Figure 5. Synergistic effect of targeting BAG-1 and HER2 on breast cancer cell growth and signaling.

(A) SKBR3 and BT474 cells were treated for 5 days with DMSO (0.125% v/v), trastuzumab (16 μg/ml), Thio-S (12.5 μM), Thio-2 (12.5 μM) or combinations of these compounds, as indicated. Cell growth was measured using crystal violet assay and is expressed as a percentage of untreated cells. Data shown on the graph are the mean ± SEM from 3 independent experiments, each with three technical replicates. Statistical significance was determined using two-way ANOVA with Holm-Sidak multiple comparisons test. *p < 0.05, ***p < 0.001. (B) MCF7, SKBR3 and BT474 cells were treated over 5 days with increasing concentrations of Thio-S, Thio-2, and/or trastuzumab at a constant ratio. Cell growth was measured by crystal violet assay and is expressed as a percentage mean ± SEM of untreated cells. Combination indexes were determined by CalcuSyn v2.11; values < 1 indicate synergism. (C) A representative immunoblot of 3 independent experiments shows the effect of single compounds and compound combinations (concentrations listed in Figure 5A), 24 h after treatment, on the phosphorylation and expression levels of molecules involved in growth factor signaling and translation/cell cycle control downstream of HER2 and BAG-1; β-actin was used as a loading control. (D) BT474 cells were treated with single compounds or compound combinations (concentrations listed in Figure 5A) for 48 h and were subsequently harvested and stained with propidium iodide for cell cycle analysis (20,000 events/sample) using ModFit LT v4.1.7. Bar graph shows the proportion of cells in each phase of the cell cycle as a percentage of the total number of cells stained. Paclitaxel (100 nM) treatment for 24 h was used as a positive control for cell cycle arrest. Data are representative of three independent experiments. (E) BT474 cells were metabolically labelled with ³⁵S to quantify global protein synthesis following 24 h treatment with the compounds indicated (concentrations listed in Figure 5A). ³⁵S incorporation corresponding to the total amount of protein synthesis is expressed as a percentage of diluent control. Data shown on graph are the mean ± SEM from 3 independent experiments, each with three technical replicates. Unpaired t-test was used for comparison between different treatments; *p < 0.05. (F) Simplified schematic diagram of PI3K/AKT and RAF/MEK/ERK signaling pathways downstream of HER2 and BAG-1. Red arrows indicate the molecular changes which occur as a result of combining trastuzumab with Thio-2, an inhibitor of BAG-1 protein-protein interactions.