ABSTRACT
<p><b>OBJECTIVE</b>To study the effects of a small interfering RNA targeting CXCR-4 (shRNA-CXCR4) on angiogenesis of human breast cancer cells.</p><p><b>METHODS</b>The expression of CXCR4 mRNA and protein in 3 breast cancer cell lines with CXCR-4 silencing mediated by shRNA-CXCR4 was detected by RT-PCR and Western blotting, respectively. The morphological changes of human umbilical vein endothelial cells (HUVECs) were observed in co-culture with human breast cancer cells after CXCR4 gene silencing.</p><p><b>RESULTS</b>CXCR4 mRNA and protein expressions decreased significantly in MCF-7, MDA-MB-231 and MDA-MB-435s breast cancer cells after the gene silencing (P<0.05). Gene silencing with shRNA-CXCR4 in human breast cancer cells significantly inhibited the ability of HUVECs to form tubular structures in the co-culture (P<0.05).</p><p><b>CONCLUSION</b>Gene silencing by shRNA-CXCR4 can obviously lower the angiogenesis-inducing ability of human breast cancer cells.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Genetics , Cell Line, Tumor , Coculture Techniques , Endothelial Cells , Cell Biology , Neovascularization, Pathologic , Genetics , RNA Interference , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Receptors, CXCR4 , Genetics , Metabolism , Umbilical Veins , Cell BiologyABSTRACT
<p><b>BACKGROUND</b>Hyaluronidase (Hyase) is an enzyme which hydrolyses hyaluronan (HA), a large nonsulfated glycosaminoglycan. Several genes have been identified to code for hyaluronidases in humans. Its role has only recently been underlined in the invasion of prostate cancer, colonic cancer, and breast cancer. Moreover, the findings were in agreement with some experimental results which showed that HA-derived oligosaccharides had angiogenesis-promoting activity. All these findings prompted us to investigate factors that had been characterized as putative invasive factors in different human breast cancer-derived cell lines.</p><p><b>METHODS</b>We selected two series of human breast cancer-derived cell lines whose expression of estrogen receptors (ER) was previously published. Hyaluronidase secretion in culture medium and expression of matrix metallo-proteinase (MMP)-9, cathepsin-D (cath-D) and vascular endothelial growth factor (VEGF) by cells were determined. We also investigated cell invasiveness in the Matrigel invasion assay, and studied the capability of cancer cells to promote in vitro formation of tubules by endothelial cells.</p><p><b>RESULTS</b>ER(-) cells secreted significantly more hyaluronidase (P < 0.001) and expressed significantly more VEGF (P < 0.01), MMP-9 (P < 0.05) and cath-D (P < 0.0001) than ER(+) cells. Invasion through Matrigel by ER(-) Hyase(+) cells was significantly higher than that by ER(+) Hyase(-) cells (P < 0.05). In both cases, invasion was decreased by heparin (P < 0.05). When ECV-304 endothelial cells were co-cultivated in millicell chambers with cancer cells, ECV-304 cells were induced to form tubules. Tubule formation was demonstrated to be more prominent with ER(-) Hyase(+) cells than with ER(+) Hyase(-) cells (P < 0.05).</p><p><b>CONCLUSION</b>Invasive features of ER(-) breast cancer cells can be characterized in vitro by an invasive Matrigel assay, as the induction of tubule formation by ECV-304 endothelial cells, higher secretion of hyaluronidase, and higher expression of proteinases MMP-9, cath-D, and the angiogenesis promoting factor VEGF.</p>