Digitoxin activates EGR1 and synergizes with paclitaxel on human breast cancer cells

Abstract

Background: Numerous studies have suggested that digitalis derivatives promise to be superior to existing adjuvant therapy for breast cancer as to effects and side-effects. In the present study, we have used gene expression analysis to determine the molecular action of digitoxin on breast cancer cells and assessed digitoxin's ability to synergize with the chemotherapy agent paclitaxel with respect to inhibition of cell proliferation

Materials and methods: We treated (Her2 overexpressing, ER low) MDA-MB-453 human breast cancer cells with digitoxin at four doses {20 ng/ml (26 nM) to 1 μg/ml} and collected RNA at 6 h and 24 h for gene expression analysis. To examine the effects on ER positive cells, we treated MCF7 cells with digitoxin at 1 μg/ml and collected RNA for RT-PCR analysis. In addition, we assayed the growth inhibitory effect of low doses of digitoxin combined with paclitaxel and determined combination index values.

Results: To reveal primary effects, we examined digitoxin's effect 6 h post-treatment with the highest dose, 1μg/ml, and found upregulation of the stress response genes EGR-1 and NAB2, lipid biosynthetic genes and the tumor suppressor gene p21, and downregulation of the mitotic cell cycle gene CDC16 and the replication gene PolR3B. RT-PCR analysis validated effects on stress response, apoptotic and cell cycle genes on MDA-MB-453 and MCF7 cells. Western blot analysis confirmed induction of EGR1 protein at 1 h and ATF3 at 24 h. Paclitaxel, as well as digitoxin, inhibited the in vitro activity of the purified Na(+)-K(+)-ATPase; digitoxin enhanced the growth inhibitory effects of paclitaxel on Her2 overexpressing breast cancer cells.

Conclusions: Our studies show the potential of digitoxin to prevent and treat breast cancer and indicate that the combination of digitoxin and paclitaxel is a promising treatment for ER negative breast cancer. These findings are the first to alert physicians to the possible dangers to patients who take a combination of digitoxin and paclitaxel. The potential dangers ensuing when paclitaxel and digitoxin are combined are dependent on the dose of digitoxin.

Keywords: Cardiac glycosides; microarrays; paclitaxel; stress response; synergy.

Figure 1

(a) Structure of digitoxin (Sigma, St. Louis, MO); (b) Effects of digitoxin on cell proliferation in MDA-MB-453 breast cancer cells, by the MTT assay. The DMSO control contained 0.33% DMSO. Similar results were obtained in an additional experiment. Bars: SD of triplicate assays. (c) Schematic diagram of genes altered after treating MDA-MB-453 cells with digitoxin at 1μg/ml for 6 h {adapted from STRING: Search Tool for Retrieving INteracting Genes/proteins (24-6)}.

Figure 2

Hierarchical clustering of differentially expressed genes analyzed on U133A 2.0 Affymetrix chips after treating MDA-MB-453 cells with digitoxin at 0.1, 0.2 or 1.0 μg/ml for 6 and 24 h. Clustering was performed with the Program Cluster 3.0 (23). We restricted probesets to those that corresponded to an absolute value of M (log fold) > 2.0 for at least one of the conditions. The threshold for color in the hierarchical clustering map is M > 3 log fold. Fold change indicates relative expression in digitoxin versus DMSO control cells. To pick the blowup region, the area containing a specific gene was expanded to include a well-defined expression pattern. A) the full hierarchical clustering map, which contains 4706 probesets (a) upregulated gene region amplified for ATF3; (b) down regulated gene region amplified for CDC16; (c) upregulated gene region amplified for EGR1; red, upregulated; green, downregulated.

Figure 3

Real-time RT-PCR analysis: a, b, c) after treating MDAMB-453 cells with digitoxin at 0.1 μg/ml for 6 and 24 h. The cells were treated with 0.1 μg/ml of digitoxin and, after 6 and 24 h, extracts were prepared and analyzed by real-time RT-PCR, as described in Materials and Methods; a, b and c display different patterns of gene expression.* indicates P<0.05; Fold change indicates relative expression in digitoxin versus DMSO control cells.

Figure 4

a) Effects of digitoxin on the level of ATF3, EGR1 protein. MDA-MB-453 cells were treated with 0, 0.1, 0.2 or 1 μg/ml of digitoxin and after 1 and 24 h extracts were prepared and analyzed by Western blotting; an antibody to β-actin was used as a loading control. b) siRNA to ERK2. Cell were pretreated with control (nonsilencing) or MAPK1 (ERK2) siRNA for 24 h, exposed to digitoxin (0.4 μg/ml) for 24 h and percent inhibition of cell proliferation determined by the MTT assay. Percentages are normalized to DMSO. c) Inhibition of Na+-K+-ATPase activity in response to increasing concentrations of paclitaxel or digitoxin. The Na+-K+-ATPase assay was performed as described in Materials and Methods. The DMSO controls contained 3.3% DMSO. BARS: SD of triplicate assays.

Figure 5

Real-time RT-PCR analysis after treating MCF7 cells with digitoxin at 1 μg/ml for 6 and 24 h, as described in Materials and Methods. The cells were treated with 1.0 μg/ml of digitoxin and, after 6 and 24 h, extracts were prepared and analyzed by real-time RT-PCR, as described in Materials and Methods. a, b and c display different patterns of gene expression. All P values were <0.05, except ATF4 at 6h; Fold change indicates relative expression in digitoxin versus DMSO control cells.

Figure 6

Effects of digitoxin alone or in combination with paclitaxel (TAX) on cell proliferation in MDA-MB-453 breast cancer cells: a) x-axis, TAX concentration, b) x-axis, digitoxin concentration. The DMSO control contained < 0.1% DMSO; Bars: SD.