Tumor Angiogenesis

Angiogenesis is essential for tumor growth and metastases. Studies in breast carcinomas suggest that microvessel quantitation as a measure of angiogenesis might be one of the most powerful prognostic tools available. Node negative breast cancer is a particular group for which better prognostic markers would be helpful. We therefore measured microvessel density in a series of well characterised node negative breast carcinomas to evaluate angiogenesis as a prognostic marker and assess its relationship to epidermal growth factor receptor (EGFR) and estrogen receptor (ER), which have previously been reported to be of value. 109 patients with a mean age of 55 years and a median follow-up of 25 months were examined. Vessels were immunohistochemically highlighted using an antibody to platelet endothelial cell adhesion molecule CD31, and microvessel density was quantified using a Chalkley point eyepiece graticule. No significant correlation was observed with patient age, tumor size, grade, ER, or EGFR expression. In a univariate analysis of survival, whereas ER expression was not a significant indicator of either relapse-free (RFS) or overall survival (OS), vascular count (VC) predicted both early RFS and OS (p=0.01 and p=0.028 respectively). Furthermore, in patients with ER positive tumors, a subgroup usually considered to have a good prognosis, there was a significant reduction in RFS and OS if tumors had high VCs (p=0.05 and p=0.002 respectively). A further statistically significant reduction in RFS (p=0.05) was observed for EGFR positive highly vascular tumors. In a Cox proportional hazard model, VC remained a significant prognostic indicator for both RFS and OS (p=0.04 and p=0.01) and conferred a 6.6 and 3.5 times respective increased risk of mortality and relapse. These findings suggest that quantitation of angiogenesis is an independent predictor of survival in node negative breast carcinomas, and due to these high hazard ratios might be more useful than other recently described prognostic markers in selecting patients who would benefit from adjuvant therapy.

might influence breast tumor angiogenesis. Although angiogenesis is currently under intense investigation in breast cancer, the expression of VEGF in this tumor has yet to be studied. We show here evidence of hypoxic induction of VEGF up-regulation in human mammary fibroblasts, suggesting the possibility of paracrine influences by fibroblasts on endothelium within hypoxic regions of these tumors. Materials and Methods Cells were grown in monolayer cultures for detection of VEGF mRNA via protection assays, following hypoxic exposure [VEGF is also called vascular permeability factor (8, 9)]. Cells were grown in multicellular sandwich cultures to study both the levels and the subcellular location of VEGF protein on a cell-by-cell basis over oxygen/nutrient gradients via immunohistochemistry and digitized imaging. RNA Detection. Monolayers of early culture (third) passage mammary fibroblast cells, isolated from both breast tumor (B437) and normal breast tissue derived from reduction mammoplasty (B106) were plated in DMEM (GIBCO)with 20%fetalbovine serum(GIBCO)at 4 X 10@ cells/60-mmglass Petri dish. Cells were obtained from Dr. Michael Gould (Department of Human Oncology, University of Wisconsin). Fibroblast origin was verified by immunostaining with a monoclonal antibody against vimentin (Dako). Hy poxia (â€oe100 ppm 02) was achieved using a Bactron IV anaerobic chamber (Sheldon) with a self-contained incubator unit that uses a 5% H2-5%C02-90% N2-injected atmosphere and a palladium catalyst. Some cultures that had undergone a 24-h hypoxic period were reoxygenated with an additional 24-h oxygen (21%) incubation. For mRNA collection cells were lysed on site in 4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% N-lauroylsarcosine and 0.1 M 2-mercapthoethanol, and total RNA was isolated using acid phenol. From human VEGF-165 cDNA (Calbio California Technology), a 146-base pair clone extending from nucleotides 440 to 585 was subcloned in a Blue script Il-KS vector (Stratagene) and utilized as the probe template. The solution hybridization RNase protection assay was performed using a high specific activity antisense riboprobe transcribed in vitro (10). For reference, cyclophilin mRNA was analyzed in each sample simultaneously with the VEGF mRNA. This technique afforded the simultaneous probing of the cyclophilin control and the VEGF bands, assuring no artifactual probing differences due to variations in gel workup. Human granulosa-lutein cells were used as positive controls (11). Total RNA was dissolved in [email protected] hybridization solution (4 Mguanidinium thiocyanate-0.! MEDTA, pH 7) and mixed with 250 pg of each probe. The protected materials were resolved through nondenatur ing 5% PAGE. Quantitation of the bands was performed using a phosphoim ager (Molecular Dynamics). Tumor-mimetic Sandwich CUltUres. Mixed-cell sandwich cultures were set up by plating on microscope slides breast-fibroblasts (isolated either from tumor or normal mammary tissue after reduction mammoplasty) along with MCF7 cells, each at a density of 1.7 X 10@ cells/slide. Single-cell sandwiches were cultured using either tumor or normal mammary fibroblasts plated at 1.7 X 10@ cells/slide. After 24-h incubation, a top slide (coated with Prosil) was placed on the cultures, sandwiching the cells and restricting cell access of medium to the 75-pm gap between the slides (Fig. 2). The medium reservoir functions as the vasculature feeding the cell population. In much the same manner as in a tumor, cell consumption and diffusion constraints (due to the narrow gap between slides) decrease the oxygen/nutrient availability as a function of distance into the sandwich.

