MnTE-2-PyP Attenuates TGF-β-Induced Epithelial-Mesenchymal Transition of Colorectal Cancer Cells by Inhibiting the Smad2/3 Signaling Pathway

Background As a key step in enhancing cancer cell invasion and metastasis, epithelial-mesenchymal transition (EMT) plays an important role in colorectal cancer progression. EMT is triggered by a variety of signaling pathways, among which the transforming growth factor β (TGF-β) signaling pathway has been implicated as a primary inducer. Accumulating evidence demonstrates that MnTE-2-PyP (chemical name: manganese(III) meso-tetrakis-(N-ethylpyridinium-2-yl), a superoxide dismutase (SOD) mimetic, inhibits TGF-β signaling; however, its ability to inhibit TGF-β-induced EMT in colorectal cancer has not yet been explored. Methods To verify our hypothesis that MnTE-2-PyP attenuates TGF-β-induced EMT, human colorectal cancer cells were treated with TGF-β in the presence or absence of MnTE-2-PyP. Cells were analyzed by several techniques including western blotting, real-time quantitative PCR, transwell assay, and wound healing assay. Results MnTE-2-PyP reverses cell phenotypes induced by TGF-β in colon cancer cells. MnTE-2-PyP treatment significantly reduced the expression of mesenchymal markers but maintained epithelial marker expression. Mechanistically, MnTE-2-PyP suppressed the phosphorylated Smad2/3 protein levels induced by TGF-β in SW480 cells, but MnTE-2-PyP failed to suppress TGF-β-induced Slug and Snail expression in colorectal cells. Furthermore, MnTE-2-PyP effectively suppressed TGF-β-mediated cell migration and invasion and the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in colorectal cells. Conclusion Taken together, we provide an in-depth mechanism by which MnTE-2-PyP inhibits colorectal cancer progression, supporting an important role for MnTE-2-PyP as an effective and innovative antitumor agent to enhance treatment outcomes in colorectal cancer.


Introduction
Colorectal cancer is one of the deadliest cancers worldwide because of its local invasion and distant metastasis [1]. As a key step in enhancing cancer cell invasion and metastasis, EMT refers to the reprogramming of epithelial cells to a mesenchymal-like phenotype [2][3][4]. EMT is mediated by a set of transcription factors such as Slug, Snail, Twist, and Zeb1/2, which can inhibit the expression of the epithelial marker E-cadherin and induce the expression of mesenchymal markers including N-cadherin, vimentin, and fibronectin [1,[5][6][7]. A variety of signaling pathways can trigger EMT by regulating those transcription factors, specifically the TGF-β signaling pathway that has been implicated as a primary inducer [8][9][10][11]. In TGF-β signaling pathways, the activated Smad2 and Smad3 combine with Smad4 to form Smad transcription factor complexes, which subsequently shuttle into the nucleus to regulate gene expression. Also, TGF-β induces EMT through non-Smad pathways such as MAPK, PI3K/AKT, GTPases, NF-κB, and HIF-1 pathways. TGF-β signaling pathway components, including the ligands, receptors, and Smad proteins, are regulated by a variety of mechanisms [11][12][13][14][15]. Oxidative stress has been proposed as one of these mechanisms that enhance TGF-β-induced EMT in numerous types of cells [16,17].

Western Blotting.
Total protein was isolated from cells, and protein concentration was determined by a Coomassie protein assay (Pierce, Rockford, IL, USA). The protein for each cell lysate was separated using SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membranes. After blocking in skim milk at room temperature for 30 min, the membranes were incubated with specific primary antibodies at 4°C overnight. Next, the membranes were incubated with a secondary antibody (1 : 5000 dilution, Wuhan Boster Biological Technology Ltd., Wuhan, China) at room temperature for 2 h. β-Actin was used as the internal control.

