**1. Introduction**

Breast cancer is the most frequently diagnosed form of cancer and the leading cause of cancer-related deaths in women worldwide [1,2]. As a result of advanced and multidisciplinary therapeutic treatments, the five-year survival rate of breast cancer has greatly improved [3]. However, an increasing trend in the incidence rate of breast cancer has been observed, and the age of patients who are diagnosed with breast cancer has been reported to be trending younger [4].

Breast cancer is categorized into three major subtypes based on the presence or absence of molecular markers for estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor 2 (ERBB2, formerly HER2/neu). The standard therapy for all patients with breast cancer is tumor eradication by surgical resection. Then, a systemic therapeutic strategy for breast cancer is determined by tumor stage and subtype. Patients with hormone-receptor-positive tumors receive endocrine therapy, such as treatment with hormone receptor antagonists, in combination with limited chemotherapy. For ERBB2-positive tumors, patients can receive ERBB2-targeted antibodies or a combination of small-molecule inhibitors and chemotherapy. Patients with triple-negative tumors (tumors without all three molecular markers) receive chemotherapy alone [5]. Previous studies have indicated that patients with triple-negative breast cancer have worse clinical outcomes compared with hormone-receptor-positive (ER+ and PR+) and ERBB2-positive (ERBB2+) breast cancer patients [6–8]. Systemic chemotherapy for patients with triple-negative breast cancer causes acute and chronic toxicities, including nausea, vomiting, neuropathy, sterility, and congestive heart failure [9,10]. Therefore, there is still an urgen<sup>t</sup> need to optimize the current therapeutic strategy for patients with triple-negative breast cancer.

Numerous natural dietary phytochemicals have been observed to inhibit carcinogenesis and have attracted growing interest as complementary and alternative medicines as well as for cancer chemoprevention due to their wide availability, mildness, and low toxicity [11]. *Glycyrrhiza* species (licorice) are widely used as herbal medicine in Asia, as they exhibit highly e ffective antitussive, expectorant, and antipyretic activities [12,13]. Among the bioactive ingredients isolated from licorice, isoliquiritigenin (ISL; 2-,4,4--trihydroxychalcone) has been reported to exert considerable biological activities. ISL has an anti-inflammatory e ffect on the inhibition of nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome-associated inflammatory diseases [14,15]; an antioxidative e ffect on the activation of the Nrf2-induced antioxidant system [16]; a hepatoprotective e ffect against CCl4-induced liver injury [17]; and a cardioprotective effect on the reduction of oxidative stress [18].

ISL has also been reported to exert potential antitumor activity on multistage carcinogenesis procession in cervical [19], ovarian [20], prostate [21], and lung cancers [22]. Previously, we found that ISL of 10 μM exhibits an inhibitory e ffect on Vascular endothelial growth factor (VEGF)-induced triple-negative breast cancer migration and invasion through downregulation of the PI3K-AKT-MAPK signaling pathway [23]. We also observed that a dose of ISL over 25 μM showed an antiproliferation effect on breast cancer cells [23]. Hence, in the present study, we used an in vitro culture system to explore the molecular mechanism underlying ISL-induced antiproliferation of triple-negative MDA-MB-231 breast cancer cells. Moreover, an MDA-MB-231 tumor xenograft mouse model was generated to examine the preventive e ffect of ISL on tumor growth in vivo.

#### **2. Materials and Methods**

#### *2.1. Cell Line and Culture Condition*

Human breast cancer MDA-MB-231 cell line was purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). MDA-MB-231 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented

with 10% fetal bovine serum (FBS; CORNING, Manassas, VA, USA), 100 units/mL of penicillin, 100 μg/mL of streptomycin (CORNING), sodium bicarbonate (2.438 g/L; BioShop, Burlington, ON, Canada), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; 5.986 g/L; BioShop) in a humidified incubator (37 ◦C, 5% CO2). Dimethyl sulfoxide (DMSO) was used as a solvent of ISL, stock concentration was 100 mM, and the dosage was according to a previous study [24].