the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.

Cannabinoids, the active components of Cannabis sativa L., act in the body by mimicking endogenous substances — the endocannabinoids — that activate specific cell surface receptors. Cannabinoids exert various palliative effects in cancer patients. In addition, cannabinoids inhibit the growth of different types of tumor cells, including glioma cells, in laboratory animals. They do so by modulating key cell signaling pathways, mostly the endoplasmic reticulum stress response, thereby inducing antitumoral actions such as the apoptotic death of tumor cells and the inhibition of tumor angiogenesis. Of interest, cannabinoids seem to be selective antitumoral compounds as they kill glioma cells but not their nontransformed astroglial counterparts. On the basis of these preclinical findings, a pilot clinical study of Δ9-tetrahydrocannabinol (Δ9-THC) in patients with recurrent glioblastoma multiforme has been recently run. The fair safety profile of Δ9-THC, together with its possible growth-inhibiting action on tumor cells, may set the basis for future trials aimed at evaluating the potential antitumoral activity of cannabinoids.

Both natural and synthetic cannabinoids have been shown to suppress the growth of tumor cells in culture and in animal models by affecting key signaling pathways including angiogenesis, a pivotal step in tumor growth, invasion, and metastasis. In our search for cannabinoid-like anticancer agents devoid of psychoactive side effects, we synthesized and evaluated the anti-angiogenic effects of a novel series of hexahydrocannabinol analogs. Among these, two analogs LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] were selected based on their anti-angiogenic activity and lack of binding affinity for cannabinoid receptors. Both LYR-7 and LYR-8 inhibited VEGF-induced proliferation, migration, and capillary-like tube formation of HUVECs in a concentration-dependent manner. The inhibitory effect of the compounds on cell proliferation was more selective in endothelial cells than in breast cancer cells (MCF-7 and tamoxifen-resistant MCF-7). We also noted effective inhibition of VEGF-induced new blood vessel formation by the compounds in the in vivo chick chorioallantoic membrane (CAM) assay. Furthermore, both LYR analogs potently inhibited VEGF production and NF-κB transcriptional activity in cancer cells. Additionally, LYR-7 or LYR-8 strongly inhibited breast cancer cellinduced angiogenesis and tumor growth. Together, these results suggest that novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8, inhibit tumor growth by targeting VEGF-mediated angiogenesis signaling in endothelial cells and suppressing VEGF production and cancer cell growth.

The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositidedependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen ® formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of ~21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen ® assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.

The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF 121 and VEGF 165 , by human glioma U87 MG cells induced tumor-associated ...

