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Human pluripotent stem cell culture conditions are constantly being optimized, thus providing insight to the environmental cues that affect cell choices. A wide variety of media, coating materials, and substrates is now available for use, serving different scientific needs. Factors such as material stiffness, roughness, and topography are being recognized to contribute or even direct the acquisition of specific phenotypes. Here, we describe the use of patterned silicon substrates coated with Matrigel for the propagation and differentiation of human pluripotent stem cells.

Human embryonic stem cells exhibit great potential as a therapeutic tool in regenerative medicine due to their self-renewal and trilineage differentiation capacity. Maintaining this unique cellular state has been shown to rely primarily on the Activin A / TGFβ signaling pathway. While most conventional culture media are supplemented with TGFβ, in the current study we utilize a modified version of the commercially available mTeSR1, substituting TGFβ for Activin A in order to preserve pluripotency. (1) Cells cultured in ActA-mTesR express pluripotency factors NANOG, OCT4 and SOX2 at comparable levels with cells cultured in TGFβ-mTeSR. (2) ActA-mTeSR cultured cells retain a physiological karyotype. (3) Cells in ActA-mTeSR maintain their trilineage differentiation capacity as shown in the teratoma formation assay. This system can be used to dissect the role of Activin A, downstream effectors and signaling cascades in human embryonic stem cell responses.

Polarization resolved second harmonic generation has been used to study the evolution of cell induced collagen remodeling in 3D volumes of collagen-based scaffolds seeded endothelial cell, at increasing days in vitro.

The two-dimensional (2D) monoculture does not recapitulate the three-dimensional (3D) environment of in vivo vasculature. Endothelial cell (ECs) spheroids have been introduced as a 3D in vitro model exhibiting angiogenic responses and sprouting behavior in vivo. However, mural cells (MCs), including the phenotypic range from pericytes to smooth muscle cells (SMCs), play an essential organotypic role in vascular homeostasis (vascular remodeling/stabilization) and disease. Therefore, the development of 3D cell structures (small-scale vascular organoids) containing both ECs and MCs subtypes provide enhanced cell–cell interactions that closely mimic the natural/physiological vascular microenvironment with beneficial effects on cell survival, phenotypic stability, and function. Due to limitations of isolation/expansion of primary MCs as well as their phenotypic plasticity during in vitro culture, human pluripotent stem cells (hPSCs) (human induced pluripotent stem cells-hiPSCs and human embryonic stem cells-hESCs) are a source for the generation of defined MC populations.

Ligand–receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A–receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.

Background Cardiac repair strategies are being evaluated for myocardial infarctions, but the safety issues regarding their arrhythmogenic potential remain unresolved. By utilizing the in-vivo rat model, we have examined the medium-term electrophysiologic effects of a biomaterial scaffold that has been cellularized with spheroids of human adipose tissue, derived from mesenchymal stem cells and umbilical vein endothelial cells. Methods Mesenchymal stem cells, which exhibit adequate differentiation capacity, were co-cultured with umbilical vein endothelial cells and were seeded on an alginate based scaffold. After in-vitro characterization, the cellularized scaffold was implanted in (n=15) adult Wistar rats 15 min post ligation of the left coronary artery, with an equal number of animals serving as controls. Two weeks thereafter, monophasic action potentials were recorded and activation-mapping was performed with a multi-electrode array. An arrhythmia score for inducible ventricular tachyarrhythmias was calculated after programmed electrical stimulation. Results The arrhythmia score was comparable between the treated animals and controls. No differences were detected in the local conduction at the infarct border and in the voltage rise in monophasic action potential recordings. Treatment did not affect the duration of local repolarization, but tended to enhance its dispersion. Conclusions The fabricated bi-culture cellularized scaffold displayed favorable properties after in-vitro characterization. Medium-term electrophysiologic assessment after implantation in the infarcted rat myocardium revealed low arrhythmogenic potential, but the long-term effects on repolarization dispersion will require further investigation.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates IRE1α, ATF6, and PERK cascades, leading to a transcriptional/translational response known as unfolded protein response (UPR). Here we show that VEGF activates UPR mediators through a PLCγ-mediated crosstalk with the mTORC1 complex without accumulation of unfolded proteins in the ER. Activation of ATF6 and PERK contributes to the survival effect of VEGF on endothelial cells (ECs) by positively regulating mTORC2-mediated phosphorylation of AKT on Ser473, which is required for full activity of AKT. Low levels of CHOP allow ECs to evade the proapoptotic effect of this UPR product. Depletion of PLCγ, ATF6, or eIF2α dramatically inhibited VEGF-induced vascularization in mouse Matrigel plugs, suggesting that the ER and the UPR machinery constitute components of the VEGF signaling circuit that regulates EC survival and angiogenesis, extending their role beyond adaptation to ER stress.

