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Fonódi M, Nagy L, Boratkó A. Role of Protein Phosphatases in Tumor Angiogenesis: Assessing PP1, PP2A, PP2B and PTPs Activity. Int J Mol Sci 2024; 25:6868. [PMID: 38999976 PMCID: PMC11241275 DOI: 10.3390/ijms25136868] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2024] [Revised: 06/19/2024] [Accepted: 06/20/2024] [Indexed: 07/14/2024] Open
Abstract
Tumor angiogenesis, the formation of new blood vessels to support tumor growth and metastasis, is a complex process regulated by a multitude of signaling pathways. Dysregulation of signaling pathways involving protein kinases has been extensively studied, but the role of protein phosphatases in angiogenesis within the tumor microenvironment remains less explored. However, among angiogenic pathways, protein phosphatases play critical roles in modulating signaling cascades. This review provides a comprehensive overview of the involvement of protein phosphatases in tumor angiogenesis, highlighting their diverse functions and mechanisms of action. Protein phosphatases are key regulators of cellular signaling pathways by catalyzing the dephosphorylation of proteins, thereby modulating their activity and function. This review aims to assess the activity of the protein tyrosine phosphatases and serine/threonine phosphatases. These phosphatases exert their effects on angiogenic signaling pathways through various mechanisms, including direct dephosphorylation of angiogenic receptors and downstream signaling molecules. Moreover, protein phosphatases also crosstalk with other signaling pathways involved in angiogenesis, further emphasizing their significance in regulating tumor vascularization, including endothelial cell survival, sprouting, and vessel maturation. In conclusion, this review underscores the pivotal role of protein phosphatases in tumor angiogenesis and accentuate their potential as therapeutic targets for anti-angiogenic therapy in cancer.
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Affiliation(s)
| | | | - Anita Boratkó
- Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary; (M.F.); (L.N.)
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Chillà A, Anceschi C, Frediani E, Scavone F, Del Rosso T, Pelagio G, Tufaro A, De Palma G, Del Rosso M, Fibbi G, Chiarugi P, Laurenzana A, Margheri F. Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling. J Transl Med 2023; 21:102. [PMID: 36759828 PMCID: PMC9912547 DOI: 10.1186/s12967-023-03954-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Accepted: 02/01/2023] [Indexed: 02/11/2023] Open
Abstract
BACKGROUND In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity. METHODS We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies. RESULTS Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF. CONCLUSIONS We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.
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Affiliation(s)
- Anastasia Chillà
- Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Viale G.B. Morgagni, 50, 50134, Florence, Italy.
| | - Cecilia Anceschi
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Elena Frediani
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Francesca Scavone
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Tommaso Del Rosso
- grid.4839.60000 0001 2323 852XDepartment of Physics, Pontifical Catholic University of Rio de Janeiro, Rio de Janeiro, RJ 22451-900 Brazil
| | - Giuseppe Pelagio
- IRCCS Istituto Tumori Giovanni Paolo II Bari, Viale Orazio Flacco 65, 70124 Bari, Italy
| | - Antonio Tufaro
- IRCCS Istituto Tumori Giovanni Paolo II Bari, Viale Orazio Flacco 65, 70124 Bari, Italy
| | - Giuseppe De Palma
- IRCCS Istituto Tumori Giovanni Paolo II Bari, Viale Orazio Flacco 65, 70124 Bari, Italy
| | - Mario Del Rosso
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Gabriella Fibbi
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Paola Chiarugi
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Anna Laurenzana
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
| | - Francesca Margheri
- grid.8404.80000 0004 1757 2304Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy
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Wang L, Ouyang B, Fan M, Qi J, Yao L. The Design, Synthesis and Evaluation of Rho-kinase Inhibitory Activity of 4-aryl-thiazole-2-amines. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH : IJPR 2021; 20:121-131. [PMID: 34903975 PMCID: PMC8653655 DOI: 10.22037/ijpr.2020.114468.14866] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Rho-associated kinases (ROCK) are a class of serine/threonine kinases that play important roles in various biological processes. ROCK are becoming attractive targets for drug designing. A novel scaffold was designed according to molecular hybridization strategy, then a series of 4-aryl-5-aminomethyl-thiazole-2-amines were synthesized, and their inhibitory activities on ROCK were screened by enzyme-linked immunosorbent assay (ELISA). The results showed that 4-aryl-5-aminomethyl-thiazole-2-amines derivatives displayed certain ROCK II inhibitory activities. The IC50 value of the most potent compound 4v was found to be 20 nM. The preliminary structure-activity-relationship investigation showed that compounds with 4-pyridine substitution were generally found to be more potent than compounds with 3-pyridine substitution. The molecular docking studies indicated that more optimization work needs to conduct to obtain more potent ROCK inhibitors.
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Affiliation(s)
- Linan Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Ben Ouyang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Meixia Fan
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Junhui Qi
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Lei Yao
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
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Morphometric Analysis of Rat Prostate Development: Roles of MEK/ERK and Rho Signaling Pathways in Prostatic Morphogenesis. Biomolecules 2021; 11:biom11121829. [PMID: 34944473 PMCID: PMC8698940 DOI: 10.3390/biom11121829] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 11/23/2021] [Accepted: 12/02/2021] [Indexed: 12/24/2022] Open
Abstract
The molecular mechanisms underlying prostate development can provide clues for prostate cancer research. It has been demonstrated that MEK/ERK signaling downstream of androgen-targeted FGF10 signaling directly induces prostatic branching during development, while Rho/Rho-kinase can regulate prostate cell proliferation. MEK/ERK and Rho/Rho kinase regulate myosin light chain kinase (MLCK), and MLCK regulates myosin light chain phosphorylation (MLC-P), which is critical for cell fate, including cell proliferation, differentiation, and apoptosis. However, the roles and crosstalk of the MEK/ERK and Rho/Rho kinase signaling pathways in prostatic morphogenesis have not been examined. In the present study, we used numerical and image analysis to characterize lobe-specific rat prostatic branching during postnatal organ culture and investigated the roles of FGF10-MEK/ERK and Rho/Rho kinase signaling pathways in prostatic morphogenesis. Prostates exhibited distinctive lobe-specific growth and branching patterns in the ventral (VP) and lateral (LP) lobes, while exogenous FGF10 treatment shifted LP branching towards a VP branching pattern. Treatment with inhibitors of MEK1/2, Rho, Rho kinase, or MLCK significantly inhibited VP growth and blocked branching morphogenesis, further supporting critical roles for MEK/ERK and Rho/Rho kinase signaling pathways in prostatic growth and branching during development. We propose that MLCK-regulated MLC-P may be a central downstream target of both signaling pathways in regulating prostate morphogenesis.
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Golla U, Ehudin MA, Annageldiyev C, Zeng Z, Bastihalli Tukaramrao D, Tarren A, Date AA, Elcheva I, Berg A, Amin S, Loughran TP, Kester M, Desai D, Dovat S, Claxton D, Sharma A. DJ4 Targets the Rho-Associated Protein Kinase Pathway and Attenuates Disease Progression in Preclinical Murine Models of Acute Myeloid Leukemia. Cancers (Basel) 2021; 13:4889. [PMID: 34638385 PMCID: PMC8508452 DOI: 10.3390/cancers13194889] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 09/15/2021] [Accepted: 09/22/2021] [Indexed: 01/22/2023] Open
Abstract
The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2-5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 μM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy.
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Affiliation(s)
- Upendarrao Golla
- Department of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (U.G.); (C.A.); (A.T.); (D.C.)
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
| | - Melanie A. Ehudin
- Division of Hematology and Oncology, Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (M.A.E.); (D.B.T.); (I.E.)
| | - Charyguly Annageldiyev
- Department of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (U.G.); (C.A.); (A.T.); (D.C.)
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
| | - Zheng Zeng
- Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (Z.Z.); (M.K.)
| | - Diwakar Bastihalli Tukaramrao
- Division of Hematology and Oncology, Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (M.A.E.); (D.B.T.); (I.E.)
| | - Anna Tarren
- Department of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (U.G.); (C.A.); (A.T.); (D.C.)
