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Background Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and “omics” approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. Methods We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Results Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. Conclusions Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs).Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay.When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-β2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs.Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

Human endostatin is an endogenous angiogenesis inhibitor, and its ability to modulate tumors vascularization is of great therapeutic interest. The antiangiogenic activity is conserved also in some of its synthetic fragments. Fragment 6-49, in particular, is even more active than full length protein. It covers an endostatin region which shows two phenylalanines unusually exposed on the surface. The effect of the mutation of these residues on fragment 6-49 structure and activity are discussed and compared to those of a similar mutation in full length endostatin. A hypothesis on the fragment active epitope is supported by data from molecular dynamics simulations.

Background and purpose: It has recently been reported that oxytocin is produced by some tumour cell types, and that oxytocin receptors, belonging to the G-protein-coupled receptor (GPCR) family, are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to oxytocin with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis. Experimental approach: We employed chemotaxis and chemoinvasion assays to characterize the effect of oxytocin on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action. Key results: We showed that oxytocin stimulates migration and invasion in HUVECs via oxytocin receptor activation. Searching for the molecular mechanism(s) responsible for oxytocin's pro-migratory effect, we identified the Gq coupling of oxytocin receptors and phospholipase C (PLC) as the main effectors of oxytocin's action in HUVECs. We also found that oxytocin stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol-3-kinase (PI-3-K)/AKT pathway, and that the activation of PI-3-K and formation of nitric oxide (NO) are required for the pro-migratory effect of oxytocin. Conclusions and implications: The ability of oxytocin to stimulate HUVEC motility and invasion suggests that the hormone can participate in physiopathological processes where activation of endothelial cells plays an important role, for example, in angiogenesis. Interestingly, both the AKT and eNOS phosphorylation induced by oxytocin receptor activation depended on PLC activity, thus suggesting the existence of a still undefined mechanism connecting PLC to the PI-3-K/AKT pathway, upon oxytocin stimulation. British Journal of Pharmacology (2008) 153, 728–736; doi:10.1038/sj.bjp.0707609; published online 3 December 2007

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y cells.

The aim of the present research was to study the relationship between chemical structure and antiangiogenic activity of endostatin. Four peptides, containing about 40 amino acid residues, designed to cover nearly the whole sequence of endostatin, were synthesized by the solid-phase method. They were termed Fragment I (sequence 6-49), II (sequence 50-92), III (sequence 93-133), and IV (sequence 134-178), with the latter bearing the original disulfide bond Cys135-Cys165. These peptides were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis assays in matrigel. Fragments I and IV inhibited cell proliferation and cell migration with a potency and an efficacy higher than that of the full length endostatin. Fragment I was also active in inhibiting in vitro the formation of tubules and in vivo the vascularization of the matrigel. Fragments II and III were devoid of antiangiogenic activity. We propose to use the peptides 6-49 and 134-178 as angiogenesis inhibitors in substitution of full length endostatin, in therapeutic applications for cancer, rheumatoid arthritis, and retinopathies.

1. Prostaglandin E(1) (PGE(1), alprostadil) is used as a vasodilator for the treatment of peripheral vascular diseases. 2. Previous reports suggested a pro-angiogenic effect for PGE(1). 3. We studied the in vitro and in vivo effect of PGE(1), complexed with alpha-cyclodextrin, on the angiogenic process. Contrary to what was expected, we found that, in human umbilical vein endothelial cells (HUVECs), PGE(1) inhibited proliferation, migration and capillary-like structure formation in Matrigel. 4. By RT-PCR studies, the expression of the EP(2) and EP(3) subtypes of the PG receptor was detected in HUVECs. 5. PGE(1) alone stimulated adenylate cyclase activity at micromolar concentrations, while at nanomolar concentrations potentiated the forskolin-induced cAMP accumulation. 6. 8-Bromoadenosine-3':5'-cyclic monophosphate (Br-cAMP) mimicked the inhibitory effect of PGE(1) on endothelial cell growth, motility and tube formation. 7. Sulprostone, an agonist at the EP(3) subtype of PG receptors, mimicked the in vitro anti-angiogenic effects of PGE(1), while butaprost, an EP(2) receptor agonist, had no effect. 8. Finally, in the plug assay model of angiogenesis in mice, PGE(1) showed a strong inhibitory effect on Matrigel neovascularization. 9. Thus, PGE(1) possesses strong anti-angiogenic activity in vitro and in vivo.

