Daidzein-rich isoflavone aglycones inhibit 17β-hydroxysteroid dehydrogenase 1 and increase estrogen sulfotransferase in endometriosis

Objective Endometriosis is an estrogen-dependent disease wherein isoflavones interact with estrogen receptors. Daidzein-rich isoflavone aglycones (DRIAs) have been shown to inhibit cell proliferation and aromatase activity in vitro and in vivo. This study aims to investigate the effects of DRIAs on the enzymes involved in estrogen metabolism in endometriosis. Study design Stromal cells isolated from ovarian endometriomas (OESCs) were cultured with DRIAs. Ovarian endometrioma (OE) specimens were obtained from patients who were treated with or without DRIAs. The gene expressions involved in estrogen metabolism and 17β-hydroxysteroid dehydrogenase (HSD17β) 1 activity were analyzed using RT-PCR and thin layer chromatography, respectively.

HSD17β1 expression in OE specimens was evaluated using immunohistochemical staining. DRIA treatment significantly suppressed HSD17β1 expression and elevated estrogen sulfotransferase (EST) levels in OESCs; however, no differences were observed in HSD17β2, HSD17β7, HSD17β12, and steroid sulfatase (STS) levels. HSD17β1 enzyme activity was inhibited by DRIAs. Furthermore, immunohistochemical analysis confirmed that HSD17β1 expression was suppressed in the OE specimens of patients undergoing treatment with DRIAs.

DRIA treatment could suppress abnormal estrogen production via EST stimulation as well as the inhibition of aromatase and HSD17β1 activities, suggesting therapeutic potential in endometriosis that needs to be confirmed by our ongoing clinical trial. ay.

Endometriosis is characterized by the presence of endometrial-like tissue outside the uterus and affects 5–10% women of reproductive age, 50–60% of women with pelvic pain, and up to 50% of women with infertility [Citation1]. Endometriosis is an undisputed hormonal disease caused by estrogen dependency and aberration in estrogen production and metabolism [Citation2]. Abnormally biosynthesized estrogens in endometriotic tissues contribute to lesion growth and worsening symptoms [Citation3,Citation4]. In endometriotic tissues, overexpression of aromatase, encoded by the CYP19A1 gene, results in the conversion of androstenedione and testosterone to estrogens, estrone, and estradiol (E2) [Citation5,Citation6]. Other important enzymes that are involved in biologically active estrogen formation include 17β-hydroxysteroid dehydrogenases (HSD17βs). Of HSD17βs, 17β-hydroxysteroid dehydrogenase 1 (HSD17β1), an enzyme that catalyzes the conversion of estrone (E1) to the highly potent E2, is highly expressed in local endometriotic lesions. Conversely, HSD17β2, an enzyme that weakens the activity of estradiol and catalyzes its conversion to the less active estrone, is undetectable or is present in lower levels than those of HSD17β1 in the tissues [Citation7]. Thus, the reaction shifts in favor of E2 production [Citation7,Citation8]. The other major source of estrogen is estrone sulfate, an inactive conjugated form that is abundant in circulation. Estrone sulfate is desulfated to estrone by steroid sulfatase (STS), and estrone is inactivated by estrogen sulfotransferase (EST) [Citation9,Citation10]. Understanding the contribution of the aberrant expression of these enzymes in endometriosis to local estrogen production and metabolism will aid the development of novel therapeutic agents. Isoflavones are naturally occurring plant-derived phytoestrogens found in soybean plants. They exert weak estrogenic-like activity by binding to estrogen receptors (ER), and their molecular structures are similar to those of estrogens. Although the estrogenic activity of isoflavones is weak, some isoflavones exert anti-estrogenic effects in reproductive-aged women with high estrogen levels [Citation11]. In the ovariectomized rat model, high-dose isoflavones promotes endometrial proliferative effects [Citation12]. In the randomized clinical trials in postmenopausal women, isoflavones improved the symptoms of menopause [Citation13,Citation14]. However, when women with endogenous estrogen levels above a certain level ingest isoflavones, their estrogenic effect is attenuated, suggesting