Figure 3

The importance of cell surface HS to trastuzumab action. (A), Emission spectra of MCF7 cells incubated with anti-HS-Alexa Fluor 546 and/or trastuzumab-Alexa Fluor 594 obtained at 543 nm excitation, and fluorescence emission detected from 550 nm to 680 nm, using 10 nm steps in a fluorometer. (B), The values represent the analysis of FRET ratios [ratio of intensity of fluorescence found in the wavelength of maximum emission of trastuzumab-Alexa 594 (617 nm) and the fluorescence intensity obtained in the maximum wavelength emission of the Anti-HS-Alexa Fluor 546 (580 nm)]. (C) Reversion of the trastuzumab effect in MCF7 and SKBR3 cell viability assay by an anti-HS antibody. 3.0x10³ SKBR3 and 5.0x10³ MCF7 cells were treated with trastuzumab (25 μg/mL) and anti-HS antibody 1:100 and cell viability was determined also by MTT on the third incubation day. Each point indicates the mean ± SD of triplicate assays. *P < 0.05, compared to respective control cells. (D), Heparin added to the medium revert the decrease in cell viability induced by trastuzumab in sensitive breast cancer cells. 3.0x10³ SKBR3 and 5.0x10³ MCF7 and MCF7-HPSE1cells were treated with trastuzumab (25 μg/mL) and/or heparin (100 μg/mL) and cell viability was determined after three days of incubation. Each point indicates the mean ± SD of triplicate assays. *P < 0.05, compared to respective control cells.