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Mark Pines - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Wilms tumor xenograft progression by Halofuginone is accompanied by activation of WT-1 gene expression.
The Journal of urology, 2020Co-Authors: Jehonathan H Pinthus, Arnon Nagler, Olga Genina, Yuval Sheffer, Eduard Fridman, Mark PinesAbstract:Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of Halofuginone, an inhibitor of collagen type I synthesis and angiogenesis, to inhibit WT development in xenografts models. WTs derived from 2 patients with favorable histology at different disease stages were implanted subcutaneously or orthotopically in the kidneys of nude mice. Halofuginone was administered intraperitoneally (2 mug per mouse every other day) or given in the diet (1 part per million). Independent of disease stage, tumor location or administration route, Halofuginone caused a decrease in angiogenesis that resulted in marked inhibition of tumor development. This result was accompanied by a reduction in collagen synthesis, reduced levels of hepatocyte growth factor receptor MET and increased levels of the tumor suppressor protein WT1. In culture Halofuginone increased the synthesis of WT1 in the human WT cell-line SK-NEP-1 and in other cancer cell lines such as hepatocellular carcinoma and prostate cancer. In SK-NEP-1 Halofuginone also lowered erb B2 levels and reduced cell proliferation. These results suggest that Halofuginone is a potent inhibitor of WT progression. Because of its unique mode of action, Halofuginone may decrease the treatment burden when combined with chemotherapy.
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Effect of Halofuginone on the development of tight skin (TSK) syndrome.
Autoimmunity, 2020Co-Authors: Tracy L Mcgaha, Mark Pines, Robert G Phelps, Harry Spiera, Takao Kodera, Constantin A BonaAbstract:The end point of pathogenic events in scleroderma is fibrosis of the skin and internal organs. Fibrosis in scleroderma results from the over synthesis and deposition of collagen in the connective tissue. The morbidity and mortality of the scleroderm is very high and presently there is no specific treatment. Halofuginone is a drug with great potential for the treatment of scleroderma since it inhibits the synthesis of collagen type I by fibroblasts. We have studied the in vivo effect of Halofuginone in tight skin (TSK) mice that spontaneously develop a scleroderma-like disease due to a genetic defect. Our results demonstrate that Halofuginone prevented the occurrence of skin sclerosis when administered to newborn mice and reduced cutaneous hyperplasia when administered in adult TSK mice. These effects correlated with a decreased number of cells synthesizing collagen gene transcripts and a reduction in the level of autoantibodies specific for human target antigens. These results indicate that Halofuginone may have use as a therapeutic in the treatment of fibrotic disease.
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hydroxy group requirement for Halofuginone dependent inhibition of muscle fibrosis and improvement of histopathology in the mdx mouse model for duchenne muscular dystrophy
Histology and Histopathology, 2019Co-Authors: Gili Wellner, Mark Pines, Olga Genin, Sharon Mordechay, Paul Evans, Orna HalevyAbstract:In Duchenne muscular dystrophy (DMD), the progressive loss of muscle and its ability to function is associated with significant fibrosis, representing the major disease complication in patients. Halofuginone, a halogenated analog of the naturally occurring febrifugine, has been shown to prevent fibrosis in various animal models, including those of muscular dystrophies. Here, two optically active enantiomers of deoxyHalofuginone—a Halofuginone analogue in which the hydroxy group in position 3 was removed from the piperidinyl entity—were evaluated with respect to their effect on muscle histopathology in mdx mice. Male mdx mice were treated with either deoxyHalofuginone (as single enantiomers or in racemic form), or Halofuginone, for 10 weeks, starting at the age of 4 weeks. Halofuginone caused a significant reduction in total collagen content, degenerative areas, as well as in utrophin and phosphorylated-Smad3 levels in the mdx diaphragms. However, neither the deoxyHalofuginone enantiomers, nor its racemic form had any effect on these parameters. A positive effect of the deoxyHalofuginone (+)-enantiomer was observed on myofiber diameters; however, it was lesser than that of Halofuginone. It is concluded that the hydroxy group plays a key role in Halofuginone’s effects related to fibrosis in DMD, and points towards the transforming growth factor β/Smad3 signaling pathway being involved in this inhibition. Elucidation of the structure–function relationship of Halofuginone, in relation to inhibiting fibrosis in muscular dystrophies, is of the utmost importance for creating the next generation of anti-fibrotic therapies that will be more efficacious and less toxic, hence improving life quality of patients.