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-β estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS.

Aims Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis. Main methods Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence. Key findings We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs. Significance Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.

Abstract —In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[ 3 H]inositol–labeled HepG2 cells, VLDL (100 μg/mL) caused a time-dependent increase in [ 3 H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P 2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% ( P

In a previous work, we showed an increased cell motility due to the accumulation and transcriptional activation of the Hypoxia Inducible Factor-1α (HIF-1α) and a reduced mitochondrial energy production in an in vitro model of endothelial dysfunction (ED) represented by human endothelial cells (ECs) chronically deprived of nitric oxide (NO) by L-NAME treatment. In the present study, in the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activation, we focused our attention on Reactive Oxygen Species (ROS). We found that ROS were partially involved in HIF-1α stabilization, but not in the pro-migratory phenotype. Regarding mitochondrial dysfunction, it did not require neither ROS generation nor HIF-1α activity, and was not due to autophagy. Very interestingly, while acute treatment with L-NAME induced a transient increase in ROS formation, chronic NO deprivation by long term L-NAME exposure drastically reduced cellular ROS content giving rise to an antioxidant environment characterized by an increase in superoxide dismutase-2 (SOD-2) expression and activity, and by nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2). These results might have important implications for our understanding of the consequences of NO deprivation in endothelium behavior and in the onset of cardiovascular diseases.

We previously reported a significant mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small-cell lung carcinoma cells (SCLC, GLC-8), mediated by both 5-HT 1D and 5-HT 1A receptors. Here we investigate possible interactions between the two receptor subtypes. Dose-effect curves obtained by simultaneously applying equipotent concentrations of the selective 5-HT 1A agonist 8-OH-DPAT and the selective 5-HT 1D receptor agonist sumatriptan are shifted to the right, although maximal effects are additive. The nonselective 5-HT antagonist metergoline displays higher potency when both receptor subtypes are activated. The 5-HT 1D receptor antagonist GR127935 is markedly more potent against sumatriptan than against the sensitive portion of 5-HT effect. Indeed, both GR127935 and the 5-HT 1A antagonist spiperone shift the EC 50 for the residual effect of 5-HT from ≈ 300 to 120–150 nM, suggesting that blocking one receptor subtype may facilitate activation of the other. Preincubation with either 8-OH-DPAT or sumatriptan suppresses the mitogenic response to the other specific receptor agonist; suppression is complete within 10 min at 37°C, and is not observed when the preincubation is done at 4°C. Measurements of adenylate cyclase activity do not help in interpreting the results. Conversely, measurements of MAP kinase activity reveals biphasic activation with a delayed activation at 1 h, and reproduce the suppression of the effect of the second drug by 15 min preincubation. These findings constitute the first evidence of a reciprocal negative interference between human 5-HT 1A and 5-HT 1D receptors, and indicate that SCLC GLC-8 cells simultaneously express both receptor subtypes. Mere reciprocal antagonism of the drugs employed cannot account for these data. We suggest that in this cell system cross-talk occurs in the transduction pathways of the two receptor subtypes.

Somatostatin possesses antisecretory and antiproliferative activity on some human tumors. We herein report that, in a human neuroblastoma cell line, the somatostatin analogue BIM 23014 inhibited mitogen-activated protein (MAP) kinase activity stimulated by either insulin-like growth factor-1, whose receptor bears a tyrosine kinase, or carbachol, which acts at a G-protein coupled receptor. In a human small cell lung carcinoma line BIM inhibited serum-stimulated MAP kinase activation. These inhibitory actions occur in a dose range quite similar to that observed for suppression of proliferation induced by the analogue in the same cell lines. The decrease in cAMP elicited by the analogue in the two cell lines is not responsible for its inhibitory action on MAP kinase and cell growth. Moreover, the analogue did not modify intracellular [Ca2+] and pH. An involvement of a phosphatase activity is suggested.

