Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19)

Toxicology and Applied Pharmacology 279 (2014) 23–32

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Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19) Jiajia Guo a,1, Yun Yuan a,c,1, Danfeng Lu a, Baowen Du a, Liang Xiong b, Jiangong Shi b, Lijuan Yang a, Wanli Liu d, Xiaohong Yuan c, Guolin Zhang a,e,⁎, Fei Wang a,e,⁎⁎ a

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China c School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, China d MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, China e Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, China b

a r t i c l e

i n f o

Article history: Received 19 February 2014 Revised 8 May 2014 Accepted 13 May 2014 Available online 20 May 2014 Keywords: Aromatase Granulosa cells Estrogen biosynthesis Phytol Ergosta-6,9,22-triene-3β,5α,8α-triol p38 MAPK

a b s t r a c t Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds— trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC50: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromataseoverexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissueselective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers. © 2014 Elsevier Inc. All rights reserved.

Introduction Estrogens play a crucial role in the normal physiology of a variety of tissues including the mammary glands, reproductive tract, central nervous system, and skeleton (Heldring et al., 2007). The biosynthesis

Abbreviations: cAMP, intracellular cylic AMP; CREB, cAMP response element-binding protein; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; FSH, follicle-stimulating hormone; PGE2, prostaglandin E2; ELISA, enzyme-linked immunosorbent assay. ⁎ Correspondence to: G. L. Zhang, Chengdu Institute of Biology, Chinese Academy of Sciences, P. O. 416, Chengdu 610041, China. Tel./fax: +86 28 82890996. ⁎⁎ Correspondence to: F. Wang, Chengdu Institute of Biology, Chinese Academy of Sciences, P. O. 416, Chengdu 610041, China. Fax: +86 28 82890651. E-mail addresses: [email protected] (G. Zhang), [email protected] (F. Wang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.taap.2014.05.008 0041-008X/© 2014 Elsevier Inc. All rights reserved.

of estrogen is catalyzed by aromatase (CYP19A1), the only enzyme that catalyzes the formation of estrogens by using androgens such as testosterone and androstenedione as substrates (Simpson et al., 2002). Overexposure to estrogens results in breast, ovarian, and endometrial carcinogenesis; therefore, reducing estrogen levels through the inhibition of aromatase becomes an option in the prevention and treatment of estrogen-mediated carcinogenesis (Bulun et al., 2005). The use of aromatase inhibitors, such as anastrozole, letrozole, and exemestane, has been demonstrated to improve outcome compared with estrogen receptor inhibitor tamoxifen in patients with hormone-dependent breast cancer in postmenopausal women (Johnston and Dowsett, 2003). However, long-term use of an aromatase inhibitor can cause side effects such as exacerbation of menopausal symptoms, increased incidences of osteoporosis and coronary heart disease, and undesirable changes in cognitive function (Smith and Dowsett, 2003). Thus, new

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aromatase modulators, preferably with tissue-specific effects, are needed to develop new therapeutic means for the prevention and treatment of estrogen-related diseases. In humans, estrogen biosynthesis occurs at a number of different sites, and transcriptional control of aromatase is tissue-specific because of the presence of different promoters (Simpson, 2004). The major site of estrogen biosynthesis in premenopausal women is the ovary, where aromatase expression results from the activation of its proximal promoter (promoter I.3/II). The primary signaling cascade through which this promoter is regulated is the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway (Hunzicker-Dunn and Maizels, 2006). CREB is the principal regulatory component in the regulation of the aromatase gene, which is stimulated by follicle-stimulating hormone (FSH)-activated cAMP-dependent PKA. FSH stimulation of aromatase is also mediated by activation of ERK, p38 MAPK, and AKT/PI3K signaling (Hunzicker-Dunn and Maizels, 2006). The switch in aromatase promoter usage is associated with the incidence of breast cancer (Simpson et al., 1997). In normal breast cells, aromatase expression is primarily derived from the tissue-specific promoter I.4 for transcription, whereas in cells from patients with breast cancer, the expression is primarily derived from the utilization of promoter I.3/II. As a result, estrogen biosynthesis switches from regulation by a promoter controlled primarily by glucocorticoids and cytokines to regulation by a promoter controlled through cAMP-mediated pathways by prostaglandin E2 (PGE2), a powerful stimulator of adenylate cyclase (Zhao et al., 1996). Thus, the inhibition of promoter II-driven aromatase expression by means of anti-inflammatory cyclooxygenase inhibitors to reduce PGE2 levels is attracting attention for the tissue-specific treatment of breast cancer (Davies et al., 2002; Subbaramaiah et al., 2012). Natural products with a long history of use, such as those from foods or from traditional medicines, are a good source of aromatase inhibitors with low associated toxicity. To date, about 300 natural compounds, mainly flavonoids, have been examined for their effects on aromatase activity in noncellular, cellular, and in vivo studies (Balunas et al., 2008). However, only a few compounds (biochanin A from red clover, genistein from soybeans, quercetin, isoliquiritigenin from licorice, resveratrol from grape peels and extracts of grape seeds) have been reported to have effects on aromatase promoter I.4, I.3/II activity, with the underlying mechanisms still unknown (Khan et al., 2011). Although many natural products interfere with the signaling pathways that are known to regulate aromatase promoter activity, further study is needed to determine whether these products can be used for the tissue-selective inhibition of aromatase expression (Wang et al., 2013). Sinocalamus affinis is widely distributed and cultivated in southwestern China, and used in traditional Chinese medicine. However, the pharmacological study of this remedy has been rarely reported (Xiong et al., 2011; Xiong et al., 2012; Zhu et al., 2012). As part of a program to study the chemical diversity of traditional Chinese medicines and their biological effects, we evaluate the effects of 63 natural compounds with diverse structures, isolated from the traditional herbal medicine S. affinis on the biosynthesis of estrogen in human ovarian granulosa-like KGN cells (Nishi et al., 2001). We further examined the mechanism whereby these potent compounds may regulate aromatase expression. Materials and methods Chemicals. The 63 natural products were isolated and identified as described previously (Xiong et al., 2011; Xiong et al., 2012; Zhu et al., 2012). Testosterone, forskolin, formestane, letrozole, SP600125, SB203580, PD98059, and LY294002 were purchased from SigmaAldrich (Shanghai, China). Cell culture and transfection. Human ovarian granulosa-like KGN cells (kindly supplied by Prof. Yiming Mu of the Chinese PLA General Hospital, Beijing, China) were maintained in DMEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum

(FBS, Invitrogen). Human embryonic kidney 293A (HEK293A) cells (Qbiogene, Carlsbad, CA, USA) were maintained in DMEM supplemented with 10% (v/v) FBS. The transient transfection of HEK293A cells using an aromatase expression vector was described previously (Lu et al., 2012). Cell proliferation assay. KGN cells were plated at 0.5 × 104 cells/well in 96-well plates with 100 μL of medium, and then treated with 10 μg/mL test chemicals. After 24 h, 10 μL of Alamar Blue reagent (SunBio Medical Biotechnology, Shanghai, China) was added to the medium in the wells and incubated for another 4 h, until the color changed from blue to pink. The relative fluorescence intensity in each well was measured using a Varioskan Flash spectral scanning multimode reader (Thermo Scientific, Waltham, MA, USA). Cell-based estrogen biosynthesis assay. Assays for estrogen biosynthesis in KGN cells or in transiently transfected HEK293A cells were conducted as described before (Lu et al., 2012). In brief, the cells were seeded overnight in 24-well plates. The following day, the medium was replaced with serum-free DMEM/F-12 medium, and the cells were pretreated with the test chemicals for 24 h. Testosterone (10 nM) was then added to each well, and the cells were incubated for an additional 24 h. Levels of 17β-estradiol in the culture medium were quantified using a magnetic particle-based 17β-estradiol enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Bio-Ekon Biotechnology, Beijing, China). The results, normalized to the total cellular protein content, were expressed as percentages of the control. Protein determination was carried out with the BCA protein assay kit (Pierce, Rockford, IL, USA). Recombinant expressed aromatase activity assay. An in vitro recombinant expressed aromatase activity assay was conducted as previously described (Lu et al., 2012). In brief, the test compounds were preincubated with an NAPDH regenerating system (Promega, Madison, WI, USA) for 10 min at 37 °C before recombinant expressed aromatase (BD Biosciences, San Jose, USA) and dibenzylfluorescein (Sigma-Aldrich) were added. The reaction mixture was then incubated for 2 h at 37 °C. The fluorescence intensity was measured at 485 nm (excitation) and 530 nm (emission). Quantitative real-time RT-PCR. Total cellular RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) with an oligo(dT)18 primer. Equal amounts of complementary DNA were subjected to real-time quantitative PCR with the fluorescent dye SYBR Green I according to the manufacturer's protocol (Fermentas, Thermo Scientific). The following primer pairs were used in the assay for aromatase, promoter II, promoter I.3, and GAPDH: 5′-ACCCTTCTGCGTCGTGTC-3′/5′-TCTGTGGAAATCCTGCGTCTT3′ (aromatase sense/antisense), 5′-TCCCTTTGATTTCCACAGGACTC3′/5′-ATGCAGTAGCCAGGACCTGGT-3′ (promoter II sense/antisense), 5′-CACTCTACCCACTCAAGGGCA-3′/5′-TTGGCTTGAATTGCAGCATTT-3′ (promoter I.3 sense/antisense), and 5′-TGCACCACCAACTGCTTAGC3′/5′-GGCATGGACTGTGGTCATGAG-3′ (GAPDH sense/antisense). The quantities of aromatase, promoter II, and promoter I.3 mRNA were normalized to the endogenous reference (GAPDH) mRNA quantity in the same samples. Western blotting. Cells were lysed in RIPA buffer (Byotime, Haimen, China) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). The cell lysates (50 μg) were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Antiaromatase (Epitomics, Burlingame, CA, USA), anti-GAPDH (Abgent, Suzhou, China), anti-phospho-CREB, anti-CREB, anti-phospho-ERK, anti-phospho-JNK (Cell Signaling Technology, Danvers, MA, USA), antiphospho-p38 and anti-phospho-AKT (Signaling Antibody LLC, College

