Butein Inhibits Cell Growth by Blocking the IL-6/IL-6Rα Interaction in Human Ovarian Cancer and by Regulation of the IL-6/STAT3/FoxO3a Pathway

Butea monosperma (Fabaceae) has been used in traditional Indian medicine to treat a variety of ailments, including abdominal tumors. We aimed to investigate the anti-IL-6 activity of butein in ovarian cancer and elucidate the underlying molecular mechanisms. Butein was isolated and identified from B. monosperma flowers, and the inhibition of IL-6 signaling was investigated using the HEK-Blue™ IL-6 cell line. The surface plasmon resonance assay was used to estimate the binding of butein to IL-6, IL-6Rα, and gp130. After treatment with butein, ovarian cancer cell migration, apoptosis, and tumor growth inhibition were evaluated in vitro and in vivo. Furthermore, we used STAT3 siRNA to identify the mechanistic effects of butein on the IL-6/STAT3/FoxO3a pathway. Butein suppressed downstream signal transduction through higher binding affinity to IL-6. In ovarian cancer, butein inhibited cell proliferation, migration, and invasion, and induced cell cycle arrest and apoptosis. In addition, it decreased the growth of ovarian cancer cells in xenograft tumor models. Butein inhibited STAT3 phosphorylation and induced FoxO3a accumulation in the nucleus by inhibiting IL-6 signaling. The anticancer activity of butein was mediated by blocking the IL-6/IL-6Rα interaction and suppressing IL-6 bioactivity via interfering with the IL-6/STAT3/FoxO3a pathway.


Introduction
Ovarian cancer is the eighth most common cancer in women worldwide and ranks fifth in cancer death among women in the United States. Standard treatment consists of cell reduction surgery and platinum-based chemotherapy. However, it is a malignant cancer with high anticancer drug resistance and recurrence rate, so the 5-year survival rate is only 10-40% [1,2]. Therefore, novel therapeutic agents are required to reduce the chemotherapy resistance and recurrence of ovarian cancer.
Butea monosperma (Fabaceae) is a medium-sized deciduous tree which grows to about 50 feet and is native to Southeastern Asian nations such as Bangladesh, India, Thailand, and Western Indonesia [3][4][5]. The flowers of B. monosperma have traditionally been used as an astringent, diuretic, depurative, aphrodisiac, and tonic [6], and a variety of flavonoids (e.g., butein, butin, coreopsin, isobutrin, and monospermoside) have been identified from this plant. Previous studies have demonstrated that butein exerts anticancer activity and inhibits the proliferation of many human cancers, including colon, breast, hepatocellular, and cervical cancers [7][8][9]. Butein is involved in cell survival, proliferation, migration, invasion, and angiogenesis, and targets many molecular pathways in various cancers.
Butein has also been shown to have immunomodulatory activity by inhibiting the expression of inflammatory mediators such as IL-6 and TNF-α in HaCaT cells, a keratinocyte cell line. It most commonly affects the expression of NF-κB and its downstream regulators [10,11]. Other important molecular targets include VEGF, STAT3, ERK, JNK, Akt, and p38 [9,[12][13][14]. However, the role of butein in ovarian cancer has not been actively studied; in particular, there have been no studies that clearly explain its molecular mechanism. The fact that it is available as a treatment for ovarian cancer requires better insight into its pharmacological mechanisms.
Interleukin-6 (IL-6) has been shown to have a direct stimulatory effect on many cancer cells through its action on several cell cycle pathways. IL-6 binds to the nonsignaling IL-6 receptor (IL-6R) and then forms a complex with the signaling co-receptor glycoprotein 130 (gp130). IL-6 induced Janus Kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) activation leads to constitutive activation of STAT3, which correlates with enhanced tumor cell growth and chemotherapy resistance [15][16][17]. This makes the IL-6/IL-6Rα/gp130 signaling pathway an attractive target for therapeutic or preventive intervention. The application of IL-6 blockers as anti-cancer agents has been investigated in many cancer types, but the only currently approved monoclonal antibodies (mAbs) in the United States are tocilizumab (anti-IL-6Rα) and siltuximab (anti-IL-6). These are intended for the treatment of rheumatoid arthritis (RA) and Castleman's disease, not for use as cancer drugs. Currently, the application of IL-6/IL-6Rα/gp130 blockers as anti-cancer agents has not been extensively studied, much less for ovarian cancer. As disease progression depends on various IL-6-related mechanisms in ovarian cancer, the IL-6 signaling pathway is an ideal target for drug development. It was recently demonstrated that STAT3 regulates the expression of forkhead box class O 3a (FoxO3a) and the cell cycle regulatory proteins p27 kip1 and p21 waf1 [18,19]. FoxO3a is a transcription factor that mediates several physiological and pathological processes by regulating gene expression in apoptosis, proliferation, cell cycle progression, and DNA damage [20]. Therefore, further studies on the effect of IL-6 blockers in ovarian cancer are needed and elucidating the correlation between IL-6 activated STAT3 phosphorylation and FoxO3a may suggest and exciting new therapeutic directions.
In this study, we investigated the therapeutic potential of butein isolated from B. monosperma flowers in ovarian cancer. We aimed to elucidate the anti-IL-6 property of butein, which blocks the interaction between IL-6 and IL-6Rα, and to elucidate the underlying molecular mechanisms involved in the regulation of the STAT3 pathway.

