Beta-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.     

Epiregulin-dependent amphiregulin expression and ERBB2 signaling are involved in luteinizing hormone-induced paracrine signaling pathways in mouse ovary

Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.     

Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma

The high affinity neurotensin receptor (NTSR1) mediates most of the biologic effects of neurotensin (NT), a 13-amino acid peptide that stimulates growth in certain cell types. NT is expressed in fetal but not differentiated colonic epithelium and is re-expressed in colonic adenocarcinoma. The cognate receptor, NTSR1, is also not expressed or is present at a low level in adult colonic epithelial cells but is expressed in most colon cancer cell lines. These observations suggest that altered NT-NTSR1 signaling may be associated with malignant transformation in the colon. To further understand the possible role of NTSR1 expression in colonic tumorigenesis and progression, we examined NTSR1 mRNA by in situ hybridization in normal colonic mucosa, adenomas, and colonic adenocarcinomas. NTSR1 mRNA expression was undetectable or weak in superficial differentiated epithelial cells of normal colonic epithelium, but adenomas and adenocarcinomas showed moderate to strong expression (p<0.05). Adenocarcinomas showed a higher level of expression compared to adenomas (p<0.05). Furthermore, adenocarcinomas that infiltrated into and beyond the muscularis propria showed a higher intensity of NTSR1 expression compared with tumors that were localized to the mucosa or submucosa. In some cases, infiltrating margins and foci of lymphovascular invasion showed a higher intensity of expression than the main mass of the tumor. These results suggest that increased NTSR1 expression may be an early event during colonic tumorigenesis and also contribute to tumor progression and aggressive behavior in colonic adenocarcinomas. NTSR1 may thus be a potential target for preventive or therapeutic strategies in colon cancer.     

The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway

Recent evidence supports a role for EphB receptor tyrosine kinases as tumour suppressors in colorectal and prostate cancer. However, it is unclear how these receptors inhibit cancer cell tumorigenicity - an activity that is highly unusual for a family of receptor tyrosine kinases. Here, we report that the EphB4 receptor can behave as a tumour suppressor in a mouse xenograft model of breast cancer when stimulated by its ligand, ephrin-B2. In breast cancer cells, EphB4 activates an antioncogenic pathway involving Abl family tyrosine kinases and the Crk adaptor protein. This Abl-Crk pathway inhibits breast cancer cell viability and proliferation in addition to motility and invasion, and also downregulates the pro-invasive matrix metalloprotease, MMP-2. Consistent with these effects, EphB4 and the Abl-Crk pathway are constitutively active in non-transformed mammary epithelial cells. These findings identify a novel Eph receptor signalling pathway with tumour-suppressor activity and predict that therapeutic intervention to activate EphB4 signalling will inhibit tumour progression.     

Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology.

The guanylin family of bioactive peptides consists of three endogenous peptides, including guanylin, uroguanylin and lymphoguanylin, and one exogenous peptide toxin produced by enteric bacteria. These small cysteine-rich peptides activate cell-surface receptors, which have intrinsic guanylate cyclase activity, thus modulating cellular function via the intracellular second messenger, cyclic GMP. Membrane guanylate cyclase-C is an intestinal receptor for guanylin and uroguanylin that is responsible for stimulation of Cl- and HCO3- secretion into the intestinal lumen. Guanylin and uroguanylin are produced within the intestinal mucosa to serve in a paracrine mechanism for regulation of intestinal fluid and electrolyte secretion. Enteric bacteria secrete peptide toxin mimics of uroguanylin and guanylin that activate the intestinal receptors in an uncontrolled fashion to produce secretory diarrhea. Opossum kidney guanylate cyclase is a key receptor in the kidney that may be responsible for the diuretic and natriuretic actions of uroguanylin in vivo. Uroguanylin serves in an endocrine axis linking the intestine and kidney where its natriuretic and diuretic actions contribute to the maintenance of Na+ balance following oral ingestion of NaCl. Lymphoguanylin is highly expressed in the kidney and myocardium where this unique peptide may act locally to regulate cyclic GMP levels in target cells. Lymphoguanylin is also produced in cells of the lymphoid-immune system where other physiological functions may be influenced by intracellular cyclic GMP. Observations of nature are providing insights into cellular mechanisms involving guanylin peptides in intestinal diseases such as colon cancer and diarrhea and in chronic renal diseases or cardiac disorders such as congestive heart failure where guanylin and/or uroguanylin levels in the circulation and/or urine are pathologically elevated. Guanylin peptides are clearly involved in the regulation of salt and water homeostasis, but new findings indicate that these novel peptides have diverse physiological roles in addition to those previously documented for control of intestinal and renal function.     

