Endoplasmic reticulum stress-induced release and binding of calreticulin from human ovarian cancer cells.

BACKGROUND: Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). METHODS: We treated OC cells with the chemotherapeutic-doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor-thapsigargin (TG)-and/or an ER stress inhibitor-TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. RESULTS: Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. CONCLUSION: Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

The role of astrocytes in brain metastasis at the interface of circulating tumour cells and the blood brain barrier.

Tumour metastasis to the brain is a complex process involving crosstalk between the circulating tumour cells and the blood brain barrier (BBB). Astrocytes, which reside in the abluminal surface of the microvasculature of the BBB, are now known to play an essential role in tumour cell migration and invasion into the brain parenchyma. For instance, pro-inflammatory astrocyte secretions, including TNF-α, IL-6, CXCL10 as well as polyunsaturated fatty acids interact with circulating tumour cells to promote migration and proliferation. Additionally, astrocyte and tumour cell derived MMPs play a vital role in tumour cell invasion through the BBB. Understanding these complex interactions between tumour cells and astrocytes in the tumour microenvironment may contribute to the development of novel therapeutics for brain metastasis. Therefore, in this review, we present key interactions within the neurovascular unit of the BBB in the tumour microenvironment that significantly aids cancer metastasis, focusing particularly on astrocytes.

Cathepsin D in the Tumor Microenvironment of Breast and Ovarian Cancers.

Cancer remains a major and leading health problem worldwide. Lack of early diagnosis, chemoresistance, and recurrence of cancer means vast research and development are required in this area. The complexity of the tumor microenvironment in the biological milieu poses greater challenges in having safer, selective, and targeted therapies. Existing strategies such as chemotherapy, radiotherapy, and antiangiogenic therapies moderately improve progression-free survival; however, they come with side effects that reduce quality of life. Thus, targeting potential candidates in the microenvironment, such as extracellular cathepsin D (CathD) which has been known to play major pro-tumorigenic roles in breast and ovarian cancers, could be a breakthrough in cancer treatment, specially using novel treatment modalities such as immunotherapy and nanotechnology-based therapy. This chapter discusses CathD as a pro-cancerous, more specifically a proangiogenic factor, that acts bi-functionally in the tumor microenvironment, and possible ways of targeting the protein therapeutically.

Cathepsin L-induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma.

BACKGROUND: New treatment options for metastasised high-grade serous carcinoma (HGSC) are urgently needed. HGSC frequently metastasises to the omentum, inducing angiogenesis in the local omental microvasculature to facilitate tumour growth. We previously showed that HGSC-secreted cathepsin L (CathL) induces pro-angiogenic changes in disease relevant human omental microvascular endothelial cells (HOMECs), suggesting a role in tumour angiogenesis. Here we investigate whether CathL acts by inducing local production of the carbohydrate-binding protein galectin-1 (Gal1), which has been reported to be involved in tumourigenesis in other tumours. METHODS: HOMECs were used for all experiments. Gal1 mRNA and protein levels were measured by RT-PCR and ELISA respectively. Gal1-induced cell proliferation was assessed using WST-1 assay, migration using a transwell assay and in vivo Gal1 expression by immunohistochemistry. RESULTS: CathL transcriptionally regulated HOMEC production and secretion of Gal1 via activation of NFκB (significantly inhibited by sulfasalazine). Gal1 significantly enhanced HOMEC migration (p < 0.001) and proliferation (p < 0.001), suggesting an autocrine action. The latter was significantly reduced by the MEK/ERK1/2 inhibitors U0126 and PD98059 suggesting downstream activation of this pathway. Immunohistochemical analysis of omenta from HGSC patients with or without metastatic disease demonstrated a positive correlation between Gal1 expression and number of microvessels (r = 0.8702, p < 0.001), and area of vessels (r = 0.7283, p < 0.001), supporting a proangiogenic role for Gal1 in omental metastases. CONCLUSION: HOMEC Gal1 transcription and release in response to CathL secreted from metastasising HGSC acts in an autocrine manner on the local microvasculature to induce pro-angiogenic changes, highlighting a potential new therapeutic target.

High Expression of Galectin-1, VEGF and Increased Microvessel Density Are Associated with MELF Pattern in Stage I-III Endometrioid Endometrial Adenocarcinoma.