a preventive effect against breast and prostate cancer development [Citation15]. Isoflavones are present in two forms as glycosides (conjugated form) and aglycones (nonconjugated form) [Citation16]. Genistin and daidzin are representative isoflavone glycosides, which are abundant in soybeans [Citation17]. Isoflavone aglycones, which are metabolites obtained by the removal of sugar chains from glycosides, can be rapidly and efficiently absorbed without intestinal bacterial degradation [Citation18]. Genistein and daidzein are the major isoflavone aglycones obtained from genistin and daidzin, respectively [Citation19]. Several studies have investigated the effects of isoflavones on endometriosis. Puerarin and parthenolide, a flavonoid, have been shown to inhibit the proliferation of ovarian endometrioma (OE) cells [Citation20,Citation21]. Genistein caused regression of endometriotic implants in the rat model [Citation22]. We recently demonstrated that daidzein-rich isoflavone aglycones (DRIAs), a commercially available supplement, inhibited cell proliferation and aromatase activity in endometriotic stromal cells derived from ovarian endometriomas (OESCs). We also previously found that in a mouse model fed with DRIA-containing food, endometriosis-like cystic lesions reduced in number and weight compared to those in mice fed with normal food [Citation23]. These observations indicated that DRIAs may be effective in treating endometriotic lesions. However, the mechanisms by which they control lesions remain unelucidated. In this study, we investigated the effect of DRIAs on the enzymes involved in estrogen metabolism in OESCs.

Isoflavones AglyMax, a DRIA-containing extract prepared via soybean germ fermentation using Koji fungus (Aspergillus awamori), followed by ethanol/water extraction and purification using a proprietary extraction procedure, was obtained from Nichimo Biotics (Tokyo, Japan) [Citation24,Citation25]. AglyMax contains the isoflavone aglycones daidzein, genistein, and glycitein in a ratio of 7:1:2 [Citation24]. Patients and samples Informed consent about our researches was obtained through paper-based consent form before surgery, and this study was approved by the institutional review board of the Kyoto Prefectural University of Medicine (ERB-E-306, ERB-C-1202-1). In the in vitro study, OE samples were obtained from eight patients of reproductive age (34.9 ± 8.2 years) who underwent surgery for OE (). The endometriosis stages of the patients were III (n = 2, 25%) and IV (n = 6, 75%) based on the revised American Society for Reproductive Medicine classification of endometriosis [Citation26]. No patient had received hormone treatment for at least 6 months before surgery. For the immunohistochemical analysis, OE specimens were obtained from patients undergoing surgery and either treated or untreated with DRIAs at an oral dose of 30 mg twice daily for 16 weeks (n = 4 for both). Placenta specimen was obtained from a woman who delivered vaginally at 38 pregnant weeks of gestation. The patients treated with DRIAs were seen once a month to check for medication. All patients were in the proliferative phase of their regular menstrual cycle when the surgery was performed. Cell preparation OE tissues were minced, digested with 2.5% type I collagenase (Sigma-Aldrich, MO, USA) and 15 IU/mL deoxyribonuclease (DNase) I (Takara Shuzo Co., Ltd, Kyoto, Japan), and filtered using a nylon cell strainer. The filtrate was centrifuged using Histo-paque-1077 (Sigma-Aldrich) to remove the red blood cells. The purity (> 95%) of all 24 OESC preparations was confirmed using positive CD10 staining and negative staining for cytokeratin. Sub-confluent primary cultured SCs were collected after treatment with 0.1% trypsin and resuspended in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% dextran-coated, charcoal-treated fetal bovine serum, and 1% penicillin and streptomycin (100 μg/mL). Cells were seeded in six-well plates for RNA extraction at a density of 10 × 103 cells per well, and in 12-well plates for the HSD17β1 enzyme assay at a density of 5 × 103 cells per well, and were cultured until the sub-confluent condition. Cells were treated with or without DRIAs (20 μM) for 72 h. The