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1.
J Steroid Biochem Mol Biol ; 178: 263-271, 2018 04.
Article in English | MEDLINE | ID: mdl-29307714

ABSTRACT

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17ß-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G2/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G2/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERß, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Apoptosis/drug effects , Brassinosteroids/pharmacology , Human Umbilical Vein Endothelial Cells/pathology , Neoplasms/pathology , Neovascularization, Pathologic/prevention & control , Angiogenesis Inhibitors/chemistry , Brassinosteroids/chemistry , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Humans , In Vitro Techniques , Neoplasms/blood supply , Neoplasms/drug therapy , Neovascularization, Pathologic/pathology , Phosphorylation , Signal Transduction , Tumor Cells, Cultured
2.
Br J Cancer ; 116(7): 912-922, 2017 Mar 28.
Article in English | MEDLINE | ID: mdl-28222068

ABSTRACT

BACKGROUND: Tumour-initiating cells (TICs) account for chemoresistance, tumour recurrence and metastasis, and therefore represent a major problem in tumour therapy. However, strategies to address TICs are limited. Recent studies indicate Cdk5 as a promising target for anti-cancer therapy and Cdk5 has recently been associated with epithelial-mesenchymal transition (EMT). However, a role of Cdk5 in TICs has not been described yet. METHODS: Expression of Cdk5 in human cancer tissue was analysed by staining of a human tissue microarray (TMA). Functional effects of Cdk5 overexpression, genetic knockdown by siRNA and shRNA, and pharmacologic inhibition by the small molecule roscovitine were tested in migration, invasion, cell death, and tumorsphere assays and in tumour establishment in vivo. For mechanistic studies, molecular biology methods were applied. RESULTS: In fact, here we pin down a novel function of Cdk5 in TICs: knockdown and pharmacological inhibition of Cdk5 impaired tumorsphere formation and reduced tumour establishment in vivo. Conversely, Cdk5 overexpression promoted tumorsphere formation which was in line with increased expression of Cdk5 in human breast cancer tissues as shown by staining of a human TMA. In order to understand how Cdk5 inhibition affects tumorsphere formation, we identify a role of Cdk5 in detachment-induced cell death: Cdk5 inhibition induced apoptosis in tumorspheres by stabilizing the transcription factor Foxo1 which results in increased levels of the pro-apoptotic protein Bim. CONCLUSIONS: In summary, our study elucidates a Cdk5-Foxo1-Bim pathway in cell death in tumorspheres and suggests Cdk5 as a potential target to address TICs.


Subject(s)
Apoptosis , Breast Neoplasms/pathology , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Epithelial-Mesenchymal Transition , Neoplastic Stem Cells/pathology , Animals , Blotting, Western , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Cell Adhesion , Cell Cycle , Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 5/genetics , Cyclin-Dependent Kinase 5/metabolism , Female , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplastic Stem Cells/enzymology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
3.
Oncotarget ; 7(19): 27108-21, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27027353

ABSTRACT

We recently introduced CDK5 as target in HCC, regulating DNA damage response. Based on this and on our previous knowledge about vascular effects of CDK5, we investigated the role of CDK5 in angiogenesis in HCC, one of the most vascularized tumors. We put a special focus on the transcription factor HIF-1α, a master regulator of tumor angiogenesis.The interaction of CDK5 with HIF-1α was tested by Western blot, PCR, reporter gene assay, immunohistochemistry, kinase assay, co-immunoprecipitation, mass spectrometry, and mutation studies. In vivo, different murine HCC models, were either induced by diethylnitrosamine or subcutaneous injection of HUH7 or HepG2 cells. The correlation of vascular density and CDK5 was assessed by immunostaining of a microarray of liver tissues from HCC patients.Inhibition of CDK5 in endothelial or HCC cells reduced HIF-1α levels in vitro and in vivo, and transcription of HIF-1α target genes (VEGFA, VEGFR1, EphrinA1). Mass spectrometry and site directed mutagenesis revealed a stabilizing phosphorylation of HIF-1α at Ser687 by CDK5. Vascular density was decreased in murine HCC models by CDK5 inhibition.In conclusion, inhibiting CDK5 is a multi-modal systemic approach to treat HCC, hitting angiogenesis, as well as the tumor cells themselves.