alpha-Conotoxins are small peptides present in the venom of different species of marine snails of the Conus genus that target nicotinic acetylcholine receptors (nAChRs), with a marked specificity for muscle-type nAChRs. alpha-Conotoxin Imperialis I (alpha-Ctx-Iml), from Conus imperialis, has been recently described as a potent antagonist of mammalian neuronal alpha-bungarotoxin (alpha-Bgtx)-sensitive nAChRs. Human small cell lung carcinoma (SCLC) is a very aggressive tumor composed of neuroendocrine secretory cells. We demonstrated that human SCLC cells express neuronal-type alpha-Bgtx-sensitive nAChRs, and that their activation causes secretion of mitogenic hormones and stimulates cell proliferation, alpha-Ctx ImI inhibits both these nicotinic effects, and could therefore be considered a new important tool for investigating human neuronal-type alpha-Bgtx-sensitive nAChRs.

Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors.

BACKGROUND: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. RESULTS: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. CONCLUSION: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.

Small cell lung carcinoma cells possess voltage-dependent calcium channels (VDCCs) of the L, omega-conotoxin-sensitive and P-like type. We hypothesized that these VDCCs might regulate the secretion of autocrine growth factors and thus influence the proliferation of these cells. We found that extracellular Ca2+ plays a stimulatory role in the proliferation of the GLC8 cell line. L-type calcium channel blockers of the dihydropyridine, phenylalkylamine and benzothiazepine classes inhibited [3H]thymidine incorporation in these cells, however at concentrations higher than those required to block L-type channel function. Moreover, the growth of murine Swiss 3T3 fibroblasts which do not possess L-type Ca2+ channels, was inhibited by the Ca2+ channel antagonists at the same effective concentrations as in small cell lung carcinoma cells. omega-conotoxin and omega-agatoxin IVA, which block the N- and P-type channel respectively, had no effect on GLC8 cell proliferation. It is concluded that the presence of extracellular Ca2+ is a positive stimulus for small cell lung carcinoma cell growth. However, under our experimental conditions, the calcium channel blockers inhibited DNA synthesis most probably by a mechanism other than VDCC antagonism.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.

Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release.

The phosphoinositide hydrolysis stimulated by agonist-receptor interaction is an universal mechanism for signal transduction that many different types of cells use to communicate with each other . It is well known that the hydrolysis of a particular phosphoinositide, phosphatidylinositol 4,5-bisphosphate, operated by a specific phospholipase, phospholipase C (PLC), generates at least two second messengers, diacylglycerol which activates proteinkinase C and inositol 1,4,5trisphosphate which mobilizes Cap+ from intracellular stores . Consequently, all the endogenous substances (hormones, neurotransmitters, growth factors) that act via this metabolic reaction activate within the cell all the proteinkinase C and calcium dependent processes . The regulation of PLC activity is therefore crucial for achieving a correct and controlled cell response . It has recently become clear that various types of PLC exist [1] . The enzymes purified from various sources possess molecular weight ranging from 56 to 154 kDa and appear to be both soluble and membrane-associated. Cloning of four of these enzymes (a, /3, y, 6) has revealed a surprisingly low degree of similarity in their primary structure, suggesting different roles and different regulation for each of them . The isoenzymes also display differential tissue locations . All four types have been found to be ubiquitously expressed, but the level of expression is highly variable among tissues and cell lines . For example, the distribution in the various rat brain areas [2] is heterogeneous (PLC-a is highest in the hippocampus, rostral raphe nuclei and cerebellar Purkinje cells ; PLC-f3 is highest in the cerebral cortex, caudate and granule cell layer of the cerebellum ; PLC-)/has a more widespread distribution while PLC-S is low throughout the brain), suggesting the possibility of a preferential association of individual PLC isoenzymes with particular postsynaptic receptors . The mechanism of the receptor-mediated activation of PLC has recently been a matter of intense research . In analogy with the adenylate cyclase system, a G protein (Gp) is considered to be physically and functionally interposed between receptors and PLC [3] . Many studies have shown that in membrane preparations or permeable cells stable analogues of GTP both activate PLC and potentiate the

