Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.


Introduction
Breast cancer is the most common form of cancer diagnosed in women worldwide. Approximately 90% of all breast cancers are sensitive to female hormones, such as estrogen and progesterone, while two-thirds of postmenopausal breast cancers are estrogen-dependent [1]. Estrogen plays a pivotal role in the proliferation of breast epithelial cells. Abnormal local overproduction of estrogen in breast tissues is implicated in breast cancer pathogenesis.
Estrogen biosynthesis is regulated by aromatase, which is encoded by the cytochrome P450 19 gene (CYP19) located on chromosome 15q21.1. CYP19 aromatase, also known as estrogen synthetase or estrogen synthase, catalyzes the aromatization of androgen to estrogen in the endoplasmic reticulum [2]. There is a significant correlation between tumor incidence and aromatase activity in breast tissue [3], and aberrant aromatase expression is closely associated with breast cancer development [1], suggesting the etiological role of estrogen in breast cancer incidence. As the expression of the estrogen receptor (ER) is considerably high in breast tumors, targeting the ER or estrogen deprivation has been (C) EGR-1 levels were measured by immunoblot analysis. GAPDH levels were used as an internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were used as an internal control. (C) CYP19 aromatase levels were determined by immunoblot analysis. GAPDH levels were used as internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n = 3). NS, not significant, ** p < 0.01, *** p < 0.001 versus control (time 0) by Dunnett's multiple comparisons test. qR-PCR, quantitative real-time PCR.

TNFα Upregulated EGR-1 Expression in MCF-7 Breast Cancer Cells
The transcription factor EGR-1 is an immediate early response protein that is rapidly induced in response to diverse extracellular signals, such as growth factors, DNA damage, and inflammatory cytokines [26][27][28][29]. Previous studies have demonstrated that EGR-1 expression is upregulated in response to TNFα stimulation in several cell types [30][31][32][33]. Consistent with previous findings, we observed the increase in EGR1 mRNA expression upon TNFα stimulation in MCF7 cells, as revealed by RT-PCR ( Figure 2A) and qR-PCR ( Figure 2B). Concordant with mRNA levels, immunoblotting studies yielded similar results ( Figure 2C). These results demonstrated that EGR-1 expression is upregulated in response to TNFα stimulation in MCF-7 breast cancer cells.

TNFα Upregulated EGR-1 Expression in MCF-7 Breast Cancer Cells
The transcription factor EGR-1 is an immediate early response protein that is rapidly induced in response to diverse extracellular signals, such as growth factors, DNA damage, and inflammatory cytokines [26][27][28][29]. Previous studies have demonstrated that EGR-1 expression is upregulated in response to TNFα stimulation in several cell types [30][31][32][33]. Consistent with previous findings, we observed the increase in EGR1 mRNA expression upon TNFα stimulation in MCF7 cells, as revealed by RT-PCR ( Figure 2A) and qR-PCR ( Figure 2B). Concordant with mRNA levels, immunoblotting studies yielded similar results ( Figure 2C). These results demonstrated that EGR-1 expression is upregulated in response to TNFα stimulation in MCF-7 breast cancer cells. (C) EGR-1 levels were measured by immunoblot analysis. GAPDH levels were used as an internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n (C) EGR-1 levels were measured by immunoblot analysis. GAPDH levels were used as an internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 by Dunnett's multiple comparisons test. NS, not significant; qR-PCR, quantitative real-time PCR. EGR-1 plays an essential role in tumor development in several cancer cells, including breast cancer [34][35][36][37]. The mitogenic chemokine growth-regulated oncogene (GROα), also known as the C-X-C motif ligand 1 (CXCL1) or melanoma growth stimulating activity alpha (MGSA-α), elicits mitogenic properties via EGR-1 induction [36]. In addition, EGR-1 enhances GROα and matrix metalloproteinase-9 (MMP-9) expression in response to TNFα stimulation in HeLa cells [38,39]. However, the role of TNFα-induced EGR-1 expression in CYP19 expression in breast cancer cells remains unknown.

