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Carducci MA, Wang D, Habermehl C, Bödding M, Rohdich F, Lignet F, Duecker K, Karpenko O, Pudelko L, Gimmi C, LoRusso P. A First-in-human, Dose-escalation Study of the Methionine Aminopeptidase 2 Inhibitor M8891 in Patients with Advanced Solid Tumors. CANCER RESEARCH COMMUNICATIONS 2023; 3:1638-1647. [PMID: 37637935 PMCID: PMC10448909 DOI: 10.1158/2767-9764.crc-23-0048] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 05/25/2023] [Accepted: 07/24/2023] [Indexed: 08/29/2023]
Abstract
Methionine aminopeptidase 2 (MetAP2) is essential to endothelial cell growth and proliferation during tumor angiogenesis. M8891 is a novel orally bioavailable, potent, selective, reversible MetAP2 inhibitor with antiangiogenic and antitumor activity in preclinical studies. The safety, tolerability, pharmacokinetics, and pharmacodynamics of M8891 monotherapy were assessed in a phase I, first-in-human, multicenter, open-label, single-arm, dose-escalation study (NCT03138538). Patients with advanced solid tumors received 7-80 mg M8891 once daily in 21-day cycles. The primary endpoint was dose-limiting toxicity (DLT) during cycle 1, with the aim to determine the maximum tolerated dose (MTD). Twenty-seven patients were enrolled across six dose levels. Two DLTs (platelet count decrease) were reported, one each at 60 and 80 mg/once daily M8891, resolving after treatment discontinuation. MTD was not determined. The most common treatment-emergent adverse event was platelet count decrease. M8891 plasma concentration showed dose-linear increase up to 35 mg and low-to-moderate variability; dose-dependent tumor accumulation of methionylated elongation factor 1α, a MetAP2 substrate, was observed, demonstrating MetAP2 inhibition. Pharmacokinetic/pharmacodynamic response data showed that preclinically defined target levels required for in vivo efficacy were achieved at safe, tolerated doses. Seven patients (25.9%) had stable disease for 42-123 days. We conclude that M8891 demonstrates a manageable safety profile, with dose-proportional exposure and low-to-moderate interpatient variability at target pharmacokinetic/pharmacodynamic levels at ≤35 mg M8891 once daily. On the basis of the data, 35 mg M8891 once daily is the recommended phase II dose for M8891 monotherapy. This study forms the basis for future development of M8891 in monotherapy and combination studies. Significance M8891 represents a novel class of reversible MetAP2 inhibitors and has demonstrated preclinical antitumor activity. This dose-escalation study assessed M8891 treatment for patients with advanced solid tumors. M8891 demonstrated favorable pharmacokinetics, tumoral target engagement, and a manageable safety profile, and thus represents a novel antitumor strategy warranting further clinical studies.
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Affiliation(s)
| | - Ding Wang
- Phase 1 Clinical Trials Program, Henry Ford Cancer Institute, Detroit, Michigan
| | | | - Matthias Bödding
- Clinical Pharmacology, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Felix Rohdich
- Pharmacokinetics, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Floriane Lignet
- Pharmacokinetics, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Klaus Duecker
- Clinical Biomarkers, the healthcare business of Merck KGaA, Darmstadt, Germany
| | | | - Linda Pudelko
- Clinical Development, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Claude Gimmi
- Clinical Development, the healthcare business of Merck KGaA, Darmstadt, Germany
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Zhang K, Hu J, Zhao Z. Fumagillin regulates stemness and malignancies in cancer stem-like cells derived from liver cancer via targeting to MetAP-2. PLoS One 2023; 18:e0289024. [PMID: 37506053 PMCID: PMC10381083 DOI: 10.1371/journal.pone.0289024] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Accepted: 07/09/2023] [Indexed: 07/30/2023] Open
Abstract
BACKGROUND Cancer relapse is associated with the presence of cancer stem-like cells (CSCs), which lead to multidirectional differentiation and unrestricted proliferative replication. Fumagillin, a myocotoxin produced by the saprophytic filamentous fungus Aspergillus fumigatus, has been reported to affect malignant characteristics in hepatocellular cancer cells. However, its exact role in CSCs is still unknown. METHODS CSCs were enriched by culturing cancer cells in serum-free medium. The effects of fumagillin on malignant cell characteristics and mitochondrial function were measured. The regulatory role of fumagillin on methionine aminopeptidase-2 (MetAP-2) was assessed. RESULTS When it was supplemented in medium, fumagillin treatment inhibited sphere formation and the maintenance of stemness of CSCs without disturbing cell growth. Fumagillin also decreased stemness-related markers and the aldehyde dehydrogenase 1 (ALDH1)-positive proportion, which demonstrated that fumagillin decreases stemness in CSCs. It was also found to inhibit malignant traits in CSCs, including cell proliferation, invasion, and tumor formation, and sensitize CSCs to chemoagents, including sorafenib and doxorubicin, by promoting chemoagent-induced apoptosis. Moreover, fumagillin treatment was found to disturb mitochondrial membrane homeostasis, ATP synthesis and mitochondrial transcriptional activity. In addition, we found that fumagillin decreased MetAP-2 protein levels and exerted anti-CSC effects potentially by regulating MetAP-2. We also found that fumagillin treatment activated p53 and its transcriptional activity and thus caused cell cycle blockade. Moreover, fumagillin treatment significantly decreased tumor formation in nude mice. CONCLUSION This work offers evidence for fumagillin as a specific inhibitor of liver cancer CSCs and proposes a novel strategy for cancer therapy.
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Affiliation(s)
- Ke Zhang
- The Second People’s Hospital of Yibin, Sichuan, China
| | - Jian Hu
- The Second People’s Hospital of Yibin, Sichuan, China
| | - Ziyi Zhao
- TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine, Sichuan, China
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Abstract
Rather than serving as a mere onlooker, adipose tissue is a complex endocrine organ and active participant in disease initiation and progression. Disruptions of biological processes operating within adipose can disturb healthy systemic physiology, the sequelae of which include metabolic disorders such as obesity and type 2 diabetes. A burgeoning interest in the field of adipose research has allowed for the elucidation of regulatory networks underlying both adipose tissue function and dysfunction. Despite this progress, few diseases are treated by targeting maladaptation in the adipose, an oft-overlooked organ. In this review, we elaborate on the distinct subtypes of adipocytes, their developmental origins and secretory roles, and the dynamic interplay at work within the tissue itself. Central to this discussion is the relationship between adipose and disease states, including obesity, cachexia, and infectious diseases, as we aim to leverage our wealth of knowledge for the development of novel and targeted therapeutics.
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Affiliation(s)
- Christopher Auger
- Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA;
| | - Shingo Kajimura
- Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA; .,Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA;
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Esa R, Steinberg E, Dagan A, Yekhtin Z, Tischenko K, Benny O. Newly synthesized methionine aminopeptidase 2 inhibitor hinders tumor growth. Drug Deliv Transl Res 2022; 13:1170-1182. [PMID: 35637333 DOI: 10.1007/s13346-022-01187-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/17/2022] [Indexed: 11/24/2022]
Abstract
Methionine aminopeptidase 2 (MetAp2) inhibition has been recognized as a promising approach for suppressing angiogenesis and cancer progression. Small molecule fumagillol derivatives with adamantane side groups were synthesized and evaluated for MetAp2 inhibition activity, and a lead molecule with superior abilities to inhibit the enzymatic activity of MetAp2 was identified. The compound, referred to as AD-3281, effectively suppressed proliferation of cancer and endothelial cells and impaired tube formation of endothelial cells in vitro. When administered systemically, AD-3281 was well tolerated and led to a significant suppression of human melanoma and mammary tumor xenografts grown in mice. The activity in vivo was associated with reduced angiogenesis and tumor proliferation as detected histologically. In order to develop a formulation that can solubilize AD-3281 with a minimal content of organic solvents, biodegradable nanoparticles comprised of poly-lactic-co-glycolic acid (PLGA) were fabricated and characterized. Compared with the free compound, AD-3281-loaded nanoparticles showed an advantageous cellular availability and uptake, leading to higher activity in cells and better transport in three-dimensional (3D) cultures. Taken together, we introduce a novel MetAp2 inhibitor with high anti-cancer activity and a stable nano-formulation with a high potential for future clinical translation.
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Affiliation(s)
- Rawnaq Esa
- Institute for Drug Research, School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
| | - Eliana Steinberg
- Institute for Drug Research, School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
| | - Arie Dagan
- Institute for Drug Research, School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
- The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
| | - Zhanna Yekhtin
- The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
| | - Katerina Tischenko
- Institute for Drug Research, School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel
| | - Ofra Benny
- Institute for Drug Research, School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel.
