Inhibitory Effects of Digoxin and Digitoxin on Cell Growth in Human Ovarian Cancer Cell Line SKOV-3

Abstract

Background: Cardiac glycosides (CGs) possess a chemical structure similar to steroids, and are inhibitors of the sodium potassium pump. An anti-tumor effect of CGs in breast and prostate cancers has been reported, but the effect of CGs on ovarian cancer is still unclear.

Aims: In this study, the effects of CGs on proliferation, cytotoxicity and cell cycle of ovarian cancer cell line (SKOV-3) have been investigated.

Procedure: The cell proliferation and cytotoxicity were detected by MTT assay and LDH activity assay, respectively. CGs, at concentrations higher than IC50, decreased cell proliferation and showed increased cytotoxicity toward SKOV-3 cells. The colony-formation ability was reduced after treatment with digoxin and digitoxin for 10 days. Furthermore, we explored the effect of digoxin and digitoxin on the distribution of cell cycle by flow cytometry.

Results: Results revealed that both digoxin and digitoxin led to cell cycle arrest in G₀/G₁ phase with 24 or 48 hours, but the arrest of G₀/G₁ phase was not observed at 72 hours. We evaluated the percentage of hypodiploid cell population as an index of the cellular fragments through flow cytometry. The data indicated that cellular fragments were significantly increased by treating with digitoxin at the concentrations of IC50 and 10^-6 M for 72 hours.

Conclusion: Taken together, these data suggest that CGs decreased cell proliferation and increased cytotoxicity through cell cycle arrest at the G₀/G₁ phase. CGs have anti-tumor effect in SKOV-3 cells and might be a potential therapeutic drug for ovarian cancer. Since this study is a preliminary investigation of CGs on SKOV-3 cells, more experiments might be performed in the future. Furthermore, more ovarian cancer cell lines might also be employed in the future studies to confirm the effect of CGs in ovarian cancer.

Keywords: SKOV-3; cell growth; digitoxin; digoxin; ouabain.

Figure 1.

Effects of CGs on proliferation of SKOV-3 cells. SKOV-3 cells were incubated with (A) digoxin (B) digitoxin and (C) ouabain at different concentrations for 24 and 48 hours. (D) Half maximal inhibitory concentration (IC50) of digoxin and digitoxin. Cell proliferation was analyzed by MTT assay. Each value represents mean ± SEM. These experiments were repeated 3 to 4 times.

Figure 2.

Effects of CGs on colonies formation of SKOV-3 cells. Cells were incubated with indicated concentrations of (A) digoxin and (B) digitoxin for 10 days. Colony-forming ability was compared with the control group. Each value represents mean ± SEM of 3 independent experiments. *P < .05, **P < .01 as compared with control group.

Figure 3.

CGs induced cytotoxicity in SKOV-3 cells. Cells were incubated in 96 well plate for 24 hours. After adhesion, cells were exposed to (A) digoxin and (B) digitoxin at indicated concentrations for 24 hours. The quantity of LDH in culture medium was measured by ELISA reader. The ratio of cytotoxicity was compared with control group. Each value represents mean ± SEM of 3 independent experiments. **P < .01 as compared with control group.

Figure 4.

Distribution of digoxin and digitoxin on cell cycle in SKOV-3 cells. Cells were exposed to (A) digoxin and (B) digitoxin at indicated concentrations for 24, 48, and 72 hours. Flow cytometric analysis was used to detect DNA contents. Percentages of cells in the G0/G1 phase were presented. (C) The percentage of hypodiploid cells incubated with digitoxin at indicated concentrations for 24, 48, and 72 hours were measured under the sub-G1 fraction. Each value represents mean ± SEM of 3 independent experiments. **P < .01 as compared with control group.

Figure 5.

Summary of the effect of digoxin and digitoxin on SKOV-3 cells. Digoxin and digitoxin treatment inhibited the ability of colonies formation and cell proliferation. The cytotoxicity was increased after administration with digoxin or digitoxin for 24 hours. CGs led to cell cycle arrest at G0/G1 phase.