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Park, MD, USA), and secondary antibodies conjugated with horseradish peroxidase (Pierce) were used for protein detection. The membranes were developed using enhanced chemiluminescence detection (Amersham Bioscience, Piscataway, NJ, USA). The protein concentration was determined by using a BCA protein assay kit (Pierce). Measurement of intracellular cAMP. The cAMP level in the KGN cells was measured using a cAMP-Glo assay kit (Promega). Briefly, 1 × 104 cells/well were seeded into a 96-well plate. On the second day, the cells were treated with the test compounds to modulate cAMP levels for the indicated time. For the cAMP-Glo assay, cells were lysed in 20 μL of cAMP-Glo lysis buffer with 20 μL of phosphate-buffered saline containing 500 μmol/L IBMX with shaking at room temperature for 15 min. Then, cAMP-Glo Detection Solution and Kinase-Glo Reagent were added to all wells according to the manufacturer's protocol. The Thermo Scientific Varioskan Flash multimode reader was used to measure the resulting luminescence. Statistical analysis. All experiments were repeated at least 3 times, and representative results are presented. Significant differences were evaluated with one-way analysis of variance at p b 0.05 using SPSS 17.0 software (SPSS Inc., Chicago, IL). Plots were generated using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Results Effects of natural products from S. affinis on estrogen biosynthesis To search for small-molecule modulators of estrogen biosynthesis, we examined the effects of 63 natural products isolated from S. affinis on estrogen biosynthesis in human ovarian granulosa-like KGN cells. The structures of these natural products, including 30 lignans, 6 flavonoids, 5 terpenoids, 6 steroids, 3 alkaloids, and 13 miscellaneous compounds are illustrated in Fig. S1. As shown in Fig. 1, forskolin (FSK), an adenylate cyclase agonist that activates the PKA/CREB pathway, significantly promoted 17β-estradiol production, whereas formestane (FOR), an aromatase inhibitor, significantly inhibited 17β-estradiol production. Among the 63 compounds, 12 showed inhibitory effects on the biosynthesis of estrogen, including 5 lignans (−)-secoisolariciresinol-9, 9′acetonide (SA-4-6), (+)-(7S,8R,7′E)-4-hydroxy-3,5′-dimethoxy-4′,7-

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epoxy-8,3′-neolign-7′-ene-9,9′-diol 9′-ethyl ether (SA-4-18), (−)(7R,7′R,7″R,8S,8′S,8″S)-4′,4″-dihydroxy-3,3′,3″,5,5′-pentamethoxy7,9′:7′,9-diepoxy-4,8″-oxy-8,8′-sesquineolignan-7″,9″-diol-7″,9″acetonide (SA-6-76a), (+)-(7R,7′R,7″R,7‴R,8S,8′S,8″S,8‴S)-4″,4‴dihydroxy-3,3′,3″,3‴,5,5′-hexamethoxy-7,9′:7′,9-diepoxy-4,8″:4′,8‴bisoxy-8,8′-dineolignan-7″,7‴,9″,9‴-tetraol (SA-6-83), (−)-(7R,8S,7′E)3,4,5′-trimethoxy-4′,7-epoxy-8,3′-neoligna-7′-ene-9,9′-diol (SA-6-91); 3 flavonoids tricin (SA-4-12), 7-methoxytricin (SA-4-27), (−)-(7″S,8″S)4″,5,7-trihydroxy-3′,3″,5′-trimethoxy-4′,8″-oxyflavonolignan-7″,9″diol (SA-6-9); 1 alkaloid N-benzoyl-O-(N′-benzoyl-L-phenylalanyl)L -phenylalaninol (SA-4-26); 1 terpenoid trans-phytol (SA-20); 1 steroid (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48); and 1 miscellaneous compound: 1-phenanthrol (SA-9). Effects of natural products on KGN cell viability To determine the effects of the natural products on KGN cell proliferation, we treated KGN cells separately with the natural compounds (10 μg/mL) for 24 h. As shown in Fig. 2, 16 compounds (SA-4-1, SA-46, SA-4-22, SA-4-26, SA-4-30, SA-6-83, SA-6-84, SA-6-84b, SA-6-85, SA-6-87, SA-12, SA-16, SA-17, SA-33, SA-35, and SA-43) promoted KGN cell proliferation, whereas 7 compounds (SA-6-3, SA-6-4, SA-6-5, SA-6-9, SA-6-37, SA-6-42, and SA-6-43) had inhibitory effects on KGN cell proliferation. Among the 12 compounds with inhibitory effects on estrogen biosynthesis (Fig. 1), only 3 compounds (SA-4-6, SA-4-26, and SA-6-83) promoted KGN cell proliferation and only 1 compound (SA-6-9) inhibited KGN cell proliferation. These results indicate that the inhibitory effects of 11 compounds on estrogen biosynthesis are not caused by their cytotoxicity on KGN cells. Effects of compound SA-20 and SA-48 on estrogen biosynthesis Considering the very limited understanding of natural terpenoids and steroids on estrogen biosynthesis, we chose two potent compounds SA-20 and SA-48 for further study. The chemical structures of compounds SA-20 and SA-48 are illustrated in Fig. 3A. KGN cells were treated with SA-20 or SA-48 at 1 μM or 10 μM for 24 h. As shown in Fig. 3B, SA-20 inhibited about 70% of the 17β-estradiol production in KGN cells as compared with that in the non-treated control cells. SA-20 also significantly inhibited forskolin-stimulated 17β-estradiol production. SA-48