Solid tumours initiate angiogenesis to support their growth by producing growth factors such as VEGF. Depriving the tumour of the excessive vessels that support its growth became the target for developing anti-angiogenic agents that could provide, in combination with chemotherapy, improved anti-cancer treatment. Naturally most agents targeted VEGF and its signalling cascades. Almost 10 years have lapsed since the first anti-angiogenic drug approved by the FDA in 2004 (a humanized antibody inhibiting VEGF-A) and several other agents followed afterwards. There is sufficient accumulated experience to conclude that the clinical results of anti-angiogenic therapy are very modest resulting in moderate improvement in overall survival. Moreover, the clinical outcome is associated with the development of resistance to the anti-angiogenic agent and the increased risk of invasion and metastasis. The initial expectations are, as yet, unfilled, and the entire concept and strategy of the anti-angiogenic intervention in cancer requires re-evaluation. In the present Mini Review we discuss these issues emphasising the underlying molecular mechanisms.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

Hydrogen peroxide (H 2 O 2 ) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular “labile iron” were able to modulate signal transduction in H 2 O 2 –induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H 2 O 2 –induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H 2 O 2 -mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H 2 O 2 , influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H 2 O 2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions.

SARA, an early endosomal protein, plays a key role in TGFβ signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFβ receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208–1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA- and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFβ and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.

We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics. Cancer Invest

The integrated roles of small GTPases in executing the transforming growth factor beta (TGFbeta) signaling pathway have attracted increasing attention in recent years. In this review, we summarize recent findings on TGFbeta signaling during receptor endocytosis, Smad trafficking and actin cytoskeleton remodeling, and emphasize the role of small GTPases in these processes. First, we give an overview of the different endocytic routes taken by TGFbeta receptors, their impact on active TGFbeta signaling versus degradation and their regulation by the small GTPases Rab, RalA/Ral-binding protein 1 and Rap2. Second, we focus on the mechanisms and regulation of Smad trafficking in the cytoplasm, through the nuclear pores and into the nucleus, and the contribution of Ran GTPase to these events. Third, we summarize the role of Rho small GTPases in early and late cytoskeleton remodeling in various cell models and diseases, and the positive and negative cross-talk between Rho GTPases and the TGFbeta/Smad pathway. The biological significance of this exciting research field, the perspectives and critical open questions are discussed.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, KDeff, values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 ( KDeff345 nM). VEGF administration affected differentially the KDeffvalues of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation.

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFA-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1R206H) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1R206H mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1R206H causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP.

The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transformation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angiogenic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof.

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID-GC-MS-SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5-5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results. (C) 2004 Elsevier Ltd. All rights reserved. Journal of Steroid Biochemistry and Molecular Biology

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC50 of about 5 μmol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3′-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G1 progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.