Characterization of Butein and Anti-IL-6 Activity In Vitro
The HEK-Blue™ IL-6 cell line was used to find isolates having an inhibitory effect on IL-6 signaling among the 14 isolates from B. monosperma. The cells were treated with increasing concentrations of the 14 isolates (0, 3.125, 6.25, 12.5, 25, and 50 µM) in the presence of 10 ng/mL IL-6. After 24 h of reaction, the secreted embryonic alkaline phosphatase (SEAP) signal of HEK-Blue™ IL-6 was inhibited in a concentration-dependent manner by the isolates (Figure S1), with butein (compound 9) achieving the maximum inhibition (Figure 2B). The HEK-Blue™ IL-6 cell bioassay showed that butein blocked IL-6 induced bioactivity. We found that butein interfered with the interaction between IL-6 and IL-6Rα and reduced it by 21.7% at 20 µM ( Figure S2A). We then performed SPR to confirm if butein could directly bind to IL-6. After each IL-6, IL-6Rα, and gp130 protein was immobilized on the CM5 chip, the binding affinity between the immobilized protein and butein was examined. We found that butein had binding affinity to immobilized IL-6 protein (KD = 91.42 µM; Figure 1C). In addition, to investigate the association between IL-6Rα and gp130 binding affinity, the RU for each concentration of butein was detected through SPR analysis, and the results were added to Figure S2B. We also compared the binding behaviors of butein to IL-6Rα with that to gp130 protein. Butein showed binding affinity to immobilized IL-6Rα and gp130 proteins (KD = 435.3 µM and KD = 615.7 µM, respectively; Figure S2B). It associates faster with IL-6 than with IL-6Rα and gp130, thus indicating that it has a lower affinity to IL-6Rα and gp130 protein. As a result, butein inhibits downstream signaling through higher binding affinity to IL-6.

