Brassinin Represses Invasive Potential of Lung Carcinoma Cells through Deactivation of PI3K/Akt/mTOR Signaling Cascade

The epithelial–mesenchymal transition (EMT) is a phenomenon that facilitates epithelial cells to acquire invasive potential to induce the initiation the metastatic spread of tumor cells. Here, we determined if brassinin (BSN) can affect the EMT process and deciphered its anti-cancer effects. BSN attenuated the levels of EMT linked genes and suppressed transforming growth factor beta (TGF-β)-mediated regulation of diverse mesenchymal markers. Additionally, BSN did increase the expression of various epithelial marker proteins in lung cancer cells. TGF-β-induced morphological changes and induction of invasive ability of tumor cells was also found to be abrogated by BSN treatment. Finally, BSN not only suppressed constitutive, but also inducible phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) phosphorylation in tumor cells.


Introduction
Lung carcinoma is a malignant tumor often characterized by abnormal growth and metastasis [1]. The aberrant growth of tumor cells can extend outside the lungs by the process of metastasis and invasion of neighboring tissues or distant organs within the body [2][3][4][5][6]. Primary lung tumors can generally metastasize to the brain, bones, liver, and adrenal glands [7]. It has been found that metastasis rather than primary tumor accounts for the majority of fatalities among cancer patients [8][9][10][11].
The epithelial-to-mesenchymal transition (EMT), although required for various physiological processes, can also contribute pathologically to fibrosis and cancer progression [12][13][14]. Even though EMT is usually tranquilized in adulthood, it can promote transformation of tumor cells to an invasive and metastatic phenotype in lung cancer once stimulated by the growth factor signaling pathway [2]. Generally, during the EMT process, an augmentation of levels of mesenchymal markers, and down modulation of epithelial markers, is frequently encountered [15][16][17].
Molecules 2019, 24 Various indole phytoalexins, identified from cruciferous (Brassica) vegetables, are important source of anti-neoplastic agents because they can exert several biological activities, such as anti-proliferative and anti-carcinogenic activities [18][19][20]. Among these phytoalexins, brassinin (BSN) was isolated from Chinese cabbage [21] and was found to suppress TNFα-induced vascular inflammation via modulation of reactive oxygen species (ROS) production in endothelial cells [22]. Its derivative, homobrassinin, has also been reported to cause ROS-dependent apoptosis in colorectal cancer cells [23]. Our group has also reported that BSN can inhibit STAT3 signaling cascades through multiple molecular mechanism(s) and also attenuate tumor growth under in vivo settings [24]. Our previous report has also indicated that BSN induced apoptosis by affecting Akt activation in prostate cancer cells [25]. The combination of BSN and capsaicin can exhibit synergistic apoptotic and anti-metastatic activities in prostate cancer cells [26]. However, there are no major studies regarding the actions of BSN on the modulation of EMT biomarkers, therefore this project aimed to decipher the effect of this phytoalexin on constitutive and inducible EMT processes in malignant cells.

BSN Modulates EMT in Tumor Cells
First, we investigated the levels of EMT markers by Western blot analysis. BSN downregulated the levels of fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail proteins ( Figure 1B). However, it was found that BSN upregulated occludin and E-cadherin expression ( Figure 1B). Moreover, we determined the levels of EMT markers by real-time quantitative PCR analysis. BSN reduced the mRNA level of fibronectin and vimentin and enhanced that of E-cadherin ( Figure 1C). Furthermore, as observed by immunocytochemistry, drug treatment caused a decrease of vimentin and N-cadherin levels, as well an increase of the E-cadherin level ( Figure 1D). The expression of various genes was studied by real-time quantitative polymerase chain reaction (PCR). Data represent means ± SD; *** p < 0.001 vs. non-treated (NT) cells, and ** p < 0.01 vs. non-treated (NT) cells. (D) Cells were treated as indicated before and levels of EMT markers were determined by immunocytochemistry. Quantitative analysis of the fluorescence intensities was performed.

BSN Regulates TGF-β-Induced EMT in Malignant Cells
The levels of EMT markers were also examined in TGF-β-treated cells. As shown in Figure 2A, TGF-β exposure augmented expression of fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail, while it downregulated occludin and N-cadherin compared with non-treated cells. BSN suppressed TGF-β-induced fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail overexpression, whereas it substantially upregulated TGF-β-induced occludin and N-cadherin reduction. Also, Figure 2B revealed that identical changes in mRNA level were also observed as noted in EMT protein expression patterns. Moreover, immunocytochemistry data showed that