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
| | - Abhijit A. Date
- The Daniel K. Inouye College of Pharmacy, University of Hawaii, Hilo, HI 96720, USA;
| | - Irina Elcheva
- Division of Hematology and Oncology, Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (M.A.E.); (D.B.T.); (I.E.)
| | - Arthur Berg
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
| | - Shantu Amin
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
- Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (Z.Z.); (M.K.)
| | - Thomas P. Loughran
- Department of Medicine, Division of Hematology and Oncology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA;
- Department of Medicine, Division of Hematology and Oncology, University of Virginia Cancer Center, Charlottesville, VA 22903, USA
| | - Mark Kester
- Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (Z.Z.); (M.K.)
- Department of Medicine, Division of Hematology and Oncology, University of Virginia Cancer Center, Charlottesville, VA 22903, USA
| | - Dhimant Desai
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
- Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (Z.Z.); (M.K.)
| | - Sinisa Dovat
- Division of Hematology and Oncology, Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (M.A.E.); (D.B.T.); (I.E.)
| | - David Claxton
- Department of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (U.G.); (C.A.); (A.T.); (D.C.)
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
| | - Arati Sharma
- Department of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (U.G.); (C.A.); (A.T.); (D.C.)
- Penn State Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (A.B.); (S.A.); (D.D.)
- Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; (Z.Z.); (M.K.)
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Wang L, Qi J, Fan M, Yao L. Design, synthesis, and biological evaluation of urea-based ROCK2 inhibitors. Chem Biol Drug Des 2021; 98:969-978. [PMID: 34581498 DOI: 10.1111/cbdd.13961] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 08/22/2021] [Accepted: 09/06/2021] [Indexed: 01/18/2023]
Abstract
A series of urea-based ROCK2 inhibitors were design and synthesized. The inhibitory activity on ROCK2 was screened by enzyme-linked immunosorbent assay (ELISA). The study results showed that the urea derivatives exhibited certain ROCK2 inhibitory activity. The most potent compound 10p showed ROCK2 inhibitory activity with the IC50 value of 0.03 μM. A preliminary structure-activity relationship was then summarized. The molecular docking studies showed that further optimization needs to conduct to obtain more potent ROCK inhibitors.
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Affiliation(s)
- Linan Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation Yantai University, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
| | - Junhui Qi
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation Yantai University, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
| | - Meixia Fan
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation Yantai University, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
| | - Lei Yao
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation Yantai University, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
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Sun H, Ma L, Chen J. Hyaluronan-mediated motility receptor expression functions as a prognostic biomarker in uterine carcinosarcoma based on bioinformatics analysis. J Int Med Res 2021; 49:3000605211021043. [PMID: 34111996 PMCID: PMC8202278 DOI: 10.1177/03000605211021043] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
OBJECTIVE Uterine carcinosarcoma (UCS) is a rare, aggressive tumour with a high metastasis rate and poor prognosis. This study aimed to explore potential key genes associated with the prognosis of UCS. METHODS Transcriptional expression data were downloaded from the Gene Expression Profiling Interactive Analysis database and differentially expressed genes (DEGs) were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses using Metascape. A protein-protein interaction network was constructed using the STRING website and Cytoscape software, and the top 30 genes obtained through the Maximal Clique Centrality algorithm were selected as hub genes. These hub genes were validated by clinicopathological and sequencing data for 56 patients with UCS from The Cancer Genome Atlas database. RESULTS A total of 1894 DEGs were identified, and the top 30 genes were considered as hub genes. Hyaluronan-mediated motility receptor (HMMR) expression was significantly higher in UCS tissues compared with normal tissues, and elevated expression of HMMR was identified as an independent prognostic factor for shorter survival in patients with UCS. CONCLUSIONS These results suggest that HMMR may be a potential biomarker for predicting the prognosis of patients with UCS.
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Affiliation(s)
- Hui Sun
- Department of Gynaecologic Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China
| | - Li Ma
- Department of Gynaecologic Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China
| | - Jie Chen
- Department of Gynaecologic Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China
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MYPT1, regulated by miR-19b-3p inhibits the progression of non-small cell lung cancer via inhibiting the activation of wnt/β-catenin signaling. Life Sci 2021; 278:119573. [PMID: 33964297 DOI: 10.1016/j.lfs.2021.119573] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Revised: 01/19/2021] [Accepted: 04/27/2021] [Indexed: 12/21/2022]
Abstract
AIMS Myosin phosphatase targeting protein 1 (MYPT1) was identified to function as a tumor suppressor in several kinds of cancers, but its role and the molecular mechanisms in non-small cell lung cancer (NSCLC) remain undiscovered. Herein, we aimed to reveal MYPT1 expression pattern and role in NSCLC, and investigate the underlying mechanisms. MAIN METHODS Sixty-eight paired NSCLC tissues and the adjacent normal tissues were included in this study. Western blotting and quantitative reverse transcription-polymerase chain (qPCR) technologies were applied for protein and RNA detection. CCK-8, colony formation, flow cytometry, wound healing, transwell chambers coated with Matrigel and in vivo experiments were applied to detect cell viability, colony formation, apoptosis, migration, invasiveness and tumorigenesis, respectively. KEY FINDINGS MYPT1 expressed at a lower level in NSCLC tissues as compared with the adjacent normal tissues, which predicted advanced clinic process and poor prognosis. Overexpression of MYPT1 resulted in obvious inhibitions in cell viability, colony formation, migration, invasiveness and tumorigenesis, and induced cell apoptotic rates, as well as decreased the expression levels of β-catenin and TCF4. Besides, overexpression of β-catenin weakened the above roles of MYPT1. In addition, the luciferase gene reporter assay verified that MYPT1 was a target of miR-19b-3p. Further experiments showed that miR-19b-3p promoted cell viability, invasiveness and migration and repressed cell apoptosis by targeting MYPT1. SIGNIFICANCE In conclusion, this study demonstrates that MYPT1, regulated by miR-19b-3p, inhibits the progression of NSCLC via inhibiting the activation of wnt/β-catenin signaling.
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Ma S, Wang L, Ouyang B, Fan M, Qi J, Yao L. Design, synthesis and biological evaluation of 4-aryl-5-aminoalkyl-thiazole-2-amines derivatives as ROCK II inhibitors. Bioorg Med Chem 2020; 28:115683. [PMID: 32912437 DOI: 10.1016/j.bmc.2020.115683] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2020] [Accepted: 07/15/2020] [Indexed: 01/21/2023]
Abstract
A series of 4-aryl-5-aminoalkyl-thiazole-2-amines were designed and synthesized, and their inhibitory activity on ROCK II was screened by enzyme-linked immunosorbent assay (ELISA). The results showed that 4-aryl-5-aminomethyl-thiazole-2-amines derivatives had certain ROCK II inhibitory activities. Compound 10l showed ROCK II inhibitory activity with IC50 value of 20 nM.
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Affiliation(s)
- Shuchao Ma
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Linan Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Ben Ouyang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Meixia Fan
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Junhui Qi
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
| | - Lei Yao
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264003, China
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Liu J, Wada Y, Katsura M, Tozawa H, Erwin N, Kapron CM, Bao G, Liu J. Rho-Associated Coiled-Coil Kinase (ROCK) in Molecular Regulation of Angiogenesis. Am J Cancer Res 2018; 8:6053-6069. [PMID: 30613282 PMCID: PMC6299434 DOI: 10.7150/thno.30305] [Citation(s) in RCA: 70] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2018] [Accepted: 10/16/2018] [Indexed: 02/06/2023] Open
Abstract
Identified as a major downstream effector of the small GTPase RhoA, Rho-associated coiled-coil kinase (ROCK) is a versatile regulator of multiple cellular processes. Angiogenesis, the process of generating new capillaries from the pre-existing ones, is required for the development of various diseases such as cancer, diabetes and rheumatoid arthritis. Recently, ROCK has attracted attention for its crucial role in angiogenesis, making it a promising target for new therapeutic approaches. In this review, we summarize recent advances in understanding the role of ROCK signaling in regulating the permeability, migration, proliferation and tubulogenesis of endothelial cells (ECs), as well as its functions in non-ECs which constitute the pro-angiogenic microenvironment. The therapeutic potential of ROCK inhibitors in angiogenesis-related diseases is also discussed.
