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In this study it was hypothesised that N-linked glycosylation might impede the binding of Herceptin to HER2 in breast cancer and alter cancer cell sensitivity to DXR and growth factors. To study Herceptin-HER2 binding a cell based quartz crystal microbalance (QCM) system with adherent cancer cells grown on the surface of a biosensor chip was used. The cell-chip enabled an evaluation of the kinetics of interaction between Herceptin and HER2 in a quasi-physiological environment25,26,27. In contrast to conventional systems, where single receptors such as glycoprotein entities are investigated, the cell based QCM enables drug-receptor interactions to be studied in the presence of the other biomolecules present at the cell surface. The interaction between Herceptin and SK-OV-3 cells (a human ovarian carcinoma cell line) was recently investigated using this approach28 and we used the QCM-based system to study Herceptin binding to the HER2-over-expressing breast cancer cell line SKBR-329 as well as to recombinant HER2 (rHER2) protein and to evaluate the effect of deglycosylation on Herceptin binding. In addition, breast cancer cells were grown in the presence of tunicamycin, an antibiotic that blocks the transfer of GlcNAc-1-P to dolichol-P, an essential early step in the intracellular production of N-linked glycans. The reduction in cell surface glycosylation following tunicamycin treatment was evaluated using the lectins wheat germ agglutinin (WGA) and Concanavilin A (Con A), carbohydrate binding proteins recognising N-linked glycans. The sensitivity of cancer cells to growth factors: EGF and IGF-1 alone and in combination, was evaluated following tunicamycin treatment and the role of glycosylation on sensitivity to DXR was determined. In summary, the current study evaluated the role of glycosylation on the binding of Herceptin and the sensitivity of breast cancer cells to DXR. In addition, the role of glycosylation on cellular sensitivity to growth factors (EGF, IGF-1) was evaluated.
The binding of Herceptin to BT474, ZR-751, MCF-7 and SKBR-3 breast cancer cells was assessed using immunofluorescence and confocal microscopy (Fig. 1). BT474 and ZR-751 showed the least binding to Herceptin while MCF-7 and SKBR-3 cells bound to Herceptin more intensely with SKBR-3 showing the most intense staining amongst these cell lines. When SKBR-3 cells were incubated with an anti-cerbB-2 antibody, as expected, the cells exhibited a much more intense staining pattern due to greater accessibility of the HER2 epitope to the antibody compared with accessibility to Herceptin. All subsequent experiments in which the kinetics of Herceptin binding was assessed were performed with SKBR-3 cells.
The changes in the frequency of the sensor surface resonance (ΔF) during the binding experiments was recorded using the Attestar software and the data were analysed using the Evaluation (Attana, Stockholm, Sweden) and TraceDrawer software (Ridgeview Instruments AB, Stockholm, Sweden). To obtain values for the specific binding response, the background binding to the reference surfaces were subtracted from the experimental surfaces. 1:1 or 1:2 binding models were used to calculate the kinetic parameters including the rate constants (ka, kd), dissociation constant (KD) and the maximum binding capacity (Bmax). All biosensor experiments were repeated using a second and third set of sensor surfaces prepared using the same procedure.
Protein expression levels were confirmed using primary anti-HER2/Neu mouse monoclonal antibody (Santa Cruz; Dallas, Texas, US sc-08, 1:500 dilution in 1% w/v BSA-Tween) and anti-EGFR mouse monoclonal antibody (Santa Cruz; Dallas, Texas, US, sc-374607 1:1000 dilution in 1% w/v BSA-Tween) followed by goat anti-mouse horseradish peroxidase conjugated antibody (Santa Cruz Dallas, Texas, US sc-2005; 1:2500 dilution in 1% v/v BSA-Tween) at room temperature. The HRP reaction was detected using ClarityTM Western ECL substrate and visualised using ChemiDoc XRS with Image Lab software (Bio-Rad, Hemel Hempstead, UK).