BACKGROUND: In this study, we investigate the expression of markers of angiogenesis and microvessel density (MVD) in cases of microcystic, elongated and fragmented (MELF) pattern, with its prognostic role in the survival of endometrioid endometrial adenocarcinomas (EA) patients. METHODS: In this study, 100 cases of EA, 49 cases with MELF pattern and 51 without, were immunohistochemically stained for galectin-1, vascular endothelial growth factor (VEGF), and MVD. Morphometry and statistical (univariate and multivariate) analyses were performed to assess overall survival (OS) and disease-free survival. RESULTS: The expression of VEGF (p<.001) and galectin-1 (p<.001), as well as MVD area (p<.001) and number of vessels/mm2 (p<.050), were significantly higher in the +MELF pattern group compared to the -MELF group. A low negative correlation between MELFpattern and the number of days of survival (p<.001, r=-0.47) was also found. A low positive correlation of MELF-pattern with galectin-1 expression (p<.001, r=0.39), area of vessels/mm2 (p<.001, r=0.36), outcome of EA (p<.001, r=0.42) and VEGF expression (p<.001, r=0.39) suggests potential pathological relevance of these factors in the prognosis of EA. A univariate survival analysis indicated a role for all parameters of survival. Multivariate Cox proportional hazard regression analysis revealed that only area of vessels/mm2 (hazard ratio [HR], 1.018; 95% confidence interval [CI], 1.002 to 1.033), galectin-1 (HR, 1.049; 95% CI, 1.025 to 1.074) and VEGF (HR, 1.049; 95% CI, 1.022 to 1.077) play key roles in OS. CONCLUSIONS: This study reports an increase in MVD, VEGF and galectin-1 expression in EA with MELF pattern and suggests that MELF pattern, along with the angiogenic profile, may be a prognostic factor in EA.

Graphene Oxide-Based Targeting of Extracellular Cathepsin D and Cathepsin L As A Novel Anti-Metastatic Enzyme Cancer Therapy.

Overexpression and secretion of the enzymes cathepsin D (CathD) and cathepsin L (CathL) is associated with metastasis in several human cancers. As a superfamily, extracellularly, these proteins may act within the tumor microenvironment to drive cancer progression, proliferation, invasion and metastasis. Therefore, it is important to discover novel therapeutic treatment strategies to target CathD and CathL and potentially impede metastasis. Graphene oxide (GO) could form the basis of such a strategy by acting as an adsorbent for pro-metastatic enzymes. Here, we have conducted research into the potential of targeted anti-metastatic therapy using GO to adsorb these pro-tumorigenic enzymes. Binding of CathD/L to GO revealed that CathD/L were adsorbed onto the surface of GO through its cationic and hydrophilic residues. This work could provide a roadmap for the rational integration of CathD/L-targeting agents into clinical settings.

Cathepsin L Induces Proangiogenic Changes in Human Omental Microvascular Endothelial Cells via Activation of the ERK1/2 Pathway.

BACKGROUND: Metastasis still remains the major cause of therapeutic failure, poor prognosis and high mortality in epithelial ovarian cancer (EOC) patients. Previously, we showed that EOC cells secrete a range of factors with potential pro-angiogenic activity, in disease-relevant human omental microvascular endothelial cells (HOMECs), including the lysosomal protease cathepsin L (CathL). Thus, the aim of this study was to examine potential pro-proliferative and pro-migratory effects of CathL in HOMECs and the activated signalling pathways, and whether these proangiogenic responses are dependent on CathL-catalytic activity. METHODS: HOMECs proliferation was investigated using WST-1, BrdU and CyQUANT assays. Cell migration was examined using a Cultrex Cell 96 transwell migration assay. Enzyme activity was assayed at various pHs using the CathL-specific fluorogenic substrate FY-CHO. Activation of cell signalling pathways was tested using a commercially available phosphokinase array and intact cellbased ELISAs. RESULTS: We showed for the first time that CathL has a potent pro-proliferative and pro-migratory effect on HOMECs. For instance, CathL significantly increases HOMEC proliferation (134.8±14.7% vs control 100%) and migration (146.6±17.3% vs control 100%). Our data strongly suggest that these proangiogenic effects of CathL are mediated via a non-proteolytic mechanism. Finally, we show that CathL-induced activation of the ERK1/2 pathway is involved in inducing these cellular effects in HOMECs. CONCLUSION: These data suggest that CathL acts as an extracellular ligand and plays an important pro-angiogenic, and thus pro-metastatic, role during EOC metastasis to the omentum, by activating the omental microvasculature, and thus can potentially be targeted therapeutically in the future.

Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways.

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum.

In vitro toxic effects of reduced graphene oxide nanosheets on lung cancer cells.

The intriguing properties of reduced graphene oxide (rGO) have paved the way for a number of potential biomedical applications such as drug delivery, tissue engineering, gene delivery and bio-sensing. Over the last decade, there have been escalating concerns regarding the possible toxic effects, behaviour and fate of rGO in living systems and environments. This paper reports on integrative chemical-biological interactions of rGO with lung cancer cells, i.e. A549 and SKMES-1, to determine its potential toxicological impacts on them, as a function of its concentration. Cell viability, early and late apoptosis and necrosis were measured to determine oxidative stress potential, and induction of apoptosis for the first time by comparing two lung cancer cells. We also showed the general trend between cell death rates and concentrations for different cell types using a Gaussian process regression model. At low concentrations, rGO was shown to significantly produce late apoptosis and necrosis rather than early apoptotic events, suggesting that it was able to disintegrate the cellular membranes in a dose dependent manner. For the toxicity exposures undertaken, late apoptosis and necrosis occurred, which was most likely resultant from limited bioavailability of unmodified rGO in lung cancer cells.

