FIGURE 2.

HS tetrasaccharides containing GlcNH₃⁺ structures inhibit heparanase activity and tumor invasion in vitro. A, the expression levels of HSPE-1 in various human breast cancer cell lines were analyzed by real-time PCR. The expression levels of HSPE-1 were normalized to G3PDH, and the means were obtained from three independent experiments. Error bars, S.D. B, structures of chemically synthesized HS tetrasaccharides. R indicates the modification of the N-position (-Ac, -SO₃H, or -H) of the GlcN residue. C, four kinds of human breast cancer cell lines were treated with either TD4-143-1, T5-67-9, or T5-60-6, and HS expression level in each cell type was analyzed as described under “Experimental Procedures.” D, the heparanase inhibitory activities of various chemically synthesized HS tetrasaccharides were compared. Heparanase activity was measured as described under “Experimental Procedures.” OGT2115 is a commercially available heparanase inhibitor. This assay was performed in triplicate, and the means were obtained from two independent experiments. Error bars, S.D. E, cell viability was measured after cells were treated with (solid bar) or without (open bar) 100 μg/ml TD4-143-1 for 22 h. F, the invasion potential of MDA-MB-231 (open bar) and BT549 cells (solid bar) treated either with 1 μm OGT2115, 100 μg/ml TD5-67-9, TD5-60-6, or TD4-143-1, was tested by a Matrigel in vitro invasion assay. This assay was performed in triplicate, and error bars represent the S.D. *, p < 0.05.