BSN Regulates TGF-β-Induced EMT in Malignant Cells
The levels of EMT markers were also examined in TGF-β-treated cells. As shown in Figure 2A, TGF-β exposure augmented expression of fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail, while it downregulated occludin and N-cadherin compared with non-treated cells. BSN suppressed TGF-β-induced fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail overexpression, whereas it substantially upregulated TGF-β-induced occludin and N-cadherin reduction. Also, Figure 2B revealed that identical changes in mRNA level were also observed as noted in EMT protein expression patterns. Moreover, immunocytochemistry data showed that vimentin and N-cadherin level increased and that of E-cadherin decreased in TGF-β-treated cells. BSN treatment also decreased TGF-β-stimulated vimentin and N-cadherin levels, whereas it increased the TGF-β-induced E-cadherin level ( Figure 2C).
Molecules 2019, 24 FOR PEER REVIEW 4 vimentin and N-cadherin level increased and that of E-cadherin decreased in TGF-β-treated cells. BSN treatment also decreased TGF-β-stimulated vimentin and N-cadherin levels, whereas it increased the TGF-β-induced E-cadherin level ( Figure 2C).

BSN Suppresses the Proliferation of Tumor Cells
To test the cytotoxic ability of BSN, we used MTT and the result showed that BSN exhibited less than 5% cytotoxicity in the cells up to 50 µM concentration ( Figure 3A). Interestingly, BSN was also found to significantly attenuate cellular growth in the tested cell lines ( Figure 3B).

BSN Inhibits Invasive and Migratory Capacity of Tumor Cells
To analyze the consequence of BSN treatment on the invasive ability of lung carcinoma cells, invasion assay was carried out. As shown in Figure 3C, BSN attenuated invasive activity in both A549 and H1299 cells. Next, we investigated the role of BSN in reducing the migratory ability of tumor cells, and as shown in Figure 3D,E, non-treated cells were noted to migrate significantly faster than BSN treated cells.
(C) The cells were treated as described above and immunocytochemistry was carried out. Quantitative analysis of the fluorescence intensities was performed.

BSN Suppresses the Proliferation of Tumor Cells
To test the cytotoxic ability of BSN, we used MTT and the result showed that BSN exhibited less than 5% cytotoxicity in the cells up to 50 μM concentration ( Figure 3A). Interestingly, BSN was also found to significantly attenuate cellular growth in the tested cell lines ( Figure 3B).

BSN Inhibits Invasive and Migratory Capacity of Tumor Cells
To analyze the consequence of BSN treatment on the invasive ability of lung carcinoma cells, invasion assay was carried out. As shown in Figure 3C, BSN attenuated invasive activity in both A549 and H1299 cells. Next, we investigated the role of BSN in reducing the migratory ability of tumor cells, and as shown in Figure 3D,E, non-treated cells were noted to migrate significantly faster than BSN treated cells.

BSN Represses TGF-β-Induced Metastatic Effects
We also analyzed how BSN and TGF-β can affect EMT process. We took a picture of the possible morphological changes with TGFβ, BSN, or combination group of the two ( Figure 4A). TGF-β-treated cells were found to be more elongated than non-treated cells and displayed a spindle like shape. However, BSN treatment prevented TGF-β-induced morphological changes, including spindle shaped morphology. In addition, we examined the role of BSN in reduction of TGF-β-activated cell invasion and migration. As shown in Figure 4B, TGFβ enhanced cell invasion, however, BSN was found to significantly down-regulate TGF-β-induced invasion. As shown in Figure 4C, wound healing assay was also investigated with TGF-β, BSN, or combination group. TGF-β-treated cells showed more wound closure and had less gap difference than non-treated cells. Moreover, BSN also inhibited TGF-β-induced migration in both lung tumor cells.

BSN Affects Activation of Multiple Oncogenic Molecules
We next investigated whether BSN modulated PI3K/Akt/mTOR/p70S6K/4E-BP1 phosphorylation in tumor cells. As shown in Figure 5A,B, BSN substantially downregulated both constitutive and TGF-β-induced Pl3K, Akt, mTOR, p70S6K, and 4E-BP1 activation. We examined whether overexpression of constitutive active Akt by pcDNA3-Akt plasmid can prevent the suppressive effect of BSN on EMT-related protein expression. First, A549 and H1299 cells were transfected with pcDNA3-Akt or pcDNA3 plasmid vectors. As shown in Figure 5C, BSN treatment in transfected cells slightly suppressed the phosphorylation of Akt, vimentin, and snail expression. Additionally, our previous study has demonstrated that treatment with BSN (200 µM) in A549 and H1299 cells suppressed the constitutive and inducible STAT3 signaling pathway [24]. Here, we found that treatment of BSN 50 µM inhibited the phosphorylation of Akt. However, BSN at 50 µM dose could not suppress both constitutive and inducible STAT3 activation ( Figure 5D).