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Human endostatin is one of the better characterized endogenous angiogenesis inhibitors, and its ability to modulate vascularization of tumours could be of great therapeutic interest. These properties are not exclusive to the full-length protein, but are shared by some of its synthetic fragments. A number of research groups have partitioned human endostatin in different peptides and have investigated their activity, in order to collect a body of experimental data which could be important in shedding new light on their structure-activity relationships. It was also reported that a small active fragment can become inactive when contained in a larger fragment, revealing an apparent discrepancy in the experimental results. Very few studies have been devoted to the computational analysis of these systems and to the rationalization of their properties using molecular modelling. Through molecular dynamics simulations of human endostatin and of four synthetic fragments, we have been able to rationalize the experimental findings. In particular, we have identified a pattern consisting of six amino acids, namely R-R(G)-A-D-R-A, which appears to be an active epitope if it is properly exposed to the solvent. Interestingly, this pattern can be already present in sequential order in the primary structure, or it can be generated by the spatial approach of two groups of residues, far apart in the primary structure, as an effect of the peptide folding. Comparing the structural features and the time evolution of all the simulated peptides we provide a coherent explanation of their activity or inactivity.

Previous studies indicate that nuclear factor kappaB (NF-κB) transcription factor is deregulated and overexpressed in several human neoplasias. The aim of this study was to test the hypothesis that the NF-κB pathway may be involved in parathyroid tumorigenesis. For this purpose, we determined the level of NF-κB activity, evaluated as phosphorylation of the transcription subunit p65, its modulation by specific and non-specific agents and its impact on cyclin D1 expression. Phosphorylated p65 levels present in parathyroid neoplasias (n = 13) were significantly lower than those found in normal tissues (n = 3; mean optical density (OD) 0.19 ± 0.1 vs 0.4 ± 0.1, P = 0.007), but there was no significant difference between adenomas and secondary and multiple endocrine neoplasia type 1 (MEN1)-related hyperplasia. Conversely, MEN2A (Cys634Arg)-related parathyroid samples showed extremely high levels of phosphorylated p65 that exhibited a nuclear localization at immunohistochemistry (n = 3). Phosphorylated p65 levels negatively correlated with menin expression (r2 = 0.42, P = 0.05). Tumor necrosis factor-α (TNFα) caused a significant increase in phosphorylated p65 levels (183 ± 13.8% of basal) while calcium sensing receptor (CaR) agonists exerted a significant inhibition (19.2 ± 3.3% of basal). Although TNFα was poorly effective in increasing cyclin D1 expression, NF-κB blockade by the specific inhibitor BAY11-7082 reduced FCS-stimulated cyclin D1 by about 60%. Finally, the inhibitory effects of CaR and BAY11-7082 on cyclin D1 expression were not additive – by blocking NF-κB CaR activation did not induce a further reduction in cyclin D1 levels. In conclusion, the study demonstrated that in parathyroid tumors: (1) p65 phosphorylation was dramatically increased by RET constitutive activation and was negatively correlated with menin expression, (2) p65 phosphorylation was increased and reduced by TNFα and CaR agonists respectively, and (3) blockade of the NF-κB pathway caused a significant decrease in cyclin D1 expression.