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Yılmaz Ö, Bayer B, Bekçi H, Uba AI, Cumaoğlu A, Yelekçi K, Küçükgüzel ŞG. Synthesis, Anticancer Activity on Prostate Cancer Cell Lines and Molecular Modeling Studies of Flurbiprofen-Thioether Derivatives as Potential Target of MetAP (Type II). Med Chem 2021; 16:735-749. [PMID: 31203805 DOI: 10.2174/1573406415666190613162322] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2019] [Revised: 04/30/2019] [Accepted: 05/03/2019] [Indexed: 12/20/2022]
Abstract
BACKGROUND Prostate cancer is still one of the serious causes of mortality and morbidity in men. Despite recent advances in anticancer therapy, there is a still need of novel agents with more efficacy and specificity in the treatment of prostate cancer. Because of its function on angiogenesis and overexpression in the prostate cancer, methionine aminopeptidase-2 (MetAP-2) has been a potential target for novel drug design recently. OBJECTIVE A novel series of Flurbiprofen derivatives N-(substituted)-2-(2-(2-fluoro-[1,1'- biphenyl]-4-il)propanoyl)hydrazinocarbothioamide (3a-c), 4-substituted-3-(1-(2-fluoro-[1,1'-biphenyl]- 4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (4a-d), 3-(substitutedthio)-4-(substituted-phenyl)- 5-(1-(2-fluoro-[1,1'-biphenyl]-4-yl)ethyl)-4H-1,2,4-triazole (5a-y) were synthesized. The purpose of the research was to evaluate these derivatives against MetAP-2 in vitro and in silico to obtain novel specific and effective anticancer agents against prostate cancer. METHODS The chemical structures and purities of the compounds were defined by spectral methods (1H-NMR, 13C-NMR, HR-MS and FT-IR) and elemental analysis. Anticancer activities of the compounds were evaluated in vitro by using MTS method against PC-3 and DU-143 (androgenindependent human prostate cancer cell lines) and LNCaP (androgen-sensitive human prostate adenocarcinoma) prostate cancer cell lines. Cisplatin was used as a positive sensitivity reference standard. RESULTS Compounds 5b and 5u; 3c, 5b and 5y; 4d and 5o showed the most potent biological activity against PC3 cancer cell line (IC50= 27.1 μM, and 5.12 μM, respectively), DU-145 cancer cell line (IC50= 11.55 μM, 6.9 μM and 9.54 μM, respectively) and LNCaP cancer cell line (IC50= 11.45 μM and 26.91 μM, respectively). Some compounds were evaluated for their apoptotic caspases protein expression (EGFR/PI3K/AKT pathway) by Western blot analysis in androgen independent- PC3 cells. BAX, caspase 9, caspsase 3 and anti-apoptotic BcL-2 mRNA levels of some compounds were also investigated. In addition, molecular modeling studies of the compounds on MetAP-2 enzyme active site were evaluated in order to get insight into binding mode and energy. CONCLUSION A series of Flurbiprofen-thioether derivatives were synthesized. This study presented that some of the synthesized compounds have remarkable anticancer and apoptotic activities against prostate cancer cells. Also, molecular modeling studies exhibited that there is a correlation between molecular modeling and anticancer activity results.
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Affiliation(s)
- Özgür Yılmaz
- TUBITAK Marmara Research Center, Materials Institute, Kocaeli, Turkey
| | - Burak Bayer
- Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpaşa 34668 İstanbul, Turkey
| | - Hatice Bekçi
- Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Biochemistry, Talas 38280 Kayseri, Turkey
| | - Abdullahi I Uba
- Kadir Has University, Faculty of Engineering and Natural Sciences, Department of Bioinformatics and Genetics, 34083 Istanbul, Turkey
| | - Ahmet Cumaoğlu
- Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Biochemistry, Talas 38280 Kayseri, Turkey
| | - Kemal Yelekçi
- Kadir Has University, Faculty of Engineering and Natural Sciences, Department of Bioinformatics and Genetics, 34083 Istanbul, Turkey
| | - Ş Güniz Küçükgüzel
- Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpaşa 34668 İstanbul, Turkey
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Xie J, Rice MA, Chen Z, Cheng Y, Hsu EC, Chen M, Song G, Cui L, Zhou K, Castillo JB, Zhang CA, Shen B, Chin FT, Kunder CA, Brooks JD, Stoyanova T, Rao J. In Vivo Imaging of Methionine Aminopeptidase II for Prostate Cancer Risk Stratification. Cancer Res 2021; 81:2510-2521. [PMID: 33637565 DOI: 10.1158/0008-5472.can-20-2969] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 12/31/2020] [Accepted: 02/24/2021] [Indexed: 11/16/2022]
Abstract
Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.
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Affiliation(s)
- Jinghang Xie
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - Meghan A Rice
- Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, California
| | - Zixin Chen
- Department of Chemistry, Stanford University, Stanford, California
| | - Yunfeng Cheng
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - En-Chi Hsu
- Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, California
| | - Min Chen
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - Guosheng Song
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - Liyang Cui
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - Kaixiang Zhou
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California
| | - Jessa B Castillo
- Department of Radiology, Cyclotron and Radiochemistry Facility, Stanford University School of Medicine, Stanford, California
| | - Chiyuan A Zhang
- Department of Urology, Stanford University School of Medicine, Stanford, California
| | - Bin Shen
- Department of Radiology, Cyclotron and Radiochemistry Facility, Stanford University School of Medicine, Stanford, California
| | - Frederick T Chin
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California.,Department of Radiology, Cyclotron and Radiochemistry Facility, Stanford University School of Medicine, Stanford, California
| | - Christian A Kunder
- Department of Urology, Stanford University School of Medicine, Stanford, California
| | - James D Brooks
- Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, California.,Department of Urology, Stanford University School of Medicine, Stanford, California
| | - Tanya Stoyanova
- Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, California.
| | - Jianghong Rao
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California. .,Department of Chemistry, Stanford University, Stanford, California
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A cross-platform approach to characterize and screen potential neurovascular unit toxicants. Reprod Toxicol 2020; 96:300-315. [PMID: 32590145 PMCID: PMC9773816 DOI: 10.1016/j.reprotox.2020.06.010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 05/28/2020] [Accepted: 06/15/2020] [Indexed: 12/24/2022]
Abstract
Development of the neurovascular unit (NVU) is a complex, multistage process that requires orchestrated cell signaling mechanisms across several cell types and ultimately results in formation of the blood-brain barrier. Typical high-throughput screening (HTS) assays investigate single biochemical or single cell responses following chemical insult. As the NVU comprises multiple cell types interacting at various stages of development, a methodology combining high-throughput results across pertinent cell-based assays is needed to investigate potential chemical-induced disruption to the development of this complex cell system. To this end, we implemented a novel method for screening putative NVU disruptors across diverse assay platforms to predict chemical perturbation of the developing NVU. HTS assay results measuring chemical-induced perturbations to cellular key events across angiogenic and neurogenic outcomes in vitro were combined to create a cell-based prioritization of NVU hazard. Chemicals were grouped according to similar modes of action to train a logistic regression literature model on a training set of 38 chemicals. This model utilizes the chemical-specific pairwise mutual information score for PubMed MeSH annotations to represent a quantitative measure of previously published results. Taken together, this study presents a methodology to investigate NVU developmental hazard using cell-based HTS assays and literature evidence to prioritize screening of putative NVU disruptors towards a knowledge-driven characterization of neurovascular developmental toxicity. The results from these screening efforts demonstrate that chemicals representing a range of putative vascular disrupting compound (pVDC) scores can also produce effects on neurogenic outcomes and characterizes possible modes of action for disrupting the developing NVU.
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The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis. Int J Mol Sci 2020; 21:ijms21145148. [PMID: 32708166 PMCID: PMC7403956 DOI: 10.3390/ijms21145148] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/14/2020] [Accepted: 07/16/2020] [Indexed: 11/17/2022] Open
Abstract
During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.
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Pottorf TS, Fagan MP, Burkey BF, Cho DJ, Vath JE, Tran PV. MetAP2 inhibition reduces food intake and body weight in a ciliopathy mouse model of obesity. JCI Insight 2020; 5:134278. [PMID: 31877115 DOI: 10.1172/jci.insight.134278] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Accepted: 12/18/2019] [Indexed: 12/13/2022] Open
Abstract
The ciliopathies Bardet-Biedl syndrome and Alström syndrome are genetically inherited pleiotropic disorders with hyperphagia and obesity as primary clinical features. Methionine aminopeptidase 2 inhibitors (MetAP2i) have been shown in preclinical and clinical studies to reduce food intake, body weight, and adiposity. Here, we investigated the effects of MetAP2i administration in a mouse model of ciliopathy produced by conditional deletion of the Thm1 gene in adulthood. Thm1 conditional knockout (cko) mice showed decreased hypothalamic proopiomelanocortin expression as well as hyperphagia, obesity, metabolic disease, and hepatic steatosis. In obese Thm1-cko mice, 2-week administration of MetAP2i reduced daily food intake and reduced body weight 17.1% from baseline (vs. 5% reduction for vehicle). This was accompanied by decreased levels of blood glucose, insulin, and leptin. Further, MetAP2i reduced gonadal adipose depots and adipocyte size and improved liver morphology. This is the first report to our knowledge of MetAP2i reducing hyperphagia and body weight and ameliorating metabolic indices in a mouse model of ciliopathy. These results support further investigation of MetAP2 inhibition as a potential therapeutic strategy for ciliary-mediated forms of obesity.
