**Inhibitory E**ff**ects of Tangeretin, a Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling**

**Yu-Chan Ko 1,**†**, Hack Sun Choi 2,**†**, Ren Liu 1, Ji-Hyang Kim 1, Su-Lim Kim 1, Bong-Sik Yun <sup>3</sup> and Dong-Sun Lee 1,2,3,4,5,\***


Academic Editor: H.P. Vasantha Rupasinghe Received: 20 May 2020; Accepted: 2 June 2020; Published: 3 June 2020

**Abstract:** Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24−) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.

**Keywords:** tangeretin; cancer stem cells; Stat3; citrus; CD44+/CD24−; phytochemicals

### **1. Introduction**

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among females [1]. Triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all diagnosed breast cancers [2], and is defined as ER-, PR- and HER2-negative breast cancer. Women with TNBC have a high risk of recurrence within three years of diagnosis, and the mortality rate is increased for five years after diagnosis [3]. Cancer stem cells (CSCs) are relatively resistant to chemotherapy, radiotherapy and hormone therapy [4]. CSCs have functional roles in self-renewal and differentiation [5]. CSCs are responsible for the processes of cancer initiation, metastasis and cancer relapse [6]. Therefore, breast CSCs may contribute to drug resistance and relapse [7]. The CD44+/CD24<sup>−</sup> being the most common cell-surface phenotype of breast CSCs can facilitate invasion, migration and proliferation [8].

Signal transducer and activator of transcription 3 (Stat3) plays a role in the inflammatory response and is a member of the seven-member Stat protein family (Stat1, 2, 3, 4, 5a, 5b, and 6) that is activated by growth factors [9]. Stat3 undergoes alternative splicing into Stat3α (92 kDa) and the isoform Stat3β

(83 kDa). Stat3 plays a role in human oncogenesis. The activation of Stat3 signaling is associated with proliferation, antiapoptotic effects and cellular transformation [10,11]. Additionally, Stat3 plays a crucial role in differentiation under normal physiological conditions [6]. Inflammatory cytokines play key roles in regulating the interaction between CSCs and the IL6 inflammatory feedback loop, leading to the expansion of the CSCs population [12]. IL-8 interacts with the CXCR1 receptor on BCSCs (breast cancer stem cells), which promotes their cellular activities such as self-renewal and invasion. IL-8 signaling is a key pathway for regulating BCSCs [13].

Citrus species are natural products containing phytochemicals, which are promising for development into cancer therapies [14,15]. Citrus flavonoids, including nobiletin, hesperidin, tangeretin and naringin, have many biological activities, including a strong antioxidant and radical scavenging activity [16]. Tangeretin, also known as 4,5,6,7,8-pentamethoxyflavone, is a major compound in citrus peels. It has been shown to possess a variety of pharmacological activities, including anti-oxidative, anti-inflammatory and anticancer properties [17]. Inflammatory cytokine IL-6 plays an important role in mediating the interaction between CSCs and the microenvironment, which can influence tumor growth by regulating CSC subpopulations. However, no studies have shown the mechanisms underlying the targeted effects of tangeretin on CSC formation and Stat3 signaling in BCSCs. In our study, we showed that tangeretin had antiproliferative effects on breast cancer cells and reduced BCSC proliferation or prevalence through a decrease in Sox2 expression by inhibiting Stat3 signaling.

#### **2. Results**

#### *2.1. CSC Inhibitor Derived from Citrus*

A CSC-inhibiting compound derived from citrus was purified by bioassay-guided isolation, as shown in Figure 1A and Supplementary Figure S10. The compound was isolated using organic solvent extraction, silica gel, gel filtration, TLC and preparatory HPLC. The isolated sample was determined to be a single compound using HPLC (Figure 1B). We assayed mammosphere formation using a purified sample (Figure 1C). The structural name of the purified compound was identified as tangeretin (Figure 2).

**Figure 1.** Purification protocol for a CSC inhibitor derived from citrus peels and a mammosphere formation assay using a purified sample. (**A**) Flowchart for the isolation of the mammosphere inhibitor. (**B**) HPLC chromatogram of the inhibitor purified from citrus. (**C**) Assay for mammosphere formation in the presence of the HPLC-purified sample. Cancer cells were treated with the HPLC-purified sample. Images show representative mammospheres, and were imaged by microscopy (scale bar: 100 μm).

