Corilagin Represses Epithelial to Mesenchymal Transition Process Through Modulating Wnt/β-Catenin Signaling Cascade

Corilagin (CLG), a major component of several medicinal plants, can exhibit diverse pharmacological properties including those of anti-cancer, anti-inflammatory, and hepatoprotective qualities. However, there are no prior studies on its potential impact on the epithelial-to-mesenchymal transition (EMT) process. EMT can lead to dissemination of tumor cells into other organs and promote cancer progression. Hence, we aimed to investigate the effect of CLG on EMT and its mechanism(s) of action in tumor cells. We noted that CLG reduced the expression of various epithelial markers and up-regulated the expression of Occludin and E-cadherin in both basal and TGFβ-stimulated tumor cells. CLG treatment also abrogated cellular invasion and migration in colon and prostate carcinoma cells. In addition, CLG effectively attenuated the Wnt/β-catenin signaling cascade in TGFβ-stimulated cells. Overall, our study suggests that CLG may function as and effective modulator of EMT and metastasis in neoplastic cells.


Introduction
Metastasis remains a vital hallmark by which cells originate and spread to distant institutions [1,2]. When cancer occurs, mutated cells undergo rapid proliferation in tissues of origin and then break through the basement membranes to migrate to other organs and can re-localize as a secondary metastatic cancer [3][4][5]. It is estimated that around 90% of the cancer cells in cancer patients are caused by metastasis [6,7]. These findings highlight the urgent requirement to further understand mechanisms regulating metastasis and to develop pharmacological strategies to target this process.
The epithelial-to-mesenchymal transition (EMT) can facilitate conversion of epithelial cells into mesenchymal cells and promote metastasis [8][9][10][11]. During the EMT process, the cancer cells generally have reduced the levels of epithelial markers, and display an augmentation in the levels of mesenchymal markers [12,13]. The EMT process can be triggered by various stimuli, including transforming growth factor-β (TGFβ), epidermal growth factor (EGF), and diverse signaling pathways such as wingless secreted glycoprotein (Wnt)/β-catenin, and have been implicated in cancer regulation [14,15]. Activation of the TGFβ signaling pathway is of major importance for the initiation of EMT [15,16]. TGFβ-stimulated cells can exhibit spindle-like morphology leading to a decrease of cellular polarity. In addition, the activation of TGFβ pathway can often lead to tumor progression and drug resistance [17].
A number of agents derived from Mother Nature have been reported to be efficacious against tumor growth and progression [18][19][20][21][22]. Corilagin (CLG) is one such unique component of the tannin family [23], which can be found in a wide range of medical plants such as Longan, Lumnitzera racemose, Terminalia catappa L, and Phyllanthus species [24][25][26]. CLG has been reported to possess many pharmacological and biological properties including anti-inflammatory, hepatoprotective, anti-microbial, antihypertensive, antidiabetic, and anti-tumor activities [27][28][29][30][31][32][33][34]. Recently, the anti-tumor effect of CLG has been focus of great interest in cancer biology [35][36][37]. It was found to attenuate cell proliferation by promoting reactive oxygen species (ROS)-dependent apoptosis and autophagy in breast and gastric cancer cells [38,39]. Jia et al. reported that CLG inhibited cell growth through TGFβ/Akt/ERK/Smad signaling pathways in ovarian cancer [36]. In addition, CLG could cause apoptosis via both the mitochondrial apoptotic pathway and death receptor pathway in hepatocellular carcinoma cells [40]. However, the actions of CLG on the regulation of EMT have not been deciphered previously, and this aspect has been studied in this report.

Cell Lines and Culture Conditions
Human colon carcinoma SNU-C2A cells, human prostate carcinoma DU145 cells, human breast carcinoma MCF-7 cells, human normal prostate RWPE-1 cells, and human normal breast MCF-10A cells were obtained from American Type Culture Collection (Manassas, VA, USA). These cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Human normal colon CCD-18Co cells were obtained from Korean Cell Line Bank (Seoul, Korea) and cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

MTT Assay
The viability of SNU-C2A and DU145 cells was measured using an MTT assay to detect NADH-dehydrogenase activity as elaborated before [41].

Western Blot Analysis
The cells were treated with CLG and TGFβ for the indicated concentrations and time points and western blotting was done as reported earlier [42] 2.5. Immunocytochemistry SNU-C2A and DU145 cells were treated with CLG and TGFβ for indicated concentrations and time points. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min and immunocytochemistry was done as described previously [43].