Fig. 1. Effects of natural products from Sinocalamus affinis on estrogen biosynthesis. KGN cells seeded in 24-well plates were pretreated with the test compounds at 10 μg/mL for 24 h. Testosterone (10 nM) was added for an addition 24 h of incubation. Concentrations of 17β-estradiol in the culture supernatants were quantified by ELISA. Cont., DMSO control; FSK, 10 μM forskolin; FOR, 50 μM formestane. (∗) p b 0.05, (∗∗) p b 0.01, and (∗∗∗) p b 0.001 compared with the control (n = 3).

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Fig. 2. Effects of natural products on KGN cell viability. KGN cells seeded in 96-well plates were pretreated with the test compounds at 10 μg/mL for 24 h. Alamar Blue reagent (10 μL/well) was added for a further 4 h of incubation, and the fluorescence intensities were measured. Cont., DMSO control. (∗) p b 0.05, (∗∗) p b 0.01, and (∗∗∗) p b 0.001 compared with the control (n = 3).

similarly inhibited about 60% of the 17β-estradiol production in KGN cells as compared with that in the non-treated control cells, and significantly inhibited the forskolin-stimulated 17β-estradiol production (Fig. 3C). These results indicate that compounds SA-20 and SA-48 are indeed potent inhibitors of estrogen biosynthesis in KGN cells.

Concentration- and time-dependent effects of SA-20 and SA-48 on estrogen biosynthesis To examine the inhibitory effects of SA-20 and SA-48 on estrogen biosynthesis in more detail, KGN cells were treated with the two compounds separately at various concentrations. As shown in Fig. 4A,

Fig. 3. Effects of compound SA-20 and SA-48 on estrogen biosynthesis. (A) The chemical structures of SA-20 and SA-48. (B) KGN cells seeded in 24-well plates were pretreated with SA-20 at the indicated concentrations for 24 h. Testosterone (10 nM) was added for a further 24 h of incubation. Concentrations of 17β-estradiol in the culture supernatants were quantified by ELISA. (C) KGN cells seeded in 24-well plates were pretreated with SA-48 at the indicated concentrations for 24 h. Testosterone (10 nM) was added for a further 24 h of incubation. Concentrations of 17β-estradiol in the culture supernatants were quantified by ELISA. Cont., DMSO control; FSK, 10 μM forskolin; FOR, 50 μM formestane. (∗∗) p b 0.01, and (∗∗∗) p b 0.001 compared with the control or FSK-treated cells (n = 3).

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Fig. 4. Concentration- and time-dependent effects of SA-20 and SA-48 on estrogen biosynthesis. (A) The concentration–response curve of SA-20 for inhibition of estrogen biosynthesis in KGN cells. (B) The concentration–response curve of SA-48 for inhibition of estrogen biosynthesis in KGN cells. (C) The time course for the inhibition of estrogen biosynthesis by SA-20 (1 μM) in KGN cells. (D) The time course for the inhibition of estrogen biosynthesis by SA-48 (1 μM) in KGN cells. (∗) p b 0.05, (∗∗) p b 0.01 compared with the control (n = 3).

