Search

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.

The combretastatins have attracted significant interest as small molecule therapies for cancer due to their ability to function as vascular disrupting agents. We have successfully prepared a range of combretastatin analogs that are based on a novel sydnone heterocycle core, and their potential as tubulin has been assessed in vitro and in vivo. The most potent candidate was found to disrupt microtubules and affect cellular morphology at sub-micromolar levels. Moreover, it was found to bind reversibly to tubulin and significantly increase endothelial cell monolayer permeability, in a similar manner to combretastatin A4. Surprisingly, the compound did not exhibit efficacy in vivo, possibly due to rapid metabolism.

Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site.

This work describes the topological analysis of the vasculature of tumours. The analysis is performed with a scale-space technique, which traces the centrelines of vessels as topological ridges of the image intensities and then obtains a series of measurements, which are used to compare the vasculatures. Besides the measurements directly associated with the centrelines, the scales obtained allow the estimation of width andthusareacoveredwithvessels. Tumours of SW1222 human colorectal carcinoma xenografts were observed when growing in dorsal skin-fold window chambers in mice. Three variants of the tumours expressing either endogenous levels of angiopoietins (WT) or over-expressing either angiopoietin-1 (Ang-1) or angiopoietin-2 (Ang-2) were assessed with/without vascular targeted therapy. The scale-space technique was able to discriminate between the vasculatures of the three different tumour types prior to treatment. Results also suggested that over-expression of Ang-2 was associated with susceptibility of the tumour vasculature to the vascular disrupting agent, combretastatin A4 phosphate (CA4P). Substantiation of this finding would point to the potential of tumour Ang-2 expression as a predictive bio-marker for response to CA4P.

The combretastatins are an important class of tubulin-binding agents. Of this family, a number of compounds are potent tumor Vascular Disrupting Agents (VDAs) and have shown promise in the clinic for cancer therapy. We have developed a modular synthetic route to combretastatin analogs based on a pyrazole core through highly-regioselective alkyne cycloaddition reactions of sydnones. These compounds show modest to high potency against human umbilical vein endothelial cell proliferation. Moreover, evidence is presented that these novel VDAs have the same mode of action as CA4P and bind reversibly to β-tubulin - believed to be a key feature in avoiding toxicity. The most active compound from in vitro studies was taken forward to an in vivo model and instigated an increase in tumor cell necrosis.

Antiangiogenic therapy based on blocking the actions of vascular endothelial growth factor-A (VEGF) can lead to "normalization" of blood vessels in both animal and human tumors. Differential expression of VEGF isoforms affects tumor vascular maturity, which could influence the normalization process and response to subsequent treatment. Fibrosarcoma cells expressing only VEGF120 or VEGF188 isoforms were implanted either subcutaneously (s.c.) or in dorsal skin-fold "window" chambers in SCID mice. VEGF120 was associated with vascular fragility and hemorrhage. Tumor-bearing mice were treated with repeat doses of SU5416, an indolinone receptor tyrosine kinase inhibitor with activity against VEGFR-2 and proven preclinical ability to induce tumor vascular normalization. SU5416 reduced vascularization in s.c. implants of both VEGF120 and VEGF188 tumors. However, in the window chamber, SU5416 treatment increased red cell velocity in VEGF120 (representing vascular normalization) but not VEGF188 tumors. SU5416 treatment had no effect on growth or necrosis levels in either tumor type but tended to counteract the increase in interstitial fluid pressure seen with growth of VEGF120 tumors. SU5416 pretreatment resulted in the normally fragile blood vessels in VEGF120-expressing tumors becoming resistant to the vascular damaging effects of the tubulin-binding vascular disrupting agent (VDA), combretastatin A4 3-O-phosphate (CA4P). Thus, vascular normalization induced by antiangiogenic treatment can reduce the efficacy of subsequent VDA treatment. Expression of VEGF120 made tumors particularly susceptible to vascular normalization by SU5416, which in turn made them resistant to CA4P. Therefore, VEGF isoform expression may be useful for predicting response to both antiangiogenic and vascular-disrupting therapy. Copyright © 2013 UICC.