The Myc family of transcription factors (c-Myc, N-Myc, and L-Myc) are central mediators of many different critical cellular processes (1–4). Additionally, alteration of Myc is one of the most common genetic abnormalities in human cancers, including neuroblastoma (5). Unfortunately, the Myc protein has proven to be difficult to target directly in anticancer strategies. Myc activity is determined not only by its DNA binding sequences but also by local chromatin histone methylation status (6). Increased H3K4 methylation (active mark), but not H3K27 methylation (repressive mark), is characteristic of Myc-binding sites (6), which is consistent with recent studies that transcriptionally active epigenetic modifications mark genomic occupancy of Myc (7–9). An emerging theory is that Myc acts as a transcriptional amplifier, increasing transcription of genes that are already turned on, while genes not actively being transcribed are unaffected (8,9). However, two recent papers clearly demonstrated that Myc is also able to repress transcription (10,11). Nevertheless, Myc appears to be required for the induction and maintenance of normal histone methylation patterns associated with active chromatin in certain settings (12). Genetic disruption of MYCN in neural progenitors alters histone modifications that result in an increase in repressive H3K9me2/me3 marks and heterochromatinization, decreased DNA accessibility, and, ultimately, silencing of genes involved in Myc signaling (12), suggesting that Myc is required to maintain a euchromatin configuration by modifying histone methylation to facilitate its function. Similar results have been shown in cancer cells in which 12-hour inactivation of c-Myc resulted in global chromatin remodeling including elevated H3K9me3 (13). However, how H3K9me3/me2 is involved in mediating Myc function is not well understood. Additionally, the genetic alteration at glycine 34 (G34) of histone H3F3A, which is believed to affect the adjacent H3K36 methylation-related function, results in statistically significant N-Myc expression in pediatric glioblastoma (14), further supporting the biological connection between Myc activity and histone methylation. The JmjC domain-containing histone demethylases, which are responsible for reversing most of the histone methyl marks in the human genome, play important roles in a number of physiologic processes such as stem cell maintenance, cell cycle regulation, and oncogenesis (15–18). Besides somatic mutations identified in the genes encoding histone demethylases such as UTX (19,20) and JARID1C (21), aberrant expression of histone demethylases has been observed in various cancers (16,18). KDM4B/JMJD2B and KDM4C/JMJD2C, which catalyze the demethylation of the repressive H3K9me3/me2 mark, are amplified in medulloblastoma (22), malignant peripheral nerve sheath tumor (23), and squamous cell carcinoma (24), suggesting a role in the pathophysiology of these tumors. However, the contribution of these histone demethylases to the activity of oncogenic drivers such as Myc is uncertain. Additionally, the opportunity to exploit this relationship as a therapeutic strategy has yet to be explored.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.

Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread. Eur J Biochem

The consumption of a plant-based diet can prevent the development and progression of chronic diseases associated with extensive neovascularization, including the progression and growth of solid malignant tumours. We have previously shown that the plant-derived isoflavonoid genistein is a potent inhibitor of cell proliferation and in vitro angiogenesis. Moreover, the concentration of genistein in the urine of subjects consuming a plant-based diet is 30-fold higher than that in subjects consuming a traditional Western diet. We have also reported that certain structurally related flavonoids are more potent inhibitors than genistein. Indeed, 3-hydroxyflavone, 3',4'-dihydroxyflavone, 2',3'-dihydroxyflavone, fisetin, apigenin and luteolin inhibit the proliferation of normal and tumour cells as well as in vitro angiogenesis at half-maximal concentrations in the lower micromolar range. The wide distribution of isoflavonoids and flavonoids in the plant kingdom, together with their anti-angiogenic and anti-mitotic properties, suggest that these phytoestrogens may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumours.

The classical view that endocytosis serves only for growth factor receptor degradation and signaling termination has recently been challenged by an increasing number of reports showing that various growth factor receptors such as epidermal growth factor receptor (EGFR) continue to activate downstream signaling molecules en route to lysosomes prior to their degradation. Moreover, the trafficking route that the ligand-receptor complexes follow to enter the cell is mutually interconnected with the final signaling output. Endosomal resident effector proteins are compartmentalized and regulate the signaling and trafficking of the ligand-bound receptor complexes. Smad anchor for receptor activation (SARA) is an early endosomal protein facilitating TGF-β signaling cascade. Even though SARA was identified as an adaptor protein that regulates SMAD2 activation and TGF-β signal propagation, an increasing number of reports in various systems describe SARA as a trafficking regulator. Recently, SARA has been shown to interact with the E3 ubiquitin ligase RNF11 (RING finger protein 11) and members of the ESCRT-0 (endosomal sorting complex required for transport) complex functionally participating in the degradation of EGFR.

