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Taeb S, Rostamzadeh D, Amini SM, Rahmati M, Golshekan M, Abedinzade M, Ahmadi E, Neha S, Najafi M. Revolutionizing Cancer Treatment: Harnessing the Power of Mesenchymal Stem Cells for Precise Targeted Therapy in the Tumor Microenvironment. Curr Top Med Chem 2025; 25:243-262. [PMID: 38797895 DOI: 10.2174/0115680266299112240514103048] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2023] [Revised: 04/03/2024] [Accepted: 04/16/2024] [Indexed: 05/29/2024]
Abstract
In recent years, mesenchymal stem cells (MSCs) have emerged as promising anti-- cancer mediators with the potential to treat several cancers. MSCs have been modified to produce anti-proliferative, pro-apoptotic, and anti-angiogenic molecules that could be effective against a variety of malignancies. Additionally, customizing MSCs with cytokines that stimulate pro-tumorigenic immunity or using them as vehicles for traditional chemical molecules with anti-cancer characteristics. Even though the specific function of MSCs in tumors is still challenged, promising outcomes from preclinical investigations of MSC-based gene therapy for a variety of cancers inspire the beginning of clinical trials. In addition, the tumor microenvironment (TME) could have a substantial influence on normal tissue stem cells, which can affect the treatment outcomes. To overcome the complications of TME in cancer development, MSCs could provide some signs of hope for converting TME into unequivocal therapeutic tools. Hence, this review focuses on engineered MSCs (En-MSCs) as a promising approach to overcoming the complications of TME.
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Affiliation(s)
- Shahram Taeb
- Department of Radiology, School of Paramedical Sciences, Guilan University of Medical Sciences, Rasht, Iran
| | - Davoud Rostamzadeh
- Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030, Connecticut, USA
| | - Seyed Mohammad Amini
- Radiation Biology Research center, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad Rahmati
- Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
| | - Mostafa Golshekan
- Guilan Road Trauma Research Center, Guilan University of Medical Sciences, Rasht, Iran
| | - Mahmoud Abedinzade
- Department of Medical Physiology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
| | - Elham Ahmadi
- Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030, Connecticut, USA
| | - Singh Neha
- Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030, Connecticut, USA
| | - Masoud Najafi
- Medical Technology Research Center, Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
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Nine Genes Mediate the Therapeutic Effects of Iodine-131 Radiotherapy in Thyroid Carcinoma Patients. DISEASE MARKERS 2020; 2020:9369341. [PMID: 32626543 PMCID: PMC7317313 DOI: 10.1155/2020/9369341] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Accepted: 06/02/2020] [Indexed: 12/19/2022]
Abstract
Background Thyroid carcinoma (THCA) is one of the most common malignancies of the endocrine system, which is usually treated by surgery combined with iodine-131 (I131) radiotherapy. Aims This study is aimed at exploring the potential targets of I131 radiotherapy in THCA. Methods The RNA-sequencing data of THCA in The Cancer Genome Atlas database (including 568 THCA samples) was downloaded. The differentially expressed genes (DEGs) between the tumour samples whether or not subjected to I131 radiotherapy were identified using edgeR package. Using the WGCNA package, the module that was relevant with I131 radiotherapy was selected. The intersection genes of the hub module nodes and the DEGs were obtained as hub genes, followed by the function and pathway enrichment analyses using the clusterProfiler package. Moreover, the protein-protein interaction (PPI) network for the hub genes was constructed using Cytoscape software. In addition, more important hub genes were analysed with function mining using the GenCLiP2 online tool. The qPCR analysis was used to verify the mRNA expression of more important hub genes in THCA tissues. Results There were 500 DEGs (167 upregulated and 333 downregulated) between the two groups. WGCNA analysis showed that the green module (428 nodes) exhibited the most significant correlation with I131 radiotherapy. A PPI network was built after the identification of 53 hub genes. In the PPI network, CDH5, KDR, CD34, FLT4, EMCN, FLT1, ROBO4, PTPRB, and CD93 exhibited higher degrees, which were mainly implicated in the vascular function. The relative expression of nine mRNAs in the THCA tissues treated with I131 was lower. Conclusion I131 radiotherapy might exert therapeutic effects by targeting CDH5, KDR, CD34, FLT4, EMCN, FLT1, ROBO4, PTPRB, and CD93 in THCA patients.
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Okamoto M, Watanabe M, Inoue N, Ogawa K, Hidaka Y, Iwatani Y. Gene polymorphisms of VEGF and VEGFR2 are associated with the severity of Hashimoto's disease and the intractability of Graves' disease, respectively. Endocr J 2020; 67:545-559. [PMID: 32074519 DOI: 10.1507/endocrj.ej19-0480] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Vascular endothelial growth factor (VEGF) is one of main regulators of angiogenesis that functions by binding to its receptors, including VEGF receptor (VEGFR) 2. There are few data available regarding the association between VEGF and VEGFR polymorphisms and the susceptibility to and prognosis of autoimmune thyroid diseases (AITDs). To elucidate this association, we genotyped four functional VEGF and two VEGFR2 polymorphisms and measured serum VEGF levels. In the four functional VEGF polymorphisms, the frequencies of the I carrier and I allele of VEGF -2549 I/D, which has lower activity, were higher in patients with severe HD than in those with mild HD. In the two functional VEGFR2 polymorphisms, the frequency of the rs2071559 CC genotype, which has higher activity, was higher in patients with intractable GD than in controls, and the proportion of GD patients with larger goiters was higher in those with the CC genotype. Moreover, the frequency of the rs1870377 TT genotype with higher activity was higher in patients with intractable GD than in those with GD in remission. Combinations of VEGF and VEGFR2 polymorphisms with stronger interactions were associated with the intractability of GD. Serum VEGF levels were higher in HD and AITD patients than those in controls. In conclusion, VEGF polymorphisms with lower activity were associated with the severity of HD, while VEGFR2 polymorphisms and the combinations of VEGF and VEGFR2 polymorphisms, which have stronger interactions, were associated with the intractability of GD. VEGF and VEGFR2 polymorphisms were associated with HD severity and GD intractability, respectively.
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Affiliation(s)
- Mami Okamoto
- Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
| | - Mikio Watanabe
- Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
| | - Naoya Inoue
- Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
- Laboratory for Clinical Investigation, Osaka University Hospital, Osaka 565-0871, Japan
| | - Kazane Ogawa
- Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
| | - Yoh Hidaka
- Laboratory for Clinical Investigation, Osaka University Hospital, Osaka 565-0871, Japan
| | - Yoshinori Iwatani
- Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
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El-Saie A, Shivanna B. Novel Strategies to Reduce Pulmonary Hypertension in Infants With Bronchopulmonary Dysplasia. Front Pediatr 2020; 8:201. [PMID: 32457857 PMCID: PMC7225259 DOI: 10.3389/fped.2020.00201] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Accepted: 04/02/2020] [Indexed: 01/10/2023] Open
Abstract
Bronchopulmonary dysplasia (BPD) is a developmental lung disorder of preterm infants primarily caused by the failure of host defense mechanisms to prevent tissue injury and facilitate repair. This disorder is the most common complication of premature birth, and its incidence remains unchanged over the past few decades. Additionally, BPD increases long-term cardiopulmonary and neurodevelopmental morbidities of preterm infants. Pulmonary hypertension (PH) is a common morbidity of BPD. Importantly, the presence of PH increases both the short- and long-term morbidities and mortality in BPD infants. Further, there are no curative therapies for this complex disease. Besides providing an overview of the pathogenesis and diagnosis of PH associated with BPD, we have attempted to comprehensively review and summarize the current literature on the interventions to prevent and/or mitigate BPD and PH in preclinical studies. Our goal was to provide insight into the therapies that have a high translational potential to meaningfully manage BPD patients with PH.