Background: Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple alternatively spliced isoforms, which display distinct receptor and matrix binding characteristics. In addition to being a major inducer of tumour angiogenesis, VEGF also has complex functions in angiogenesis-independent aspects of tumour growth,but the role of individual VEGF isoforms remains poorly understood. Here we investigated the effects of endogenous VEGF isoform expression on tumour cell migration and invasion. \ud \ud Method: We used a panel of mouse fibrosarcoma cells we developed (fs188, fs164 and fs120) that express single VEGF isoforms (188, 164 or 120 respectively), under endogenous promoter control. We investigated adhesion to different matrices, 2D migration and invasion through 3D collagen. \ud \ud Results: Fs188 cells, are typically mesenchymal, form ruffles, display strong integrin-dependent adhesion and express high levels of pERK1/2 and pAKT. In contrast, fs164 and fs120 cells are not typically mesenchymal in morphology; they display weak binding to collagen, lack ruffles and align longitudinally forming long multicellular chains and abundant cell-cell contacts. On 3D collagen, fs188 cells remain mesenchymal while fs164 and fs120 cells adopt a rounded/amoeboid and a mix of rounded/mesenchymal morphologies respectively. Cell morphology and migration are dependent on the cytoskeleton and actinomyosin contractility, to provide traction force in mesenchymal movement, and cortical contraction for rounded amoeboid motility. Consistent with their mesenchymal characteristics, fs188 cells migrated faster in 2D and invaded 3D collagen more efficiently than fs164 or fs120 cells. Contractility inhibitors caused fs164/fs120 cells to switch to a mesenchymal morphology and accelerated their migration but not that of fs188 cells. \ud \ud Conclusion: VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results suggest that individual VEGF isoforms influence the migration and invasion strategies of tumour cells thus adding to the complexity of VEGF signaling within the tumor microenvironment. Acknowledgements This work was funded by Cancer Research UK.

The heart is a critical dose-limiting organ during radiotherapy. In patients treated for breast cancer or Hodgkin's disease, regions of the heart can receive radiation doses in excess of 40 Gy. More recently, epidemiological and clinical studies suggested that even moderate to low radiation doses increase the risk of cardiovascular disease that can present years after the initial exposure. Radiation can cause cardiac microvascular damage but the mechanisms of pathogenesis of radiation-induced heart disease have not been clearly defined. Cardiac injury triggers repair processes in the myocardium requiring neovascularisation. Here, we investigated latent effects of ionizing radiation on angiogenic responses in the mouse heart. We also assessed the effects of radiation on cardiac endothelial responses and angiogenesis in vitro. The hearts of C57BL/6 and ApoE-/- mice were locally irradiated with 0.2 to 16 Gy and animals were sacrificed at 20, 40 and 60 weeks post-irradiation. Formation of angiogenic sprouts was assessed in ventricular heart explants embedded in fibrin gels. The angiogenic capacity of irradiated hearts was also assessed through a novel fibroblast-endothelial "self-assembling assay" we developed. Mouse hearts were enzymatically digested into a single cell suspension and cultured for 10 days. Within 5 to 7 days, endothelial cells began to form capillary-like structures surrounded by fibroblasts and pericytes, modeling the angiogenic process. Cardiac endothelial cells were also irradiated in vitro and effects on migration and capillary-tube formation on a fibroblast bed were established. Radiation caused a dose-dependent inhibition of endothelial sprout formation in explants from both mouse models. Significant changes were observed at 8 and 16 Gy after 20 weeks and at ≥2 Gy after 40 or 60 weeks post irradiation. Irradiation also significantly reduced the adherence and growth of extracted cardiac cells and inhibited the quantity and quality of the capillary-like structures formed in the "self-assembling" model. In vitro, irradiation (≥0.2 Gy) inhibited the migration of cardiac endothelial cells in scratch wound assays and capillary-like formation on a fibroblast bed. Our data show that radiation causes persistent and progressive vascular damage and suppresses angiogenic activity in the heart. Angiogenesis was inhibited at doses likely to be delivered during thoracic radiation in cancer patients. Impairment of the angiogenic response could lead to further ischemia and degeneration in the myocardium and contribute to the development of heart disease.