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

We present a method for the quantitative determination of the phytoestrogens formononetin, biochanin A, daidzein, genistein, and coumestrol and simultaneously the lignans secoisolariciresinol (SECO) and matairesinol in plant-derived foods. These compounds are measured by isotope dilution gas chromatography-mass spectrometry in the selected ion monitoring mode (ID/GC/MS/SIM) using synthesized deuterated internal standards for the correction of losses during the procedure. A three-step hydrolysis--a rehydration with distilled H2O, followed by enzymatic and acid hydrolysis--has been applied in order to convert the diphenolic glycosides into their respective aglycones. Purification and separation are carried out in two ion-exchange chromatographic steps followed by derivatization and GC-MS. The within-assay imprecision values vary 3.1-9.6% and the between-assay imprecision 7.0-21.2%. The mean recovery of authentic standards processed through the whole procedure varied from 95.5 to 105.5%. Values for some different food samples are presented. The simultaneous determination of the biologically most interesting phytoestrogens and lignans in foods has not been carried out previously and the method will be useful for screening of important foods in populations with different risk of cancer and coronary heart disease, and for metabolic studies. Anal Biochem

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFA-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.

1. The effect of the nitric oxide (NO)-producing nitrovasodilators isosorbide mononitrate (ISMN) and isosorbide dinitrate (ISDN) were assessed on (a) the in vivo model of angiogenesis of the chick chorioallantoic membrane (CAM) and (b) on the growth and metastatic properties of the Lewis Lung carcinoma (LLC) in mice. 2. Isosorbide 5-mononitrate (ISMN) and isosorbide dinitrate (ISDN), inhibited angiogenesis in the CAM dose-dependently. ISMN was more potent in inhibiting this process. Both compounds were capable of completely reversing the angiogenic effect of alpha-thrombin. These effects of ISMN and ISDN on angiogenesis were comparable to those previously observed with sodium nitroprusside which generates NO non-enzymatically. 3. Mice, implanted intramuscularly with LLC, received daily i.p. injections of ISMN for 14 days resulting in a significant decrease in the size of the primary tumour and a reduction in the number and size of metastatic foci in the lungs. ISDN had a similar but less pronounced effect than that observed with ISMN. 4. Addition of ISMN or ISDN to cultures of bovine, rabbit and human endothelial cells and to cultures of LLC cells had no effect on their growth characteristics. 5. These results indicate that ISMN and ISDN inhibit angiogenesis and tumor growth and metastasis in an animal tumour model. The possibility should therefore be considered that these nitrovasodilators which are widely used therapeutically and have well characterized pharmacological profiles, may also possess antitumour properties in the clinic.

Diphenolic compounds belonging to the classes of lignans and isoflavonoids have been identified in urine of man and animals, including the chimpanzee. Some of these compounds, formed by intestinal bacteria from plant lignans and phytoestrogens, have been shown in animal studies to exhibit biological activities that suggest they could function as cancer-protective compounds. The effect of diet on urinary excretion of these compounds in the adult male chimpanzee has been studied. It was found that the chimpanzees consuming their regular food excreted large amounts of the isoflavonoid phytoestrogens, equol (mean ± SE) (127.5 ± 34.0 nmol/mg cr.) and daidzein (20.7 ± 9.0 nmol/mg cr.) and the lignan, enterolactone(14.1 + 3.5 nmol/mg cr.). Small amounts of the lignan, enterodiol, (0.4 ± 0.2 nmol/mg cr.) were also excreted. On all other four test diets (high protein, high carbohydrate, high vegetable, and high fat), the excretion was less, particularly on a high fat diet where the excretion of all diphenolic compounds was reduced by more than 90% to a level observed in omnivorous human subjects or women with breast cancer. These results suggest that diet profoundly influences the excretion of both animal lignans and phytoestrogens in urine. Because non-human primates are particularly resistant to mammary and genital carcinoma on estrogen treatment, the present data suggest that the very high levels of phytoestrogens and lignans as found during exposure to the regular diet may partially account for why these primates are so resistant to hormonal manipulations to induce cancer.