The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

Loss of the endothelial glycocalyx is associated with increased E-selectin mediated adhesion of lung tumour cells to the brain microvascular endothelium.

BACKGROUND: Arrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells. METHODS: Endothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm(2)). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC). RESULTS: A549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion. CONCLUSION: These data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.

Clinical Relevance of Increased Endothelial and Mesothelial Expression of Proangiogenic Proteases and VEGFA in the Omentum of Patients with Metastatic Ovarian High-Grade Serous Carcinoma.

Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.

Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 µM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 µM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

Epithelial ovarian cancer-induced angiogenic phenotype of human omental microvascular endothelial cells may occur independently of VEGF signaling.

Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA165-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.

Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions.

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.

An improved and reliable method for isolation of microvascular endothelial cells from human omentum.

OBJECTIVES: Despite an increasing research demand for human microvascular endothelial cells, isolation of primary endothelial cells from human tissue remains difficult. The omentum, a highly vascular visceral adipose tissue, could provide an excellent source of these cells. METHODS: A reliable method to isolate HOMECs has been developed. It consists of initial enzymatic digestion (to deplete cell contaminants), followed by further digestion, selective filtration, and immunoselection using Dynabeads coated with CD31 antibody. Cultures were characterized for expression of endothelial cell markers and their ability to undergo VEGF-dependent in vitro tube structure formation. RESULTS: Omental-derived cultures of microvascular endothelial cells were achieved with <5% contamination of other cell types. The endothelial origin of cells was confirmed by the constitutive expression of a range of vascular endothelial markers (CD31, CD105, vWF) and internalization of DiI-AcLDL. Furthermore, cultures were negative for lymphatic endothelial markers, underwent in vitro angiogenesis, and exhibited typical endothelial morphology. CONCLUSIONS: This isolation method produces homogeneous HOMEC cultures that can be maintained in vitro for at least six passages without loss of cellular features characterizing endothelial cells.

Tetrahydroxanthones by sequential Pd-catalyzed C-O and C-C bond construction and use in the identification of the "antiausterity" pharmacophore of the kigamicins.

Readily available C-acylated cycloalkanones undergo efficient Pd catalyzed ring closure/cross-coupling providing 7-substituted tetrahydroxanthones in a single operation. One of the synthesized derivatives (depicted) is shown to selectively kill pancreatic cancer (PANC-1) cells under conditions of nutrient deprivation indicating that the tetrahydroxanthone is responsible, in part, for the "antiausterity" effects of the naturally occurring kigamicins.

Synthesis and evaluation of prodrugs for anti-angiogenic pyrrolylmethylidenyl oxindoles.

Potential prodrugs of inhibitors of VEGF-induced angiogenesis have been investigated. The prodrug systems studied were the 4-nitrobenzyl, 2-nitrophenylacetyl and 3-methyl-3-(3,6-dimethylbenzo-1,4-quinon-2-yl)butanoyl groups, readily attached to acidic OH or NH groups in drug molecules, and released upon bioreductive activation. The anti-angiogenic compounds studied were the pyrrolylmethylidenyl oxindole SU5416 (semaxanib) and its novel 6-hydroxy derivative. The potentially pro-anti-angiogenic compounds were assayed for their ability to block VEGF-induced angiogenesis in HUVECS in comparison to the free agents.

Comparative study of isoflavone, quinoxaline and oxindole families of anti-angiogenic agents.

A study designed to compare the effects on VEGF-induced angiogenesis of a number of known anti-angiogenic agents together with some novel derivatives thereof was undertaken. Thus the isoflavone biochanin A 1[structure: see text], indomethacin 2[structure: see text], the 3-arylquinoxaline SU1433 and its derivatives 3-6[structure: see text], the benzoic acid derivative 7[structure: see text], the oxindoles SU5416 8[structure: see text] and SU6668 11[structure: see text], together with their simple N-benzyl derivatives 9, 10, and 12[structure: see text] were selected for study. Using an in vitro assay the compounds were evaluated for their ability to inhibit VEGF-induced angiogenesis in HUVECs, and the cytotoxicity of representative compounds was also studied in tumour cell lines using 24-h exposure. The results indicate that the SU compounds, SU1433, SU 5416 and SU6668, are more potent inhibitors of VEGF-induced angiogenesis than indomethacin or the naturally occurring biochanin A, presumably because they inhibit VEGF receptor signalling. Blocking one of the phenolic OH groups of SU1433 reduced anti-angiogenic activity, as did blocking the NH groups of SU5416 and SU6668. Cytotoxicity studies indicate that none of the compounds examined exhibited cytotoxicity at anti-angiogenic concentrations.