SA-20 inhibited 17β-estradiol biosynthesis in a concentrationdependent manner, with a calculated IC50 value of 1.093 μM. Similarly, concentration-dependent inhibition of SA-48 was also observed, with a

calculated IC50 value of 0.517 μM (Fig. 4B). SA-20 and SA-48 did not show cytotoxicity in KGN cells at the concentrations examined (Fig. S2). The inhibition of 17β-estradiol biosynthesis by SA-20 at 1

Fig. 5. SA-20 and SA-48 inhibit aromatase expression in KGN cells. KGN cells were treated with the test compounds at the indicated concentrations for 24 h. (A) Aromatase mRNA was measured in total cellular RNA by using real-time qPCR. The results are expressed as fold increase relative to levels in untreated cells. GAPDH was used as an internal control. (B) Cell lysates were immunoblotted with anti-aromatase or anti-GAPDH antibodies. The quantitative results are depicted. Cont., DMSO control; FSK, 10 μM forskolin; FOR, 50 μM formestane. (∗) p b 0.05, (∗∗) p b 0.01 and (∗∗∗) p b 0.001 compared with the control (n = 3).

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μM was first evident at 12 h and was sustained until 36 h, when 17βestradiol biosynthesis was inhibited by approximately 60% as compared with that in the control (Fig. 4C). The SA-48 inhibition (at 1 μM) of 17βestradiol biosynthesis was most prominent at 24 h, when 17β-estradiol biosynthesis was inhibited by approximately 50% as compared with that in the control (Fig. 4D).

SA-20 and SA-48 inhibit aromatase expression in KGN cells KGN cells lack endogenous 17α-hydroxylase and cannot synthesize androgens or estrogens de novo (Nishi et al., 2001). Therefore, the effect of SA-20 and SA-48 on 17β-estradiol biosynthesis in the presence of testosterone might be caused by aromatase, the only enzyme able to convert testosterone to 17β-estradiol. To determine whether SA-20 and SA-48 inhibit 17β-estradiol biosynthesis through an effect on aromatase, we examined aromatase mRNA and protein levels in KGN cells treated with SA-20 and SA-48 at various concentrations (1–50 μM). As shown in Fig. 5A, forskolin significantly increased aromatase mRNA levels in KGN cells, whereas formestane inhibited aromatase mRNA levels, consistent with reported results (Cavaliere et al., 2010; Gonzalez-Robayna et al., 1999). SA-20 and SA-48 decreased aromatase transcript levels in a concentration-dependent manner (Fig. 5A). At a concentration of 10 μM, SA-20 decreased 40% of the aromatase mRNA levels and SA-48 decreased 50% of the aromatase mRNA levels as compared with those in the DMSO-treated control cells. Aromatase protein expression was also examined following treatment with SA-20 and SA-48 at various concentrations (10–50 μM). Consistent with their

effects on aromatase transcription, SA-20 and SA-48 significantly decreased the aromatase protein expression (Fig. 5B). These results indicate that SA-20 and SA-48 inhibit estrogen biosynthesis in KGN cells by decreasing the expression of aromatase. Effects of SA-20 and SA-48 on aromatase catalytic activity To examine whether SA-20 and SA-48 inhibit the estrogen biosynthesis through the direct effect on the catalytic activity of aromatase protein, HEK293A cells, which do not express aromatase, were transiently transfected with an aromatase expression vector and treated with SA-20 and SA-48 at various concentrations (1–50 μM). Formestane significantly inhibited the 17β-estradiol biosynthesis, but SA-20 and SA-48 had no apparent effect on 17β-estradiol biosynthesis in aromatase-overexpressing HEK293A cells (Fig. 6A). We then conducted an in vitro assay by using recombinant expressed aromatase. As shown in Fig. 6B, formestane inhibited aromatase activity; however, SA-20 and SA-48 (1–50 μM) had no effect on aromatase activity. These results indicate that SA-20 and SA-48 do not directly inhibit aromatase activity. Effects of SA-20 and SA-48 on promoter I.3/II-driven aromatase transactivity Aromatase transcription is primarily controlled by promoter I.3/II in ovarian granulosa cells. Thus, we examined whether SA-20 and SA-48 exert their inhibitory effects on aromatase transcription through these

Fig. 6. Effects of SA-20 and SA-48 on aromatase catalytic activity. (A) HEK293A cells were co-transfected with pCMV6-aromatase and pSV-β-galactosidase expression plasmids. After 24 h, the cells were incubated with the various concentrations of SA-20 and SA-48 for an additional 24 h, and then testosterone (10 nM) was added for a further 24 h. 17β-Estradiol concentrations in the culture supernatants were quantified by ELISA. (B) An in vitro aromatase assay using recombinant expressed aromatase and dibenzylfluorescein as a substrate was conducted as described under Materials and methods. Various concentrations of SA-20 and SA-48 were added into the mixtures containing recombinant expressed aromatase and NADPH regenerating system, and fluorescence intensity induced by the aromatase-catalyzed dibenzylfluorescein hydrolysis was detected. Cont., DMSO control; FOR, 10 μM formestane. (∗∗∗) p b 0.001 compared with the control (n = 3).