Edible berry anthocyanins possess a broad spectrum of therapeutic and anti-carcinogenic properties. Berries are rich in anthocyanins, compounds that provide pigmentation to fruits and serve as natural antioxidants. Anthocyanins repair and protect genomic DNA integrity. Earlier studies have shown that berry anthocyanins are beneficial in reducing age-associated oxidative stress, as well as in improving neuronal and cognitive brain function. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds, and strawberry) were studied for antioxidant efficacy, cytotoxic potential, cellular uptake, and anti-angiogenic (the ability to reduce unwanted growth of blood vessels, which can lead to varicose veins and tumor formation) properties. We evaluated various combinations of edible berry extracts and developed a synergistic formula, OptiBerry IH141, which exhibited high ORAC (Oxygen-Radical Absorbing Capacity) value, low cytotoxicity, and superior anti-angiogenic properties compared to the other combinations tested. Anti-angiogenic approaches to treat cancer represent a priority area in vascular tumor biology. OptiBerry significantly inhibited both H2O2- and TNF-α-induced VEGF (Vascular Endothelial Growth Factor) expression by human keratinocytes. VEGF is a key regulator of tumor angiogenesis. Matrigel assay using human microvascular endothelial cells showed that OptiBerry impaired angiogenesis. In anin vivo model of angiogenesis, OptiBerry significantly inhibited basal MCP-1 and inducible NF-κB transcriptions. Endothelioma cells pretreated with OptiBerry showed a diminished ability to form hemangioma and markedly decreased tumor growth by more than 50%. In essence, these studies highlight the novel anti-angiogenic, antioxidant, and anti-carcinogenic potential of a novel anthocyanin-rich berry extract formula, OptiBerry.

The maintenance of vascular integrity and control of blood loss are regulated by a sophisticated system of circulating and cell-associated hemostatic factors. These factors control local platelet aggregation, the conversion of soluble fibrinogen to an insoluble fibrin polymer, and the dissolution of fibrin. However, hemostatic factors are also involved in a number of physiologic processes, including development, tissue remodeling, wound repair, reproduction, inflammation, and angiogenesis. In this review, we outline ways in which angiogenesis is coordinated with and regulated by hemostasis. We focus on inhibitors of angiogenesis contained within platelets or harbored as cryptic fragments of hemostatic proteins and assess the experimental and preclinical evidence for their ability to inhibit tumor angiogenesis and, thus, their potential to be anticancer agents. Finally, we review the results of recent clinical trials involving angiogenesis inhibitors and the evidence that antiangiogenic therapy may be associated with hemostatic complications. [J Natl Cancer Inst 2003;95:1660 -73]

Coagulation abnormalities occur frequently in cancer patients. It is becoming evident that blood platelets have an important function in this process. However, understanding of the underlying mechanisms is still very modest. In this review, we discuss the role of platelets in tumor angiogenesis and growth and suggest their potential significance in malignancies. Platelets contain various pro-and antiangiogenic molecules, which seem to be endocytosed and sequestered in different populations of α-granules. Furthermore, tumor endothelial cells are phenotypically and functionally different from endothelial cells in healthy tissue, stimulating local platelet adhesion and subsequent activation. As a consequence, platelets are able to secrete their angiogenic and angiostatic content, most likely in a regulated manner. The overall effect of these platelet-endothelium interactions appears to be proangiogenic, stimulating tumor angiogenesis. We favor the view that local adhesion and activation of blood platelets and dysregulation of coagulation represent underestimated pathways in the progression of cancer.

Background: Germ cell testicular tumors have survival rate that diminishes with high tumor marker levels, such as human chorionic gonadotropin (hCG). hCG may regulate vascular neoformation through vascular endothelial growth factor (VEGF). Our purpose was to determine the relationship between hCG serum levels, angiogenesis, and VEGF expression in germ cell testicular tumors.

Angiogenesis has a critical role in physiologic and disease processes. For the growth of tumors, angiogenesis must occur to carry sufficient nutrients to the tumor. In addition to growth, development of new blood vessels is necessary for invasion and metastases of the tumor. A number of strategies have been developed to inhibit tumor angiogenesis and further understanding of the interplay between tumors and angiogenesis should allow new approaches and advances in angiogenic therapy. One such promising angiogenic approach is to target and inhibit angiogenesis with vaccines. This review will discuss recent advances and future prospects in vaccines targeting aberrant angiogenesis of tumors. The strategies utilized by investigators have included whole endothelial cell vaccines as well as vaccines with defined targets on endothelial cells and pericytes of the developing tumor endothelium. To date, several promising anti-angiogenic vaccine strategies have demonstrated marked inhibition of tumor growth in pre-clinical trials with some showing no observed interference with physiologic angiogenic processes such as wound healing and fertility.