Transwell
Assay. The invasive ability was evaluated using a transwell assay (8 μm pore size; Corning Inc., Corning, NY, USA). The transwell top chambers were coated with 100 μl of 1 mg/ml Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). A cell suspension was prepared in serum-free medium; then, 1 × 10 4 cells/well were seeded into the upper chamber of each insert and medium containing 10% FBS was placed into the lower chambers. After incubation for 24 h at 37°C in a 5% CO 2 humidified incubator, noninvasive cells in the upper chamber were carefully removed with a cotton swab and invasive cells on the bottom were fixed and stained with 0.05% crystal violet. Cell numbers from five random fields were counted and expressed as the average number of cells/field. Three independent experiments were performed.

Wound Healing Assay.
For the wound healing assay, all cell fractions were cultured in six-well plates. Cell monolayers were wounded by scratching with sterile micropipette tips. The migration of cells to cover the wound space was examined and photographed at indicated time points (0, 24, and 48 hours) in five random microscopic regions with the Carl Zeiss Axio Observer Z1 microscope. The migrated distance was analyzed by measuring distances between wounded margins at three randomly chosen points.

Statistical Analysis.
Values were presented as means ± SD of measurements of at least three independently performed experiments to avoid possible variation of cell cultures. An ANOVA was performed, and a secondary test between two groups was determined by Student's t-test using the SPSS 19 software program. Statistical significance was defined as P value < 0.05.

MnTE-2-PyP Inhibits TGF-β-Induced Morphological
Changes in Colorectal Cancer Cells. TGF-β has been implicated as a primary inducer of EMT in colorectal cancer. As shown in Figure 1, morphologic changes in LOVO, HT29, and SW480 cells were observed after two days of TGF-β (5 ng/ml) treatment. The cells lost their polarized epithelial phenotype with increased cell-cell close connections and acquired mesenchymal traits. They became dispersed and displayed a fibroblast-like appearance with a long shape and a central nucleus. treatment significantly inhibited TGF-β-induced morphologic changes in LOVO, HT29, and SW480 cells. After treatment with MnTE-2-PyP, these cells kept a more epithelial-like appearance even if induced by TGF-β ( Figure 1). Based on these phenomena, we speculated that MnTE-2-PyP might attenuate TGF-β-induced EMT, which has been demonstrated to have an important role in cancer cell migration and invasion.

MnTE-2-PyP Does Not Inhibit Slug and Snail Expression
Induced by TGF-β. Because the downregulation of E-cadherin is mediated by transcription factors like Slug, Snail, Twist, and Zeb1/2, we investigated the regulation of these factors by the TGF-β signaling pathway. In TGF-β-treated SW480 cells, no changes in the expression of Twist and Zeb1/2 were observed compared with untreated controls (Figure 4). By contrast, TGF-β increased the expression levels of Slug and Snail (Figure 4). This is consistent with the recent publications that Snail and Slug are highly expressed in colorectal cancer [8,10]. In addition, the contribution of Snail and Slug to the repression of E-cadherin and to EMT in vivo and in vitro has been demonstrated in colorectal cancer models [8,10,11,28]. While the administration of MnTE-2-PyP could not reverse the changes in Snail and Slug expression induced by TGF-β, our results suggest that the induction of Snail and Slug upon TGF-β treatment is not impaired by MnTE-2-PyP.
3.6. MnTE-2-PyP Reverses the Expression of MMP-2 and MMP-9 Induced by TGF-β. To clarify the mechanism of TGF-β-induced migration and invasion, we performed the comprehensive analysis of a public colorectal cancer dataset (GSE17538). Here, we identified various TGF-β correlated genes in colorectal cancer. Among them, we discovered matrix metalloproteinases, such as MMP-2 and MMP-9, which are widely involved in tumor metastasis. In line with other studies that TGF-β is strongly correlated with MMP-2 and MMP-9, a positive correlation between TGF-β