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two promoters. SA-20 and SA-48 at 50 μM decreased 20–30% of the promoter I.3 (Fig. 7A). However at that same concentration (50 μM), SA-20 and SA-48 decreased 60–70% of the promoter II (Fig. 7B). These results indicate that the inhibition of aromatase transcription by SA-20 and SA-48 is mediated through promoter I.3/II, with promoter II playing a more prominent role.

Effects of SA-20 and SA-48 on cAMP signaling Cyclic AMP homeostasis plays an important role in the control of PKA-mediated aromatase transcription. Thus, we examined whether SA-20 and SA-48 disrupt the cAMP levels in KGN cells. As shown in Fig. 8A, forskolin rapidly increased the intracellular cAMP levels in KGN cells, consistent with the function of forskolin in activating adenylate cyclase. However, neither SA-20 nor SA-48 had any effect on intracellular cAMP levels (Fig. 8A) or on the forskolin-stimulated production of cAMP (Fig. 8B). These results indicate that SA-20 and SA-48 do not inhibit aromatase expression through intracellular cAMP homeostasis. CREB is the key transcription factor in ovarian granulosa cells that responds to cAMP signaling, which recognizes the specific binding sites in promoter II to promote aromatase transcription. Therefore, we next examined whether SA-20 and SA-48 could affect CREB. As shown in Fig. 8C, SA-20 significantly inhibited the phosphorylation and protein expression of CREB in a time-dependent manner. In comparison, SA-40

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inhibited the phosphorylation of CREB, but had no effect on the protein expression of CREB (Fig. 8D). These results indicate that SA-20 and SA-48 may inhibit the activation or expression of CREB to decrease the aromatase transcription. Effects of SA-20 and SA-48 on protein kinase signaling Because the transcriptional factors CREB or AP-1, which are implicated in the control of aromatase expression in the ovary, could be regulated by phosphorylation, we then investigated the effects of SA-20 and SA-48 on the activation states of the related signaling protein kinases. SA-20 inhibited the phosphorylation of JNK, ERK, and p38 MAPK, but had no effect on AKT phosphorylation (Fig. 9A). Similarly, SA-40 also inhibited the phosphorylation of JNK, ERK, and p38 MAPK, without affecting AKT phosphorylation (Fig. 9B). To further examine the effects of these protein kinases on aromatase in KGN cells, we treated cells with specific protein kinase pathway inhibitors. The inhibitors of aromatase (letrozole), AKT/PI3K (LY294002), and p38 MAPK (SB203580) significantly inhibited the estrogen biosynthesis in KGN cells, while inhibitors of JNK (SP600125) and ERK (PD98059) MAPK had no such effect (Fig. 9C). We then examined the effects of these inhibitors on aromatase expression. Only inhibitors of AKT/PI3K and p38 MAPK significantly decreased the aromatase expression in KGN cells, whereas inhibitors of JNK (SP600125) and ERK (PD98059) MAPK had no such inhibitory effects (Fig. 9D). Unlike formestane, letrozole did not affect the transcription of aromatase, consistent with a previous report (Santner et al., 1997). SA-20 and SA-48 had no effect on AKT/PI3K, indicating that these two compounds inhibited aromatase expression through the inhibition of p38 MAPK. Discussion

Fig. 7. Effects of SA-20 and SA-48 on promoter I.3/II-driven aromatase transactivity. KGN cells were treated with the test compounds at the indicated concentrations for 24 h. Aromatase promoter I.3 mRNA (A) or promoter II mRNA (B) was measured in total cellular RNA by using real-time qPCR. The results are expressed as fold increase relative to levels in untreated cells. GAPDH was used as an internal control. Cont., DMSO control; FSK, 10 μM forskolin; FOR, 50 μM formestane. (∗∗) p b 0.01 and (∗∗∗) p b 0.001 compared with the control (n = 3).

Natural products with a low incidence of side effects are a good source of aromatase inhibitors. Of these natural products, flavonoids are studied most because they have a structure similar to androgens and estrogens (Balunas et al., 2008). Although most bioactive flavonoids inhibit the catalytic activity of aromatase, tricin (SA-4-12), a constituent of rice bran, potently inhibits cyclooxygenase enzymes (Cai et al., 2005). Thus, it would be interesting to determine whether tricin or its analog 7-methoxytricin (SA-4-27) inhibit PGE2-stimulated aromatase transcription in breast cancer. Several lignans have been shown by using microsomes or aromatase overexpressing HEK293 cells to inhibit aromatase (Adlercreutz et al., 1993; Saarinen et al., 2003). A further investigation will be needed to determine whether the bioactive lignans identified in the present study (SA-4-6, SA-4-18, SA-6-76a, SA-6-83, and SA-6-91) also inhibit aromatase catalytic activity. Previously, a total of 36 terpenoids were tested on microsomes or breast cancer SK-BR-3 cells, but only one diterpenoid inflexin (IC50, 9.2 μg/mL), three sesquiterpenoid deoxycumambrin derivatives (IC50, 2–10 μM) potently inhibited aromatase (Balunas et al., 2008). Recently, two natural terpenoids 2-methoxy-5-acetoxyfruranogermacr-1(10)-en-6-one and dehydroabietic acid exhibited significant aromatase inhibiting activity with IC50 values at 0.2 μM and 0.3 μM, respectively (Su et al., 2009). Although synthetic steroidal aromatase inhibitors are well developed to bind aromatase and produce enzyme inactivation, natural steroids have been rarely reported to modulate aromatase activity or expression (Balunas et al., 2008). Previously, the steroid hydroperoxy24-vinyllathosterol from Vernonia anthelmintica was found to promote estrogen biosynthesis in human granulosa cells (Hua et al., 2012). In the present study out of only 5 terpenoids and 6 steroids examined, one acyclic diterpenoid, SA-20, and one functionalized ergostane steroid, SA-48, potently inhibited estrogen biosynthesis in KGN cells. Currently, these two compounds are the most potent terpenoid and steroid reported to inhibit aromatase in a cell-based assay. These results indicate that natural terpenoids or steroids may affect aromatase mainly at the transcriptional or post-transcriptional level, and that using a