Ghrelin is a novel gastrointestinal hormone involved in several metabolic functions. It has been identified previously in several normal and tumoral neuroendocrine tissues, including human medullary thyroid carcinomas (MTCs). The aim of the study was to evaluate ghrelin levels in patients with MTC and nontoxic goitre (NTG) with elevated calcitonin (CT) levels, as an additional marker of the disease.The study included 22 patients with MTC (four before and 18 after thyroidectomy), 12 patients with NTG with basal CT levels exceeding 10 ng/l and 15 healthy subjects matched for age, sex and body mass index (BMI). After thyroidectomy, MTC patients were considered cured when basal and pentagastrin-stimulated CT levels were0.2 and10 ng/l, respectively. A pentagastrin-induced CT peak over 50 ng/l was considered as an abnormal response while 100 ng/l was the cut-off accepted for the diagnosis of C-cell hyperplasia or tumour. Circulating ghrelin and CT levels were evaluated at baseline in patients and controls and at -10, 0, 1, 2, 5 and 15 min after pentagastrin injection (0.5 microg/kg body weight) in 12 patients with MTC and nine with NTG. Four surgically removed MTCs were tested for ghrelin expression.Total plasma ghrelin and CT levels were measured with a commercially available radioimmunoassay (RIA) and two-site chemiluminescence immunometric assays, respectively. In paraffin-embedded MTC samples ghrelin immunostaining was performed with a polyclonal antibody (1:1000) and the reaction visualized by an indirect immunoperoxidase system.Plasma ghrelin levels found in cured or not cured MTC and in NTG patients were similar to those of BMI-matched healthy controls. No correlation between ghrelin and CT levels, thyroid disease or previous thyroidectomy was observed. The administration of pentagastrin caused a 17% increase in ghrelin levels (basal ghrelin vs. peak: 162 +/- 62 pmol/l vs. 189 +/- 58 pmol/l, P0.05) that was particularly evident (33% increase) in patients with an abnormal CT response to the test (CT50 ng/l). Immunohistochemistry showed positivity for ghrelin in a small proportion of CT positive cells from the four MTCs removed.Patients with MTC, NTG and controls showed similar ghrelin levels, ruling out this parameter as a marker of MTC. The increase in ghrelin levels in patients with a positive CT response to pentagastrin, together with the immunopositivity for ghrelin in some MTC cells, suggests C cells as minor source of ghrelin production.

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y cells.

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself.

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [3H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.

Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter and vasoactive agent, is contained in two small cell lung carcinoma cell lines GLC8 and NCI-N-592 and is released in the culture medium. It also stimulates DNA synthesis in the same cell lines. In GLC8 cells this mitogenic effect is not counteracted by ketanserin, ICS 205-930 and GR 113-808 which are antagonists of the 5-HT2, 5-HT3 and 5-HT4 receptors, respectively. On the contrary, the antagonists metergoline, methysergide, SDZ 21-009 and methiothepin inhibit the 5-HT-stimulated incorporation of [3H]thymidine in GLC8 cells. The 5-HT1D agonist sumatriptan is capable of mimicking 5-HT action on cell proliferation. Both sumatriptan and 5-HT inhibit adenylate cyclase activity at doses which correlate with the mitogenic effect. We conclude that a 5-HT1D receptor type contributes to the mitogenic effect of 5-HT in GLC8 cells. This is the first demonstration of an involvement of the 5-HT1D receptor type in human cell proliferation. The design of specific antagonists for this type of receptor might be useful for the growth control of this very aggressive tumor.

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small-cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose-dependent manner; this effect is antagonized by alpha-bungarotoxin (alpha Bgtx) and alpha-conotoxin MI (alpha Ctx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both alpha Bgtx and alpha Ctx. By PCR analysis, we demonstrate the presence in SCLC of both the alpha 7 and the beta 2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that alpha Bgtx-sensitive receptors of the alpha 7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

