Physiological Concentrations of Cimicifuga racemosa Extract Do Not Affect Expression of Genes Involved in Estrogen Biosynthesis and Action in Endometrial and Ovarian Cell Lines

In postmenopausal women, estrogen levels exclusively depend on local formation from the steroid precursors dehydroepiandrosterone sulfate and estrone sulfate (E1-S). Reduced estrogen levels are associated with menopausal symptoms. To mitigate these symptoms, more women nowadays choose medicine of natural origin, e.g., Cimicifuga racemosa (CR), instead of hormone replacement therapy, which is associated with an increased risk of breast cancer, stroke, and pulmonary embolism. Although CR treatment is considered safe, little is known about its effects on healthy endometrial and ovarian tissue and hormone-dependent malignancies, e.g., endometrial and ovarian cancers that arise during menopause. The aim of our study was to examine the effects of CR on the expression of genes encoding E1-S transporters and estrogen-related enzymes in control and cancerous endometrial and ovarian cell lines. CR affected the expression of genes encoding E1-S transporters and estrogen-related enzymes only at very high concentrations, whereas no changes were observed at physiological concentrations of CR. This suggests that CR does not exert estrogenic effects in endometrial and ovarian tissues and probably does not affect postmenopausal women’s risks of endometrial or ovarian cancer or the outcomes of endometrial and ovarian cancer patients.


Introduction
Around menopause, most women experience symptoms such as hot flashes, difficulty sleeping, and mood changes. To improve the quality of life of women with menopausal symptoms, hormone replacement therapy is routinely prescribed [1]. However, the correlation between hormone replacement therapy and an increased risk of breast cancer [2] and stroke [3] has led to the reduced use of such therapy [4]. As an alternative to hormone replacement therapy, plant-derived drugs, such as Cimicifuga racemosa (CR, black cohosh) extract, are used to alleviate menopausal symptoms. CR is a perennial dicot plant from the Ranunculaceae family, native to Canada and the Eastern United States, from which several different compounds have been isolated, including phenols, chromones, triterpenoids, and nitrogen-containing constituents [5]. CR extract has been used for centuries, and clinical studies have shown that CR reduces the occurrence of menopausal symptoms [6,7] and is safe, with no systemic [8,9] or breast-specific estrogenic effects [9,10]. Furthermore, no changes in endometrial thickness [10][11][12] or the occurrence of endometrial hyperplasia or cancer were observed [13]. However, the latest Cochrane report from 2012 based on data from 2027 peri-or postmenopausal women concluded that "there is currently insufficient evidence to support the use of black cohosh for menopausal symptoms, although there is adequate justification for conducting further studies in this area" [14].
In vivo studies on ovariectomized rats showed that CR extract prevents hot flashes [15] and exerts osteoprotective effects without estrogenic effects in the uterus [16] or mammary sues but preferentially in adipose tissue, where it can be synthesized either by the aromatase pathway from dehydroepiandrosterone sulfate and dehydroepiandrosterone or by the sulfatase pathway from E1-S. We recently showed that in menopausal EC patients, E2 formation more likely results from the sulfatase pathway, which is also the case in the adjacent control endometrium, with increased levels of E2 seen in cancerous tissues [36]. Here, steroid sulfatase (STS) and sulfotransferase 1E1 (SULT1E1) direct the formation of E1-S or estrone (E1), and 17β-hydroxysteroid dehydrogenase 1 (HSD17B1) and 2 (HSD17B2) direct the formation of E2 or E1 (Figure 1). The active forms of estrogens can promote cell proliferation, for which estrogen receptors α and β play a crucial role. Active estrogens promote rapid cell multiplication, and thus errors in DNA sequences can appear, consequently causing carcinogenesis [34,35]. To date, the effects of CR are still poorly understood, including its effects on steroid precursor import, estradiol synthesis, estrogen metabolism, metabolite elimination, and active estrogen concentrations in hormonally dependent endometrial and ovary tissues. The aim of this study was to elucidate the mechanism of action and possible protective or stimulating effects of CR extracts on EC and OC development.