Silencing of EGR-1 Abrogated TNFα-Induced CYP19 Expression
To investigate whether TNFα-induced EGR-1 expression is linked to CYP19 aromatase expression, we used MCF-7 variant cell lines expressing lentiviral short-hairpin RNA (shRNA) against EGR-1 (shEgr1) or the scrambled control (shCT). The stable knockdown of EGR-1 after TNFα stimulation was confirmed by immunoblot analysis ( Figure 3A). RT-PCR analysis revealed that EGR-1 silencing abrogated TNFα-induced CYP19 mRNA expression ( Figure 3B). Quantitation of mRNA using qR-PCR revealed that compared to the MCF7/shCT cells, basal CYP19 mRNA levels were significantly reduced to 0.600 ± 0.100-fold, while TNFα-induced CYP19 mRNA levels were significantly reduced from 3.97 ± 0.503-fold to 1.37 ± 0.153-fold in MCF7/shEgr1 cells ( Figure 3C). Immunoblot analysis revealed that the level of CYP19 aromatase reduced significantly in MCF7/shEgr1 cells compared to that in MCF7/shCT cells ( Figure 3D). These results suggest that EGR-1 plays a critical role in both basal and TNFα-induced CYP19 expression in MCF-7 cells.  Exponentially growing MCF-7 variant cells were cultured in the presence or absence of 10 ng/mL TNFα for 1 h. The cells were then harvested and analyzed by immunoblotting using anti-EGR-1 antibody. GAPDH was used as an internal control. (B-D) MCF-7 cells expressing shCT or shEgr1 were treated with 10 ng/mL TNFα for 24 h. CYP19 mRNA levels were determined by RT-PCR (B) and qR-PCR (C). GAPDH mRNA levels were used as an internal control. CYP19 aromatase levels were measured by immunoblot analysis (D). GAPDH levels were used as an internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n = 3). * p < 0.05, *** p < 0.001 by Dunnett's multiple comparisons test. NS, not significant; qR-PCR, quantitative real-time PCR.

Chrysoeriol Inhibited the ERK1/2 MAPK Pathway to Block TNFα-Induced CYP19 Expression in MCF-7 Cells
We next determined the effect of MAPK inhibition on TNFα-induced CYP19 mRNA expression. Pretreatment with U0126 inhibited the ability of TNFα to induce CYP19 mRNA expression, as revealed by RT-PCR ( Figure 7A) and qR-PCR ( Figure 7B). Notably, SB203580 and SP600125 significantly inhibited TNFα-induced CYP19 mRNA expression as well. These data suggest that all three MAPK pathways are involved in TNFα-induced CYP19 mRNA expression. It seems likely that ERK1/2 mediates EGR-1-dependent CYP19 expression, whereas JNK1/2 and p38 kinase induce CYP19 expression independent of EGR-1.
It has been demonstrated that prostaglandin E2 stimulates all three MAPKs (ERK, JNK, and p38 kinase); however, JNK and p38 kinase, though not ERK, are necessary for CYP19 aromatase expression in adipocyte fibroblasts [48]. In contrast, follicle-stimulating hormone-induced CYP19 aromatase expression is mediated by the phosphatidylinositol 3-kinase signaling pathway, whereas the ERK MAPK pathway inhibits estrogen production in testis Sertoli cells [49]. As CYP19 transcription is controlled in a tissue-specific manner [50], it seems likely that ERK, JNK, and p38 MAPKs might contribute to tissue-specific regulation of CYP19 transcription.
Finally, we sought to address the involvement of the MAPK pathways in chrysoeriol-induced suppression of CYP19 expression. Pretreatment with chrysoeriol significantly abrogated TNFα-induced ERK1/2 phosphorylation in a dose-dependent manner, while the same was not applicable to JNK1/2 and p38 kinase ( Figure 7C). These data suggest that chrysoeriol inhibits TNFα-induced CYP19 expression via selective inhibition of ERK1/2-mediated EGR-1 expression.

