Anti-metastatic Effects of Baicalein by Targeting STAT3 Activity in Breast Cancer Cells

Abstract

Signal transducer and activator of transcription 3 (STAT3) is considered a potential target for cancer treatment because of its relationship with cellular transformation and tumor initiation and progression. In this study, we aimed to identify a new anti-cancer drug candidate from natural products by targeting STAT3 activity. Using STAT3-luciferase reporter cell line, we screened the chemical library of natural products and found that baicalein, a flavone isolated from the roots of Scutelleria baicalensis, strongly suppressed STAT3 activity in breast cancer cells. Baicalein inhibited STAT3 transcriptional activity and its phosphorylation, and further exhibited anti-proliferative effects in breast cancer cells. Moreover, baicalein suppressed the production of interleukin (IL)-6 and the metastatic potential of breast cancer cells both in vitro and in vivo. Collectively, our study suggests baicalein as an attractive phytochemical compound for reducing metastatic potential of breast cancer cells by regulating STAT3 activity.

INTRODUCTION

The inflammatory tumor microenvironment (TME) is known to promote oncogenic transformation and malignant progression through genetic or epigenetic mechanisms.¹⁾ This pro-tumor role of cancer-associated inflammation is mediated by a variety of inflammatory immune cells through their production of cytokines and chemokines.¹⁾ Among these pro-inflammatory cytokines, members of the interleukin (IL)-6 family are known to play essential roles in tumor progression through the activation of signal transducer and activator of transcription 3 (STAT3).^2–7) STAT3 is a latent cytosolic transcription factor activated by the phosphorylation of tyrosine705 in its SH2 domain.⁸⁾ Binding of IL-6 to its receptor leads to the activation of Janus kinase (JAK) family of protein tyrosine kinases, which results in the phosphorylation and activation of STAT3.⁹⁾ In addition to STAT3 activation by IL-6, the over-activation of STAT3 plays an important role in different type of cancer, including breast cancers.¹⁰⁾ It plays an essential role in selectively inducing and maintaining a pro-carcinogenic inflammatory microenvironment, both at the initiation of malignant transformation and during cancer progression^11–18); therefore, STAT3 is considered a promising target for cancer therapy.

Natural products are used for medical treatment and as valuable resources for drug discovery and development. Baicalein is an active constituent of the dried root of Scutellaria baicalensis Georgi, which is widely used as a traditional medicine in many countries. Baicalein is a flavonoid compound and has a numerous biological activities.¹⁹⁾ In this study, we identified that baicalein suppresses STAT3 activity and has anti-metastatic effects in the breast cancer cell lines.

MATERIALS AND METHODS

Reagents

Baicalein and baicalin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). JSI-124 (cucurbitacin I hydrate) was purchased from Sigma Aldrich (St. Louis, MO, U.S.A.). These compounds were dissolved in dimethyl sulfoxide (DMSO, FUJIFILM Wako Pure Chemical Corporation) and stored at −20 °C. The stock solution diluted in the relevant assay medium and 0.1% DMSO served as vehicle controls. D-Luciferin was obtained from Promega (Madison, WI, U.S.A.). The primary antibodies against STAT3 and p-STAT3 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.), and the antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Cells

The murine breast cancer cell line 4T1 (ATC C) and human breast cancer cell line MDA-MB-231 (ATC C) were maintained in RPMI-1640 medium containing 10% fetal bovine serum 1% antibiotics (penicillin and streptomycin) in 5% CO₂ at 37 °C. 4T1 cells stably expressing an STAT3-mediated luciferase gene (4T1-STAT3-C4) were established previously.²⁰⁾ Briefly, the tandem-repeated binding sites for STAT3 were subcloned into the pGL4.26 vector (Promega) and 4T1 cells were transfected with the reporter plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.). The cells were selected with Hygromycin B (100 µg/mL) and cloned by limiting dilution.

Cell Viability Assay

Cell viability was quantified using the WST-8 Cell Counting kit (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer’s instructions. Cells (2 × 10⁴ cells/well) were seeded onto 96-well plates and co-cultured with test compounds. After the incubation with test compounds (48 h), WST-8 reagent was added and the absorbance at 450/620 nm was measured using a microplate reader.