Cell Culture
The HEC-1-A (CVCL_0293) cell line was originally established from moderately differentiated endometrial adenocarcinoma from a 71-year-old patient and was purchased from the American Type Culture Collection (ATCC_HTB-112 TM ) as p125 on May 31, 2012. McCoy's 5A Medium (M4892, Sigma-Aldrich St. Louis, MO, USA) supplemented with 10% foetal bovine serum (FBS) was used as growth medium. McCoy's 5A Medium without phenol red (SH30270.01, GE Healthcare Life Sciences, Piscataway, NJ, USA) was used as treatment medium. HEC-1-A passage 15 (p+15) cells were authenticated by short tandem repeats (STR) profiling performed by ATCC on February 22, 2018.
The Ishikawa (CVCL_2529) cell line was originally established from a well differentiated endometrial adenocarcinoma from a 39-year-old patient and was purchased from Sigma-Aldrich (ECACC 99040201) as p+3 on December 18, 2012. Minimum Essential Medium Eagle (#M5650) with 2 mM L-glutamine (#G7513) and 5% FBS (#F9665, Sigma-Aldrich St. Louis, MO, USA) was used as growth medium. MEM without phenol red To date, the effects of CR are still poorly understood, including its effects on steroid precursor import, estradiol synthesis, estrogen metabolism, metabolite elimination, and active estrogen concentrations in hormonally dependent endometrial and ovary tissues. The aim of this study was to elucidate the mechanism of action and possible protective or stimulating effects of CR extracts on EC and OC development.

Cell Culture
The HEC-1-A (CVCL_0293) cell line was originally established from moderately differentiated endometrial adenocarcinoma from a 71-year-old patient and was purchased from the American Type Culture Collection (ATCC_HTB-112 TM ) as p125 on 31  The Ishikawa (CVCL_2529) cell line was originally established from a well differentiated endometrial adenocarcinoma from a 39-year-old patient and was purchased from Sigma-Aldrich (ECACC 99040201) as p+3 on 18 December 2012. Minimum Essential Medium Eagle (#M5650) with 2 mM L-glutamine (#G7513) and 5% FBS (#F9665, Sigma-Aldrich St. Louis, MO, USA) was used as growth medium. MEM without phenol red (#51200-038, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 2 mM L-glutamine (#G7153) was used as treatment medium. Ishikawa p+13 cells were authenticated by STR profiling performed by ATCC on 22 February 2018.
The control cell line HIEEC was obtained from Michael A. Fortier (Laval University, Quebec, QC, Canada) as p14 on 4 April 2014. It was originally generated from a primary culture prepared from an endometrial biopsy from a 37-year-old woman with confirmed absence of neoplasia and endometriosis, on day 12 of her menstrual cycle [38]. All cell lines were negative for mycoplasma infection, which was periodically tested with the MycoAlert TM mycoplasma detection kit (Lonza, Basel, Switzerland). STR profiling was performed by ATCC on cell lines that were purchased from culture collections several years prior or obtained from other laboratories.