Reverse Transcription PCR (RT-PCR)
MCF-7 cells were treated with TNFα or chrysoeriol, and total RNA was extracted using a TRIzol RNA extraction kit (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was reverse-transcribed into complementary DNA (cDNA) using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). RT-PCR was performed using the reverse transcriptase enzyme according to the manufacturer's instructions (Promega). The gene-specific PCR primers used were as follows: PCR conditions were as follows: hold for 5 min at 94 • C, followed by 30 cycles consisting of denaturation at 94 • C (30 s), annealing at 55 • C (30 s), and elongation at 72 • C (1 min). The amplified products were electrophoresed on a 2% agarose gel using ethidium bromide and were detected under UV light.

Quantitative Real-Time PCR (qR-PCR)
Quantitation of mRNA levels was conducted using the quantitative real-time PCR (qR-PCR) approach using an iCycler iQ system with an iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's recommendations. Validated commercial qR-PCR primers and SYBR Green-based fluorescent probes specific for CYP19 mRNA (id: qHsaCIP0026454) and GAPDH mRNA (id: qHsaCEP0041396) were obtained from Bio-Rad. PCR conditions were as follows: denaturation at 95 • C for 2 min, followed by 40 cycles using a step program (95 • C for 10 s and 60 • C for 45 s). The relative expression levels of CYP19 mRNA were normalized to those of GAPDH using the software provided by the manufacturer.

Immunoblotting
Cells were lysed in a buffer consisting of 20 mM Hydroxyethyl piperazine Ethane Sulfonicacid (HEPES, pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The blots were incubated with the appropriate primary and secondary antibodies and developed using an Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Science, Chicago, IL, USA).

EGR1 Promoter Reporter Assay
The MCF-7 cells seeded onto 12-well plates were transfected with 0.1 µg of the EGR1 promoter construct p-668egrLuc(-668/+ 1) [52] using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. To monitor the transfection efficiency, we included a pRL ® -null plasmid (50 ng) encoding Renilla luciferase in all transfections. At 48 h post-transfection, the levels of firefly luciferase and Renilla luciferase activities were measured sequentially from the same sample using the Dual-Glo Luciferase Assay System (Promega), as described previously [38]. The relative luciferase activity in the untreated cells was designated 1. The luminescent signal was detected and measured using a dual luminometer Centro LB960 (Berthold Tech, Bad Wildbad, Germany). For the screening of flavonoid compounds inhibiting TNFα-induced EGR1 promoter activity, we calculated the inhibitory activity by the following formula: where P F = flavonoid-induced EGR1 promoter activity, Pb = unstimulated basal EGR1 promoter activity, and P T = TNFα-induced EGR1 promoter activity.

Immunofluorescence
MCF-7 cells cultured on coverslips were either treated with phosphate-buffered saline (PBS) or 10 ng/mL TNFα in the presence or absence of chrysoeriol for 24 h, followed by fixation, permeabilization, and incubation with primary antibodies. Samples were probed with primary antibodies against α/β-tubulin and CYP19 aromatase for 2 h, followed by probing with Alexa Fluor 555-(for α/β-tubulin; red signal) and Alexa Fluor 488-conjugated (for CYP19; green signal) secondary antibodies for an additional 30 min. Nuclear DNAs were stained with Hoechst 33258 for 10 min (blue signal). Fluorescent cells were examined under an EVOS FL fluorescence microscope (Advanced Microscopy Group; Bothell, WA, USA).

Statistical Analysis
The data are plotted as means with SD. Statistical comparisons were performed using a one-way ANOVA followed by Dunnett's or Sidak's multiple comparisons test with the GraphPad Prism V8.3.1 software (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