Western Blotting

Cells (10⁶ cells/well) were treated with tested compounds varying concentrations for 24 h. The treated cells were trypsinized and collected by adding phosphate buffered saline (PBS) and centrifuged for 10 min at 2000 rpm and 4 °C. Then, the supernatant was discarded and the cells were lysed in whole-cell lysis buffer (25 mmol/L N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.7, 300 mmol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 20 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 10 mg/mL aprotinin, and 10 mg/mL leupeptin). Cell lysates were subjected to electrophoresis by 7.5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred to Immobilon-P nylon membranes (Millipore, Bedford, MA, U.S.A.). The membranes were treated with Block Ace (Dainippon Pharmaceutical, Co., Ltd., Osaka, Japan) for at least 2 h, and probed with the indicated primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1 : 1000 dilutions). Bands were visualized using enhanced chemiluminescence (ECL) reagents (Amersham Bioscience, Piscataway, NJ, U.S.A.). Primary antibodies used (at a dilution of 1 : 1000) were specific to STAT3 (79D7, #4904), p-STAT3 (S727, #9134, Cell Signaling Technology) and also specific to β-actin (C4, sc-47778, Santa Cruz).

Wound Healing Assay

Cells were seeded on 24-well plates (2 × 10⁵cells/well) and when they reached confluency, the cell monolayer was scraped by sterile 200-µL pipette tips, and fresh medium was added containing different concentrations of baicalein and JSI-124. After a 24-h incubation, cells were fixed and photographed. Images were acquired using inverted microscope and the percentage of inhibition of migrated cells was expressed using 100% as the value of the untreated group.

Transwell Migration Assay

A total of 10⁶ cells/well were pre-treated with tested compounds at varying concentrations for 24 h. A 100-µL aliquot of cell suspension containing 3 × 10⁴ cells were added to Transwell cell culture insert with filter (8 µm pore size; Whatman Japan K K, Tokyo, Japan) and incubated for 6 to 8 h. Non-migrated cells in the upper chamber was discarded using a cotton swab. The migrated cells were fixed in methanol and stained with hematoxylin and eosin. Migrated cells in five randomly selected fields were counted and photographed under an inverted microscope. The results were expressed as the percentage relative migrated cells compared with the untreated group.

Enzyme-Linked Immunosorbent Assay (ELISA) Assay

Cells seeded in 6-well plates were pre-treated with test compounds for 24 h and cell-free culture supernatants were collected. The amounts of cytokines in the supernatants for IL-6 were measured using a specific ELISA kit (Biolegend) according to the manufacturer’s instructions. Three replicates were carried out for each of the different treatments.

Experimental Metastasis Model

BALB/c mice (6-week old) were purchased from Japan SLC Inc. (Hamamatsu, Japan). All experiments were approved (A2016INM-7, A2019INM-5) and performed according to the guidelines of the Care and Use of Laboratory Animals of University of Toyama. Cells were inoculated intravenously (6 × 10⁵ cells/100-µL PBS/mouse) into mice. D-Luciferin (3 mg, In Vivo Glo, Promega) was intraperitoneally injected into mice at 24 h after the tumor inoculation. After 10 min, the lungs were removed for bioluminescence assay using an in vivo imaging system (IVIS Lumina II, Caliper Life Sciences, Hopkinton, MA, U.S.A.).

Statistical Analysis

All data are expressed as the mean ± standard deviation (S.D.) of at least two or three independent experiments unless otherwise stated. Significance was analyzed using the Mann–Whitney U-test or one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests. p < 0.05 was considered significant.

RESULTS

Baicalein to Suppressed STAT3 Activity in Breast Cancer Cells

In order to identify a novel anti-cancer agent from natural products by targeting STAT3 activity, we used 4T1-STAT3-C4 cells expressing the firefly luciferase gene under the control of the STAT3 reporter. By screening the INMUT natural compounds library (96 natural compounds shown in Table 1, http://www.inm.u-toyama.ac.jp/jp/collabo/h28_collabo_02.html) using 4T1-STAT3-C4 cells, we found 5 compounds, bisdemethoxy curcumin, costunolide, alisol A, curcumin, and baicalein, as potential candidates that suppress STAT3 activity without largely affecting cell viability (Fig. 1A). Among these natural compounds, baicalein (at 10 µM) most strongly suppressed STAT3 activity in 4T1 cells, similar to the STAT3 inhibitor JSI-124 (at 10 nM, Fig. 1B).

