Inhibition of neuropilin-1 by RNA-interference and its angiostatic potential in the treatment of hepatocellular carcinoma.
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.
Raskopf
E
E
Department of Inner Medicine I, University of Bonn, Medical School, Bonn Germany. esther.raskopf@ukb.uni-bonn.de
Vogt
A
A
Standop
J
J
Sauerbruch
T
T
Schmitz
V
V
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor / Q000187:drug effects / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000473:pathology; D005786:Gene Expression Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000098:blood supply* / Q000235:genetics* / Q000473:pathology; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008833:Microcirculation / Q000187:drug effects / Q000235:genetics; D009368:Neoplasm Transplantation; D039942:Neuropilin-1 / Q000037:antagonists & inhibitors* / Q000235:genetics*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D020533:Angiogenesis Inhibitors; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D039942:Neuropilin-1