Figure 6.

Addition of Targaprimir-515 (2) sensitizes MCF-7 cells to Herceptin and Kadcyla by stimulating the expression of HER2. (A) Addition of 2 to MCF-7 cells for 48 h enhances HER2 protein expression as measured via ELISA. Dotted line represents HER2 protein expression in untreated cells. (B) Addition of 2 to MCF-7 cells enhances cell surface expression of HER2 via flow cytometry. Dotted line represents HER2 surface expression in untreated cells. (C) 2 sensitizes MCF-7 cells to Herceptin-mediated loss of cell viability in a dose-dependent fashion. “Cell Viability Ratio (Herceptin:Untreated)” is the ratio between the cell viability of Herceptin treated cells and untreated cells. (D) Forced overexpression of miR-515 on a doxycycline inducible plasmid ablated Herceptin (20 nM) sensitivity in the presence of doxycycline (+Dox) but not in its absence (−Dox). (E) 2 stimulated Kadcyla-mediated cell death in otherwise insensitive MCF-7 cells. (F) Increased apoptotic activity in MCF-7 cells treated with 2 and Kadcyla as measured by Caspase 3/7 activity. Dotted line represents apoptotic activity in untreated cells. (G) Addition of SKI-178, a SK1 inhibitor, or a siRNA targeting SK1, ablated Herceptin sensitivity, whereas a scrambled control did not. 515-VM, Anti-515–5p Vivo-Morpholino; SCR-VM, scrambled control Vivo-Morpholino. (H) Transfection of SK1 plasmid into MCF-7 enhanced Herceptin (20 nM) sensitivity. Measurements were taken after 24 h incubation with 2, followed by 48 h of either Herceptin or Kadcyla treatment. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated or vehicle samples, as determined by a two-tailed Student t test.