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Affiliation(s)
- Ahmed El-Saie
- Department of Pediatrics, Section of Neonatology, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, United States
| | - Binoy Shivanna
- Department of Pediatrics, Section of Neonatology, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, United States
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Takano S, Ishikawa E, Matsuda M, Sakamoto N, Akutsu H, Yamamoto T, Matsumura A. The anti-angiogenic role of soluble-form VEGF receptor in malignant gliomas. Int J Oncol 2016; 50:515-524. [PMID: 28000842 DOI: 10.3892/ijo.2016.3810] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2016] [Accepted: 11/21/2016] [Indexed: 11/05/2022] Open
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Soluble Flt-1 gene delivery in acute myeloid leukemic cells mediating a nonviral gene carrier. BIOMED RESEARCH INTERNATIONAL 2013; 2013:752603. [PMID: 23509773 PMCID: PMC3581295 DOI: 10.1155/2013/752603] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 10/28/2012] [Accepted: 11/15/2012] [Indexed: 11/17/2022]
Abstract
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in angiogenesis-mediated progression of acute myeloid leukemia (AML). Studies have reported the role of soluble form of fms-like tyrosine kinase (sFlT-1) delivery as an antitumor agent by inhibiting VEGF. This study investigates the outcome of delivery of a VEGF165 antagonist, soluble vascular endothelial growth factor receptor, namely sFLT-1, mediating lipofectamine 2000 in acute myeloid leukemic cells. A recombinant plasmid expressing sFLT-1 was constructed and transfected into the K562 and HL60 cells using lipofectamine 2000 transfection reagent. sFLT-1 expression/secretion in pVAX-sFLT-1 transfected cells was verified by RT-PCR and western blot. MTS assay was carried out to evaluate the effect of sFLT-1 on human umbilical vein endothelial cells and K562 and HL60 cells in vitro. Treatment with pVAX-sFLT-1 showed no association between sFLT-1 and proliferation of infected K562 and HL60 cells, while it demonstrated a significant inhibitory impact on the proliferation of HUVECs. The results of the current study imply that the combination of nonviral gene carrier and sFLT-1 possesses the potential to provide efficient tool for the antiangiogenic gene therapy of AML.
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Zhu H, Li Z, Mao S, Ma B, Zhou S, Deng L, Liu T, Cui D, Zhao Y, He J, Yi C, Huang Y. Antitumor effect of sFlt-1 gene therapy system mediated by Bifidobacterium Infantis on Lewis lung cancer in mice. Cancer Gene Ther 2011; 18:884-96. [PMID: 21921942 PMCID: PMC3215997 DOI: 10.1038/cgt.2011.57] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Soluble fms-like tyrosine kinase receptor (sFlt-1) is a soluble form of extramembrane part of vascular endothelial growth factor receptor-1 (VEGFR-1) that has antitumor effects. Bifidobacterium Infantis is a kind of non-pathogenic and anaerobic bacteria that may have specific targeting property of hypoxic environment inside of solid tumors. The aim of this study was to construct Bifidobacterium Infantis-mediated sFlt-1 gene transferring system and investigate its antitumor effect on Lewis lung cancer (LLC) in mice. Our results demonstrated that the Bifidobacterium Infantis-mediated sFlt-1 gene transferring system was constructed successfully and the system could express sFlt-1 at the levels of gene and protein. This system could not only significantly inhibit growth of human umbilical vein endothelial cells induced by VEGF in vitro, but also inhibit the tumor growth and prolong survival time of LLC C57BL/6 mice safely. These data suggest that Bifidobacterium Infantis-mediated sFlt-1 gene transferring system presents a promising therapeutic approach for the treatment of cancer.
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Affiliation(s)
- H Zhu
- Department of Abdominal Cancer, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China
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Soluble vascular endothelial growth factor (VEGF) receptor-1 inhibits migration of human monocytic THP-1 cells in response to VEGF. Inflamm Res 2011; 60:769-74. [PMID: 21487788 DOI: 10.1007/s00011-011-0332-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2010] [Accepted: 03/28/2011] [Indexed: 12/15/2022] Open
Abstract
OBJECTIVE We aimed to investigate the regulation and contribution of vascular endothelial growth factor (VEGF) and sFlt-1(1-3) to human monocytic THP-1 migration. MATERIALS AND METHODS Ad-sFlt-1/FLAG, a recombinant adenovirus carrying the human sFlt-1(1-3) (the first three extracellular domains of FLT-1, the hVEGF receptor-1) gene, was constructed. L929 cells were infected with Ad-sFlt-1/FLAG and the expression of sFlt-1 was detected by immunofluorescent assay and ELISA. Corning(®) Transwell(®) Filter Inserts containing polyethylene terephthalate (PET) membranes with pore sizes of 3 μm were used as an experimental model to simulate THP-1 migration. Five VEGF concentrations (0, 0.1, 1, 10 and 100 ng/ml), four concentrations of sFlt-1(1-3)/FLAG expression supernatants (0.1, 1, 10 and 100 ng/ml), and monocyte chemoattractant protein-1 (MCP-1, 10 ng/ml) were used to test the ability of THP-1 cells to migrate through PET membranes. RESULTS The sFlt-1(1-3) gene was successfully recombined into Ad-sFlt-1/FLAG. sFlt-1(1-3) was expressed in L929 cells transfected with Ad-sFlt-1/FLAG. THP-1 cell migration increased with increasing concentrations of VEGF, while cell migration decreased with increasing concentrations of sFlt1(1-3)/FLAG. sFlt1(1-3)/FLAG had no effect on MCP-1-induced cell migration. CONCLUSIONS This study demonstrated that VEGF is able to elicit a migratory response in THP-1 cells, and that sFlt-1(1-3) is an effective inhibitor of THP-1 migration towards VEGF.
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Yang F, Jin C, Jiang YJ, Li J, Di Y, Fu DL. Potential role of soluble VEGFR-1 in antiangiogenesis therapy for cancer. Expert Rev Anticancer Ther 2011; 11:541-549. [PMID: 21504321 DOI: 10.1586/era.10.171] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Antiangiogenesis therapy for cancer may inhibit tumor growth and metastasis when combined with chemotherapy, and has received a great deal of attention over recent years. However, accurate assessments of biological efficacy and toxicity are major hurdles for this approach. Soluble VEGF receptor-1 (sFlt-1) has been reported to have a role in the pathogenesis of preeclampsia, the hallmark of which is similar to the toxicities related to antiangiogenesis therapy. Clinical evidence and animal studies support the hypothesis that sFlt-1 may contribute to hypertension and proteinuria in patients treated with anti-VEGF agents. The intratumoral imbalance between sFlt-1 and VEGF levels correlates with the malignancy grades of tumors, survival and responsiveness to therapy. The therapeutic potential of sFlt-1 as an antiangiogenic agent has been validated by an increasing number of preclinical studies. Furthermore, antiangiogenesis therapy changes the concentration of circulating VEGF, PlGF, sFlt-1, soluble VEGFR-2 and even soluble VEGFR-3, with some of these being identified as potential biomarkers of response and toxicity. All these factors suggest that sFlt-1 may prove invaluable for driving the future development of molecular therapeutics with novel targets and mechanisms of action, and its impact on antiangiogenesis therapy in cancers needs further investigation.