test compounds were dissolved in dimethyl sulfoxide and added to the culture medium at a final concentration of 0.1%. RNA extraction, cDNA preparation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA was extracted from OESCs using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). RNA concentrations were quantified by ultraviolet absorption (optical density, 260 nm/280 nm) using a NanoDrop instrument (Thermo Scientific, Waltham, MA). Total RNA (100 ng) was reverse transcribed in a 10 μL volume using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) and GeneAmp PCR 9700 (Applied Biosystems, Foster City, CA). Real-time PCR was performed using Thunderbird Master Mix (Toyobo, Osaka, Japan) and the CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA). The total PCR mixture of 20 µL contained 1 μL of cDNA samples and 0.3 μM of each primer for the target genes. Quantitative real-time PCR was performed under the following thermal cycling conditions: a denaturing step at 95 °C for 60 s, and 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Threshold cycle (Ct) values were calculated using the ΔΔCt method. The sequences of the primer pairs were shown in . HSD17β1 enzyme assays HSD17β1 activity was measured using thin layer chromatography, as described previously [Citation27]. Briefly, cells were washed twice with phenol red-free DMEM/Ham’s F-12 and then incubated at 37 °C/5% carbon dioxide for 3 h with 0.5 ml of serum-free medium containing [4-14C] estrone (5.0 × 104 dpm; Perkin Elmer, MA, USA). The reaction was stopped via transferring the medium to test tubes containing 2 mL chloroform and the corresponding carrier steroids: [6,7-3H] estradiol (5.0 × 104 dpm; Perkin Elmer, MA, USA) and nonradioactive estrone and estradiol (0.2 mg each). Steroids were isolated via thin-layer chromatography using silica gel 60 F254 (0.25 mm; Merck, Darmstadt, Germany) in a chloroform-ethyl acetate system (4:1, v/v). The aliquot was mixed with Clear-sol I (Nacalai Tesque Inc., Kyoto, Japan), and radioactivity was measured using a scintillation counter (Beckman Coulter, CA, USA). The enzyme activity was calculated from the 14C/3H ratio. Immunohistochemistry Tissues were embedded in paraffin, cut into 4-µm-thick sections, and mounted onto slides. The slides were deparaffinized, dehydrated through a series of xylene and ethanol washes, and then incubated in 1% hydrogen peroxide for 20 min. After few minutes, the slides were steamed in 10 mM sodium citrate buffer for 20 min, cooled for 2 h, and rinsed for another few minutes. Immunohistochemical staining was performed as described previously [Citation28]. The antibody against HSD17β1 was used as a primary antibody (1:200 dilution; Cat no. ab51045; Lot no. GR5360-8, Abcam, Cambridge, UK). Slides were incubated with the primary antibody overnight at 4 °C. Biotinylated anti-rabbit secondary antibody (1:200 dilution; Cat no. MKB-2225; Lot no. ZF-0122; Vector Laboratories, Burlingame, CA) was used and applied for 1 h at room temperature. Thereafter, the slides were rinsed and incubated in ABC Elite (Vector Laboratories) for 30 min at room temperature. Immune complexes were visualized by incubation with 3, 3′-diaminobenzidine tetrahydrochloride, and the nuclei were counterstained with hematoxylin. Normal term placental tissue was used as the positive control (). Rabbit IgG isotype was used was negative control (1:200 dilution; Cat no. 30000-0-AP; Lot no. 22000010, Proteintech, Tokyo, Japan). HSD17β1 immunoreactivity in OE was evaluated using the H-score, a semi-quantitative index involving an algorithm described previously [Citation28]. Briefly, two independent observers evaluated approximately 500 cells/slide and scored them as follows: 3 × percentage of strongly stained cells + 2 × percentage of moderately stained cells + percentage of weakly stained cells. The H-score was calculated as the mean of the two scores. Statistical analysis Statistical analysis of the results of each procedure was performed using the Mann–Whitney U test. Data are presented as mean ± standard error of the mean. GraphPad Prism (GraphPad Software, La Jolla, CA) was used for all statistical analyses. P values < 0.05 were considered as statistically significant.