Lexibulin is a small-molecule tubulin-depolymerizing agent that is being developed by \ud YM BioSciences\ud as a\ud vascular-disrupting agent (VDA) for the potential treatment of cancer. In vitro cell culture and in v\ud ivo tumor\ud models established that lexibulin has vascular-disrupting activity. Lexibulin is currently undergoing\ud clinical\ud development and can be administered either intravenously or orally. In two phase I clinical trials in\ud patients\ud with solid tumors, lexibulin induced changes in tumors that were consistent with vascular disruption \ud and\ud associated blood-flow modifications. Pharmacokinetics of lexibulin were favorable and generally dose-\ud linear\ud for both the oral and intravenous formulations. The side-effect profile of lexibulin was similar to t\ud hat of other\ud VDAs and included cardiovascular, respiratory and hematological toxicities. Lexibulin was also to be \ud assessed\ud in a phase Ib/II trial in combination with \ud carboplatin\ud in patients with relapsed glioblastoma multiforme and\ud a phase II trial as monotherapy in patients with multiple myeloma. However, both trials were terminat\ud ed,\ud although preliminary efficacy results are available for the combination trial. At the time of publica\ud tion,\ud lexibulin remained listed as being in phase II development on \ud YM BioSciences\ud ' pipeline.

This AGIR report (RCE-16) reviews the epidemiological, experimental and medical evidence for radiation exposure elevating the incidence of circulatory diseases.\ud \ud This report is based on work conducted by the Advisory Group on Ionising Radiation (AGIR) subgroup on circulatory disease risk.\ud \ud Increased evidence of an association between circulatory diseases and radiation exposures has become available over recent years.\ud \ud Radiological protection does not currently include circulatory diseases in the calculation of health detriment from low dose exposures. It is therefore timely to review the evidence for radiation association and causation of circulatory diseases and consider the likely need and implications for radiological protection to take these diseases into account in protection of health from low dose exposures and medical exposures.

Background: Combretastatin A4 phosphate (CA4P) is a tumour vascular disrupting agent (VDA) that targets endothelial microtubules, triggering remodelling of the actin cytoskeleton, contractility and disruption of VE-cadherin junctions through RhoGTPase/ROCK-dependent pathways. These events lead to a rise in endothelial monolayer permeability. A rise in permeability is considered crucial for vascular shutdown elicited by CA4P in vivo. CA4P also inhibits endothelial migration and induces mitotic arrest and apoptosis, so potentially it could also target tumour angiogenesis. \ud \ud Method: In this study, the nature of signalling interactions between Rho/ROCK and cAMP/cGMP and their influence on cytoskeletal and functional responses of endothelial cells to CA4P were investigated. \ud \ud Results: Several cAMP/cGMP analogues inhibited CA4P-induced Rho/ROCK activation and prevented actin remodeling, disruption of cell-to-cell junctions and permeability rise in endothelial monolayers. cAMP inhibits Rho by either protein kinase A (PKA)-dependent mechanisms or via activation of Epac1/Rap1. O-Me-cAMP, an analogue that selectively activates Epac1/Rap1 abolished activation of Rho/ROCK by CA4P while selective PKA activator 6-Bnz-cAMP only partially inhibited Rho/ROCK activation and actin remodelling by CA4P. Inhibitors of PKA did not alter endothelial responses to CA4P in the presence of cAMP analogues suggesting that cAMP acts primarily via Epac1/Rap1 to inhibit Rho/CA4P interactions. CA-4-P also inhibited endothelial migration and abolished lamellipodia at the leading edge of migrating cells in injured monolayers. Rho inhibitor C3 exoenzyme and ROCK inhibitor Y27632 as well as cAMP analogues re-established cell movement and formation of lamellipodia in wounded monolayers exposed to CA-4-P, suggesting that inhibitory effects on migration were mediated via Rho/ROCK. \ud \ud Conclusion: Deciphering molecular pathways that modulate endothelial responses to VDAs is important for further targeting. Our data demonstrate that interactions between cGMP/cAMP and Rho influence both the vascular disrupting and anti-angiogenic activities of CA-4-P and point to cAMP/cGMP as potential targets for improving VDA activity. Acknowledgements Funded by Cancer Research UK