Subject(s)
Carcinoma, Hepatocellular/genetics , Cyclin-Dependent Kinase 5/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Liver Neoplasms/genetics , Neovascularization, Pathologic/genetics , RNA Interference , Animals , Base Sequence , Carcinoma, Hepatocellular/blood supply , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase 5/metabolism , Disease Models, Animal , Female , Hep G2 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/blood supply , Liver Neoplasms/metabolism , Male , Mice, Inbred C57BL , Mice, SCID , Neovascularization, Pathologic/metabolism , Protein Stability , Sequence Homology, Nucleic Acid , Transplantation, Heterologous
4.
Eur J Med Chem ; 112: 347-366, 2016 Apr 13.
Article in English | MEDLINE | ID: mdl-26907157

ABSTRACT

Several selective and potent EphB4 inhibitors have been discovered, optimized and biophysically characterized by our groups over the past years. On the outset of these discoveries high throughput docking techniques were applied. Herein, we review the optimization campaigns started from three of these hits (Xan-A1, Pyr-A1 and Qui-A1) with emphasis on their in depth in vitro and in vivo characterization, together with previously unpublished angiogenesis and fluorescence based assays.


Subject(s)
Drug Design , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor, EphB2/antagonists & inhibitors , Animals , Computer Simulation , Humans , Isoquinolines/chemistry , Isoquinolines/pharmacology , Neoplasms/drug therapy , Neoplasms/enzymology , Quinoxalines/chemistry , Quinoxalines/pharmacology , Receptor, EphB2/metabolism , Structure-Activity Relationship , Xanthine/chemistry , Xanthine/pharmacology
5.
Oncotarget ; 7(5): 6088-104, 2016 Feb 02.
Article in English | MEDLINE | ID: mdl-26755662

ABSTRACT

Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels. Here we pin down the in vivo impact of endothelial Cdk5 inhibition in angiogenesis and elucidate the underlying mechanism in order to judge the potential of Cdk5 as a novel anti-angiogenic and anti-cancer target. By the use of endothelial-specific Cdk5 knockout mouse models and various endothelial and tumor cell based assays including human tumor xenograft models, we show that endothelial-specific knockdown of Cdk5 results in excessive but non-productive angiogenesis during development but also in tumors, which subsequently leads to inhibition of tumor growth. As Cdk5 inhibition disrupted Notch function by reducing the generation of the active Notch intracellular domain (NICD) and Cdk5 modulates Notch-dependent endothelial cell proliferation and sprouting, we propose that the Dll4/Notch driven angiogenic signaling hub is an important and promising mechanistic target of Cdk5. In fact, Cdk5 inhibition can sensitize tumors to conventional anti-angiogenic treatment as shown in tumor xenograft models. In summary our data set the stage for Cdk5 as a drugable target to inhibit Notch-driven angiogenesis condensing the view that Cdk5 is a promising target for cancer therapy.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/drug therapy , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Glioblastoma/blood supply , Glioblastoma/drug therapy , Human Umbilical Vein Endothelial Cells , Humans , Melanoma, Experimental/blood supply , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , Neoplasms, Experimental/enzymology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/enzymology , Receptors, Notch/metabolism , Signal Transduction
6.
Oncotarget ; 6(41): 43508-28, 2015 Dec 22.
Article in English | MEDLINE | ID: mdl-26496038