Estrone sulphate (E1S) may be an important estrogen source in breast cancers, particularly in postmenopausal women. Recent studies have shown that tamoxifen inhibits the uptake and metabolism of E1S to estradiol (E2) in cell cultures. To evaluate a possible influence of tamoxifen on E1S dispositionin vivo, we measured plasma levels of E1S together with unconjugated estrogens (E1 and E2), androgens (T, A, DHEA and DHEAS), SHBG, FSH and LH in 32 postmenopausal breast cancer patients before and during tamoxifen treatment. In a subgroup of 10 patients, we measured 24 h urinary excretion of estrogen metabolites to evaluate the influence of tamoxifen treatment on estrogen metabolism and total estrogen production. Tamoxifen increased plasma levels of E1 S (mean increase of 18.1%, P < 0.05) and the ratio of E1/SE1 (mean increase of 25.7%, P < 0.01) and E1/SE2 (mean increase of 34.7%, P < 0.0005). No significant change in plasma E1 was seen, but plasma E2 was reduced (mean reduction of 12.1%, P < 0.005). The fall in plasma E2 was probably secondary to a fall in plasma T (mean reduction of 11.9%, P < 0.05) due to a reduced ovarian excretion of this androgen. The mechanism may be a reduced gonadotrophin stimulation of the ovary, as plasma FSH and LH fell by mean values of 45.5 and 48.1%, respectively (P < 0.0001 for both). The increase in plasma E1S was accompanied by a reduced ratio of 2OHE1/E1 in urine (mean reduction of 38.2%, P < 0.025) indicating reduced 2-hydroxylation. Possible mechanisms for these alterations are discussed.

OBJECTIVE Oestrogens, androgens and anti-endocrine drugs such as tamoxifen and aminoglutethimide influence plasma insulin-like growth factor-I (IGF-I). IGF-I, in turn, has been found to stimulate the peripheral aromatase in vitro. The aim of this study was to examine relations between sex hormones, IGF-I and insulin-like growth factor binding protein-1 (IGFBP-1) in post-menopausal women with breast cancer. DESIGN To measure plasma sex steroids, sex hormone binding globulin (SHBG), IGF-I, IGFBP-1, insulin and urinary oestrogen metabolites in post-menopausal women with breast cancer not receiving any endocrine therapy. PATIENTS Thirty-two patients had fasting blood samples obtained between 0800 and 1000 h. A sub-group of 10 patients had 24-hour urine oestrogen metabolites determined. MEASUREMENTS Plasma steroids and proteins were measured by radioimmunoassays. Urinary oestrogens were measured by GC-MS. RESULTS SHBG correlated negatively with plasma androstenedione (P < 0.001), insulin (P < 0.001), IGF-I, height and plasma oestrone sulphate (P < 0.025 for all), but positively with plasma IGFBP-1 (P < 0.025). IGFBP-1 correlated negatively with IGF-I (P < 0.001) and the testosterone/SHBG ratio (P < 0.05). Neither IGF-I nor IGFBP-1 correlated with any of the plasma or urinary sex hormones or with the oestrone/androstenedione and oestradiol/testosterone ratios. Multivariate analysis revealed plasma SHBG to correlate positively with IGFBP-1 (P = 0.029) and negatively with insulin (P = 0.031). Plasma IGFBP-1 correlated negatively with IGF-I (P < 0.0001) but not with insulin. CONCLUSION Our results do not suggest any influence of plasma sex steroids in physiological concentrations on IGF-I or IGFBP-1 in post-menopausal breast cancer patients, nor do they indicate IGF-I at physiological concentrations influences the ratios between plasma oestrogens and their androgen precursors.