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis.

Cyclooxygenase (COX) and lipoxygenase (LO) metabolic pathways are emerging as key regulators of cell proliferation and neo-angiogenesis. COX and LO inhibitors are being investigated as potential anticancer drugs and results from clinical trials seem to be encouraging. In this article we will review evidence of COX-2 and 5-LO involvement in cancer pathobiology, propose a model of integrated control of cell proliferation by these enzymes, and discuss the pharmacologic implications of this model.-Romano, M., Clària, J. Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and tumor angiogenesis: implications for cancer therapy. FASEB J. 17, 1986 -1995 (2003) Key Words: arachidonic acid ⅐ cancer ⅐ cyclooxygenase and lipoxygenase inhibitors ⅐ eicosanoids

Destruction of the neovasculature is essential for tumor eradication by photodynamic therapy. Since the over-expression of integrins is correlated with tumor angiogenesis, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenylchlorin or porphyrin) to the a v b 3 integrin specific peptide RGD (H-Arg-Gly-Asp-OH) motif as a common sequence. We reported an efficient solid-phase synthesis of a new family of peptidic photosensitizers with linear or cyclic[RGDfK] RGD motif and compared conjugates in vitro selectivity and photodynamic activity. The conjugates were characterized by 1 H NMR, MALDI, UV-visible spectroscopy and singlet oxygen formation was performed. Chlorins containing linear and constrained RGD motif were incorporated up to 98-and 80-fold more, respectively, than the unconjugated photosensitizer over a 24-h exposure in human umbilical vein endothelial cells (HUVEC) over-expressing a v b 3 integrin. Peptidic moiety also led to a non-specific increased cellular uptake by murine mammary carcinoma cells (EMT-6), lacking RGD binding 0045-2068/$ -see front matter Ó (M. Barberi-Heyob). Bioorganic Chemistry 35 (2007) 205-220 www.elsevier.com/locate/bioorg

We recently developed a cysteinecontaining peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test 99m Tc-HYNIC-C-tagged VEGF (99m Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. Methods: HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. 99m Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 μCi, 1-2 μg protein) followed by microSPECT imaging 1 h later. Results: Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with 99m Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3±5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o. d. ×4; 1.14±0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. × 7; 1.03±0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of 99m Tc/biotin-inactivated VEGF, as compared with 99m Tc-HYNIC-VEGF. Conclusion: 99m Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. 99m Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24 h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 lM-1 mM) for various time periods (0-24 h) confirm its angioinhibitory action at the level of gene expression. Because of its nontoxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.

Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one electron-oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment during reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.

New blood vessel formation, known as angiogenesis, is a fundamental event in the process of tumor growth and metastatic dissemination. Due to its central role in tumor angiogenesis, the vascular endothelial growth factor (VEGF) and its receptor have been a major focus of basic research and drug development in the field of oncology, including the treatment of non-small cell lung cancer (NSCLC). Approaches targeting VEGF include monoclonal antibodies and vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs). To date, bevacizumab, an anti-VEGF recombinant humanized monoclonal antibody, is the first targeted agent that, when combined with chemotherapy, reported superior efficacy versus chemotherapy alone in the treatment of advanced NSCLC. Several angiogenetic agents are being evaluated in clinical research and some of them, such as ZD6474, sorafenib and sunitinib, seem to be promising targeted agents for the treatment of NSCLC because of evidence of antitumor activity, an excellent toxicity profile and oral administration. The complexity of the signaling process leading to cancer cell proliferation and to the acquisition of a neoplastic phenotype supports the necessity to interfere at different stages to avoid an escape of potential resistance mechanisms. Targeting multiple path-ways in tumor cells should be an effective strategy of treatment in NSCLC. Clinical trials of multiple targeted therapy may represent the second generation of studies in this setting.