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Role of rho-kinase (ROCK) in tonic but not phasic contraction in the frog stomach smooth muscle. Life Sci 2018; 198:46-55. [PMID: 29455004 DOI: 10.1016/j.lfs.2018.02.023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Revised: 02/14/2018] [Accepted: 02/15/2018] [Indexed: 01/17/2023]
Abstract
AIMS Rho/Rho-kinase (ROCK) signaling has extensively been shown to take part in mammalian smooth muscle contractions in response to diverse agents yet its role in the contraction of amphibian smooth muscle has not been investigated. Therefore, we aimed to explore any role of this pathway in the contractions of frog stomach smooth. MAIN METHODS The strips were prepared and suspended in organ baths filled with Ringer solution. Changes in the circular strips of the frog stomach muscle length were recorded isotonically with a force transducer in organ baths. KEY FINDINGS Carbachol (CCh) exerted both phasic and tonic contractions. In contrast, atropin abolished all types of contractions by CCh. The phasic contractions were suppressed by a Ca2+ channel blocker, nifedipine but not by the ROCK inhibitor, Y-27632. However, the tonic contractions were markedly attenuated by Y-27632. Selective M1 receptor blocker, pirenzepin, selective M3 receptor blocker and DAMP had no effects on CCh-elicited contractions. On the other hand, selective M2 receptor blocker, AF-DX suppressed all types of contractile activity by CCh. SIGNIFICANCE These data suggest that M2 receptor activation could mainly mediate CCh-induced phasic and tonic contractions, and ROCK seems to be involved in the CCh-induced tonic but not phasic contractions of the frog stomach smooth muscle.
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Liang Y, Zhuo Y, Lin Z, Jiang F, Dai Q, Lu J, Dong W, Zhu X, Han Z, Zhong W. Decreased Expression of MYPT1 Contributes to Tumor Angiogenesis and Poor Patient Prognosis in Human Prostate Cancer. Curr Mol Med 2018; 18:100-108. [PMID: 29974831 PMCID: PMC6302349 DOI: 10.2174/1566524018666180705111342] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2017] [Revised: 05/29/2018] [Accepted: 07/03/2018] [Indexed: 12/22/2022]
Abstract
BACKGROUND Our previous study demonstrated that Myosin Phosphatase Targeting subunit 1 (MYPT1) may function as a direct target of microRNA-30d, which promotes tumor angiogenesis and tumor growth of prostate cancer (PCa). Here, we aimed to investigate the clinical significance of MYPT1 expression and its functions in PCa. METHODS Roles of MYPT1 deregulation in tumor angiogenesis of PCa was determined in vitro and in vivo experiments. Expression patterns of MYPT1 and CD31 proteins were examined by immunohistochemistry and immunofluorescence, respectively. Associations of MYPT1/CD31 combination with various clinicopathological features and patients' prognosis of PCa were also statistically evaluated. RESULTS Through gain- and loss-of-function experiments, MYPT1 inhibited capillary tube formation of endothelial cells and in vivo tumor angiogenesis in a mouse model with the downregulation of VEGF and CD31 expression. In addition, MYPT1 expression was significantly decreased, while CD31 expression was dramatically increased in PCa tissues compared to benign prostate tissues. Notably, MYPT1 expression levels in PCa tissues were negatively correlated with that of CD31. Statistically, MYPT1-low/CD31- high expression was distinctly associated with high Gleason score, positive biochemical recurrence, and reduced overall survival of PCa patients. Moreover, PCa patients with MYPT1-low/CD31-high expression more frequently had shorter overall, biochemical recurrence-free and metastasis-free survivals. MYPT1/CD31 combination was identified as an independent factor to predict biochemical recurrence-free and metastasis-free survivals of PCa patients. CONCLUSIONS Our findings indicate that MYPT1 may inhibit angiogenesis and contribute favorable prognosis in PCa patients, implying that MYPT1 might be a potential drug candidate in anticancer therapy.
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Affiliation(s)
- Y Liang
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - Y Zhuo
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - Z Lin
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, Guangdong 510260, China
| | - F Jiang
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - Q Dai
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - J Lu
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - W Dong
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - X Zhu
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - Z Han
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
| | - W Zhong
- Department of Urology, Guangzhou First People's Hospital, The Second Affliated Hospital of South China University of Technology, South China University of Technology, Guangzhou, Guangdong 510180, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China
- Department of Urology, Huadu District People's Hospital, Southern Medical University, Guangzhou 510800, China
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Yao Y, Li R, Liu X, Yang F, Yang Y, Li X, Shi X, Yuan T, Fang L, Du G, Jiao X, Xie P. Discovery of Novel N-Substituted Prolinamido Indazoles as Potent Rho Kinase Inhibitors and Vasorelaxation Agents. Molecules 2017; 22:E1766. [PMID: 29048389 PMCID: PMC6151428 DOI: 10.3390/molecules22101766] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Accepted: 10/16/2017] [Indexed: 01/04/2023] Open
Abstract
Inhibitors of Rho kinase (ROCK) have potential therapeutic applicability in a wide range of diseases, such as hypertension, stroke, asthma and glaucoma. In a previous article, we described the lead discovery of DL0805, a new ROCK I inhibitor, showing potent inhibitory activity (IC50 6.7 μM). Herein, we present the lead optimization of compound DL0805, resulting in the discovery of 24- and 39-fold more-active analogues 4a (IC50 0.27 μM) and 4b (IC50 0.17 μM), among other active analogues. Moreover, ex-vivo studies demonstrated that 4a and 4b exhibited comparable vasorelaxant activity to the approved drug fasudil in rat aortic rings. The research of a preliminary structure-activity relationship (SAR) indicated that the target compounds containing a β-proline moiety have improved activity against ROCK I relative to analogues bearing an α-proline moiety, and among the series of the derivatives with a β-proline-derived indazole scaffold, the inhibitory activity of the target compounds with a benzyl substituent is superior to those with a benzoyl substituent.
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Affiliation(s)
- Yangyang Yao
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Renze Li
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Xiaoyu Liu
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Feilong Yang
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Ying Yang
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Xiaoyu Li
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Xiang Shi
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Tianyi Yuan
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Lianhua Fang
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Guanhua Du
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Xiaozhen Jiao
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Ping Xie
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
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14
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Lin ZY, Chen G, Zhang YQ, He HC, Liang YX, Ye JH, Liang YK, Mo RJ, Lu JM, Zhuo YJ, Zheng Y, Jiang FN, Han ZD, Wu SL, Zhong WD, Wu CL. MicroRNA-30d promotes angiogenesis and tumor growth via MYPT1/c-JUN/VEGFA pathway and predicts aggressive outcome in prostate cancer. Mol Cancer 2017; 16:48. [PMID: 28241827 PMCID: PMC5327510 DOI: 10.1186/s12943-017-0615-x] [Citation(s) in RCA: 74] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 02/20/2017] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Even though aberrant expression of microRNA (miR)-30d has been reported in prostate cancer (PCa), its associations with cancer progression remain contradictory. The aim of this study was to investigate clinical significance, biological functions and underlying mechanisms of miR-30d deregulation in PCa. METHODS Involvement of miR-30d deregulation in malignant phenotypes of PCa was demonstrated by clinical sample evaluation, and in vitro and in vivo experiments. The mechanisms underlying its regulatory effect on tumor angiogenesis were determined. RESULTS miR-30d over-expression was observed in both PCa cells and clinical specimens. High-miR-30d was distinctly associated with high pre-operative PSA and Gleason score, advanced clinical and pathological stages, positive metastasis and biochemical recurrence (BCR), and reduced overall survival of PCa patients. Through gain- and loss-of-function experiments, we found that miR-30d promoted PCa cell proliferation, migration, invasion, and capillary tube formation of endothelial cells, as well as in vivo tumor growth and angiogenesis in a mouse model. Simulation of myosin phosphatase targeting subunit 1 (MYPT1), acting as a direct target of miR-30d, antagonized the effects induced by miR-30d up-regulation in PCa cells. Notably, miR-30d/MYPT1 combination was identified as an independent factor to predict BCR of PCa patients. Furthermore, miR-30d exerted its pro-angiogenesis function, at least in part, by inhibiting MYPT1, which in turn, increased phosphorylation levels of c-JUN and activated VEGFA-induced signaling cascade in endothelial cells. CONCLUSIONS miR-30d and/or its target gene MYPT1 may serve as novel prognostic markers of PCa. miR-30d promotes tumor angiogenesis of PCa through MYPT1/c-JUN/VEGFA pathway.