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Affiliation(s)
- Tana S Pottorf
- Jared Grantham Kidney Institute and.,Department of Anatomy and Cell Biology, University of Kansas Medical Center (KUMC), Kansas City, Kansas, USA
| | | | | | - David J Cho
- Jared Grantham Kidney Institute and.,Department of Anatomy and Cell Biology, University of Kansas Medical Center (KUMC), Kansas City, Kansas, USA
| | | | - Pamela V Tran
- Jared Grantham Kidney Institute and.,Department of Anatomy and Cell Biology, University of Kansas Medical Center (KUMC), Kansas City, Kansas, USA
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Affiliation(s)
- Kamal Kumar
- Max-Planck-Institut für molekulare PhysiologieAbteilung Chemische Biologie Otto-Hahn Str. 11 44227- Dortmund Germany
| | - Herbert Waldmann
- Max-Planck-Institut für molekulare PhysiologieAbteilung Chemische Biologie Otto-Hahn Str. 11 44227- Dortmund Germany
- Technische Universität DortmundFakultät Chemie, Chemische Biologie Otto-Hahn-Straße 6 Dortmund 44221 Germany
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11
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MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state. Oncotarget 2018; 7:63306-63323. [PMID: 27542228 PMCID: PMC5325365 DOI: 10.18632/oncotarget.11216] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2016] [Accepted: 07/19/2016] [Indexed: 12/17/2022] Open
Abstract
Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.
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Metallic gold and bioactive quinacrine hybrid nanoparticles inhibit oral cancer stem cell and angiogenesis by deregulating inflammatory cytokines in p53 dependent manner. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2018; 14:883-896. [PMID: 29366881 DOI: 10.1016/j.nano.2018.01.007] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/25/2017] [Revised: 12/26/2017] [Accepted: 01/09/2018] [Indexed: 01/13/2023]
Abstract
Complete eradication of aggressive oral cancer remains a challenge due to the presence of CSCs. They resist conventional chemotherapeutic agents due to their self-renewal, drug efflux, and efficient DNA repair capacity. Here, we formulated a hybrid-nanoparticle (QAuNP) using quinacrine and gold and characterized/investigated its anti-angiogenic and anti-metastatic effect on OSCC-CSCs. QAuNP significantly inhibited cellular proliferation, caused apoptosis in vitro, and disrupted angiogenesis in vivo and tumor regression in xenograft mice model. It not only inhibited crucial angiogenic markers Ang-1, Ang-2 and VEGF but also depleted MMP-2 in H-357-PEMT cells in a p53 and p21-dependent manner. QAuNP also increased the ROS and NO generation in OSCC-CSCs and reduced the mitochondrial membrane potential. It altered the level of inflammatory cytokines IL-6, IL-1β, TNF-α and metastasis-associated markers (CD-44, CD-133) in H-357-PEMT and CM-treated endothelial cells (HUVEC) in p53/p21-dependent manner. Therefore, QAuNP will be a useful therapeutic agent against metastatic OSCC.
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Saint-Germain E, Mignacca L, Vernier M, Bobbala D, Ilangumaran S, Ferbeyre G. SOCS1 regulates senescence and ferroptosis by modulating the expression of p53 target genes. Aging (Albany NY) 2017; 9:2137-2162. [PMID: 29081404 PMCID: PMC5680560 DOI: 10.18632/aging.101306] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Accepted: 10/15/2017] [Indexed: 05/25/2023]
Abstract
The mechanism by which p53 suppresses tumorigenesis remains poorly understood. In the context of aberrant activation of the JAK/STAT5 pathway, SOCS1 is required for p53 activation and the regulation of cellular senescence. In order to identify p53 target genes acting during the senescence response to oncogenic STAT5A, we characterized the transcriptome of STAT5A-expressing cells after SOCS1 inhibition. We identified a set of SOCS1-dependent p53 target genes that include several secreted proteins and genes regulating oxidative metabolism and ferroptosis. Exogenous SOCS1 was sufficient to regulate the expression of p53 target genes and sensitized cells to ferroptosis. This effect correlated with the ability of SOCS1 to reduce the expression of the cystine transporter SLC7A11 and the levels of glutathione. SOCS1 and SOCS1-dependent p53 target genes were induced during the senescence response to oncogenic STAT5A, RasV12 or the tumor suppressor PML. However, while SOCS1 sensitized cells to ferroptosis neither RasV12 nor STAT5A mimicked the effect. Intriguingly, PML turned cells highly resistant to ferroptosis. The results indicate different susceptibilities to ferroptosis in senescent cells depending on the trigger and suggest the possibility of killing senescent cells by inhibiting pathways that mediate ferroptosis resistance.
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Affiliation(s)
- Emmanuelle Saint-Germain
- Département de Biochimie et Médecine Moléculaire; Université de Montréal, Montréal, Québec, H3C 3J7; Canada
| | - Lian Mignacca
- Département de Biochimie et Médecine Moléculaire; Université de Montréal, Montréal, Québec, H3C 3J7; Canada
| | - Mathieu Vernier
- Department of Biochemistry, Medicine & Oncology, Faculty of Medicine, McGill University, Goodman Cancer Research Centre, Montreal, Quebec, H3A 1A3, Canada
| | - Diwakar Bobbala
- Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1K 2R1, Canada
| | - Subburaj Ilangumaran
- Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1K 2R1, Canada
| | - Gerardo Ferbeyre
- Département de Biochimie et Médecine Moléculaire; Université de Montréal, Montréal, Québec, H3C 3J7; Canada
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14
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Purushothaman P, Uppal T, Sarkar R, Verma SC. KSHV-Mediated Angiogenesis in Tumor Progression. Viruses 2016; 8:E198. [PMID: 27447661 PMCID: PMC4974533 DOI: 10.3390/v8070198] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2016] [Revised: 06/18/2016] [Accepted: 07/07/2016] [Indexed: 12/14/2022] Open
Abstract
Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is a malignant human oncovirus belonging to the gamma herpesvirus family. HHV-8 is closely linked to the pathogenesis of Kaposi's sarcoma (KS) and two other B-cell lymphoproliferative diseases: primary effusion lymphoma (PEL) and a plasmablastic variant of multicentric Castleman's disease (MCD). KS is an invasive tumor of endothelial cells most commonly found in untreated HIV-AIDS or immuno-compromised individuals. KS tumors are highly vascularized and have abnormal, excessive neo-angiogenesis, inflammation, and proliferation of infected endothelial cells. KSHV directly induces angiogenesis in an autocrine and paracrine fashion through a complex interplay of various viral and cellular pro-angiogenic and inflammatory factors. KS is believed to originate due to a combination of KSHV's efficient strategies for evading host immune systems and several pro-angiogenic and pro-inflammatory stimuli. In addition, KSHV infection of endothelial cells produces a wide array of viral oncoproteins with transforming capabilities that regulate multiple host-signaling pathways involved in the activation of angiogenesis. It is likely that the cellular-signaling pathways of angiogenesis and lymph-angiogenesis modulate the rate of tumorigenesis induction by KSHV. This review summarizes the current knowledge on regulating KSHV-mediated angiogenesis by integrating the findings reported thus far on the roles of host and viral genes in oncogenesis, recent developments in cell-culture/animal-model systems, and various anti-angiogenic therapies for treating KSHV-related lymphoproliferative disorders.
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Affiliation(s)
- Pravinkumar Purushothaman
- Department of Microbiology and Immunology, University of Nevada, Reno, School of Medicine, 1664 N Virginia Street, MS 320, Reno, NV 89557, USA.
| | - Timsy Uppal
- Department of Microbiology and Immunology, University of Nevada, Reno, School of Medicine, 1664 N Virginia Street, MS 320, Reno, NV 89557, USA.
| | - Roni Sarkar
- Department of Microbiology and Immunology, University of Nevada, Reno, School of Medicine, 1664 N Virginia Street, MS 320, Reno, NV 89557, USA.
| | - Subhash C Verma
- Department of Microbiology and Immunology, University of Nevada, Reno, School of Medicine, 1664 N Virginia Street, MS 320, Reno, NV 89557, USA.
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15
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Morgen M, Jöst C, Malz M, Janowski R, Niessing D, Klein CD, Gunkel N, Miller AK. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem Biol 2016; 11:1001-11. [PMID: 26686773 DOI: 10.1021/acschembio.5b00755] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivatives of fumagillin have shown promise in clinical studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivatives has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's molecular scaffold but have improved pharmacological profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochemical enzymatic assays against three MetAP isoforms. Inhibitors with the same relative and absolute stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallographic analysis revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochemically active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clinically investigated fumagillin derivative beloranib.