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Fig. 8. Effects of SA-20 and SA-48 on cAMP signaling. (A) KGN cells seeded in 96-well plates were treated with the test compounds (10 μM) for different times. Cell lysates were measured for total cAMP concentration as described in the Materials and methods. (B) KGN cells seeded in 96-well plates were pretreated with the test compounds (10 μM) for 4 h, forskolin was added for an additional 5 min. Cell lysates were measured for total cAMP concentration. (C) KGN cells were treated with SA-20 (10 μM) at the indicated time. Cell lysates were immunoblotted with anti-phospho-CREB, anti-CREB, or anti-GAPDH antibodies. (D) KGN cells were treated with SA-48 (10 μM) at the indicated time. Cell lysates were immunoblotted with anti-phospho-CREB, anti-CREB, or anti-GAPDH antibodies. Cont., DMSO control; FSK, 10 μM forskolin. (∗∗) p b 0.01 and (∗∗∗) p b 0.001 compared with the control (A) or FSK (B) (n = 3).

cell line-based assay is more appropriate than using the conventional recombinant expressed aromatase or microsomes for the evaluation of terpenoids and steroids to find aromatase modulators. Phytol (SA-20) is an acyclic diterpenoid categorized as a branched-chain

fatty alcohol (3,7,11,15-tetramethylhexadec-2-en-1-ol). It is found abundantly in nature as part of the chlorophyll molecule, and a relatively high amount of free phytol is present in dairy products (Brown et al., 1993). The finding in this study that phytol is a potent aromatase

Fig. 9. Effects of SA-20 and SA-48 on protein kinase signaling. KGN cells were treated with 10 μM SA-20 (A) or 10 μM SA-48 (B) at the indicated time. Cell lysates were immunoblotted with anti-phospho-JNK, anti-phospho-ERK, anti-phospho-p38, anti-phospho-AKT, or anti-GAPDH antibodies. (C) KGN cells seeded in 24-well plates were pretreated with the specific inhibitors for 24 h. Testosterone (10 nM) was added for a further 24 h of incubation. Concentrations of 17β-estradiol in the culture supernatants were quantified by ELISA. (D) KGN cells were treated with the specific inhibitors for 24 h. Aromatase mRNA was measured in total cellular RNA by using real-time qPCR. The results are expressed as fold increases relative to levels in untreated cells. GAPDH was used as an internal control. Cont., DMSO control; Let, 1 μM letrozole; LY, 20 μM LY294002; SB, 1 μM SB203580; SP, 20 μM SP600125; PD, 20 μM PD98059. (∗∗) p b 0.01 and (∗∗∗) p b 0.001 compared with the control (n = 3).