ABSTRACT

Prognosis for patients suffering from T-ALL is still very poor and new strategies for T-ALL treatment are urgently needed. Our study shows potent anti-leukemic effects of the myxobacterial V-ATPase inhibitor Archazolid A. Archazolid A reduced growth and potently induced death of leukemic cell lines and human leukemic samples. By inhibiting lysosomal acidification, Archazolid A blocked activation of the Notch pathway, however, this was not the mechanism of V-ATPase inhibition relevant for cell death induction. In fact, V-ATPase inhibition by Archazolid A decreased the anti-apoptotic protein survivin. As underlying mode of action, this work is in line with recent studies from our group demonstrating that Archazolid A induced S-phase cell cycle arrest by interfering with the iron metabolism in leukemic cells. Our study provides evidence for V-ATPase inhibition as a potential new therapeutic option for T-ALL.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Macrolides/pharmacology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Thiazoles/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Immunoblotting , Mice , Mice, Inbred NOD , Mice, SCID , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Xenograft Model Antitumor Assays
7.
Oncotarget ; 6(26): 21799-801, 2015 Sep 08.
Article in English | MEDLINE | ID: mdl-26327394

ABSTRACT

Lymphatic vessel dysfunction is associated with various pathologic conditions, including immunologic disorders, lymphedema, as well as tumor dissemination. Yet, the knowledge about the regulation of lymphatic vessel development is still limited. Our study elucidates cyclin dependent kinase 5 (Cdk5) as an essential player in the development of lymphatic vessels. Deletion of Cdk5 in the mouse endothelium results in severe lymphedema formation and embryonic lethality. On the mechanistic level, we show that Cdk5 phosphorylates the forkhead transcription factor Foxc2 which regulates Foxc2-dependent transcription. In summary, our study elucidates the Cdk5-Foxc2 interaction as a critical regulator of lymphatic vessel development.


Subject(s)
Cyclin-Dependent Kinase 5/physiology , Forkhead Transcription Factors/metabolism , Lymphangiogenesis/physiology , Lymphatic Vessels/cytology , Lymphatic Vessels/physiology , Animals , Mice , Mice, Knockout
8.
Nat Commun ; 6: 7274, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-26027726

ABSTRACT

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling.


Subject(s)
Cyclin-Dependent Kinase 5/genetics , Endothelial Cells/metabolism , Forkhead Transcription Factors/metabolism , Lymphangiogenesis/genetics , Lymphatic Vessels/metabolism , Animals , Cyclin-Dependent Kinase 5/metabolism , Endothelial Cells/cytology , Forkhead Transcription Factors/genetics , Human Umbilical Vein Endothelial Cells , Humans , Lymphatic Vessels/pathology , Mice , Mice, Knockout , Phosphorylation , Real-Time Polymerase Chain Reaction , Stress, Mechanical
9.
Cancer Res ; 75(14): 2863-74, 2015 Jul 15.
Article in English | MEDLINE | ID: mdl-26018087

ABSTRACT

Generalized strategies to improve breast cancer treatment remain of interest to develop. In this study, we offer preclinical evidence of an important metabolic mechanism underlying the antitumor activity of inhibitors of the vacuolar-type ATPase (V-ATPase), a heteromultimeric proton pump. Specifically, our investigations in the 4T1 model of metastatic breast cancer of the V-ATPase inhibitor archazolid suggested that its ability to trigger metabolic stress and apoptosis associated with tumor growth inhibition related to an interference with hypoxia-inducible factor-1α signaling pathways and iron metabolism. As a consequence of disturbed iron metabolism, archazolid caused S-phase arrest, double-stranded DNA breaks, and p53 stabilization, leading to apoptosis. Our findings link V-ATPase to cell-cycle progression and DNA synthesis in cancer cells, and highlight the basis for the clinical exploration of V-ATPase as a potentially generalizable therapy for breast cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Iron/metabolism , Macrolides/pharmacology , Thiazoles/pharmacology , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Breast Neoplasms/pathology , Enzyme Inhibitors/therapeutic use , Female , Humans , MCF-7 Cells , Macrolides/therapeutic use , Metabolic Networks and Pathways/drug effects , Mice , Mice, Inbred BALB C , Thiazoles/therapeutic use , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
10.
J Hepatol ; 63(1): 102-13, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25660209