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Affiliation(s)
- Michael Morgen
- Cancer
Drug Development Group, German Cancer Research Center (DKFZ), Im Neunheimer
Feld 280, D-69120 Heidelberg, Germany
| | - Christian Jöst
- Medicinal
Chemistry, Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
| | - Mona Malz
- Cancer
Drug Development Group, German Cancer Research Center (DKFZ), Im Neunheimer
Feld 280, D-69120 Heidelberg, Germany
| | - Robert Janowski
- Institute
of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), D-85764 Neuherberg, Germany
| | - Dierk Niessing
- Institute
of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), D-85764 Neuherberg, Germany
- Biomedical Center of the Ludwig-Maximilians-Universität München, D-82152 Planegg-Martinsried, Germany
| | - Christian D. Klein
- Medicinal
Chemistry, Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
| | - Nikolas Gunkel
- Cancer
Drug Development Group, German Cancer Research Center (DKFZ), Im Neunheimer
Feld 280, D-69120 Heidelberg, Germany
| | - Aubry K. Miller
- Cancer
Drug Development Group, German Cancer Research Center (DKFZ), Im Neunheimer
Feld 280, D-69120 Heidelberg, Germany
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16
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Fumagillin, a potent angiogenesis inhibitor, induces Kaposi sarcoma-associated herpesvirus replication in primary effusion lymphoma cells. Biochem Biophys Res Commun 2015; 463:1267-72. [PMID: 26093300 DOI: 10.1016/j.bbrc.2015.06.100] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Accepted: 06/14/2015] [Indexed: 01/11/2023]
Abstract
Kaposi sarcoma and primary effusion lymphoma cells are infected with Kaposi sarcoma-associated herpesvirus (KSHV), predominantly in the latent form, and KSHV replication is observed rarely. Angiogenesis plays a crucial role in the pathogenesis of both Kaposi sarcoma and primary effusion lymphoma. In this study, we found that fumagillin, a potent angiogenesis inhibitor, induced replication of KSHV in primary effusion lymphoma cell lines. The transcript and protein product of replication transcriptional activator (RTA) were induced by 1-10 μM fumagillin at 24 and 48 h, respectively. Western blot analysis demonstrated that 10 μM fumagillin induced not only RTA expression but also other KSHV-encoded lytic proteins. A real-time PCR array detecting KSHV gene expression demonstrated that the expression profiles of KSHV induced by fumagillin were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), but the amounts of each transcript were lower than those induced by TPA. Finally, real-time PCR demonstrated an increase in that viral DNA copy number per cell in fumagillin-stimulated primary effusion lymphoma cell lines, indicating replication of KSHV. In addition to TPA, 10 μM fumagillin resulted in growth inhibition of primary effusion lymphoma cell lines. These observations suggest that an angiogenesis inhibitor is an agent with potent effects on cell growth and KSHV reactivation in primary effusion lymphoma cells.
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17
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Dual inhibition of cyclooxygenase-2 and soluble epoxide hydrolase synergistically suppresses primary tumor growth and metastasis. Proc Natl Acad Sci U S A 2014; 111:11127-32. [PMID: 25024195 DOI: 10.1073/pnas.1410432111] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.
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18
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Abstract
In recent years, small interference RNAs (siRNAs) have greatly enhanced our understanding of protein functions by allowing knockdown of targeted proteins at the mRNA level. Similarly, in an effort to achieve degradation of targeted proteins at the post-translational level, chimeric small molecules called "PROTACs" (PROteolysis TArgeting Chimeric molecules) have been developed. The PROTAC approach utilizes chimeric small molecules which recruit targeted proteins to the ubiquitin-proteasome pathway, a major intracellular protein degradation system. Unlike conventional small molecules that bind to protein and inhibit its function, the PROTAC approach induces destruction of target protein via the ubiquitin-proteasome system. This article presents a typical strategy for PROTAC design and preparation and biological characterization. Curr. Protoc. Chem Biol. 2:71-87. © 2010 by John Wiley & Sons, Inc.
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19
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Friis T, Engel AM, Bendiksen CD, Larsen LS, Houen G. Influence of levamisole and other angiogenesis inhibitors on angiogenesis and endothelial cell morphology in vitro. Cancers (Basel) 2013; 5:762-85. [PMID: 24202320 PMCID: PMC3795364 DOI: 10.3390/cancers5030762] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2013] [Revised: 06/11/2013] [Accepted: 06/14/2013] [Indexed: 02/06/2023] Open
Abstract
Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.
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Affiliation(s)
- Tina Friis
- Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark.
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20
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Zhang F, Bhat S, Gabelli SB, Chen X, Miller MS, Nacev BA, Cheng YL, Meyers DJ, Tenney K, Shim JS, Crews P, Amzel LM, Ma D, Liu JO. Pyridinylquinazolines selectively inhibit human methionine aminopeptidase-1 in cells. J Med Chem 2013; 56:3996-4016. [PMID: 23634668 DOI: 10.1021/jm400227z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.
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Affiliation(s)
- Feiran Zhang
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA
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21
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Wettersten HI, Hee Hwang S, Li C, Shiu EY, Wecksler AT, Hammock BD, Weiss RH. A novel p21 attenuator which is structurally related to sorafenib. Cancer Biol Ther 2013; 14:278-85. [PMID: 23298903 DOI: 10.4161/cbt.23374] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
p21 is a member of the cyclin kinase inhibitor family of proteins and plays pivotal roles in cellular proliferation as well as in the regulation of apoptosis, and thus has diverse functions in diseases as varied as cancer and atherosclerosis. In light of its pleiotropic effects and potential clinical relevance, new methods of attenuation of p21 protein levels by selective inhibitors are therefore powerful tools to probe malignant, infectious and other diseases. Here we introduce a novel p21 attenuator, UC2288, which possesses consistent and relatively selective activity for p21. UC2288 was synthesized based on the chemical model of sorafenib, a multikinase inhibitor that also attenuates p21, but unlike sorafenib, UC2288 did not inhibit Raf kinases or alter p-ERK protein levels. UC2288 decreased p21 mRNA expression independently of p53, and attenuated p21 protein levels with minimal effect on p21 protein stability. In addition, UC2288 inhibits cell growth in the kidney cancer cell lines (GI50 = approximately 10 µM) as well as multiple other cancer cell lines. Thus, this novel p21 inhibitor will be indispensable for exploring the function of p21, and upon further study may be translatable to the clinic.
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Affiliation(s)
- Hiromi I Wettersten
- Division of Nephrology, Department of Internal Medicine, University of California, Davis, Davis, CA, USA
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22
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Napione L, Strasly M, Meda C, Mitola S, Alvaro M, Doronzo G, Marchiò S, Giraudo E, Primo L, Arese M, Bussolino F. IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism. Angiogenesis 2012; 15:713-25. [PMID: 22797886 DOI: 10.1007/s10456-012-9286-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2012] [Accepted: 06/25/2012] [Indexed: 11/28/2022]
Abstract
Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.
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Affiliation(s)
- Lucia Napione
- Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino, 10060, Candiolo, Torino, Italy.
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23
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Jang ER, Lee NR, Han S, Wu Y, Sharma LK, Carmony KC, Marks J, Lee DM, Ban JO, Wehenkel M, Hong JT, Kim KB, Lee W. Revisiting the role of the immunoproteasome in the activation of the canonical NF-κB pathway. MOLECULAR BIOSYSTEMS 2012; 8:2295-302. [PMID: 22722901 DOI: 10.1039/c2mb25125f] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The discovery of NF-κB signaling pathways has greatly enhanced our understanding of inflammatory and immune responses. In the canonical NF-κB pathway, the proteasomal degradation of IκBα, an inhibitory protein of NF-κB, is widely accepted to be a key regulatory step. However, contradictory findings have been reported as to whether the immunoproteasome plays an obligatory role in the degradation of IκBα and activation of the canonical NF-κB pathway. Such results were obtained mainly using traditional gene deletion strategies. Here, we have revisited the involvement of the immunoproteasome in the canonical NF-κB pathway using small molecule inhibitors of the immunoproteasome, namely UK-101 and LKS01 targeting β1i and β5i, respectively. H23 and Panc-1 cancer cells were pretreated with UK-101, LKS01 or epoxomicin (a prototypic inhibitor targeting both the constitutive proteasome and immunoproteasome). We then examined whether these pretreatments lead to any defect in activating the canonical NF-κB pathway following TNFα exposure by monitoring the phosphorylation and degradation of IκBα, nuclear translocation of NF-κB proteins and DNA binding and transcriptional activity of NF-κB. Our results consistently indicated that there is no defect in activating the canonical NF-κB pathway following selective inhibition of the immunoproteasome catalytic subunits β1i, β5i or both using UK-101 and LKS01, in contrast to epoxomicin. In summary, our current results using chemical genetic approaches strongly support that the catalytic activity of the immunoproteasome subunits β1i and β5i is not required for canonical NF-κB activation in lung and pancreatic adenocarcinoma cell line models.
