XJP Inhibits Apoptosis, Invasion, EMT, and Wnt/Β-Catenin Pathway in Triple-Negative Breast Cancer


 Background: Triple-negative breast cancer (TNBC) progresses at a rapid pace. Chemotherapy is a major clinical application. However, resistance and metastases are key barriers to chemotherapy. Xiaojin pills (XJP) have been used clinically for treating TNBC for decades. However, the potential molecular mechanisms of the effect of XJP on breast cancer is still not understood.Methods: The cell viability was analyzed using Cell Counting Kit-8 (CCK-8). Flow cytometry was used to detect apoptosis, and the migration and invasion abilities of TNBC were assessed using Transwell assay. For molecular mechanisms, the protein expression levels were determined by Western blot analysis. The expression of β-catenin in the Wnt/β-serial protein (β-catenin) pathway was detected with immunofluorescence (IF).Results: XJP inhibited the viability and proliferation of the TNBC cell line in vitro. Flow cytometry analysis showed that apoptosis increased in both MDA-MB-231 and MDA-MB-468 cells induced by XJP. The expression of the proteins associated with invasion, for example, matrix metalloproteinase (MMP) and MMP9, was reduced. Among epithelial–mesenchymal transition markers, E-cadherin was upregulated and N-cadherin was downregulated. The apoptosis-related proteins caspase-8, caspase-3, caspase-9, and Parp were all upregulated. Additionally, XJP effectively suppressed the expression of β-catenin, which belonged to the Wnt/β-catenin pathway.Conclusions: These results suggested that XJP suppressed the progression of TNBC cells by suppressing apoptosis, invasion, EMT, and Wnt/β-catenin pathway.

Apoptosis can lead to certain changes in cellular properties. These changes are activation of caspases, mitochondrial depolarization, cell volume loss, and DNA fragmentation (10). Caspase cascade plays an important role in cellular death, ensuring normal tissue renewal (11). Caspases were recruited at the death-inducing signaling complex; caspase-8 acts as "initiating" caspases (12). When caspase 8 is activated, downstream effector caspases, such as caspase-3 and caspase-9, are subsequently activated, causing the cleavage of the proteins involved in the execution of apoptosis (11). Caspase-3 is a critical effector of apoptosis and is responsible for the proteolytic cleavage of key proteins such as poly(ADPribose) polymerase 1 (PARP1) (13). PARPs are DNA-dependent nuclear enzymes. PARP1 is considered a suitable therapeutic target for the potential treatment of cancers (14).
Epithelial-mesenchymal transition (EMT) is a continuous process of transformation of epithelial cells into motile mesenchymal cells with invasive properties; it plays an important and necessary role in cellular development and the metastasis of breast cancer (15). A decrease in the levels of the epithelial marker E-cadherin (CDH1) and an increase in the levels of the mesenchymal marker N-cadherin are the key changes during EMT (16). CDH1 is downregulated in some malignant tumors, especially undifferentiated tumors with metastatic characteristics; however, CDH2 has the opposite expression.
CDH1 has a critical role in β-catenin function and stabilization; β-catenin may disassociate from Ecadherin/β-catenin complexes and translocate to the nucleus (17). β-Catenin, the core of the canonical Wnt/β-catenin pathway, plays two roles in simple epithelia: it acts as a binding partner for adherens junction proteins, such as E-cadherin, or as a messenger in the signaling pool (18). Wnt signaling regulates various cellular functions, such as cell proliferation, differentiation, and development, and processes in disease progression, such as EMT (19). In the absence of Wnt ligand, β-catenin is usually degraded by the proteasome system, including axin, glycogen synthase kinase 3 beta (GSK3β), and casein kinase 1, while the binding of Wnt to a frizzled receptor blocks the activity of destruction complex to degrade β-catenin in the presence of Wnt ligand and so β-catenin is translocated into the nucleus (20).
Additionally, the dysfunctioning of Wnt/catenin signaling promotes the proliferation of mammary and colorectal cancers (21). Meanwhile, the progression of a tumor in situ to an invasive phenotype requires an increase in tumor cell plasticity (22). Matrix metalloproteinase (MMP) families, such as MMP2 and MMP9 (23), also play essential roles in breast cancer metastasis.
Therefore, a drug that in uences the apoptosis and EMT of breast cancer has a signi cant value in the treatment. Many Chinese herbs are applied to the treatment of breast cancer. So far, XJP is a traditional Chinese medical formula used as adjuvant therapy to treat breast and thyroid cancers (24). Rong et al. (25) proved that XJP could effectively inhibit the migration and invasion of highly metastatic breast cancer cells. They also inferred that the mechanism might be related to regulating the p38 mitogenactivated protein kinase (MAPK), Jun N-terminal Kinase(JNK) MAPK pathway and the reversal of tumor cell EMT. In this study, the function and cytotoxicity of XJP in TNBC cells were investigated and its antitumor mechanism was further explored. All the herbals were purchased in the A liated Hospital of Shandong University of Traditional Chinese Medicine, and all of them were identi ed by Chuanjiang Ma, who was devoted to the research of traditional medicine. The ratio of these 10 herbs was 1:5:5:5:2.5:2.5:5:2.5:5:0.4. The extract of XJP was prepared by decocting the dried prescription of herbs. Brie y, raw materials of XJP formulation were mixed and crushed into small pieces. Ten-time volumes of water were added with raw components and boiled for 2 h. Then, a centrifuge (Eppendorf, Germany) was used to precipitate at 3000 rpm for 5 min, ve times. The decoction was concentrated by removing a portion of water in a vacuum at 60℃. Finally, the concentration of XJP was 1 g/mL. Before analysis, the decoction was ltered through a 0.22-µm polytetra uoroethylene lter (Millipore, MA, USA). The ltered decoction was stored at 4℃ before treating cells.