DRIAs suppressed HSD17β1 and elevated EST expression at the mRNA level To investigate the effect of DRIAs on enzymes involved in estrogen metabolism, we assessed the HSD17β1, HSD17β2, HSD17β7, HSD17β12, STS, and EST expression levels with qRT-PCR in the presence of DRIAs (n = 7, respectively). DRIAs significantly suppressed HSD17β1 expression () and elevated EST expression in OESCs (); however, no changes were observed in the expression levels of HSD17β12, STS in OESCs treated with or without DRIAs (). DRIAs inhibited HSD17β1 enzyme activity To confirm the change in HSD17β1 enzyme activity by DRIAs in OESCs, we also performed thin layer chromatography (n = 4, respectively). Consistent with the results of qRT-PCR, DRIAs significantly inhibited HSD17β1 enzymatic activity (). HSD17β1 expression was suppressed in the OE of patients with DRIAs treatment To verify DRIAs treatment-induced suppression of HSD17β1 expression in OE resected from patients with endometriosis, we performed immunohistochemical analysis of the OE tissues with and without DRIAs treatment (n = 4, respectively). Patient characteristics are shown in . No significant differences were observed between patients treated and not treated with DRIAs. The immunostaining intensity of HSD17β1 was significantly lower in the OE of patients treated with DRIAs than the patients who were untreated ().

In this study, we revealed that DRIAs suppressed HSD17β1 expression and elevated EST expression in endometriotic lesions, which were the enzymes contributing to E2 biosynthesis. Additionally, thin layer chromatography verified that DRIAs significantly inhibited HSD17β1 enzymatic activity. Furthermore, as a clinical trial, we confirmed the suppressed expression of HSD17β1 in OE tissues of the patients treated with DRIAs using immunohistochemical analysis. These actions can reduce the local progression of endometriotic lesions via suppressing E2 production, suggesting that DRIAs is a therapeutic agent on endometriosis. Endometriosis is known to be an estrogen-dependent disease [Citation29]. Symptoms such as dysmenorrhea and pelvic pain are often relieved in many cases after menopause. In addition, endometriotic lesions become atrophic in low-estrogen environments, such as after treatment with gonadotropin-releasing hormone agonists. Thus, estrogens, particularly E2, significantly contribute the development and progression of endometriosis. A series of enzymes is involved in in situ estrogen production in local endometriotic lesions (). Three principal estrogen sources in the final step of its formation are as follows: One is produced by aromatase, a key enzyme that converts androstenedione or testosterone into E1 or E2. Another is produced by HSD17β1 and HSD17β2. HSD17β1 catalyzes E2 reduction of biologically inactive E1 to E2, whereas HSD17β2 preferentially catalyzes the oxidation of E2 to E1. The last source is using STS and EST. STS hydrolyzes circulating estrone sulfate (E1-S), a biologically inactive form of estrogen in the peripheral blood, to E1, whereas EST hydrolyzes E1 to E1-S [Citation30]. These enzymes control estrogen levels and regulate estrogenic actions in estrogen-targeted tissues [Citation8,Citation30]. Several studies have reported that aromatase is more abundant in endometriotic tissues than in the normal endometrium (NE) [Citation31,Citation32]. Additionally, we recently showed that the aromatase mRNA expression levels were greater than those in the NE and eutopic endometrium in endometriosis (EE) [Citation7]. Abnormally elevated aromatase in local tissues induces E2 overproduction, which stimulates endometriosis development and upregulates cyclooxygenase-2 (COX-2) expression, leading to excessive production of PGE2. This stimulates aromatase expression and activity. Alternatively, HSD17β1 is highly expressed in endometriotic lesions compared with that in NE [Citation7,Citation9]. The RT-PCR results of this study showed that HSD17β1 was highly expressed in endometriotic lesions compared to that in NE and EE, whereas HSD17β2 was expressed to a lesser extent in endometriotic lesions than in NE and EE [Citation33]. Immunohistochemistry analysis also showed HSD17β1 was expressed in the cytoplasm of both epithelial and stromal cells of not only endometriotic tissues but also NE and EE [Citation33]. HSD17β1 intensity was greater in the