Subscribe to RSS
DOI: 10.1055/s-0028-1109907
© Georg Thieme Verlag KG Stuttgart · New York
Inhibition of Neuropilin-1 by RNA-Interference and its Angiostatic Potential in the Treatment of Hepatocellular Carcinoma
Angiostatische Wirkungen einer Inhibition von Neuropilin-1 durch RNA-Interferenz im hepatozellulären KarzinomPublication History
manuscript received: 24.8.2009
manuscript accepted: 29.10.2009
Publication Date:
13 January 2010 (online)
Zusammenfassung
Neuropilin-1 (Nrp1) wurde erst vor Kurzem als weiterer Rezeptor für den endothelialen Wachstumsfaktor VEGF beschrieben. Daher wird Nrp1 auch eine Rolle in der Tumorangiogenese und Progression zugeschrieben, was die nachgewiesene Expression von Nrp1 auf Endothel- und Tumorzellen bestätigt. Hier wurde daher die potenzielle Rolle eines Nrp1-knock-down durch siRNA auf Hepatom- und Endothelzell-Funktionen in vitro und das Tumorwachstum in vivo untersucht. Der Einfluss von siRNA-Nrp1 auf die Expression angiogener Faktoren sowie auf die intrazelluläre Signaltransduktion in Endothel-(SVEC4 – 10) und Hepatom (Hepa129)-Zellen wurde mittels semiquantitativer Real-Time-PCR und Western-Blot untersucht. Funktionelle Effekte auf Endothelzellen wurden im Matrigel-Assay in vitro und in vivo analysiert. Diese Ergebnisse wurden daraufhin in vivo in ein subkutanes Hepatom-Modell transferiert und mittels Immunhistochemie (Proliferation, Apoptose und Blutgefäßdichte) nachgewiesen. Die Transfektion von SVEC4 – 10 und Hepa129 mit siRNA-Nrp1 führte zu einer starken Herabregulation von Nrp1 auf RNA- und Protein-Ebene, wobei sich kein Einfluss auf die intrazelluläre Signaltransduktion zeigte. Trotzdem wurde die Bildung endothelialer Strukturen in Matrigel durch siRNA-Nrp1 in vitro um 59 % und in vivo um 94 % im Vergleich zur Kontroll-siRNA gehemmt. Dies korrelierte mit einer verringerten Expression des Zelladhäsionsmoleküls VCAM. Andererseits konnten subkutanes Tumorwachstum und Tumorzellproliferation durch siRNA-Nrp1 nicht gebremst werden, obwohl auch die Blutgefäßdichte deutlich reduziert war. Damit führte die Herabregulation von Nrp1 mittels siRNA zu deutlichen antiangiogenen Effekten, ohne Tumorwachstum und -proliferation zu beeinflussen.
Abstract
Neuropilin-1 (Nrp1) was recently described as a novel receptor for the pro-angiogenic molecule vascular endothelial growth factor (VEGF), indicating a role in tumor angiogenesis and tumor progression. Recent data confirm this assumption by demonstrating that some tumor and endothelial cells express Nrp1. Therefore, we wanted to investigate the potential role of Nrp1-knockdown on hepatoma and endothelial cell function in vitro and tumor growth in vivo. Nrp1 knockdown in SVEC4 – 10 and Hepa129 cells and its influence on signal transduction (MAPK pP38, pAKT, pERK1 / 2) was analyzed by Western blot. Effects on endothelial tube formation were assayed in an in vitro and in vivo matrigel assay. In vivo, effects of siRNA-Nrp1 were analyzed in a subcutaneous hepatoma model. To verify effects on endothelial and tumor cells in vivo, immunohistochemistry for proliferation, apoptosis and endothelial vessels was performed. LightCycler and Western blot analysis showed efficient inhibition of gene expression in SVEC4 – 10 and Hepa129 cells following siRNA-Nrp1 transfection. Signal transduction pathways were not influenced after siRNA-Nrp1 treatment compared to the controls. Endothelial tube formation was reduced by 59 % and 94 % in vitro and in vivo compared to controls, corresponding to reduced VCAM expression. Subcutaneous tumor growth was not influenced after siRNA treatment. Intratumoral proliferation was not altered after treatment with siRNA-Nrp1, whereas microvessel density and apoptosis were reduced after treatment with siRNA-Nrp1 compared to siRNA-Ctrl. In conclusion, inhibition of Nrp1 expression led to strong anti-endothelial effects, whereas tumor cells and tumor growth were not affected.
Schlüsselwörter
hepatozelluläres Karzinom - RNA-Interferenz - Neuropilin-1 - VEGF Signalling - antiangiogene Effekte
Key words
hepatocellular carcinoma - RNA interference - neuropilin-1 - VEGF signaling - anti-endothelial effects
References
- 1 Soker S, Takashima S, Miao H Q. et al . Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell. 1998; 92 735-745
- 2 Guttmann-Raviv N, Kessler O, Shraga-Heled N. et al . The neuropilins and their role in tumorigenesis and tumor progression. Cancer Lett. 2006; 231 1-11
- 3 Robinson C J, Stringer S E. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001; 114 (Pt 5) 853-865
- 4 Soker S. Neuropilin in the midst of cell migration and retraction. Int J Biochem Cell Biol. 2001; 33 433-437
- 5 Pan Q, Chanthery Y, Liang W C. et al . Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell. 2007; 11 53-67
- 6 Miao H Q, Lee P, Lin H. et al . Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression. Faseb J. 2000; 14 2532-2539
- 7 Quante M, Raskopf E, Stahl S. et al . No functional and transductional significance of specific neuropilin 1 siRNA inhibition in colon carcinoma cell lines lacking VEGF receptor 2. Oncol Rep. 2009; 21 1161-1168
- 8 Llovet J M, Bruix J. Novel advancements in the management of hepatocellular carcinoma in 2008. J Hepatol. 2008; 48 (Suppl 1) S20-S37
- 9 Llovet J M, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology. 2008; 48 1312-1327
- 10 Raskopf E, Vogt A, Sauerbruch T. et al . siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo. J Hepatol. 2008; 49 977-984
- 11 Kornek M, Lukacs-Kornek V, Limmer A. et al . DOTAP formulated (immune-stimulatory) VEGF-A siRNA increased antitumoral efficacy in orthotopic HCC in murine liver fibrosis. Mol Med. 2008; 14 (7–8) 365-373
- 12 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65 55-63
- 13 Pan Q, Chathery Y, Wu Y. et al . Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting. J Biol Chem. 2007; 282 24 049-24 056
- 14 Schmitz V, Raskopf E, Gonzalez-Carmona M A. et al . Plasminogen fragment K 1 – 5 improves survival in a murine hepatocellular carcinoma model. Gut. 2007; 56 271-278
- 15 Zachary I. VEGF signalling: integration and multi-tasking in endothelial cell biology. Biochem Soc Trans. 2003; 31 (Pt 6) 1171-1177
- 16 Rajan S, Ye J, Bai S. et al . NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells. J Cell Biochem. 2008; 105 477-486
- 17 Robbins M, Judge A, Ambegia E. et al . Misinterpreting the therapeutic effects of siRNA caused by immune stimulation. Hum Gene Ther. 2008; 19 (10) 991-999
Dr. Esther Raskopf
Department of Inner Medicine I, University of Bonn, Medical School
Sigmund-Freud-Straße 25
53127 Bonn
Germany
Phone: ++ 49/2 28/28 71 64 69
Fax: ++ 49/2 28/28 71 14 83
Email: esther.raskopf@ukb.uni-bonn.de