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Affiliation(s)
- Zhuo-Yuan Lin
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Department of Urology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, 510260, China
| | - Guo Chen
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
| | - Yan-Qiong Zhang
- Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China
- Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
- Department of Urology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
| | - Hui-Chan He
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
| | - Yu-Xiang Liang
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
| | - Jian-Heng Ye
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
- Department of Urology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
| | - Ying-Ke Liang
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Ru-Jun Mo
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Jian-Ming Lu
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Yang-Jia Zhuo
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Yu Zheng
- Department of Urology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, 510260, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Fu-Neng Jiang
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
| | - Zhao-Dong Han
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China
| | - Shu-Lin Wu
- Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
- Department of Urology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
| | - Wei-de Zhong
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China.
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
- Urology Key Laboratory of Guangdong Province, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, 510230, China.
- Graduate school of Jinan University, Guangzhou, 510632, China.
| | - Chin-Lee Wu
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, 510180, China.
- Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA.
- Department of Urology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA.
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15
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Te Boekhorst V, Friedl P. Plasticity of Cancer Cell Invasion-Mechanisms and Implications for Therapy. Adv Cancer Res 2016; 132:209-64. [PMID: 27613134 DOI: 10.1016/bs.acr.2016.07.005] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Cancer cell migration is a plastic and adaptive process integrating cytoskeletal dynamics, cell-extracellular matrix and cell-cell adhesion, as well as tissue remodeling. In response to molecular and physical microenvironmental cues during metastatic dissemination, cancer cells exploit a versatile repertoire of invasion and dissemination strategies, including collective and single-cell migration programs. This diversity generates molecular and physical heterogeneity of migration mechanisms and metastatic routes, and provides a basis for adaptation in response to microenvironmental and therapeutic challenge. We here summarize how cytoskeletal dynamics, protease systems, cell-matrix and cell-cell adhesion pathways control cancer cell invasion programs, and how reciprocal interaction of tumor cells with the microenvironment contributes to plasticity of invasion and dissemination strategies. We discuss the potential and future implications of predicted "antimigration" therapies that target cytoskeletal dynamics, adhesion, and protease systems to interfere with metastatic dissemination, and the options for integrating antimigration therapy into the spectrum of targeted molecular therapies.
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Affiliation(s)
- V Te Boekhorst
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - P Friedl
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, United States; Radboud University Medical Centre, Nijmegen, The Netherlands; Cancer Genomics Center (CGC.nl), Utrecht, The Netherlands.
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16
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Hong J, Li D, Cao W. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells. PLoS One 2016; 11:e0149735. [PMID: 26901778 PMCID: PMC4764682 DOI: 10.1371/journal.pone.0149735] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Accepted: 01/27/2016] [Indexed: 12/13/2022] Open
Abstract
Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.
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Affiliation(s)
- Jie Hong
- Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America
- Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
| | - Dan Li
- Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America
| | - Weibiao Cao
- Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America
- Department of Pathology, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America
- * E-mail:
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17
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Shafqat-Abbasi H, Kowalewski JM, Kiss A, Gong X, Hernandez-Varas P, Berge U, Jafari-Mamaghani M, Lock JG, Strömblad S. An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes. eLife 2016; 5:e11384. [PMID: 26821527 PMCID: PMC4749554 DOI: 10.7554/elife.11384] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2015] [Accepted: 12/16/2015] [Indexed: 12/16/2022] Open
Abstract
Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration.
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Affiliation(s)
| | - Jacob M Kowalewski
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Alexa Kiss
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Xiaowei Gong
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | | | - Ulrich Berge
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | | | - John G Lock
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Staffan Strömblad
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
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18
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Chin VT, Nagrial AM, Chou A, Biankin AV, Gill AJ, Timpson P, Pajic M. Rho-associated kinase signalling and the cancer microenvironment: novel biological implications and therapeutic opportunities. Expert Rev Mol Med 2015; 17:e17. [PMID: 26507949 PMCID: PMC4836205 DOI: 10.1017/erm.2015.17] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The Rho/ROCK pathway is involved in numerous pivotal cellular processes that have made it an area of intense study in cancer medicine, however, Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors are yet to make an appearance in the clinical cancer setting. Their performance as an anti-cancer therapy has been varied in pre-clinical studies, however, they have been shown to be effective vasodilators in the treatment of hypertension and post-ischaemic stroke vasospasm. This review addresses the various roles the Rho/ROCK pathway plays in angiogenesis, tumour vascular tone and reciprocal feedback from the tumour microenvironment and explores the potential utility of ROCK inhibitors as effective vascular normalising agents. ROCK inhibitors may potentially enhance the delivery and efficacy of chemotherapy agents and improve the effectiveness of radiotherapy. As such, repurposing of these agents as adjuncts to standard treatments may significantly improve outcomes for patients with cancer. A deeper understanding of the controlled and dynamic regulation of the key components of the Rho pathway may lead to effective use of the Rho/ROCK inhibitors in the clinical management of cancer.
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Affiliation(s)
- Venessa T. Chin
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
| | - Adnan M. Nagrial
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
- The Department of Medical Oncology, Crown Princess Mary Cancer Centre, Westmead Hospital, NSW, Australia
| | - Angela Chou
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
- Anatomical Pathology, Sydpath, St Vincent's Hospital, Sydney, Australia
| | - Andrew V. Biankin
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
- Department of Surgery, Bankstown Hospital, Eldridge Road, Bankstown, Sydney, NSW 2200, Australia
- Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden, Glasgow, Scotland G61 1BD, UK
| | - Anthony J. Gill
- Department of Anatomical Pathology, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia
- University of Sydney, Sydney, NSW 2006, Australia
| | - Paul Timpson
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
- Faculty of Medicine, St Vincent's Clinical School, University of NSW, Australia
| | - Marina Pajic
- The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia
- Faculty of Medicine, St Vincent's Clinical School, University of NSW, Australia
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19
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Cheng XB, Sato N, Kohi S, Koga A, Hirata K. Receptor for Hyaluronic Acid-Mediated Motility is Associated with Poor Survival in Pancreatic Ductal Adenocarcinoma. J Cancer 2015; 6:1093-8. [PMID: 26516356 PMCID: PMC4615344 DOI: 10.7150/jca.12990] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 08/09/2015] [Indexed: 01/10/2023] Open
Abstract
Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) is a nonintegral cell surface receptor involved in the aggressive phenotype in a wide spectrum of human malignancies, but the significance of RHAMM in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, we investigated the expression of RHAMM and its clinical relevance in PDAC. RHAMM mRNA expression was examined in 8 PDAC cell lines and in primary pancreatic cancer and adjacent non-tumor tissues from 14 patients using real-time RT-PCR. Western blotting was carried out to analyze the expression of RHAMM protein in PDAC cell lines. We also investigated the expression patterns of RHAMM protein in tissue samples from 70 PDAC patients using immunohistochemistry. The RHAMM mRNA expression was increased in some PDAC cell lines as compared to a non-tumorous pancreatic epithelial cell line HPDE. The RHAMM mRNA expression was significantly higher in PDAC tissues as compared to corresponding non-tumorous pancreatic tissues (P < 0.0001). The RHAMM protein expression was higher in the vast majority of PDAC cell lines relative to the expression in HPDE. The immunohistochemical analysis revealed strong expression of RHAMM in 52 (74%) PDAC tissues. Strong expression of RHAMM was significantly associated with a shorter survival time (P = 0.038). In multivariate analysis, tumor stage (P = 0.039), residual tumor (P = 0.015), and strong RHAMM expression (P = 0.034) were independent factors predicting poor survival. Strong expression of RHAMM may predict poor survival in PDAC patients and may provide prognostic and, possibly, therapeutic value.