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Affiliation(s)
- Feng Yang
- Pancreatic Disease Institute, Department of Pancreatic Surgery, Huashan Hospital, 12 Central Urumqi Road, Shanghai Medical College, Fudan University, Shanghai 200040, China
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Samaranayake H, Määttä AM, Pikkarainen J, Ylä-Herttuala S. Future prospects and challenges of antiangiogenic cancer gene therapy. Hum Gene Ther 2010; 21:381-96. [PMID: 20163246 DOI: 10.1089/hum.2010.017] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
In 1971 Judah Folkman proposed the concept of antiangiogenesis as a therapeutic target for cancer. More than 30 years later, concept became reality with the approval of the antivascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab as a first-line treatment for metastatic colorectal cancer. Monoclonal antibodies and small molecular drugs are the most widely applied methods for inhibition of angiogenesis. The efficacy of these antiangiogenic modalities has been proven, in both preclinical and clinical settings. Although angiogenesis plays a major role in wound healing, hypoxia, and in the female reproductive cycle, inhibition of angiogenesis seems to be a relatively safe therapeutic option against cancers, and has therefore become a logical arena for a wide range of experimentation. The twentieth century has shown the boom of gene therapy and thus it has been applied also in the antiangiogenic setting. This review summarizes methods to induce antiangiogenic responses with gene therapy and discusses the obstacles and future prospects of antiangiogenic cancer gene therapy.
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Vachutinsky Y, Oba M, Miyata K, Hiki S, Kano MR, Nishiyama N, Koyama H, Miyazono K, Kataoka K. Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles. J Control Release 2010; 149:51-7. [PMID: 20138936 DOI: 10.1016/j.jconrel.2010.02.002] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2009] [Revised: 12/21/2009] [Accepted: 02/01/2010] [Indexed: 01/06/2023]
Abstract
Disulfide crosslinked polyplex micelles with RGD peptides were formed through ion complexation of thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(L-lysine) (c(RGDfK)-PEG-P(Lys-SH)) and plasmid DNA encoding sFlt-1 and tested for their therapeutic effect in BxPC3 pancreatic adenocarcinoma tumor bearing mice. These micelles, systemically injected, demonstrated significant inhibition of tumor growth up to day 18, as a result of the antiangiogenic effect that was confirmed by vascular density measurements. Significant therapeutic activity of the 15% crosslinked micelle (c(RGDfK)-PEG-P(Lys-SH15)) was achieved by combined effect of increased tumor accumulation, interaction with endothelial cells and enhanced intracellular uptake through receptor-mediated endocytosis. These results suggest that RGD targeted crosslinked polyplex micelles can be effective plasmid DNA carriers for antiangiogenic gene therapy.
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Affiliation(s)
- Yelena Vachutinsky
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
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Wu FTH, Stefanini MO, Mac Gabhann F, Kontos CD, Annex BH, Popel AS. A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use. J Cell Mol Med 2009; 14:528-52. [PMID: 19840194 PMCID: PMC3039304 DOI: 10.1111/j.1582-4934.2009.00941.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Angiogenesis is the growth of new capillaries from pre-existent microvasculature. A wide range of pathological conditions, from atherosclerosis to cancer, can be attributed to either excessive or deficient angiogenesis. Central to the physiological regulation of angiogenesis is the vascular endothelial growth factor (VEGF) system – its ligands and receptors (VEGFRs) are thus prime molecular targets of pro-angiogenic and anti-angiogenic therapies. Of growing interest as a prognostic marker and therapeutic target in angiogenesis-dependent diseases is soluble VEGF receptor-1 (sVEGFR1, also known as sFlt-1) – a truncated version of the cell membrane-spanning VEGFR1. For instance, it is known that sVEGFR1 is involved in the endothelial dysfunction characterizing the pregnancy disorder of pre-eclampsia, and sVEGFR1’s therapeutic potential as an anti-angiogenic agent is being evaluated in pre-clinical models of cancer. This mini review begins with an examination of the protein domain structure and biomolecular interactions of sVEGFR1 in relation to the full-length VEGFR1. A synopsis of known and inferred physiological and pathological roles of sVEGFR1 is then given, with emphasis on the utility of computational systems biology models in deciphering the molecular mechanisms by which sVEGFR1’s purported biological functions occur. Finally, we present the need for a systems biology perspective in interpreting circulating VEGF and sVEGFR1 concentrations as surrogate markers of angiogenic status in angiogenesis-dependent diseases.
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Affiliation(s)
- Florence T H Wu
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Wu FTH, Stefanini MO, Mac Gabhann F, Popel AS. A compartment model of VEGF distribution in humans in the presence of soluble VEGF receptor-1 acting as a ligand trap. PLoS One 2009; 4:e5108. [PMID: 19352513 PMCID: PMC2663039 DOI: 10.1371/journal.pone.0005108] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2008] [Accepted: 03/10/2009] [Indexed: 12/25/2022] Open
Abstract
Vascular endothelial growth factor (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis--new capillary growth from existing microvasculature--at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1)--a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains--has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis-dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF(121) and VEGF(165), signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 - acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization--did not significantly lower interstitial VEGF, nor inhibit signaling potential in tissues. Additionally, the sensitivity of plasma VEGF and sVEGFR1 to physiological fluctuations in transport rates may partially account for the heterogeneity in clinical measurements of these circulating angiogenic markers, potentially hindering their diagnostic reliability for diseases.
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Affiliation(s)
- Florence T H Wu
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
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Wu FTH, Stefanini MO, Mac Gabhann F, Kontos CD, Annex BH, Popel AS. Computational kinetic model of VEGF trapping by soluble VEGF receptor-1: effects of transendothelial and lymphatic macromolecular transport. Physiol Genomics 2009; 38:29-41. [PMID: 19351908 DOI: 10.1152/physiolgenomics.00031.2009] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Vascular endothelial growth factor (VEGF) signal transduction through the cell surface receptors VEGFR1 and VEGFR2 regulates angiogenesis-the growth of new capillaries from preexistent microvasculature. Soluble VEGF receptor-1 (sVEGFR1), a nonsignaling truncated variant of VEGFR1, has been postulated to inhibit angiogenic signaling via direct sequestration of VEGF ligands or dominant-negative heterodimerization with surface VEGFRs. The relative contributions of these two mechanisms to sVEGFR1's purported antiangiogenic effects in vivo are currently unknown. We previously developed a computational model for predicting the compartmental distributions of VEGF and sVEGFR1 throughout the healthy human body by simulating the molecular interaction networks of the VEGF ligand-receptor system as well as intercompartmental macromolecular biotransport processes. In this study, we decipher the dynamic processes that led to our prior prediction that sVEGFR1, through its ligand trapping mechanism alone, does not demonstrate significant steady-state antiangiogenic effects. We show that sVEGFR1-facilitated tissue-to-blood shuttling of VEGF accounts for a counterintuitive and drastic elevation in plasma free VEGF concentrations after both intramuscular and intravascular sVEGFR1 infusion. While increasing intramuscular VEGF production reduces free sVEGFR1 levels through increased VEGF-sVEGFR1 complex formation, we demonstrate a competing and opposite effect in which increased VEGF occupancy of neuropilin-1 (NRP1) and the corresponding reduction in NRP1 availability for internalization of sVEGFR1 unexpectedly increases free sVEGFR1 levels. In conclusion, dynamic intercompartmental transport processes give rise to our surprising prediction that VEGF trapping alone does not account for sVEGFR1's antiangiogenic potential. sVEGFR1's interactions with cell surface receptors such as NRP1 are also expected to affect its molecular interplay with VEGF.
