• The Korean Journal of Physiology and Pharmacology
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• v.19 no.4
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• pp.335-340
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• 2015

Here, we investigated the role of zerumbone, a natural cyclic sesquiterpene of Zingiber zerumbet Smith, on angiogenesis using human umbilical vein endothelial cells (HUVECs). Zerumbone inhibited HUVECs proliferation, migration and tubule formation, as well as angiogenic activity by rat aorta explants. In particular, zerumbone inhibited phosphorylation of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1, which are key regulators of endothelial cell function and angiogenesis. In vivo matrigel plug assay in mice demonstrated significant decrease in vascularization and hemoglobin content in the plugs from zerumbone-treated mice, compared with control mice. Overall, these results suggest that zerumbone inhibits various attributes of angiogenesis, which might contribute to its reported antitumor effects.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.3
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• pp.193-201
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• 2006

Squamous cell carcinoma(SCC) of head and neck(SCCHN) is the sixth most common human malignant tumor. However, despite advances in prevention and treatment of SCC, the five-year survival rates for patients remain still low. To improve the outcome for patients with SCCHN, novel treatment strategies are needed. Overexpression of the epidermal growth factor(EGF) and activation of its receptor(EGFR) are associated with progressive growth of SCCHN. Vascular endothelial growth factor(VEGF) signaling molecules are related with neoangiogenesis and vascular metastasis of SCC. In this study, we determined the therapeutic effect of AEE788(Novartis Pharma AG, Basel, Switzerland), which is a dual inhibitor of EGFR/ErbB2 and VEGFR tyrosine kinases, on human oral SCC. At first, we screened the expression of EGFR, c-ErbB2(HER-2) and VEGFR-2 in a series of human oral SCC cell lines. And then we evaluated the effects of AEE788 on the phosphorylation of EGFR and VEGFR-2 in a oral SCC cell line expressing EGFR/HER-2 and VEGFR-2. We also evaluated the effects of AEE788 alone, or with paclitaxel(Taxol) on the oral SCC cell growth and apoptosis. As a result, all oral SCC cells expressed EGFR and VEGFR-2. Treatment of oral SCC cells with AEE788 led to dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation, growth inhibition, and induction of apoptosis. Moreover, AEE788 sensitizes the cells to paclitaxel-mediated toxicity and apoptosis. These data mean EGFR and VEGFR-2 can be reliable targets for molecular therapy of oral SCC, and therefore warrant clinical use of EGFR/VEGFR inhibition in the treatment of patients with recurrent or metastatic oral SCC.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.2
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• pp.66-73
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• 2009

Tumor angiogenesis is a process leading to formation of blood vessels within tumors and is crucial for maintaining a supply of oxygen and nutrients to support tumor growth and metastasis. Vascular endothelial growth factor(VEGF) plays a key role in tumor angiogenesis including induction of endothelial cell proliferation, migration, survival and capillary tube formation. VEGF binds to two distinct receptors on endothelial cells. VEGFR-2 is considered to be the dominant signaling receptor for endothelial cell permeability, proliferation, and differentiation. Bevacizumab(Avastin, Genetech, USA) is a monoclonal antibody against vascular endothelial growth factor. It is used in the treatment of cancer, where it inhibits tumor growth by blocking the formation of new blood vessels. The goal of this study is to identify the anti-tumor effect of Bevacizumab(Avastin) for oral squamous cell carcinoma cell lines. Human squamous cell carcinoma cell line(HN4) was used in this study. We examined the sensitivity of HN4 cell line to Bevacizumab(Avastin) by using in vitro proliferation assays. The results were as follows. 1. In the result of MTT assay according to concentration of Bevacizumab(Avastin), antiproliferative effect for oral squamous cell carcinoma cell lines was observed. 2. The growth curve of cell line showed the gradual growth inhibition of oral squamous cell carcinoma cell lines after exposure of Bevacizumab(Avastin). 3. In the apoptotic index, groups inoculated Bevacizumab(Avastin) were higher than control groups. 4. In condition of serum starvation, VEGFR-2 did not show any detectable autophosphorylation, whereas the addition of VEGF activated the receptor. Suppression of phosphorylated VEGFR-2 and phosphorylated MAPK was observed following treatment with Bevacizumab(Avastin) in a dose-dependent manner. 5. In TEM view, dispersed nuclear membrane, scattered many cytoplasmic vacuoles and localized chromosomal margination after Bevacizumab(Avastin) treatment were observed. These findings suggest that Bevacizumab(Avastin) has the potential to inhibit MAPK pathway in proliferation of oral squamous cell carcinoma cell lines via inhibition of VEGF-dependent tumor growth.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.2
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• pp.110-122
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• 2005

