Betavulgarin Isolated from Sugar Beet (Beta vulgaris) Suppresses Breast Cancer Stem Cells through Stat3 Signaling

Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24− subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.


Isolation of a BCSC Inhibitor from Beta vulgaris
To screen and purify BCSC inhibitors from Beta vulgaris rubra, a mammosphere formation assay using MDA-MB-231 cells was performed, and a BCSC inhibitor was purified using methanol extracts of Beta vulgaris rubra generated by ethyl acetate extraction, silica gel filtration, Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) chromatography, preparatory thin-layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) ( Figure 1A). The purified compound suppressed BCSC formation ( Figure 1B) and was analyzed using HPLC ( Figure 1C). The molecule identified using nuclear magnetic resonance (NMR) data was determined to be betavulgarin ( Figure 2).

Betavulgarin Inhibits Breast Cancer Cell Growth and Mammosphere Formation
To ascertain whether betavulgarin has an inhibitory effect on breast cancer growth, we assessed the inhibitory effect of betavulgarin at increased concentrations in MDA-MB-231 and MCF-7 cells. Betavulgarin had an antiproliferative effect on the MDA-MB-231 and MCF-7 cells at ≥ 100 µM and 50 µM, after 24 hrs of treatment ( Figure 3A,B). To confirm whether betavulgarin can suppress mammosphere formation, it was added to mammospheres derived from MDA-MB-231 or MCF-7 cells. As shown in Figure 3C,D, betavulgarin decreased not only the sphere numbers of MDA-MB-231 and MCF-7 cells by 78% and 68%, respectively, but also the sizes of the mammospheres. In addition, betavulgarin inhibited migration and colony formation of MDA-MB-231 and MCF-7 cells ( Figure 3E,F). We showed that betavulgarin inhibits mammosphere formation, migration, colony formation, and breast cancer growth.

Betavulgarin Inhibits Breast Cancer Cell Growth and Mammosphere Formation
To ascertain whether betavulgarin has an inhibitory effect on breast cancer growth, we assessed the inhibitory effect of betavulgarin at increased concentrations in MDA-MB-231 and MCF-7 cells. Assay for mammosphere formation inhibition using beet extracts. Mammospheres were incubated with beet extracts or DMSO. MDA-MB-231 cells were treated with beet extracts or DMSO in BCSC culture medium for seven days. Images were obtained by microscopy at 10× magnification and show representative mammospheres (scale bar=100 µm). (C) HPLC chromatogram of the inhibitor isolated from beet extracts.
. Identification of a breast cancer stem cell (BCSC) inhibitor isolated from beet extr sphere formation assay. (A) Isolation procedure for the mammosphere formation y for mammosphere formation inhibition using beet extracts. Mammospheres were i t extracts or DMSO. MDA-MB-231 cells were treated with beet extracts or DMSO edium for seven days. Images were obtained by microscopy at 10× magnification a tative mammospheres (scale bar=100 µm). (C) HPLC chromatogram of the inhibito t extracts.
. Molecular structure of the BCSC inhibitor isolated from beet extracts. Molecular ulgarin. mammosphere formation, it was added to mammospheres derived from MDA-MB-231 or MCF-7 cells. As shown in Figure 3C and D, betavulgarin decreased not only the sphere numbers of MDA-MB-231 and MCF-7 cells by 78% and 68%, respectively, but also the sizes of the mammospheres. In addition, betavulgarin inhibited migration and colony formation of MDA-MB-231 and MCF-7 cells ( Figure 3E and F). We showed that betavulgarin inhibits mammosphere formation, migration, colony formation, and breast cancer growth.