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Affiliation(s)
- Florence T H Wu
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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Zhu CS, Hu XQ, Xiong ZJ, Lu ZQ, Zhou GY, Wang DJ. Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) inhibits experimental autoimmune encephalomyelitis in dark Agouti (DA) rats. Life Sci 2008; 83:404-12. [DOI: 10.1016/j.lfs.2008.07.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2008] [Revised: 06/10/2008] [Accepted: 07/04/2008] [Indexed: 10/21/2022]
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Gautron L. [Gustave Roussy's (1874-1948) contribution to neuroendocrinology]. ANNALES D'ENDOCRINOLOGIE 2007; 68:400-402. [PMID: 17996213 DOI: 10.1016/j.ando.2007.09.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2007] [Revised: 09/26/2007] [Accepted: 09/27/2007] [Indexed: 05/25/2023]
Abstract
Gustave Roussy (1874-1948) is remembered as a distinguished French neuropathologist and a leader in the field of oncology. However, his original contribution to the study of hypothalamic functions and neuroendocrinology remains ignored. Among Roussy's discoveries is the first experimental demonstration of the hypothalamic origin of diabetes insipidus and adiposo-genital dystrophy. Also, based on his own histological work, he pioneered the concept of neurosecretion, which was termed by him "neuricrinie".
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Affiliation(s)
- L Gautron
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9077, USA.
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VEGF in physiological process and thyroid disease. ANNALES D'ENDOCRINOLOGIE 2007; 68:438-48. [PMID: 17991452 DOI: 10.1016/j.ando.2007.09.004] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2007] [Revised: 09/26/2007] [Accepted: 09/27/2007] [Indexed: 01/04/2023]
Abstract
Angiogenesis is a physiological process involving the growth of new vessels from pre-existing vasculature. Vascular endothelial growth factor (VEGF) is an important regulator of both benign and malignant disease processes in the thyroid gland. The VEGF family includes seven members respectively named VEGF-A, also known as VPF (vascular permeability factor), VEGF-B, VEGF-C, VEGF-D, all described in mammals, VEGF-E (found in Parapoxviridae), VEGF-F (also called svVEGF, for snake venom VEGF, found in viper venom) and PlGF (placental growth factor). Thyrocytes are able to synthesize and secrete VEGF. VEGF-A is implicated in tumour growth and metastasis via blood vessels while VEGF-C and VEGF-D, involved in lymphangiogenesis, favour metastasis to the cervical lymph nodes in papillary thyroid carcinomas. High levels of VEGF expression in thyroid tumour cells may correlate with a poorer outcome in papillary thyroid carcinomas. Because of its important role in malignant angiogenesis, VEGF is the preferential target of a new variety of therapeutic agents called angiogenesis inhibitors.
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Jebreel A, England J, Bedford K, Murphy J, Karsai L, Atkin S. Vascular endothelial growth factor (VEGF), VEGF receptors expression and microvascular density in benign and malignant thyroid diseases. Int J Exp Pathol 2007; 88:271-7. [PMID: 17696908 PMCID: PMC2517322 DOI: 10.1111/j.1365-2613.2007.00533.x] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Angiogenesis is critical for the growth and metastatic spread of tumours. Vascular endothelial growth factor (VEGF) is the most potent inducer of neovasculature, and its increased expression has been related to a worse clinical outcome in many diseases. The purpose of this study was to evaluate the relation between VEGF, its receptors (VEGFR-1 and VEGFR-2) and microvessel density (MVD) in thyroid diseases. Immunostaining for VEGF and VEGF receptors was performed in 66 specimens of thyroid tissue, comprising 17 multinodular goitre (MNG), 14 Graves' disease, 10 follicular adenoma, 8 Hashimoto's thyroiditis, 7 papillary carcinoma and 10 normal thyroid specimens. Thyrocyte positivity for VEGF and VEGF receptors was scored 0-3. Immunohistochemistry for CD31, and CD34 on the same sections was performed to evaluate MVD. Immunohistochemical staining of VEGF in thyrocytes was positive in 92% of all the thyroid tissues studied. Using an immunostaining intensity cut off of 2, increased thyrocyte staining was seen in follicular adenoma specimens, MNG and normal thyroids compared with Hashimoto's thyroiditis and Graves' disease (P < 0.05). Similarly, VEGF thyrocyte expression in Graves' disease was less than other pathologies (P < 0.05). VEGFR-1 expression and the average MVD score did not differ between the different thyroid pathologies. VEGF expression was lower in autoimmune pathologies compared to autonomous growth processes. Conversely, both VEGFR-1 and VEGFR-2 were widely expressed in benign and neoplastic thyroid disease, suggesting that the up-regulation of VEGF and not its receptors occurs as tissue becomes autonomous. There was no clear relationship between MVD measurement and thyroid pathology.
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Kundra P, Burman KD. Thyroid cancer molecular signaling pathways and use of targeted therapy. Endocrinol Metab Clin North Am 2007; 36:839-53, viii. [PMID: 17673131 DOI: 10.1016/j.ecl.2007.06.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Several agents are currently being tested that target thyroid molecular signaling and cancer cell biology. The pathways involved include but are not limited to the Ras pathway, vascular endothelial growth factor and epidermal growth factor receptors and antibodies, angiogenesis inhibitors, tyrosine kinase inhibitors, heat shock protein inhibitors, demethylating agents, histone deacetylase inhibitors, and gene therapy. Each of these targeted approaches holds promise for our future ability to treat patients with thyroid cancer unresponsive to traditional therapy.
