RESUMEN
Oligosaccharides of hyaluronan (o-HA) can induce angiogenesis and the growth and tube formation of vascular endothelial cells (ECs) in particular. As the major o-HA receptor, CD44 has been implicated in EC function, but its role in mediating o-HA-induced EC proliferation and tube formation remains unclear. In this study, we investigated the role of CD44 in o-HA-induced proliferation and tube formation of human umbilical vein endothelial cells (HUVECs) and explored the molecular mechanisms underlying the angiogenesis process. A CD44 siRNA was delivered into HUVECs by electroporation and o-HA-induced proliferation and tube formation capacity of CD44-silenced or control HUVECs were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Matrigel assays. Furthermore, the changes in Src, focal adhesion kinase (FAK) and extracellular signal-regulated kinase1 and 2 (ERK1/2) phosphorylation, as well as the expression of c-jun and c-fos were examined by Western blot and realtime-polymerase chain reaction assays. Our results demonstrated that 10 µg/mL o-HA obviously induced the proliferation and tube formation in HUVECs, and stimulated the phosphorylation of Src, FAK and ERK1/2 and upregulation of c-jun and c-fos, which could be inhibited by CD44 silencing. Altogether our data suggest that CD44 functions to initiate tyrosine phosphorylation of Src, FAK and ERK1/2, and upregulates the expression of c-jun and c-fos, thus mediating o-HA-induced proliferation and tube formation in HUVECs.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , Receptores de Hialuranos/fisiología , Ácido Hialurónico/farmacología , Oligosacáridos/fisiología , Transducción de Señal/fisiología , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Genes fos/efectos de los fármacos , Genes fos/fisiología , Genes jun/efectos de los fármacos , Genes jun/fisiología , Humanos , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/metabolismo , Oligosacáridos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluate the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. METHODS: A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. RESULTS: Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. CONCLUSIONS: Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.