressor of fused (Sufu), was shown to be involved in the activation of fibrotic diseases. However, the exact role of Sufu in fibrosis has not been investigated so far. In our study, we aimed to define the role of sufu in the process of fibrosis using dermal fibroblasts of healthy donors that were cultured in vitro. Cyclopamine, a Smo antagonist, and Sufu lentivector were used to treat or transfect cells. The expression of fibrosis markers and ERK1/2, Smad2, and GSK3β at the protein level was determined by Western blot. Fibroblast migration was measured by in vitro wound healing assay. Bleomycin-induced dermal fibrosis mouse model was introduced to assess the effect of cyclopamine on dermal fibrosis in vivo. We found that cyclopamine significantly upregulated the expression of Sufu. Both cyclopamine and Sufu lentivector reduced migration and myofibroblast differentiation of human dermal fibroblasts at a statistically significant level. Furthermore, cyclopamine reversed dermal fibrosis induced by TGF-β1. Cyclopamine and the overexpression of Sufu inhibited the phosphorylation of GSK-3β and restrained the migration of fibroblasts. Dermal fibrosis was inhibited by intraperitoneal injection of cyclopamine in a mouse model of scleroderma. Our findings suggest that cyclopamine and Sufu-overexpression may effectively inhibit the endogenous as well as the TGF-β1-induced activation of fibroblasts through subsequent activation of GSK-3β. Sufu agonists may be a promising approach in the development of antifibrotic medications for dermal fibrosis and systemic sclerosis.
Topics: Animals; Bleomycin; Cell Movement; Disease Models, Animal; Fibrosis; Hedgehog Proteins; Humans; Immunoblotting; In Vitro Techniques; Mice; Mice, Inbred C57BL; Phosphorylation; Real-Time Polymerase Chain Reaction; Repressor Proteins; Skin; Skin Diseases; Veratrum Alkaloids
PubMed: 28009972
DOI: No ID Found -
Clinical Cancer Research : An Official... Sep 2012The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).
PURPOSE
The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).
EXPERIMENTAL DESIGN
The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice.
RESULTS
A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors.
CONCLUSIONS
An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.
Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Anilides; Animals; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Mesothelioma; Mice; Middle Aged; NIH 3T3 Cells; Phosphoproteins; Pleural Effusion, Malignant; Pyridines; RNA, Small Interfering; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Survivin; Tomatine; Transcription Factors; Transplantation, Heterologous; Veratrum Alkaloids; YAP-Signaling Proteins; Zinc Finger Protein GLI1
PubMed: 22733539
DOI: 10.1158/1078-0432.CCR-12-0599 -
Japanese Journal of Pharmacology Nov 1994Itopride is a gastroprokinetic benzamide derivative. This agent inhibited both electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE)....
Itopride is a gastroprokinetic benzamide derivative. This agent inhibited both electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE). The IC50 of itopride with AChE (2.04 +/- 0.27 microM) was, however, 100-fold less than that with BuChE, whereas in the case of neostigmine with AChE (11.3 +/- 3.4 nM), it was 10-fold less. The recovery of AChE activity inhibited by 10(-7) M neostigmine was partial, but that inhibited by up to 3 x 10(-5) M itopride was complete when the reaction mixture was subjected to ultrafiltration. Double reciprocal plots of the experimental data showed that both Km and Vmax were affected by itopride, suggesting that the inhibition is a "mixed" type, although primarily being an uncompetitive one. The inhibitory effect of itopride on cholinesterase (ChE) activity in guinea pig gastrointestine was much weaker than that on pure AChE. However, in the presence of a low dose of diisopropyl fluorophosphate, just enough to inhibit BuChE but not AChE, the IC50s of itopride against ChE activities were found to be about 0.5 microM. In conclusion, itopride exerts reversible and a "mixed" type of inhibition preferably against AChE. The IC50 of itopride for electric eel and guinea pig gastrointestinal AChE inhibition was 200 times and 50 times as large as that of neostigmine, respectively.
Topics: Acetylcholine; Animals; Benzamides; Cholinesterase Inhibitors; Digestive System; Dose-Response Relationship, Drug; Gastrointestinal Motility; Guinea Pigs; Male; Neostigmine; Veratrum Alkaloids
PubMed: 7869618
DOI: 10.1254/jjp.66.317 -
Developmental Biology Dec 2004Taste papillae are ectodermal specializations that serve to house and distribute the taste buds and their renewing cell populations in specific locations on the tongue....