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Affiliation(s)
- Priya Kundra
- Endocrine Sections, Washington Hospital Center, Georgetown University Medical Center, 110 Irving Street, NW, Washington, DC 20010, USA
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Liu J, Li J, Su C, Huang B, Luo S. Soluble Fms-like tyrosine kinase-1 expression inhibits the growth of multiple myeloma in nude mice. Acta Biochim Biophys Sin (Shanghai) 2007; 39:499-506. [PMID: 17622469 DOI: 10.1111/j.1745-7270.2007.00310.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Angiogenesis is an essential factor in the growth and progression of hematological malignancies including multiple myeloma (MM). Vascular endothelial growth factor and its receptors have been shown to be targets for treating tumors. This study explores the effect of adenovirus-mediated delivery of soluble vascular endothelial growth factor receptor Fms-like tyrosine kinase-1 (sFLT-1) on the growth of MM cell line KM3 in nude mice. sFLT-1 cDNA was amplified by reverse transcription-polymerase chain reaction from human umbilical vein endothelial cells and was used as a transgene to construct an adenoviral vector carrying sFLT-1 (ADV-sFLT). Cell proliferation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays were carried out to evaluate the effect of ADV-sFLT on human umbilical vein endothelial cells and KM3 cells in vitro. Eighteen female BALB/c nude mice were inoculated subcutaneously with KM3 cells, and they were randomly divided into three groups and injected intravenously with ADV-sFLT, ADV-LacZ, or phosphate-buffered saline (PBS). The volume of KM3 xenografts was measured twice a week. Three weeks after the initial treatment, the volume of MM xenografts in the mice treated with ADV-sFLT, ADV-LacZ, or PBS was 770.32+/-28.73 mm3, 1983.36+/-43.72 mm3, and 2042.05+/-82.31 mm3, respectively (P<0.01, ADV-sFLT versus ADV-LacZ or PBS). The value of microvessel density was 29.17+/-6.85, 79.17+/-7.35, and 78.83+/-8.54 in the tumors treated with ADV-sFLT, ADV-LacZ, and PBS, respectively (P<0.01, ADV-sFLT versus ADV-LacZ or PBS). This study suggested that the adenovirus-mediated sFLT-1 gene greatly inhibits MM-derived tumor growth and angiogenesis in mouse xenograft, and might serve as a new therapy for MM.
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Affiliation(s)
- Junru Liu
- Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
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21
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Ambati BK, Patterson E, Jani P, Jenkins C, Higgins E, Singh N, Suthar T, Vira N, Smith K, Caldwell R. Soluble vascular endothelial growth factor receptor-1 contributes to the corneal antiangiogenic barrier. Br J Ophthalmol 2006; 91:505-8. [PMID: 17151056 PMCID: PMC1994740 DOI: 10.1136/bjo.2006.107417] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
PURPOSE Pathological neovascularisation within the normally avascular cornea is a serious event that can interfere with normal vision. Upregulation of vascular endothelial growth factor (VEGF) has been associated with neovascularisation in the eye, suggesting that maintaining low levels of VEGF is important for corneal avascularity and intact vision. This study aims to determine the expression profile and possible contribution of sVEGFR-1 to the corneal avascular barrier. DESIGN Experimental case series investigating VEGF and soluble fms-like tyrosine kinase (sFlt) levels in normal and neovascularised human corneas. PARTICIPANTS Four normal human corneas, five human corneas with alkali burns, three human corneas with aniridia, one with ocular cicatricial pemphigoid and two with interstitial keratitis were examined. METHODS Western blot and immunohistochemical analyses were performed to determine sFlt and VEGF levels in normal and neovascularised human corneas. Immunoprecipitation was utilised to demonstrate sFlt-VEGF binding. RESULTS Normal human corneas strongly express sFlt in the corneal epithelium and weakly in the corneal stroma close to the limbus. VEGF is bound by sFlt in the normal human cornea. Neovascularised human corneas have greatly reduced expression of sFlt and significantly less VEGF bound by sFlt. CONCLUSIONS sFlt is highly expressed in the human cornea and normally sequesters VEGF.
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Affiliation(s)
- Balamurali K Ambati
- Augusta Veterans Affairs Medical Center, One Freedon Way, Augusta, GA 30912, USA.
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Kim WJ, Yockman JW, Jeong JH, Christensen LV, Lee M, Kim YH, Kim SW. Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice. J Control Release 2006; 114:381-8. [PMID: 16884805 DOI: 10.1016/j.jconrel.2006.05.029] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2006] [Revised: 05/25/2006] [Accepted: 05/31/2006] [Indexed: 11/30/2022]
Abstract
Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.
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Affiliation(s)
- Won Jong Kim
- Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, 20 S 2030 E RM 205 BPRB, University of Utah, Salt Lake City, UT 84112-5820, USA
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23
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Rodrigues A. Perspectivas de novos tratamentos para o carcinoma tireoidiano avançado. Rev Col Bras Cir 2006. [DOI: 10.1590/s0100-69912006000300011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
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Younes MN, Yazici YD, Kim S, Jasser SA, El-Naggar AK, Myers JN. Dual Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Inhibition with NVP-AEE788 for the Treatment of Aggressive Follicular Thyroid Cancer. Clin Cancer Res 2006; 12:3425-34. [PMID: 16740767 DOI: 10.1158/1078-0432.ccr-06-0793] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Patients with radioiodine-resistant follicular thyroid cancer (FTC) have a poor prognosis, if metastasized, with currently available treatment modalities. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) and their receptors (EGFR and VEGFR) have been reported to be overexpressed in FTC and have been implicated in FTC development. We hypothesized that inhibiting the phosphorylation of EGFR and VEGFR by treatment with NVP-AEE788 (AEE788), a novel dual specific EGFR and VEGFR inhibitor, either alone or in combination with paclitaxel, would inhibit the growth of FTC xenografts in an orthotopic nude mouse model. EXPERIMENTAL DESIGN To confirm previous reports, EGF and EGFR expression and vascularity were analyzed in human samples of FTC, Hürthle cell carcinoma, and normal thyroid tissues. EGFR expression in four FTC cell lines was measured using Western blotting. The antitumor effect of AEE788 on FTC cells in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and Western blotting. The effect of AEE788, alone and in combination with paclitaxel, on FTC tumor growth in an orthotopic nude mouse model was also investigated. Immunohistochemical analysis of EGFR and VEGFR signaling status, cell proliferation, apoptosis, and microvessel density was done. RESULTS EGF, EGFR, and vascularity were increased in human thyroid tumor samples and EGFR was increased in FTC cells. AEE788 inhibited FTC cell growth in vitro and reduced the phosphorylation status of EGFR, VEGFR, and two downstream targets, AKT and mitogen-activated protein kinase, in FTC cells. AEE788 alone and, to a greater extent, AEE788 plus paclitaxel suppressed FTC tumor growth in the thyroids of nude mice. CONCLUSION Dual inhibition of EGFR and VEGFR by AEE788 could represent a novel approach to the treatment of radioiodine-resistant FTC.
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Affiliation(s)
- Maher N Younes
- Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030-4009, USA
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Abstract
Vascular endothelial growth factor (VEGF) is an essential peptide in new vessel growth in physiology (endometrial growth, embryonic development); pathological conditions (diabetic retinopathy, rheumatoid arthritis); as well as in tumor cell growth, particularly distant metastases. This study focused on VEGF structure, receptors, and angiogensis in tumors, especially their roles in thyroid cancer. The VEGF mRNA undergoes alternative splicing events that generate four homodimeric isoforms, including VEGF121, VEGF165, VEGF189, or VEGF206. Using VEGF purified from a culture medium conditioned by A-431 human epidermoid carcinoma cells, VEGF-binding site complexes of 230, 170, and 125 kDa were detected on human umbilical vein endothelial cells. The VEGF specifically induced the tyrosine phosphorylation of a 190-kDa polypeptide, which had similar mass to the largest binding site detected through affinity cross-linking. A transmembrane receptor belongs to the tyrosine kinase family, fms-like tyrosine kinase (FLT). These receptor tyrosine kinases encoded by the FLT gene family have distinct functions in regulating blood vessel growth and differentiation. Regulation of VEGF is a complex, multistep mechanism in various kinds of cells and tissues. Hypoxia-dependent and -independent mechanisms are illustrated in different cancer tissues. Hypoxic tumor cells may switch to a proangiogenic phenotype, which increases VEGF transcription. Clinical applications of VEGF in cancer have included diagnosis, prediction of prognosis, and treatment in different solid tumors, including thyroid tumors. Studies involving thyroid cancer cell lines, serum level determination, immunohistocytochemical staining, molecular biological studies, and gene therapy to the in vivo clinical trials, have shown that antiangiogensis therapy can provide another treatment modality for thyroid cancer. Future studies focused on recombinant human anti-VEGF research involving patients with advanced thyroid cancer, and investigation of the protection of high-risk patients by using novel antiangiogenic vaccines, are warranted.