**Figure 2.** Chemical structure of the compound purified from citrus. Chemical structure of tangeretin.

*2.2. Tangeretin Suppresses the Proliferation of MDA-MB-231 and MCF-7 Cells and the Formation of Mammospheres*

Breast cancer cells were incubated with various concentrations of tangeretin for 24 h. The antiproliferative function of tangeretin was assayed. Tangeretin inhibited cell proliferation, as shown in Figure 3A. To test whether tangeretin can suppress mammosphere formation, we treated cancer cells with tangeretin. Tangeretin inhibited mammosphere formation, as shown in Figure 3B. Tangeretin suppressed the migration and colony formation of cancer cell lines (Figure 3C,D). Our data indicated that tangeretin inhibited cancer hallmarks (proliferation, migration and colony formation) and mammosphere formation.

**Figure 3.** The effects of tangeretin on cell proliferation and mammosphere formation. (**A**) MDA-MB-231 and MCF-7 cells were treated with tangeretin for 24 h in a medium supplemented with 10% FBS. The cytotoxicity of tangeretin was measured with an EZ-Cytox kit. (**B**) Tangeretin inhibits the formation of mammospheres. To establish mammospheres, 1 <sup>×</sup> 104 MDA-MB-231 cells and 5 <sup>×</sup> <sup>10</sup><sup>4</sup> MCF-7 cells were seeded in ultralow-attachment 6-well plates with CSC culture medium. The mammospheres were incubated with increasing concentrations of tangeretin. Photos show representative mammospheres, and were captured by microscopy (scale bar: 100 μm). Mammosphere formation efficiency (MFE) was examined. (**C**) The effect of tangeretin on the migration of breast cancer cells (MDA-MB-231 and MCF-7 cells) was evaluated. Migration with or without tangeretin was captured at 0 and 24 h (scale bar: 100 μm). (**D**) Tangeretin inhibits colony formation by cancer cells. Breast cancer cells (MDA-MB-231 and MCF-7 cells) were incubated and treated with tangeretin. Representative data were collected. The data from triplicate experiments are represented as the mean ± SD; \* *p* < 0.05, \*\* *p* < 0.01.

#### *2.3. Tangeretin Modestly Induces Apoptosis in Mammospheres and Inhibits Mammosphere Proliferation*

Late apoptosis in mammospheres was modestly induced by 80 μM of tangeretin (Figure 4A). Tangeretin reduced the transcript level of stem cell marker genes (Oct3/4, Sox2, and Nanog gene) (Figure 4B). To test whether tangeretin suppresses mammosphere growth, we treated

mammospheres with tangeretin and counted the number of cancer cells derived from mammospheres. Tangeretin treatment inhibited mammosphere growth (Figure 4C). Our data showed that tangeretin, which disregulates the Stat3/Sox2 signaling pathway, was essential for inhibiting the proliferation of BCSCs.

**Figure 4.** The effects of tangeretin on apoptosis and mammosphere growth. (**A**) Tangeretin modestly induced apoptosis in mammospheres treated with tangeretin. We induced mammosphere formation and treated mammospheres with tangeretin. After treatment, apoptotic cells were examined using Annexin V/PI staining. (**B**) Transcription levels of CSC markers, including the Nanog, Sox2 and Oct4 genes, determined in tangeretin- and DMSO-treated mammospheres using CSC marker-specific primers and real-time PCR. β-actin acts as an internal control. The data shown represents the mean ± SD of three independent experiments. \* *p* < 0.05 vs. DMSO-treated control. (**C**) Tangeretin inhibited mammosphere growth. Mammospheres with or without tangeretin were dissociated into single cells, and the single cells were plated in 6 cm dishes in equal numbers. The cells were examined one, two and three days later.

#### *2.4. Tangeretin Decreases Tumor Growth In Vivo*

As tangeretin has antiproliferative effects, we examined whether tangeretin suppresses tumor formation in a nude mouse model. There was no significant body weight difference between control and tangeretin-treated mice (Figure 5A). At each time point, the tumor volume (Figure 5B) and weight (Figure 5C) of the tangeretin-treated nude mice were smaller than those of the untreated nude mice.

**Figure 5.** The effect of tangeretin on tumor growth in a xenograft model. (**A**) MDA-MB-231 cells (2 <sup>×</sup> 106 cells/mouse) were inoculated into the mammary fat pad of female nude mice and treated with tangeretin (2.5 mg/kg) or DMSO (n = 6). Nude mice were intraperitoneally injected with/without Tangeretin (2.5 mg/kg) once a week for a total of four time injections. The body weights of the tangeretin-treated group were comparable to those of the control group. (**B**) Tumor volume was calculated as (width<sup>2</sup> <sup>×</sup> length)/2 at the indicated time points. (**C**) The tumor weights of the control and tangeretin-treated mice were assayed after sacrifice at day 35. The data are presented as the mean ± SD of three independent experiments. \*\* *p* < 0.05 versus the DMSO-treated control group.
