Synthesis and evaluation of nicotinamide derivative as anti-angiogenic agents

Previously, we have found that BRN-103, a nicotinamide derivative, inhibits vascular endothelial growth factor (VEGF)-mediated angiogenesis signaling in human endothelial cells. During our continuous efforts to identify more potent anti-angiogenic agents, we synthesized various nicotinamide derivatives and evaluated their anti-angiogenic effects. We found that 2-{1-[1-(6-chloro-5-fluoropyrimidin-4-yl)ethyl]piperidin-4-ylamino}-N-(3-chlorophenyl) pyridine-3-carboxamide (BRN-250) significantly inhibited human umbilical vascular endothelial cells (HUVECs) proliferation, migration, tube formation, and microvessel growth in a concentration range of 10–100 nM. Furthermore, BRN-250 inhibited the VEGF-induced phosphorylation and intracellular tyrosine kinase activity of VEGF receptor 2 (VEGFR2) and the activation of its downstream AKT pathway. Taken together, these findings suggest that BRN-250 be considered a potential lead compound for cancer therapy.     

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Plays a Key Role in Vasculogenic Mimicry Formation,Neovascularization and Tumor Initiation by Glioma Stem-like Cells

Human glioblastomas (GBM) are thought to be initiated by glioma stem-like cells (GSLCs). GSLCs also participate in tumor neovascularization by transdifferentiating into vascular endothelial cells. Here, we report a critical role of GSLCs in the formation of vasculogenic mimicry (VM), which defines channels lined by tumor cells to supply nutrients to early growing tumors and tumor initiation. GSLCs preferentially expressed vascular endothelial growth factor receptor-2 (VEGFR-2) that upon activation by VEGF, mediated chemotaxis, tubule formation and increased expression of critical VM markers by GSLCs. Knockdown of VEGFR-2 in GSLCs by shRNA markedly reduced their capacity of self-renewal, forming tubules, initiating xenograft tumors, promoting vascularization and the establishment of VM. Our study demonstrates VEGFR-2 as an essential molecule to sustain the “stemness” of GSLCs, their capacity to initiate tumor vasculature, and direct initiation of tumor.     

NSK-01105, a Novel Sorafenib Derivative,Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.     

A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2⁺ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals.     

Down‐regulating Myoferlin inhibits the vasculogenic mimicry of melanoma via decreasing MMP‐2 and inducing mesenchymal‐to‐epithelial transition

Vasculogenic mimicry (VM) constitutes a novel approach for tumour blood supply and contributes to tumour metastasis and poor prognosis in patients with melanoma. Myoferlin (MYOF), a type II membrane protein involved in membrane regeneration and repair, is elevated in several malignant tumours, especially in advanced melanomas. This study aims to investigate the role and mechanism of MYOF in the regulation of VM. VM structures were found in 14 of 52 tested melanoma samples, and high MYOF expression correlated with VM structures. According to Kaplan–Meier survival curves, VM channels and elevated MYOF expression both correlated with poor prognosis in melanoma patients. Down‐regulation of MYOF by siRNA severely impaired the capability of A375 cells to form VM structures in vitro. Further studies demonstrated MYOF knockdown inhibited cell migration and invasion, which is required for VM formation, via decreasing MMP‐2 expression as evidenced by Western blotting, RT‐RCP and ELISA results. SB‐3CT, a specific inhibitor of MMP‐2, showed similar inhibiting effects with siMYOF, further supporting that MYOF down‐regulation inhibits MMP‐2 expression to affect VM formation. Moreover, MYOF knockdown suppress VM formation by A375 cells by inducing mesenchymal‐to‐epithelial transition (MET). After down‐regulating MYOF, focal adhesions were enlarged and A375 cells developed into a clear epithelial morphology. Such cells acquired the expression of E‐cadherin at adherens junctions along with a loss of mesenchymal markers, such as Vimentin and Twist1. In conclusion, MYOF plays an important role in VM and knockdown of MYOF suppresses VM formation via decreasing MMP‐2 and inducing MET in A375 melanoma cells.     

Exploring stemness gene expression and vasculogenic mimicry capacity in well- and poorly-differentiated hepatocellular carcinoma cell lines

Vasculogenic mimicry (VM) is the phenomenon where cancer cells mimic endothelial cells by forming blood vessels. A stem cell-like phenotype has been proposed to be involved in this tumor plasticity. VM seems to correlate with metastasis rate, but there have been no reports on the effects of pro-metastatic and pro-angiogenic factors or hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on VM formation of hepatocellular carcinoma (HCC) cells. Here, we determine VM capacity and expression of stemness genes (Oct4, Sox2, Nanog and CD133) in well- and poorly-differentiated HCC cell lines. The poorly-differentiated cell line SK-Hep-1 with mesenchymal features (high invasiveness and expressing Vimentin, with no E-cadherin) could form VM in vitro, while the well-differentiated cell line HepG2 did not form VM. There was no correlation between expression of stemness genes and intrinsic VM capacity. However, HGF but not VEGF, could induce VM formation in HepG2, concomitant with epithelial-mesenchymal transition (EMT), de-differentiation and increased expression of stemness genes. Our results show that the role of stemness genes in VM capacity of HCC cells is likely to depend on differentiation status.     

