Oleandrin-mediated inhibition of human tumor cell proliferation: Importance of Na,K-ATPase α subunits as drug targets

Expression of α3 isoform in BxPC3 and Panc-1 human pancreatic cancer cell lines. Cell lines were exposed to diluent (Control) or oleandrin (50 nmol/L) and MitoTracker Orange CM-H₂XRos (red-orange) to detect the presence and location of mitochondria for 2 h prior to fixation. After processing for α3 immunostaining, cells were counterstained with DAPI to detect nuclei (blue). BxPC3 cells contain low α3 levels whereas Panc-1 cells exhibit high α3 content. Also shown is a change in cell morphology to a more differentiated morphology upon exposure of Panc-1 cells to oleandrin. Insets, higher magnification of representative cells. B, exposure of Panc-1 cells to oleandrin did not affect the expression of α3 protein for at least 48 h at concentrations up to 50 nmol/L.

Uptake of fluorescent oleandrin analogue (BODIPY-oleandrin) by living cells. A, concentration-dependent uptake of oleandrin within BxPC-3 human pancreatic cells was observed at concentrations as high as 50 nmol/L over the 24 h observation period. In contrast, drug uptake was easily noted within Panc-1 cells with concentrations as low as 5 nmol/L. B, time-dependent uptake of oleandrin was evident in Panc-1 cells as early as 2 h, whereas at 24 h, nearly all cells contained marked concentrations of the drug. In contrast, no drug is observed in BxPC3 cells. Living cells were counterstained with MitoTracker orange CM-H₂XRos (red) to detect viable mitochondria and DAPI, which is excluded from viable cells with intact plasma membranes. More yellow signals indicate where mitochondria and BODIPY-oleandrin colocalize. DAPI uptake was minimal over this 2-h timeframe. Insets, a higher magnification of representative cells.

Decreasing the expression of α3 protein in Panc-1 cells affects cell sensitivity to oleandrin. A, cells were transfected for 24 h with either control siRNA or α3 siRNA. As shown, this resulted in an approximately 50% reduction of α3 protein. The extent of the reduction was dependent on the concentration of the α3 siRNA agent. 1, nontransfected control; 2, cells transfected with transfection agent alone; 3, control siRNA–transfected cells; 4, α3 siRNA–transfected cells. B, oleandrin uptake or association with Panc-1 cells is clearly evident in the nontransfected cells (1) yet significantly reduced in the siRNA-treated cells (2). C, the cells exhibiting knockdown of α3 protein were less sensitive to oleandrin implying that the α3 target is necessary to retain sensitivity to this cardiac glycoside.