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Halofuginone promotes satellite cell activation and survival in muscular dystrophies
Biochimica et Biophysica Acta, 2016Co-Authors: Hila Barzilaitutsch, Mark Pines, Anna Bodanovsky, Hadar Maimon, Orna HalevyAbstract:Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, Halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf(-/-) mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of Halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating Halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf(-/-) mice, Halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the Halofuginone-induced cell survival, suggesting this pathway's involvement in mediating Halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, Halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.
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Halofuginone improves muscle cell survival in muscular dystrophies
Biochimica et Biophysica Acta, 2014Co-Authors: Anna Bodanovsky, Mark Pines, Noga Guttman, Hila Barzilaitutsch, Ola Genin, Oshrat Levy, Orna HalevyAbstract:Abstract Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that Halofuginone would reduce apoptosis—the presumed cause of satellite-cell depletion during muscle degradation—in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when Halofuginone was added to cultured myofibers. No apparent effect of Halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by Halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating Halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which Halofuginone improves muscle pathology and function in muscular dystrophies.
Arnon Nagler - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Wilms tumor xenograft progression by Halofuginone is accompanied by activation of WT-1 gene expression.
The Journal of urology, 2020Co-Authors: Jehonathan H Pinthus, Arnon Nagler, Olga Genina, Yuval Sheffer, Eduard Fridman, Mark PinesAbstract:Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of Halofuginone, an inhibitor of collagen type I synthesis and angiogenesis, to inhibit WT development in xenografts models. WTs derived from 2 patients with favorable histology at different disease stages were implanted subcutaneously or orthotopically in the kidneys of nude mice. Halofuginone was administered intraperitoneally (2 mug per mouse every other day) or given in the diet (1 part per million). Independent of disease stage, tumor location or administration route, Halofuginone caused a decrease in angiogenesis that resulted in marked inhibition of tumor development. This result was accompanied by a reduction in collagen synthesis, reduced levels of hepatocyte growth factor receptor MET and increased levels of the tumor suppressor protein WT1. In culture Halofuginone increased the synthesis of WT1 in the human WT cell-line SK-NEP-1 and in other cancer cell lines such as hepatocellular carcinoma and prostate cancer. In SK-NEP-1 Halofuginone also lowered erb B2 levels and reduced cell proliferation. These results suggest that Halofuginone is a potent inhibitor of WT progression. Because of its unique mode of action, Halofuginone may decrease the treatment burden when combined with chemotherapy.
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inhibition of matrix metalloproteinase 2 by Halofuginone is mediated by the egr1 transcription factor
Anti-Cancer Drugs, 2012Co-Authors: Eyal Zcharia, Avichai Shimoni, Israel Vlodavsky, Ruth Atzmon, Adi Nagler, Tamar Peretz, Arnon NaglerAbstract:Halofuginone, a low-molecular-weight quinazolinone alkaloid that inhibits collagen α1(I), has been shown to suppress cancer growth, metastasis, and angiogenesis. These activities were attributed in part to the inhibition of matrix metalloproteinase-2 (MMP-2). The present study was carried out to explore the molecular mechanism underlying this effect. We found a marked (50%) inhibition in MMP-2 gelatinolytic activity in human breast cancer MDA-MB-435 cells pretreated with as little as 50 ng/ml of Halofuginone, a concentration that markedly inhibited their invasive and proliferative capacities. We further show that both early growth response 1 (Egr-1) and Nab-2 (corepressor of Egr1 activation) are upregulated by Halofuginone in a dose-dependent and time-dependent (up to 5 h) manner. Using MMP-2 reporter gene and chromatin immunoprecipitation analyses, we found that Egr-1 binds to the MMP-2 promoter and inhibits its activity. Altogether, our results identify the downstream elements (Egr-1, Nab-2, and MMP-2) by which Halofuginone exerts its antitumoral effect, thereby advancing its potential therapeutic application as an anticancer drug.
