Figure 1.
ATPase Assay of breast cell membranes treated with cardiac glycosides; (A) ouabain, (B) digitoxin, and (C) bufalin.
The Na,K-ATPase activity in total membranes isolated from non-tumorous 184D, 184A, and MCF10A (10A) cells, as well as cancerous MDA-MB-231 (MDA), MCF7, and MCF10CA1 (CA1) cells is inhibited in a concentration dependent manner for all cells types. Na,K-ATPase activity (µmol Pi liberated per mg protein per hour) was determined and displayed as the percentage of activity relative to the untreated sample for each cell type. There was no statistically significant difference in activity between normal and tumor cells treated with similar concentrations of ouabain, digitoxin, or bufalin (p>0.05).
Figure 2.
Proliferation of CTS-treated cells.
A) Normal MCF10A cells exhibited a concentration-dependent decrease in BrdU incorporation (proliferation) when treated with 500 nM ouabain, 50 nM bufalin or 100 nM bufalin for 2 hours (p<0.01). MCF7, MCF10CA1, and MDA-MB-231 cells showed no significant decrease (p>0.05) in proliferation under comparable conditions. MDA-MB-231 cells treated with 50 nM or 100 nM bufalin had p-values of 0.057 and 0.061, respectively. B) High ouabain treatment (500 nM) for 24 hours caused a decrease in proliferation of MDA-MB-231 and MCF10CA1 cells, but no change to proliferation of MCF7 cells. Low ouabain or bufalin treatment had no effect on proliferation of any cancer cells tested.
Figure 3.
Viability of CTS-treated cells.
Normal 184D, 184A1, and MCF10A cells and tumorous MCF10CA1, MCF7, and MDA-MB-231 cells were treated with (A) ouabain (B) bufalin, or (C) digitoxin for 24 hours, and viability was determined by PrestoBlue Viability assay. Normal cells exhibited a concentration-dependent decrease in viability when treated with all CTS compounds tested. Tumor MCF7 and MDA-MB-231 cells were more resistant to CTS-induced cell death than MCF10A cells. Level of significance was determined (p<0.05) using ANOVA for group comparisons or Student’s t-test for individual comparisons.
Figure 4.
CTS-treated MCF10A cell viability is time-dependent and serum-sensitive.
A) MCF10A (dashed line) cells incubated with 500 nM ouabain showed a time-dependent decrease in viability during CTS treatment. MDA-MB-231 (solid line) viability was unaffected during the 48 hour duration of ouabain treatment. B) MCF10A cells grown with serum deprivation or with serum concentrations less than 5% had reduced viability when treated with 500 nM ouabain for 24 hours (dashed line). MDA-MB-231 cell viability was not affected by ouabain treatment when grown in 0-10% serum concentrations (solid line). C) MCF10A isolated membranes were incubated with 0-10% serum with or without 500 nM ouabain prior to ATPase assay. The difference in ATPase activity between untreated and ouabain treated was measured and displayed according to serum level. No significant difference in ATPase inhibition by ouabain exists from altering serum levels (p > 0.05).
Figure 5.
Apoptosis in normal and cancerous cells.
Ouabain causes MCF10A cells to undergo apoptosis, but not MCF7, MDA-MB-231, or MCF10CA1 cells. DNA fragmentation (free 3’-OH ends) was visible by FITC (green) staining using the TUNEL assay when MCF10A cells were treated with 500 nM ouabain or 50 nM bufalin for 6 hours. DNaseI was included as a positive control for DNA fragmentation of in MCF10A and MCF10CA1 cells. DAPI (blue) was used to counter-stain the nucleus, and images were collected using Zeiss Confocal Microscope and Imaging Software.
Figure 6.
Protein expression in cells with or without CTS treatment.
Lysates from MCF10A, MCF10CA1, and MDA-MB-231 cells treated with ouabain (Oua) or staurosporine (St) were analyzed with western blot using anti-actin, p53, total ERK1/2, or p-ERK antibodies. Western blot shows tumor cells contain altered levels of p53, and p-ERK after long-term ouabain treatment.
Figure 7.
Src kinase does not coimmunoprecipitate with α-subunit in MCF10A or MDA-MB-231 cells.
MCF10A and MDA-MB-231 cells were treated with (+) or without (-) 500 nM ouabain for 24 hours. Lysates were collected and subject to immunoprecipitation using a rabbit anti-α antibody, raised against Na,K-ATPase α-subunit’s M4/M5 cytoplasmic loop. 10% of the initial lysate (IN), the co-immunoprecipitated proteins (IP), and 10% of the unbound supernatant (Sup) after IP were used for western blots. Western blot analysis was performed and probed using mouse anti-Na,K-ATPase α1-subunit, mouse anti-Src, and mouse anti-Na,K-ATPase β1 subunit antibodies.