Background: Tubulin binding microtubule depolymerising agents form a growing group of tumour vascular disrupting agents (VDAs) in clinical trial, with combretastatin A-4-phosphate (CA4P) the lead compound. Signalling through RhoGTPase/ROCK-dependent pathways is central to CA4P-induced effects on endothelial cells in vitro (Kanthou C. & Tozer GM, 2002, Blood 99: 2060-2069) . Here, we tested the hypothesis that RhoGTPase/ROCK signalling is also important in vivo. \ud \ud Method: SW1222 human colorectal carcinoma cells were grown as solid sub-cutaneous tumours in SCID mice. The Rho kinase (ROCK) inhibitor, Y-27632 (50 mg/kg) or saline control, was administered intraperitoneally (i.p.), 5 minutes prior to 100 mg/kg CA4P or saline i.p.. Laser Doppler flowmetry was used to assess tumour vascular response from 0 – 2h post-treatment. Intravenous administration of fluorescent tomato lectin was used for assessing tumour perfusion at 1, 3, 6 and 24 hours post-treatment. Necrosis (H&E) and leukocyte infiltration (immunohistochemistry) were assessed at 24h. \ud \ud Results: Y-27632 alone did not significantly increase necrosis at 24 hours (17±4% versus 10±3% of tumour sectional area). However, prior administration of Y-27632 significantly reduced CA4P-induced tumour necrosis from 61±5% to 35±7%, accompanied by a decrease in staining for the myeloid markers, myeloperoxidase and GR-1. Y-27632 pre-treatment did not affect laser Doppler and perfused vascular volume measurements in the first few hours after CA4P but significantly reduced the effect of CA4P on perfused vascular volume measured at 6 and 24 hours. \ud \ud Conclusion: Our data indicate that RhoGTPase/ROCK-dependent signalling is a critical factor in determining extent of tumour necrosis induction by CA4P and suggest that ROCK inhibition is acting downstream from initial vascular shut-down, potentially via modulation of myeloid cell recruitment. These mechanisms also have significance for similar VDAs in development. Funded by Cancer Research UK

An image-processing algorithm for the analysis of migration of vascular endothelial cells in culture is presented. The algorithm correctly detected the cellular regions on either side of an artificial ‘wound’ made by dragging a sterile pipette tip across the monolayer of cells (scratch wound assay). Frequency filtering and mathematical morphology were used to approximate the boundaries of cellular regions. This allowed the measurement of the distance between the regions, and therefore the migration rates, regardless of the orientation of the wound and even in cases where the cells were sparse and not tightly packed.