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Halofuginone induces post transcriptional down regulation of cyclin d1 cell cycle arrest and apoptosis in mantle cell lymphoma cells through activation of integrated stress response pathways
Blood, 2010Co-Authors: Rodrigo Protosiqueira, Melina G Santos, Valdemir M Carvalho, Yumi H Maekawa, Leonardo A Testagrossa, Zelia R Andrade, Jose Salvador Oliveira, Maria De Lourdes Lopes Ferrari Chauffaille, Marco Antonio Zago, Arnon NaglerAbstract:Abstract 773 Mantle cell lymphoma (MCL) remains an incurable disease and has the worst outcome among B-cell lymphomas. Patients generally have a good response to first line treatment but most relapse and tend to have shorter responses or resistant disease. Thus, novel treatment strategies capable of providing and sustaining durable responses are clearly needed. The translocation t(11;14), a hallmark of MCL, leads to cyclin D1 overexpression and is invariably accompanied by different secondary genetic lesions that collaborate for lymphomagenesis. In a previous study, we found that several genes related to the AKT, WNT and TGFβ signaling pathways were aberrantly expressed in MCL. The role of the AKT and WNT pathways in MCL pathogenesis has been well established by other groups, but little is known about the role of the TGFβ pathway. To address this issue, we tested whether Halofuginone, a small molecule with recognized anti-TGFβ and antifibrotic activity, would have cytotoxic effect against a panel of MCL cell lines. We found that Halofuginone at nanomolar levels had significant cytotoxic activity against MCL cell lines as measured by the MTT assay. The IC 50 9s for Mino and HBL-2 cell lines were 30 and 61 ng/mL at 48h, respectively, with IC 50 9s for Jeko-1, JVM-2 and Granta-519 falling in between. Halofuginone induced apoptosis in Mino and HBL-2 cells in a time- and concentration-dependent fashion, as evidenced by annexin V/7-AAD staining by flow cytometry and electron microscopy studies. However, Halofuginone failed to inhibit SMAD2 phosphorylation induced by recombinant TGFβ1 in Mino and HBL-2 cells, as shown by Western blot analysis, and co-treatment experiments with TGFβ1 failed to show antagonism, suggesting that the effect of Halofuginone in MCL is not mediated by TGFβ inhibition. Cell cycle analysis of Mino and HBL-2 cells exposed to Halofuginone revealed time- and concentration-dependent accumulation in G1 (83% of Mino cells at G1 upon exposure to 50 ng/mL for 24h vs. 48% in untreated Mino cells), and immunocytochemical analysis showed that this effect was accompanied by striking down-regulation of cyclin D1 protein levels starting as early as 3h after exposure to Halofuginone, a finding that was reproduced in primary MCL cells. Real-time RT-PCR experiments, however, revealed up-regulation of cyclin D1 mRNA levels by Halofuginone over time, suggesting a post-transcriptional mechanism for the observed down-regulation of cyclin D1 protein levels. Western blot analysis of Mino and HBL-2 cells exposed to Halofuginone for 24h showed a concentration-dependent phosphorylation of GCN2, PERK and EIF2α, and up-regulation of ATF4. These findings point to an activation of integrated stress response pathways (amino acid starvation response and endoplasmic reticulum stress response) that causes a general shutdown in protein synthesis and explain, at least partially, the down-regulation in cyclin D1 levels. To further characterize the proteins targeted by Halofuginone in MCL we employed a proteomic profiling approach in which differentially expressed proteins were revealed by label-free liquid chromatography tandem mass spectrometry (LC-MSE) analysis on a nanoAcquity system coupled to a Synapt MS Q-Tof mass spectrometer. A comprehensive catalogue representing 147 proteins was generated from this analysis and we found that several members of the heat shock protein family are up-regulated in Mino cells exposed to 100 ng/mL of Halofuginone for 14h, the relevance of which is currently under investigation. Together, our data demonstrate that Halofuginone at nanomolar levels has significant antiproliferative and cytotoxic effects in MCL cells that are induced by the activation of integrated stress response pathways. More importantly, our study provides a rationale for exploring the clinical activity of this oral agent in patients with MCL. Disclosures: No relevant conflicts of interest to declare.