Taste papillae are ectodermal specializations that serve to house and distribute the taste buds and their renewing cell populations in specific locations on the tongue. We previously showed that Sonic hedgehog (Shh) has a major role in regulating the number and spatial pattern of fungiform taste papillae on embryonic rat tongue, during a specific period of papilla formation from the prepapilla placode. Now we have immunolocalized the Shh protein and the Patched receptor protein (Ptc), and have tested potential roles for Shh in formation of the tongue, emergence of papilla placodes, development of papilla number and size, and maintenance of papillae after morphogenesis is advanced. Cultures of entire embryonic mandible or tongues from gestational days 12 to 18 [gestational or embryonic days (E)12-E18] were used, in which tongues and papillae develop with native spatial, temporal, and molecular characteristics. The Shh signaling pathway was disrupted with addition of cyclopamine, jervine, or the 5E1 blocking antibody. Shh and Ptc proteins are diffuse in prelingual tissue and early tongue swellings, and are progressively restricted to papilla placodes and then to regions of developing papillae. Ptc encircles the dense Shh immunoproduct in papillae at various stages. When the Shh signal is disrupted in cultures of E12 mandible, tongue formation is completely prevented. At later stages of tongue culture initiation, Shh signal disruption alters development of tongue shape (E13) and results in a repatterned fungiform papilla distribution that does not respect normally papilla-free tongue regions (E13-E14). Only a few hours of Shh signal disruption can irreversibly alter number and location of fungiform papillae on anterior tongue and elicit papilla formation on the intermolar eminence. However, once papillae are well formed (E16-E18), Shh apparently does not have a clear role in papilla maintenance, nor does the tongue retain competency to add fungiform papillae in atypical locations. Our data not only provide evidence for inductive and morphogenetic roles for Shh in tongue and fungiform papilla formation, but also suggest that Shh functions to maintain the interpapilla space and papilla-free lingual regions. We propose a model for Shh function at high concentration to form and maintain papillae and, at low concentration, to activate between-papilla genes that maintain a papilla-free epithelium.
Topics: Animals; Embryo, Mammalian; Female; Gestational Age; Hedgehog Proteins; Mandible; Membrane Proteins; Microscopy, Electron, Scanning; Morphogenesis; Patched Receptors; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Signal Transduction; Taste Buds; Tissue Culture Techniques; Tongue; Trans-Activators; Veratrum Alkaloids
PubMed: 15581865
DOI: 10.1016/j.ydbio.2004.07.042 -
Molecules (Basel, Switzerland) Aug 2017Rapid, non-destructive, and accurate quantitative determination of the effective components in traditional Chinese medicine (TCM) is required by industries, planters,...
Determination and Visualization of Peimine and Peiminine Content in Fritillaria thunbergii Bulbi Treated by Sulfur Fumigation Using Hyperspectral Imaging with Chemometrics.
Rapid, non-destructive, and accurate quantitative determination of the effective components in traditional Chinese medicine (TCM) is required by industries, planters, and regulators. In this study, near-infrared hyperspectral imaging was applied for determining the peimine and peiminine content in bulbi under sulfur fumigation. Spectral data were extracted from the hyperspectral images. High-performance liquid chromatography (HPLC) was conducted to determine the reference peimine and peiminine content. The successive projection algorithm (SPA), weighted regression coefficient (), competitive adaptive reweighted sampling (CARS), and random frog (RF) were used to select optimal wavelengths, while the partial least squares (PLS), least-square support vector machine (LS-SVM) and extreme learning machine (ELM) were used to build regression models. Regression models using the full spectra and optimal wavelengths obtained satisfactory results with the correlation coefficient of calibration (), cross-validation () and prediction () of most models being over 0.8. Prediction maps of peimine and peiminine content in bulbi were formed by applying regression models to the hyperspectral images. The overall results indicated that hyperspectral imaging combined with regression models and optimal wavelength selection methods were effective in determining peimine and peiminine content in bulbi, which will help in the development of an online detection system for real-world quality control of bulbi under sulfur fumigation.
Topics: Cevanes; Fritillaria; Fumigation; Regression Analysis; Spectrum Analysis; Sulfur
PubMed: 28832506
DOI: 10.3390/molecules22091402