Urolithin A suppresses the proliferation of endometrial cancer cells by mediating estrogen receptor-α-dependent gene expression

Abstract

Scope: Obese and overweight women are at high risk of developing endometrial cancer; indeed, many of endometrial cancer patients are obese. The increased number and size of adipocytes due to obesity elevate levels of circulating estrogens that stimulate cell proliferation in the endometrium. However, black raspberries are a promising approach to preventing endometrial cancer.

Methods and results: We examined 17 black raspberry constituents and metabolites (10 μM or 10 μg/mL, 48 h) for their ability to prevent endometrial cancer cells from proliferating. Urolithin A (UA) was most able to suppress proliferation in a time- and dose-dependent manner (p < 0.05). It arrested the G2/M phase of the cell cycle by upregulating cyclin-B1, cyclin-E2, p21, phospho-cdc2, and CDC25B. UA also acted as an estrogen agonist by modulating estrogen receptor-α (ERα) dependent gene expression in ER-positive endometrial cancer cells. UA enhanced the expression of ERβ, PGR, pS2, GREB1 while inhibiting the expression of ERα and GRIP1. Coincubating UA-treated cells with the estrogen antagonist ICI182,780 abolished UA's estrogenic effects. Knocking down ERα suppressed PGR, pS2, and GREB gene expression but increased GRIP1 expression. Thus, UA's actions appear to be mediated through ERα.

Conclusion: This study suggests that UA modulates ERα-dependent gene expression, thereby inhibiting endometrial cancer proliferation.

Keywords: Black raspberry; Cell proliferation; Endometrial cancer; Estrogen receptor; Urolithin A.

Figure 1

Urolithin A and B inhibit endometrial cancer cell proliferation. (A) List of 17 constituents and metabolites in BRBs. (B) ECC-1 and (C) Ishikawa cells were treated with the indicated compounds for 48h, and cell proliferation was measured using the CellTiter solution. Relative cell number was compared against vehicle-only (gray dotted line). *: P<0.05.

Figure 2

Urolithin A suppresses endometrial cancer cell proliferation. Human endometrial cancer (ECC-1, Ishikawa, and HEC1A) and normal (T HESCs) cells were treated with UA at 0-50μM for 48h (top panels) or at 10μM for 1-7 days (lower panels). Cell proliferation was measured by the CellTiter solution. ^#: P<0.05 UA compared with untreated. *: P<0.05 UA compared with vehicle treatment.

Figure 3

Urolithin A arrests the cell cycle at the G2/M phase. (A-B) ECC-1, Ishikawa, and HEC1A cells were treated with 10μM or 50μM UA or with vehicle for 48h. Cell cycles were determined by flow cytometry. (C) Expression of cell cycle regulators was analyzed by western blotting. β-actin was the loading control. *: P<0.05; **: P<0.01; ***: P<0.001 compared with vehicle only in the same cell line. ^###: P<0.001 compared between two treatment groups in the same cell line.

Figure 4

Urolithin A modulates estrogen receptor-regulated gene expression. ECC-1 and Ishikawa cells were pretreated with E2 (10nM) or ICI182,780 (ICI, 10μM) for 1h and then exposed to UA (10μM) for 48h. Total RNA was isolated, and real-time PCR was conducted using 36B4 as an internal control. *: P<0.05 compared with control (CT = vehicle-treated) cells.

Figure 5

Urolithin A's actions on estrogen-regulated gene expression and cell proliferation are ERα dependent. ECC-1 and Ishikawa cells were transfected with ERα siRNA (siRNA) and/or treated with UA (10μM) for 48h. mRNA (A) and protein (B) levels. Relative cell numbers (C) are shown as a function of these variables. Total RNA was isolated and measured following real-time PCR, using 36B4 as an internal control. Cell proliferation was detected using the CellTiter solution. *: P<0.05 compared with control (CT = vehicle-treated) cells.