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Halofuginone a novel antimyeloma agent upregulates c jun jnk and p 53 protein in vitro and inhibits tumor growth and improves survival in in vivo multiple myeloma mm animal models
Blood, 2008Co-Authors: Merav Leiba, Arnon Nagler, Jana Jakubikova, Steffen Klippel, Constantine S Mitsiades, Teru Hideshima, Paul G Richardson, Yutaka Okawa, Hiroshi Ikeda, Kenneth C AndersonAbstract:Halofuginone, a synthetic derivative of quinazolinone alkaloid, previously has been shown to have anti-cancer effects in various solid and hematological malignancies. Halofuginone inhibits mainly collagen type I synthesis, and extracellular matrix formation, via the inhibition of TGFβ signaling, matrix metalloproteinase 2(MMP2), and angiogenesis. Last year, we first reported, that Halofuginone in a low doses (IC50 of 50—100 nM) induces cytotoxicity in multiple MM cell lines, including cells resistant to conventional (e.g., dexamethasone, alkylating agents, and anthracyclines) or novel (e.g. thalidomide and bortezomib) anti-MM agents and overcomes the survival and growth advantages conferred by interleukin-6, insulin-like growth factor-1 and by bone marrow stroma cells. Halofuginone induced apoptosis in a caspase 3, 8, and 9 dependent mechanisms, reduced mitochondrial membrane potential, and down regulated MCL1 protein. We now assessed the cytotoxic effect of Halofuginone in primary MM patient cells in vitro and, its effect on tumor growth and survival in in vivo models. We found that Halofuginone also induces growth inhibition and cell death in primary MM cells (n=4, IC50: 100–200nM). Importantly, Halofuginone demonstrated additive or synergistic effects with some of the established anti-MM agents such as Melphalan, Dexamethasone, and Lenalidomide. In addition, Halofuginone inhibits IL6 production in the supernatant of a co-culture of MM.1S cells with HS-5 stromal cell line. Mechanistically, Halofuginone induces MM cell death, which involves the up-regulation of c-jun NH2-terminal kinase signaling (JNK), c-Jun, as well as the p-53 proapoptotic protein. Additionally, the in vivo anti-MM activity of Halofuginone was evaluated in 2 separate in vivo models, a xenograft model in SCID mice (subcutaneous injection of MM1S cells), and a model of diffuse MM lesions in SCID-beige mice (generated by i.v. injections of OPM-2 cells). In both models, mice were first sublethally irradiated (200 rads), injected s.c or i.v., respectively, with 1×106 MM cells and then randomly assigned to receive, either treatment with 0.75mg/kg Halofuginone (IP or by oral gavage, respectively; n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. In both models, Halofuginone inhibited MM tumor growth and improved survival, 70% vs 40% at 140 days (NS) in the treated vs control group respectively (figure). Clinical evidence of adverse events (weight loss, vomiting) were not observed. Halofuginone may, thus represents a promising novel orally bioavailable anti-MM agent that needs further evaluation for possible clinical trials in MM. Diffuse MM lesions in SCID-beige mice. Animals were sub lethally irradiated (200rads), injected i.v. with 1×106 OPM2 cells and then randomly assigned to receive, either Halofuginone treatment (by oral gavage; 0.75mg/kg (n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. ![Figure][1] Figure [1]: pending:yes
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prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by Halofuginone
Neuromuscular Disorders, 2008Co-Authors: Tidhar Turgeman, Orna Halevy, Arnon Nagler, Olga Genin, Yosey Hagai, Kyla D Huebner, Davinder S Jassal, Judy E Anderson, Mark PinesAbstract:Abstract Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of Halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFβ in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, Halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.
Olga Genina - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Wilms tumor xenograft progression by Halofuginone is accompanied by activation of WT-1 gene expression.
The Journal of urology, 2020Co-Authors: Jehonathan H Pinthus, Arnon Nagler, Olga Genina, Yuval Sheffer, Eduard Fridman, Mark PinesAbstract:Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of Halofuginone, an inhibitor of collagen type I synthesis and angiogenesis, to inhibit WT development in xenografts models. WTs derived from 2 patients with favorable histology at different disease stages were implanted subcutaneously or orthotopically in the kidneys of nude mice. Halofuginone was administered intraperitoneally (2 mug per mouse every other day) or given in the diet (1 part per million). Independent of disease stage, tumor location or administration route, Halofuginone caused a decrease in angiogenesis that resulted in marked inhibition of tumor development. This result was accompanied by a reduction in collagen synthesis, reduced levels of hepatocyte growth factor receptor MET and increased levels of the tumor suppressor protein WT1. In culture Halofuginone increased the synthesis of WT1 in the human WT cell-line SK-NEP-1 and in other cancer cell lines such as hepatocellular carcinoma and prostate cancer. In SK-NEP-1 Halofuginone also lowered erb B2 levels and reduced cell proliferation. These results suggest that Halofuginone is a potent inhibitor of WT progression. Because of its unique mode of action, Halofuginone may decrease the treatment burden when combined with chemotherapy.
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gene expression during chemically induced liver fibrosis effect of Halofuginone on tgf β signaling
Cell and Tissue Research, 2007Co-Authors: Yulia Gnainsky, Arnon Nagler, Olga Genina, Gadi Spira, Yosey Hagai, Z Kushnirsky, G Bilu, Hanne Volpin, Rafael Bruck, Norifumi KawadaAbstract:Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-β (TGF-β), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-β-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of Halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-β. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, Halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-β-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by Halofuginone. This study thus highlights the role of TGF-β signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.