Smad anchor for receptor activation (SARA) is highly enriched on endocytic membranes via binding to phosphatidylinositol 3-phosphates through its FYVE (Fab1p-YOTB-Vps27p-EEA1) domain. SARA was originally identified as a protein that recruits non-phosphorylated SMAD2/3 to the activated TGFβ receptors for phosphorylation, but later reports suggested a regulatory role in endocytic trafficking. Here we demonstrate that the ubiquitin ligase RNF11 is a SARA-interacting protein residing on early and late endosomes, as well as the fast recycling compartment. RNF11 and SARA interact with the ESCRT-0 subunits STAM2 and Eps15b, but only RNF11 associates with the core subunit Hrs. Both gain- and loss-of-function perturbation of RNF11 and SARA levels result in delayed degradation of epidermal growth factor (EGF)-activated EGF receptor (EGFR), while loss-of-function sustained/enhanced EGF-induced ERK1/2 phosphorylation. These findings suggest that RNF11 and SARA are functional components of the ESCRT-0 complexes. Moreover, SARA interacts with clathrin, the ESCRT-I subunit Tsg101 and ubiquitinated cargo exhibiting all the properties of Hrs concerning ESCRT-0 function, indicating that it could substitute Hrs in some ESCRT-0 complexes. These results suggest that RNF11 and SARA participate structurally and functionally in the ESCRT-dependent lysosomal degradation of receptors. As a consequence, the negative influence that perturbation of RNF11 and SARA levels exerts on the lysosomal degradation of EGFRs could underscore the reported overexpression of RNF11 in several cancers. In these cancers, deficient termination of the oncogenic signaling of mutated receptors, such as the EGFRs, through suboptimal lysosomal degradation could contribute to the process of malignant transformation.

There are conflicting results on whether the 16α−hydroxylation or the 2−hydroxylation pathway in estrogen metabolism, or neither, is a risk factor of breast cancer (BC). We studied diet and measured the urinary, plasma and fecal estrogens in 7 groups of premenopausal women with presumed different risk of breast cancer. Ten women were currently healthy with a past history of BC. In this report we present some preliminary results of correlations between dietary macronutrients and estrogen pattern. Our results support the view that high 2−hydroxylation rather than high 16α−hydroxylation of estrogens is a risk factor of BC. High catecholestrogen excretion may be a marker of a Western diet. Methylation of catecholestrogens, on the other hand, may be protective for BC. However, the main risk factor of BC may be higher circulating levels of biologically active estrogens in women consuming a Western diet with secondary high urinary catecholestrogens and insufficient methylation. There appears to be a pos...

Background Increased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions. Results In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P Conclusions 6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.

The first quantitative method for the determination of both lignans and isoflavonoid phytoestrogens in plasma is presented. Using ion-exchange chromatography the diphenols are separated into two fractions 1) the biologically "active" fraction containing the free compounds + mono- and disulfates and 2) the biologically "inactive" fraction containing the mono- and diglucuronides and the sulfoglucuronides. After hydrolysis the fractions are further purified by solid phase extraction and ion exchange chromatography. Losses during the complete procedure are corrected for using radioactive estrogen conjugates during the first steps and later by adding deuterated internal standards of all compounds measured (matairesinol, enterodiol, enterolactone, daidzein, O-desmethylangolensin, equol, and genistein). The final determination is carried out by isotope dilution gas chromatography-mass spectrometry in the selected ion monitoring mode (GC/MS/SIM). The diphenols may be measured at concentrations as low as 0.2 to 1.0 nmol/l. Results of plasma analyses of all compounds in 27 pre- and postmenopausal omnivorous and vegetarian women are presented for the first time. The most important findings are that the free+sulfate fraction is low for genistein (3.8% of total), but as much as 21-25% of enterolactone and enterodiol occurs in this fraction. A good correlation between plasma and urine values was found. Total concentrations of individual compounds vary greatly between the subjects (from pmol/l to mumol/l), the vegetarians having higher values, particularly one vegan subject. The highest total enterolactone concentration value exceeded 1 mumol/l. It is concluded that a highly specific method for the assay of 3 lignans and 4 isoflavonoids in plasma has been developed. This method will be useful in future studies of lignan and isoflavonoid metabolism. Scand J Clin Lab Invest Suppl

The effect of red and white wine total extracts and phenolic fractions on heat shock protein (Hsp) levels in tumor cells and on tumor and endothelial cell populations in vitro has been investigated. Total extracts of red wines decreased Hsp70 and Hsp27 levels and the numbers of tumor and endothelial cells. Several red and white wine fractions significantly decreased Hsp27 levels, and some of them had also an effect on Hsp70 levels. A red wine fraction rich in polymeric flavanols and a white wine one rich in phenolic acids, flavonols, and tyrosol strongly lowered Hsp27 levels. Some red and white wine fractions strongly reduced tumor cell numbers, whereas most of them decreased endothelial cell numbers to variable extents. The present results indicate that wine phenolics decrease Hsp levels in tumor cells and tumor and endothelial cell populations. These properties may be important in the potent anticarcinogenic action of wine phenolics. J Agric Food Chem