Characterization of Butein and Anti-IL-6 Activity In Vitro
The HEK-Blue™ IL-6 cell line was used to find isolates having an inhibitory effect on IL-6 signaling among the 14 isolates from B. monosperma. The cells were treated with increasing concentrations of the 14 isolates (0, 3.125, 6.25, 12.5, 25, and 50 µM) in the presence of 10 ng/mL IL-6. After 24 h of reaction, the secreted embryonic alkaline phosphatase (SEAP) signal of HEK-Blue™ IL-6 was inhibited in a concentration-dependent manner by the isolates ( Figure S1), with butein (compound 9) achieving the maximum inhibition ( Figure 2B). The HEK-Blue™ IL-6 cell bioassay showed that butein blocked IL-6 induced bioactivity. We found that butein interfered with the interaction between IL-6 and IL-6Rα and reduced it by 21.7% at 20 µM ( Figure S2A). We then performed SPR to confirm if butein could directly bind to IL-6. After each IL-6, IL-6Rα, and gp130 protein was immobilized on the CM5 chip, the binding affinity between the immobilized protein and butein was examined. We found that butein had binding affinity to immobilized IL-6 protein (K D = 91.42 µM; Figure 1C). In addition, to investigate the association between IL-6Rα and gp130 binding affinity, the RU for each concentration of butein was detected through SPR analysis, and the results were added to Figure S2B. We also compared the binding behaviors of butein to IL-6Rα with that to gp130 protein. Butein showed binding affinity to im-mobilized IL-6Rα and gp130 proteins (K D = 435.3 µM and K D = 615.7 µM, respectively; Figure S2B). It associates faster with IL-6 than with IL-6Rα and gp130, thus indicating that it has a lower affinity to IL-6Rα and gp130 protein. As a result, butein inhibits downstream signaling through higher binding affinity to IL-6.

Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells
To investigate the effects of butein on ovarian cancer in vitro, A2780 and SKOV3 cell lines were used. Cell viability of A2780 and SKOV3 cells was inhibited with IC50 values of 64.7 ± 6.27 µM and 175.3 ± 61.95 µM, respectively, upon butein treatment ( Figure 3A). Similarly, their clonogenicity was significantly inhibited in the butein-treated cells compared with the control siltuximab (anti-IL-6)-treated cells ( Figure 3B). The wound healing assay indicated that butein reduced cell migration at 24 h and 48 h in a concentration-dependent manner compared with the control, 0.1% DMSO-, and siltuximab-treated group ( Figure 3C). Cell infiltration was similarly affected as observed in the Matrigel cell invasion assay ( Figure 3D).

Butein Suppresses the Cell Viability, Migration, and Invasion of Ovarian Cancer Cells
To investigate the effects of butein on ovarian cancer in vitro, A2780 and SKOV3 cell lines were used. Cell viability of A2780 and SKOV3 cells was inhibited with IC50 values of 64.7 ± 6.27 µM and 175.3 ± 61.95 µM, respectively, upon butein treatment ( Figure 3A). Similarly, their clonogenicity was significantly inhibited in the butein-treated cells compared with the control siltuximab (anti-IL-6)-treated cells ( Figure 3B). The wound healing assay indicated that butein reduced cell migration at 24 h and 48 h in a concentrationdependent manner compared with the control, 0.1% DMSO-, and siltuximab-treated group ( Figure 3C). Cell infiltration was similarly affected as observed in the Matrigel cell invasion assay ( Figure 3D).

Butein Induced Ovarian Cancer Cell Cycle Arrest and Cell Apoptosis
Cell cycle progression was monitored using flow cytometry. Exposure to butein resulted in an increase in G1-phase cells along with a decrease in S-phase cells. The effect was observed with 25 µM butein treatment, leading to 55.9% of A2780 cells in G1-phase vs. 45.3% under control conditions. Similarly, 65.8% of SKOV3 cells upon butein treatment and 58.3% under control conditions were in G1-phase ( Figure 4A). Butein was also found to significantly increase total apoptosis in both A2780 and SKOV3 cell lines in a dosedependent manner (p < 0.005) ( Figure 4B). Consequently, our results demonstrated that treatment with butein inhibits the growth of ovarian cancer cells by inducing cell cycle arrest and apoptosis.
In accordance with the above data, Western blot analysis indicated that the expression levels of the cell cycle proteins CDK4, CDK6, and Cyclin D1 were reduced, while that of p27kip1 was enhanced, in the butein-treated A2780 and SKOV3 cell lines in a dosedependent manner ( Figure 4C). Meanwhile, the pro-apoptosis protein Bax was upregulated while Bcl-2 and Mcl-1 were downregulated in the butein-treated A2780 and SKOV3 cell lines in a dose-dependent manner ( Figure 4D). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S3. These data suggest that butein regulates cell fate by modulating the expression of cell cycle and apoptosis proteins.

Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a through Inhibition of IL-6 Signaling
To further elucidate the effect of IL-6 inhibition by butein, the expression of the downstream gene, STAT3, was analyzed by Western blotting of proteins from the A2780 and SKOV3 cell lines after treatment with varying concentrations of butein. No significant change was observed in the total amount of STAT3. However, the phosphorylation of STAT3 was found to be inhibited in a concentration-dependent manner in the butein-treated cells relative to the IL-6 treated cells ( Figure 5A). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S4. Western blot analysis of the nuclear and cytoplasmic protein extracts revealed that butein also induced FoxO3a accumulation in the nucleus and decreased cytoplasmic FoxO3a. Consistent with this, expression of the proliferation-related gene p27 kip1 was also found to be enhanced in the nucleus ( Figure 5B). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S5. These results indicate that butein induces STAT3 inactivation through the inhibition of IL-6 signaling in ovarian cancer and increases FoxO3a and p27 kip1 levels in the nucleus following STAT3 inactivation. Butein induced cycle arrest in ovarian cancer cells, which were stained by PI, and cell cycle distribution was analyzed by flow cytometry. (B) A2780 and SKOV3 cells were treated with various concentrations of butein in triplicate for 48 h to detect cell apoptosis using the FITC Annexin V apoptosis Kit (** p < 0.01 and *** p < 0.001). (C) Butein decreased the expression of Cyclin D1, CDK 4, CDK 6, and increased the expression of p27 kip1 in A2780 and SKOV3 cells. (D) Butein decreased the expression of Bcl-2, Mcl-1, and increased the expression of Bax in A2780 and SKOV3 cells.

Butein Inhibited STAT3 Phosphorylation and Induced Intranuclear Accumulation of FoxO3a through Inhibition of IL-6 Signaling
To further elucidate the effect of IL-6 inhibition by butein, the expression of the downstream gene, STAT3, was analyzed by Western blotting of proteins from the A2780 and SKOV3 cell lines after treatment with varying concentrations of butein. No significant change was observed in the total amount of STAT3. However, the phosphorylation of STAT3 was found to be inhibited in a concentration-dependent manner in the buteintreated cells relative to the IL-6 treated cells ( Figure 5A). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S4. Western blot analysis of the nuclear and cytoplasmic protein extracts revealed that butein also induced FoxO3a accumulation in the nucleus and decreased cytoplasmic FoxO3a. Consistent with this, expression of the proliferation-related gene p27 kip1 was also found to be enhanced in the nucleus ( Figure 5B). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S5. These results indicate that butein induces STAT3 inactivation through the inhibition of IL-6 signaling in ovarian cancer and increases FoxO3a and p27 kip1 levels in the nucleus following STAT3 inactivation.

Butein Increased Protein Expression of FoxO3a and p27 kip1 through Inactivation of STAT3
To elucidate the mechanism of butein's effect on FoxO3a and p27 kip1 , we used siRNAmediated knock-down of STAT3. Based on the Western blot results, siSTAT3-5-which had the highest knock-down efficiency among the siSTAT3 primers-was selected ( Figure 6A). When the ovarian cancer cell line was treated with siSTAT3, the cell growth inhibition was similar to that in the butein-treated group. The inhibitory effect was enhanced when butein and siSTAT3 were administered together ( Figure 6B). Treatment of butein and siSTAT3 collectively in ovarian cancer cell lines inhibited colony formation, while no significant difference was found between groups treated separately with siSTAT3 and butein ( Figure 6C). These data demonstrate that butein exerts antiproliferative effects on ovarian cancer cell lines through STAT3. Additionally, FoxO3a and p27 kip1 protein levels were found to be upregulated upon STAT3 knock-down, as in the butein-treated group ( Figure 6D). Whole membrane and protein expression levels detected by Western blotting are shown in Figure S6. These data show that butein treatment not only mimics the downstream effects of STAT3 suppression but also enhances them, suggesting that butein affects FoxO3a and p27 kip1 through STAT3 inactivation.

