• Biomolecules & Therapeutics
• /
• v.27 no.1
• /
• pp.117-125
• /
• 2019

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.29 no.6
• /
• pp.499-508
• /
• 2007

Background and Purpose: Some subtypes of malignant salivary gland tumors such as adenoid cystic carcinoma (ACC) frequently result in distant metastasis of vascular origin, which are main causes of treatment failure. The reasons for the affinity for vascular metastatic potential are unclear. Therefore, molecular characteristics that influence the dissemination of metastatic tumor cells are important for the design of more effective treatment of salivary ACC. Tumor angiogenesis has been known to be essential for the distant metastasis of malignant cells. So, we determined expressions of vascular metastasis related factors in orthotopic (parotid) murine models of parotid ACC and compared with those in ectopic (subcutis) tumors of athymic mice. Experimental Design: Using specimens from murine parotid (orthotopic, experimental group) and subcutaneous (ectopic, control group) tumors, which have developed via transplantation of tumor cells, originated from human parotid ACC, we performed immunohistochemical assays with anti-vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF, FGF2), matrix metalloproteinase (MMP)-9, and interleukin (IL)-8 antibodies. We also performed immunohistochemical assays with VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and phosphorylated VEGFR-2. Results: Transplantation of human ACC tumor cell $(5{\times}10^5)$ into the parotid and subcutis successfully resulted in orthotopic (parotid) and ectopic (subcutaneous) tumors in athymic mice. Immunohistochemical staining demonstrated higher expression of major angiogenic factors (VEGF, bFGF, MMP-9) in the orthotopic tumors than in ectopic tumors (P<0.05). But the expression level of angiogenic receptors were same in orthotopic and ectopic tumors of parotid ACC. Conclusion: VEGF, bFGF, and MMP-9 could be a good candidates for antiangiogenic therapy for the contol of vascular metastatic lesions of salivary ACC.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.5
• /
• pp.459-466
• /
• 2016

Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than $Lnk^{-/-}$ MSCs. An ex vivo adipogenic differentiation assay showed that $Lnk^{-/-}$ MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) and its adipogenic target genes (LPL and FABP4) significantly decreased in $Lnk^{-/-}$ MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the $IGF-1/Akt/PPAR-{\gamma}$ pathway.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.3
• /
• pp.1187-1192
• /
• 2014

Background: To correlate breast cancer subtypes with prognostic factors, microvessel density (MVD), vascular endothelial growth factor (VEGF) expression and clinical features. Materials and Methods: One hundred cases of primary breast carcinoma were classified using biomarkers on tissue microarray as: luminal A [estrogen receptor (ER)+, HER2-, $Ki-67{\leq}14%$], luminal B [ER+, HER2+ or ER+, HER2-, Ki-67>14%], HER2, triple negative basal-like (TNB) [any basal cytokeratins (CKs, 5, 14, 17) and/or endothelial growth factor receptor (EGFR) expression], and TN without such markers [TNN, null], and assessed for p53, vimentin, VEGF and CD31 immunoperoxidase. Results: Of the 100 cases (mean age, 51 years; mean tumor size, 3.2cm; 56% with nodal metastasis; 89 invasive ductal carcinomas, not otherwise specified, 4 invasive lobular carcinomas, 3 metaplastic carcinomas, and 4 other types) there were 39 luminal A, 18 luminal B, 18 HER2, 15 TNB and 10 TNN. The positivities of basal-like markers in the basal-like subtype were 78.3% for CK5, 40% for CK14, 20% for CK17, 46.7% for EGFR. There was no significant difference in age distribution, tumor size, degree of tubular formation, pleomorphism, lymphovascular invasion, nodal metastasis, MVD, VEGF expression and survival among subgroups. TNs demonstrated significantly higher tumor grade, mitotic count, Ki-67 index, p53 and vimentin and decreased overall survival compared with nonTN. Conclusions: The distribution of breast cancer subtypes in this study was similar to other Asian countries with a high prevalence of TN. The high grade character of TN was confirmed and CK5 expression was found to be common in our basal-like subtype. No significant elevation of MVD or VEGF expression was apparent.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.33 no.1
• /
• pp.11-19
• /
• 2007

This study was to evaluate the expression of vascular endothelial growth factor receptors (VEGFRs) in tumor and stromal cells of tougue squamous cell carcinoma (SCC). We also wanted to characterize the differences, from the angiogenic aspect, between cancer-associated stromal cells and non-malignant stromal cells. Paraffin-embedded tumor specimens from eleven patients with tongue SCCs were studied. Immunohistochemical staining for VEGFR-1,-2, and -3 was performed on the tumor cells, stromal fibroblasts and tumor-associated macrophages of the specimens. The expression of all 3 receptors was detected in the tumor cells themselves of the biopsy specimens. All 3 receptors were also expressed on stromal cells, except that VEGFR-2 was not expressed in stromal fibroblasts. In radical excision specimens, the staining intensity for VEGFR-1, -2 in the tumor cells and VEGFR-1,-3 in the tumor-associated macrophages was significantly lower than that in the biopsy specimens (P < 0.05). By using the general marker of fibroblast and macrophage, 5B5 and CD68, respectively, we performed double immunofluorescence staining for 5B5 and each VEGFR in the stromal fibroblasts and for CD68 and each VEGFR in the tumor-associated macrophages of the radical excision specimens. We used 4 cases of fibroma and 4 cases of chronic inflammation tissue as the controls. It was found that only each marker was expressed in the control group, however, 5B5/VEGFR-1 and 5B5/VEGFR-3 in the stromal fibroblasts, and CD68/VEGFR-1 and CD68/VEGFR-3 in the tumor-associated macrophages were double stained in the radical excision specimens. Although our study used small number of specimens, the results of our study showed that in tongue SCC, in association with the angiogenesis, the stromal cells showed the activated phenotype and this was different from the nonmalignant stromal cells.