Betavulgarin Inhibits the Nuclear Translocation of Stat3 in BCSCs
To examine the biochemical mechanism underlying the suppression of mammosphere formation by betavulgarin, we examined the total protein levels of Stat3, p-Stat3, and NF-κB p65. Our data showed that the levels of Stat3 and p-Stat3 were significantly decreased following betavulgarin treatment ( Figure 5A). The levels of nuclear Stat3, p-Stat3, and p65 were determined, and these results showed that the nuclear Stat3 and p-Stat3 levels were significantly reduced by betavulgarin but those of p65 were not ( Figure 5B). Furthermore, an immunofluorescence (IF) assay assessing pStat3 was performed in MDA-MB-231 cells, and the level of nuclear pStat3 in betavulgarin-treated cancer cells was lower than that in control cells ( Figure 5C). Moreover, we examined the direct binding of a Stat3 binding probe to Stat3 proteins under betavulgarin treatment, using an electrophoretic mobility shift assay (EMSA) ( Figure 5D). We examined nuclear Stat3-specific DNA binding using an Infrared Dye (IRDye)-labeled Stat3 probe that bound to Stat3 proteins under betavulgarin treatment. Our data showed that the amounts of nuclear Stat3 proteins bound to the IRDye-labeled Stat3 probe (indicated by arrow) were significantly decreased by betavulgarin treatment ( Figure 5D, line 3). The specific binding of the Stat3 proteins/probe was confirmed using a self-competitor ( Figure 5D, line 4) and a mutated Stat3 oligo ( Figure 5D, line 5). Recently, it was reported that Stat3 protein binds to the promoter region of the SOX2 gene and increases SOX2 transcription. Stat3/SOX2 regulates the selfrenewal of lung CSCs [17][18][19][20]. After betavulgarin treatment, we checked the Sox2 level because the Stat3 dimer activates the Sox2 gene. Our data showed that betavulgarin decreased the transcript and

Betavulgarin Inhibits the Nuclear Translocation of Stat3 in BCSCs
To examine the biochemical mechanism underlying the suppression of mammosphere formation by betavulgarin, we examined the total protein levels of Stat3, p-Stat3, and NF-κB p65. Our data showed that the levels of Stat3 and p-Stat3 were significantly decreased following betavulgarin treatment ( Figure 5A). The levels of nuclear Stat3, p-Stat3, and p65 were determined, and these results showed that the nuclear Stat3 and p-Stat3 levels were significantly reduced by betavulgarin but those of p65 were not ( Figure 5B). Furthermore, an immunofluorescence (IF) assay assessing pStat3 was performed in MDA-MB-231 cells, and the level of nuclear pStat3 in betavulgarin-treated cancer cells was lower than that in control cells ( Figure 5C). Moreover, we examined the direct binding of a Stat3 binding probe to Stat3 proteins under betavulgarin treatment, using an electrophoretic mobility shift assay (EMSA) ( Figure 5D). We examined nuclear Stat3-specific DNA binding using an Infrared Dye (IRDye)-labeled Stat3 probe that bound to Stat3 proteins under betavulgarin treatment. Our data showed that the amounts of nuclear Stat3 proteins bound to the IRDye-labeled Stat3 probe (indicated by arrow) were significantly decreased by betavulgarin treatment ( Figure 5D, line 3). The specific binding of the Stat3 proteins/probe was confirmed using a self-competitor ( Figure 5D, line 4) and a mutated Stat3 oligo ( Figure 5D, line 5). Recently, it was reported that Stat3 protein binds to the promoter region of the SOX2 gene and increases SOX2 transcription. Stat3/SOX2 regulates the self-renewal of lung CSCs [17][18][19][20]. After betavulgarin treatment, we checked the Sox2 level because the Stat3 dimer activates the Sox2 gene. Our data showed that betavulgarin decreased the transcript and protein levels of Sox2 ( Figure 5E). Our data showed that Stat3/Sox2 signaling was important in mammosphere formation.

Betavulgarin Inhibits the mRNA Levels of BCSC-Specific Marker Genes and Mammosphere Growth
To examine whether betavulgarin reduced the mRNA levels of BCSC marker genes, we determined the mRNA levels of these genes. Betavulgarin reduced the transcriptional levels of the BCSC marker genes ( Figure 6A). To check whether betavulgarin decreased mammosphere growth, we cultured mammospheres with betavulgarin and counted the number of mammosphere cancer cells. Betavulgarin increased cell death and reduced mammosphere growth ( Figure 6B). protein levels of Sox2 ( Figure 5E). Our data showed that Stat3/Sox2 signaling was important in mammosphere formation. cells. Betavulgarin increased cell death and reduced mammosphere growth ( Figure 6B).