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Affiliation(s)
- Xiao-Bo Cheng
- 1. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan ; 2. Department of Breast Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
| | - Norihiro Sato
- 1. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan
| | - Shiro Kohi
- 1. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan
| | - Atsuhiro Koga
- 1. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan
| | - Keiji Hirata
- 1. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan
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20
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The regulatory roles of ROCK and MRCK kinases in the plasticity of cancer cell migration. Cancer Lett 2015; 361:185-96. [DOI: 10.1016/j.canlet.2015.03.017] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Revised: 03/09/2015] [Accepted: 03/11/2015] [Indexed: 12/29/2022]
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21
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Matsuoka T, Yashiro M. Rho/ROCK signaling in motility and metastasis of gastric cancer. World J Gastroenterol 2014; 20:13756-13766. [PMID: 25320513 PMCID: PMC4194559 DOI: 10.3748/wjg.v20.i38.13756] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2014] [Revised: 04/21/2014] [Accepted: 06/13/2014] [Indexed: 02/06/2023] Open
Abstract
Gastric cancer is one of the most frequent and lethal malignancies worldwide because of high frequency of metastasis. Tumor cell motility and invasion play fundamental roles in cancer metastasis. Recent studies have revealed that the Rho/Rho-associated protein kinases (ROCK) pathway plays a critical role in the regulation of cancer cell motility and invasion. In addition, the Rho/ROCK pathway plays important roles in invasion and metastasis on the basis of its predominant function of cell cytoskeletal regulation in gastric cancer. According to the current understanding of tumor motility, there are two modes of tumor cell movement: mesenchymal and amoeboid. In addition, cancer cell movement can be interchangeable between the mesenchymal and amoeboid movements under certain conditions. Control of cell motility through the actin cytoskeleton creates the potential for regulating tumor cell metastasis. In this review we discuss Rho GTPases and ROCK signaling and describe the mechanisms of Rho/ROCK activity with regard to motility and metastasis in gastric cancer. In addition, we provide an insight of the therapeutic potential of targeting the Rho/ROCK pathway.
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22
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Chen W, Mao K, Liu Z, Dinh-Xuan AT. The role of the RhoA/Rho kinase pathway in angiogenesis and its potential value in prostate cancer (Review). Oncol Lett 2014; 8:1907-1911. [PMID: 25289078 PMCID: PMC4186560 DOI: 10.3892/ol.2014.2471] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Accepted: 07/23/2014] [Indexed: 11/29/2022] Open
Abstract
Prostate cancer (PCa) remains a major cause of mortality among males in western countries, with little change in mortality rates observed over the past 25 years. Despite recent advances in therapy, treatment options for metastatic castration-resistant disease remain limited. In terms of chemotherapy, only the combination of docetaxel and prednisone has been shown to improve survival in these patients, but duration of response to therapy is short. There is a continuing unmet need for new systemic interventions that act either alone or synergistically with chemotherapy in patients with progressive PCa. Angiogenesis plays a critical role in tumor growth and metastasis in PCa. Several strategies have been used to target angiogenesis; however, it is becoming increasingly apparent that current anti-angiogenic therapies frequently achieve only modest effects in clinical settings. The RhoA/Rho kinase (ROCK) pathway plays a crucial role in the process of angiogenesis in PCa, and studies have demonstrated that ROCK inhibitors decrease VEGF-induced angiogenesis and tumor cell growth. However, further research is required to fully elucidate the molecular mechanisms involved in this pathway, and the potential value of modulating these mechanisms in the treatment of PCa. This study reviews the current understanding of the role of the RhoA/ROCK pathway in the process of angiogenesis in PCa, and the potential of this pathway as a therapeutic target in the future.
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Affiliation(s)
- Weihua Chen
- Department of Clinical Physiology, Medical School, Cochin Hospital, Paris Descartes University, EA 2511, Paris 75014, France ; Department of Urology, Tongji University, School of Medicine, Shanghai East Hospital, Shanghai 200120, P.R. China
| | - Kaili Mao
- Department of Clinical Physiology, Medical School, Cochin Hospital, Paris Descartes University, EA 2511, Paris 75014, France ; Department of Urology, Tongji University, School of Medicine, Shanghai East Hospital, Shanghai 200120, P.R. China
| | - Zhongmin Liu
- Clinical and Translational Research Center, Tongji University, School of Medicine, Shanghai East Hospital, Shanghai 200120, P.R. China
| | - Anh Tuan Dinh-Xuan
- Department of Clinical Physiology, Medical School, Cochin Hospital, Paris Descartes University, EA 2511, Paris 75014, France ; Clinical and Translational Research Center, Tongji University, School of Medicine, Shanghai East Hospital, Shanghai 200120, P.R. China
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Abstract
Peripheral arterial disease (PAD) is a common vascular disease that reduces blood flow capacity to the legs of patients. PAD leads to exercise intolerance that can progress in severity to greatly limit mobility, and in advanced cases leads to frank ischemia with pain at rest. It is estimated that 12 to 15 million people in the United States are diagnosed with PAD, with a much larger population that is undiagnosed. The presence of PAD predicts a 50% to 1500% increase in morbidity and mortality, depending on severity. Treatment of patients with PAD is limited to modification of cardiovascular disease risk factors, pharmacological intervention, surgery, and exercise therapy. Extended exercise programs that involve walking approximately five times per week, at a significant intensity that requires frequent rest periods, are most significant. Preclinical studies and virtually all clinical trials demonstrate the benefits of exercise therapy, including improved walking tolerance, modified inflammatory/hemostatic markers, enhanced vasoresponsiveness, adaptations within the limb (angiogenesis, arteriogenesis, and mitochondrial synthesis) that enhance oxygen delivery and metabolic responses, potentially delayed progression of the disease, enhanced quality of life indices, and extended longevity. A synthesis is provided as to how these adaptations can develop in the context of our current state of knowledge and events known to be orchestrated by exercise. The benefits are so compelling that exercise prescription should be an essential option presented to patients with PAD in the absence of contraindications. Obviously, selecting for a lifestyle pattern that includes enhanced physical activity prior to the advance of PAD limitations is the most desirable and beneficial.
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Affiliation(s)
- Tara L Haas
- Angiogenesis Research Group, Muscle Health Research Centre, Faculty of Health, York University, Toronto, Ontario, Canada
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24
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Mardilovich K, Olson MF, Baugh M. Targeting Rho GTPase signaling for cancer therapy. Future Oncol 2012; 8:165-77. [PMID: 22335581 DOI: 10.2217/fon.11.143] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Accumulating evidence from basic and clinical studies supports the concept that signaling pathways downstream of Rho GTPases play important roles in tumor development and progression. As a result, there has been considerable interest in the possibility that specific proteins in these signal transduction pathways could be potential targets for cancer therapy. A number of inhibitors targeting critical effector proteins, activators or the Rho GTPases themselves, have been developed. We will review the strategies currently being used to develop inhibitors of Rho GTPases and downstream signaling kinases and discuss candidate entities. Although molecularly targeted drugs that inhibit Rho GTPase signaling have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to drive considerable pharmaceutical research and development.
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Affiliation(s)
- Katerina Mardilovich
- Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK
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25
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Pireddu R, Forinash KD, Sun NN, Martin MP, Sung SS, Alexander B, Zhu JY, Guida WC, Schönbrunn E, Sebti SM, Lawrence NJ. Pyridylthiazole-based ureas as inhibitors of Rho associated protein kinases (ROCK1 and 2). MEDCHEMCOMM 2012; 3:699-709. [PMID: 23275831 PMCID: PMC3531244 DOI: 10.1039/c2md00320a] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Potent ROCK inhibitors of a new class of 1-benzyl-3-(4-pyridylthiazol-2-yl)ureas have been identified. Remarkable differences in activity were observed for ureas bearing a benzylic stereogenic center. Derivatives with hydroxy, methoxy and amino groups at the meta position of the phenyl ring give rise to the most potent inhibitors (low nM). Substitutions at the para position result in substantial loss of potency. Changes at the benzylic position are tolerated resulting in significant potency in the case of methyl and methylenehydroxy groups. X-Ray crystallography was used to establish the binding mode of this class of inhibitors and provides an explanation for the observed differences of the enantiomer series. Potent inhibition of ROCK in human lung cancer cells was shown by suppression of the levels of phosphorylation of the ROCK substrate MYPT-1.