RNA Isolation and Quantitative Real-Time PCR
Total RNA from cells was isolated and purified after cultivation in treatment medium using Nucleospin RNA isolation kits (Macherey-Nagel GmbH & Co. KG, Düren, Germany), according to the manufacturer's instructions. The quality of RNA was determined using the Agilent 2100 Bioanalyzer and RNA 600 Nanokit (Agilent Technologies Inc., Santa Clara, CA, USA). The measured RNA integrity number values were above 9.0, indicating that the RNA was of good quality. Total RNA was reverse transcribed into cDNA using the SuperScript ® VILO™ cDNA Synthesis kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer's instructions. The cDNA samples were stored at −20 • C.
The expressions of genes that encode estrogen receptors and proteins involved in estradiol biosynthesis and oxidative metabolism were examined using quantitative PCR (qPCR). The following was used: exon-spanning hydrolysis probes commercially available as 'Assay on Demand' (Applied Biosystems; Foster City, CA, USA) ( Table 1), using TaqMan ® Fast Advanced Master Mix. The expressions of genes that encode for transporters were examined using SYBR Green I Master (Roche, Basel, Switzerland) and primers that were designed in our laboratory (Table 2). Quantification was accomplished with the Applied Biosystems ® ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). All the cDNA samples were run in triplicates, using 0.25 µL of cDNA, and the reactions were performed in Applied Biosystems ® MicroAmp ® Optical 384-well plates (Thermo Fisher Scientific, Waltham, MA, USA) in a reaction volume of 5 µL. For gene expression analysis, the normalization factor for each sample was calculated based on the geometric mean of the three most stably expressed reference genes (POLR2A, HPRT1, RPLP0). Gene expression for each sample was calculated from the crossing-point value (Cq) as E −Cq , divided by the normalization factor and multiplied by 10 12 . The Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines were considered in the performance and interpretation of the qPCR reactions [39].

Median Cytotoxic Concentration (CC50)
After the cells were grown in treatment medium for 72 h, the medium was aspirated, and the cells were washed with PBS and detached with trypsin. Detached cells were resuspended in 1 mL of growth medium. Cell suspensions were mixed with trypan blue dye (1:1) and used for cell counting with an automated cell counter. Cell viability was calculated as a ratio between live treated (with CR extract dissolved in 50% ethanol, final concentration of ethanol was 0.5%) and control cells (only 50% ethanol). Experiments were performed in at least two independent replicates.

xCELLigence
For real-time proliferation monitoring using the xCELLigence RTCA DP system (Agilent, Santa Clara, CA, USA), KLE cells were seeded onto E-plates 16 (ACEA Biosciences, San Diego, CA, USA) at cell densities of 5000 cells/well in growth medium without phenol red and with charcoal-stripped FBS (#F6765, Sigma-Aldrich). The next day, KLE cells were treated with different concentrations of CR extract (20-400 µg/mL; Table S2), only 50% ethanol, or only medium. The final concentration of ethanol was 2.5%. Experiments were repeated in two independent experiments, each time with four technical replicates for individual treatments. Cell proliferation was monitored for 180 h after treatment.

Statistical Analysis
Statistical analysis was performed in Graph Pad Prism 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA). Values of gene expression in individual cell lines for treated and untreated (control) samples was normalized with the expression of reference genes. Data were statistically analysed via the Kruskal-Wallis test with Dunn's multiple comparisons test. p < 0.05 was considered statistically significant.

CC50 Values of CR Were Higher in EC and OC Cell Lines Compared to Those in Control Cell Lines
We first examined the CC 50 values of the CR extract in all nine cell lines (Table S3). Cell viability decreased with higher CR concentrations in a dose-dependent manner. In EC cell lines, CC 50 values were in the range of 20.16-58.23 µg/mL (Figure 2,  [24]. For KLE cells, which represent a model of poorly differentiated EC with bad prognosis, cell proliferation after CR treatment was measured in real time. Cell death occurred with 400 or 300 µg/ml of CR extract, whereas lower CR concentrations only reduced the growth rate, while 100% confluence could still be reached (Figure 3).    For KLE cells, which represent a model of poorly differentiated EC with bad prognosis, cell proliferation after CR treatment was measured in real time. Cell death occurred with
When the control endometrial cell line HIEEC was treated with CR extract (5 and 500 ng/mL), no changes in the expression of the investigated genes were detected (Figures 4-6). The Ishikawa and HEC-1-A cell lines represent well-differentiated EC and were established from a premenopausal and postmenopausal EC patient, respectively. Physiological CR concentrations exhibited no effects on gene expression. Only at 500 ng/mL, Ishikawa cells exhibited downregulated expression of SLCO1A2, which encodes an influx transporter. RL-95-2 cells are derived from Grade 2 moderately differentiated EC. Similarly, CR did not affect gene expression, except for ESR1, which was downregulated at very high CR concentrations (100 µg/mL). Our results suggest that CR does not influence estrogen concentration and actions in either normal endometrial cells or well-to moderately differentiated endometrial cancer.

