Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

BACKGROUND. Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. METHODOLOGY/PRINCIPAL FINDINGS. Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. CONCLUSIONS/SIGNIFICANCE. Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.Canadian Institutes of Health Research (16512, Cancer Consortium Training Grant Award, CancerConsortium Training Grant Award); US Lymphoma Foundation Award; National Institute of Health (RO1 GM073855); National Crime Information Center (017099); Cole Foundation Awar

Understanding the function of endoglin through the generation and characterization of endoglin deficient mice and the study of patients with hereditary hemorrhagic telangiectasia

grantor: University of TorontoEndoglin, (CD105), an integral membrane glycoprotein, is a member of the TGF-ß receptor superfamily. It is expressed at high levels on vascular endothelium and also found in several human hematopoietic cells. Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder associated with mutations in the 'Endoglin' gene, leading to loss of a functional allele (haploinsufficiency). To define the role of endoglin in normal and pathological blood vessel formation, we first investigated its expression in vascular endothelium of HHT patients. We then generated ' Endoglin' deficient mice and analyzed their vascular and immune systems. The haploinsufficiency model proposed for HHT was first based on ' in vitro' experiments. This thesis further confirms this model. It demonstrates that all blood vessels of HHT1 patients express reduced endoglin ' in situ', and that severe lesions such as arteriovenous malformations (AVMs) are not attributed to loss of heterozygosity. To generate an animal model for BHT and ascertain the role of endoglin in development, we generated mice lacking one and two copies of the gene. We show that 'Endoglin' null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development suggesting that endoglin is critical for both angiogenesis and heart valve formation. Some mice expressing a single allele of 'Endoglin' spontaneously developed clinical signs of HHT. The analysis of disease onset and progression revealed that clinical manifestations were heterogeneous, leading to severe vascular abnormalities and fatal hemorrhages. Our results demonstrate that the HHT mouse model reproduces well the heterogeneous phenotype of the human disease. The data suggest that in addition to reduced expression of endoglin, modifier genes can contribute to heterogeneity and severity of HHT. As expression of a single allele of 'Endoglin' leads to a vascular disorder, we investigated if it could alter the immune system of ' Endoglin' heterozygous mice. We show that reduced endoglin expression either on vascular endothelium or on subsets of hematopoietic cells leads to alteration of T and B lymphocyte homeostasis.Ph.D

Pierre Laffitte, un disciple très discipliné

National audienc

Cell-cell interactions influence vascular reprogramming by Prox1 during embryonic development.

Lymphangiogenesis is a highly regulated process that involves the reprogramming of venous endothelial cells into early lymphatic endothelial cells. This reprogramming not only displays a polarized expression pattern from the cardinal vein, but also demonstrates vascular specificity; early lymphatics only develop from the cardinal vein and not the related dorsal aorta. In our transgenic model of lymphangiogenesis, we demonstrate that Prox1 overexpression has the ability to reprogram venous endothelium but not early arterial endothelial cells in vivo, in spite of the fact that Prox1 expression is forced onto both vascular beds. Our observations suggest that this specificity during embryogenesis may be due to cell-cell interactions between the developing arterial endothelial cells and smooth muscle cells. These conclusions have far reaching implications on how we understand the vascular specificity of lymphangiogenesis

Vasculotide, an Angiopoietin-1 mimetic, ameliorates several features of experimental atopic dermatitis-like disease