Real-Time Cell Proliferation Analysis
To measure cell growth, proliferation assay was performed by xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany) as elaborated previously [44].

Boyden Chamber Assay
Invasion capacity of SNU-C2A and DU145 cells was determined using a 48-well micro chemotaxis Boyden chamber (Nuero Probe, Cabin John, MD, USA) as described before [45].

Wound Healing Assay
To measure cell migration, wound healing assay was performed. Monolayer of SNU-C2A, DU145, and PC-3 cells were scratched and treated with CLG as per the protocol described earlier [46].

Cell Transfection with β-Catenin siRNA
SNU-C2A and DU145 cells were transfected with β-catenin siRNA or scrambled siRNA using iN-fect TM in vitro Transfection Reagent (iNtRON Biotechnology, Seongnam, KOREA) for 24 h, and thereafter, western blot analysis was carried out.

Statistical Analysis
The results were expressed as means ± SD, and an analysis of variance (ANOVA) with Bonferroni's test was used for the statistical analysis.

CLG Modulated the Expression of EMT Markers in Tumor Cells
The structure of CLG has been depicted in Figure 1A. The cytotoxic action of CLG in SNU-C2A, CCD-18Co, DU145, RWPE-1, MCF-7, and MCF-10A cells was first determined. Interestingly, CLG did not exhibit any marked effect on cell viability in human colon, prostate, breast carcinoma, and normal epithelial cells. The results demonstrated that the cytotoxicity of CLG was less than 10% at 20 µM concentration ( Figure 1B). Hence, in vitro experiments were carried out below the 20 µM dose. We also determined whether CLG can modulate the levels of EMT markers. Interestingly, the levels of MnSOD, Fibronectin, Vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail was suppressed by CLG. However, the levels of Occludin and E-cadherin were increased ( Figure 1C-F). Immunohistochemical analysis as shown in Figure 1G,H, also revealed that the expression of MnSOD and Snail was down-regulated, whereas expression of Occludin was increased in SNU-C2A and DU145 cells.

CLG Effectively Attenuated Cellular Invasion and Migration
Invasive activity of SNU-C2A and DU145 cells was investigated by Boyden chamber assay. As shown in Figure 2A,C, CLG was observed to attenuate invasion of cells.

CLG Effectively Attenuated Cellular Invasion and Migration
Invasive activity of SNU-C2A and DU145 cells was investigated by Boyden chamber assay. As shown in Figure 2A and C, CLG was observed to attenuate invasion of cells. In addition, migration was examined by wound healing assay. The results showed that CLG significantly reduced migratory ability in the treated cells ( Figure 2B and D). In addition, migration was examined by wound healing assay. The results showed that CLG significantly reduced migratory ability in the treated cells ( Figure 2B,D).

CLG Abrogated TGFβ-Induced EMT Cascade
Next, we determined whether CLG can also modulate TGFβ-driven EMT in SNU-C2A and DU145 cells. Western blotting data revealed that exposure to TGFβ up-regulated levels of various proteins regulating EMT and invasion processes. Interestingly, CLG treatment reversed TGFβ-induced up-regulation of these oncogenic proteins ( Figure 2E,G). Moreover, TGFβ down-regulated occludin and E-cadherin expression and CLG treatment could reverse these changes effectively ( Figure 2F,H). In addition, immunocytochemistry data showed that, CLZ suppressed TGFβ-induced MnSOD and Snail levels, whereas it enhanced occludin levels ( Figure 3A,B).

CLG Abrogated TGFβ-Induced EMT Cascade
Next, we determined whether CLG can also modulate TGFβ-driven EMT in SNU-C2A and DU145 cells. Western blotting data revealed that exposure to TGFβ up-regulated levels of various proteins regulating EMT and invasion processes. Interestingly, CLG treatment reversed TGFβinduced up-regulation of these oncogenic proteins ( Figure 2E and G). Moreover, TGFβ downregulated occludin and E-cadherin expression and CLG treatment could reverse these changes effectively ( Figure 2F and H). In addition, immunocytochemistry data showed that, CLZ suppressed TGFβ-induced MnSOD and Snail levels, whereas it enhanced occludin levels ( Figure 3A and B).

CLG Suppressed TGFβ-Induced Metastatic Effects
It was also noted that CLG suppressed TGFβ-induced cellular invasion in SNU-CA and DU145 cells ( Figure 3C,D). TGFβ-treated cells displayed enhanced invasive property, whereas CLG treatment prevented TGFβ-induced invasion. In addition, wound healing assay data revealed that TGFβ induced migration, but CLG suppressed this activity significantly ( Figure 4A,B).
Biomolecules 2020, 10, x 7 of 13 It was also noted that CLG suppressed TGFβ-induced cellular invasion in SNU-CA and DU145 cells ( Figure 3C and D). TGFβ-treated cells displayed enhanced invasive property, whereas CLG treatment prevented TGFβ-induced invasion. In addition, wound healing assay data revealed that TGFβ induced migration, but CLG suppressed this activity significantly ( Figure 4A and B).

CLG Down-Regulated TGFβ-Induced Activation of Wnt/β-Catenin Pathway
We investigated whether CLG could affect the Wnt/β-catenin signaling pathway in SNU-C2A and DU145 cells. As shown in Figure 4C,D, CLG substantially suppressed TGF β-induced the levels of β-catenin, Wnt3a, FZD-1, Axin-1, and GSK-3β activation. We also performed a β-catenin knockdown study to confirm the relationship between CLG-induced modulation of Wnt/β-catenin pathway and EMT proteins. As shown in Figure 4E, β-catenin, Fibronectin, and Vimentin expression was suppressed in β-catenin siRNA transfected cells. However, the expression of these proteins was not altered substantially in the cells transfected with scrambled siRNA, thus indicating that β-catenin may act as a potential molecular target primarily affected by CLG.

Discussions
Corilagin (CLG) can be isolated from wide range of medical plants [23,24] and can display many biological activities including those of anti-inflammatory, hepatoprotective, anti-microbial, antihypertensive, antidiabetic, and anti-tumor [27][28][29][30][31][32][33][34], but its impact on EMT has not been well characterized yet. We report here for the first time that CLG reduced levels of different mesenchymal markers and enhanced that of epithelial markers (occludin and E-cadherin) in both SNU-C2A and DU145 cells. In addition, we found that CLG abrogated TGFβ-induced expression of various proteins regulating EMT, whereas it increased TGFβ-promoted reduction in occludin and N-cadherin levels. Interestingly, CLG-induced alteration of EMT was observed to be associated with attenuation of invasion and migration. In addition, CLG mitigated constitutive as well as TGFβ-induced Wnt/β-catenin signaling pathway activation in tumor cells.
EMT can alter the levels of different epithelial markers and then cause transformation into mesenchymal state via up-regulating the levels of important markers such as Fibronectin, Vimentin, N-cadherin [47,48]. Consequently, these mesenchymal cells can exhibit enhanced motility and have increased ability to undergo metastasis [49]. In addition, previous reports have found that polyphenol and resveratrol can effectively suppress cell migration and invasion in human prostate and colorectal carcinoma cells [50,51]. Moreover, resveratrol also modulated the expression of EMT-related markers such as E-cadherin and Vimentin in DU145 and PC3 cells [50]. We observed that CLG down-regulated MnSOD, fibronectin, Vimentin, and N-cadherin in SNU-C2A and DU145 cells. The levels of occludin and E-cadherin were also effectively up-regulated by CLG in these cells. In addition, CLG down-regulated the levels of E-cadherin repressor proteins, Twist and Snail. Matrix metalloproteinases (MMP) family such as MMP-9 and MMP-2 can play an important role in cancer proliferation, invasion, and metastasis [52][53][54]. We observed that CLG inhibited the level of both MMP-9 and MMP-2 proteins, thus implicating that the negative regulation of invasion and metastasis by CLG may be mediated by down-regulation of these two proteins. Overall, alteration of diverse mesenchymal markers and epithelial markers by CLG can abrogate EMT in tumor cell lines.
TGFβ can regulate EMT process and tumorigenesis, thus leading to enhanced motility and invasion [55]. Previous studies have reported that mesenchymal markers were found to be substantially overexpressed and epithelial markers were suppressed in TGFβ-stimulated cells [8,43,44]. We also noted that CLG suppressed the levels of up-regulated EMT-associated proteins and down-regulated E-cadherin and occludin expression in TGFβ-induced SNU-C2A and DU-145 cells. We further observed that CLG altered the morphological transformation as well as migration and invasion induced by TGFβ. Our findings suggest that CLG can effectively target TGFβ-induced EMT and subsequent downstream phenotypic changes in tumor cells.