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Affiliation(s)
- Eun Ryoung Jang
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone, Lexington, Kentucky 40536-0596, USA
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24
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Kass DJ, Rattigan E, Kahloon R, Loh K, Yu L, Savir A, Markowski M, Saqi A, Rajkumar R, Ahmad F, Champion HC. Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats. PLoS One 2012; 7:e35388. [PMID: 22509410 PMCID: PMC3324555 DOI: 10.1371/journal.pone.0035388] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2010] [Accepted: 03/16/2012] [Indexed: 01/30/2023] Open
Abstract
Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.
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MESH Headings
- Aminopeptidases/antagonists & inhibitors
- Aminopeptidases/genetics
- Aminopeptidases/metabolism
- Animals
- Cell Proliferation/drug effects
- Cells, Cultured
- Cyclohexanes/administration & dosage
- Disease Models, Animal
- Fatty Acids, Unsaturated/administration & dosage
- Gene Expression Regulation
- Glycoproteins/antagonists & inhibitors
- Glycoproteins/genetics
- Glycoproteins/metabolism
- Heart Ventricles/drug effects
- Heart Ventricles/physiopathology
- Hemodynamics
- Humans
- Hypertension, Pulmonary/chemically induced
- Hypertension, Pulmonary/pathology
- Hypertension, Pulmonary/prevention & control
- Male
- Monocrotaline/pharmacology
- Myocytes, Cardiac/drug effects
- Myocytes, Cardiac/metabolism
- Myocytes, Smooth Muscle/cytology
- Myofibroblasts/drug effects
- Myofibroblasts/pathology
- Platelet-Derived Growth Factor/genetics
- Platelet-Derived Growth Factor/metabolism
- Pulmonary Artery/cytology
- Pulmonary Artery/drug effects
- Rats
- Rats, Sprague-Dawley
- Sesquiterpenes/administration & dosage
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Affiliation(s)
- Daniel J Kass
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine and the Dorothy P and Richard P Simmons Center for Interstitial Lung Disease, Pittsburgh, Pennsylvania, United States of America.
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25
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Weiss A, den Bergh HV, Griffioen AW, Nowak-Sliwinska P. Angiogenesis inhibition for the improvement of photodynamic therapy: the revival of a promising idea. Biochim Biophys Acta Rev Cancer 2012; 1826:53-70. [PMID: 22465396 DOI: 10.1016/j.bbcan.2012.03.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2011] [Revised: 03/13/2012] [Accepted: 03/14/2012] [Indexed: 12/31/2022]
Abstract
Photodynamic therapy (PDT) is a minimally invasive form of treatment, which is clinically approved for the treatment of angiogenic disorders, including certain forms of cancer and neovascular eye diseases. Although the concept of PDT has existed for a long time now, it has never made a solid entrance into the clinical management of cancer. This is likely due to secondary tissue reactions, such as inflammation and neoangiogenesis. The recent development of clinically effective angiogenesis inhibitors has lead to the initiation of research on the combination of PDT with such angiostatic targeted therapies. Preclinical studies in this research field have shown promising results, causing a revival in the field of PDT. This review reports on the current research efforts on PDT and vascular targeted combination therapies. Different combination strategies with angiogenesis inhibition and vascular targeting approaches are discussed. In addition, the concept of increasing PDT selectivity by targeted delivery of photosensitizers is presented. Furthermore, the current insights on sequencing the therapy arms of such combinations will be discussed in light of vascular normalization induced by angiogenesis inhibition.
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Affiliation(s)
- Andrea Weiss
- Medical Photonics Group, Institute of Bioengineering, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland
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26
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Machado MJC, Watson MG, Devlin AH, Chaplain MAJ, McDougall SR, Mitchell CA. Dynamics of angiogenesis during wound healing: a coupled in vivo and in silico study. Microcirculation 2011; 18:183-97. [PMID: 21166934 DOI: 10.1111/j.1549-8719.2010.00076.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
OBJECTIVE The most critical determinant of restoration of tissue structure during wound healing is the re-establishment of a functional vasculature, which largely occurs via angiogenesis, specifically endothelial sprouting from the pre-existing vasculature. MATERIALS AND METHODS We used confocal microscopy to capture sequential images of perfused vascular segments within the injured panniculus carnosus muscle in the mouse dorsal skin-fold window chamber to quantify a range of microcirculatory parameters during the first nine days of healing. This data was used to inform a mathematical model of sequential growth of the vascular plexus. The modeling framework mirrored the experimental circular wound domain and incorporated capillary sprouting and endothelial cell (EC) sensing of vascular endothelial growth factor gradients. RESULTS Wound areas, vessel densities and vessel junction densities obtained from the corresponding virtual wound were in excellent agreement both temporally and spatially with data measured during the in vivo healing process. Moreover, by perturbing the proliferative ability of ECs in the mathematical model, this leads to a severe reduction in vascular growth and poor healing. Quantitative measures from this second set of simulations were found to correlate extremely well with experimental data obtained from animals treated with an agent that targets endothelial proliferation (TNP-470). CONCLUSION Our direct combination and comparison of in vivo longitudinal analysis (over time in the same animal) and mathematical modeling employed in this study establishes a useful new paradigm. The virtual wound created in this study can be used to investigate a wide range of experimental hypotheses associated with wound healing, including disorders characterized by aberrant angiogenesis (e.g., diabetic models) and the effects of vascular enhancing/disrupting agents or therapeutic interventions such as hyperbaric oxygen.
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Affiliation(s)
- Maria J C Machado
- Centre for Molecular Biosciences, University of Ulster, Coleraine, UK
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Hong J. Role of natural product diversity in chemical biology. Curr Opin Chem Biol 2011; 15:350-4. [PMID: 21489856 DOI: 10.1016/j.cbpa.2011.03.004] [Citation(s) in RCA: 95] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2011] [Accepted: 03/15/2011] [Indexed: 12/31/2022]
Abstract
Through the natural selection process, natural products possess a unique and vast chemical diversity and have been evolved for optimal interactions with biological macromolecules. Owing to their diversity, target affinity, and specificity, natural products have demonstrated enormous potential as modulators of biomolecular function, been an essential source for drug discovery, and provided design principles for combinatorial library development.
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Affiliation(s)
- Jiyong Hong
- Department of Chemistry, Duke University, Durham, NC 27708, USA.
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Lijnen HR, Frederix L, Van Hoef B. Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity. Obesity (Silver Spring) 2010; 18:2241-6. [PMID: 20094042 DOI: 10.1038/oby.2009.503] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The effect of fumagillin (a methionine aminopeptidase-type 2 (Met-AP2) inhibitor, with antiangiogenic properties) was investigated in murine models of diet-induced obesity. Eleven-week-old male C57Bl/6 mice (group 1) were given fumagillin by oral gavage at a dose of 1 mg/kg/day during 4 weeks while fed a high-fat diet (HFD) (20.1 kJ/g), and control mice (group 2) received solvent and were pair-fed. At the end of the experiment, body weights in group 1 were significantly lower as compared to group 2 (P < 0.0005). The subcutaneous (SC) and gonadal (GON) fat mass was also significantly lower in group 1 (P < 0.005 and P < 0.05, respectively). Adipocytes were smaller in adipose tissues of mice in group 1, associated with higher adipocyte density. Blood vessel density normalized to adipocyte density was lower in group 1 adipose tissues. However, in mice with established obesity monitored to maintain the same body weight and fat mass as controls, short-term fumagillin administration was also associated with adipocyte hypotrophy (P = 0.01) without affecting blood vessel size or density. Thus, treatment with fumagillin impaired diet-induced obesity in mice, associated with adipocyte hypotrophy but without marked effect on adipose tissue angiogenesis.
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Affiliation(s)
- Henri R Lijnen
- Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium.
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Shim JS, Matsui Y, Bhat S, Nacev BA, Xu J, Bhang HEC, Dhara S, Han KC, Chong CR, Pomper MG, So A, Liu JO. Effect of nitroxoline on angiogenesis and growth of human bladder cancer. J Natl Cancer Inst 2010; 102:1855-73. [PMID: 21088277 DOI: 10.1093/jnci/djq457] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Angiogenesis plays an important role in tumor growth and metastasis; therefore, inhibition of angiogenesis is a promising strategy for developing new anticancer drugs. Type 2 methionine aminopeptidase (MetAP2) protein is likely a molecular target of angiogenesis inhibitors. METHODS Nitroxoline, an antibiotic used to treat urinary tract infections, was identified from a high-throughput screen of a library of 175,000 compounds for MetAP2 inhibitors and from a parallel screen using the Johns Hopkins Drug Library to identify currently used clinical drugs that can also inhibit human umbilical vein endothelial cells (HUVEC) proliferation. To investigate the mechanism of action of nitroxoline, inhibition of MetAP2 activity and induction of senescence were assessed in HUVEC. To test the antiangiogenic activity of nitroxoline, endothelial tube formation in Matrigel and microvessel formation in Matrigel plugs in vivo were assessed. Antitumor efficacy of nitroxoline was evaluated in mouse models of human breast cancer xenograft (n = 10) and bladder cancer orthotopic xenograft (n = 11). Furthermore, the mechanism of action of nitroxoline was investigated in vivo. RESULTS Nitroxoline inhibited MetAP2 activity in vitro (half maximal inhibitory concentration [IC(50)] = 54.8 nM, 95% confidence interval [CI] = 22.6 to 132.8 nM) and HUVEC proliferation (IC(50) = 1.9 μM, 95% CI = 1.54 to 2.39 μM). Nitroxoline inhibited MetAP2 activity in HUVEC in a dose-dependent manner and induced premature senescence in a biphasic manner. Nitroxoline inhibited endothelial tube formation in Matrigel and reduced microvessel density in vivo. Mice (five per group) treated with nitroxoline showed a 60% reduction in tumor volume in breast cancer xenografts (tumor volume on day 30, vehicle vs nitroxoline, mean = 215.4 vs 86.5 mm(3), difference = 128.9 mm(3), 95% CI = 32.9 to 225.0 mm(3), P = .012) and statistically significantly inhibited growth of bladder cancer in an orthotopic mouse model (tumor bioluminescence intensities of vehicle [n = 5] vs nitroxoline [n = 6], P = .045). CONCLUSION Nitroxoline shows promise as a potential therapeutic antiangiogenic agent.
