Ana R. Quesada

Angiogenesis and signal transduction in endothelial cells.

Endothelial cells receive multiple information from their environment that eventually leads them to progress along all the stages of the process of formation of new vessels. Angiogenic signals promote endothelial cell proliferation, increased resistance to apoptosis, changes in proteolytic balance, cytoskeletal reorganization, migration and, finally, differentiation and formation of a new vascular lumen. We aim to review herein the main signaling cascades that become activated in angiogenic endothelial cells as well as the opportunities of modulating angiogenesis through pharmacological interference with these signaling mechanisms. We will deal mainly with the mitogen-activated protein kinases pathway, which is very important in the transduction of proliferation signals; the phosphatidylinositol-3-kinase/protein kinase B signaling system, particularly essential for the survival of the angiogenic endothelium; the small GTPases involved in cytoskeletal reorganization and migration; and the kinases associated to focal adhesions which contribute to integrate the pathways from the two main sources of angiogenic signals, i.e. growth factors and the extracellular matrix.

Relevance of glutamine metabolism to tumor cell growth

Tumor cells are characterized as rapidly dividing cells, and consequently they need a constant supply of both energy and nitrogen substrates. To resolve their energy requirements, they are able to use virtually any substrate: glucose [see 1 for a review; 2-4], lipids [5-7], ketone bodies [3], even amino acids [2-4, 8-10]. Nevertheless, the glucose and amino acid consumption by malignant tumor cells overcomes their own needs for their metabolic requirements; thus, tumor cells apparently waste glucose and amino acids without any profit [1, ll]. In this context, tumor has been described as a trap for glucose and nitrogen [12-13]. Tumors compete with the host for glucose [13-14]; this competence results in a progressive hypoglycemia [15] and host hepatic glycogen depletion [13]. In the same way, tumors compete for nitrogen compounds; this process produces in the host a negative nitrogen balance and a characteristic weight loss, and in the tumor a reciprocal nitrogen increase. The biochemical mechanisms underlying these phenomena still remain unclear. There is consensus that tumors increase protein degradation and reduce protein synthesis in the host tissues [16]. Alanine and glutamine are two efficient vehicles for the transport of nitrogen and carbon-skeletons between the different tissues in the living organism [17-18]. When a tumor develops, there is a net flux of amino acids from host tissues to the tumor [19]. Since ammonium ions are very toxic for most of the cells, glutamine is the physiological non-toxic ammonium vehicle between different mammalian tissues; therefore, glutamine is the main source of nitrogen for tumor cells [2, 20-21]. Thus, the presence of a tumor must produce great changes in the metabolism of glutamine in host tissues in such a way that host nitrogen metabolism is accomodated to tumor enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria, where it is metabolized [21]. This implies two transport processes: the transport of glutamine across the plasma membrane and across the inner mitochondrial membrane. Once glutamine has been incorporated into tumor cells, this amino acid is quickly metabolized [12, 16, 19].

Antiangiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge

(+)‐Aeroplysinin‐1, an antibacterial brominated compound produced by certain sponges, was selected during a blind high‐throughput screening for new potential antiangiogenic compounds obtained from marine organisms. In a variety of experimental systems, representing the sequential events of the angiogenic process, aeroplysinin‐1 treatment of endothelial cells resulted in strong inhibitory effects. Aeroplysinin‐1 inhibited the growth of endothelial cells in culture and induced endothelial cell apoptosis. Capillary tube formation on Matrigel was completely abrogated by addition of aeroplysinin‐1 at the low micromolar range. Aeroplysinin‐1 also exhibited a clear inhibitory effect on the migration capabilities of endothelial cells. Zymographic assays showed that aeroplysinin‐1 treatment produced a decrease in the concentration of matrix metalloproteinase‐2 and urokinase in conditioned medium from endothelial cells. Finally, aeroplysinin‐1 exhibited a dose‐dependent inhibitory effect on the in vivo chorioallantoic membrane assay, showing potent apoptosis‐inducing activity in the developing endothelium. The in vivo inhibition of angiogenesis by aeroplysinin‐1 was confirmed by the Matrigel plug assay. Together, our data indicate that aeroplysinin‐1 is a compound that interferes with key events in angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis‐related pathologies.

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.

Histamine, polyamines, and cancer

Mammalian ornithine decarboxylase and histidine decarboxylase present common structural and functional features, and their products also share pharmacological and physiological properties. Although accumulated evidence pointed for years to a direct involvement of polyamines and histamine in tumour growth, it has been only in the last few years that new molecular data have contributed to the clarification of this topic. The aim of this commentary is to review the molecular grounds of the role of histamine and polyamines in cancer and to point to possible directions for future research in emerging areas of interest.

Nitrogen Metabolism in Tumor Bearing Mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.