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Affiliation(s)
- Jen-Der Lin
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University, Taiwan, Republic of China.
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Ohlfest JR, Demorest ZL, Motooka Y, Vengco I, Oh S, Chen E, Scappaticci FA, Saplis RJ, Ekker SC, Low WC, Freese AB, Largaespada DA. Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma. Mol Ther 2005; 12:778-88. [PMID: 16150649 DOI: 10.1016/j.ymthe.2005.07.689] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2005] [Revised: 07/24/2005] [Accepted: 07/25/2005] [Indexed: 11/21/2022] Open
Abstract
Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.
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Affiliation(s)
- John R Ohlfest
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA
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Reinblatt M, Pin RH, Bowers WJ, Federoff HJ, Fong Y. Herpes simplex virus amplicon delivery of a hypoxia-inducible soluble vascular endothelial growth factor receptor (sFlk-1) inhibits angiogenesis and tumor growth in pancreatic adenocarcinoma. Ann Surg Oncol 2005; 12:1025-36. [PMID: 16244806 DOI: 10.1245/aso.2005.03.081] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2004] [Accepted: 07/08/2005] [Indexed: 01/03/2023]
Abstract
BACKGROUND Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates angiogenesis and tumor proliferation. The VEGF signaling pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase 1; sFlk-1), which bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV) amplicons are replication-incompetent viruses used for gene delivery. We attempted to attenuate angiogenesis and inhibit pancreatic tumor growth through HSV amplicon-mediated expression of sFlk-1 under hypoxic control. METHODS A multimerized hypoxia-responsive enhancer (10 x HRE) was cloned upstream of the sFlk-1 gene (10 x HRE/sFlk-1). A novel HSV amplicon expressing 10 x HRE/sFlk-1 was genetically engineered (HSV10 x HRE/sFlk-1).Human pancreatic adenocarcinoma cells (AsPC1) were transduced with HSV10 x HRE/sFlk-1 and incubated in normoxia (21% oxygen) or hypoxia (1% oxygen). Capillary inhibition was evaluated by human umbilical vein endothelial cell assay. Western blot assessed sFlk-1 expression. AsPC1 flank tumor xenografts (n = 24) were transduced with HSV10 x HRE/sFlk-1. RESULTS Media from normoxic AsPC1 transduced with HSV10 x HRE/sFlk-1 yielded a 36% reduction in capillary formation versus controls (P < .05), whereas hypoxic AsPC1 yielded a 76% reduction (P < .005). Western blot of AsPC1 transduced with HSV10 x HRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia versus normoxia. AsPC1 flank tumors treated with HSV10 x HRE/sFlk-1 exhibited a 59% reduction in volume versus controls (P < .000001). CONCLUSIONS HSV amplicon delivery of a hypoxia-inducible soluble VEGF receptor significantly reduces new vessel formation and tumor growth. Tumor hypoxia can thus be used to direct antiangiogenic therapy to pancreatic adenocarcinoma.
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Affiliation(s)
- Maura Reinblatt
- Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
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Rüegg C, Hasmim M, Lejeune FJ, Alghisi GC. Antiangiogenic peptides and proteins: from experimental tools to clinical drugs. Biochim Biophys Acta Rev Cancer 2005; 1765:155-77. [PMID: 16263219 DOI: 10.1016/j.bbcan.2005.09.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2005] [Revised: 09/18/2005] [Accepted: 09/20/2005] [Indexed: 12/27/2022]
Abstract
The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.
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Affiliation(s)
- Curzio Rüegg
- Centre Pluridisciplinaire d'Oncologie, Faculty of Biology and Medicine, University of Lausanne, Epalinges s/Lausanne, Switzerland.
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Zhang Z, Zou W, Wang J, Gu J, Dang Y, Li B, Zhao L, Qian C, Qian Q, Liu X. Suppression of tumor growth by oncolytic adenovirus-mediated delivery of an antiangiogenic gene, soluble Flt-1. Mol Ther 2005; 11:553-62. [PMID: 15771958 DOI: 10.1016/j.ymthe.2004.12.015] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2004] [Accepted: 12/24/2004] [Indexed: 12/14/2022] Open
Abstract
Armed oncolytic adenoviruses represent an appealing tumor treatment approach, as they can attack tumors at multiple levels. In this study, considering that angiogenesis plays a central role in tumor growth, we inserted an antiangiogenic gene, sflt-1(1-3) (the first three extracellular domains of FLT1, the hVEGF receptor-1), into an E1B-55-kDa-deleted oncolytic adenovirus (ZD55) to construct ZD55-sflt-1. Although soluble (s) Flt-1 did not affect tumor cell growth, ZD55-sflt-1 could specifically induce a cytopathic effect in tumor cells, like ONYX-015. The secretion of sFlt-1 from ZD55-sflt-1 was much higher than that from replication-deficient Ad-sflt-1 upon infection of SW620 human colon tumor cells, leading to a stronger inhibitory effect on VEGF-induced proliferation and tube formation ability of HUVECs. Moreover, marked reduction of tumor growth and long-term survival rates were observed in ZD55-sflt-1-treated nude mice with subcutaneous SW620 tumor. Its efficacy correlated with a decrease in microvessel density and an increase in apoptotic tumor cells. In addition, ZD55-sflt-1 showed a synergic effect with the chemotherapeutic agent 5-FU. These results indicate that ZD55-sflt-1, combining the advantages of oncolytic adenovirus and antiangiogenic gene therapy, is a powerful agent for human tumor treatment.
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Affiliation(s)
- Zilai Zhang
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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Schmitz V, Kornek M, Hilbert T, Dzienisowicz C, Raskopf E, Rabe C, Sauerbruch T, Qian C, Caselmann WH. Treatment of metastatic colorectal carcinomas by systemic inhibition of vascular endothelial growth factor signaling in mice. World J Gastroenterol 2005; 11:4332-6. [PMID: 16038030 PMCID: PMC4434658 DOI: 10.3748/wjg.v11.i28.4332] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Tumor angiogenesis has been shown to be promoted by vascular endothelial growth factor (VEGF) via stimulating endothelial cell proliferation, migration, and survival. Blockade of VEGF signaling by different means has been demonstrated to result in reduced tumor growth and suppression of tumor angiogenesis in distinct tumor entities. Here, we tested a recombinant adenovirus, AdsFlt1-3, that encodes an antagonistically acting fragment of the VEGF receptor 1 (Flt-1), for systemic antitumor effects in pre-established subcutaneous CRC tumors in mice.