Adenoid cystic carcinoma (ACC) of salivary glands has a protracted clinical course with perineural invasion, delayed onset of hematogenous metastasis, and poor responses to classical cytotoxic chemotherapic agents. Most deaths from salivary ACC are caused by lung metastases that are resistant to conventional therapy. Therefore, knowledge of cellular properties and tumor-host interactions that influence the dissemination of metastatic cells is important for the design of more effective therapy of salivary cancer. I determined in vitro expression of epidermal growth factor receptor (EGFR) and its downstream effectors and vascular endothelial growth factor receptor (VEGFR)-2 on a human salivary ACC cell line (ACC2). I also evaluated the expression of EGF and VEGF signaling molecules and metastasis-related proteins on human salivary ACC cells orthotopically growing in nude mice. In Western blot and immunohistochemical analyses, EGFR and VEGFR-2 were presented and phosphorylated in ACC2 cells. In human parotid cancer xenografts in nude mice, EGF and VEGF signaling molecules, IL-8, and MMP-9 were expressed at markedly higher levels than in normal parotid tissues. Moreover, tumor-associated endothelial cells of this orthotopic parotid tumor expressed phosphorylated VEGFR-2 and phosphorylated Akt, which is a cell-survival protein. These data show that those biomarkers can be molecular targets for therapy of salivary ACC, which has a propensity for delayed lung metastasis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.2
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• pp.55-65
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• 2009

Purpose: We determined the therapeutic effects of blockade of epidermal growth factor(EGF) and vascular endothelial growth factor(VEGF) receptor tyrosine kinases on the growth of oral squamous cell carcinoma(OSCC) xenografted in athymic nude mice. Experimental Design: We investigated the in vivo antitumor effects of a tyrosine kinase inhibitor for EGFR and VEGFR-2, AEE788 in a mouth floor(orthotopic) tumor model. Nude mice with orthotopic tumors were randomized to receive AEE788, paclitaxel, a combination of AEE788 and paclitaxel, or control. Antitumor mechanisms of AEE788 were determined by immunohistochemical/immunofluorescent and apoptosis assays. Results: Tumors of mice treated with AEE788 demonstrated down-regulation of phosphorylated EGFR, phosphorylated VEGFR and their downstream mediators(pMAPK and pAkt), decreased proliferative index, decreased microvessel density(MVD). As a result, growth of the primary tumor and nodal metastatic potentials were inhibited by AEE788. Conclusion: These data show that EGFR and VEGFR can be molecular targets for the treatment of OSCC.

• Archives of Plastic Surgery
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• v.37 no.1
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• pp.7-11
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• 2010

Purpose: Vascular endothelial growth factor (VEGF) is known as a growth factor of endothelium and fibroblast. The purpose is to know the VEGF effects on fibroblast proliferation and fibroblast's notch receptor expression. Methods: CCD-986sk fibroblast was purchased from the Korean Cell Bank and was used in XTT assay for proliferation and wound healing assay for migration. Immunofluorescent (IF) staining and western blotting were used in testing notch expression of fibroblast. Semiquantitative RT-PCR was used in checking notch 1 mRNA production by fibroblast. Student-t test was used for analyzing results. Results: Cell proliferation assay using XTT showed significant higher proliferation in VEGF treated fibroblast, $2.324{\pm}0.0026$ vs. $2.463{\pm}0.017$ (p=0.002). Wound healing assay showed longer migration in VEGF treated fibroblast (p=0.062). The fluorescence was brighter in VEGF treated cells of notch 1 IF staining. Notch 1 expressions and mRNA productions increased more in VEGF treated cells. Conclusion: VEGF stimulates fibroblast to proliferate, migrate and to express Notch 1 simultaneously. Notch receptor could be related to VEGF mediated wound healing.