Serum stimulation of cultured human fibroblasts activates a Na+ influx via an amiloride-sensitive Na+/H+ exchange system. Evidence is presented which indicates that phospholipase activity is an important component of the mechanism by which mitogen receptor occupation leads to activation of Na+/H+ exchange. Serum stimulation of Na+ flux is effectively blocked by inhibitors of phospholipase activity such as mepacrine and U-1002. The Ki values for inhibition of Na+ flux by these agents (10 microM and 18 microM, respectively) are comparable to their Ki values for inhibition of serum-stimulated arachidonic acid release. In contrast, the activation of Na+ influx produced by the divalent cation A23187 is not affected by phospholipase inhibitors indicating that these agents specifically block mitogen activation of Na+ flux rather than nonspecifically disrupting the membrane's ability to perform Na+/H+ exchange. The phospholipase activator melittin stimulates Na+ influx in the absence of mitogens at concentrations that cause a comparable stimulation of arachidonic acid release. The melittin-stimulated Na+ influx is inhibited by amiloride and mepacrine, suggesting that melittin activation of phospholipase activity leads to the activation of the Na+/H+ exchange system. In addition, chronic treatment of cells with dexamethasone, which is known to induce an endogenous phospholipase inhibitor in human fibroblasts, leads to a substantial inhibition of the serum stimulation of both Na+ influx and arachidonic acid release. When taken together, these data support the involvement of phospholipase activity in the serum stimulation of the Na+/H+ exchange system.

Intracerebroventricular administration of muscimol, a potent GABA-receptor agonist, counteracted the antinociceptive effect of morphine or beta-endorphin in rats as measured by the "tail flick" method. Muscimol's activity was reversed by bicuculline. Isoguvacine, another GABA agonist, as well as nipecotic acid and guvacine, two inhibitors of neuronal and glial uptake of GABA, also antagonized morphine's antinociceptive effect. A role of the central GABA-ergic system in mediating opiate antinociception is proposed.

The EGF receptor, which is homologous to the ν-erb-B oncogene product, has intrinsic tyrosine kinase activity, and mediates an increase in polyphosphoinositide turnover and [Ca 2+ ] i . Recently, great progress has been made in understanding the mechanism of signal transduction at this receptor. Jacopo Meldolesi and colleagues discuss how this knowledge may lead to a better understanding of the control of cell proliferation.

The changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. The lack of Ca2+ rise seen in attached cells loaded with quin2 and treated with EGF in Ca2+-free medium was probably the result of a Ca2+ buffer artifact. Concomitantly to the Ca2+ signal, EGF induced phosphoinositide hydrolysis, with stimulated accumulation of inositol 1,3,4,trisphosphate and -1,3,4,5-tetrakisphosphate. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store (possibly mediated by generation of inositol trisphosphate) and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin (growth factors that cause much greater phosphoinositide hydrolysis and Ca2+ redistribution responses than EGF). It is therefore suggested that the Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5′-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5′-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF.

The role played by brain serotonin (5-HT) neurotransmission in mediating the PRL-releasing effect of enkephalins was investigated in the rat. Both MetG-enkephalin (Metenk) and (D-Met2, Pro5)-enkephalinamide (EKNH2), an analog with long lasting analgesic activity, were used in freely moving rats bearing a cannula chronically implanted into the jugular vein. Met-enk injected intracerebroventricularly (IVT; 200 and 400 μg/rat) induced a marked and dose-related increase in plasma PRL; EKNH2 (0.2 mg/kg, iv) induced a rise in plasma PRL similar to that evoked by Met-enk (400μg/rat). Metergoline (1 mg/kg, iv), a 5-HT receptor blocker, significantly reduced the PRL-releasing effect of Met-enk (200 μg/rat); these results were duplicated by using another 5-HT receptor blocker, i.e. methysergide (2.5 mg/kg, iv). Metergoline and methysergide also significantly reduced the increase in plasma PRL due to EKNH2. 5,6-Dihydroxytryptamine (50 μg, IVT), a neurotoxic drug which destroys 5-HT nerve terminals, almost completely...

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.

SummarySystemic administration of parachlorophenylalanine (PCPA, 100 mg/ kg sc on alternate days x two times), a blocker of serotonin (5-HT) synthesis, considerably decreased brain 5-HT and plasma prolactin (PRL) levels in young male rats. Intraventricular (IVT) administration of 5,7-dihydroxytryptamine (5,7-DHT, 200 μg/20 μl), a neurotoxic drug which destroys 5-HT nerve terminals, induced, 3, 12, and 30 days after treatment, a marked depletion of brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and considerably reduced plasma PRL levels at each time interval. Feeding of rats for up to 4 days with a tryptophan (TP)-deficient diet, caused a depletion of brain 5-HT and 5-HIAA contents and did not modify plasma PRL levels. Addition of TP (2 g/kg of diet) to the TP-deficient diet resulted in increased brain 5-HT and 5-HIAA contents and significantly increased PRL levels. These data provide evidence for the role of the 5-HT system in the maintenance of tonic PRL secretion.We are grateful for the technical as...