Table 1. List of Natural Compounds in Institute of Natural Medicine, University of Toyama (INMUT) Wakanyaku Library

No.	Compound name	No.	Compoud name
1	Aconitine	49	Ginsenoside-Rb1
2	Albiflorin	50	Ginsenoside-Rc
3	Alisol A	51	Ginsenoside-Rd
4	Alisol B	52	Ginsenoside-Re
5	Alkannin	53	Ginsenoside-Rg1
6	Amygdalin	54	Glabridin
7	Arbutin	55	Glycyrrhizic acid
8	Astragaloside IV	56	Gomisin A
9	Atractylenolide III	57	Gomisin N
10	Atractylodin	58	Hesperidin
11	Atropine sulfate	59	Hirsutine
12	Aucubin	60	Honokiol
13	Baicalein	61	Hypaconitine
14	Baicalin	62	Icariin
15	Barbaloin	63	Isofraxidine
16	Benzoylmesaconine hydrochloride	64	Isorhynchophylline
17	Berberine chloride	65	(Z)-Ligustilide
18	Bergenin	66	Limonin
19	Bisdemethoxycurcumin	67	Liquiritin
20	Bufalin	68	Loganin
21	Bufotalin	69	Luteolin
22	Capillarisin	70	Magnolol
23	(E)-Capsaicin	71	Mesaconitine
24	Catalpol	72	Naringin
25	(E)-Chlorogenic acid	73	Nodakenin
26	(E)-Cinnamic acid	74	Osthole
27	Cinobufagin	75	Oxymatrine
28	Cinobufotalin	76	Paeoniflorin
29	Coptisine chloride	77	Paeonol
30	Corydaline	78	Palmatine chloride
31	Costunolide	79	Perillaldehyde
32	Curcumin	80	Praeruptorin A
33	Dehydrocorydaline nitrate	81	Puerarin
34	Dehydrocostuslactone	82	Rhynchophylline
35	demethoxycurcumine	83	Rosmarinic acid
36	Dihydrocapsaicin	84	Saikosaponin a
37	Dimethylesculetin	85	Saikosaponin b2
38	Eleutheroside B	86	Saikosaponin c
39	(−)-Epigallocatechin gallate	87	Saikosaponin d
40	Epihesperidin	88	Schizandrin
41	Ergosterol	89	Sennoside A
42	β-Eudesmol	90	Sennoside B
43	Evodiamine	91	Shikonin
44	(E)-Ferulic acid	92	[6]-Shogaol
45	Geniposide	93	Sinomenine
46	Geniposidic acid	94	Swertiamarin
47	Gentiopicroside	95	Timosaponin A-III
48	[6]-Gingerol	96	Wogonin

Fig. 1. Identification of Baicalein as a Suppressor of STAT3 Activity in Murine Cancer Cells

(A) 4T1STAT3C4 cells (2 × 10⁴ cells/well) were seeded onto 96-well plates and co-cultured with 96 natural products (at 50 µM, Table 1) for 24 h. The inhibitory effects against STAT3 transcriptional activity were measured as %STAT3 inhibition to the untreated control. Cell viability was assessed by WST-8 assay and shown as % inhibition to the untreated control.(B) The compounds in the square of panel A were subjected to the second test under the same conditions except for the dose (10 µM). JSI-124 (10 nM) was used as a positive control. The data are presented as the mean ±S.D. (* p < 0.01).

To further assess whether baicalein and its glycoside baicalin have similar STAT3 inhibition activity, we evaluated both compounds in the STAT3 reporter assay. As baicalein demonstrated much stronger STAT3 inhibition than baicalin (Fig. 2B), the STAT3 inhibitory activity of baicalein depends on its flavonoid structure. Moreover, baicalein inhibited STAT3 activity in 4T1 cells as early as 1 h after treatment and inhibition was maintained 24 h thereafter (Fig. 2C). In order to clarify the mechanism by which baicalein inhibits the transcriptional activity of STAT3, we next examined the expression of STAT3 and its phosphorylation status upon baicalein treatment. As shown in Fig. 3, the expression of phosphorylated STAT3, but not that of STAT3, was inhibited by baicalein; therefore, baicalein likely inhibit STAT3 transcriptional activity inhibiting its phosphorylation.

