Comparison of hyaluronidase expression,invasiveness and tubule formation promotion in ER （-） and ER （＋） breast cancer cell lines in vitro

Background 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. Methods 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. Results 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）. Conclusion 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.

Comparison of hyaluronidase expression, invasiveness and tubule formation promotion in ER (-) and ER (+) breast cancer cell lines in vitro

Background 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.Methods 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.Results 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 Matdgel 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).Conclusion 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.