Mazindol, a new anorexigenic agent which possesses a different chemical structure from phenylethylamine derivatives such as amphetamine, causes anorexia along with increases in locomotor activity and body temperature. Mazindol also induces stereotyped behaviour and, if injected into rats with unilateral nigro-striatal lesions, causes turning towards the lesioned side. Mazindol-induced anorexia is antagonized by pretreatment with α-methyl-p-tyrosine or pimozide. Pimozide pretreatment prevents the rotation induced by Mazindol in rats with unilateral nigro-striatal lesions. The involvement of dopamine in the mechanism whereby Mazindol elicits anorexia and turning behaviour is discussed.

Summary In the attempt to further characterize the role of the GABA-ergic system on morphine analgesia, nipecotic acid and guvacine, inhibitors of GABA reuptake, were administered intraventricularly in rats pretreated with morphine. Under these conditions the animals showed a decreased response to the antinociceptive effect of morphine when compared to control rats.

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.

Summary When muscimol was injected intraventricularly or intraperitoneally to rats at the dose of 0.6 μg/ventricle and 4 mg/kg, respectively, dihydroxyphenylacetic acid (DOPAC) levels, determined by a radioenzymatic method, were statistically increased in a qualitative similar way in the striatum and olfactory tubercles after both routes of administration. The results, in agreement with other Authors' findings, support the hypothesis of a stimulation of dopamine turnover by muscimol.

The data presented in this paper suggest that we have identified several key steps in the sequence of events occurring between binding of growth factors to their surface receptors and the activation of the Na + -H + exchange system. Our current working model is shown in Fig. 4. The initial step after binding of peptide rnitogens appears to be the release of inositol trisphosphate from membrane pools of phosphatidylinositol 4′,5′-bisphosphate (PIP 2 ). This inositol trisphosphate then interacts with an internal Ca2 + storage site to mobilize intracellular Ca 2+ thereby elevating the intracellular Ca 2+ activity. An elevation of Ca 2+ activity then acts through a Ca 2+ -dependent regulatory protein which somehow leads to the activation of the Na + -H + exchanger. The current data are most consistent with calmodulin being the Ca 2+ -dependent regulatory protein involved in the activation process; however, the synergism between A23187 and TPA suggests that protein kinase C may also play some role in this process, although the lack of effect of TPA alone suggests that activation of protein kinase C is not a sufficient stimulus for activation of the Na + -H + exchanger. The current data on phospholipase involvement are most readily explained on the basis of a mitogen activation of phospholipase C activity which acts to release inositol trisphosphate, which in turn mobilizes intracellular Ca 2+ . However, it should be pointed out that in general the compounds (mepacrine and melittin) that we have used to perturb phospholipase activity have been traditionally thought of as interacting with phospholipase A 2 instead of phospholipase C. However, recent results in our laboratory indicate that mepacrine will inhibit the mitogen-induced release of inositol trisphosphate and that melittin will stimulate its release in the absence of mitogens (Jamieson and Villereal, in preparation), suggesting either that these compounds do interact with phospholipase C or that there is some regulation of phospholipase C activity by the breakdown products of phoshpolipase A 2 activity. A report from Majerus's laboratory indicating that mepacrine directly inhibits phospholi pase C activity suggests that effects of mepacrine in the HSWP cell are probably at the level of phospholipase C. In the summary scheme (Fig. 4), the possibility that the release of inositol trisphosphate could be potentiated via the stimulation of a kinase activity which would increase the substrate (PIP2) available to phospholipase C is presented. This possibility is suggested by the observation that certain oncogene products with kinase activity can phosphorylate phosphatidylinositol to the polyphosphorylated forms (Sugimoto et al. , 1984; Macara et al. , 1984).