The formation of new blood vessels (angiogenesis) is critical for the growth of tumours and is a dominant feature in various angiogenic diseases such as diabetic retinopathy, arthritis, haemangiomas and psoriasis. Recognition of the potential therapeutic benefits of controlling pathological angiogenesis has led to a search for angiogenesis inhibitors. Here we report that 2-methoxyoestradiol, an endogenous oestrogen metabolite of previously unknown function, is a potent inhibitor of endothelial cell proliferation and migration as well as angiogenesis in vitro. Moreover, when administered orally in mice, it strongly inhibits the neovascularization of solid tumors and suppresses their growth. Unlike the angiostatic steroids of corticoid structure, it does not require the co-administration of heparin or sulphated cyclodextrins for activity. Thus, 2-methoxyoestradiol is the first steroid to have high antiangiogenic activity by itself. Our results suggest that this compound may have therapeutic potential in cancer and other angiogenic diseases. Nature

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.

Luteolin is a flavonoid that has been shown to reduce proinflammatory molecule expression in vitro. In the present study, we have tested the ability of luteolin to inhibit lipopolysaccharide (LPS)- induced lethal toxicity and proinflammatory molecule expression in vivo. Mice receiving LPS (Salmonella enteriditis LPS, 32 mg/kg, intraperitoneally) exhibited high mortality with only 4.1% of the animals surviving seven days after the LPS challenge. On the contrary, mice that had received luteolin (0.2 mg/kg, intraperitoneally) before LPS showed an increased survival rate with 48% remaining alive on Day 7. To investigate the mechanism by which luteolin affords protection against LPS toxicity we measured intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-alpha) production in response to LPS in the presence or absence of luteolin pretreatment. Treatment of animals with LPS increased serum TNF-alpha levels in a time-dependent manner. The increase in peak serum TNF-alpha levels was sensitive to luteolin pretreatment. Luteolin pretreatment also reduced LPS-stimulated ICAM-1 expression in the liver and abolished leukocyte infiltration in the liver and lung. We conclude that luteolin protects against LPS-induced lethal toxicity, possibly by inhibiting proinflammatory molecule (TNF-alpha, ICAM-1) expression in vivo and reducing leukocyte infiltration in tissues.

Transforming growth factor beta (TGFbeta) receptors require SARA for phosphorylation of the downstream transducing Smad proteins. SARA, a FYVE finger protein, binds to membrane lipids suggesting that activated receptors may interact with downstream signaling molecules at discrete endocytic locations. In the present study, we reveal a critical role for the early endocytic compartment in regulating Smad-dependent signaling. Not only is SARA localized on early endosomes, but also its minimal FYVE finger sequence is sufficient for early endosomal targeting. Expression of a SARA mutant protein lacking the FYVE finger inhibits downstream activin A signaling in endothelial cells. Moreover, a dominant-negative mutant of Rab5, a crucial protein for early endosome dynamics, causes phosphorylation and nuclear translocation of Smads leading to constitutive (i.e. ligand independent) transcriptional activation of a Smad-dependent promoter in endothelial cells. As inhibition of endocytosis using the K44A negative mutant of dynamin and RN-tre did not lead to activation of Smad-dependent transcription, the effects of the dominant-negative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGFbeta/activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGFbeta/activin signal transduction pathways.

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas. Oncogene

Vascular endothelial growth factor (VEGF) activates unfolded protein response sensors in the endoplasmic reticulum through phospholipase C gamma (PLCγ)-mediated crosstalk with mammalian target of rapamycin complex 1 (mTORC1). Activation of transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) activate mTORC2, ensuring maximal endothelial cell survival and angiogenic activity through phosphorylation of AKT on Ser473. As mTORC1 is a metabolic sensor, metabolic signals may be integrated with signals from VEGF in the regulation of angiogenesis.