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Involvement of the tyrosine phosphatase early gene of liver regeneration (PRL–1) in cell cycle and in liver regeneration and fibrosis effect of Halofuginone
Cell and Tissue Research, 2006Co-Authors: Yulia Gnainsky, Orna Halevy, Arnon Nagler, Olga Genina, Gadi Spira, Melia Paizi, Raffael Bruck, Rebbeca Taub, Mark PinesAbstract:Tyrosine phosphatase PRL–1 is one of the immediate-early genes up-regulated during liver regeneration and is apparently involved in cell proliferation. Previously, we have demonstrated that Halofuginone, an inhibitor of collagen type I synthesis, prevents liver fibrosis and improves cirrhotic liver regeneration. In this study, we evaluated the effect of Halofuginone on PRL–1 expression, its cellular localization in vitro and during liver regeneration, and fibrosis progression in vivo. In culture, Halofuginone increased PRL–1 expression in primary rat hepatocytes and in hepatocellular carcinoma (HCC) cell lines, the former being more sensitive to Halofuginone. The Halofuginone-dependent increase in PRL–1 gene expression was correlated with an increase in the transcription factor early growth response–1 (Egr–1) and inversely correlated with the inhibition of cell proliferation. Halofuginone arrested HepG2 and Huh7 cell lines at the G1 phase, whereas Hep3B cells were arrested at G2/M, probably because of a reduction in the synthesis of cyclins D1 and B1 in all HCC cells and increased cyclin A in Hep3B cells. Halofuginone also affected the PRL–1 sub-cellular localization that was cell-cycle-dependent. In addition, Halofuginone augmented PRL–1 expression in the remnant liver after partial hepatectomy and in chemically induced fibrosis in rats; this was accompanied by increased expression of insulin-like growth factor binding protein 1 (IGFBP–1), another immediate-early gene of regeneration. The regulation of the expression of the early genes of regeneration such as PRL–1 and IGFBP–1 is thus part of the mode of action of Halofuginone and results in the prevention of liver fibrosis and improved cirrhotic liver regeneration.
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involvement of the tyrosine phosphatase early gene of liver regeneration prl 1 in cell cycle and in liver regeneration and fibrosis effect of Halofuginone
Cell and Tissue Research, 2006Co-Authors: Yulia Gnainsky, Orna Halevy, Arnon Nagler, Olga Genina, Gadi Spira, Melia Paizi, Raffael Bruck, Rebbeca Taub, Mark PinesAbstract:Tyrosine phosphatase PRL–1 is one of the immediate-early genes up-regulated during liver regeneration and is apparently involved in cell proliferation. Previously, we have demonstrated that Halofuginone, an inhibitor of collagen type I synthesis, prevents liver fibrosis and improves cirrhotic liver regeneration. In this study, we evaluated the effect of Halofuginone on PRL–1 expression, its cellular localization in vitro and during liver regeneration, and fibrosis progression in vivo. In culture, Halofuginone increased PRL–1 expression in primary rat hepatocytes and in hepatocellular carcinoma (HCC) cell lines, the former being more sensitive to Halofuginone. The Halofuginone-dependent increase in PRL–1 gene expression was correlated with an increase in the transcription factor early growth response–1 (Egr–1) and inversely correlated with the inhibition of cell proliferation. Halofuginone arrested HepG2 and Huh7 cell lines at the G1 phase, whereas Hep3B cells were arrested at G2/M, probably because of a reduction in the synthesis of cyclins D1 and B1 in all HCC cells and increased cyclin A in Hep3B cells. Halofuginone also affected the PRL–1 sub-cellular localization that was cell-cycle-dependent. In addition, Halofuginone augmented PRL–1 expression in the remnant liver after partial hepatectomy and in chemically induced fibrosis in rats; this was accompanied by increased expression of insulin-like growth factor binding protein 1 (IGFBP–1), another immediate-early gene of regeneration. The regulation of the expression of the early genes of regeneration such as PRL–1 and IGFBP–1 is thus part of the mode of action of Halofuginone and results in the prevention of liver fibrosis and improved cirrhotic liver regeneration.
