Flavonoids Quercetin and Kaempferol Are NR4A1 Antagonists and Suppress Endometriosis in Female Mice

Abstract

Nuclear receptor 4A1 (NR4A1) plays an important role in endometriosis progression; levels of NR4A1 in endometriotic lesions are higher than in normal endometrium, and substituted bis-indole analogs (NR4A1) antagonists suppress endometriosis progression in mice with endometriosis. In addition, the flavonoids kaempferol and quercetin are natural products that directly bind NR4A1 and significantly repress the intrinsic NR4A1-dependent transcriptional activity in human endometriotic epithelial and stromal cells and Ishikawa endometrial cancer cells. NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin suppressed proliferation of human endometriotic epithelial cells and Ishikawa cells by inhibiting epidermal growth factor receptor/c-Myc/survivin-mediated growth-promoting and survival pathways, The mammalian target of rapamycin (mTOR) signaling and αSMA/CTGF/COL1A1/FN-mediated fibrosis signaling but increasing Thioredoxin domain Containing 5/SESN2-mediated oxidative/estrogen receptors stress signaling. In human endometriotic stromal cells, NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin primarily inhibited mTOR signaling by suppressing proliferation of human endometrial stromal cells. In addition, kaempferol and quercetin treatment also effectively suppressed the growth of endometriotic lesions in mice with endometriosis compared with the vehicle without any body weight changes. Therefore, kaempferol and quercetin are NR4A1 antagonists with potential as nutritional therapy for endometriosis.

Keywords: NR4A1 antagonists; endometriosis; kaempferol; quercetin.

Figure 1.

Kaempferol and quercetin as NR4A1 ligands. Quercetin (A) and kaempferol (B) interactions with the LBD of NR4A1 using a modeling approach as described (42-44) and critical interacting amino acids are indicated. Effects of kaempferol and quercetin on luciferase activity were determined in IHEECs (C), IHESCs (D), and Ishikawa cells (E) transfected with a chimeric GAL4-NR4A1 construct and a UAS-luc reporter gene. Luciferase activity was determined as outlined in “Materials and Methods.” Results (C-E) are expressed as means ± SE for at least 3 determinations, and significant (P < .05) changes compared with DMSO (control) are indicated (*).

Figure 2.

Endometriotic cell growth inhibition by quercetin and kaempferol. (A) Knockdown efficiencies of NR4A1 in IHEECs, IHESCs, and Ishikawa cells determined by Western blots of whole cell lysates as outlined in “Materials and Methods.” (B-D) IHEECs (B), IHESCs (C), and Ishikawa cells (D) were transfected with 2 oligonucleotides targeting NR4A1, and levels of NR4A1 were determined with Western blot analysis. (E-G) The proliferation of IHEECs (E), IHESCs (F), and Ishikawa cells (G) transfected with siNR4A1 oligonucleotides and control siRNA (siCtrl) was determined by the MTT assays. (E-H) The proliferation of IHEECs (E), IHESCs (F), Ishikawa cells (G), and NEM-4 (H) cells treated with quercetin or kaempferol for 24 hours was determined by MTT assays. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 3.

siNR4A1 knockdown, quercetin, and kaempferol decrease expression of growth-promoting and survival genes. (A-C) The levels of EGFR, c-Myc, and survivin in IHEECs (A), IHESCs (B), and Ishikawa cells (C) transfected with siNR4A1 oligonucleotides or treated with quercetin or kaempferol for 24 hours were analyzed by Western blotting. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 4.

siNR4A1, quercetin, and kaempferol modulate oxidative/ER stress pathway genes in endometriotic cells. (A-C) Levels of TXNDC5, SESN2, β-actin in IHEECs (A), IHESCs (B), and Ishikawa cells (C) transfected with siNR4A1 oligonucleotides or treated with quercetin or kaempferol for 24 hours were determined by Western blot analysis. (D) Levels of SESN2 and β-actin in IHEECs, IHESCs, and Ishikawa cells treated with DMSO, GSH, quercetin, kaempferol, GSH plus quercetin, and GSH plus kaempferol for 24 hours were determined by Western blotting. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 5.

siNR4A1, quercetin, and kaempferol inhibit mTOR signaling in human endometriotic cells. (A-C) Levels of mTOR marker proteins in IHEECs (A), IHESCs (B), and Ishikawa cells (C) transfected with siNR4A1 oligonucleotides or treated with kaempferol or quercetin for 24 hours were determined by Western blotting. (D) Quantitation of bands generated in Western blots (A-C) using β-actin as a loading control and compared with siCtrl band intensities. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 5.

siNR4A1, quercetin, and kaempferol inhibit mTOR signaling in human endometriotic cells. (A-C) Levels of mTOR marker proteins in IHEECs (A), IHESCs (B), and Ishikawa cells (C) transfected with siNR4A1 oligonucleotides or treated with kaempferol or quercetin for 24 hours were determined by Western blotting. (D) Quantitation of bands generated in Western blots (A-C) using β-actin as a loading control and compared with siCtrl band intensities. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 6.

siNR4A1, quercetin and kaempferol inhibit fibrosis in human endometriotic cells. (A-C) Levels of fibrosis marker proteins in IHEECs (A), IHESCs (B), and Ishikawa cells (C) treated with quercetin or kaempferol or transfected with siRNAs were determined by Western blotting. (D-E). Levels of α-SMA in IHEECs (D) and Ishikawa cells (E) treated with 75 µM quercetin and kaempferol for 24 hours were determined by immunofluorescence. Hoechst-stained nucleus. Results are expressed as means ± SE for at least 3 replicate determinations significant (P < .05) inhibition of cell growth is indicated (*).

Figure 7.

Quercetin and kaempferol treatment suppressed the growth of endometriotic lesions in mice with endometriosis. (A) Luciferase activity of ectopic lesions in mice with endometriosis treated with vehicle, quercetin, and kaempferol before drug treatment and 14th day after drug treatment. (B) Quantification of luciferase activity shown in A. (C) The body weight changing of mice treated with vehicle, quercetin, and kaempferol for 14 days. (D) aberrant levels of NR4A1 in endometriotic lesions compared with normal endometrium. The expression levels of NR4A1 in control endometrium, control peritoneum, endometrium, and peritoneum of patients with endometriosis, as well as peritoneal and deep infiltration ectopic lesions and ovarian endometriomas, were determined using EndometDB.