**3. Results**

#### *3.1. ISL Suppressed Breast Cancer MDA-MB-231 Cell Growth*

The MTT assay was performed to examine whether ISL can suppress breast cancer MDA-MB-231 cell growth. The results showed that cell viability was reduced by treatment of cells with ISL at 25 and 50 μM for 48 and 72 h (Figure 1a), whereas treatment of cells with ISL at 25 and 50 μM for 48 and 72 h was shown to increase cytotoxicity by LDH activity analysis (Figure 1b). The cell morphology and number were also monitored, and we found that the cell morphology switched from the spindle type to irregular by treatment of cells with ISL at 25 and 50 μM for 48 h (Figure 1c). Furthermore, a reduction of the cell number was observed by treatment of cells with 25 and 50 μM for 48 h (Figure 1d).

**Figure 1.** Inhibitory e ffects of isoliquiritigenin (ISL) on the proliferation of breast cancer MDA-MB-231 cells. (**a**) MDA-MB-231 cells were seeded in 96-well plates (3000 cells per well) with 100 μL per well culture medium. Cells were treated with ISL in various doses for 24, 48, and 72 h. At the end of incubation, cell viability was measured by MTT assay (*n* = 3). (**b**) MDA-MB-231 cells (2 × 10<sup>4</sup> cells per well) seeded in 96-well plates. Cells were treated with various doses for 24, 48, and 72 h. The cytotoxic effects of ISL was detected using the lactate dehydrogenase (LDH) assay kit (*n* = 3). (**<sup>c</sup>**,**d**) MDA-MB-231 cells (2 × 10<sup>5</sup> cells per well) were seeded in six-well plates. After treatment of cells with ISL for 48 h, the cell number was monitored (*n* = 4). Data are represented as mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group.

#### *3.2. ISL Suppressed the Cell Cycle Progression of MDA-MB-231 Cells*

Here, we studied whether ISL suppressed cell growth by inflecting the cell cycle progression. MDA-MB-231 cells were treated with ISL and then the cell cycle distribution was monitored using flow cytometry analysis. The results showed that treatment with ISL at 25 and 50 μM for 48 h reduced the cell population in the G1 phase in MDA-MB-231 cells (Figure 2a,b). In addition, the expression of G1/S gate-associated proteins CDK4 and cyclin D was detected. The results showed that treatment with ISL did not alter the expression of CDK4 (Figure 2c), but the expression of cyclin D1 was reduced by treatment with ISL at 25 and 50 μM for 48 h (Figure 2d).

**Figure 2.** Cell cycle progression of MDA-MB-231 cells was suppressed by treatment with ISL. (**a**) MDA-MB-231 cells were treated with ISL for 48 h. Cells were stained with propidium iodide, and the cell cycle distributions were analyzed by the flow cytometry. (**b**) The quantitative results of cell distributions in MDA-MB-231 cells are shown. Cell-cycle-associated proteins (**c**) CDK4 and (**d**) CyclinD1 were analyzed using Western blot. Each target protein was normalized to GAPDH expression. Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group.

#### *3.3. ISL Induced Apoptotic Cell Death*

To further examine whether ISL induced cell apoptosis in MDA-MB-231 cells, the cells were stained with annexin V FITC, and then the apoptotic cell population was analyzed by flow cytometry. The results showed that the annexin-V-positive (early apoptotic phase) and annexin-plus-PI-positive (later apoptotic phase) cell populations were increased by ISL treatment at 25 and 50 μM for 48 h (Figure 3a,b). The expression of Bcl-2 protein decreased from ISL treatment at 50 μM for 48 h (Figure 3c,d), whereas treatment with ISL increased the expression of Bax protein (Figure 3c,e). In addition, the cleaved form of caspase-3 was induced by treatment with ISL at 25 and 50 μM for 48 h (Figure 3f,g), and the expression of cleaved PARP was also increased by treatment with ISL at 50 μM for 48 h (Figure 3f,h).

**Figure 3.** ISL induced cell apoptosis by upregulating apoptotic protein expression in MDA-MB-231 cells. (**a**) MDA-MB-231 cells were treated with ISL for 48 h. Cells were stained with propidium iodide and annexin V fluorescein isothiocyanate (FITC), and the apoptosis rates were analyzed by flow cytometry. (**b**) The quantitative data of apoptotic cell death in early and late phases are shown. (**c**) After treatment as indicated above, the anti- and proapoptotic proteins were monitored using Western blotting in MDA-MB-231 cells. (**d**,**<sup>e</sup>**) Each target protein was normalized to GAPDH or β-actin expression. (**f**) The expression of caspase-3 and its downstream molecule, PAPR, was monitored using Western blotting in MDA-MB-231 cells. (**g**,**h**) Each target protein was normalized to β-actin expression.

Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group. + *p* < 0.05 compared with the early apoptotic phase of the control group. # *p* < 0.05, ## *p* < 0.01 compared with the later apoptotic phase of the control group.