Anti-LRP/LR-specific antibody IgG1-iS18 significantly reduces adhesion and invasion of metastatic lung, cervix, colon and prostate cancer cells

The 37-kDa/67-kDa laminin receptor [laminin receptor precursor/high-affinity laminin receptor (LRP/LR)] is thought to play a major role in invasion and adhesion, key components of metastatic cancer. Lung cancer, cervical cancer, colon cancer and prostate cancer are among the top 10 cancer types worldwide. Here, we report that LRP/LR levels on the surface of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells are significantly increased compared to non-tumorigenic fibroblasts. Adhesion of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells to laminin-1 is significantly reduced, employing the anti-LRP/LR-specific antibody IgG1-iS18. Invasion of these cell lines into the Matrigel? matrix was significantly impeded with IgG1-iS18. The Pearson's correlation coefficient proves a correlation between LRP/LR cell-surface levels and invasion potential, as well as adhesion and invasion, respectively. Our findings suggest that IgG1-iS18 antibody might act as alternative therapeutic tool for treatment of various metastatic cancer types.     

15-lipoxygenase-1 metabolites down-regulate peroxisome proliferator-activated receptor gamma via the MAPK signaling pathway.

Human colon tumors have elevated levels of 15-lipoxygenase-1 (15-LO-1), suggesting that 15-LO-1 may play a role in the development of colorectal cancer. Also, 15-LO-1 metabolites can up-regulate epidermal growth factor signaling pathways, which results in an increase in mitogenesis. However, metabolites of 15-LO-1 can serve as ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), and activation of this receptor causes most colon cancer cell lines to undergo a differentiative response and reverse their malignant phenotype. Hence, the role 15-LO-1 plays in colon cancer is not clear. To clarify the role of 15-LO-1 in carcinogenesis, the effect of 15-LO-1 and its metabolites on epidermal growth factor signaling and PPARgamma was investigated. In HCT-116 cells, exogenously added 15-LO-1 metabolites, 13-(S)-hydroxyoctadecadienoic acid, 13-(R)-hydroxyoctadecadienoic acid, and 13-(S)-hydroperoxyoctadecadienoic acid, up-regulated the MAPK signaling pathway, and an increase in PPARgamma phosphorylation was observed. Furthermore, in stable overexpressing 15-LO-1 HCT-116 cells, which produce endogenous 15-LO-1 metabolites, an up-regulation in mitogen-activated protein kinase and PPARgamma phosphorylation was observed. Incubation with a MAPK inhibitor ablated MAPK and PPARgamma phosphorylation. The 15-LO-1 up-regulates MAPK activity and increases PPARgamma phosphorylation, resulting in a down-regulation of PPARgamma activity. Thus, 15-LO-1 metabolites may not only serve as ligands for PPARgamma but can down-regulate PPARgamma activity via the MAPK signaling pathway.     

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research tools and potential therapeutic agents

The 17-amino acid neuropeptide nociceptin/Orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid receptor-like (ORL1) receptor, a fourth member of the classical mu, delta, and kappa opioid receptor family. Although ORL1 clearly belongs to the opioid receptor family, it does not bind classical opiates and the ORL1-N/OFQ system has pharmacological actions distinct from the opioid receptor system. This new ligand-receptor system has generated active interest in the opioid community because of its wide distribution and involvement in a myriad of neurological pathways. The past two years have witnessed tremendous advances in the design and discovery of very potent and selective peptide and nonpeptide agonist and antagonist ligands at ORL1. These discoveries have facilitated the understanding of the role of the ORL1-N/OFQ system in a variety of processes such as pain modulation, anxiety, food intake, learning, memory, neurotransmitter release, reward pathways, and tolerance development. The ORL1 receptor therefore represents a new molecular target for the design of novel agents for anxiety, analgesia, and drug addiction. Indeed, there is tremendous interest in the pharmaceutical industry in the development of nonpeptide ligands such as the potent ORL1 agonist, Ro 64-6198, as anxiolytics and the ORL1 antagonist JTC-801 as novel analgesics. This review presents an overview of the various peptide and nonpeptide ORL1 ligands with an emphasis on their potential therapeutic utility in various human disorders.     