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Affiliation(s)
- Joong Sup Shim
- Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
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Hines J, Ju R, Dutschman GE, Cheng YC, Crews CM. Reversal of TNP-470-induced endothelial cell growth arrest by guanine and guanine nucleosides. J Pharmacol Exp Ther 2010; 334:729-38. [PMID: 20571059 PMCID: PMC2939662 DOI: 10.1124/jpet.110.169110] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2010] [Accepted: 06/21/2010] [Indexed: 01/22/2023] Open
Abstract
The mechanism of action of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21(WAF1/CIP1), respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21(WAF1/CIP1) by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid, showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced after TNP-470 treatment. Addition of guanosine at the start of G(1) phase causes a doubling in GTP levels that persists to the G(1)/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather than a restoration of a drug-induced GTP deficit. Determining the mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.
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Affiliation(s)
- John Hines
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA
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Activation of the planar cell polarity formin DAAM1 leads to inhibition of endothelial cell proliferation, migration, and angiogenesis. Proc Natl Acad Sci U S A 2010; 107:6906-11. [PMID: 20351293 DOI: 10.1073/pnas.1001075107] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The Wnt/planar cell polarity (PCP) pathway regulates directed cell movement during development and was recently found to play a critical role in endothelial cell proliferation and angiogenesis [Zhang Y, et al. (2006) Chem Biol 13:1001-1009; Masckauchan TN, et al. (2006) Mol Biol Cell 17:5163-5172]. However, the mechanisms by which PCP signaling components regulate angiogenesis remain unknown. We report that expression of a constitutively active C-terminal domain of Dishevelled-associated activator of morphogenesis 1 (DAAM1) selectively inhibited endothelial cell proliferation. Moreover, this activated construct suppressed endothelial cell migration and the ability to form coordinated networks in vivo and in vitro. Although constitutively active DAAM1 (CDAAM1) induced both actin polymerization and microtubule (MT) stabilization, the stabilization of MTs alone was sufficient to inhibit endothelial cell growth selectively. Inhibition of actin polymerization alone by jasplakinolide treatment failed to reproduce the inhibitory effects of CDAAM1. These results indicate that DAAM1 regulates endothelial cell growth through MT stabilization in a cell type-selective manner and suggest that PCP signaling plays a pivotal role in angiogenesis by regulating MT stabilization.
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van Wijngaarden J, Snoeks TJA, van Beek E, Bloys H, Kaijzel EL, van Hinsbergh VWM, Löwik CWGM. An in vitro model that can distinguish between effects on angiogenesis and on established vasculature: actions of TNP-470, marimastat and the tubulin-binding agent Ang-510. Biochem Biophys Res Commun 2009; 391:1161-5. [PMID: 20004648 DOI: 10.1016/j.bbrc.2009.11.097] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2009] [Accepted: 11/16/2009] [Indexed: 02/08/2023]
Abstract
In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature; one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach.
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Affiliation(s)
- Jens van Wijngaarden
- Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands
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Frottin F, Espagne C, Traverso JA, Mauve C, Valot B, Lelarge-Trouverie C, Zivy M, Noctor G, Meinnel T, Giglione C. Cotranslational proteolysis dominates glutathione homeostasis to support proper growth and development. THE PLANT CELL 2009; 21:3296-314. [PMID: 19855051 PMCID: PMC2782297 DOI: 10.1105/tpc.109.069757] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/01/2009] [Revised: 09/17/2009] [Accepted: 10/05/2009] [Indexed: 05/18/2023]
Abstract
The earliest proteolytic event affecting most proteins is the excision of the initiating Met (NME). This is an essential and ubiquitous cotranslational process tightly regulated in all eukaryotes. Currently, the effects of NME on unknown complex cellular networks and the ways in which its inhibition leads to developmental defects and cell growth arrest remain poorly understood. Here, we provide insight into the earliest molecular mechanisms associated with the inhibition of the NME process in Arabidopsis thaliana. We demonstrate that the developmental defects induced by NME inhibition are caused by an increase in cellular proteolytic activity, primarily induced by an increase in the number of proteins targeted for rapid degradation. This deregulation drives, through the increase of the free amino acids pool, a perturbation of the glutathione homeostasis, which corresponds to the earliest limiting, reversible step promoting the phenotype. We demonstrate that these effects are universally conserved and that the reestablishment of the appropriate glutathione status restores growth and proper development in various organisms. Finally, we describe a novel integrated model in which NME, protein N-alpha-acylation, proteolysis, and glutathione homeostasis operate in a sequentially regulated mechanism that directs both growth and development.
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Affiliation(s)
- Frédéric Frottin
- Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Unité Propre de Recherche2355, Protein Maturation, Cell Fate, and Therapeutics, F-91198 Gif-sur-Yvette cedex, France
| | - Christelle Espagne
- Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Unité Propre de Recherche2355, Protein Maturation, Cell Fate, and Therapeutics, F-91198 Gif-sur-Yvette cedex, France
| | - José A. Traverso
- Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Unité Propre de Recherche2355, Protein Maturation, Cell Fate, and Therapeutics, F-91198 Gif-sur-Yvette cedex, France
| | - Caroline Mauve
- Université Paris-Sud, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, F-91405 Orsay, France
- Centre National de la Recherche Scientifique, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, Unité Mixte de Recherche 8618, F-91405 Orsay, France
| | - Benoît Valot
- Université Paris-Sud, Plateforme de Protéomique, Institut Fédératif de Recherche87, Centre National de la Recherche Scientifique/Université Paris-Sud/Institut National de la Recherche Agronomique, F-91190 Gif-sur-Yvette, France
- Centre National de la Recherche Scientifique, Plateforme de Protéomique, Institut Fédératif de Recherche87, F-91190 Gif-sur-Yvette, France
- Institut National de la Recherche Agronomique, Plateforme de Protéomique, Institut Fédératif de Recherche87, F-91190 Gif-sur-Yvette, France
| | - Caroline Lelarge-Trouverie
- Université Paris-Sud, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, F-91405 Orsay, France
- Centre National de la Recherche Scientifique, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, Unité Mixte de Recherche 8618, F-91405 Orsay, France
| | - Michel Zivy
- Université Paris-Sud, Plateforme de Protéomique, Institut Fédératif de Recherche87, Centre National de la Recherche Scientifique/Université Paris-Sud/Institut National de la Recherche Agronomique, F-91190 Gif-sur-Yvette, France
- Centre National de la Recherche Scientifique, Plateforme de Protéomique, Institut Fédératif de Recherche87, F-91190 Gif-sur-Yvette, France
- Institut National de la Recherche Agronomique, Plateforme de Protéomique, Institut Fédératif de Recherche87, F-91190 Gif-sur-Yvette, France
| | - Graham Noctor
- Université Paris-Sud, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, F-91405 Orsay, France
- Centre National de la Recherche Scientifique, Institut Fédératif de Recherche87, Institut de Biotechnologie des Plantes, Plateforme Métabolisme-Métabolome, Unité Mixte de Recherche 8618, F-91405 Orsay, France
| | - Thierry Meinnel
- Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Unité Propre de Recherche2355, Protein Maturation, Cell Fate, and Therapeutics, F-91198 Gif-sur-Yvette cedex, France
| | - Carmela Giglione
- Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Unité Propre de Recherche2355, Protein Maturation, Cell Fate, and Therapeutics, F-91198 Gif-sur-Yvette cedex, France
- Address correspondence to
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Datta B. Roles of P67/MetAP2 as a tumor suppressor. Biochim Biophys Acta Rev Cancer 2009; 1796:281-92. [PMID: 19716858 DOI: 10.1016/j.bbcan.2009.08.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2009] [Revised: 08/12/2009] [Accepted: 08/22/2009] [Indexed: 12/17/2022]
Abstract
A precise balance between growth promoting signals and growth inhibitory signals plays important roles in the maintenance of healthy mammalian cells. Any deregulation of this critical balance converts normal cells into abnormal or cancerous cells. Several macromolecules are being identified and characterized that are involved in the regulation of cell signaling pathways that connect to the cell cycle and thus they play roles as tumor promoters or tumor suppressors. In situ tumor formation needs active angiogenesis, a process that generates new blood vessels from existing ones either by splitting or sprouting. Several small molecule inhibitors and proteins have been identified as inhibitors of angiogenesis. One such protein, p67/MetAP2 also known as methionine aminopeptidase 2 (MetAP2), has been shown to bind covalently to fumagillin and its derivatives that have anti-angiogenic activity. In addition to fumagillin or its derivatives, several other small molecule inhibitors of p67/MetAP2 have been recently identified and some of these drugs are in phase III trials for cancer therapy. Although molecular details of actions toward tumor suppression by these drugs are largely unknown, a significant progress has been made to understand the structure-function relationship of p67/MetAP2 and its roles in the maintenance of the levels of phosphorylation of the proportional, variant-subunit of eukaryotic initiation factor 2 (eIF2 proportional, variant) and extracellular signal-regulated kinases 1 and 2 (ERK1/2). In this article, roles of p67/MetAP2 in the suppression of cancer development are also discussed.