• Molecules and Cells
• /
• v.38 no.6
• /
• pp.528-534
• /
• 2015

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-$1{\alpha}$ stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-$1{\alpha}$ in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-$1{\alpha}$, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-$1{\alpha}$ and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-$1{\alpha}$ stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.32 no.1
• /
• pp.1-8
• /
• 2010

Background and Purpose: Vascular endothelial growth factor (VEGF)-C and VEGF receptor (VEGFR)-3 are involved in tumor lymphangiogenesis. Oral mucosal squamous cell carcinoma (OMSCC) preferentially metastasizes to cervical lymph nodes, so we investigated the expression and distribution of VEGFR-3 signaling proteins in OMSCC. Materials and Methods: Tissue samples of 18 OMSCC, 10 oral mucosal leukoplakia, and 3 normal oral mucosa were evaluated for expression of VEGF-C, VEGF-D, and VEGFR-3 by immunohistochemical staining. The presence of lymphatic vessels was determined using D2-40 staining, by which we also measured lymphatic vessel density (LVD). Results: 72% (13/18) and 56% (10/18) of tissue samples showed VEGF-C and VEGF-D immunopositivity in tumor cells and tumor-associated endothelial cells. VEGFR-3 was also expressed in most of OMSCC, which was up-regulated when compared with normal mucosa or with leukoplakia. Furthermore, LVD was higher in OMSCC than in leukoplakia. Conclusion: Taken together, our results suggest that autocrine activation of lymphatic endothelial cell via VEGFR-3 by VEGF-C and/or VEGF-D could be involved in progression of OMSCC. Therefore, VEGF-C/VEGFR-3 signaling pathway can be a molecular target for anti-metastatic therapy in OMSCC.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.35 no.1
• /
• pp.1-12
• /
• 2013

Purpose: Vascular endothelial growth factor (VEGF)-C and its tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. In this study, we determined whether the expression of lymphangiogenic factors correlate with nodal metastasis or survival in a nude mouse model of oral squamous cell carcinoma (OSCC). Methods: Three OSCC cells (KB, SCC4, SCC9) were xenografted into the right mandibular gland of athymic nude mice. The mice were followed for tumor development and growth, and the mice were sacrificed when they had lost more than 20% of their initial body weight, or the diameter of the induced tumor exceeds 20 mm. After necropsy, the murine tumors were examined histologically and radiologically (micro-positron emission tomography computed tomography) for regional or distant metastasis. We performed immunohistochemical assays with anti-VEGF-C, VEGFR-3, CD105, and D2-40 antibodies. Immunofluorescence double staining for LYVE-1/CD31 was also performed. To quantify the VEGF-C and VEGFR-3 level in the cancer tissue, Western blotting was performed. Finally, we determined the correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time. Results: OSCC tumor cells into the mandibular gland of the nude mice successfully resulted in the formation of recapitulating orthotopic tumor. Tumor cells of the induced tumor did not express VEGF-C. VEGF-C/VEGFR-3 expression was mainly distributed in the endothelial cells of the stromal area. There were no correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time of mice injected with different OSCC cell lines. Conclusion: An recapitulating orthotopic model of OSCC in nude mice was established, which copies the cervical nodal metastasis of human OSCC. Overexpression of lymphangiogenic factors seems to have no effect on survival of hosts in this in vivo experiment.

• Biomedical Science Letters
• /
• v.20 no.4
• /
• pp.209-220
• /
• 2014

Vascular endothelial growth factor (VEGF) is a key factor involved in the induction of angiogenesis and has become an attractive target for anti-angiogenesis therapies. The purpose of this study was to elucidate the anti-angiogenic activity of Rubus coreanus Miquel water extract (RCME). Rubus coreanus Miquel has long been employed as a traditional medicine, and recent studies have demonstrated that it has measureable biological activities. Thus, we investigated for the first time the effect of RCME on angiogenesis and its underlying signaling pathways. The effects of RCME were tested on in vitro models of angiogenesis, namely, proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells as well as an ex vivo model of vessel sprouting from the rat aorta in response to VEGF. We observed that VEGF-induced angiogenesis was strongly suppressed by RCME treatment compared to that of the control group. Moreover, we found that RCME inhibited VEGF-induced activation of matrix metalloproteinases and phosphorylation of extracellular signal-regulated kinase and p38, and also effectively inhibited phosphorylation of VEGF receptor 2. These results indicated that RCME inhibits angiogenesis by suppressing phosphorylation of the VEGF receptor and may be useful for the treatment of angiogenesis-dependent diseases such as cancer and diabetic retinopathy.

• Biomolecules & Therapeutics
• /
• v.29 no.5
• /
• pp.506-518
• /
• 2021

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3'-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the three-dimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.