Delta Opioid Receptors Regulate Temporoammonic-Activated Feedforward Inhibition to the Mouse CA1 Hippocampus

The opioid system influences learning and memory processes. However, neural mechanisms underlying the modulation of hippocampal activity by opioid receptors remain largely unknown. Here, we compared how mu and delta receptors operate within the mouse CA1 network, and used knock-in mice expressing functional delta opioid receptors fused to the green fluorescent protein (DOR-eGFP) to determine how delta opioid receptor-expressing interneurons integrate within the hippocampal circuitry. Through whole cell patch-clamp recording of CA1 pyramidal neurons from wild-type and DOR-eGFP mice, we found that mu and delta receptors both modulate spontaneous GABAergic inhibition received by these cells. Interestingly, mu but not delta receptor activation decreased the feed-forward inhibitory input evoked by Schaffer collateral stimulation. However, mu and delta agonists modulated GABAergic feed-forward inhibition when evoked upon stimulation of the temporoammonic pathway. In addition, anterograde tracing using biotinylated dextran amine injected into the entorhinal cortex of DOR-eGFP mice suggests the existence of synaptic contacts between temporoammonic afferents and delta receptor-expressing interneurons processes in CA1. Altogether, our data demonstrate a distinct modulatory role of the hippocampal network activity by mu and delta opioid receptors, and show for the first time that delta receptor-expressing interneurons in the CA1 are recruited by the temporoammonic pathway rather than the Schaffer collateral.     

Bivalent opioid peptide analogues with reduced distances between pharmacophores

To investigate the role of distance between two opioid peptide pharmacophores on in vitro and in vivo activities, three new bivalent opioid analogues have been synthesized in which the dipeptide Tyr-D-Phe was connected with diamine moieties ("bridges"). The analogue with a hydrazine bridge has high receptor affinity to mu, kappa, and delta receptor types, as well as potent and long acting antinociceptive activity after intraperitoneal administration.     

A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells

Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome.     

Syndecan-2 Functions as a Docking Receptor for Pro-matrix Metalloproteinase-7 in Human Colon Cancer Cells

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.     

BCL-2-induced glioma cell invasiveness depends on furin-like proteases

Migration and invasion are prerequisites for the neoplastic phenotype of malignant glioma. Ectopic expression of BCL-2 enhances migration and invasion of glioma cells and promotes their synthesis of transforming growth factor-beta2 (TGF-beta2). We here report that BCL-2-expressing cells show enhanced expression and activity of the proprotein convertase, furin, which processes metalloproteinases (MMP) and TGF-beta. Consistent with a biological role for a BCL-2-dependent increase in furin-like protease (FLP) activity, BCL-2-expressing cells exhibit enhanced MMP activity. Both a pseudosubstrate furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), or alpha 1-anti-trypsin Portland (PDX), a recombinant furin-inhibitory protein, suppress constitutive and BCL-2-mediated MMP activity and invasion. This inhibition is not overcome by TGF-beta or hepatocyte growth factor (HGF). A neutralizing TGF-beta antibody attenuates, but not abrogates, the invasive properties conferred by exogenous expression of BCL-2, whereas the MMP inhibitor o-phenantroline (o-PA) abolishes the pro-invasive action of BCL-2. Exogenous HGF results in enhanced, and expression of dominant-negative ezrin in reduced, FLP activity, and dec-RVKR-cmk blunts the HGF-induced expression of mature TGF-beta2. Consequently, HGF and BCL-2 family proteins use a furin-dependent pathway to promote invasion via TGF-beta and MMP in human malignant glioma cells and the pro-invasive properties of TGF-beta require furin- dependent MMP activity.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