ABSTRACT

BACKGROUND & AIMS: For a long time cyclin dependent kinase 5 (Cdk5) was thought to be exclusively important in neuronal cells. However, increasing evidence recently suggests a function of Cdk5 in cancer progression. In this study, we examined the role of Cdk5 and its therapeutic accessibility in hepatocellular carcinoma (HCC), a highly chemoresistant cancer with poor prognosis and paramount clinical importance in order to develop novel targeted therapies for systemic treatment. METHODS: Expression and activity of Cdk5 was analyzed in a human HCC tissue microarray, human patient samples and HCC cell lines. To characterize Cdk5 functions and signaling pathways in HCC, we applied genetic downregulation and pharmacologic inhibition in various approaches including cell based assays and mouse xenograft models. RESULTS: Expression and activity of Cdk5 was increased in human HCC tissues as compared to normal liver tissues. Functional ablation of Cdk5 significantly decreased HCC cell proliferation and clonogenic survival. Moreover, genetic and pharmacological inhibition of Cdk5 showed in vivo efficacy in HCC xenograft mouse models. Investigating the mechanisms behind these functional effects revealed that Cdk5 is most active in the nucleus of cells in G2/M phase. Cdk5 regulates DNA damage response by phosphorylating ataxia telangiectasia mutated (ATM) kinase and thereby influencing its downstream cascade. Consequently, combination of Cdk5 inhibition with DNA-damage-inducing chemotherapeutics synergistically inhibited HCC tumor progression in vitro and in vivo. CONCLUSIONS: In summary, we introduce Cdk5 as a novel drugable target for HCC treatment and suggest the combination of Cdk5 inhibition and DNA damaging agents as a novel therapeutic approach.


Subject(s)
Camptothecin/analogs & derivatives , Carcinoma, Hepatocellular/genetics , Cyclin-Dependent Kinase 5/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Purines/therapeutic use , RNA, Neoplasm/genetics , Animals , Antineoplastic Agents/therapeutic use , Camptothecin/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Cyclin-Dependent Kinase 5/biosynthesis , Cyclin-Dependent Kinase 5/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Female , Humans , Irinotecan , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Mice , Mice, SCID , Roscovitine , Treatment Outcome
11.
Cardiovasc Res ; 104(2): 303-14, 2014 Nov 01.
Article in English | MEDLINE | ID: mdl-25239826

ABSTRACT

AIMS: Inhibiting angiogenesis is a major approach in tumour therapy. To combat angiogenesis, the tubulin cytoskeleton has emerged as an interesting target in many pre- and clinical studies. Contrarily, the actin cytoskeleton has been largely neglected as a potential drug target in angiogenesis. However, due to the development of drug resistances, new therapeutic strategies are always needed in tumour treatment. Therefore, the therapeutic potential of actin-binding small molecules is of particular interest. METHODS AND RESULTS: We investigate the impact of chondramide (Ch), an actin polymerizing myxobacterial compound, on angiogenesis and underlying signalling. Chondramide treatment not only reduces the migration of endothelial cells but also the maturation of endothelial tube networks on matrigel. These observations can partly be explained by a disintegration of stress fibres due to aggregation and subsequent accumulation of actin in cellular structures known as 'aggresomes'. Chondramide treatment impairs the maturation of focal adhesions and reduces the amount of active ß1 integrin at the cell surface. Accordingly, signalling events downstream of focal adhesions are reduced. Thus, we observed that the activity of Src and downstream factors Rho-GTPases Rac1 and Rho is reduced upon Ch treatment. In vivo, Ch was well tolerated in mice and vascularization of a tumour xenograft as well as of the developing retina was significantly reduced. CONCLUSION: Chondramide diminishes angiogenesis via two ways: (i) the disintegration of stress fibres and (ii) the reduction of promigratory signals. Our findings highlight Ch as a novel class of therapeutic lead compound with anti-angiogenic potential.