High Concentrations of CR Extract Greatly Affected the Expression of Estrogen-Related Genes in KLE Cells
KLE cells are derived from poorly differentiated endometrial carcinoma Grade 3 and were the most affected by CR extract, which influenced both genes encoding influx and

High Concentrations of CR Extract Greatly Affected the Expression of Estrogen-Related Genes in KLE Cells
KLE cells are derived from poorly differentiated endometrial carcinoma Grade 3 and were the most affected by CR extract, which influenced both genes encoding influx and efflux transporters and genes involved in estradiol biosynthesis, metabolism, and action. At 100 µg/mL of CR extract, one gene encoding influx transporters (SLCO2A1) was downregulated, whereas two (SLC10A6 and SLCO4A1) were upregulated. Similarly, three of the five examined efflux transporters (ABCC1, ABCC4, and ABCG2) were upregulated at higher CR concentrations. ESR1 was upregulated at both 50 and 100 µg/mL of CR, and STS was downregulated at the highest CR concentration (Figure 7). This suggests a higher flux of estrogens through cells, but lower concentrations of the active estrogen estradiol. Transporters are not specific for E1-S transport, and their differential expression can also affect other processes such as ion transport, signal transduction, and toxin secretion.
higher CR concentrations. ESR1 was upregulated at both 50 and 100 µg/ml of CR, and STS was downregulated at the highest CR concentration (Figure 7). This suggests a higher flux of estrogens through cells, but lower concentrations of the active estrogen estradiol. Transporters are not specific for E1-S transport, and their differential expression can also affect other processes such as ion transport, signal transduction, and toxin secretion.
Although lower CR concentrations did not affect control or cancer cell lines, our results show that the poorer the differentiation, the more affected the expressions of the investigated genes were. The concentrations that affected gene expression were mostly higher than the CC50 values for individual cell lines, except for SLCO2A1 downregulation in Ishikawa cells and ABCG2 upregulation in KLE cells.
In EC, estrogens promote proliferation via estrogen receptor α, which is encoded by the ESR1 gene. With cancer progression, ESR1 levels drop, the ratio between ERα and ERβ shifts, and the loss of ERα is associated with shorter disease-free survival [41,42]. Thus, higher ESR1 levels would be beneficial, especially in poorly differentiated cancers.

Higher Concentrations of CR Extract Upregulated the Expression of Influx and Efflux Transporter Genes in Ovarian Control Cell Line HIO-80
HI0-80, a control cell line established from the ovarian surface, was treated with CR extract (5 ng/ml, 500 ng/ml, 25 µg/ml, and 50 µg/ml). Physiological CR concentrations (5 ng/ml) did not affect gene expression. The concentrations that affected gene expression were four-fold higher than the CC50 values for HIO-80 cells. While the expression of most genes encoding influx and efflux transporters was higher at 25 and 50 µg/ml of CR extract, statistically significant changes were demonstrated for SLCO2B1, SLC22A11, and SLC51B. At 50 µg/ml of CR, SULT1E1 expression was significantly downregulated (Figure 8). This suggests the higher flux of estrogens into the cells. If mRNA levels of SULT1E1 correlate to protein levels, less estrone and estradiol is sulphated and higher levels of active estrogens could promote the proliferation of cells. Although lower CR concentrations did not affect control or cancer cell lines, our results show that the poorer the differentiation, the more affected the expressions of the investigated genes were. The concentrations that affected gene expression were mostly higher than the CC 50 values for individual cell lines, except for SLCO2A1 downregulation in Ishikawa cells and ABCG2 upregulation in KLE cells.
In EC, estrogens promote proliferation via estrogen receptor α, which is encoded by the ESR1 gene. With cancer progression, ESR1 levels drop, the ratio between ERα and ERβ shifts, and the loss of ERα is associated with shorter disease-free survival [41,42]. Thus, higher ESR1 levels would be beneficial, especially in poorly differentiated cancers.