Discussion
Red beetroot (Beta vulgaris var. rubra L.) contains many bioactive compounds, including anthocyanin, betacyanin, folic acid, phenolic compounds, ascorbic acid, flavonoids, vitamin C, and other biologically active components. The most important bioactive phytochemicals in red beetroot are betalains, a class of tyrosine-derived pigments obtained from betalamic acid, whose members are grouped into yellow betaxanthins and red betacyanins. Red dye E162 extract from beetroot is approved for use in the food industry by the European Food Safety Authority. Betalains have been demonstrated to have strong free radical scavenging, antioxidant [5,21,22], and anti-inflammatory activities [23,24]. In this report, we isolated a BCSC inhibitor, betavulgarin, based on activity-guided fractionation. Betavulgarin was reported to be a fungus infection response molecule and an antifungal molecule in beetroot [25]. For the first time, we report that betavulgarin inhibits BCSCs.
Breast cancer is the most frequent cancer among women [8]. Breast cancer is a systemic disease characterized by early tumor cell dissemination and displays a high degree of intratumor

Discussion
Red beetroot (Beta vulgaris var. rubra L.) contains many bioactive compounds, including anthocyanin, betacyanin, folic acid, phenolic compounds, ascorbic acid, flavonoids, vitamin C, and other biologically active components. The most important bioactive phytochemicals in red beetroot are betalains, a class of tyrosine-derived pigments obtained from betalamic acid, whose members are grouped into yellow betaxanthins and red betacyanins. Red dye E162 extract from beetroot is approved for use in the food industry by the European Food Safety Authority. Betalains have been demonstrated to have strong free radical scavenging, antioxidant [5,21,22], and anti-inflammatory activities [23,24]. In this report, we isolated a BCSC inhibitor, betavulgarin, based on activity-guided fractionation. Betavulgarin was reported to be a fungus infection response molecule and an antifungal molecule in beetroot [25]. For the first time, we report that betavulgarin inhibits BCSCs.
Breast cancer is the most frequent cancer among women [8]. Breast cancer is a systemic disease characterized by early tumor cell dissemination and displays a high degree of intratumor heterogeneity that is important for therapeutic resistance, recurrence, and tumor progression [26,27]. Recently, a BCSC model was proposed and has received increasing interest in the field. CSCs are characterized by the common features of stem cells, including static behaviors, self-renewal, and differentiation.
Achieving efficacious breast cancer treatment is challenging because of the existence of BCSCs. Numerous pathways and factors that could be targeted to inhibit BCSCs were identified. Our results showed that betavulgarin inhibits the proliferation of MDA-MB-231 and MCF-7 cells ( Figure 3A,B) and the size and number of mammospheres derived from MDA-MB-231 or MCF-7 cells ( Figure 3C,D). To address changes in the diverse biological properties of breast cancer cells under betavulgarin treatment, cell migration and colony formation were tested in the context of betavulgarin treatment. Our results showed that betavulgarin inhibits the migration and colony formation of human breast cancer cells ( Figure 3E,F). Additionally, betavulgarin reduced the size of the CD44 + /CD24 − subpopulation in breast cancer cells (Figure 4). It is known that BCSCs are substantially regulated by a multitude of signaling pathways and transcription factors (such as Notch, Hedgehog, Wnt pathways, NF-kB, and Stat3), and that targeting these pathways represents a potential therapeutic approach [9]. In this regard, we explored the role of betavulgarin in the inhibition of BCSCs. Interestingly, the expression levels of Stat3 and p-Stat3 were downregulated by betavulgarin, as was the nuclear localization of Stat3 ( Figure 5). It was reported that natural products such as quercetin, apigenin, oroxylin A (flavones), butein (chalcone), piperlongumine, and caffeic acid (hydoxycinnamic acid) act as stat3 inhibitors [28]. Betavulgarin belongs to isoflavone and might be a small-molecule inhibitor of Stat3 because of a similar structure of flavone. The activation of several transcriptional factors related to embryonic stem cell growth and differentiation, such as sex determining region Y (SRY)-box 2 (SOX2), could explain the enhanced stemness of BCSCs, compared to that of non-BCSCs [9]. One key transcription factor regulating SOX2 expression is Stat3, which directly binds to the promoter of SOX2 [29]. Subsequently, after treatment with betavulgarin, the mRNA transcription and protein expression of SOX2 were assessed, and the results showed that betavulgarin inhibited SOX2 through Stat3 inhibition ( Figure 5). Betavulgarin reduced the transcriptional levels of the C-Myc, Nanog, and Oct4 genes and decreased mammosphere growth ( Figure 6). Our data suggest that betavulgarin, which targets Sox2/Stat3 signaling, might be used as an anti-cancer agent.