Abstract Background Earlier studies by our group have demonstrated that a transgenic animal engineered to express Tie2 under the control of the Tie2 promoter produced animals with a scaly skin phenotype that recapitulated many of the hallmarks of atopic dermatitis (AT-Derm). To test the hypothesis that this model of AT-Derm is driven by dysregulated Tie2-signalling, we have bred AT-Derm transgenic (TG) animals with TG-animals engineered to overexpress Angiopoietin-1 or -2, the cognate Tie2 ligands. These two ligands act to antagonize one another in a context-dependent manner. To further evaluate the role of Ang1-driven-Tie2 signalling, we examined the ability of Vasculotide, an Ang1-mimetic, to modulate the AT-Derm phenotype. Results AT-Derm+Ang2 animals exhibited an accentuated phenotype, whereas AT-Derm+Ang1 presented with a markedly reduced skin disease, similarly VT-treated AT-Derm animals present with a clear decrease in the skin phenotype. Moreover, a decrease in several important inflammatory cytokines and a decrease in the number of eosinophils was noted in VT-treated animals. Bone marrow differentiation in the presence of VT produced fewer CFU-G colonies, further supporting a role for Tie2-signalling in eosinophil development. Importantly, we demonstrate activation of Tie2, the VT-target, in lung tissue from naïve animals treated with increasing amounts of VT. Conclusions The AT-Derm phenotype in these animals is driven through dysregulation of Tie2 receptor signalling and is augmented by supplemental Ang2-dependent stimulation. Overexpression of Ang1 or treatment with VT produced a similar amelioration of the phenotype supporting the contention that VT and Ang1 have a similar mechanism of action on the Tie2 receptor and can both counteract the signalling driven by Ang2. Our results also support a possible role for Tie2-signalling in the development of eosinophilic diseases and that activation of Tie2 may directly or indirectly modulate the differentiation of eosinophils, which express Tie2. In summary, these data support the hypothesis that this AT-Derm mouse model is driven by dysregulation of the Tie2 signalling pathway and increased Ang2 levels can aggravate it, whereas it can be reversed by either Ang1-overexpression or VT treatment. Moreover, our data supports the contention that VT acts as an Angiopoietin-1 mimetic and may provide a novel entry point for Tie2-agonist-based therapies for atopic diseases

Overexpression of Prox1 results in the expression of lymphatic markers on the jugular vein.

<p>(A) Normally, the expression of Podoplanin (FITC) on the jugular vein is downregulated by E13.5 and upregulated in lymph sacs, along with Prox1 (Cy3). (B) Prox1 overexpression results in its' expression on the jugular vein as well as the lymph sac. Furthermore, Podoplanin is now found expressed on the jugular vein (arrows). Note that the lymph sac has become significantly enlarged. Similarly, immunohistochemistry on (C) control and (D) double transgenic E13.5 embryos show an increase in staining of LYVE-1 (arrows) on the lymph sac and jugular vein. Scale bar = 25 µm. JV: jugular vein; LS: lymph sac.</p

Reprogramming via Prox1 in double transgenics is restricted to veins.

<p>Immunohistochemistry on E13.5 controls and double transgenics stained with (A and C) Podoplanin or (B and D) LYVE-1. While the jugular veins of DT embryos stained positive for both markers (C and D, arrows), the dorsal aortas did not (arrowheads). (E) Furthermore, Prox1 expression is absent on the dorsal aorta (the DA identified using smooth muscle actin-FITC) in E13.5 double transgenics (arrowhead), in contrast to the clear presence of Prox1 (Cy3, arrows) on the jugular vein. The absence of Prox1 on the dorsal aorta does not appear to be due to transgene functionality given that the constructs express within the dorsal aorta, assessed in <i>tie1 tTA:tetOS nls-LacZ</i> transgenics by β-gal staining at (F) E9.5 and (G) E13.5. (H) Furthermore, transgene expression on <i>tie2 tTA:tetOS prox1</i> double transgenics at E13.5 was directly assessed via VP16 (green) expression on the dorsal aorta (arrowheads) as well as the jugular vein (arrows). Staining in panel E, Prox1: Cy3; SMA: FITC. Staining in H, Prox1: Cy3; VP16: FITC. Scale bar = 25 µm (A–D, F, G); Scale bar = 50 µm (E and H). JV: jugular vein; CV: cardinal vein; DA: dorsal aorta.</p

Ectopic expression of Prox1 in arterial endothelial cells is not suppressed by the presence of smooth muscle cell conditioned media.

<p>Transfection of Prox1 in cultured arterial and venous endothelial cells demonstrate that they are both amenable to reprogramming. (A) RT-PCR analysis of targets such as Neuropilin-1 (NRP1) and STAT6, or western analysis of VEGFR-2 or Tie2 show a typical profile associated with Prox1 expression on vascular endothelial cells. (B) Furthermore, the overexpression of Prox1 in VECs and AECs result in an increase in VEGFR-3 and CyclinE2 transcript levels. (C) Smooth muscle cells associate in a timely and specific manner to the dorsal aorta, but not to the cardinal vein on wild type E9.5 and E10.5 embryos. (D) Prox1 overexpressing arterial endothelial cells were incubated with SMC conditioned media for 24 hours. Scale bar = 50 µm. DA: dorsal aorta; CV: cardinal vein.</p