Fig. 2. Baicalein Suppresses STAT3 Transcriptional Activity in 4T1 Breast Cancer Cells

(A) Chemical structure of baicalein and baicalin. (B)4T1-STAT3-C4 cells (2 × 10⁴ cells/well) were seeded onto 96-well plates and co-cultured with baicalein or baicalin at different doses (0.01, 0.1, 1, 10, 100 µM) for 24 h. STAT3 transcriptional activity was evaluated by luciferase gene reporter assay. (C) 4T1-STAT3-C4 cells (2 × 10⁴ cells/well) were seeded onto 96-well plates and co-cultured with 10 µM baicalein for 24 h. STAT3 transcriptional activity was examined at the indicated times by measuring the luminescence. The data are presented as the mean ±S.D. (* p < 0.01).

Fig. 3. Effects of Baicalein on STAT3 Phosphorylation in 4T1 Cells

4T1 cells (1 × 10⁶ cells/well) were seeded in 6-well plates and treated with baicalein (0.01, 0.1, 1, 10 µM). After 24 h cells were harvested, and equal amounts of protein in cell lysates were analyzed by Western blotting. The actin protein levels were used to confirm that equal amounts of protein were subjected to electrophoresis.

Anti-metastatic Properties of Baicalein against Breast Cancer Cells

STAT3 is a key transcription factor that responds to the pro-inflammatory cytokine IL-6, which is produced by different types of cancer cells. It plays an important role as a oncogenic molecule to regulate tumor initiation, progression, and metastasis. Therefore, to examine the biological significance of baicalein as a potential anti-cancer agent, we first evaluated its effects on 4T1 and MDA-MB-231 cell growth. As shown in Fig. 4, a high dose of baicalein (100 µM) showed its cytotoxic effect on 4T1 cells (Fig. 4A) or MDA-MB-231 (Fig. 4B) at 24 h and a low dose (10 µM) had anti-proliferative effect at 48 h. These results demonstrated the direct anti-tumor activity of baicalein against 4T1 and MDA-MB-231 cells. To further examine the effects of baicalein on the production of IL-6 from 4T1 cells, we measured the IL-6 level in the cell culture supernatant of 4T1 cells with or without baicalein. As shown in Fig. 5, baicalein inhibited IL-6 production in a dose-dependent manner. As such IL-6 inhibition was also seen in the JSI-124 treatment, the suppression of STAT3 activity may be one of the underlying mechanisms of baicalein activity.

Fig. 4. Effects of Baicalein on the Growth of 4T1 and MDA-MB-231 Cells

(A) 4T1 cells or (B) MDA-MB-231 cells (2 × 10⁴ cells/well) were treated with baicalein for 24 and 48 h at the indicated dose, and cell viability was evaluated as the relative cell viability to untreated control. The data are presented as the mean ±S.D. (* p < 0.01).

Fig. 5. Effects of Baicalein on IL-6 Production by 4T1 Cells

4T1 cells (2 × 10⁵ cells/well) were seeded in 24-well plates, and treated with baicalein and JSI-124. After 24 h, culture supernatants were collected and cytokine IL-6 was quantified using the ELISA kit according to the manufacturer’s instructions. The data are presented as the mean ±S.D. (* p < 0.01).

As STAT3 activity is related to the distant metastasis of cancer cells, we next assessed the effects of baicalein on the cellular migration of 4T1 cells. Baicalein treatment, similar to JSI-124 treatment, inhibited the migration of 4T1 cells in a dose-dependent manner in the wound-healing assay (Figs. 6A, C) or transwell migration assay (Figs. 6B, D). Lastly, we examined the anti-metastatic effect of baicalein using an experimental metastasis model of 4T1 breast cancer. 4T1 cells were treated with or without 10 µM baicalein, and then intravenously inoculated into mice to investigate whether baicalein impairs the metastatic capacity of 4T1 cells in vivo. As shown in Fig. 7, baicalein significantly inhibited the formation of lung metastasis by 4T1 breast cancer cells. Collectively, our study demonstrated the biological significance of baicalein to inhibit the pro-tumor function of STAT3 in breast cancer cells and its ability to control their metastatic potential.