Subject(s)
Actin Cytoskeleton/drug effects , Angiogenesis Inhibitors/pharmacology , Bacterial Proteins/pharmacology , Breast Neoplasms/drug therapy , Depsipeptides/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Neovascularization, Pathologic , Neovascularization, Physiologic/drug effects , Actin Cytoskeleton/metabolism , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , Focal Adhesions/drug effects , Focal Adhesions/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Integrin beta1/metabolism , Mice, SCID , Signal Transduction/drug effects , Stress Fibers/drug effects , Stress Fibers/metabolism , Time Factors , Xenograft Model Antitumor Assays , rac1 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/metabolism , src-Family Kinases/metabolism
12.
Angiogenesis ; 17(3): 587-601, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24254321

ABSTRACT

The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach.


Subject(s)
Cell Movement , Endothelial Cells/cytology , Endothelial Cells/enzymology , Signal Transduction , Vacuolar Proton-Translocating ATPases/metabolism , Cell Adhesion/drug effects , Cell Cycle/drug effects , Cell Death/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Enzyme Inhibitors/pharmacology , Gene Silencing/drug effects , Humans , Macrolides/pharmacology , Phosphorylation/drug effects , Protein Subunits/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism , rac1 GTP-Binding Protein/metabolism
13.
Cancer Res ; 73(19): 6004-12, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23946383

ABSTRACT

While metastasis is the chief cause of cancer mortality, there nonetheless remains a lack of antimetastatic therapies that are clinically available. In this study, we present the indirubin derivative 6-bromo-indirubin-3'-oxime (6BIO) as a promising antimetastatic agent. 6BIO strongly reduced formation of lung metastasis in the well-established 4T1 mouse model of aggressive breast cancer. Several major hallmarks of the metastatic process were affected by subtoxic concentrations of 6BIO, which inhibited adhesion, migration, and invasion of a variety of metastatic cell types in vitro. Mechanistic analyses focused on known targets of 6BIO, which were silenced by this compound. Unexpectedly, RNAi-mediated silencing of glycogen synthase kinase 3ß (GSK3ß) and phosphoinositide-dependent protein kinase 1 (PDK1), both modulators of cellular metastasis targeted by 6BIO, were not found to affect invasive migration in this study. Instead, the Jak/STAT3 signaling pathway appeared to play a major role through modulation of its downstream migration regulators C-terminal tensin-like protein and matrix metalloproteinase 2. However, PDK1 and GSK3ß contributed to the overall response to 6BIO, as silencing of all three pathways resulted in almost complete inhibition of migration, phenocopying the 6BIO response. Taken together, our findings illustrate the antimetastatic activity of 6BIO on the basis of its ability to simultaneously inhibit several kinase cascades involved in metastasis of cancer cells, supporting the concept of "polypharmacology" in developing drugs to attack metastasis, the most deadly aspect of cancer.


Subject(s)
Breast Neoplasms/prevention & control , Cell Movement/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Indoles/pharmacology , Lung Neoplasms/prevention & control , Oximes/pharmacology , Spheroids, Cellular/drug effects , Animals , Apoptosis/drug effects , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Chemotaxis , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , STAT Transcription Factors/metabolism , Signal Transduction
14.
Mol Cell Biol ; 33(19): 3749-61, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23878394

ABSTRACT

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.


Subject(s)
Endothelial Cells/metabolism , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Transcription, Genetic/genetics , Amino Acid Sequence , Animals , Binding Sites/genetics , COS Cells , Cells, Cultured , Chlorocebus aethiops , Forkhead Transcription Factors/metabolism , HEK293 Cells , Hep G2 Cells , Humans , Immunoblotting , Mice , Mice, Transgenic , Microscopy, Confocal , Molecular Sequence Data , Mutation , Oligonucleotide Array Sequence Analysis , Phosphorylation , Proline/genetics , Proline/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serine/genetics , Serine/metabolism , Threonine/genetics , Threonine/metabolism
15.
PLoS One ; 8(1): e54607, 2013.
Article in English | MEDLINE | ID: mdl-23336010