Butein Exerts an Antitumor Effect In Vivo on Ovarian Cancer Cells
To test the effect of butein on tumor growth inhibition in vivo, we generated xenograft mice using the A2780 cell line. The butein-treated group showed a significant inhibition of tumor growth compared with the vehicle as well as the siltuximab-treated group ( Figure 7A). The tumor mass also showed a similar trend ( Figure 7B,C). There was no difference in the total mouse weight during the experimental period ( Figure 7D). To evaluate the production of IL-6, IL-1β, and TNF-α in serum in mouse blood, the total amount of IL-6, IL-1β, and TNF-α was normalized to the total amount of vehicle ( Figure 7E). In the vehicle group, tumor cells were closely arranged into complete and atypical structures. In contrast, the group treated with a high concentration of butein displayed a greater degree of tumor cell death, characterized by an incomplete cell membrane, a pyknotic nucleus, and condensed cytoplasm. Supporting the in vitro experimental results, we identified similar changes in protein expression upon butein treatment in vivo ( Figure 7G,H). Buteinmediated inhibition of STAT3 phosphorylation as well as increased nuclear FoxO3a were observed in mouse tumor tissues. Whole membrane and protein expression levels detected by Western blotting are shown in Figure S7. These data suggested that butein treatment inhibited tumor growth in ovarian cancer cells by increasing the nuclear accumulation of FoxO3a through the inhibition of STAT3 phosphorylation in vivo.

Discussion
Ovarian cancer is the most lethal gynecological malignancy, and inflammation has been shown to play a large role in ovarian cancer growth. IL-6 is a cytokine that acts on chronic inflammation as a major tumor-promoting inflammatory mediator. IL-6 has been shown to activate signaling pathways leading to tumor proliferation, the most studied of which are the JAK and STAT3 pathways. Many drugs were found to inhibit IL-6 signaling, including siltuximab and sirukumab [31], although, none of them currently show promising outcomes in ovarian cancer treatment. Therefore, we aimed to discover new small molecule inhibitors of IL-6 signaling and to elucidate their mechanisms of action.
The 14 compounds isolated and identified from Butea monosperma flowers are in accordance with previous studies [21][22][23][24][25][26][27][28][29][30]. Of these, butein shows anti-inflammatory activity and has been shown to be a potential therapeutic agent for the treatment of chronic inflammatory diseases and cancers [12]. Recent evidence suggests that it inhibits the activities of anti-inflammatory cytokines such as IL-6, IL-1β, and TNF-α [13]. However, whether it directly inhibits anti-inflammatory cytokines has not been studied. Using the SPR assay, we confirmed that butein binds to IL-6, IL-6Rα, and gp130 through intermolecular interactions. We further validated that the binding force between butein and IL-6 was higher, and that butein inhibited IL-6 downstream signaling using the HEK-Blue™ IL-6 cell line. Ours is the first study to report that butein binds to IL-6 and inhibits its downstream signaling. Furthermore, our data indicating the low expression of these cytokines in the mouse blood serum following butein treatment supports these previous findings.
Our results showed that survival, migration, and invasion of ovarian cancer cells were inhibited by butein, and that this occurred in a concentration-dependent manner. Furthermore, treatment with increasing concentrations of butein resulted in increased cell cycle arrest as well as increased apoptosis. Further investigation revealed that butein affects the expression levels of cell cycle proteins as well as apoptosis proteins. Along similar lines, previous studies have shown that butein inhibits the activation of various oncogenes through many signaling mechanisms [12,32]. A recent report revealed that butein can exert a chemosensitizing effect through the miR-186-5p-TWIST1 axis, suggesting that butein exerts its chemosensitizing effect, at least in part, through microRNA modulation [33].
Butein is known to exhibit anticancer effects by the inhibition of STAT3, Akt, and PI3K signaling in other cancers as well [9,13,34]. Our in vitro as well as in vivo data suggests that the same molecular pathways are affected by butein via similar mechanisms in ovarian cancer. Additionally, it has been reported that, following IL-6 signaling, phosphorylated STAT3 regulates the nuclear translocation of FoxO3a, leading to increased expression of p27 kip1 in T cells [19], similar to the mechanisms we found in ovarian cancer cells. Furthermore, recent evidence suggests that butein leads to increased p27 kip1 expression by promoting not only the nuclear localization of FoxO3a, but also by enhancing its binding to the p27 kip1 promoter, resulting in cell cycle arrest and thereby inhibiting cell proliferation [18,35]. We found that butein increased the expression of p27 kip1 by regulating the mechanism of STAT3 and FoxO3a in ovarian cancer. According to a recent study, FoxO3a is an important regulator during the development of drug resistance and may show great potential as a novel biomarker for prognostic evaluation and therapeutic targets in cancer patients [36][37][38]. Additionally, FoxO3a exhibits great therapeutic potential due to its essential role in cancer progression, particularly in drug resistance.
In summary, butein from Butea monosperma flower isolates show excellent therapeutic potential in ovarian cancer cells. It has shown anti-IL-6 activity, which induces inactivation of STAT3 through IL-6 binding and nuclear accumulation of FoxO3a in ovarian cancer cells. This suggests that butein, along with current front-line chemotherapy drugs, may represent a promising approach towards ovarian cancer treatment; however, this requires further research.