Discussion
Manganese(III) porphyrins (MnPs) have been extensively studied due to their anticancer effects, and they have the ability to protect nontumor tissues from ROS-mediated side effects. Recent studies have shown that the Mn porphyrin analogs MnTnHex-2-PyP(5+) and MnTnBuOE-2-PyP(5+) can modulate the metastatic ability of cancers [19,20]. A previous study showed that MnTnHex-2-PyP(5+), by suppressing the phosphorylation of several MAP kinases, enhances radiation-induced apoptosis in mouse breast tumor cells [29]. Furthermore, in the inflammatory breast cancer cell line, the involvement of MnTnBuOE-2-PyP(5+) in ERK and NF-κB signaling led to cell death [30]. Recently, our group revealed that MnTE-2-PyP reduces prostate cancer growth and metastasis in combination with radiation [7,31]. MnTE-2-PyP has also been shown to sensitize tumor tissue to chemotherapy agents [32][33][34]. In a breast cancer model, MnTE-2-PyP suppresses tumor growth via antiangiogenic effects at the level of tumor vasculature [35]. These studies demonstrate that MnTE-2-PyP is an effective antitumor agent. The present study provides novel evidence that MnTE-2-PyP also inhibits cancer cell progression by reversing the EMT process.
have recently shown in a radiation-induced fibrosis model that MnTE-2-PyP inhibits the fibroblast activation pathway by downregulating the expression of TGFβRII, which in turn reduces the activation and/or expression of Smad2, Smad3, and Smad4. In this study, we showed that TGF-β induced an increase in phosphorylated Smad2/3 protein levels in SW480 cells. Our results indicated that MnTE-2-PyP attenuated TGF-β-induced EMT in colorectal cancer cells by inhibiting the Smad2/3 signaling pathway. One of the major negative regulators of the TGF-β-Smad system is Smad7, which has been assumed to serve as a brake of this system [4]. Smad4 and Smad6 are also involved in TGF-β-induced EMT [13,14]. Further studies would aim at determining the effects of MnTE-2-PyP on Smad7, Smad6, and Smad4 in TGF-β-induced EMT. In addition, the effects of MnTE-2-PyP on non-Smad pathways, including MAPK, PI3K/AKT, and GTPase pathways, should also be investigated. Consistent with the important roles of Smads, the loss of epithelial phenotypes and acquisition of mesenchymal features are achieved through a set of transcription factors involving Slug, Snail, Twist, and Zeb1/2 [8][9][10][11]. In our study, changes in the expression of Slug and Snail were observed in TGF-β-treated SW480 cells. The transcription factors Slug and Snail bind to the E-cadherin promoter and repress E-cadherin expression in epithelial cells, leading to EMT. TGF-β induces Slug and Snail expression either through a Smad-dependent mechanism or indirectly through the activation of other transcription factors or relief of repression. We found that MnTE-2-PyP could not suppress TGF-β-induced Slug and Snail expression in colorectal cells. Thus, MnTE-2-PyP reverses TGF-β-induced EMT mainly through the inhibition of the Smad signaling pathway and not through the Slug and Snail in colorectal cells.
The expression of the gelatinases MMP-2 and MMP-9 is increased in colorectal cancer tissues when compared to concomitant normal tissues [36,37]. It has been assumed that MMP-mediated E-cadherin disruption is a key step in tubular cell EMT, and loss of E-cadherin is an important cellular event observed during EMT. MMP-9-mediated degradation of E-cadherin promotes head and neck squamous carcinoma cell migration and invasion [38]. In ovarian carcinoma cells, MMP-9 regulates the posttranslational modification of E-cadherin [39]. Furthermore, MMP-2 and MMP-9 are induced by TGF-β in SW480 colorectal cancer cells [40,41]. Thus, we explored the potential role of MMP-2 and MMP-9 in TGF-β-mediated EMT in colorectal cancer. A positive correlation between TGF-β and MMP-2 or MMP-9 transcript levels was noted in the publicly available dataset GSE17538. Consistent with previous research findings, TGF-β treatment of SW480 cells induced MMP-2 and MMP-9 expression. Treatment of the cells with MnTE-2-PyP markedly inhibited TGF-β-induced MMP-2 and MMP-9 expression. Furthermore, MMP-2 and MMP-9 were closely associated with the poor outcome of cancer patients. These studies indicate that MnTE-2-PyP may increase E-cadherin expression via inhibiting TGF-β-induced MMP-2 and MMP-9.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
Dr. Oberley-Deegan is a consultant with BioMimetix Pharmaceutical Inc. and holds equities in BioMimetix Pharmaceutical Inc. There are no conflicts of interest for the other authors.