ABSTRACT

Current inhibitors of angiogenesis comprise either therapeutic antibodies (e.g. bevacicumab binding to VEGF-A) or small molecular inhibitors of receptor tyrosin kinases like e.g. sunitinib, which inhibits PDGFR and VEGFR. We have recently identified cyclin-dependent kinase 5 (Cdk5) as novel alternative and pharmacologically accessible target in the context of angiogenesis. In the present work we demonstrate that trisubstituted pyrazolo[4,3-d]pyrimidines constitute a novel class of compounds which potently inhibit angiogenesis. All seven tested compounds inhibited endothelial cell proliferation with IC(50) values between 1 and 18 µM. Interestingly, this seems not to be due to cytotoxicity, since none of them showed acute cytotoxic effects on endothelial cells at a concentration of 10 µM,. The three most potent compounds (LGR1404, LGR1406 and LGR1407) also inhibited cell migration (by 27, 51 and 31%, resp.), chemotaxis (by 50, 70 and 60% in accumulative distance, resp.), and tube formation (by 25, 60 and 30% of total tube length, resp.) at the non-toxic concentration of 10 µM. Furthermore, angiogenesis was reduced in vivo in the CAM assay by these three compounds. A kinase selectivity profiling revealed that the compounds prevalently inhibit Cdk2, Cdk5 and Cdk9. The phenotype of the migrating cells (reduced formation of lamellipodia, loss of Rac-1 translocation to the membrane) resembles the previously described effects of silencing of Cdk5 in endothelial cells. We conclude that especially LGR1406 and LGR1407 are highly attractive anti-angiogenic compounds, whose effects seem to largely depend on their Cdk5 inhibiting properties.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/toxicity , Cell Death/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Cyclin-Dependent Kinase 5/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Protein Transport/drug effects , Pseudopodia/drug effects , Pseudopodia/metabolism , Pyrazoles/chemistry , Pyrazoles/toxicity , Pyrimidines/chemistry , Pyrimidines/toxicity , rac1 GTP-Binding Protein/metabolism
16.
Cancer Res ; 72(22): 5976-87, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22986742

ABSTRACT

The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.


Subject(s)
Breast Neoplasms/drug therapy , Macrolides/pharmacology , Thiazoles/pharmacology , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , rac1 GTP-Binding Protein/antagonists & inhibitors , Animals , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Polarity/drug effects , Down-Regulation , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Xenograft Model Antitumor Assays , rab5 GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/metabolism
17.
Steroids ; 77(13): 1502-9, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22974826

ABSTRACT

Antiangiogenic activity of the brassinosteroid plant hormones (BRs) and their derivative cholestanon was investigated in human umbilical vein endothelial cells (HUVEC) and in human microvascular endothelial cells (HMEC-1). 24-Epibrassinolide and 28-homocastasterone from group of 21 tested natural BRs inhibited migration of HUVEC cells. Seven tested BRs decreased the number of tubes significantly. Synthetic analogue cholestanon inhibited angiogenesis in vitro more effectively than natural BRs. Because of the similarity of BRs to human steroids, we have also studied interactions of BRs with human steroid receptors. Synthetic BRs cholestanon showed agonistic effects on estrogen-receptor-α, estrogen-receptor-ß and androgen receptor. Of the natural BRs, 24-epibrassinolide was found to be a weak antagonist of estrogen-receptor-α (ERα). Our results provide the first evidence that large group of BRs can inhibit in vitro angiogenesis of primary endothelial cells. BRs constitute a novel group of human steroid receptor activators or inhibitors with capacity to inhibit angiogenesis.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Brassinosteroids/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Neovascularization, Physiologic/drug effects , Angiogenesis Inhibitors/metabolism , Brassinosteroids/metabolism , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Humans
18.
Mol Cancer ; 11: 9, 2012 Mar 08.
Article in English | MEDLINE | ID: mdl-22401581