Surface Plasmon Resonance Assay
The surface plasmon resonance (SPR) assay was measured using the BIAcore T200 model (

Clonogenic Formation Assay
A2780 and SKOV3 cell lines were seeded at approximately 5000 cells/well in 24-well plates. Butein was added to the cell medium at various concentrations (5, 10, and 25 µM) together with control siltuximab (CTNO328; EUSA Pharma, Inc., Hemel Hempstead, UK) for 12 h. After 2 weeks, the colonies formed were fixed with fixer buffer, stained with crystal violet, and imaged with a ChemiDoc imaging system (Bio-Rad, Herles, CA, USA).

Wound-Healing Assay
A2780 and SKOV3 cells were cultured to 90% confluence in 6-well plates. After changing to a serum-free medium, the cell monolayer was scraped to artificially form homogeneous wound with uniform thickness. The drug treatment for each well was carried out in the following manner: siltuximab at 50 µM; butein at 10, 25, and 50 µM. The cells were imaged at 0, 24, and 48 h.

Matrigel Invasion Assay
Matrigel invasion assays were performed using a BioCoat Matrigel Invasion Chamber (BD Bioscience, Bedford, MA, USA). A2780 and SKOV3 cells (50,000 cells) were placed inside the chamber in serum-free medium containing 500 µL of drugs, and 700 µL of medium containing serum were placed outside the chamber. After incubation for 48 h, the infiltrating cells were stained using the Differential Quick Stain Kit (Cat.no. 26096-50, Electron Microscopy Sciences, Hatfield, PA, USA). Cells were imaged with a Slide Scanner (Aperio CS2, Leica Microsystems, Wetzlar, Germany) and the number of invading cells was quantified using Image J.

Flow Cytometric Analysis of Cell Cycle and Apoptosis
A2780 and SKOV3 cell lines were treated with control (siltuximab 10 µM) and butein (5, 10, and 25 µM). After 6 h, the cells were fixed with 70% ethanol for cell cycle analysis and stained with propidium iodide (PI). Cell death was analyzed using the FITC Annexin V Apoptosis Detection kit with 7-AAD (Cat.no. 640922, BioLegend, San Diego, CA, USA). Cells were sorted using a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) and cell cycle and apoptosis were analyzed using BD FACS Diva software version 6.2. Analysis (BD Biosciences, Franklin Lakes, NJ, USA) was performed in triplicate.