Fig. 6. Effects of Baicalein on the Migratory Activity of 4T1 Cells

(A, C) 4T1 cells (2 × 10⁵ cells/well) were seeded in 24-well plate and incubated until confluency. A vertical scratch was made on the cell monolayer using a 200-µL pipette tip. After washing the media, the cells were treated with baicalein and JSI-124, and a snapshot was taken using an inverted microscope. After a 24-h incubation, snapshots were taken again. The black lines indicate the section occupied by the initial scraping. (B, D) 4T1 cells (10⁶ cells/well) were seeded in 6-well plates and treated with baicalein and JSI-124. After 24 h, the cells (3 × 10⁴ cells/chamber) were seeded in transwell chambers and incubated for 6–8 h. Snapshots were taken using an inverted microscope. The migratory activity was measured as the relative % of migrated cells to untreated cells. The data are presented as the mean ±S.D. (* p < 0.05). (Color figure can be accessed in the online version.)

Fig. 7. Anti-metastatic Effects of Baicalein on 4T1 Cells

4T1 cells were pretreated with 10 µM baicalein. After a 24-h incubation, cells were inoculated intravenously (6 × 10⁵cells/100 µL of PBS/mouse) into mice. Mice were sacrificed 24 h after the tumor inoculation and lungs were removed for bioluminescent assay using an in vivo imaging system. The data are presented as the mean luminescence ±S.D. (n = 3) * p < 0.01 compared with the untreated control. (Color figure can be accessed in the online version.)

DISCUSSION

Breast cancer is highly malignant because of its aggressive metastatic potential. In general, metastasis is one of the key factors for the survival of cancer patients; therefore, novel potential drug candidates are needed to prevent tumor metastasis. In this study, we identified baicalein as a promising natural compound that has anti-metastatic activity, possibly through the inhibition of STAT3 activity in breast cancer cells.

Flavones, such as baicalein and baicalin, are the major bioactive compounds obtained from the dried roots of Scutelleria baicalensis Georgi, a species of flowering plant in the family Lamiaceae. Baicalein and baicalin were reported to have numerous pharmacological actions, including anti-cancer activity.^21–26) STAT proteins have dual roles: they transduce signals through the cytoplasm and function as transcription factors in the nucleus. Although some STAT proteins, such as STAT1, increase anti-tumor immunity, STAT3 is known to be involved in cancer-promoting inflammation. The activation of STAT3 increases tumor cell proliferation, survival, and invasion, while suppressing anti-tumor immunity.²⁷⁾ STAT3 can be activated by inflammatory mediators, such as cytokines and chemokines, as well as by oncogenic proteins, and its target genes are involved in multiple steps of metastasis.^27–29) In particular, the pro-inflammatory cytokine IL-6 drives cancer cell proliferation, survival and metastasis through the activation of the STAT3 pathway while also suppress the anti-tumor immune responses.³⁰⁾ There are several reports regarding the anti-inflammatory effect of baicalein and their relationship with STAT3 inhibition in different types of cells,^22,31–33) including hematological malignancy.³⁴⁾ Of note, Ke et al. recently reported that baicalein and baicalin decreased STAT3 activity to downregulate interferon-γ (IFN-γ)-induced programmed cell death-ligand 1 (PD-L1) expression and restored anti-tumor T cell activity.³⁵⁾ However, we newly demonstrated the anti-metastatic action of baicalein through the inhibition of cancer-intrinsic STAT3 activity in breast cancer cells. Our study suggests baicalein as a potential therapeutic agent against breast cancer growth and metastasis through the inhibition of STAT3 activity.

Acknowledgments

This study was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas (17H06398), The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Y.H.) and the Cooperative Research Project from the Institute of Natural Medicine, University of Toyama. G. D. S. was supported by a Toyama Prefectural Scholarship for ASEAN Study Abroad Students and K.M. was supported by a scholarship from the Shoshisha Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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