Angiogenesis inhibitors have shown clinical benefits in patients with advanced cancer, but further therapeutic improvement is needed. We have previously shown that the zinc finger protein 36, C3H type-like 1 (ZFP36L1) enhances vascular endothelial growth factor (VEGF) mRNA decay through its interaction with AU-rich elements within VEGF 3 0 -untranslated region. In this study, we evaluated the possibility to develop an antiangiogenic and antitumoral strategy using the mRNA-destabilizing activity of ZFP36L1. We engineered a cell-penetrating ZFP36L1, by fusing it to the protein transduction domains (PTDs) TAT derived from HIV, or the polyarginine peptides R7 or R9. PTD-ZFP36L1 fusion proteins were expressed in bacterial cells and affinity-purified to homogeneity. TAT-, R7-and R9-ZFP36L1 were efficiently internalized into living cells and decreased both endogenous VEGF mRNA half-life and VEGF protein levels in vitro. Importantly, a single injection of R9-TIS11b fusion protein into a high-VEGF expressing tissue in vivo (in this study, the mouse adrenal gland) markedly decreased VEGF expression. We further evaluated the effect of R9-ZFP36L1 on tumor growth using Lewis Lung Carcinoma (LL/2) cells implanted subcutaneously into nude mice. Intratumoral injection of R9-ZFP36L1 significantly reduced tumor growth and markedly decreased the expression of multiple angiogenic and inflammatory cytokines, including VEGF, acidic fibroblast growth factor, tumor necrosis factor a, interleukin (IL)-1a and IL-6, with a concomitant obliteration of tumor vascularization. These findings indicate that R9-ZFP36L1 fusion protein may represent a novel antiangiogenic and antitumoral agent, and supports the emerging idea that modulation of mRNA stability represents a promising therapeutic approach to treat cancer.

Microsomal prostaglandin (PG) E synthase 1 (mPGES-1) is a major PGE synthase and has recently been reported to be expressed at high levels in several cancer types. We previously reported that the PGE receptor EP3 is expressed in bone marrow (BM) derived cells, enriched in stromal tissue, and enhances the potential for tumor angiogenesis. In the present study, we examined the role of mPGES-1-expressing BM cells on tumor angiogenesis using BM chimeric mice. After lethal radiation, wild-type (WT) BMs were excised and replaced with BM cells isolated from mPGES-1 knockout mice (mPGES-1 À/À ). Lewis lung carcinoma cell were implanted subcutaneously and the levels of neoangiogenesis were measured tumor growth in mPGES-1 À/À BM chimeric mice was significantly reduced compared to that observed in WT BM chimeric mice. Tumor-associated angiogenesis as measured by histological analysis was localized to tumor stroma, and was significantly lower in mPGES-1 À/À BM chimeric mice compared to that in WT BM chimeric mice. Tumor sections probed by immunohistochemistry revealed that vascular endothelial growth factor (VEGF) that was present in the stromal tissue was markedly reduced in mPGES-1 À/À BM chimeric mice compared to WT BM chimeras. These results suggest that the recruitment of mPGES-1expressing BM cells to tumor-associated stromal tissue is crucial for tumor growth and angiogenesis, and correlates with gene expression of host VEGF in stroma. Taken together, these data suggest that regulation of mPGES-1-expressing BM cell recruitment to the site of primary tumors may be a novel strategy for the treatment of solid tumors. ß

Despite major advances in the field of tumor angiogenesis, relatively little attention has been paid to the permeability of blood vessels in tumors. The leakiness of tumor vessels is well documented in experimental tumor models and in human cancer, but the mechanism is poorly understood, as are the implications to the rate of cancer growth, predisposition to metastasis, and delivery of macromolecular therapeutics to tumor cells. Sixteen experts in the fields of cancer biology and vascular biology gathered at the William Guy Forbeck "Focus on the Future" Conference to discuss this topic. The meeting was the first of its kind focused on the significance of blood vessel leakiness in tumors. The participants discussed the cellular basis of tumor vessel leakiness, endothelial barrier function of blood vessels, monitoring tumor vessel leakiness, mediators of endothelial leakiness, consequences of tumor vessel leakiness, genomic analysis of vascular targets, targeting drugs to tu...

The vascular basement membrane (BM) is a thin and dense cross-linked extracellular matrix layer that covers and protects blood vessels. Understanding how cells cross the physical barrier of the vascular BM will provide greater insight into the potentially critical role of vascular BM breaching in cancer extravasation, leukocyte trafficking and angiogenic sprouting.
In the last year, new evidence has mechanistically linked the breaching of vascular BM with the formation of specific cellular micro-domains known as podosomes and invadopodia. These structures are specialized cell-matrix contacts with an inherent ability to degrade the extracellular matrix. Specifically, the formation of podosomes or invadopodia was shown as an important step in vascular sprouting and tumor cell extravasation, respectively. Here, we review and comment on these recent findings and explore the functions of podosomes and invadopodia within the context of pathological processes such as tumor dissemination and tumor angiogenesis.

