Figure 3

NRC-03 and NRC-07 bind to anionic structures on breast cancer cells. (a) MDA-MB-231 breast cancer cells or normal dermal fibroblasts were cultured in the absence or presence of 50 μM biotinylated-NRC-03 or -NRC-07 for 10 minutes, stained with Texas Red-streptavidin, and visualized by fluorescence microscopy. Peptide binding was quantified by using NIS-Elements software. Statistical significance was determined with the Bonferroni multiple comparisons test; *p < 0.05 relative to NRC-03- and NRC-07-treated MDA-MB-231 cells, and †p < 0.05 relative to NRC-03-treated MDA-MB-231 cells. (b) MDA-MB-231 cells were incubated in the absence or presence of O-sialoglycoprotein endopeptidease (OSGE) for 30 minutes and then cultured in the absence or presence of 50 μM NRC-03 or NRC-07. Cell viability was determined with the MTT assay after 24 hours. Statistical significance was determined with the Bonferroni multiple comparisons test; *p < 0.05 relative to vehicle-treated cells. (c) Wild-type L cells, gro2C cells (heparan sulfate proteoglycan-deficient L cells), or sog9 cells (heparan sulfate proteoglycan- and chondroitin sulfate proteoglycan-deficient L cells) were cultured in the absence or presence of the indicated concentrations of NRC-03 and NRC-07. Cell viability was determined with the MTT assay after 24 hours. Statistical significance was determined with the Bonferroni multiple comparisons test; *p < 0.05 relative to L cells; †p < 0.05 relative to gro2C cells. (d) Biotinylated-NRC-03 and biotinylated-NRC-07 binding to heparan sulfate (HeS) and chondroitin sulfate (CS) proteoglycans was determined by using the solid-phase heparan sulfate- and chondroitin sulfate-binding assays. Data shown are significant by the Bonferroni multiple comparisons test; *p < 0.05 in comparison with the medium control. All data shown are the mean of three independent experiments ± SEM.