Discussion
This study deciphers the possible actions of BSN on the EMT-related cell signaling cascade. We found that BSN downregulated the levels of various mesenchymal markers and upregulated that of epithelial markers, and it can also substantially suppress TGF-β-induced fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail expression, whereas it upregulated TGF-β-induced occludin and N-cadherin levels. Moreover, the suppression of EMT by BSN was found to cause an abrogation of proliferation and invasion. Interestingly, BSN inhibited not only constitutive but also inducible PI3K/Akt/mTOR/p70S6K/4E-BP1 phosphorylation in tumor cells.
EMT can cause loss of polarity in epithelial cells and downregulate the levels of epithelial markers, such as occluding, and then lead to the formation of mesenchymal cells via upregulation of the levels of markers, such as fibronectin, vimentin, etc. [27,28]. We observed for the first time that BSN downregulated fibronectin, vimentin, N-cadherin, Twist, and Snail levels at 50 µM concentration, while no toxic effect was detected at these concentrations. In addition, BSN substantially reduced the levels of the E-cadherin repressor proteins, Twist and Snail. Overall, findings suggest that the modulation of mesenchymal markers and epithelial markers by BSN can lead to blockage of EMT in lung carcinoma cells.
Matrix metalloproteinases (MMP)-9 and MMP-2 proteins can actively mediate the dissemination of tumor cells to distant sites [29,30]. Overexpression of MMP-9 and MMP-2 can also act as markers of metastasis and poor prognosis in patients [31,32]. We found that BSN substantially inhibited the MMP-9 and MMP-2 levels, thereby suggesting that the anti-metastatic effects displayed by BSN could be possibly mediated through the reduced activity of these two endopeptidases.
It was noted that this phytoalexin also suppressed the levels of upregulated mesenchymal markers and downregulated E-cadherin and occludin levels upon TGF-β treatment. We observed that BSN attenuated the conversion into a spindle-like morphology caused by TGF-β exposure Additionally, cellular invasion and migration promoted by TGF-β was attenuated by exposure to BSN in tumor cells.
TGF-β has been found to cause induction of multiple oncogenic cascades, including PI3K, as well as Ras and Rho GTPases cascades [33], and thus drive the EMT process by controlling levels of diverse genes that can control metastasis. Our previous reports already suggest that PI3K/Akt/mTOR signaling cascade may have a crucial role in TGF-β-driven EMT phenomena [4,6]. In this study, we also found that BSN can substantially abrogate TGF-β-induced activation of these oncogenic kinases ( Figure 6). However, additional experiments are required to decipher the different mechanisms underlying the beneficial actions of BSN against the metastatic spread of malignant cells.

Immunocytochemistry
A549 and H1299 cells were seeded in an 8-well glass chamber slide, and were treated with BSN for 24 h. Both cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, washed three times with 1× phosphate-buffered saline (PBS), and permeabilized with 0.2% Triton-X-100. Then, they were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h and incubated overnight at 4 • C with anti-Vimentin, anti-N-cadherin, and anti-E-cadherin (1:100; Santa Cruz, CA, USA). The next day, cells were washed three times by 1× PBS and incubated with Alexa Fluor ® 488 donkey anti-goat IgG (H+L) antibody and Alexa Fluor ® 594 donkey anti-rabbit IgG (H+L) antibody for 1 h at room temperature. The cells were stained with 4 ,6-diamidino-2-phenylindole (DAPI) (1 µg/mL) for 3 min at room temperature and mounted on glass slides using Fluorescent Mounting Medium (Golden Bridge International Labs, Mukilteo, WA, USA). Finally, the fluorescence signal was detected by using an Olympus FluoView FV1000 confocal microscope (Tokyo, Japan) [4].

Real-Time Cell Proliferation Analysis
Cell growth behavior was performed using the Roche xCELLigence Real-Time Cell Analyzer (RTCA) dual purpose (DP) instrument (Roche Diagnostics GmbH, Germany), as described previously. The cell growth pattern was determined as elaborated previously [4].

Invasion Assay
Instrument real-time monitoring of cellular invasion was performed using the Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany). The RTCA DP instrument used CIM (cellular invasion/migration)-plate 16 and measured electrical measurements. The upper chamber of the CIM-plates was coated with matrigel (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA) and medium containing 10% FBS was placed in the lower chamber as a chemoattractant. Then, upper and lower chamber plates were assembled. Serum-free medium was placed in the upper chamber and incubated for 1 h at 37 • C and background impedance measurement was performed. After background measurement, A549 and H1299 cells (1 × 10 5 cells/well) were seeded onto the upper chamber and the electrical impedance of the membrane was recorded every 15 min [4].

Wound Healing Assay
Wound healing assay was performed in the monolayers of cells. The cells (2 × 10 5 cells/well) were plated in a 12-well plate and incubated until 80% confluence was reached. Then, the cell monolayer was scratched by a 200 µL micropipette tip and washed with serum-free medium. Next, cells were treated with BSN and incubated with serum-free medium for 24 h. The width of the wound was observed by using a microscope (Nikon ECLIPSE Ts2., Tokyo, Japan) at time 0 and 24 h. Gap distance of the wound was measured at four different sites and non-treated samples were used as controls [4].

Statistical Analysis
All experiments are presented as the mean ± standard deviation (SD). Statistical significance was analyzed by Mann-Whitney U test.