Eukaryotic Initiation Factor 4E (eIF4E) and angiogenesis: prognostic markers for breast cancer

BACKGROUND: The overexpression of eukaryotic translation initiation factor 4E (eIF4E), a key regulator of protein synthesis, is involved in the malignant progression of human breast cancer. This study investigates the relationship between eIF4E and angiogenesis, as well as their prognostic impact in patients with human breast cancer. METHODS: Immunohistochemical staining was used to determine protein expression of eIF4E, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and CD105 in a set of 122 formalin-fixed, paraffin-embedded primary breast cancer tissues. Expression of eIF4E in positive cells was characterized by cytoplasmic staining. Evaluation of VEGF and IL-8 in the same tissue established the angiogenic profiles, while CD105 was used as an indicator of microvessel density (MVD). RESULTS: A significant relationship was found between the level of eIF4E expression and histological grade (P = 0.016). VEGF, IL-8, and MVD were closely related to tumor grade (P = 0.003, P = 0.022, and P < 0.001, respectively) and clinical stage (P = 0.007, P = 0.048, and P < 0.001, respectively). Expression of eIF4E was also significantly correlated with VEGF (P = 0.007), IL-8 (P = 0.007), and MVD (P = 0.006). Patients overexpressing eIF4E had significantly worse overall (P = 0.01) and disease-free survival (P = 0.006). When eIF4E, histological grade, tumor stage, ER, PR, Her-2 status and the levels of VEGF, IL-8, MVD were included in a multivariate Cox regression analysis, eIF4E emerged as an independent prognostic factor for breast cancer (P = 0.001), along with stage (P = 0.005), node status (P = 0.046), and MVD (P = 0.004). CONCLUSION: These results suggest that higher eIF4E expression correlates with both angiogenesis and vascular invasion of cancer cells, and could therefore serve as a useful histological predictor for less favorable outcome in breast cancer patients, as well as represent a potential therapeutic target

Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action

Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer

Correction to Kleiner HE, Krishnan P, Tubbs J, Smith M, Meschonat C, Shi R, Lowery-Nordberg M, Adegboyega P, Unger M, Cardelli J et al: Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer. J Exp Clin Cancer Res 2009, 28:5

The Integrin Antagonist Cilengitide Activates αVβ3, Disrupts VE-Cadherin Localization at Cell Junctions and Enhances Permeability in Endothelial Cells

Cilengitide is a high-affinity cyclic pentapeptdic αV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through αVβ3/αVβ5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the β1 family, and little is known on the effect of cilengitide on endothelial cells expressing αVβ3 but adhering through β1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on αVβ3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the β1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface αVβ3, stimulated phosphorylation of FAK (Y397 and Y576/577), Src (S418) and VE-cadherin (Y658 and Y731), redistributed αVβ3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density β1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of αVβ3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent

Tight Junction-Related Barrier Contributes to the Electrophysiological Asymmetry across Vocal Fold Epithelium

Electrophysiological homeostasis is indispensable to vocal fold hydration. We investigate tight junction (TJ)-associated components, occludin and ZO-1, and permeability with or without the challenge of a permeability-augmenting agent, histamine. Freshly excised ovine larynges are obtained from a local abattoir. TJ markers are explored via reverse transcriptase polymerase chain reaction (RT-PCR). Paracellular permeabilities are measured in an Ussing system. The gene expression of both TJ markers is detected in native ovine vocal fold epithelium. Luminal histamine treatment significantly decreases transepithelial resistance (TER) (N = 72, p<0.01) and increases penetration of protein tracer (N = 35, p<0.001), respectively, in a time-, and dose-dependent fashion. The present study demonstrates that histamine compromises TJ-related paracellular barrier across vocal fold epithelium. The detection of TJ markers indicates the existence of typical TJ components in non-keratinized, stratified vocal fold epithelium. The responsiveness of paracellular permeabilities to histamine would highlight the functional significance of this TJ-equivalent system to the electrophysiological homeostasis, which, in turn, regulates the vocal fold superficial hydration

A model for co-expression pattern analysis of genes implicated in angiogenesis and tumour cell invasion in cervical cancer

To date, numerous genes have been identified which are involved in both tumour neovascularisation (angiogenesis) and tumour cell invasion, and most of them are also expressed to some extent under normal physiological conditions. However, little is known about how these genes co-express in these settings. This study was undertaken to quantitate mRNA levels in normal and malignant cervical tissues of nine selected genes (VEGF121, VEGF165, VEGF189, VEGF-C, eIF-4E, b-FGF, TSP-2, MMP-2 and MMP-9) implicated in the above processes using real-time quantitative RT–PCR. In addition, the Spearman's rank correlation was used to determine their co-expression patterns. The transcript levels for the different VEGF-A splice variants (VEGF121, VEGF165, VEGF189) were at least 10-fold higher in the cancer cases, with the highest levels in the primary tumours demonstrating lympho-vascular space involvement. The lymphangiogenic factor VEGF-C and MMP-9 were upregulated 130- and 80-fold respectively in cervical cancers. The highest levels of VEGF-C mRNA were found in the lymph-node positive group. The transcript levels for b-FGF were similar in normal cervical tissue and early-stage cervical cancer, however, higher levels were found in the cervical cancers with advanced stage disease. Comparing gene transcript levels between recurrent and non-recurrent cervical cancer patients revealed significant differences (P=0.038) in transcript levels for the angiogenesis inhibitor TSP-2, with the highest levels in non-recurrent cases. Co-expression pattern analysis in normal cervical tissue revealed highly significant co-expressions (P<0.0001) between TSP-2 and most other genes analysed (VEGF121, VEGF165, VEGF-C, b-FGF and MMP-2). In cervical cancer, TSP-2 appears only to be highly co-expressed with MMP-2 (P<0.0001). In contrast to normal cervical tissue, we found a highly significant co-expression (P<0.0001) between MMP-9 and VEGF189 in cervical cancer. The combined application of real-time quantitative RT–PCR and Spearman's rank correlation identifies gene transcripts which are simultaneously co-expressed. Our results revealed a significant co-expression between the angiogenesis inhibitor TSP-2 and most other genes analysed in normal cervical tissue. In cervical cancer, we found a strong upregulation of VEGF-C and MMP-9 mRNA, with a highly significant co-expression between MMP-9 and VEGF189

The Heart Is an Early Target of Anthrax Lethal Toxin in Mice: A Protective Role for Neuronal Nitric Oxide Synthase (nNOS)

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS−/− mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS−/− mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS−/− and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS−/− mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS−/− and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage

Vascular permeability, vascular hyperpermeability and angiogenesis

The vascular system has the critical function of supplying tissues with nutrients and clearing waste products. To accomplish these goals, the vasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. Physiologists and many vascular biologists differ as to the definition of vascular permeability and the proper methodology for its measurement. We review these conflicting views, finding that both provide useful but complementary information. Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). We demonstrate that three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, and the anatomic pathways by which molecules of different size cross-vascular endothelium. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. Finally, we list the numerous (at least 25) gene products that different authors have found to affect vascular permeability in variously engineered mice and classify them with respect to their participation, as far as possible, in BVP, AVH and CVH. Further work will be required to elucidate the signaling pathways by which each of these molecules, and others likely to be discovered, mediate the different types of vascular permeability