Properties of a human insulin receptor with a COOH-terminal truncation. II. Truncated receptors have normal kinase activity but are defective in signaling metabolic effects

We have previously shown that a mutant human insulin receptor with a COOH-terminal 43-amino acid deletion (HIR delta CT), when expressed in Rat 1 fibroblasts, binds insulin normally, autophosphorylates, and undergoes endocytosis after insulin binding in a manner comparable to the normal human insulin receptor (HIRc). In this paper we have examined the biologic activity of the truncated and normal insulin receptors. In vitro, the HIR delta CT receptors caused a 1.8-fold greater phosphorylation of a Glu4/Tyr1 polypeptide than did the HIRc receptors, but the two receptor types were nearly equivalent in their ability to phosphorylate a src-derived peptide. Furthermore, insulin preactivation of HIRc and HIR delta CT receptors in intact cells led to equivalent stimulation of tyrosine kinase activity as subsequently determined for histone in vitro. Expression of HIRc receptors in cells led to enhanced sensitivity to insulin of 2-deoxy-D-glucose uptake and glycogen synthase activation. This increased sensitivity was proportional to receptor number at low (Ro = 6400) but not at high (Ro = 1.25 X 10(6] levels of receptor expression. However, expression of HIR delta CT receptors (Ro = 2.5 X 10(5] led to little, if any, increase in insulin sensitivity of either 2-deoxy-D-glucose uptake or glycogen synthase activation. Furthermore, compared with HIRc cells, HIR delta CT cells respond poorly to an agonistic monoclonal antibody specific for the human insulin receptor. In conclusion, the HIR delta CT receptor retains intact protein kinase activity in vitro. Despite this, however, the receptor displays low activity in mediating the metabolic effects of insulin.     

Dynorphin A analogs containing a conformationally constrained phenylalanine derivative in position 4: reversal of preferred stereochemistry for opioid receptor affinity and discrimination of kappa vs. delta receptors

Analogs of the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2 containing optically pure (R)- and (S)-2-aminotetralin-2-carboxylic acid (Atc) in position 4 were synthesized and evaluated for opioid receptor affinity. These peptides are the first reported dynorphin A analogs containing a conformationally constrained amino acid in place of the important aromatic residue Phe4. By incorporating resolved Atc isomers, the opioid receptor affinity and the stereochemistry of the constrained residue could be unambiguously correlated. Both Dyn A analogs containing Atc in position 4 retained nanomolar affinity for kappa and mu opioid receptors. Unexpectedly the peptide containing (R)-Atc, corresponding to a conformationally constrained D-Phe analog, displaying higher affinity for both kappa and mu receptors than the peptide containing (S)-Atc. In contrast [D-Phe4,D-Ala8]Dyn A-(1-11)NH2 exhibited significantly lower affinity for kappa and mu receptors than the parent peptide, as expected. Conformational restriction of the Phe4 sidechain or incorporation of D-Phe in position 4 had the largest effect on delta receptor affinity, yielding compounds with negligible affinity for these receptors. Thus, there appear to be distinctly different structural requirements for this residue for kappa vs. delta receptors, and it is possible to completely distinguish between these two receptors by changing a single residue in Dyn A.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumors. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM), which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic- ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this Commentary & View, we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.Key words: brain tumor, blood coagulation, hypoxia, MAP kinase, cancer stem cells, tumor invasion     

An opioid growth factor regulates the replication of microorganisms.

An opioid growth factor (OGF), [Met5]-enkephalin, interacts with the zeta (zeta) opioid receptor to modulate development of eukaryotes. We have found that [Met5]-enkephalin, an endogenous opioid peptide serves to inhibit the growth of S. aureus. This effect on growth involves cell proliferative events and is under tonic control, since potent opioid antagonists accelerate cell replication. Both the OGF and zeta opioid receptor were associated with these microorganisms. Other opioid receptors (mu, delta and kappa) were not detected. OGF also controlled the growth of other bacteria: P. aeruginosa and S. marcesans. These results indicate that OGF and its receptor, known to be important in the regulation of mammalian development, also function in the growth of simple unicellular organisms. We suggest that the endogenous opioid system related to growth originated billions of years ago.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.