Federal University of Minas Gerais – Belo Horizonte/MG, Brazil PhD degree in Biological Science: Physiology All the experimental part of the thesis were done at Max Delbrück Center for Molecular Medicine (MDC-Berlin, Germany) as part of a collaborative project between Brazil and Germany (CAPES/PROBAL/ DAAD) Federal University of Minas Gerais – Belo Horizonte/MG, Brazil Master degree in Biological Science: Physiology

Angiogenesis is the development of new blood vessels from the existing vascular bed. In normal conditions this tightly regulated process occurs only during embryonic development, the female reproductive cycle and wound repair. In contrast, in pathological conditions such as malignant growth, atherosclerosis and diabetic retinopathy, angiogenesis becomes persistent due to an imbalance in the interplay between the positive and negative regulatory signals controlling the process. Thus, the control of tumor neovascularization may lead to new therapeutic approaches. Indeed, several anti-angiogenic drugs are currently undergoing preclinical characterization and/or clinical investigation. Recent achievement has clarified the mechanisms of action leading to pathological angiogenesis and has highlighted the role of hypoxia, growth factors, growth factor-receptors, enzymes and cell adhesion molecules involved in the process. This knowledge has permitted the design of receptor antagonists, adh...

Diffuse optical imaging and spectroscopy use near-infrared light to derive physiological parameters such as total hemoglobin concentration and tissue oxygen saturation. Numerous clinical studies have been carried out, either using stand-alone optical methods or in combination with alternative imaging techniques. Studies have demonstrated that diffuse optical imaging and spectroscopy are able to distinguish malignant lesions from benign tissues. Breast cancer is characterized by an increase in total hemoglobin and a decrease in tissue oxygen saturation. Benign lesions such as cysts and fibroadenomas have also been studied, with less conclusive results. As diffuse optical imaging and spectroscopy do not use ionizing radiation, they are a suitable technique for performing repeated scans, such as for monitoring treatment response. This provides a unique functional and dynamic imaging method that reflects changes in tumor angiogenesis and hypoxia. When breast cancers are treated with primary medical therapy, this can result in a selective antiangiogenic effect that could help predict response to treatment earlier than by assessment of tumor size. Diffuse optical imaging and spectroscopy have been used to scan women at several points prior to and during their neoadjuvant chemotherapy treatment, with images and data showing physiological changes in the tumor in response to treatment. In the women who respond to therapy, the total hemoglobin concentration decreases and the level of oxygenation increases in the tumor over the course of the treatment. It is possible to predict a response to treatment as little as 4 days after the start of treatment. These findings demonstrate that optical techniques could play a role in the monitoring of changes in angiogenesis, apoptosis and hypoxia due to neoadjuvant chemotherapy.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that process growth factors, growth factor binding proteins, cell surface proteins, degrade extracellular matrix (ECM) components and thereby play a central role in tissue remodeling and tumor progression. Membrane-type matrix metalloproteinases (MT-MMPs) are a recently discovered subgroup of intrinsic plasma membrane proteins. Their functions have been extended from pericellular proteolysis and control of cell migration to cell signaling, control of cell proliferation and regulation of multiple stages of tumor progression including growth and angiogenesis. This review sheds light on the new functions of MT-MMPs and their inhibitors in tumor development and angiogenesis, and presents recent investigations that document their influence on various cell functions.

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.

Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs. The glioma CM effect was significantly reduced by cytosolic (cPLA 2 ) and Ca ++ -independent (iPLA 2 ) phospholipase A 2 , cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA 2 and iPLA 2 enzyme activities and phosphorylation of cPLA 2 , significantly stimulated after 24 h CM co-incubation, were attenuated by PLA 2 , PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA 2 , phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA 2 was in parallel observed. Electroporation of anti-iPLA 2 and cPLA 2 antibodies and siRNAs directed against iPLA 2 and cPLA 2 significantly inhibited cell proliferation and migration. Incubation of CM-or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA 2 and iPLA 2 protein levels. The findings suggest that PLA 2 activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA 2 is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis.

