**4. Discussion**

Patients with triple-negative breast cancer are insensitive to endocrine therapy and ERBB2-targeted antibody treatment. Systemic chemotherapy for patients with triple-negative breast cancer is the predominant clinical treatment strategy [27,28]. Triple-negative breast cancer cells have been reported to exhibit genetic and phenotypical shifts in subpopulations, leading to resistance to the available standard

drugs [29,30]. However, the functional di fferences made by genomically heterogeneous triple-negative breast cancer cells to chemoresistance remain poorly understood [31]. Natural ingredients from plants are widely explored to identify their availability for application in patients with breast cancer as an adjuvant approach [32]. For example, curcumin, a bioactive ingredient of the plant *Curcuma longa*, exerts an inhibitory e ffect on triple-negative MDA-MB-231 breast cancer cells through upregulation of p21 protein expression and enhancement of the Bax-to-Bcl-2 ratio [33,34]. A clinical phase I trial of curcumin has been reported to increase the sensitivity of breast cancer patients to docetaxel chemotherapy [35]. Similar e ffectiveness was observed for licorice roots (*Glycyrrhiza glabra*). The compounds derived from licorice have been widely investigated for their anticancer e ffects on several cancer cell types. Among the chalcone-type derivatives from licorice, ISL has been identified for cancer prevention in the last decade [12,36].

In the present study, we found that treatment of triple-negative MDA-MB-231 breast cancer cells with ISL inhibited cancer cell growth through blocking the cell cycle by reduction of cyclin D1 expression. The enhancement of the sub-G1 cell population by ISL treatment indicated that the apoptotic cell death pathway was active. Flow cytometry analysis was used to further confirm whether ISL induced MDA-MB-231 cell apoptosis. Our results showed that treatment with ISL increased the annexin-V- and PI-positive cell population. The expression of the anti-apoptotic protein Bcl-2 was reduced, whereas expression of the proapoptotic protein Bax was increased. Furthermore, the downstream molecules caspase-3 and PARP were active. In agreemen<sup>t</sup> with our findings, Peng et al. reported that treatment of MDA-MB-231 cells with ISL induced cell apoptosis, and they observed an enhancement of the ratio of Bax to Bcl-2 by ISL treatment [37]. Moreover, ISL treatment was also observed to induce cancer cell apoptosis in lung cancer [38], uterine leiomyoma [39], and endometrial cancer [40].

A clinical study reported that the expression of Beclin1 is lower in patients with breast and ovarian cancers [41]. Beclin1, a Bcl-2 homology 3 (BH3)-domain-only protein, is an important protein in initial autophagy. The anti-apoptotic Bcl-2 protein has been reported to interact with Beclin1 via its BH3 domain [42]. The Beclin1 and Bcl-2 complex suppresses the pre-autophagosomal structure combination, thereby inhibiting autophagy progression. In this study, the results showed that treatment with ISL reduced the expression of anti-apoptotic Bcl-2 and induced autophagy-induced- apoptosis. Our results found that ISL may have p62 accumulation. Additionally, p62 may activate caspase-8, which can induce apoptosis by autophagy inhibition. Caspase-8 activation promotes cell apoptosis [43]. In addition, we found that treatment with ISL induced autophagy-associated signaling proteins mTOR and ULK1 were downregulated by ISL treatment, while the expressions of p62, Beclin1, and LC3 autophagy-promoting proteins were all increased. However, during autophagy progression, p62 was enclosed by autolysosome and degraded by cathepsin B in lysosomes [44], although both p62 and LC3 II levels were increased by ISL. Therefore, ISL influences p62 accumulation by reduced cathepsin B to inhibit autophagy by blocked phagolysosome formation. Furthermore, ISL can activate apoptosis by increased caspase-8 protein expression.

Cathepsin B is a lysosomal protease and is related to autophagy progression and p62 degradation [45]. Cancer studies have shown that cathepsin B is related to cancer cell proliferation [46], and its ability to break down the cell base membrane can increase cancer cell invasion. Our results show that ISL can decrease cathepsin B protein expression to suppress p62 accumulation, increasing autophagy-mediated apoptosis and decreasing cancer cell proliferation-related Ki67 expression.

Cuendet et al. reported that administration of ISL for 1 week delays 7,12-dimethylbenz (*a*)anthracene (DMBA)-induced mammary carcinogenesis in female rats. However, this e ffect does not reduce the incidence of tumor formation [36,47]. In the present study, to examine whether ISL has preventive e ffectiveness for anti-proliferation of breast cancer cells, mice were administered with ISL by oral gavage for 2 weeks before implantation of MDA-MB-231 breast cancer cells. Our results showed that pretreatment of mice with ISL reduced the success rate of breast cancer cell implantation, thereby reducing tumor tissue formation. Despite tumor formation from the ISL-treated group, the expression of the Ki-67 protein, a hallmark of proliferation, was reduced, whereas the expressions of caspase-3 and

p62 proteins were increased, suggesting that these tumors were undergoing apoptotic and autophagic cell death progression. In addition, we further evaluated the e ffects of ISL on anti-angiogenesis in breast cancer cells. Our results show that the protein expression and secretion of VEGF were reduced by treatment of mice with ISL. Consistent with our results, Wang et al. reported that treatment with ISL suppresses VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) through interaction with the VEGF2R receptor and the reduction of VEGF-regulated downstream signaling [48]. Moreover, we also found that the capability of capillary-like tube formation of SVEC4-10 cells was abolished by treatment with ISL. ISL has a potential role in antiangiogenesis, which might contribute to the inhibitory e ffect of ISL on the migration and invasion ability of cancer cells.

The e ffectiveness of ISL in combination with traditional chemotherapeutic drugs has also been investigated. Wang et al. reported that ISL exerts synergistic e ffects with first-line chemotherapeutic drugs for breast cancer therapy [49]. Specifically, ISL chemosensitizes breast cancer stem cells by docking into the adenosine triphosphate (ATP) domain of GRP78 directly, which in turn suppresses β-catenin/ATP-binding cassette subfamily G2 (ABCG2) signaling [49]. The side-e ffects of systemic chemotherapeutic therapy were also investigated. ISL has been reported to enhance the toxicity-induced cell death of bladder cancer cells. Furthermore, ISL has been shown to attenuate cisplatin-induced normal renal proximal tubular cell death via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) regulatory pathway [50]. Similarly, the protective e ffect of ISL on the kidney and liver against cisplatin-induced colon cancer cell death has also been demonstrated [51]. These results sugges<sup>t</sup> that the promotion of chemosensitivity and the lower toxicity of ISL should be further considered in clinical trials.