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inhibitor gives new insight on our understanding of the beneficial effects of vegetables and fruit on human health. We found that SA-20 and SA-48 exhibited their inhibitory effect in KGN cells until 12–24 h, indicating that they modulate aromatase at the transcriptional level. This is further supported by the findings that they inhibited aromatase mRNA and protein expression. However, unlike other terpenoids, SA-20 and SA-48 had no effect on the catalytic activity of aromatase protein. In ovarian granulosa cells, aromatase transcription is primarily controlled by the cAMP/PKA/CREB pathway through promoter I.3/II (Simpson, 2004). We determined that the level of these promoters was potently decreased by SA-20 and SA-48. In breast cancer cells, aromatase transcription is also regulated by promoter I.3/II. For developing a new prodrug for the tissue-selective treatment of breast cancer, it would be interesting to determine whether SA-20 and SA-48 inhibit aromatase expression in breast cancer cells. Intracellular cAMP homeostasis plays important role in the activation of PKA in response to FSH in ovarian granulosa cells, which is mediated by adenylate cyclases and phosphodiesterases (Conti, 2002; Hunzicker-Dunn and Maizels, 2006). We found that SA-20 and SA-48 did not affect the cAMP level in KGN cells, indicating they did not act on cAMP homeostasis or PKA activation. CREB is the key transcriptional factor in the regulation of aromatase in the ovary and is phosphorylated and activated by PKA or other protein kinases (Mayr and Montminy, 2001). We found that SA-20 not only inhibited CREB phosphorylation, but also unexpectedly decreased its expression. In contrast, SA-48 inhibited CREB phosphorylation, but had no effect on the CREB expression. These results indicate that SA-20 and SA-48 may regulate factors other than PKA to inhibit the CREB phosphorylation and expression, and that the mechanism of the two compounds on the regulation of CREB may be different. The phosphorylation of CREB is reportedly regulated by ERK, p38 MAPK, and PI3K pathways (Mayr and Montminy, 2001). FSH activates several signaling pathways to exert its full function in granulosa cells, including ERK, p38 MAPK, and PI3K (Hunzicker-Dunn and Maizels, 2006). We found that SA-20 and SA-48 inhibited p38, ERK, and JNK MAPKs with different potencies and time courses, while specific inhibitors of ERK and JNK had no effect on aromatase activity and transcription, which was significantly decreased by inhibitor of p38. The finding here indicates that p38 MAPK, but not ERK and JNK, plays the key role in the basal control of aromatase in granulosa cells, which may be different from the FSH-stimulated aromatase expression. Although inhibition of AKT/PI3K by specific inhibitor significantly suppressed the activity and transcription of aromatase in KGN cells, SA-20 and SA-48 had no effect on the AKT/PI3K pathway, indicating that these two compounds do not exert their inhibitory effect on aromatase transcription through this pathway. In addition to CRE elements, the aromatase promoter I.3/II also contains the binding sites for AP-1 transcription factor families (c-Jun/c-fos/ATF), which can also be regulated by p38 MAPK (Li et al., 2011; Shaulian and Karin, 2002). Thus, SA-20 and SA-48 may decrease aromatase expression by the inhibition of p38 MAPK to suppress the activation of CREB and the AP-1 transcriptional factors-mediated promoter I.3/II activation. In breast cancer cells, the switch from the aromatase promoter I.4 to promoter I.3/II is considered critical in breast tumorigenesis (Simpson et al., 1997). The potent pro-inflammatory PGE2 induced aromatase promoter I.3/II via activation of p38 and JNK in adipose fibroblasts; thus, p38 and JNK have been proposed as potential new drug targets for tissue-specific ablation of aromatase expression in breast cancer (Chen et al., 2007). In granulosa cells, we found that inhibition of JNK did not decrease but moderately increased the basal aromatase activity and transcription. The difference may reflect the tissue-specific complexity of basal aromatase expression control and regulation in response to extracellular stimuli in ovarian cells as compared with breast cancer cells. Several natural products modulate MAPK pathway signaling to regulate aromatase expression. The soy isoflavone genistein promotes

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aromatase promoter I.3/II in hepatoma HepG2 cells by increasing the phosphorylation of ERK and p38 MAPK (Ye et al., 2009). The dietary flavone luteolin inhibits aromatase transcription by inhibition of JNK in breast cancer cells (Li et al., 2011). Recently, the environmental pollutant 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) was found to activate ERK in brain cells, which stimulated the brain-specific aromatase promoter I.f (Tan et al., 2013). MAPK may regulate aromatase promoters through different mechanisms in a cell-specific manner, allowing the possibility of tissue-selective aromatase modulators. Many natural terpenoids were also found to inhibit MAPKs including p38 MAPK to exert their anti-inflammatory effects (Salminen et al., 2008), and so it is of interest to investigate whether these compounds are able to inhibit aromatase expression. Interestingly, phytol (SA-20) was indeed found to inhibit inflammatory responses by reducing cytokine production (Silva et al., 2013). Estrogen has been implicated in inflammation modulation, but the mechanism for this action is not clear (Harnish, 2006). Thus, further study of terpenoids on aromatase expression is important not only for discovering new tissue-selective aromatase modulators, but also for elucidating the unknown mechanism for the interaction between inflammation and estrogen anabolism. In summary, this study identified two natural products, SA-20 and SA-48, which potently decrease aromatase expression in human ovarian granulosa-like KGN cells by inhibiting p38 MAPK. Terpenoids or steroids may be a new source for tissue-selective aromatase modulators, which will be superior to the phytoestrogens in the inhibition of aromatase catalytic activity. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention of treatment of estrogendependent cancers.

Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 20932007, 21372214), the West Light Foundation Chinese Academy of Sciences, the Pillar Program of Science & Technology Department of Sichuan Province (No. 2012SZ0219), and the National New Drug Innovation Major Project of China (No. 2011ZX09307-00202).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2014.05.008.

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