Cell viability assay
The sensitivity of breast cancer cells to XJP and epirubicin was detected using the Cell Counting Kit-8

XJP-induced apoptosis of TNBC cells
MDA-MB-231 and MDA-MB-468 cells were exposed to XJP at different concentrations to explore whether the growth inhibition of TNBC cells by XJP was due to the apoptotic response, and ow cytometry was performed to assess their apoptotic rate (Fig. 2). and 6B, P < 0.01 at 3.75 mg/mL, P < 0.001 at 7.5 mg/mL) and MDA-MB-468 ( Fig. 6C and 6D, P < 0.001 at 2 and 4 mg/mL) cells compared with that in the control group (0 mg/mL). Therefore, XJP inhibited the invasive and migratory abilities of MDA-MB-231 and MDA-MB-468 cells in a dose-dependent manner.

XJP affected the expression of EMT-associated proteins and MMPs
Since EMT-inducing transcription factors promote cancer metastasis, the present study examined whether XJP inhibited breast cancer migration and invasion by regulating the expression of EMTassociated proteins. As markers of EMT, the expression levels of CDH1 and CDH2 were detected by Western blot analysis. The results showed that the expression of CDH1 in MDA-MB-231 (Fig. 7A) and MDA-MB-468 (Fig. 7B) cells signi cantly increased following XJP treatment. However, the expression of CDH2 decreased compared with that in the control group. In addition, the relative expression level of CDH1 decreased in XJP-treated cells compared with that in the untreated control group (Fig. 7C and 7D).
The expression levels of gelatinases MMP2 and MMP9, which serve crucial roles in maintaining extracellular matrix homeostasis and tumor invasion, were also signi cantly inhibited by XJP compared with those in the control group ( Fig. 7A and 7B). These results indicated that the inhibition of XJPinduced migration and invasion might occur through the suppression of EMT and MMP expression.

XJP inhibited the Wnt/β-catenin pathway in TNBC cells
A recent study reported that Wnt signaling was associated with tumor cell migration and metastasis (26). β-catenin is the core molecule of the canonical Wnt pathway. As a consequence of its stabilization, it is translocated to the nucleus, where it controls gene expression and regulates the activities of downstream signals (27). In this study, β-catenin was downregulated in XJP-treated cells. As shown in Figure 8 A and 8B, Western blot analysis indicated that the downregulation of β-catenin was induced by XJP in a concentration-dependent manner in MDA-MB-231 and MDA-MB-468 cells, meanwhile the quantitative analysis of IF expression proved this result also. In addition, immuno uorescence results clearly showed that β-catenin displayed noticeable nuclear localization in control cells. However, when cells were treated with XJP, the intracellular distribution of β-catenin changed notably, as was characterized by decreased intranuclear uorescence intensity (Fig. 8D). This phenomenon only appears in MDA-MB-231 cells. In MDA-MB-468 cells, the expression of β-catenin is poor at the nuclear position, but the expression in cytoplasm position is downregulated after exposure to XJP. These results indicated that XJP inhibits the Wnt/β-catenin pathway.

Discussion
TNBC is associated with higher metastasis and a poorer prognosis compared with other breast cancer subtypes due to the lack of effective chemotherapeutic drugs and frequently acquired chemoresistance (28, 29). Late diagnosis, presence of metastasis, and adverse effects of chemotherapeutic drugs bring a huge challenge in the treatment of TNBC (30).
Apoptosis of cancer cells is essential to prevent the proliferation, metastasis, and development of chemoresistance among cancers (31). In tumor therapy, apoptosis is a well-known mechanism of cell death, involving the activation of caspases (32). Caspases are key mediators in programmed cell death or apoptosis (33). Studies showed that the caspase family was involved in extrinsic and intrinsic apoptotic pathways (34)(35)(36)(37). The extrinsic apoptotic pathway involved CASP8 activation (38). CASP9 and CASP3 are two members of a family of proteases, and primary mediators of apoptosis (39). CASP3 is mainly activated through CASP8-and CASP9-dependent apoptotic pathways (40,41); when the CASP3 is activated, PARP1 is degraded (42).  (39). However, the mechanism of XJP for TNBC cells has not been reported. The present study filled this gap, revealing that the TNBC cells treated with XJP were found to be signi cantly involved in signaling pathways such as apoptosis, suggesting that XJP could activate the apoptosis of TNBC cells.
In patients with TNBC, the TNBC cells had strong invasion and migration abilities, resulting in a poor prognosis (43). MMPs are major components involved in metastasis. Especially, the increased levels of MMP2 and MMP9, two important members of MMPs, were associated with cancer aggressiveness and metastasis in TNBC (44,45). The ndings of this study showed that XJP reduced the expression of MMP2 and MMP9 in both MDA-MB-231 and MDA-MB-468 cell lines, suggesting that XJP might inhibit cell invasion and migration through the suppression of MMPs in TNBC. Studies also showed that EMT was an initial step in cancer metastasis, CDH2, which is a positive regulator and a canonical marker of EMT, has a correlation with aggressive clinical phenotype in TNBC (46). Besides, it has been proved that E-cadherin promotes metastasis in diverse models of invasive ductal carcinomas. While the loss of CDH1 increased invasion, it also reduced cancer cell proliferation and survival, circulating tumor cell number, seeding of cancer cells in distant organs, and metastasis outgrowth (47      Experiments were conducted in triplicate. ***P < 0.001, ****P < 0.0001. ACTB, β-actin; CDH1, E-cadherin; CDH2, N-cadherin; MMP, matrix metallopeptidase. CTNNB1, β-catenin. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.