Chemical and Reagents
Silica gel 60 and TLC plates were purchased from Merck (Darmstadt, Germany), and Sephadex LH-20 was obtained from Pharmacia (Uppsala, Sweden). Cell viability was measured using the EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Korea). Other compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Plant Material
A sample of beet was obtained from verified market sources (Seogwipo, Jeju, Korea). The beets were washed and freeze-dried, and the dried beet was ground. A voucher specimen (No. 2018_010) was deposited in the Department of Biomaterial, Jeju National University (Jeju-Si, Korea).

Isolation of a Mammosphere Formation Inhibitor form Beat
The ground samples of beet were extracted with methanol. The isolation method is summarized in Figure 1A. The beet powder was solubilized with 10 L of methanol. The methanol extracts were concentrated and mixed with equal volumes of water, and the methanol part was evaporated. The water-suspended part was extracted with equal volumes of ethyl acetate. The solubilized ethyl acetate-concentrated part was loaded onto a silica gel column (3 × 35 cm) and eluted with a solvent (chloroform-methanol, 10:1) ( Figure S1). Five fractions were divided and assayed by evaluating mammosphere formation. The #2 fraction potentially suppressed mammosphere formation. The #2 fraction was loaded onto a Sephadex LH-20 open column (2.5 × 30 cm) and fractionated into four fractions ( Figure S2). The four fractions were further fractionated and analyzed by evaluating the mammosphere formation. Part #4 showed inhibition of mammosphere formation. Part #4 was isolated using preparatory TLC (glass plate; 20 × 20 cm) and developed in a TLC chamber. Individual bands were separated, and each fraction was assayed by evaluating the mammosphere formation ( Figure S3). The #1 fraction was loaded onto a Shimadzu HPLC instrument (Shimadzu, Tokyo, Japan). HPLC was performed with an ODS 10 × 250-mm column (flow rate; 2 mL/min). For elution, the acetonitrile proportion was initially set at 30%, increased to 60% at 20 min, and finally increased to 100% at 30 min ( Figure S4).

Structure Analysis of the Purified Compound
The chemical structure of the compound was determined by ESI-mass spectrometry and NMR spectroscopy measurements. The molecular weight was estimated to be 312 by ESI-mass spectrometry, which showed a quasi-molecular ion peak at m/z 313.3 [M + H] + in the positive mode ( Figure S5). The 1 H NMR spectrum measured in CDCl 3 exhibited signals due to a hydroxyl proton at δ 9.02, and four aromatic methine protons at δ 7.32, 7.09, 7.07, and 6.93, which could be attributed to a 1,2-disubstituted benzene ring; two aromatic singlet methines at δ 7.90 and 6.70; a dioxymethylene at δ 6.10; and a methoxy group at δ 4.11. In the 13 Figure S6). All proton-bearing carbons were assigned by the HMQC spectrum, and the 1 H-1 H COSY spectrum revealed a partial structure of 1,2-disubstituted benzene (Figures S7 and S8). Further structural elucidation was performed with the aid of the HMBC spectrum, which showed long-range correlations from the methine proton at δ 7.90 to the carbons at δ 178.7, 154.7, 125.7, and 120.8; from the methine proton at δ 7.09 to the carbons at δ 156.7 and 125.7; from the methine proton at δ 6.70 to the carbons at δ 154. 7, 153.8, 135.8, and 112.8; and from the dioxymethylene protons to the carbons at δ 153.8 and 135.8. Finally, a methyl proton showed a long-range correlation to the carbon at δ 141.4 ( Figure S9 and S10). Therefore, the structure of the isolated compound was identified as that of betavulgarin ( Figure 2).

