Therapeutic utility of natural estrogen receptor beta agonists on ovarian cancer

Abstract

Ovarian cancer is the deadliest of all gynecologic cancers. Despite success with initial chemotherapy, the majority of patients relapse with an incurable disease. Development of chemotherapy resistance is a major factor for poor long-term survival in ovarian cancer. The biological effects of estrogens are mediated by estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Emerging evidence suggests that ovarian cancer cells express ERβ that functions as a tumor suppressor; however, the clinical utility of ERβ agonists in ovarian cancer remains elusive. We tested the utility of two natural ERβ agonists liquiritigenin (Liq), which is isolated from Glycyrrhiza uralensis and S-equol, which is isolated from soy isoflavone daidzein, for treating ovarian cancer. Both natural ERβ ligands had significant growth inhibition in cell viability and survival assays, reduced migration and invasion, and promoted apoptosis. Further, ERβ agonists showed tumor suppressive functions in therapy-resistant ovarian cancer model cells and sensitized ovarian cancer cells to cisplatin and paclitaxel treatment. Global RNA-Seq analysis revealed that ERβ agonists modulate several tumor suppressive pathways, including downregulation of the NF-κB pathway. Immunoprecipitation assays revealed that ERβ interacts with p65 subunit of NF-κB and ERβ overexpression reduced the expression of NF-κB target genes. In xenograft assays, ERβ agonists reduced tumor growth and promoted apoptosis. Collectively, our findings demonstrated that natural ERβ agonists have the potential to significantly inhibit ovarian cancer cell growth by anti-inflammatory and pro-apoptotic actions, and natural ERβ agonists represent novel therapeutic agents for the management of ovarian cancer.

Keywords: NF-κB; natural ERβ agonists; ovarian cancer.

Figure 1. ERβ agonists reduce OCa cell viability, survival and promote apoptosis in vitro

(A-B) ES2, BG-1, SKOV3, and SKOV3-TR cells were treated with vehicle or S-equol or Liq for 72 h, and the cell viability was measured by using a MTT assay. (C-D) ES2 and SKOV3 cells were treated with vehicle, S-equol, or Liq for 72 h and then cultured in growth medium for additional 7 days. The number of colonies for each group was counted. (E) ES2 cells were transduced with either empty or ERβ-FLAG expression vector and cell proliferation rates were measured using MTT assay. (F) ES2, SKOV3, and SKOV3-TR cells were treated with vehicle, S-equol, or Liq for 48 h, and the Caspase-3/7 activity was measured as described in Methods. Data are represented as mean ± SE. * p<0.05; ** p<0.01; *** p<0.001.

Figure 2. ERβ agonists sensitize OCa cells to paclitaxel and cisplatin treatment and reduce migration and invasion of OCa cells

(A-B) ES2 cells were pretreated with Liq (25 μM) for 48 h followed by treatment with varying doses of cytotoxic drugs paclitaxel or cisplatin for an additional 5 days. Cell viability was determined using MTT assay. (C) ES2 and SKOV3 cells were treated with vehicle, Liq or S-equol for 24 h and then used in transwell migration assays. Optical density was measured 16 h after migration. Photomicrographs of migrated cells in various treatments are shown. (D) Cell invasion potential of ES2 and SKOV3 cells treated with ERβ agonists was analyzed by using Matrigel invasion chamber assays. Photomicrographs of invaded cells in various treatments are shown. Data are represented as mean ± SE. ** p<0.01; *** p<0.001.

Figure 3. Analysis of global transcriptional changes modulated by ERβ agonists in OCa cells

Total RNA was isolated from the ES2 cells that were treated with either vehicle or Liq (100 μM) for 24 h and subjected to RNA sequencing. (A) Heat map of differentially expressed genes between vehicle and Liq is shown. (B) Differentially expressed genes were subjected to pathway analysis using IPA software, and the selected top canonical pathways are shown. Analysis of molecular and cellular functions of differentially expressed genes are shown. (C) Gene set enrichment analysis (GSEA) testing correlation of Liq-regulated genes with signatures of NF-κB signaling gene set and inflammatory response gene set. (D) ES2 and SKOV3 cells were treated with either vehicle or Liq or S-equol for 24 h, and the selective genes representing each pathway were validated by using RT-qPCR. Data are represented as mean ± SE. * p<0.05; ** p<0.01; *** p<0.001.

Figure 4. ERβ agonists attenuate NF-κB pathway activation

(A) ES2, SKOV3, and SKOV3-TR cells were transfected with NF-κB-luc reporter plasmid and grown for 24 h. Then, cells were treated with ERβ agonists and reporter activity was measured after 24 h. ES2 (B) and SKOV3 (C) cells were treated with Liq or S-equol (100 μM) for 24 h, and the expression status of NF-κB target genes was analyzed by using RT-qPCR. (D) ES2 cell lysates were used in pull-down assays using GST or ERβ-GST, and interaction of ERβ with p65 was analyzed by Western blotting. (E) ES2-ERβ-FLAG cell lysates were subjected to immunoprecipitation with IgG or FLAG antibodies and the interaction of ERβ with p65 was confirmed by Western blotting. (F) SKOV3 cells were transduced with empty vector or ERβ-FLAG expression vector and the expression of NF-κB target genes was analyzed by using RT-qPCR. Data are represented as mean ± SE. * p<0.05; ** p<0.01; *** p<0.001.

Figure 5. Effect of ERβ agonist Liq on OCa progression in vivo

(A) Athymic nude mice were implanted with SKOV3ip1-Luc cells intra-peritoneally and treated with vehicle or Liq for 3 weeks. Luciferase intensity detected by the Xenogen in vivo imaging system was used to measure tumor growth. (B) Representative images of tumor-bearing mice from control and treatment groups are shown. The tumor weight (C) and number of nodules (D) from control and treatment group are shown. Ki67 expression (E) as a marker of proliferation and TUNEL staining (F) as a marker of apoptosis were analyzed by performing immunohistochemistry (IHC) on tumor sections. For quantitation, Ki-67-positive and apoptotic cells from five different fields were counted and plotted as histogram. Data are represented as mean ± SE. * p<0.05. **p<0.01. (G) The expression of NF-κB target genes IL-1 beta and Cox-2 was determined on vehicle or Liq treated tumor sections by performing IHC.