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Affiliation(s)
- Roberta Pireddu
- The Department of Drug Discovery, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, Florida, 33612, USA
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26
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Carbajo-Lozoya J, Lutz S, Feng Y, Kroll J, Hammes HP, Wieland T. Angiotensin II modulates VEGF-driven angiogenesis by opposing effects of type 1 and type 2 receptor stimulation in the microvascular endothelium. Cell Signal 2012; 24:1261-9. [PMID: 22374305 DOI: 10.1016/j.cellsig.2012.02.005] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2012] [Revised: 02/14/2012] [Accepted: 02/14/2012] [Indexed: 11/19/2022]
Abstract
Vascular endothelial growth factor (VEGF) is a main stimulator of pathological vessel formation. Nevertheless, increasing evidence suggests that Angiotensin II (Ang II) can play an augmentory role in this process. We thus analyzed the contribution of the two Ang II receptor types, AT(1)R and AT(2)R, in a mouse model of VEGF-driven angiogenesis, i.e. oxygen-induced proliferative retinopathy. Application of the AT(1)R antagonist telmisartan but not the AT(2)R antagonist PD123,319 largely attenuated the pathological response. A direct effect of Ang II on endothelial cells (EC) was analyzed by assessing angiogenic responses in primary bovine retinal and immortalized rat microvascular EC. Selective stimulation of the AT(1)R by Ang II in the presence of PD123,319 revealed a pro-angiogenic activity which further increased VEGF-driven EC sprouting and migration. In contrast, selective stimulation of the AT(2)R by either CGP42112A or Ang II in the presence of telmisartan inhibited the VEGF-driven angiogenic response. Using specific inhibitors (pertussis toxin, RGS proteins, kinase inhibitors) we identified G(12/13) and G(i) dependent signaling pathways as the mediators of the AT(1)R-induced angiogenesis and the AT(2)R-induced inhibition, respectively. As AT(1)R and AT(2)R stimulation displays opposing effects on the activity of the monomeric GTPase RhoA and pro-angiogenic responses to Ang II and VEGF requires activation of Rho-dependent kinase (ROCK), we conclude that the opposing effects of the Ang II receptors on VEGF-driven angiogenesis converge on the regulation of activity of RhoA-ROCK-dependent EC migration.
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MESH Headings
- Angiotensin II/metabolism
- Angiotensin Receptor Antagonists/pharmacology
- Animals
- Cattle
- Cell Movement
- Cells, Cultured
- Endothelial Cells/cytology
- Endothelial Cells/metabolism
- Endothelium, Vascular/cytology
- Endothelium, Vascular/growth & development
- Endothelium, Vascular/metabolism
- GTP-Binding Protein alpha Subunits, G12-G13/metabolism
- Mice
- Mice, Inbred C57BL
- Microvessels/cytology
- Microvessels/growth & development
- Microvessels/metabolism
- Neovascularization, Pathologic
- Neovascularization, Physiologic
- Rats
- Receptor, Angiotensin, Type 1/metabolism
- Receptor, Angiotensin, Type 2/metabolism
- Retina/pathology
- Retina/ultrastructure
- Vascular Endothelial Growth Factor A/metabolism
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Affiliation(s)
- Javier Carbajo-Lozoya
- Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Maybachstrasse 14, D-68169 Mannheim, Germany
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27
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Hudson CA, Heesom KJ, López Bernal A. Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1). Mol Hum Reprod 2011; 18:265-79. [PMID: 22155728 PMCID: PMC3339637 DOI: 10.1093/molehr/gar078] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Force generation in smooth muscle is driven by phosphorylation of myosin light chains (MYL), which is regulated by the equilibrium between the activities of myosin light chain kinase (MYLK) and myosin phosphatase (MYLP). MYLK is activated by Ca2+-calmodulin whereas MYLP is inhibited by phosphorylation of its myosin-binding subunit (MYPT1) by Ca2+-independent mechanisms, leading to generation of increased MYL phosphorylation and force for a given intracellular Ca2+ concentration, a phenomenon known as ‘calcium-sensitization’. The regulation of MYPT1 phosphorylation in human myometrium, which shows increasing phasic contractility at the onset of labour, has yet to be fully investigated. Here, we explore phosphorylation of MYPT1 at Thr696 and Thr853, alongside phosphorylation of MYL, in fresh human myometrial tissue and cultured myometrial cells. We report that pMYPT1 (Thr853) levels are dependent on the activity of Rho-associated kinase (ROCK), determined using the ROCK inhibitor g-H-1152 and siRNA-mediated knockdown of ROCK1/2, and are highly correlated to ppMYL (Thr18/Ser19) levels. Pharmacological inhibition of ROCK was associated with a decrease in oxytocin (OXT)-stimulated contractility of myometrial strips in vitro. Moreover, we have measured pMYPT1 and pMYL levels between and during spontaneous and OXT-stimulated phasic contractions by rapidly freezing contracting muscle, and demonstrate for the first time functional coupling between increases in pMYPT1 (Thr853), ppMYL (Thr18/Ser19) and phasic contractility that is ROCK-dependent. The combined approach of measuring contractility and phosphorylation has demonstrated that the phosphorylation of MYPT1 (Thr853) changes dynamically with each contraction and has elucidated a defined role for ROCK in regulating myometrial contractility through MYLP, providing new insights into uterine physiology which will stimulate further research into treatments for preterm labour.
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Affiliation(s)
- Claire A Hudson
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (Obstetrics and Gynaecology), School of Clinical Sciences, University of Bristol, Bristol BS1 3NY, UK
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28
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Heikkila T, Wheatley E, Crighton D, Schroder E, Boakes A, Kaye SJ, Mezna M, Pang L, Rushbrooke M, Turnbull A, Olson MF. Co-crystal structures of inhibitors with MRCKβ, a key regulator of tumor cell invasion. PLoS One 2011; 6:e24825. [PMID: 21949762 PMCID: PMC3176812 DOI: 10.1371/journal.pone.0024825] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2011] [Accepted: 08/18/2011] [Indexed: 12/20/2022] Open
Abstract
MRCKα and MRCKβ (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKβ influence cell shape and motility. We report further evidence for MRCKα and MRCKβ contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKβ together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKβ kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC(50) values for MRCKα and MRCKβ. We also describe the crystal structure of MRCKβ in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.
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Affiliation(s)
- Timo Heikkila
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Edward Wheatley
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Diane Crighton
- Beatson Institute for Cancer Research, Glasgow, United Kingdom
| | - Ewald Schroder
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Alexandra Boakes
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Sarah J. Kaye
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Mokdad Mezna
- Beatson Institute for Cancer Research, Glasgow, United Kingdom
| | - Leon Pang
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Mathew Rushbrooke
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
| | - Andrew Turnbull
- Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, United Kingdom
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29
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Momotani K, Artamonov MV, Utepbergenov D, Derewenda U, Derewenda ZS, Somlyo AV. p63RhoGEF couples Gα(q/11)-mediated signaling to Ca2+ sensitization of vascular smooth muscle contractility. Circ Res 2011; 109:993-1002. [PMID: 21885830 DOI: 10.1161/circresaha.111.248898] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
RATIONALE In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. Although it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. OBJECTIVE We examine whether p63RhoGEF, known to couple specifically to Gα(q/11) in vitro, is functional in blood vessels as a mediator of RhoA activation and if it is selectively activated by Gα(q/11) coupled agonists. METHODS AND RESULTS We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly Gα(12/13)-mediated thromboxane analog U46619. This is because endothelin-1 acts on Gα(q/11) as well as Gα(12/13). Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331-580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through Gα(q/11). CONCLUSION We demonstrate that p63RhoGEF selectively couples Gα(q/11) but not Gα(12/13), to RhoA activation in blood vessels and cultured cells and thus mediates the physiologically important Ca2+ sensitization of force induced with Gα(q/11)-coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.