Anti-angiogenic activity of tocotrienol

The anti-angiogenic property of vitamin E compounds, with particular emphasis on tocotrienol, has been investigated in vitro. Tocotrienol, but not tocopherol, inhibited both the proliferation and tube formation of bovine aortic endothelial cells, with delta-tocotrienol appearing the highest activity. Also, delta-tocotrienol reduced the vascular endothelial growth factor-stimulated tube formation by human umbilical vein endothelial cells. Our findings suggest that tocotrienol has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

Rictor regulates the vasculogenic mimicry of melanoma via the AKT‐MMP‐2/9 pathway

Vasculogenic mimicry (VM)‐positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin‐insensitive complex of mTOR (mTORC2), is up‐regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan–Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM‐2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down‐regulation. Western blotting assays indicated that down‐regulating Rictor significantly inhibited the phosphorylation of AKT at Ser⁴⁷³ and Thr³⁰⁸, which subsequently inhibited the expression and activity of downstream MMP‐2/9, as confirmed by real‐time PCR and gelatin Zymography. MK‐2206, a small‐molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP‐2/9, further supporting that Rictor down‐regulation inhibits the phosphorylation of AKT and activity of downstream MMP‐2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor—AKT—MMP‐2/9 signalling pathway.     

MMP9 Processing of HSPB1 Regulates Tumor Progression

Matrix metalloproteinases regulate pathophysiological events by processing matrix proteins and secreted proteins. Previously, we demonstrated that soluble heat shock protein B1 (HSPB1) is released primarily from endothelial cells (ECs) and regulates angiogenesis via direct interaction with vascular endothelial growth factor (VEGF). Here we report that MMP9 can cleave HSPB1 and release anti-angiogenic fragments, which play a key role in tumorprogression. We mapped the cleavage sites and explored their physiological relevance during these processing events. HSPB1 cleavage by MMP9 inhibited VEGF-induced ECs activation and the C-terminal HSPB1 fragment exhibited more interaction with VEGF than did full-length HSPB1. HSPB1 cleavage occurs during B16F10 lung progression in wild-type mice. Also, intact HSPB1 was more detected on tumor endothelium of MMP9 null mice than wild type mice. Finally, we confirmed that secretion of C-terminal HSPB1 fragment was significantly inhibited lung and liver tumor progression of B16F10 melanoma cells and lung tumor progression of CT26 colon carcinoma cells, compared to full-length HSPB1. These data suggest that in vivo MMP9-mediated processing of HSPB1 acts to regulate VEGF-induced ECs activation for tumor progression, releasing anti-angiogenic HSPB1 fragments. Moreover, these findings potentially explain an anti-target effect for the failure of MMP inhibitors in clinical trials, suggesting that MMP inhibitors may have pro-tumorigenic effects by reducing HSPB1 fragmentation.     

Anti-Cancer Activity of an Osthole Derivative,NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

Cyperenoic acid,a sesquiterpene derivative from Croton crassifolius,inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

BackgroundCyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated.PurposeTo investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis.Study designTargeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated.MethodsThe in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model.ResultsCyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2.ConlusionCyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.     

Vascular Mimicry: A Novel Neovascularization Mechanism Driving Anti-Angiogenic Therapy (AAT) Resistance in Glioblastoma

Glioblastoma (GBM) is a hypervascular neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT therapy resistance. Alternative neovascularization mechanisms were shown to be at work in these resilient tumors to counter the AAT therapy insult. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor in the event of the ensuing hypoxia resulting from AAT. In our previous studies, we have demonstrated that AAT accelerates VM in GBM. In this review, we will focus on the origins of VM, visualizing VM in AAT-treated tumors and the development of VM as a resistance mechanism to AAT.     

Regulation of the pro-angiogenic microenvironment by carboxyamido-triazole

Anti-angiogenic agents regulate tumor growth by inhibiting endothelial cell proliferation and invasion. Carboxyamido-triazole (CAI), an inhibitor of non-voltage-operated calcium entry and calcium influx-mediated pathways, has angiogenesis and invasion inhibitory activity. We hypothesized that CAI may express its anti-angiogenic effects through negative regulation of pro-angiogenic cytokine production and/or function. In vivo, orally administered CAI prevented A2058 human melanoma xenograft growth and concomitantly resulted in a marked reduction in circulating vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In vitro, A2058 cell secretion of VEGF was inhibited by CAI treatment under limiting micronutrient conditions that approximate the tumor microenvironment, media restriction, and acidification to pH 6.8 (P=0.0003 and P=0.0006, respectively). VEGF and HIF-1alpha message and protein were also reduced by CAI treatment. Oral CAI treatment reduced vascular ingrowth in vivo into VEGF-containing Matrigel plugs. Commensurate with those findings, human umbilical vein endothelial cell (HUVEC) migration towards VEGF was reduced below background by exposure to CAI in the migration chamber (P<0.0001). An 88% reduction in circulating IL-8 concentration was measured in CAI-treated animals. However, IL-8 protein secretion and gene expression were increased by CAI treatment in culture (P< or =0.01), where CAI caused a dose-dependent acidification of the culture milieu (P< or =0.005). This paradox suggests that IL-8 production in vitro may be more sensitive to ambient pH than cytosolic calcium. These observations suggest that CAI inhibition of tumor cell VEGF production and endothelial cell response to VEGF results in disruption of signaling between the tumor and its microenvironment, causing a net anti-angiogenic effect.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.     

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro^1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype^6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)⁹. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis^{6, 10-13}. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients^{8, 10, 11}. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.Download video file.^{(24M, mov)}     

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.