ABSTRACT

BACKGROUND: Hepatoblastoma (HB) is an embryonal liver neoplasm of early childhood with a poor prognosis for patients with distant metastases and vascular invasion. We and others have previously shown that the overexpression of insulin-like growth factor 2 (IGF2), loss of imprinting at the IGF2/H19 locus, and amplification of pleomorphic adenoma gene 1 (PLAG1) are common features in HB, suggesting a critical role of the IGF axis in hepatoblastomagenesis. In this study, we investigated the role of the insulin-like growth factor binding protein 3 (IGFBP3), a known competitor of the IGF axis, in pediatric liver cancers. RESULTS: The IGFBP3 gene was highly expressed in normal pediatric livers but was heavily downregulated in four HB cell lines and the majority of HB primary tumors (26/36). Detailed methylation analysis of CpG sites in the IGFBP3 promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of IGFBP3 in HB cell lines. Consequently, the treatment of HB cell lines with 5-aza-2'-deoxycytidine resulted in DNA demethylation and reactivation of the epigenetically silenced IGFBP3 expression. Interestingly, IGFBP3 promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring IGFBP3 expression in HB cells resulted in reduced colony formation, migration, and invasion. CONCLUSION: This study provides the first direct evidence that the reactivation of IGFBP3 decreases aggressive properties of pediatric liver cancer cells and that IGFBP3 promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.


Subject(s)
Epigenesis, Genetic/genetics , Insulin-Like Growth Factor Binding Protein 3/genetics , Liver Neoplasms/genetics , Apoptosis/genetics , Apoptosis/physiology , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cell Survival/genetics , Cell Survival/physiology , Humans , In Vitro Techniques , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
19.
Circulation ; 124(17): 1848-59, 2011 Oct 25.
Article in English | MEDLINE | ID: mdl-21969013

ABSTRACT

BACKGROUND: Urokinase-type plasminogen activator (uPA) has recently been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury. The underlying mechanisms remain largely unclear. METHODS AND RESULTS: Using in vivo microscopy on the mouse cremaster muscle, I/R-elicited firm adherence and transmigration of neutrophils were found to be significantly diminished in uPA-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)-deficient mice. Interestingly, postischemic leukocyte responses were significantly reduced on blockade of the integrin CD11b/Mac-1, which also serves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a selectively reduced transmigration in wild-type animals. On I/R or stimulation with recombinant uPA, >90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence microscopy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. CONCLUSIONS: Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the subsequent paracellular transmigration of neutrophils during I/R. This process is regulated via CD11b/Mac-1, and does not require uPAR. Pharmacological blockade of uPA interferes with these events and effectively attenuates postischemic tissue injury.


Subject(s)
Macrophage-1 Antigen/physiology , Neutrophils/cytology , Neutrophils/physiology , Receptors, Urokinase Plasminogen Activator/physiology , Transendothelial and Transepithelial Migration/physiology , Urokinase-Type Plasminogen Activator/physiology , Animals , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration/physiology , Random Allocation
20.
Cell Signal ; 23(11): 1698-707, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21741478

ABSTRACT

Cdk5 (cyclin-dependent kinase 5 or initially NCLK for neuronal CDC2-like kinase) was switched twice at its birth nearly twenty years ago: first it was thought to be cyclin-dependent, second it was assumed to be primarily of importance in neuronal cells-both turned out not to be the case. In this review we want to discuss issues of pharmacological inhibition, to highlight the versatile roles, and to summarize the growing evidence for the functional importance of Cdk5 in non-neuronal tissues, such as blood cells, tumor cells, epithelial cells, the vascular endothelium, testis, adipose and endocrine tissues. The organizing principles we follow are apoptosis/cell death, migration/motility, aspects of inflammation, and, finally, secretion/metabolism.


Subject(s)
Cyclin-Dependent Kinase 5 , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation/physiology , Nociception/physiology , Signal Transduction/genetics , Animals , Apoptosis/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 5/physiology , Endothelial Cells/cytology , Epithelial Cells/cytology , Humans , Inflammation/genetics , Inflammation/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Neovascularization, Physiologic/genetics , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Neurons/metabolism , Organ Specificity/genetics , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Roscovitine , Signal Transduction/drug effects
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