#### *3.4. ISL-Mediated p62 Accumulation Causes Autophagy-Mediated Apoptosis*

The total and phosphorylated protein levels of mTOR decreased after treatment with ISL at 50 μM for 48 h (Figure 4a,b). The downstream molecule ULK1 was activated by mTOR regulation in the autophagy pathway [25]. We found that the total and phosphorylated protein levels of ULK1 decreased after ISL treatment at 10, 25, and 50 μM for 48 h (Figure 4a,c). The autophagosome-associated proteins p62, Beclin1, and LC3 were increased by treatment with ISL for 48 h (Figure 4d–f). In our results observing that p62 accumulation, with autophagy inhibitor bafilomycin (BAF), blocked p62 accumulation and caused cell toxicity (Supplementary Figure S1). In the autophagy process, lysosomal proteases are needed, such as Cathepsins degrades p62 [26]. Treatment with ISL at 50 μM for 48 h significant decreased cathepsin B protein expression (Figure 4g), which explains the p62 accumulation. Moreover, p62 accumulation was related to the increased apoptosis through the increase of caspase-8. Furthermore, we used siRNA to eliminate p62 function. After 48 h, ISL increased caspase-8 protein expression. However, ISL combined with siRNA treatment have a lower expression of caspase-8 than ISL treatment (Figure 4h).

**Figure 4.** ISL treatment induced the expression of autophagy-associated proteins in MDA-MB-231 cells.

MDA-MB-231 cells were treated with ISL for 48 h. (**a**) The expression levels of mTOR and ULK1 protein were analyzed by Western blotting. The total and phosphorylated forms of (**b**) mTOR and (**c**) ULK1 were normalized to β-actin expression. The expression levels of (**d**) p62, (**e**) Beclin1, (**f**) LC3, and (**g**) Cathepsin B were also analyzed using Western blotting. MDA-MB-231 cells were used lipofectamine 3000 (Thermo Fisher Scientific) to transfect SignalSilence® SQSTM1/p62 (Cell Signaling), and treated with ISL for 48 h. The expression of (**h**) caspase-8 proteins were also analyzed using Western blotting. Data are represented as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 compared with the control group. # *p* < 0.05, ## *p* < 0.01 compared with the phosphorylated protein level of the control group.

#### *3.5. Preventive E*ff*ects of ISL on Breast Cancer Cell Growth in a Xenograft Mouse Model*

A breast cancer MDA-MB-231 cell xenograft mouse model was generated to evaluate whether ISL has a potential role in antitumor proliferation in vivo. After pretreatment with PBS or ISL at 2.5 and 5.0 mg/kg for 2 weeks, MDA-MB-231 cells were inoculated into mice, which were constitutively treated with ISL for an additional 25 days. The results showed that MDA-MB 231 tumors formed in all five mice by treatment with PBS. However, the number of MDA-MB 231 tumors was reduced by pretreatment with ISL at 2.5 mg/kg (three of five mice) and 5.0 mg/kg (two of five mice) (Figure 5a,c). Measuring the tumor volume elicited by MDAMB-231 cells showed that the tumor volume increased by treatment with PBS in a time-dependent manner, and ISL treatments significantly reduced them (Figure 5b). At the end of the experiment, tumor tissues were isolated and weighed. Treatments of mice with ISL at 2.5 and 5.0 mg/kg showed significantly reduced tumor weight compared with the vehicle group (Figure 5d). According to the IHC results, expression of the Ki-67 protein level was observed in tumor tissues for mice treated with ISL at 2.5 and 5.0 mg/kg, separately (Figure 6, top panel). Treatment with ISL at 5.0 mg/kg increased the caspase-3 expression level in tumor tissues (Figure 6, middle panel). Moreover, the expression of p62 was increased in tumor tissues by treatment with ISL at 2.5 and 5.0 mg/kg, separately (Figure 6, bottom panel).

**Figure 5.** ISL suppressed tumor growth, as shown by the human MDA-MB-231 breast tumor xenograft

mouse model. Mice were pretreated with ISL at 2.5 and 5.0 mg/kg or PBS as control by oral gavage once daily for 2 weeks. Then, MDA-MB-231 tumor cells (5 × 10<sup>6</sup> cells per mouse) were implanted into mice by subcutaneous injection on the right flank and the mice received oral gavage of PBS and/or ISL for 25 days. At the end of the experiment, (**a**) the mice were photographed, (**b**) tumor size was monitored, (**c**) tumors were isolated and photographed, and (**d**) tumor weight was recorded. Data are represented as mean ± SEM (*n* = 5). \*\* *p* < 0.01 compared with the control group.

**Figure 6.** Representative results of IHC staining for Ki-67, caspase-3, and p62 protein expression levels in ISL-treated MDA-MB-231 breast tumor xenograft mouse model. MDA-MB-231 tumor cells (5 × 10<sup>6</sup> cells per mouse) were implanted and mice were treated with ISL for 25 days Then, tumor tissues were isolated and immunohistochemistry was performed to analyze Ki-67, caspase-3, and p62 protein levels. Images were photographed (200× magnification; scale bar = 200 μm).

#### *3.6. ISL Suppressed VEGF Production and Capillary-Like Tube Formation of SVEC4-10 Cells*

IHC analysis was performed to evaluate the expression of VEGF in tumor tissues by treatment with ISL. The results showed that the expression of VEGF was significantly reduced in treatment of mice with ISL at 2.5 and 5.0 mg/kg, separately (Supplementary Figure S2a). Measuring the serum VEGF level showed that treatment with ISL at 2.5 and 5.0 mg/kg reduced the serum VEGF level compared with the vehicle group (Supplementary Figure S2b). Additionally, to further investigate whether ISL has a potential role in antiangiogenesis, we performed a capillary-like tube formation assay. The results showed that the capability of capillary-like tube formation in SVEC4-10 cells was inhibited by treatment with ISL at 10 μM for 5 h (Supplementary Figure S3).