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Affiliation(s)
- Ko Momotani
- University of Virginia, Charlottesville, VA 22908-0736, USA
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30
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OLFERT IMARK, BIROT OLIVIER. Importance of Anti-angiogenic Factors in the Regulation of Skeletal Muscle Angiogenesis. Microcirculation 2011; 18:316-30. [DOI: 10.1111/j.1549-8719.2011.00092.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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31
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van der Meel R, Symons MH, Kudernatsch R, Kok RJ, Schiffelers RM, Storm G, Gallagher WM, Byrne AT. The VEGF/Rho GTPase signalling pathway: A promising target for anti-angiogenic/anti-invasion therapy. Drug Discov Today 2011; 16:219-28. [DOI: 10.1016/j.drudis.2011.01.005] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2010] [Revised: 11/26/2010] [Accepted: 01/14/2011] [Indexed: 12/17/2022]
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32
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Hara Y, Wakino S, Tanabe Y, Saito M, Tokuyama H, Washida N, Tatematsu S, Yoshioka K, Homma K, Hasegawa K, Minakuchi H, Fujimura K, Hosoya K, Hayashi K, Nakayama K, Itoh H. Rho and Rho-Kinase Activity in Adipocytes Contributes to a Vicious Cycle in Obesity That May Involve Mechanical Stretch. Sci Signal 2011; 4:ra3. [DOI: 10.1126/scisignal.2001227] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
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33
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Paleček J, Zweigerdt R, Olmer R, Martin U, Kirschning A, Dräger G. A practical synthesis of Rho-Kinase inhibitor Y-27632 and fluoro derivatives and their evaluation in human pluripotent stem cells. Org Biomol Chem 2011; 9:5503-10. [DOI: 10.1039/c1ob05332a] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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34
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Pathak A, Kumar S. Biophysical regulation of tumor cell invasion: moving beyond matrix stiffness. Integr Biol (Camb) 2011; 3:267-78. [DOI: 10.1039/c0ib00095g] [Citation(s) in RCA: 161] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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35
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Nakabayashi H, Shimizu K. HA1077, a Rho kinase inhibitor, suppresses glioma-induced angiogenesis by targeting the Rho-ROCK and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signal pathways. Cancer Sci 2010; 102:393-9. [DOI: 10.1111/j.1349-7006.2010.01794.x] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
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36
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Jia Y, Wu SL, Isenberg JS, Dai S, Sipes JM, Field L, Zeng B, Bandle RW, Ridnour LA, Wink DA, Ramchandran R, Karger BL, Roberts DD. Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton. Cell Stress Chaperones 2010; 15:165-81. [PMID: 19579057 PMCID: PMC2866983 DOI: 10.1007/s12192-009-0130-0] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2009] [Revised: 06/12/2009] [Accepted: 06/17/2009] [Indexed: 10/20/2022] Open
Abstract
Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.
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Affiliation(s)
- Yifeng Jia
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Shiaw-Lin Wu
- Barnett Institute, Northeastern University, Boston, MA 02115 USA
| | - Jeff S. Isenberg
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
- Hemostasis and Vascular Biology Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA
| | - Shujia Dai
- Barnett Institute, Northeastern University, Boston, MA 02115 USA
| | - John M. Sipes
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Lyndsay Field
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Bixi Zeng
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Russell W. Bandle
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Lisa A. Ridnour
- Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - David A. Wink
- Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
| | - Ramani Ramchandran
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
- Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226 USA
| | - Barry L. Karger
- Barnett Institute, Northeastern University, Boston, MA 02115 USA
| | - David D. Roberts
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
- NIH, Building 10 Room 2A33, 10 Center Dr MSC1500, Bethesda, MD 20892-1500 USA
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Tatsumiya K, Yamanishi T, Watanabe M, Masuda A, Mizuno T, Kamai T, Yoshida KI. Effects of fasudil, a Rho-kinase inhibitor, on contraction of pig bladder tissues with or without urothelium. Int J Urol 2010; 16:959-66. [PMID: 19817915 DOI: 10.1111/j.1442-2042.2009.02397.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
OBJECTIVES To investigate the effects of fasudil, a Rho-associated serine-threonine protein kinase inhibitor, on contraction of the pig urinary bladder tissues with or without urothelium. METHODS Cumulative concentration-response curves (CRCs) to carbachol were obtained with and without 3-10 microM fasudil. Drug effects were evaluated in detrusor with and without urothelium. Inhibitory responses to fasudil were also examined in tissues precontracted with KCl and carbachol, and in response to electrical field stimulation, in pig bladder with and without urothelium. RESULTS In detrusor without urothelium, maximum contraction (E(max)) decreased after administration of fasudil at 3 or 10 micromol/L (both P < 0.01), or 30 micromol/L (72.5 + or - 7.43%, 58.4 + or - 8.04% and 68.4 + or - 9.6%, respectively, of the first curve). In detrusor with urothelium, E(max) decreased significantly (all P < 0.05) after the addition of 3, 10 or 30 micromol/L of fasudil (84.9 + or - 6.7%, 67.9 + or - 5.2% and 35.2 + or - 4.1%, respectively). In tissues precontracted with 80 mmol/L KCl or 100 micromol/L carbachol, tension after administration of fasudil (1 nmol/L to 100 micromol/L) decreased (by approximately 40%), only after administration of fasudil at high concentration (>1 micromol/L), in detrusor both with and without urothelium. In tissues with and without urothelium, responses to electrical field stimulation at 1-50 Hz decreased significantly in a concentration-dependent manner after addition of fasudil (3 to 30 micromol/L). CONCLUSIONS Fasudil seems to provoke relaxation of the bladder detrusor via both urothelium-dependent and independent pathways.
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Hahmann C, Schroeter T. Rho-kinase inhibitors as therapeutics: from pan inhibition to isoform selectivity. Cell Mol Life Sci 2010; 67:171-7. [PMID: 19907920 PMCID: PMC11115778 DOI: 10.1007/s00018-009-0189-x] [Citation(s) in RCA: 120] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2009] [Revised: 09/08/2009] [Accepted: 09/17/2009] [Indexed: 01/02/2023]
Abstract
The emerging critical implications of Rho/Rho-kinase (ROCK) signaling in neurodegenerative diseases, glaucoma, renoprotection, diabetes and cancer have sparked growing interest in the pharmacological potential of ROCK inhibitors beyond their current application in cardiovascular disease. This article discusses the therapeutic benefits of novel ROCK inhibitors in development, and highlights the recent advances in the current understanding of disease-dependent and isoform-specific functions of ROCK and their potential impact on future therapeutic strategies.
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Affiliation(s)
- C. Hahmann
- Discovery Biology, Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458 USA
| | - T. Schroeter
- Discovery Biology, Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458 USA
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Nakanishi K, Kamai T, Mizuno T, Arai K, Yamanishi T. Expression of RhoA mRNA and activated RhoA in urothelium and smooth muscle, and effects of a rho-kinase inhibitor on contraction of the porcine urinary bladder. Neurourol Urodyn 2009; 28:521-8. [DOI: 10.1002/nau.20694] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Isayeva T, Moore LD, Chanda D, Chen D, Ponnazhagan S. Tumoristatic effects of endostatin in prostate cancer is dependent on androgen receptor status. Prostate 2009; 69:1055-66. [PMID: 19301304 PMCID: PMC5087284 DOI: 10.1002/pros.20952] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
BACKGROUND Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR.
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Affiliation(s)
- Tatyana Isayeva
- Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294
| | - Lakisha D. Moore
- Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294
| | - Diptiman Chanda
- Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294
| | - Dongquan Chen
- Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294
| | - Selvarangan Ponnazhagan
- Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294
- Corresponding author: Selvarangan Ponnazhagan, Ph.D., Department of Pathology, LHRB 513, 701, 19 Street South, University of Alabama at Birmingham, Birmingham, AL 35294-0007, Phone: (205) 934-6731, Fax: (205) 975-9927,
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Roudier E, Chapados N, Decary S, Gineste C, Le Bel C, Lavoie JM, Bergeron R, Birot O. Angiomotin p80/p130 ratio: a new indicator of exercise-induced angiogenic activity in skeletal muscles from obese and non-obese rats? J Physiol 2009; 587:4105-19. [PMID: 19546164 DOI: 10.1113/jphysiol.2009.175554] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Skeletal muscle capillarisation responds to physiological and pathological conditions with a remarkable plasticity. Angiomotin was recently identified as a new pro-angiogenic molecule. Angiomotin is expressed as two protein isoforms, p80 and p130. Whereas p80 stimulates endothelial cell migration and angiogenesis, p130 is rather characteristic of stabilized and matured vessels. To date, how angiomotin expression is physiologically regulated in vivo remains largely unknown. We thus investigated (1) whether angiomotin was physiologically expressed in skeletal muscle; (2) whether exercise training, known to stimulate muscle angiogenesis, affected angiomotin expression; and (3) whether such regulation was altered in obesity, a pathological situation often associated with an impaired angiogenic activity and some capillary rarefaction in skeletal muscle. Two models of obesity were used: a high fat diet regime and Zucker Diabetic Fatty rats (ZDF). Our results provide evidence that angiomotin was expressed both in capillaries and myofibres. In non-obese rats, the p80 isoform was increased in plantaris muscle in response to endurance training whereas p130 was unaffected. In obese animals, no change was observed for p80 whereas training significantly decreased p130 expression. Exercise training induced angiogenesis in plantaris from both obese and non-obese rats, possibly through the modulation of angiomotin level and its consequences on RhoA-ROCK signalling. In conclusion, any increase in p80 or decrease in p130, as respectively observed in non-obese and obese animals, led to an increased ratio between p80 and p130 isoforms. This increased angiomotin p80/p130 ratio might then directly reflect the enhanced angiogenic ability of skeletal muscle in response to exercise training.