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Affiliation(s)
- Bansidhar Datta
- Department of Chemistry, Kent State University, Kent, OH 44242, USA.
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Selvakumar P, Lakshmikuttyamma A, Das U, Pati HN, Dimmock JR, Sharma RK. NC2213: a novel methionine aminopeptidase 2 inhibitor in human colon cancer HT29 cells. Mol Cancer 2009; 8:65. [PMID: 19703310 PMCID: PMC2740849 DOI: 10.1186/1476-4598-8-65] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2009] [Accepted: 08/24/2009] [Indexed: 11/10/2022] Open
Abstract
Methionine aminopeptidase 2 (MetAP2) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. MetAP2 is overexpressed in human colon cancer. In this report we screened various MetAP2 inhibitors and treated HT29 cells with various concentrations of compounds. We evaluated the expression of MetAP2 and pp60c-src expressions in HT29 cells. In addition we also carried out the cell proliferation and cell cycle analysis in the MetAP2 inhibitor-treated HT29 cells. The cell cycle analysis of HT29 treated with 1.0 microM of NC2213 showed an arrest in the G2 phase followed by an induction in the percentage of cells undergoing apoptosis in the sub-G1 phase. Western blot analysis revealed that the MetAP2 expression was dose-dependently decreased when the HT29 cells were treated with the 3,5-bis(benzylidene)-4-piperidone derivative (NC2213). In addition, phosphorylation of Src, a myristoylated oncoprotein was significantly decreased by 1.0 microM of NC2213 as revealed by Western blot analysis. Furthermore, NC2213 also inhibits the expression of pp60c-src in HT29 cells. Interestingly, this compound also inhibits the phosphorylation at Tyr416 of pp60c-src while increasing the phosphorylation at Tyr527 of pp60c-src. NC2213 inhibits the growth of HT29 cells by inducing apoptosis and might be useful for the treatment of human colon cancer.
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Affiliation(s)
- Ponniah Selvakumar
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N4H4, Canada.
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Dey A, Tergaonkar V, Lane DP. Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-kappaB pathways. Nat Rev Drug Discov 2008; 7:1031-40. [PMID: 19043452 DOI: 10.1038/nrd2759] [Citation(s) in RCA: 123] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The p53 and nuclear factor-kappaB (NF-kappaB) pathways play crucial roles in human cancer, in which inactivation of p53 and hyperactivation of NF-kappaB is a common occurrence. Activation of p53 and inhibition of NF-kappaB promotes apoptosis. Although drugs are being designed to selectively activate p53 or inhibit NF-kappaB, there is no concerted effort yet to deliberately make drugs that can simultaneously do both. Recent results suggest that a surprising selection of small molecules have this desirable dual activity. In this Review we describe the principles behind such dual activities, describe the current candidate molecules and suggest mechanisms and approaches to their further development.
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Affiliation(s)
- Anwesha Dey
- Laboratory of Cell Cycle Control, Institute of Molecular and Cell Biology, Proteos, 138673 Singapore
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Stevanovic J, Stanimirovic Z, Radakovic M, Stojic V. In vitro evaluation of the clastogenicity of fumagillin. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2008; 49:594-601. [PMID: 18613037 DOI: 10.1002/em.20409] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
Fumagillin, an antibiotic compound produced by Aspergillus fumigatus, is effective against microsporidia and various Amoeba species, but is also toxic when administered systemically to mammals. Furthermore, a recent in vivo study by Stanimirovic Z et al. 2007: (Mutat Res 628:1-10) indicated genotoxic effects of fumagillin. The aim of the present study was to investigate and explain the clastogenic effects of fumagillin (in the form of fumagillin dicyclohexylamine salt) on human peripheral blood lymphocytes in vitro by sister-chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests. The mitotic index (MI), proliferation index (PI), and nuclear division index (NDI) were calculated to evaluate the cytotoxic potential of fumagillin. Five concentrations of fumagillin (0.34, 0.68, 1.02, 3.07, and 9.20 microg/ml) were applied to lymphocyte cultures. All the tested concentrations of fumagillin increased the frequency of SCE per cell significantly (P < 0.001 or P < 0.01) compared with the negative control. A significant (P < 0.001) increase in frequency of structural CA was observed at the three highest concentrations in comparison with the negative control. In addition, the three highest test concentrations increased MN formation and decreased MI, PI, and NDI significantly compared with the negative control. The present results indicate that fumagillin is clastogenic and cytotoxic to cultured human lymphocytes.
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Affiliation(s)
- Jevrosima Stevanovic
- Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia.
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Martinez A, Traverso JA, Valot B, Ferro M, Espagne C, Ephritikhine G, Zivy M, Giglione C, Meinnel T. Extent of N-terminal modifications in cytosolic proteins from eukaryotes. Proteomics 2008; 8:2809-31. [PMID: 18655050 DOI: 10.1002/pmic.200701191] [Citation(s) in RCA: 128] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.
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Affiliation(s)
- Aude Martinez
- Institut des Sciences du Végétal, UPR2355, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
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Cirone P, Lin S, Griesbach HL, Zhang Y, Slusarski DC, Crews CM. A role for planar cell polarity signaling in angiogenesis. Angiogenesis 2008; 11:347-60. [PMID: 18798004 DOI: 10.1007/s10456-008-9116-2] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2008] [Accepted: 08/20/2008] [Indexed: 11/26/2022]
Abstract
The planar cell polarity (PCP) pathway is a highly conserved signaling cascade that coordinates both epithelial and axonal morphogenic movements during development. Angiogenesis also involves the growth and migration of polarized cells, although the mechanisms underlying their intercellular communication are poorly understood. Here, using cell culture assays, we demonstrate that inhibition of PCP signaling disrupts endothelial cell growth, polarity, and migration, all of which can be rescued through downstream activation of this pathway by expression of either Daam-1, Diversin or Inversin. Silencing of either Dvl2 or Prickle suppressed endothelial cell proliferation. Moreover, loss of p53 rescues endothelial cell growth arrest but not the migration inhibition caused by PCP disruption. In addition, we show that the zebrafish Wnt5 mutant (pipetail (ppt)), which has impaired PCP signaling, displays vascular developmental defects. These findings reveal a potential role for PCP signaling in the coordinated assembly of endothelial cells into vascular structures and have important implications for vascular remodeling in development and disease.
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Affiliation(s)
- Pasquale Cirone
- Department of Molecular, Cellular and Developmental Biology, Yale University, Kline Biology Tower, P.O. Box 208103, New Haven, CT 06520-8103, USA
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Hines J, Groll M, Fahnestock M, Crews CM. Proteasome inhibition by fellutamide B induces nerve growth factor synthesis. CHEMISTRY & BIOLOGY 2008; 15:501-12. [PMID: 18482702 PMCID: PMC2485210 DOI: 10.1016/j.chembiol.2008.03.020] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/06/2008] [Revised: 03/20/2008] [Accepted: 03/25/2008] [Indexed: 01/26/2023]
Abstract
Neurotrophic small molecules have the potential to aid in the treatment of neuronal injury and neurodegenerative diseases. The natural product fellutamide B, originally isolated from Penicillium fellutanum, potently induces nerve growth factor (NGF) release from fibroblasts and glial-derived cells, although the mechanism for this neurotrophic activity has not been elucidated. Here, we report that fellutamide B potently inhibits proteasome catalytic activity. High-resolution structural information obtained from cocrystallization of the 20S proteasome reveals novel aspects regarding beta-subunit binding and adduct formation by fellutamide B to inhibit their hydrolytic activity. We demonstrate that fellutamide B and other proteasome inhibitors increased NGF gene transcription via a cis-acting element (or elements) in the promoter. These results demonstrate an unrecognized connection between proteasome inhibition and NGF production, suggesting a possible new strategy in the development of neurotrophic agents.