• BMB Reports
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• v.51 no.2
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• pp.73-78
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• 2018

Vascular endothelial growth factor and its receptor (VEGF-VEGFR) system play a critical role in the regulation of angiogenesis and lymphangiogenesis in vertebrates. Each of the VEGF has specific receptors, which it activates by binding to the extracellular domain of the receptors, and, thus, regulates the angiogenic balance in the early embryonic and adult stages. However, de-regulation of the VEGF-VEGFR implicates directly in various diseases, particularly cancer. Moreover, tumor growth needs a dedicated blood supply to provide oxygen and other essential nutrients. Tumor metastasis requires blood vessels to carry tumors to distant sites, where they can implant and begin the growth of secondary tumors. Thus, investigation of signaling systems related to the human disease, such as VEGF-VEGFR, will facilitate the development of treatments for such illnesses.

• International Journal of Oncology
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• v.54 no.2
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• pp.753-763
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• 2019

The mutation of isocitrate dehydrogenase (IDH)1 (R132H) and IDH2 (R172K) and the induction of hypoxia in various solid tumors results in alterations in metabolic profiles, including the production of the d- or l-forms of 2-hydroxyglutarate (2HG) from α-ketoglutarate in aerobic metabolism in the tricarboxylic acid (TCA) cycle. However, it is unclear whether the oncometabolite d-2HG increases angiogenesis in endothelial cells. Therefore, in this study, we analyzed the levels of various metabolites, including d-2HG, under hypoxic conditions and in IDH2R172K mutant breast cancer cells by mass spectrometry. We then further evaluated the effects of this metabolite on angiogenesis in breast cancer cells. The results revealed that treatment with d-2HG increased the levels of secreted vascular endothelial growth factor (VEGF) in cancer cells and enhanced endothelial cell proliferation in a concentration-dependent manner. Wound healing and cell migration (examined by Transwell assay) were significantly increased by d-2HG to a level similar to that induced by VEGF. Tube formation was significantly stimulated by d-2HG, and chick chorioallantoic membrane angiogenesis was also enhanced by d-2HG. d-2HG activated VEGF receptor (VEGFR)2 and VEGFR2 downstream signaling, extracellular signal-regulated kinase 1/2, focal adhesion kinase, AKT and matrix metalloproteinase (MMP)2. Taken together, the findings of this study suggested that d-2HG induced angiogenic activity via VEGFR2 signaling and increased MMP2 activity.

• BMB Reports
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• v.39 no.5
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• pp.469-478
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• 2006

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-$C{\gamma}$-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.

• Molecules and Cells
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• v.41 no.8
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• pp.771-780
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• 2018

Angiogenesis must be precisely controlled because uncontrolled angiogenesis is involved in aggravation of disease symptoms. Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) signaling is a key pathway leading to angiogenic responses in vascular endothelial cells (ECs). Therefore, targeting VEGF/VEGFR-2 signaling may be effective at modulating angiogenesis to alleviate various disease symptoms. Oleanolic acid was verified as a VEGFR-2 binding chemical from anticancer herbs with similar binding affinity as a reference drug in the Protein Data Bank (PDB) entry 3CJG of model A coordination. Oleanolic acid effectively inhibited VEGF-induced VEGFR-2 activation and angiogenesis in HUVECs without cytotoxicity. We also verified that oleanolic acid inhibits in vivo angiogenesis during the development and the course of the retinopathy of prematurity (ROP) model in the mouse retina. Taken together, our results suggest a potential therapeutic benefit of oleanolic acid for inhibiting angiogenesis in proangiogenic diseases, including retinopathy.