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reduction in dermal fibrosis in the tight skin tsk mouse after local application of Halofuginone
Biochemical Pharmacology, 2001Co-Authors: Mark Pines, Olga Genina, Meir Ohana, A J Domb, Jacob Inbar, Rosaly Alexiev, Arnon NaglerAbstract:The effect of dermal application of Halofuginone—an inhibitor of collagen type I synthesis—on skin collagen and collagen α1(I) gene expression in an animal model of scleroderma and chronic graft versus host disease (cGvHD) was evaluated. Halofuginone-containing cream was applied on the tight-skin mouse (Tsk) and skin biopsies were taken for collagen staining by sirius red and for collagen α1(I) gene expression by in situ hybridization. In addition, cell proliferation was evaluated by immunostaining for proliferation cell nuclear antigen (PCNA) alone or in combination with collagen α1(I) probe. The number of mast cells was assessed by toluidine blue. Dermal application of Halofuginone (0.01%) for 60 days was as good as systemic administration (1 μg/mouse/day) in reducing collagen α1(I) gene expression in skin biopsy and almost as good in reducing skin width. Halofuginone was stable and effective only at acidic pH. The effect of Halofuginone (0.03%) was time-dependent. After 40 days of daily treatment, a significant reduction in the collagen α1(I) gene expression was observed and further decrease was observed after 60 days. The reduction in collagen α1(I) gene expression and the reduction in the proliferation of dermal fibroblasts probably occur in the same subset of cells. No effect of Halofuginone on the proliferation of keratinocytes or on mast cell number was observed. These results suggest that target-oriented application of Halofuginone may become a novel therapy for fibrotic disorders in general and for scleroderma in particular.
Orna Halevy - One of the best experts on this subject based on the ideXlab platform.
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hydroxy group requirement for Halofuginone dependent inhibition of muscle fibrosis and improvement of histopathology in the mdx mouse model for duchenne muscular dystrophy
Histology and Histopathology, 2019Co-Authors: Gili Wellner, Mark Pines, Olga Genin, Sharon Mordechay, Paul Evans, Orna HalevyAbstract:In Duchenne muscular dystrophy (DMD), the progressive loss of muscle and its ability to function is associated with significant fibrosis, representing the major disease complication in patients. Halofuginone, a halogenated analog of the naturally occurring febrifugine, has been shown to prevent fibrosis in various animal models, including those of muscular dystrophies. Here, two optically active enantiomers of deoxyHalofuginone—a Halofuginone analogue in which the hydroxy group in position 3 was removed from the piperidinyl entity—were evaluated with respect to their effect on muscle histopathology in mdx mice. Male mdx mice were treated with either deoxyHalofuginone (as single enantiomers or in racemic form), or Halofuginone, for 10 weeks, starting at the age of 4 weeks. Halofuginone caused a significant reduction in total collagen content, degenerative areas, as well as in utrophin and phosphorylated-Smad3 levels in the mdx diaphragms. However, neither the deoxyHalofuginone enantiomers, nor its racemic form had any effect on these parameters. A positive effect of the deoxyHalofuginone (+)-enantiomer was observed on myofiber diameters; however, it was lesser than that of Halofuginone. It is concluded that the hydroxy group plays a key role in Halofuginone’s effects related to fibrosis in DMD, and points towards the transforming growth factor β/Smad3 signaling pathway being involved in this inhibition. Elucidation of the structure–function relationship of Halofuginone, in relation to inhibiting fibrosis in muscular dystrophies, is of the utmost importance for creating the next generation of anti-fibrotic therapies that will be more efficacious and less toxic, hence improving life quality of patients.
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Halofuginone promotes satellite cell activation and survival in muscular dystrophies
Biochimica et Biophysica Acta, 2016Co-Authors: Hila Barzilaitutsch, Mark Pines, Anna Bodanovsky, Hadar Maimon, Orna HalevyAbstract:Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, Halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf(-/-) mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of Halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating Halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf(-/-) mice, Halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the Halofuginone-induced cell survival, suggesting this pathway's involvement in mediating Halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, Halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.
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Halofuginone improves muscle cell survival in muscular dystrophies
Biochimica et Biophysica Acta, 2014Co-Authors: Anna Bodanovsky, Mark Pines, Noga Guttman, Hila Barzilaitutsch, Ola Genin, Oshrat Levy, Orna HalevyAbstract:Abstract Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that Halofuginone would reduce apoptosis—the presumed cause of satellite-cell depletion during muscle degradation—in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when Halofuginone was added to cultured myofibers. No apparent effect of Halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by Halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating Halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which Halofuginone improves muscle pathology and function in muscular dystrophies.