Cell Proliferation Assay
Breast cancer cells were seeded at 1.5 × 10 4 cells per well in a 96-well plate for 24 h and incubated with betavulgarin (0, 50, 100, 200, 300, 400, or 500 µM) for 24 h. Then, proliferation was assayed using the EZ-Cytox Kit (DoGenBio, Seoul, Korea) in accordance with the manufacturer's protocol. The optical density at 450 nm (OD 450 ) was measured using a VERSA max microplate reader (Molecular Device, San Jose, CA, USA).

Colony Formation Assay
MDA-MB-231 and MCF-7 cells were cultured at a low density (2 × 10 3 and 3 × 10 3 cells/well) in a six-well plate and treated with betavulgarin in DMEM. After 7 days of incubation, the medium was replaced, and the cells were washed with PBS, fixed with 3.7% formaldehyde, and stained for 15 min with 0.05% crystal violet. Images were acquired using a scanner.

Transwell Assay
We followed a previously described method [33]. Migration assays were performed with 12-well hanging inserts (Merck Millipore, Darmstadt, Germany). MDA-MB-231 cells were suspended in 200 µL of DMEM containing 1% FBS and added to the upper chamber (2 × 10 5 cells/chamber). The bottom chamber was filled with 750 µL of DMEM containing 20% FBS. The cells were incubated for 24 h at 37 • C in a 5% CO 2 incubator. The lower surface of the inserts was fixed with 3.7% paraformaldehyde and stained with 0.03% crystal violet. Images were captured with a light microscope.

Real-Time RT-qPCR
We used a previously described method [34]. RNA was extracted from MDA-MB-231 cancer cells and mammospheres and purified. Real-time RT-qPCR was performed with a one-step RT-qPCR kit (Enzynomics, Daejeon, Korea). The specific primers are described in Supplementary Table S1.

EMSA
Nuclear extracts were prepared as described previously [35]. An EMSA for Stat3 binding was performed using an IRDye 700-labeled Stat3 DNA (LI-COR). Samples were run on a nondenaturing 5% PAGE gel, and EMSA data were captured with an ODYSSEY CLx instrument (LI-COR).

Statistical Analysis
All presented data are the mean ± standard deviation (SD). Data were analyzed using Student's t-test. A p-value less than 0.05 was considered statistically significant (GraphPad Prism 5 software).

Conclusions
A BCSC-inhibiting compound from beet extracts was purified using silica gel, gel filtration, TLC, and HPLC. The compound was identified as Betavulgarin, a mammosphere formation inhibitor, was isolated from beetroot and identified by mass and NMR spectroscopy. Betavulgarin inhibited cell proliferation, BCSC formation, and reduced the size of the CD44 + /CD24 − subpopulation and the transcript levels of the C-myc, Nanog, and Oct4 gene. This compound decreased the nuclear localization of Stat3 and reduced the mRNA and protein levels of SOX2 in mammospheres. Our results in this study showed that betavulgarin inhibited the Stat3/Sox2 signaling pathway and induced BCSC death, indicating that betavulgarin might be a potential natural compound that targets breast cancer and BCSCs.
Supplementary Materials: The following are available online. Specific primer sequences for real-time RT-qPCR are described in Table S1. Isolation and structure analysis of BCSC inhibitor are described in Figures S1-S10.