Chemokines are a large group of low molecular weight cytokines that are known to selectively attract and activate different cell types. Although the primary function of chemokines is well recognized as leukocyte attractants, recent evidences indicate that they also play a role in number of tumor-related processes, such as growth, angiogenesis and metastasis. Chemokines activate cells through cell surface seven trans-membranes, G-proteincoupled receptors (GPCR). The role played by chemokines and their receptors in tumor pathophysiology is complex as some chemokines favor tumor growth and metastasis, while others may enhance anti-tumor immunity. These diverse functions of chemokines establish them as key mediators between the tumor cells and their microenvironment and play critical role in tumor progression and metastasis. In this review, we present some of the recent advances in chemokine research with special emphasis on its role in tumor angiogenesis and metastasis.

Viscum album (VA) preparations consist of aqueous extracts of different types of lectins of VA. Mistletoe lectins have both cytotoxic and immunomodulatory properties that support their study for the development for cancer therapy. However, the mechanisms of the anti- tumoral properties in vivo of mistletoe lectins are not fully understood. Because endothelial cells (EC) play a pivotal role in tumor angiogenesis, we tested the hypothesis that VA extracts induce endothelial cell death and apoptosis. We investigated the effect of various VA preparations on both human venous endothelial cell (HUVEC) and immortalized human venous endothelial cell line (IVEC) using morphologic assessment of EC, FACScan analysis after propidium iodine and annexin V labeling, and detection of cleavage of poly(A)DP-ribose polymerase (PARP). All tested VA preparations, except Iscador P, were cytotoxic in IVEC. Apoptosis, assessed by morphologic examination, annexin V labeling, and Western blot analysis for PA...

Cell adhesion and migration are essential for embryonic development, tissue regeneration, but also for tumor development. The physical link between the extracellular matrix (ECM) and the actin cytoskeleton is mainly mediated by receptors of the integrin family. Through signals transduced upon integrin ligation to ECM proteins, this family of proteins plays key roles in regulating tumor growth and metastasis as well as tumor angiogenesis. During melanoma development, changes in integrin expression, intracellular control of integrin functions and signals perceived from integrin ligand binding impact upon the ability of tumor cells to interact with their environment and enable melanoma cells to convert from a sessile, stationary to a migratory and invasive phenotype. Antagonists of several integrins are now under evaluation in clinical trials to determine their potential as therapeutics for malignant melanoma and other kinds of cancer.

Vessel co-option is the movement of cancer cells towards and along the pre-existing vasculature and is an alternative to angiogenesis to gain access to nutrients. Vessel co-option has been shown as a strategy employed by some glioblastoma (GBM) cells to invade further into the brain, leading to one of the greatest challenges in treating GBM. In GBM, vessel co-option may be an intrinsic feature or an acquired mechanism of resistance to anti-angiogenic treatment. Here, we describe the histological features and the dynamics visualized through intravital microscopy of vessel co-option in GBM, as well as the molecular players discovered until now. We also highlight key unanswered questions, as answering these is critical to improve understanding of GBM progression and for developing more effective approaches for GBM treatment.

Triptolide is confirmed to suppress angiogenesis of anaplastic thyroid carcinoma. Here we further expound the precise mechanism involved in this activity. Triptolide downregulated nuclear factor kappa B (NF-κB) pathway and its targeting genes associated with endothelial cell mobilization in human umbilical vein endothelial cells (HUVECs) and impaired VEGF expression in thyroid carcinoma TA-K cells. Furthermore, both triptolide and the conditioned medium from triptolide-treated TA-K cells (CMT) significantly attenuated proliferation, migration and tube formation of HUVECs. In vivo, triptolide inhibited TA-K cellinduced tumor growth, vascular formation and VEGF expression. Our data establish that triptolide inhibits tumor angiogenesis by the dual action on vascular endothelial cells and tumor cells, thus providing a novel and overall explanation for the anti-angiogenesis action of triptolide. The multicellular targets emphasize triptolide as a high-performance and potential angiogenesis inhibitor.

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC).