Inhibition of Wnt3a/FOXM1/β-Catenin Axis and Activation of GSK3β and Caspases are Critically Involved in Apoptotic Effect of Moracin D in Breast Cancers

Although Moracin D derived from Morus alba was known to have anti-inflammatory and antioxidant activities, the underlying antitumor mechanism of Moracin D has not been unveiled thus far. Thus, in the recent study, the apoptotic mechanism of Moracin D was elucidated in breast cancer cells. Herein, Moracin D exerted significant cytotoxicity in MDA-MB-231 and MCF-7 cells. Furthermore, Moracin D increased sub G1 population; cleaved poly (Adenosine diphosphate (ADP-ribose)) polymerase (PARP); activated cysteine aspartyl-specific protease 3 (caspase 3); and attenuated the expression of c-Myc, cyclin D1, B-cell lymphoma 2 (Bcl-2), and X-linked inhibitor of apoptosis protein (XIAP) in MDA-MB231 cells. Of note, Moracin D reduced expression of Forkhead box M1 (FOXM1), β-catenin, Wnt3a, and upregulated glycogen synthase kinase 3 beta (GSK3β) on Tyr216 along with disturbed binding of FOXM1 with β-catenin in MDA-MB-231 cells. Conversely, GSK3β inhibitor SB216763 reversed the apoptotic ability of Moracin D to reduce expression of FOXM1, β-catenin, pro-caspase3, and pro-PARP in MDA-MB-231 cells. Overall, these findings provide novel insight that Moracin D inhibits proliferation and induces apoptosis via suppression of Wnt3a/FOXM1/β-catenin signaling and activation of caspases and GSK3β.

Emerging evidences reveal that transcription factor Forkhead box M1(FOXM1) is involved in chemoresistance, carcinogenesis, and metastasis [7,8], and so suppression of FOXM1 can be a good strategy for cancer therapy [9]. Certainly, thiostrepton and casticin induced apoptosis and suppressed cell growth in cancer cells by inhibiting FOXM1 [10,11].
Additionally, dysfunction of Wnt/catenin signaling promoted proliferation of mammary and colorectal cancers [12]. In the absence of Wnt ligand, β-catenin is usually degraded by the proteosome system including axin, glycogen synthase kinase 3 beta (GSK3β), and casein kinase 1, while 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 nucleus [13]. Phosphorylation of GSK3β on Tyr216 induces GSK3β activation and function [14], which is followed by its dephosphorylation on Ser9 [15], though the role of GSK3β is controversial in cancer progression or apoptosis [16]. GSK3β is a primary target of Akt, which inhibits GSK3β function by phosphorylating it on Ser9 in proliferating cells.
For efficient cancer chemoprevention, it is considered one of the potent anticancer strategies to find out effective natural chemicals that selectively induce apoptosis and inhibit proliferation in human breast cancer cells [17][18][19][20].
Nevertheless, the underlying antitumor mechanism of Moracin D was not clearly understood so far. Thus, in the present study, the antitumor mechanism of Moracin D was elucidated in breast cancer cells in association with FOXM1 and β-Catenin/GSK3β signaling with the possibility of a potent pharmaceutical for future agricultural commercialization.

Cytotoxic Effect of Moracin D in Human Breast Cancer Cells
The cytotoxicity of Moracin D (Figure 1a  presence of Wnt ligand and so β-catenin is translocated into nucleus [13]. Phosphorylation of GSK3β on Tyr216 induces GSK3β activation and function [14], which is followed by its dephosphorylation on Ser9 [15], though the role of GSK3β is controversial in cancer progression or apoptosis [16]. GSK3β is a primary target of Akt, which inhibits GSK3β function by phosphorylating it on Ser9 in proliferating cells. For efficient cancer chemoprevention, it is considered one of the potent anticancer strategies to find out effective natural chemicals that selectively induce apoptosis and inhibit proliferation in human breast cancer cells [17][18][19][20].
Nevertheless, the underlying antitumor mechanism of Moracin D was not clearly understood so far. Thus, in the present study, the antitumor mechanism of Moracin D was elucidated in breast cancer cells in association with FOXM1 and β-Catenin/GSK3β signaling with the possibility of a potent pharmaceutical for future agricultural commercialization.

Cytotoxic Effect of Moracin D in Human Breast Cancer Cells
The cytotoxicity of Moracin D (Figure 1a

Moracin D Effectively Attenuated the Expression of FOXM1 Related Proteins in MDA-MB-231 Cells
To confirm whether or not the anticancer effect of Moracin D is related to FOXM1 and Wnt3a/β-

Moracin D Disturbed the Binding between FOXM1 and Β-Catenin in MDA-MB-231 Cells
To confirm the inhibitory effect of Moracin D on interaction between FOXM1 and β-catenin, immunoprecipitation was performed in the MDA-MB-231 cell treated by Moracin D. The score of protein-protein interaction (PPI) between FOXM1 and β-catenin was known 0.747 by String database (Figure 4a). As shown in Figure 4b

GSK3β Inhibitor SB216763 Blocked the Apoptosis Induced by Moracin D in MDA-MB-231 Cells
To explain the downstream role of Wnt signaling in FOXM1-mediated tumorigenesis, GSK3β inhibitor SB216763 was used in MDA-MB-231 cells. Pretreatment of SB216763 blocked the apoptotic effect of Moracin D to inhibit FOXM1, β-catenin, pro-PARP, and pro-caspase3 in treated MDA-MB-231 cells (Figure 5a,b).

Moracin D Disturbed the Binding between FOXM1 and Β-Catenin in MDA-MB-231 Cells
To confirm the inhibitory effect of Moracin D on interaction between FOXM1 and β-catenin, immunoprecipitation was performed in the MDA-MB-231 cell treated by Moracin D. The score of protein-protein interaction (PPI) between FOXM1 and β-catenin was known 0.747 by String database (Figure 4a). As shown in Figure 4b

GSK3β Inhibitor SB216763 Blocked the Apoptosis Induced by Moracin D in MDA-MB-231 Cells
To explain the downstream role of Wnt signaling in FOXM1-mediated tumorigenesis, GSK3β inhibitor SB216763 was used in MDA-MB-231 cells. Pretreatment of SB216763 blocked the apoptotic effect of Moracin D to inhibit FOXM1, β-catenin, pro-PARP, and pro-caspase3 in treated MDA-MB-231 cells (Figure 5a,b).

GSK3β Inhibitor SB216763 Blocked the Apoptosis Induced by Moracin D in MDA-MB-231 Cells
To explain the downstream role of Wnt signaling in FOXM1-mediated tumorigenesis, GSK3β inhibitor SB216763 was used in MDA-MB-231 cells. Pretreatment of SB216763 blocked the apoptotic effect of Moracin D to inhibit FOXM1, β-catenin, pro-PARP, and pro-caspase3 in treated MDA-MB-231 cells (Figure 5a,b).

Discussion
In the current study, the antitumor mechanism of Moracin D, a constituent of FOXM1, which belongs to the Forkhead box (Fox) protein superfamily, is one of the proliferation-associated transcription factors [32,33]. Previous evidence reveals that overexpression of FOXM1 has been implicated in proliferation, metastasis, epithelial-mesenchymal transition (EMT), chemoresistance, and poor prognosis of cancers [8,34,35]. Hence, suppression of FOXM1 is regarded to reduce proliferation and induce apoptosis [36,37]. Previous studies demonstrated that FOXM1 is a key regulator of G1, S, and G2/M progression [38], and so overexpression of FOXM1 has been shown to promote cell cycle progression [39,40]. Also, FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the improvement of cell cycle progression and cell proliferation [41,42]. Here, Moracin D inhibited the expression of FOXM1 and cyclin D1 in MDA-MB-231 cells, but not in MCF-7 cells, indicating FOXM1 mediated inhibition of cyclin D1. Emerging evidences suggest that FOXM1 upregulates antiapoptotic genes such as Bcl-2 [43] and XIAP [44]. In our study, Moracin D decreased the expression of Bcl-2 and XIAP in MDA-MB-231 cells.
Wnt/β-catenin signaling plays an important role in cancer progression, including regulation of transformation, cell proliferation, and invasion [45,46]. Wnt3a increases the expression and nuclear translocation of FOXM1, which directly binds to β-catenin for nuclear localization and transcriptional activity [47,48]. Additionally, Yaohui et al reported that GSK3β phosphorylates FOXM1 on serine 474, which induces FOXM1 ubiquitination mediated by FBXW7 [49], while Wnt activation inhibits FOXM1 phosphorylation by GSK3β-Axin complex for deubiquitination and stabilization of FOXM1 [49]. Consistently, Moracin D suppressed the expression of Wnt3a and β-catenin, and induced the phosphorylation of GSK3β (Tyr 216) in MDA-MB-231 cells, but not in MCF-7 cells (data not shown), implying that Moracin D inhibits proliferation and induces apoptosis via inhibition of Wnt3a and βcatenin and activation of GSK3β in MDA-MB-231cells.

Discussion
In the current study, the antitumor mechanism of Moracin D, a constituent of Morus alba, was examined in breast cancer cells in association with FOXM1 and β-Catenin/GSK3β signaling. FOXM1, which belongs to the Forkhead box (Fox) protein superfamily, is one of the proliferation-associated transcription factors [32,33]. Previous evidence reveals that overexpression of FOXM1 has been implicated in proliferation, metastasis, epithelial-mesenchymal transition (EMT), chemoresistance, and poor prognosis of cancers [8,34,35]. Hence, suppression of FOXM1 is regarded to reduce proliferation and induce apoptosis [36,37]. Previous studies demonstrated that FOXM1 is a key regulator of G1, S, and G2/M progression [38], and so overexpression of FOXM1 has been shown to promote cell cycle progression [39,40]. Also, FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the improvement of cell cycle progression and cell proliferation [41,42]. Here, Moracin D inhibited the expression of FOXM1 and cyclin D1 in MDA-MB-231 cells, but not in MCF-7 cells, indicating FOXM1 mediated inhibition of cyclin D1. Emerging evidences suggest that FOXM1 upregulates antiapoptotic genes such as Bcl-2 [43] and XIAP [44]. In our study, Moracin D decreased the expression of Bcl-2 and XIAP in MDA-MB-231 cells.

Moracin D Preparation
Moracin D was purchased from Chem Faces Biochemical (Wuhan, China).

Cytotoxicity Assay
The cytotoxicity of Moracin D was evaluated using MTT assay. Cells (1 × 10 4 cells/well) were seeded onto 96-well plate and exposed to various concentrations of Moracin D for 24 h. The cells were incubated with MTT (1 mg/mL) (Sigma Chemical, St. Louis, MO, USA) for 2 h and then exposed to dimethyl sulfoxide (DMSO) for 20 min. Finally, optical density (OD) was measured using a microplate reader (Molecular Devices Co., San Jose, CA, USA) at 570 nm.

Cell Cycle Analysis
Cells (1 × 10 6 cells/mL) were treated with Moracin D (0, 8 or 16 µM) for 24 h, washed with phosphate buffered saline (PBS) and fixed in 70% ethanol at −20 • C. The cells were treated with RNase A (10 mg/mL) for 40 min at 37 • C and stained with propidium iodide (50 µg/mL). The stained cell DNA contents were performed in a fluorescence-activated cell sorting (FACS) calibur (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest Software.

Co-Immunoprecipitation
MDA-MB-231 cells were lyzed in lysis buffer and then were immuneprecipitated with FOXM1 antibody or normal immunoglobulin G antibody. Protein A/G sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied. The last precipitated proteins were subjected to immunoblotting with the indicated antibodies.

Statistical Analysis
For statistical analysis of the data, Sigmaplot version 12 software (Systat Software Inc., San jose, CA, USA) was used. Student's t-test was used for comparison of two groups. The statistically significant difference was set at p values of <0.05 between control and Moracin-D treated groups. All data were expressed as means ± standard deviation (SD).

Conflicts of Interest:
The authors declare no conflict of interest.