Small Interfering RNA (siRNA) Transfection
The siRNA siNC was used as a negative control, and siSTAT3 was used as a target siRNA. The siRNAs used were produced by Genolution (Genolution Pharmaceutical lnc., Seoul, Korea) and were transfected according to the manufacturer's instructions. Target sequences for siRNAs are listed in Table S1.

Western Blotting
After each drug treatment of the cells, the cells were dissolved in 100 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and centrifuged to separate the supernatant containing protein. Isolated protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Aliquots of proteins quantified at 30 ng were boiled at 100 • C in 5× sample buffer, separated on a 12% SDS-polyacrylamide gel and electrophoresed onto polyvinylidene difluoride (PDVF) membranes (Millipore, Billerica, MA, USA). The PDVF membranes were blocked for 1 h at 25 • C with 5% BSA in 1× tris buffered saline containing 0.05% Tween20 (TBST) (Sigma-Aldrich, St. Louis, MO, USA). The membrane was incubated in the primary antibody at 4 • C overnight. PDVF membranes were washed with 1× TBST and incubated with AffiniPure goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. After washing again with TBST buffer, enhanced signals were detected using the SuperSignal™ West Femto Maximum Sensitivity Substrate kit (cat.no. 34094, Thermo Scientific, Rockford, IL, USA). The blot was imaged using the ChemiDoc imaging system (Bio-Rad, Herles, CA, USA), and protein expression was quantified with the ImageJ software (v1.8.0).
The following primary antibodies were used: anti-β-actin (cat.

Mouse Xenografts
Adult BALB/c nude mice aged 5 weeks (n = 5, body weight 17−19 g) were purchased from Orient Bio (Seongnam, Republic of Korea) and reared in aseptic conditions with 55 ± 10% humidity and 25 ± 2 • C temperature under a 12 h/12 h light-dark cycle (Catholic University protocol). All animal experimental work was carried out in compliance with the legal obligations and federal guidelines and legal obligations for the care and maintenance of laboratory animals. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Catholic University of Korea (approval number: CUK-IACUC-2019-026-01). A2780 cell suspension (1 × 10 7 cells/200 µL in 1 × PBS) was injected subcutaneously into the dorsal scapula area of each mouse. The tumor developed 14 days after implantation to a size of approximately 150 mm 3 . The mice were then divided randomly into vehicle control (5% DMSO), siltuximab (10 mg/kg), and butein (2 or 4 mg/kg) groups. All drugs were dissolved in 0.05% carboxymethylcellulose sodium salt (CMC) (Sigma-Aldrich, St. Louis, MO, USA) and injected intraperitoneally five times a week for three weeks. The tumor size was measured using calipers once every 2 days. The tumor volume was calculated using a simplified equation (length × width 2 × 0.5). Each tumor was harvested 22 days post-treatment.

Hematoxylin and Eosin (H&E) Staining in Mouse Tumor Tissues
After the experiment was completed, mice were sacrificed and tumor tissues were collected. Collected tumor tissues were fixed in 4% paraformaldehyde for 24 h. Fixed tissues were washed in 1 × PBS and embedded in paraffin. The paraffin block was sectioned at a thickness of 2 µm and sections placed on a slide glass. After hematoxylin and eosin staining, the sections were dehydrated, deparaffinized with mineral oil, and a cover slip was added.

Data Analysis
All data, except that from the cell cycle analysis, were evaluated using GraphPad Prism software (version 7.00; GraphPad Software, Inc., San Diego, CA, USA). Results are presented as mean ± standard deviation (SD). Following two-way ANOVA, Bonferroni's post hoc tests were performed with GraphPad Prism 7.0.

Conclusions
Based on our findings, we can conclude that butein from Butea monosperma flower isolates inhibits ovarian cancer growth by binding to IL-6 and suppressing its activity, which results in inactivation of STAT3 and nuclear accumulation of FoxO3a and p27 kip1, thereby limiting tumor growth. Our work highlights butein as a promising therapeutic agent for ovarian cancer treatment.