METHODS: Murine colorectal carcinoma cells (CT26) were inoculated subcutaneously into Balb/c mice for in vivo studies. Tumor size and survival were determined. 293 cell line was used for propagation of the adenoviral vectors. Human lung cancer line A549 and human umbilical vein endothelial cells were transfected for in vitro experiments.
RESULTS: Infection of tumor cells with AdsFlt1-3 resulted in protein secretion into cell supernatant, demonstrating correct vector function. As expected, the secreted sFlt1-3 protein had no direct effect on CT26 tumor cell proliferation in vitro, but endothelial cell function was inhibited by about 46% as compared to the AdLacZ control in a tube formation assay. When AdsFlt1-3 (5×109 PFU/animal) was applied to tumor bearing mice, we found a tumor inhibition by 72% at d 12 after treatment initiation. In spite of these antitumoral effects, the survival time was not improved. According to reduced intratumoral microvessel density in AdsFlt1-3-treated mice, the antitumor mechanism can be attributed to angiostatic vector effects. We did not detect increased systemic VEGF levels after AdsFlt1-3 treatment and liver toxicity was low as judged by serum alanine aminotransferase determination.
CONCLUSION: In this study we confirmed the value of a systemic administration of AdsFlt1-3 to block VEGF signaling as antitumor therapy in an experimental metastatic colorectal carcinoma model in mice.
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Affiliation(s)
- Volker Schmitz
- Medizinische Klinik I, Sigmund-Freud-Str. 25, Bonn 53105, Germany.
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Ramsden JD, Buchanan MA, Egginton S, Watkinson JC, Mautner V, Eggo MC. Complete inhibition of goiter in mice requires combined gene therapy modification of angiopoietin, vascular endothelial growth factor, and fibroblast growth factor signaling. Endocrinology 2005; 146:2895-902. [PMID: 15817662 DOI: 10.1210/en.2005-0168] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
In goiter, increased expression of growth factors and their receptors occurs. We have inhibited the action of some of these growth factors, alone and in combination, to determine which are important in goitrogenesis. Recombinant adenovirus vectors (RAds) expressing truncated, secreted forms of human Tie2 (RAd-sTie2) and vascular endothelial growth factor receptor 1 (RAd-sVEGFR1) or a truncated, dominant-negative fibroblast growth factor receptor 1 (RAdDN-FGFR1) were used. Goiters in mice were induced by feeding an iodide-deficient diet, containing methimazole and sodium perchlorate. RAds were administered to mice simultaneously with the goitrogenic regimen, which was continued for 14 d. RAd treatment did not significantly affect increases in TSH or reductions in thyroid hormone or thyroid hyperactivity seen in goitrogen-treated controls mice, suggesting no effect on pituitary or thyroid responses to hypothyroidism. In control goiters, a 4-fold increase in vascular volume accompanied a 2-fold increase in thyroid mass. Complete inhibition of these increases was found when animals were treated with the three RAds in combination. In thyroids from three RAd-treated animals, there was marked, significant inhibition of Tie2, FGFR1, VEGFR1, FGF-2, and VEGF expression, compared with control goiters. When used individually, RAdDN-FGFR1 partially prevented goiter and RAd-sVEGFR1 partially reduced vascular volume. Their effects were not additive. RAd-sTie2 did not reduce goiter mass or vascular volume when used alone but was essential for complete goiter inhibition. VEGF and VEGFR1 expression was reduced in these thyroids. Limitation of physiologic organ growth is complex, requiring inhibition of multiple, interdependent growth factor axes.
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Affiliation(s)
- James D Ramsden
- Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, UK
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De Nicola H, Szejnfeld J, Logullo AF, Wolosker AMB, Souza LRMF, Chiferi V. Flow pattern and vascular resistive index as predictors of malignancy risk in thyroid follicular neoplasms. JOURNAL OF ULTRASOUND IN MEDICINE : OFFICIAL JOURNAL OF THE AMERICAN INSTITUTE OF ULTRASOUND IN MEDICINE 2005; 24:897-904. [PMID: 15972703 DOI: 10.7863/jum.2005.24.7.897] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
OBJECTIVES The purpose of this study was to evaluate whether flow pattern and resistive index (RI) are useful parameters for distinguishing benign from malignant thyroid follicular neoplasms (FNs). METHODS Eighty-six thyroid nodules that underwent sonographically guided fine-needle aspiration and were diagnosed as cases of FN were evaluated by power and duplex Doppler sonography. Pathologic correlation was available for all nodules. The flow pattern seen via power Doppler examination was ranked for each nodule on a scale of 0 to 4, in increasing flow order. For each nodule, the RI value was considered the average of 1 to 3 values obtained with different flow signals. RESULTS Ten nodules (11.63%) were malignant (3 follicular carcinomas, 5 follicular variants of papillary carcinoma, and 2 papillary carcinomas). Fourteen nodules (16.27%) were adenomas, and 62 (72%) were non-neoplastic nodules. The average RI in non-neoplastic nodules was 0.588 (P < .001, chi(2) test): 0.662 in adenomas and 0.763 in malignant nodules. None of the nodules had flow pattern type 0. Flow patterns 1 and 2 (peripheral flow only or predominantly) were present in 58 non-neoplastic nodules (93.5%), 10 adenomas (71.4%), and 2 malignant nodules (20%). Flow pattern type 3 (predominantly central flow) was present in 7 malignant nodules (70%), 4 adenomas (28.6%), and 4 non-neoplastic nodules (6.5%). Only 1 nodule, a papillary carcinoma, had flow pattern type 4 (internal flow only). CONCLUSIONS In FNs, there were significant positive associations between predominantly central flow and malignancy and between predominantly peripheral flow and benign disease (P < .0001, Fisher exact test). However, power Doppler characteristics could not be used to rule out malignancy because 20% of malignant nodules had predominantly peripheral flow. For predicting malignancy, an RI cutoff of 0.75 had good accuracy, specificity, and negative predictive value but had low sensitivity and positive predictive value (respectively, 91%, 97%, 92%, 40%, and 67%). Resistive index values in non-neoplastic nodules were lower than in adenomas and malignant nodules (P < .001, chi(2) test).