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Affiliation(s)
- John Hines
- Dept. of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA
| | - Michael Groll
- Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstrasse 4, Garching D-85747, Germany
| | - Margaret Fahnestock
- Dept. of Psychiatry & Behavioural Neurosciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Craig M. Crews
- Dept. of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA
- Dept. of Chemistry, Yale University, New Haven, CT 06511, USA
- Dept. of Pharmacology, Yale University, New Haven, CT 06511, USA
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Bainbridge J, Madden L, Essex D, Binks M, Malhotra R, Paleolog EM. Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition. Arthritis Res Ther 2008; 9:R127. [PMID: 18072970 PMCID: PMC2246249 DOI: 10.1186/ar2340] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2007] [Revised: 10/24/2007] [Accepted: 12/11/2007] [Indexed: 12/19/2022] Open
Abstract
The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.
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Affiliation(s)
- John Bainbridge
- Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College London, 1, Aspenlea Road, London W6 8LH, UK.
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Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation. Oncogene 2008; 27:3967-76. [PMID: 18264137 DOI: 10.1038/onc.2008.14] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Methionine aminopeptidase-2 (MetAP2) processes N-terminal methionine from nascent cellular proteins. Inhibition of MetAP2 has been shown to block angiogenesis and suppress tumor growth in preclinical tumor models. However, the biological role of MetAP2 in cancer is not well understood. We examined the effect of three distinct chemical classes of MetAP2 inhibitors on the growth of a panel of human cancer cells in vitro. All MetAP2 inhibitors caused inhibition of tumor cell growth in both anchorage-dependent and, particularly, in anchorage-independent manner. These data prompted us to examine the possible roles of MetAP2 in cancers. Ectopic expression of MetAP2 in NIH-3T3 cells caused transformation, evidenced by the formation of foci in monolayer culture and growth of large colonies in soft agar. Overexpression of MetAP2 in an immortalized bronchial epithelial cell line NL20 accelerated growth. These phenotypes induced by the overexpression of MetAP2 were reversed by the treatment with MetAP2 inhibitors, indicating that the catalytic function of MetAP2 was essential. Accordingly, overexpression of a catalytically inactive MetAP2 resulted in growth retardation of HT1080 tumor cells, suggesting a dominant-negative role of the inactive MetAP2 mutant. Finally, we analysed the expression of MetAP2 in patient cancer samples by immunohistochemistry. Moderate-to-high staining was identified in the majority of breast, colon, lung, ovarian and prostate carcinomas examined. These data suggest that MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis.
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Correlation of tumor growth suppression and methionine aminopetidase-2 activity blockade using an orally active inhibitor. Proc Natl Acad Sci U S A 2008; 105:1838-43. [PMID: 18252827 DOI: 10.1073/pnas.0708766105] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
This laboratory and others have shown that agents that inhibit the in vitro catalytic activity of methionine aminopeptidase-2 (MetAP2) are effective in blocking angiogenesis and tumor growth in preclinical models. However, these prototype MetAP2 inhibitors are clearly not optimized for therapeutic use in the clinic. We have discovered an orally active class of MetAP2 inhibitors, the anthranilic acid sulfonamides exemplified by A-800141, which is highly specific for MetAP2. This orally bioavailable inhibitor exhibits an antiangiogenesis effect and a broad anticancer activity in a variety of tumor xenografts including B cell lymphoma, neuroblastoma, and prostate and colon carcinomas, either as a single agent or in combination with cytotoxic agents. We also have developed a biomarker assay to evaluate in vivo MetAP2 inhibition in circulating mononuclear cells and in tumors. This biomarker assay is based on the N-terminal methionine status of the MetAP2-specific substrate GAPDH in these cells. In cell cultures in vitro, the sulfonamide MetAP2 inhibitor A-800141 caused the formation of GAPDH variants with an unprocessed N-terminal methionine. A-800141 blocked tumor growth and MetAP2 activity in a similar dose-response in mouse models, demonstrating the antitumor effects seen for A-800141 are causally connected to MetAP2 inhibition in vivo. The sulfonamide MetAP2 inhibitor and GAPDH biomarker in circulating leukocytes may be used for the development of a cancer treatment.
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TNP-470: The Resurrection of the First Synthetic Angiogenesis Inhibitor. Angiogenesis 2008. [DOI: 10.1007/978-0-387-71518-6_35] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Hu X, Dang Y, Tenney K, Crews P, Tsai CW, Sixt KM, Cole PA, Liu JO. Regulation of c-Src nonreceptor tyrosine kinase activity by bengamide A through inhibition of methionine aminopeptidases. ACTA ACUST UNITED AC 2007; 14:764-74. [PMID: 17656313 PMCID: PMC3165037 DOI: 10.1016/j.chembiol.2007.05.010] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2007] [Revised: 05/07/2007] [Accepted: 05/24/2007] [Indexed: 11/17/2022]
Abstract
Methionine aminopeptidases (MetAPs) remove the N-terminal initiator methionine during protein synthesis, a prerequisite step for N-terminal myristoylation. N-myristoylation of proto-oncogene c-Src is essential for its membrane association and proper signal transduction. We used bengamides, a family of general MetAP inhibitors, to understand the downstream physiological functions of MetAPs. c-Src from bengamide A-treated cells retained its N-terminal methionine and suffered a decrease in N-terminal myristoylation, which was accompanied by a shift of its subcellular distribution from the plasma membrane to the cytosol. Furthermore, bengamide A decreased the tyrosine kinase activities of c-Src both in vitro and in vivo and eventually delayed cell-cycle progression through G(2)/M. Thus, c-Src is a physiologically relevant substrate for MetAPs whose dysfunction is likely to account for the cell-cycle effects of MetAP inhibitors including bengamide A.
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Affiliation(s)
- Xiaoyi Hu
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
| | - Yongjun Dang
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
| | - Karen Tenney
- Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064
| | - Phillip Crews
- Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064
| | - Chiawei W. Tsai
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
| | - Katherine M. Sixt
- Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
| | - Philip A. Cole
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
| | - Jun O. Liu
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
- Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St. Baltimore, MD 21205
- Correspondence: Dr. Jun O. Liu, , (410)-955-4619
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Klein C. Aspergillus fumigatus und Angiogenese. Von einer Zufallsentdeckung zum Rationalen Drug-Design. ACTA ACUST UNITED AC 2007; 36:450-1. [DOI: 10.1002/pauz.200700242] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Hu XV, Chen X, Han KC, Mildvan AS, Liu JO. Kinetic and Mutational Studies of the Number of Interacting Divalent Cations Required by Bacterial and Human Methionine Aminopeptidases. Biochemistry 2007; 46:12833-43. [DOI: 10.1021/bi701127x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Xiaoyi V. Hu
- Departments of Pharmacology and Molecular Sciences and Biological Chemistry and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
| | - Xiaochun Chen
- Departments of Pharmacology and Molecular Sciences and Biological Chemistry and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
| | - Kee Chung Han
- Departments of Pharmacology and Molecular Sciences and Biological Chemistry and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
| | - Albert S. Mildvan
- Departments of Pharmacology and Molecular Sciences and Biological Chemistry and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
| | - Jun O. Liu
- Departments of Pharmacology and Molecular Sciences and Biological Chemistry and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
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Kass D, Bridges RS, Borczuk A, Greenberg S. Methionine aminopeptidase-2 as a selective target of myofibroblasts in pulmonary fibrosis. Am J Respir Cell Mol Biol 2007; 37:193-201. [PMID: 17446530 DOI: 10.1165/rcmb.2006-0352oc] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive, scarring lung disease characterized by fibroblast accumulation and deposition of collagen. Factors that promote growth and/or survival of fibroblasts are potential therapeutic targets. Methionine aminopeptidase 2 (MetAP2), a member of the aminopeptidase family of proteases, has been implicated in cell proliferation in a variety of cell types, but its expression and function in the lung is not known. By immunohistochemistry, MetAP2 was expressed in many cell types, including fibroblasts, in IPF lungs. Fumagillin, an irreversible inhibitor of the enzymatic activity of MetAP2, attenuated collagen deposition in the bleomycin model of acute lung injury in mice. Treatment with fumagillin caused a selective reduction in the numbers of bromodeoxyuridine (BrdU)-positive myofibroblasts, but not type II alveolar epithelial cells, macrophages, or B- and T-lymphocytes in the lungs of bleomycin-treated mice. Incubation of primary rat lung fibroblasts with either fumagillin or with short interfering RNA that targeted MetAP2 led to reduced proliferation, as assessed by incorporation of BrdU. The profibrotic growth factor, platelet-derived growth factor, increased expression of MetAP2 in rat lung fibroblasts. We propose that MetAP2 plays a role in the proliferation of fibroblasts and myofibroblasts in fibrotic lung diseases and may serve as a novel pharmacologic target in IPF.
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Affiliation(s)
- Daniel Kass
- Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
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