Background\ud \ud A functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P) is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia). Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1), controlling angiogenic and metastatic pathways.\ud \ud Methods\ud \ud We investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro.\ud \ud Results\ud \ud CA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor κB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A), was increased.\ud \ud Conclusion\ud \ud Our findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.\ud \ud

We have previously proposed the plant enzyme horseradish peroxidase (HRP) and the plant hormone indole-3-acetic acid (IAA) as an enzyme/prodrug combination for cancer gene therapy. In the current study, we evaluated the potential of HRP/IAA for gene-directed enzyme/prodrug therapy in three human tumor cell lines (T24 bladder carcinoma, MCF-7 breast adenocarcinoma, and FaDu nasopharyngeal squamous carcinoma) and one endothelial cell line (HMEC-1). The action of 10 IAA analogues in combination with HRP was studied in vitro in normoxic conditions as well as in the extreme tumor conditions of anoxia. Compounds characterized by prompt normoxic or anoxic cytotoxic activation and high HRP transfectant killing or selectivity were identified. Some variations were observed in the response of cells of different origin, with IAA, 1-Me-IAA, and 5-Br-IAA representing the most promising candidates for HRP gene therapy. In particular, 5-Br-IAA showed a very prompt and selective activation in anoxia. A strong bystander effect was produced by activated IAA and analogues because 70-90% cell kill was obtained when only 5% of the cells expressed the HRP enzyme. These results indicate that HRP/IAA represents an effective system for enzyme/prodrug-based anticancer approaches, and further improvements could be achieved by the use of novel IAA derivatives.

BACKGROUND: Combretastatin A-4-phosphate (CA-4-P) is a microtubule depolymerising agent currently in clinical trial as a tumour vascular-targeting agent. In vivo, CA-4-P causes rapid shutdown of tumour blood flow (within minutes) and a significant neutrophil infiltration at later times. MATERIALS AND METHODS: Using an in vitro flow-cell assay, we investigated neutrophil-endothelial cell interactions and associated mechanisms, following endothelial cell exposure to CA-4-P. Cellular adhesion molecule (CAM) expression was examined using immunoblotting and immunofluorescence, and the role of CAM in neutrophil recruitment was investigated using specific blocking antibodies. RESULTS: Exposure of HUVEC to CA-4-P, resulted in significant neutrophil recruitment, and increased expression of endothelial CAM. Results of antibody studies demonstrated that endothelial CAM expression induced by CA-4-P is responsible for the observed neutrophil recruitment. DISCUSSION: This study demonstrated that the tumour vascular targeting agent, CA-4-P, directly induces endothelial CAM expression and subsequent neutrophil recruitment. In vivo, neutrophil infiltration probably contributes to CA-4-P-induced tumour cell kill. Therefore, increasing neutrophil recruitment into tumours may have potential for optimising vascular-targeted strategies for cancer therapy.

Segmentation and morphological analysis of microvessels in immunostained histological tumour sectionsA fully automatic segmentation and morphological analysis algorithm for the analy- sis of microvessels from CD31 immunostained histological tumour sections is presented. The algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, which provided the seeds for a region-growing algorithm in the 3D Hue, Saturation, Value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing ob- jects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels.

In a controlled study of 15 pregnant patients undergoing therapeutic termination of pregnancy, seven received subcutaneously 5,000 anti-FXa units of low molecular weight (LMW) heparin 15 and 3 h prior to the termination, and eight patients acted as controls. Paired maternal and fetal blood samples were taken (before or immediately after the termination) for assay of heparin activity by a chromogenic anti-FXa method sensitive to levels of 0.02 anti-FXa U/ml. LMW heparin was detected in all maternal samples of the test patients but was not detected in any of the fetal samples. The use of LMW heparin as a thromboprophylactic agent was then evaluated in 11 patients who were known to have a severe thromboembolic tendency, had suffered recurrent miscarriages and had responded poorly to conventional anticoagulation (oral anticoagulant, conventional heparin). All patients receiving LMW heparin in thromboprophylactic doses completed uneventful pregnancies and gave birth to healthy babies (three for the first time) without complication. Bone density scans performed in all patients shortly after the delivery showed normal mineral mass. We conclude that LMW heparin does not cross the placental barrier, and in addition offers satisfactory antithrombotic protection for both maternal and placental circulation. In addition, this study provides preliminary data from 11 patients suggesting LMWH may not give rise to maternal osteoporosis, a finding that now needs further investigation.