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Affiliation(s)
- Harley De Nicola
- Diagnostic Imaging Department, Federal University of São Paulo-Escola Paulista de Medicina, São Paulo, Brazil
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Younes MN, Yigitbasi OG, Park YW, Kim SJ, Jasser SA, Hawthorne VS, Yazici YD, Mandal M, Bekele BN, Bucana CD, Fidler IJ, Myers JN. Antivascular Therapy of Human Follicular Thyroid Cancer Experimental Bone Metastasis by Blockade of Epidermal Growth Factor Receptor and Vascular Growth Factor Receptor Phosphorylation. Cancer Res 2005; 65:4716-27. [PMID: 15930290 DOI: 10.1158/0008-5472.can-04-4196] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Patients suffering from bone metastases of follicular thyroid carcinoma (FTC) have a poor prognosis because of the lack of effective treatment strategies. The overexpression of epidermal growth factor receptor (EGFR) associated with increased vascularity has been implicated in the pathogenesis of FTC and subsequent bone metastases. We hypothesized that inhibiting the phosphorylation of the EGFR and vascular endothelial growth factor receptor (VEGFR) by AEE788, a dual tyrosine kinase inhibitor of EGFR and VEGFR, in combination with paclitaxel would inhibit experimental FTC bone lesions and preserve bone structure. We tested this hypothesis using the human WRO FTC cell line. In culture, AEE788 inhibited the EGF-mediated phosphorylation of EGFR, VEGFR2, mitogen-activated protein kinase, and Akt in culture. AEE788, alone and in combination with paclitaxel, inhibited cell growth and induced apoptosis. When WRO cells were injected into the tibia of nude mice, tumor and endothelial cells within the lesions expressed phosphorylated EGFR, VEGFR, Akt, and mitogen-activated protein kinase that were inhibited by the oral administration of AEE788. Therapy consisting of orally given AEE788 and i.p. injected paclitaxel induced a high level of apoptosis in tumor-associated endothelial cells and tumor cells with the inhibition of tumor growth in the bone and the preservation of bone structure. Collectively, these data show that blocking the phosphorylation of EGFR and VEGFR with AEE788 combined with paclitaxel can significantly inhibit experimental human FTC in the bone of nude mice.
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Affiliation(s)
- Maher Nabil Younes
- Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030-4009, USA
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Abstract
CONTEXT Angiogenesis has been recognized as an important process contributing to the pathophysiology of many benign and malignant diseases. It is not surprising, therefore, that this complex process is proving to be an important regulator of both benign and malignant disease processes in the thyroid gland. This paper will review the general principles of angiogenesis and lymphangiogenesis, as well as the importance of the balance between angiogenic stimulators and inhibitors in the normal thyroid gland. We will also review how this balance is disturbed in benign and malignant thyroid conditions. Finally, we will address the role manipulation of this process may play in the development of novel treatment strategies for diseases of the thyroid. OBJECTIVE To review the literature concerning the role of angiogenesis in the thyroid gland. CONCLUSIONS Angiogenesis is an important process which has been shown to be involved in the pathophysiology of benign and malignant diseases of the thyroid gland. Manipulation of this process holds great promise for the development of novel treatments for these disorders. As the mechanisms regulating angiogenesis in the thyroid become increasingly clear, researchers will come ever closer to turning this promise into clinical reality.
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Affiliation(s)
- Jamie C Mitchell
- Department of Surgery, Section of Endocrine Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA
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Hebert C, Siavash H, Norris K, Nikitakis NG, Sauk JJ. Endostatin inhibits nitric oxide and diminishes VEGF and collagen XVIII in squamous carcinoma cells. Int J Cancer 2005; 114:195-201. [PMID: 15540202 DOI: 10.1002/ijc.20692] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Low pO(2) values are a common finding among oral squamous cell carcinomas (SCC). Our objective was to determine the role that oxygen tension plays on the direct tumor effect of endostatin (ES). Squamous carcinoma cell lines were grown under normoxic or hypoxic conditions and treated with endostatin (ES), nitric oxide (NO) donors, NO scavengers, NO synthase inhibitors, or transduced with AdenoVec-hEndo or AdenoVec Null vectors. The expression of vascular endothelial growth factor (VEGF) and collagen XVIII were determined by RT-PCR and protein levels assessed by Western blot analyses. Our studies demonstrated that collagen XVIII and VEGF are expressed and responsive to ES in a limited number of SCC cell lines during normoxia but were most responsive when grown under hypoxic conditions. VEGF and collagen XVIII were downregulated by both ES and transduction of cells with AdenoVec-hEndo. The effects of ES on SCC cells were enhanced by aminoguanidine (Ag), L-NAME, and diphenyleneiodonium chloride (DPI). Endostatin and transduced with ES vectors diminished the levels of NO whereas NO donors enhanced VEGF expression and collagen XVIII expression. In conclusion, the direct effect of endostatins on tumor cells is most effective under conditions of low oxygen tension and can be potentiated by the use of nitric oxide synthase inhibitors or NO scavengers.
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Affiliation(s)
- Carla Hebert
- Department of Diagnostic Sciences and Pathology, University of Maryland-Baltimore, 666 W. Baltimore Street, Baltimore, MD 21201, USA
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Schmidt K, Hoffend J, Altmann A, Strauss LG, Dimitrakopoulou-Strauss A, Engelhardt B, Koczan D, Peter J, Vorwald S, Eskerski H, Eisenhut M, Metz J, Kinscherf R, Haberkorn U. Transfer of thesFLT-1Gene in Morris Hepatoma Results in Decreased Growth and Perfusion and Induction of Genes Associated with Stress Response. Clin Cancer Res 2005; 11:2132-40. [PMID: 15788658 DOI: 10.1158/1078-0432.ccr-04-2133] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Therefore, monitoring of antiangiogenic approaches with functional imaging and histomorphometrical analyses are desirable to evaluate the biological effects caused by this treatment modality. EXPERIMENTAL DESIGN Using a bicistronic retroviral vector for transfer of the soluble receptor for the vascular endothelial growth factor (sFLT) hepatoma (MH3924A) cell lines with sFLT expression were generated. In human umbilical vein endothelial cells cultured with conditioned medium of sFLT-expressing hepatoma cells, the inhibitory action of secreted sFLT was determined using a Coulter counter and a thymidine incorporation assay. Furthermore, in vivo experiments were done to measure the effects on tumor growth and perfusion. Finally, the tumors were examined by immunohistochemistry (including computer-assisted morphometry) and DNA chip analysis. RESULTS Stable sFLT-expressing hepatoma cells inhibited endothelial cell proliferation in vitro. In vivo, growth and perfusion, as measured by H(2)(15)O positron emission tomography, were reduced in genetically modified tumors. However, the immunohistochemically quantified microvascularization and macrovascularization, as indicated by CD31- and alpha-actin-positive area, revealed no significant changes, whereas the number of apoptotic cells was increased in sFLT-expressing tumors, although not significantly. DNA chip analysis of tumors with gene transfer showed an increase of genes related to apoptosis, signal transduction, and oxidative stress. CONCLUSION Our results suggest that sFLT expression inhibits tumor growth and perfusion and enhances expression of apoptosis-related genes in this model. Enhanced expression of genes for signal transduction, stress, and metabolism indicates tumor defense reactions.
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Affiliation(s)
- Kerstin Schmidt
- Department of Anatomy and Cell Biology III, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany
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Abstract
Thyroid carcinomas are suitable targets for gene therapy because they can be highly lethal on one hand, while being susceptible to specific tumour targeting on the other hand. Several gene therapy modalities have been evaluated so far in experimental models of thyroid cancer, including tumour suppressor gene replacement, oncogene inhibition, suicide gene therapy, immunotherapy, antiangiogenesis, and viral oncolysis. All of these strategies have shown promising results, but clinical studies are lacking. Based on the clinical experience achieved in a pilot study in patients with advanced thyroid cancer and on clinical results in other types of solid cancer, it is suggested that combined gene therapy approaches, as well as multimodality therapeutic regimens, including gene therapy and conventional treatments, should be pursued to achieve clinically significant results.
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Affiliation(s)
- Luisa Barzon
- Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via Gabelli 63, I-35121 Padova, Italy.
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