Investigation of Physiological Effects Induced by Dehydroepiandrosterone in Human Endothelial Cells and Ovarian Cancer Cell Line

Abstract

Objectives: Dehydroepiandrosterone (DHEA) is an endogenous hormone that acts as a ligand for several cellular receptors. An age-dependent decline in circulating levels of DHEA is linked to changes in various physiological functions. In gynecological clinical practice, DHEA is commonly prescribed to induce ovulation. Some clinical studies report a positive association between high serum concentrations of DHEA and an increased risk of developing ovarian cancer. However, the in vitro physiological effects of DHEA on ovarian cancerous cells have not been explored thus far. In this study, we aimed to investigate the physiological effects of DHEA treatment (0-200 μM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells.

Materials and methods: The physiological effects of DHEA treatment (0-200 μM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells were investigated with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, acridine orange/ethidium bromide staining, and scratch assay.

Results: DHEA treatment promoted proliferation of the MDAH-2774 cancer cell line in a dose-dependent manner (r=0.6906, p<0.0001, for 24 hours) (r=0.6802, p<0.0001, for 48 hours) (r=0.7969, p<0.0001, for 72 hours). In contrast, DHEA inhibited proliferation of the primary HuVeC cells (r=0.9490, p<0.0001, for 24 hours) (r=0.9533, p<0.0001, for 48 hours) (r=0.9584, p<0.0001, for 72 hours). In agreement with these observations, DHEA treatment resulted in a dose-dependent increase in the number of necrotic cells in the primary HuVeC cells (r=0.97, p<0.0001). However, the number of necrotic or apoptotic cells did not change significantly when the MDAH-2774 cells was exposed to DHEA. Moreover, we found that DHEA treatment reduced the migration rate of HuVeC cells in a dose-dependent manner (r=0.9868, p<0.0001), whereas only a slight increase was observed in the MDAH-2774 ovarian cancer cell line (r=0.8938, p<0.05).

Conclusion: Our findings suggest that DHEA promotes the proliferation of ovarian cancer cells in a dose-dependent manner in vitro. Moreover, DHEA induced necrosis and inhibited proliferation in endothelial cells. Although mechanistic evidence is required, our preliminary findings imply that exposure to high doses of DHEA may be associated with an increased risk of developing ovarian cancer.

Keywords: Dehydroepiandrosterone; HuVeC; MDAH-2774; ovarian cancer.

Figure 1

The effect of DHEA on cell proliferation. a, c, e) MDAH-2774 ovarian cancer and b, d, f) HuVeC endothelial cell lines were treated with dehydroepiandrosterone for 24, 48, and 72 hours in an incubator. MTT assays were performed 24, 48, and 72 hours after treatment with the indicated doses of DHEA relative % changes in proliferation rates were compared against the non-treated control group (0 μM), and statistical significance was tested using One-Way ANOVA followed by Tukey’s multiple-comparison test (*, **, *** p<0.0001, n=6). The correlation between DHEA dose and its effect on proliferation was analyzed by linear regression (GraphPad Prism program). MDAH: r=0.6906, p<0.0001, for 24 hours; r=0.6802, p<0.0001, for 48 hours; and r=0,7969, p<0.0001 for 72 hours. HuVeC: r=0.9490, p<0.0001 for 24 hours; r=0.9533, p<0.0001, for 48 hours; and r=0,9584, p<0.0001 for 72 hours. DHEA: Dehydroepiandrosterone, MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

Figure 2

DHEA induces necrosis at high concentrations. a) MDAH-2774, and b) HuVeC endothelial cell lines were treated with 10-200 μM DHEA for 48 hours in an incubator. AO/EtBr double staining was performed 48 hours after treatment with the indicated doses of DHEA. Percent changes in the ratio of necrotic cells were compared against the non-treated control group (0 μM), and statistical significance was tested using One-Way ANOVA followed by Tukey’s multiple-comparisons test. **, ***: p<0.05, n=3, DHEA: Dehydroepiandrosterone, AO/EtBr: Acridine orange/ethidium bromide

Figure 3

Representative microscope images from AO/EtBr double staining. a) Magnification: 10x, MDAH-2774 cells (control: 0 μM DHEA), b) magnification: 40x, MDAH-2774 cells (100 μM DHEA), c) magnification: 10x, HuVeC cells (100 μM DHEA), d) magnification: 40x, HuVeC cells (100 μM DHEA). Arrows point to apoptotic cells, arrow heads point to necrotic cells, and double arrow heads point to live cells. AO/EtBr: Acridine orange/ethidium bromide

Figure 4

DHEA reduces the cell migration rate at high concentrations in HuVeC and increases the migration rate in MDAH-2774. a) MDAH- 2774, b) HuVeC endothelial cell lines were treated with 10-200 μM DHEA for 48 hours in an incubator. A scratch assay was performed 48 hours after treatment with the indicated doses of DHEA. The rate of migration (how soon the gap closes) in 48 hours was calculated by measuring the gap at 0 and 48 hours after scratching the plates. Percent changes in the migration rates were compared against the non-treated control group (0 μM), and statistical significance was tested using One-Way ANOVA followed by Tukey’s multiple-comparison test. *, **, ***: p<0.05, n=3, DHEA: Dehydroepiandrosterone

Figure 5

Representative microscope images from in vitro scratch assay (magnification 10x). a) 0 hour MDAH-2774 cells, b) 48 hour 0 μM DHEA, MDAH-2774 cells, c) 48 hour 100 μM DHEA, MDAH-2774 cells, d) 0 hour HuVeC cells, e) 48 hour 0 μM DHEA, HuVeC cells, f) 24 hour 100 μM DHEA, HuVeC cells. DHEA: Dehydroepiandrosterone