Hyperforin, a bio‐active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process

Hyperforin, a phloroglucinol derivative found in St. Johns wort related mainly to its antidepressant effects, has been reported recently to induce apoptosis in tumour cells and to inhibit cancer invasion and metastasis. We show that hyperforin inhibits angiogenesis in vitro in bovine aortic endothelial cells and in vivo in the chorioallantoic membrane assay. In a variety of experimental systems representing the sequential events of the angiogenic process, hyperforin treatment of endothelial cells resulted in strong inhibitory effects. Hyperforin inhibited the growth of endothelial cells in culture. Capillary tube formation on Matrigel was abrogated completely by addition of hypeforin at the low micromolar range. Hyperforin also exhibited a clear inhibitory effect on the invasive capabilities of endothelial cells. Zymographic assays showed that hyperforin treatment produced a complete inhibition of urokinase and a remarkable inhibition of matrix metalloproteinase 2. Our data indicates that hyperforin is a compound that interferes with key events in angiogenesis, confirming the recent and growing evidence about a potential role of this compound in cancer and metastasis inhibition and making it a promising drug for further evaluation in the treatment of angiogenesis‐related pathologies.

Anti-angiogenic and anti-inflammatory properties of kahweol, a coffee diterpene.

Background Epidemiological studies have shown that unfiltered coffee consumption is associated with a low incidence of cancer. This study aims to identify the effects of kahweol, an antioxidant diterpene contained in unfiltered coffee, on angiogenesis and key inflammatory molecules. Methodology/Principal Findings The experimental procedures included in vivo angiogenesis assays (both the chicken and quail choriallantoic membrane assay and the angiogenesis assay with fluorescent zebrafish), the ex vivo mouse aortic ring assay and the in vitro analysis of the effects of treatment of human endothelial cells with kahweol in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Additionally, two inflammation markers were determined, namely, the expression levels of cyclooxygenase 2 and the levels of secreted monocyte chemoattractant protein-1. We show for the first time that kahweol is an anti-angiogenic compound with inhibitory effects in two in vivo and one ex vivo angiogenesis models, with effects on specific steps of the angiogenic process: endothelial cell proliferation, migration, invasion and tube formation on Matrigel. We also demonstrate the inhibitory effect of kahweol on the endothelial cell potential to remodel extracellular matrix by targeting two key molecules involved in the process, MMP-2 and uPA. Finally, the anti-inflammatory potential of this compound is demonstrated by its inhibition of both COX-2 expression and MCP-1 secretion in endothelial cells. Conclusion/Significance Taken together, our data indicate that, indeed, kahweol behaves as an anti-inflammatory and anti-angiogenic compound with potential use in antitumoral therapies. These data may contribute to the explanation of the reported antitumoral effects of kahweol, including the recent epidemiological meta-analysis showing that drinking coffee could decrease the risk of certain cancers.

Anti-angiogenic properties of carnosol and carnosic acid, two major dietary compounds from rosemary

BackgroundThe use of rosemary (Rosmarinus officinalis) leaves and their constituents as a source of dietary antioxidants and flavoring agents is continuously growing. Carnosol and carnosic acid, two major components of rosemary extracts, have shown activity for cancer prevention and therapy.Aim of the studyIn this study, we investigate the cytotoxic and anti-angiogenic activities of carnosol and carnosic acid, in order to get further insight into their mechanism of action.ResultsOur results demonstrate that the mentioned diterpenes inhibit certain functions of endothelial cells, namely, differentiation, proliferation, migration and proteolytic capability. Our data indicate that their growth inhibitory effect, exerted on proliferative endothelial and tumor cells, could be due to, at least in part, an induction of apoptosis. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallantoic membrane assay.ConclusionsThe anti-angiogenic activity of carnosol and carnosic acid could contribute to the chemopreventive, antitumoral and antimetastatic activities of rosemary extracts and suggests their potential in the treatment of other angiogenesis-related malignancies.

Evaluation of the anti-angiogenic effect of aloe-emodin.

Abstract.The present study identified aloe-emodin (AE, a hydroxyanthraquinone from Aloe vera and other plants) as a new anti-angiogenic compound with inhibitory effects in an in vivo angiogenesis assay and evaluates its effects on specific key steps of the angiogenic process. AE inhibits endothelial cell proliferation, but this effect is not cell specific, since AE also inhibits tumor cell proliferation. Cell migration and invasion are not remarkably affected by AE. On the other hand, AE has different effects on endothelial and tumor cell gelatinases. Two main targets of the pharmacological action of AE as an anti-angiogenic compound seem to be urokinase secretion and tubule formation of endothelial cells. Finally, AE produces a remarkable photocytotoxic effect on tumor cells. Taken together, our data indicate that AE can behave both as an anti-tumor and an anti-angiogenic compound and suggest that AE could be a candidate drug for photodynamic therapy.