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inhibition of muscle fibrosis and improvement of muscle histopathology in dysferlin knock out mice treated with Halofuginone
Histology and Histopathology, 2013Co-Authors: Orna Halevy, Hila Barzilaitutsch, Olga Genin, Yaniv Pima, Oshrat Levi, Itai Moshe, Mark PinesAbstract:Absence of, or loss-of-function mutations in the dysferlin gene (dysf) result in dysferlinopathy, characterized by increased muscle inflammation, collagen deposition and deterioration in muscle function. We evaluated Halofuginone efficacy in improving muscle histopathology in mice with deleted dysf transmembrane domain. Quadriceps sublumbar and longissimus muscles of 9-month-old dysf-/- mice treated with Halofuginone for 4 months exhibited a reduction in centrally-nucleated myofibers, inflammatory infiltrates and collagen content. Late onset of dysferlinopathy makes it ideal for evaluating the efficacy of early treatments on late outcome. The dysf-/- mice were treated with Halofuginone for 3 to 4 months starting at 1, 5 or 9 months of age, and quadricep muscle histopathology was evaluated at 12 months. Collagen content and number of centrally nucleated myofibers decreased after early Halofuginone treatment, administered when myofibers with central nuclei and inflammatory infiltrates are evident, but there was almost no fibrosis. When administered at the beginning of fibrosis it resulted in a further decrease in the number of centrally-nucleated myofibers with no additional decrease in collagen levels. Cardiac fibrosis was almost completely abolished following early Halofuginone treatment. Halofuginone inhibited Smad3 phosphorylation and its translocation to the nucleus and increased the activity of matrix metalloproteinases 9 and 2 responsible for resolution of pre-existing collagen. Macrophage and myofibroblast invasion into the dystrophic muscle at the site of myofibers with central nuclei was inhibited by Halofuginone. These results suggest that early Halofuginone treatment can prevent the late outcome of dysferlinopathy and can cause resolution of the established fibrosis when administered at later stages.
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Halofuginone inhibits smad3 phosphorylation via the pi3k akt and mapk erk pathways in muscle cells effect on myotube fusion
Experimental Cell Research, 2010Co-Authors: Suzy Roffe, Mark Pines, Yosey Hagai, Orna HalevyAbstract:Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that Halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating Halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, Halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, Halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in Halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.
Israel Vlodavsky - One of the best experts on this subject based on the ideXlab platform.
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inhibition of matrix metalloproteinase 2 by Halofuginone is mediated by the egr1 transcription factor
Anti-Cancer Drugs, 2012Co-Authors: Eyal Zcharia, Avichai Shimoni, Israel Vlodavsky, Ruth Atzmon, Adi Nagler, Tamar Peretz, Arnon NaglerAbstract:Halofuginone, a low-molecular-weight quinazolinone alkaloid that inhibits collagen α1(I), has been shown to suppress cancer growth, metastasis, and angiogenesis. These activities were attributed in part to the inhibition of matrix metalloproteinase-2 (MMP-2). The present study was carried out to explore the molecular mechanism underlying this effect. We found a marked (50%) inhibition in MMP-2 gelatinolytic activity in human breast cancer MDA-MB-435 cells pretreated with as little as 50 ng/ml of Halofuginone, a concentration that markedly inhibited their invasive and proliferative capacities. We further show that both early growth response 1 (Egr-1) and Nab-2 (corepressor of Egr1 activation) are upregulated by Halofuginone in a dose-dependent and time-dependent (up to 5 h) manner. Using MMP-2 reporter gene and chromatin immunoprecipitation analyses, we found that Egr-1 binds to the MMP-2 promoter and inhibits its activity. Altogether, our results identify the downstream elements (Egr-1, Nab-2, and MMP-2) by which Halofuginone exerts its antitumoral effect, thereby advancing its potential therapeutic application as an anticancer drug.
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Halofuginone inhibits nf κb and p38 mapk in activated t cells
Journal of Leukocyte Biology, 2006Co-Authors: Merav Leiba, Liora Cahalon, Avichai Shimoni, Iris Hecht, Uri Sela, Israel Vlodavsky, O Lider, Alexandra Zaninzhorov, Arnon NaglerAbstract:Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, Halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that Halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of Halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of Halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of Halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml Halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng Halofuginone, P = 0.002). In addition, 40 ng/ml Halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, Halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent.
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Halofuginone inhibits NF-κB and p38 MAPK in activated T cells
Journal of Leukocyte Biology, 2006Co-Authors: Merav Leiba, Liora Cahalon, Avichai Shimoni, Lider O, Alexandra Zanin-zhorov, Iris Hecht, Uri Sela, Israel Vlodavsky, Arnon NaglerAbstract:Halofuginone, a low molecular weight plant alkaloid, inhibits collagen 1 (I) gene expres- sion in several animal models and in patients with fibrotic disease, including scleroderma and graft- versus-host disease. In addition, Halofuginone has been shown to inhibit angiogenesis and tumor pro- gression. It was demonstrated recently that Halofuginone inhibits transforming growth factor- (TGF-), an important immunomodulator. The present study was undertaken to explore the effects of Halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclo- nal antibodies in the absence and presence of Halofuginone and assessed for nuclear factor (NF)-B activity, production of tumor necrosis fac- tor (TNF-) and interferon- (IFN-), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A de- layed-type hypersensitivity (DTH) model was ap- plied to investigate the effect of Halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml Halofuginone re- sulted in a significant dose-dependent decrease in NF-B activity (80% inhibition following incuba- tion with 40 ng Halofuginone, P0.002). In addi- tion, 40 ng/ml Halofuginone inhibited secretion of TNF-, IFN-, interleukin (IL)-4, IL-13, and TGF- (P
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Suppression of hepatocellular carcinoma growth in mice by the alkaloid coccidiostat Halofuginone.
European Journal of Cancer, 2004Co-Authors: Arnon Nagler, Mark Pines, Israel Vlodavsky, Meir Ohana, Oren Shibolet, M Y Shapira, Ruslana Alper, Y. IlanAbstract:Halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen α1 (I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and fibroblast proliferation. It was recently shown to be effective in suppression of bladder carcinoma and glioma. This study sought to evaluate the effect of treatment with Halofuginone on growth of hepatocellular carcinoma (HCC) in mice. Athymic Balb/c mice were injected subcutaneously with 107 human hepatoma cells (Hep3B), followed by treatment with Halofuginone administered in the diet (750 μg/kg) starting on day 3, before tumour innoculation. The control group was received a normal diet. Mice were followed for survival, tumour volume and serum α-fetoprotein (αFP). The mechanism of the anti-tumour effect of Halofuginone was determined in vitro by assessing tumour cell growth, and by measuring the serum concentrations of interferon-γ (IFNγ) and interleukin 2 (IL2). Halofuginone treatment induced almost complete tumour suppression in treated mice. Mortality rates were 10% and 50%, in Halofuginone-treated and control mice, respectively (P
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suppression of hepatocellular carcinoma growth in mice by the alkaloid coccidiostat Halofuginone
European Journal of Cancer, 2004Co-Authors: Arnon Nagler, Mark Pines, Israel Vlodavsky, Meir Ohana, Oren Shibolet, M Y Shapira, Ruslana Alper, Y. IlanAbstract:Halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen α1 (I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and fibroblast proliferation. It was recently shown to be effective in suppression of bladder carcinoma and glioma. This study sought to evaluate the effect of treatment with Halofuginone on growth of hepatocellular carcinoma (HCC) in mice. Athymic Balb/c mice were injected subcutaneously with 107 human hepatoma cells (Hep3B), followed by treatment with Halofuginone administered in the diet (750 μg/kg) starting on day 3, before tumour innoculation. The control group was received a normal diet. Mice were followed for survival, tumour volume and serum α-fetoprotein (αFP). The mechanism of the anti-tumour effect of Halofuginone was determined in vitro by assessing tumour cell growth, and by measuring the serum concentrations of interferon-γ (IFNγ) and interleukin 2 (IL2). Halofuginone treatment induced almost complete tumour suppression in treated mice. Mortality rates were 10% and 50%, in Halofuginone-treated and control mice, respectively (P<0.001). No visible tumour was observed in treated mice, as compared with a 364 mm3 tumour in control mice. Serum αFP were 0.1 and 212 ng/ml in treated and control mice, respectively (P<0.005). Halofuginone significantly inhibited HCC proliferation in vitro. Maximal inhibition of 64% of tumour cell growth was observed at a concentration of 10−8 M. The anti-tumour effect was mediated via a significant increase in IFNγ and IL2 (90 vs. 35, and 210 vs. 34 pg/ml in treated and control groups, respectively, P<0.005). Treatment with Halofuginone effectively suppressed the progression of HCC in mice. This effect may be associated with a direct anti-tumour effect, and/or enhancement of a systemic immune response.