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Affiliation(s)
- Emilie Roudier
- York University, Muscle Health Research Center, School of Kinesiology and Health Science, Norman Bethune College (Room 353), 4700 Keele Street, Toronto, ON, M3J 1P3, Canada
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De Smet F, Segura I, De Bock K, Hohensinner PJ, Carmeliet P. Mechanisms of Vessel Branching. Arterioscler Thromb Vasc Biol 2009; 29:639-49. [DOI: 10.1161/atvbaha.109.185165] [Citation(s) in RCA: 283] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Filopodia, “the fingers that do the walking,” have been identified on endothelial cells at the tip of sprouting vessels for half a century, but the key role of the tip cell in vessel branching has been recognized only in the past few years. A model is emerging, whereby tip cells lead the way in a branching vessel, stalk cells elongate the sprout, and a very recently discovered phalanx cell ensures quiescence and perfusion of the newly formed branch. Recent genetic studies have shed light on the molecular signature of these distinct endothelial phenotypes; this provides a novel conceptual framework of how vessel morphogenesis occurs. Here, we will discuss the molecular candidates that participate in the decision of endothelial cells to adapt these distinct fates and highlight the emerging insights on how these cells send out filopodia while navigating.
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Affiliation(s)
| | | | - Katrien De Bock
- From the Vesalius Research Center, VIB, K.U. Leuven, Belgium
| | | | - Peter Carmeliet
- From the Vesalius Research Center, VIB, K.U. Leuven, Belgium
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van Nieuw Amerongen GP, van Hinsbergh VWM. Role of ROCK I/II in vascular branching. Am J Physiol Heart Circ Physiol 2009; 296:H903-5. [PMID: 19218507 DOI: 10.1152/ajpheart.00125.2009] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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Kroll J, Epting D, Kern K, Dietz CT, Feng Y, Hammes HP, Wieland T, Augustin HG. Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis. Am J Physiol Heart Circ Physiol 2009; 296:H893-9. [PMID: 19181962 DOI: 10.1152/ajpheart.01038.2008] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.
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Affiliation(s)
- Jens Kroll
- Center for Biomedicine and Medical Technology Mannheim, Joint Research Division Vascular Biology Medical Faculty Mannheim, Univ. of Heidelberg and the German Cancer Research Center, Ludolf-Krehl-Str. 13-17, 68167 Mannheim, Germany.
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Santos AF, Zaltsman AB, Martin RC, Kuzmin A, Alexandrov Y, Roquemore EP, Jessop RA, Erck MGMV, Verheijen JH. Angiogenesis: An Improved In Vitro Biological System and Automated Image-Based Workflow to Aid Identification and Characterization of Angiogenesis and Angiogenic Modulators. Assay Drug Dev Technol 2008; 6:693-710. [DOI: 10.1089/adt.2008.146] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
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Current status of experimental therapeutics for prostate cancer. Cancer Lett 2008; 266:116-34. [DOI: 10.1016/j.canlet.2008.02.065] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2007] [Revised: 02/22/2008] [Accepted: 02/22/2008] [Indexed: 11/17/2022]
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Mizuno Y, Isotani E, Huang J, Ding H, Stull JT, Kamm KE. Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo. Am J Physiol Cell Physiol 2008; 295:C358-64. [PMID: 18524939 DOI: 10.1152/ajpcell.90645.2007] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.
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Affiliation(s)
- Yusuke Mizuno
- Dept. Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040, USA
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Lin SL, Chiang A, Chang D, Ying SY. Loss of mir-146a function in hormone-refractory prostate cancer. RNA (NEW YORK, N.Y.) 2008; 14:417-24. [PMID: 18174313 PMCID: PMC2248249 DOI: 10.1261/rna.874808] [Citation(s) in RCA: 288] [Impact Index Per Article: 16.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/12/2007] [Accepted: 11/14/2007] [Indexed: 05/25/2023]
Abstract
The pattern of microRNA (miRNA) expression is associated with the degree of tumor cell differentiation in human prostate cancer. MiRNAs bind complementarily to either oncogenes or tumor suppressor genes, which are consequently silenced, resulting in alterations of tumorigenecity. We have detected eight down-regulated and three up-regulated known miRNAs in androgen-independent human prostate cancer cells compared to those in androgen-dependent cells, using miRNA microarray analyses. These identified miRNAs showed the same expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive noncancerous prostate epithelium as determined by fluorescent in situ hybridization assays in human prostate cancer tissue arrays. One of the eight down-regulated miRNAs, mir-146a, was selected and constitutively expressed to examine its effects on suppression of prostate cancer transformation from androgen-dependent to -independent cells as determined by in vitro tumorigenecity assays. Transfection of mir-146a, which perpetually express the miRNA, suppressed >82% of the expression of the targeted protein-coding gene, ROCK1, in androgen-independent PC3 cells, consequently markedly reducing cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers. Given that ROCK1 is one of the key kinases for the activation of hyaluronan (HA)-mediated HRPC transformation in vivo and in PC3 cells, mir-146a may function as a tumor-suppressor gene in modulating HA/ROCK1-mediated tumorigenecity in androgen-dependent prostate cancer.
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Affiliation(s)
- Shi-Lung Lin
- Department of Cell and Neurobiology, Keck School of Medicine, BMT-403, University of Southern California, 1333 San Pablo Street, Los Angeles, CA 90033, USA.
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Lin SL, Chang D, Chiang A, Ying SY. Androgen receptor regulates CD168 expression and signaling in prostate cancer. Carcinogenesis 2008; 29:282-90. [PMID: 18174258 DOI: 10.1093/carcin/bgm259] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Dysregulation of the androgen receptor (AR) and its signaling in the prostate often occurs during normal aging or after androgen ablation, consequently leading to the development of hormone-refractory prostate cancer (HRPC). Hyaluronan (HA) plays an important role in this transformation of androgen-independent cancer. Previous studies have shown that activation of the receptor for hyaluronan-mediated motility, CD168, was correlated with the Gleason's score, cancer stage, transformation and metastasis in >90% of HRPC patients. However, the relationship between loss of AR dependency and HA-mediated CD168 signaling remains unclear. We report here that AR regulates normal CD168 expression and its downstream signaling in androgen-dependent (AD) prostatic epithelial cell lines. Furthermore, we observed that the concurrent treatments of HA and dihydrotestosterone (DHT), a native androgen, significantly promoted the tumorigenicity of AD prostate cancer cell lines, which showed elevated rates of cell proliferation, invasion and metastasis to the human bone marrow endothelial cell layer. Inhibition of CD168 downstream Rho-activated protein kinases completely prevented this type of tumorigenicity. These findings suggest that the interaction of androgen and AR is essential for regulating HA-mediated cancer progression via the CD168/ROCK signal transduction pathway and also indicate that the loss of AR regulation not only causes CD168 overexpression but it also activates HA-mediated CD168 signaling in malignant cancer progression and metastasis of HRPC.
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Affiliation(s)
- Shi-Lung Lin
- Department of Cell and Neurobiology, Keck School of Medicine, BMT-403, University of Southern California, 1333 San Pablo Street, Los Angeles, CA 90033, USA
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Yin L, Morishige KI, Takahashi T, Hashimoto K, Ogata S, Tsutsumi S, Takata K, Ohta T, Kawagoe J, Takahashi K, Kurachi H. Fasudil inhibits vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Mol Cancer Ther 2007; 6:1517-25. [PMID: 17513600 DOI: 10.1158/1535-7163.mct-06-0689] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer.
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Affiliation(s)
- Limei Yin
- Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan
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