endometriotic lesions than in NE and EE. These data suggest that E2, the most biologically potent estrogen, is more likely to be produced in local endometriotic lesions. DRIAs are a subgroup of phytoestrogens found in soybean plants that possess weak estrogenic activity. DRIAs are commercially available as dietary supplements. DRIAs have been reported to reduce hot flashes in peri-/post-menopausal women without causing serious adverse events [Citation14]. DRIAs have also been reported to inhibit tumor growth and weight loss in a mouse model of cachexia in human gastric cancer [Citation34]. Other studies have recently shown that mice fed DRIAs show attenuated muscle fiber atrophy [Citation25]. We previously reported that DRIAs inhibited cell growth, expression, and enzymatic activity of aromatase and inflammatory cytokines, such as interleukin 6, interleukin 8, and COX-2 in SCs cultured from endometriotic lesions. We also confirmed that DRIAs reduced the extent of endometriosis-like lesions in a mouse model [Citation23]. Thus, DRIAs exert inhibitory effects on cell proliferation and aromatase activity during endometriosis; however, a comprehensive understanding of the beneficial effects of DRIAs at the molecular level is currently unavailable. A report showed that an ER antagonist and a MAPK inhibitor abolished the increased gene expression induced by DRIAs, suggesting an antagonistic effect of DRIAs on ERs [Citation35]. Other studies have demonstrated that DRIAs stimulate ER-mediated histone acetylation [Citation36]. We previously demonstrated that the inhibitory effect of DRIAs on cell proliferation in OESCs was suppressed by ERβ antagonist, 4-(2-phenyl-5,7-bis(trifluoromethyl) pyrazolo(1,5-a)pyrimidin-3-yl)phenol (PHTPP). In addition, the effect of DRIA was significantly suppressed in cells with ERβ knockdown. Conversely, the ERα antagonist, methylpiperidino pyrazole (MPP), did not reverse the effects of DRIAs, indicating that DRIA activity was mediated by ERβ but not ERα in endometriotic lesions [Citation23]. However, the effects of DRIAs on enzymes involved in estrogen metabolism remain unknown. Our results of DRIA effects on HSD17β1 and EST revealed the mechanism for endometriosis lesion suppression by DRIAs via estrogen metabolism, in addition to the ERβ-mediated pathway (). The main limitation of this study is that DRIAs were not verified to improve endometriotic lesions in patients. Our previous and present study confirmed the suppressive effects of DRIAs on endometriosis lesion via the ERβ-mediated pathway and estrogen metabolism in vitro and in vivo. However, the verification of endometriosis lesion improvement and the altered ERβ-mediated pathway the patients treated with DRIAs remain unknown. Additionally, the suppressive effect of DRIAs on the endometriosis lesion was investigated in the four patients with the treatment as the preliminary study. Therefore, double-blind and multi-institutional clinical trial comparing DRIAs with a placebo in patients with endometriosis is currently ongoing (jRCTs051200074). In conclusion, this study found that DRIAs inhibited HSD17β1 and increased EST. Furthermore, as a preliminary clinical trial, DRIAs suppressed HSD17β1 expression in OE tissues of the patients treated with DRIAs. Coupled with our previous findings that DRIAs suppressed aromatase activity and inhibited endometriotic lesion growth in vitro and in vivo, DRIAs could suppress abnormal estrogen production via these enzymes and be a novel therapeutic agent for endometriosis. Ethical statements This study was approved by the Kyoto Prefectural University of Medicine institutional review board (IRB) (ERB-E-306, ERB-C-1202-1). Informed consent was obtained from all patients in accordance with a protocol approved by the IRB of the Kyoto Prefectural University of Medicine, and this study was approved by the IRB. Acknowledgment We thank Ayaka Miura, Makoto Kazui, and Naoko Matsunaga for technical assistance. AglyMax, a DRIA-containing extract, was obtained from Nichimo Biotics. Disclosure statement The authors report there are no competing interests to declare. Data availability statement The data that support the findings of this study are available from the corresponding author, Y. T., upon reasonable request. Additional information Funding

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