Higher Concentrations of CR Extract Upregulated the Expression of Influx and Efflux Transporter Genes in Ovarian Control Cell Line HIO-80
HI0-80, a control cell line established from the ovarian surface, was treated with CR extract (5 ng/mL, 500 ng/mL, 25 µg/mL, and 50 µg/mL). Physiological CR concentrations (5 ng/mL) did not affect gene expression. The concentrations that affected gene expression were four-fold higher than the CC 50 values for HIO-80 cells. While the expression of most genes encoding influx and efflux transporters was higher at 25 and 50 µg/mL of CR extract, statistically significant changes were demonstrated for SLCO2B1, SLC22A11, and SLC51B. At 50 µg/mL of CR, SULT1E1 expression was significantly downregulated (Figure 8). This suggests the higher flux of estrogens into the cells. If mRNA levels of SULT1E1 correlate to protein levels, less estrone and estradiol is sulphated and higher levels of active estrogens could promote the proliferation of cells.

Higher CR Concentrations Affected the Expression of Influx Transporter Genes and Genes Involved in Estradiol Biosynthesis, Metabolism, and Action in the High-Grade Serous OC Cell Lines Kuramochi and COV362
We next studied the effects of CR in the OC cell lines Kuramochi, COV362, and OVSAHO. Although all three cancer cell lines were established from high-grade serous OC, they differ in aggressiveness and chemoresistance. In 2016, Haley et al. compared the ability of high-grade serous OC cell lines to migrate, invade, proliferate, and form colonies. OVSAHO cells exhibited the lowest functional activity, migration time, invasion time, and colony formation time. COV362 and Kuramochi cells did not differ in migration or invasion time, whereas colony formation time was shorter in Kuramochi cells, indicating a more aggressive type of cancer cell line. When chemoresistance was examined, COV362 cells were the most resistant against platinum-based drugs and had the highest IC50 value, whereas OVSAHO cells had the lowest IC50 value [43].
Similar to EC cancer cell lines and the control cell line HIO-80, no gene expression changes were observed at physiological CR concentrations. The most abundant genes encoding influx transporters in all three OC cell lines were SLCO3A1, SLCO4A1, and SLCO4C1 (Figure 9), whereas the most highly expressed efflux transporter genes were ABCC1 and ABCC4 ( Figure 10). In Kuramochi cells, SLCO4A1 and SLCO1B3 were downregulated at 100 µg/ml and 50 µg/ml of CR, respectively, whereas SLCO4C1 was upregulated at 500 ng/ml of CR. In COV362 cells, SLCO2B1 levels were lower at 100 µg/ml of CR. No changes were observed regarding influx transporters in OVSAHO cells. Out of all efflux transporter genes, only ABCC1 levels were higher in COV362 cells.
Among genes involved in estradiol biosynthesis, metabolism, and action, ESR1 was downregulated in COV362 cells at 50 and 100 µg/ml of CR, and SULT1E1 was downregulated at 50 µg/ml of CR. In Kuramochi cells, STS was downregulated at 100 µg/ml of CR ( Figure 11). The concentrations that affected gene expression were close to or higher than the CC50 value for the cell line, except for SLCO4C1 in Kuramochi cells.
Overall, if protein levels correlate with mRNA levels, Kuramochi cells would have lower E1-S influx and less E2 formed in the cell after CR treatment, COV362 cells would exhibit reduced estrogen action due to decreased ESR1 levels, and OVSAHO cells would exhibit unaltered estrogenic properties.
To date, to the best of our knowledge, no studies have explored the effects of CR on genes involved in estrogen action in endometrial and ovarian cell lines; however, such a study has been carried out in the breast cancer cell line MCF-7. Gaube et al. showed that 15 µg/ml of lipophilic CR extract affected the expression of antiproliferative and proapop-