Conclusions
CYP19 aromatase overexpression is associated with malignant phenotypes in the human breast [1]. However, aberrant expression of CYP19 aromatase is not regulated by the proliferation of tumor cells or clinical course; instead, it occurs as a result of interaction between tumor cells and stromal cells [53,54]. Accordingly, we observed that untreated MCF-7 cells expressed low levels of CYP19 aromatase, while TNFα stimulation resulted in an increase in CYP19 mRNA levels. Additionally, we observed that pretreatment with chrysoeriol prior to TNFα treatment substantially prevented TNFα-induced CYP19 expression via the downregulation of ERK MAPK-mediated EGR-1 expression. TNFα is the major inflammatory cytokine produced by adipose tissues, tumor-associated fibroblasts, and various inflammatory cells infiltrating the tumor tissues [22]. It is well established that localized TNFα in the tumor microenvironment promotes inflammation-associated tumors in most solid tumors, including breast cancer [55,56]. Additionally, estrogen induces EGR-1 expression in MCF-7 cells [57]. This gives rise to the possibility that EGR-1-dependent signaling loop and the TNFα-EGR-1-CYP19-estrogen-EGR-1 axis could result in persistent activation of estrogen production, thereby facilitating the development of ER-positive breast cancer in the tumor microenvironment. Besides CYP19 aromatase, EGR-1 enhances paclitaxel-induced multi-drug resistance by upregulation of P-glycoprotein, an ATP-dependent efflux pump [58], and mediates TNFα-induced GROα and MMP-9 expression [38,39]. Furthermore, inhibition of EGR-1 by DNAzyme inhibits fibroblast growth factor-dependent angiogenesis in breast cancer [37]. Therefore, suppression of EGR-1 and CYP19 aromatase expression by dietary chrysoeriol in the breast tumor microenvironment may be beneficial as a preventive agent or as a chemotherapeutic adjuvant against estrogen receptor-positive breast cancer.
Chrysoeriol is present in several foods and vegetables [41][42][43][44][45]. Many dietary phenolics are generally poorly bioavailable, limiting their distribution in systemic tissues of their native form, mainly as glycosides and complex oligomeric structures [59]. In general, methylation protects the flavonoids from widespread conjugation, and methylated flavonoids have improved intestinal absorption and metabolic stability compared to unmethylated forms [60]. Chrysoeriol contains a methoxy group attached to the C3' atom of the flavonoid backbone. The pharmacokinetics profile of circulating chrysoeriol is not fully characterized yet. However, the oral bioavailability and tissue distribution of chrysoeriol could be indirectly predicted through the study of luteolin (3 ,4 ,5,7-tetrahydroxyflavone). Luteolin is a common catechol-type flavonoid present in many types of edible plants and medicinal herbs [61], possessing a wide range of biological effects, including anti-inflammatory and anti-cancer activities [62]. However, luteolin is hardly detected and is mainly present in the metabolites of glucuronides and methylated forms in vivo [63]. Glucuronidation by uridine 5 -diphospho-glucuronosyltransferases (UGTs) and methylation by catechol-O-methyltransferases (COMTs) are two main metabolic pathways of luteolin in animals and humans [64,65]. Chrysoeriol is generated by the methoxylation of luteolin at the C3' atom (luteolin-3 -methoxy ether) by catechol-O-methyltransferase (COMT) [66]. Chrysoeriol has been detected in rat plasma samples after oral administration of luteolin [63] and is broadly distributed to the lungs, kidney, spleen, muscle, and heart, but found to be undetectable in the brain, within 1 h after oral administration [67], suggesting that circulating chrysoeriol can reach various target tissues. The anti-breast cancer efficacy of chrysoeriol in vivo is closely related to its concentration in breast cancer tissues. The distribution of chrysoeriol in the breast tissue has not been well characterized. As the distribution of phenolic metabolites in breast tissue is similar to that observed in plasma [59] and the fact that chrysoeriol exerts chemopreventive cancer effects in mouse mammary organ culture [68], we suggest that circulating chrysoeriol could reach primary breast cancer tissues and exert anti-cancer effects. To provide evidence-based potential of chrysoeriol as a functional food or nutrition supplement in the prevention or treatment of breast cancer, further physiologically relevant in vivo studies, including bioavailability, metabolism, and tissue distribution of dietary chrysoeriol and its derived metabolites, are necessary [59].
In conclusion, the present study demonstrates that chrysoeriol reduces TNFα-induced upregulation of CYP19 aromatase expression via inhibition of ERK MAPK-mediated EGR-1 expression in MCF-7 breast cancer cells. Chrysoeriol might serve as a beneficial supplement or adjuvant for the prevention or treatment of estrogen receptor-positive breast cancer with the potential to inhibit local estrogen production in the tumor microenvironment in breast cancer.