Higher CR Concentrations Affected the Expression of Influx Transporter Genes and Genes Involved in Estradiol Biosynthesis, Metabolism, and Action in the High-Grade Serous OC Cell Lines Kuramochi and COV362
We next studied the effects of CR in the OC cell lines Kuramochi, COV362, and OVSAHO. Although all three cancer cell lines were established from high-grade serous OC, they differ in aggressiveness and chemoresistance. In 2016, Haley et al. compared the ability of high-grade serous OC cell lines to migrate, invade, proliferate, and form colonies. OVSAHO cells exhibited the lowest functional activity, migration time, invasion time, and colony formation time. COV362 and Kuramochi cells did not differ in migration or invasion time, whereas colony formation time was shorter in Kuramochi cells, indicating a more aggressive type of cancer cell line. When chemoresistance was examined, COV362 cells were the most resistant against platinum-based drugs and had the highest IC 50 value, whereas OVSAHO cells had the lowest IC 50 value [43].
Similar to EC cancer cell lines and the control cell line HIO-80, no gene expression changes were observed at physiological CR concentrations. The most abundant genes encoding influx transporters in all three OC cell lines were SLCO3A1, SLCO4A1, and SLCO4C1 (Figure 9), whereas the most highly expressed efflux transporter genes were ABCC1 and ABCC4 ( Figure 10). In Kuramochi cells, SLCO4A1 and SLCO1B3 were downregulated at 100 µg/mL and 50 µg/mL of CR, respectively, whereas SLCO4C1 was upregulated at 500 ng/mL of CR. In COV362 cells, SLCO2B1 levels were lower at 100 µg/mL of CR. No changes were observed regarding influx transporters in OVSAHO cells. Out of all efflux transporter genes, only ABCC1 levels were higher in COV362 cells.
Among genes involved in estradiol biosynthesis, metabolism, and action, ESR1 was downregulated in COV362 cells at 50 and 100 µg/mL of CR, and SULT1E1 was downregulated at 50 µg/mL of CR. In Kuramochi cells, STS was downregulated at 100 µg/mL of CR ( Figure 11). The concentrations that affected gene expression were close to or higher than the CC 50 value for the cell line, except for SLCO4C1 in Kuramochi cells.
Overall, if protein levels correlate with mRNA levels, Kuramochi cells would have lower E1-S influx and less E2 formed in the cell after CR treatment, COV362 cells would exhibit reduced estrogen action due to decreased ESR1 levels, and OVSAHO cells would exhibit unaltered estrogenic properties.
To date, to the best of our knowledge, no studies have explored the effects of CR on genes involved in estrogen action in endometrial and ovarian cell lines; however, such a study has been carried out in the breast cancer cell line MCF-7. Gaube et al. showed that 15 µg/mL of lipophilic CR extract affected the expression of antiproliferative and proapoptotic genes, including downregulating ESR1; the authors concluded that this could contribute to the antitumor activity of CR [44]. Biomolecules 2022, 12, x 15 of 20

Conclusions
Our research presents insight into the effects of CR on model cell lines of normal endometrial and ovarian tissue and EC and OC. Our results reveal that CR affects the

Conclusions
Our research presents insight into the effects of CR on model cell lines of normal endometrial and ovarian tissue and EC and OC. Our results reveal that CR affects the expression of genes encoding E1-S transporters and estrogen-related enzymes only at very high concentrations (50 and 100 µg/mL). However, no changes in the expression of genes involved in E1-S influx or efflux, estrogen synthesis, or estrogen receptors were observed with concentrations similar to those detected in the plasma of CR users. These findings support previously published studies that demonstrated that CR extract most likely does not exert estrogenic effects or affect postmenopausal women's risks of EC and OC or the outcomes of EC and OC patients.