Antroquinonol Targets FAK-Signaling Pathway Suppressed Cell Migration, Invasion, and Tumor Growth of C6 Glioma

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine that is overexpressed in many types of tumors and plays a pivotal role in multiple cell signaling pathways involved in cell survival, migration, and proliferation. This study attempts to determine the effect of synthesized antroquinonol on the modulation of FAK signaling pathways and explore their underlying mechanisms. Antroquinonol significantly inhibits cell viability with an MTT assay in both N18 neuroblastoma and C6 glioma cell lines, which exhibits sub G1 phase cell cycle, and further induction of apoptosis is confirmed by a TUNEL assay. Antroquinonol decreases anti-apoptotic proteins, whereas it increases p53 and pro-apoptotic proteins. Alterations of cell morphology are observed after treatment by atomic force microscopy. Molecular docking results reveal that antroquinonol has an H-bond with the Arg 86 residue of FAK. The protein levels of Src, pSrc, FAK, pFAK, Rac1, and cdc42 are decreased after antroquinonol treatment. Additionally, antroquinonol also regulates the expression of epithelial to mesenchymal transition (EMT) proteins. Furthermore, antroquinonol suppresses the C6 glioma growth in xenograft studies. Together, these results suggest that antroquinonol is a potential anti-tumorigenesis and anti-metastasis inhibitor of FAK.


Introduction
Focal adhesion kinase (FAK), a protein tyrosine kinase, localizes to focal adhesions and is involved in several cellular functions such as survival, invasion, motility, adhesion, metastasis, proliferation, and angiogenesis [1]. FAK is autophosphorylated at tyrosine 397, which results in a high binding affinity site for the SH2 domain of the src family kinases [2,3]. The mutually activated FAK/Src complex then activates a cascade of phosphorylation events in new protein -protein interactions to trigger several signaling pathways that eventually lead to different cellular responses. FAK can recruit SOS into the complex that activates the downstream Ras-MAPK pathway and/or transduces the signal through the activation of the PI3K-Akt cascade [4][5][6]. Recent work shows the active Src/FAK complex stimulates Rac1 activity through the DMEM supplemented with 10% FBS and 1% antibiotics. These cells were tested and authenticated by the provider.

Cell viability assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen) assay is a colorimetric technique that is performed to analyze cell proliferation. Cells (1 x 10 4 cells) were seeded in 96-well plates overnight. Cells were treated with various concentrations of the drug for an additional 24 h, and then 20 μL (5 mg/mL) of MTT solution was added per well and further incubated for 4 h. The medium was removed, and formazan was solubilized by adding 100 μL/well of DMSO (Sigma-Aldrich) and the OD was measured at 570 nm using a microplate reader (ELISA reader, Thermo Labsystems). The percentage of viable cells was compared to untreated control cells.

Cell cycle analysis
The cells (2 x 10 5 ) were seeded in 6-well plates and incubated overnight. Cells were treated with various concentrations of antroquinonol (5 and 10 μM) for an additional 24 h. After treatment, cells were harvested and fixed with ice cold 70% ethanol for 1 h at -20°C. Next, the cells were washed twice with cold PBS and resuspended in 1 mL (v/v) of staining solution containing 20 μg/mL propidium iodide, 0.1% Triton X-100, and 0.2 mg/mL RNase (Bionovas). They were incubated in a water bath at 37°C for 30 min. Lastly, the results were analyzed using a flow cytometer (Beckman Coulter).

TUNEL assay
A terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay was performed according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA). The cells were seeded in chamber slides (Nalge Nunc International, Rochester, NY, USA) and treated with 10 μM antroquinonol for 24 h. After treatment, the cells were fixed in 3.7% formaldehyde at room temperature (RT) for 25 min, and permeabilized in 0.1% Triton 1 X-100 at RT for 5 min. The cells were incubated with the TUNEL reaction mixture (Equilibration Buffer, Nucleotide Mix, and rTdT Enzyme) for 60 min at 37°C in a humidified atmosphere, and DNA fragmentation was detected immuno-histochemically using the DeadEnd TM Fluorometric TUNEL System.

Tapping-mode atomic force microscopy (TM-AFM) scanning
This experiment was measured as previously described [28] with slight modification. Cells were seeded on a cover slip and incubated for 24 h. After treatment with different concentrations of antroquinonol for 24 h, cells were washed with PBS, and fixed with 1% glutaraldehyde for 5 min. The cells were imaged with a bio-atomic force microscope (Bio-AFM, Nanowizard, JPK, Germany) that was mounted on an inverted microscope, TE-2000-U (Nikon, Tokyo, Japan). Silicon nitride non-sharpened probes with a nominal cantilever force constant of 0.06 Nm-1 (DNP-20, Veeco, CA, USA) were used. Imaging was performed using contact mode. Line scan rates varied from 0.5 to 2 Hz.

In vitro FAK [pY397] assay
The pFAK (Y397) kit was purchased from Invitrogen (www.invtrogen.com) and the procedure was performed according to the manufacturer's protocol. The absorbance of samples was read at 450 nm and the readout was plotted on a graph against standard concentrations. One unit is equivalent to the amount of FAK (pY397) autophosphorylated at 300 pg of total FAK protein.

Gelatin zymography for MMP2 and MMP9
The activities of MMP2 and MMP9 were determined by gelatin zymography. Cells (2 x 10 5 ) were seeded in 6-well plates and incubated until reaching 80% confluence. Cells were starved in DMEM containing 0.1% BSA for 6 h, and then they were treated with various concentrations of antroquinonol for 24 h. The supernatants were collected and the protein concentration was quantified by Bradford dye (Bio-Rad). Next, 8% SDS-PAGE gels containing 10% gelatin were prepared for the detection of MMP2 and MMP9. After electrophoresis, gels were washed twice with washing buffer containing 2.5% Triton X-100, and then incubated in developing buffer (0.05 M Tris-HCl, pH 8.8, 5 mM CaCl 2 , 0.02% NaN 3 ) at 37°C for 16 h. Finally, the gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 h, and then destained by fixing buffer (45% methanol, 10% acetic acid). The gels were scanned using an Epson scanner and quantified using multi-gauge software (Fujifilm).

Western blotting
The cells (5 x 10 5 ) were seeded in a 6 cm dish, grown until 80% confluent, and then incubated with various concentrations of antroquinonol for 24 h. Cells were collected and lysed with RIPA buffer. Protein samples were loaded and separated with SDS-PAGE then transferred to the PVDF membrane (PerkinElmer, Turku, Finland). The membranes were incubated with appropriate primary antibodies at 4°C overnight. The membranes were washed 3 times with TBST, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. Finally, the membranes were exposed to ECL reagents (PerkinElmer) for 1 min and the results were analyzed with LAS-3000 film (Fujifilm, Tokyo, Japan). Beta actin was used as an internal control.

Molecular modeling and docking
The experiment was performed as described previously [28]. The ligand (antroquinonol) was docked into the active site using the 'Ligand Fit' option. The docked proteins with low energy were recorded and validated.

Animal xenograft tumor model
Six-week-old athymic male nude mice were purchased from LASCO (Charles River technology, Taipei, Taiwan). All animal experiments were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" of National Dong-Hwa University (Hualien, Taiwan). A total of 5x10 5 C6 cells were injected subcutaneously on the back of nude mice. When the tumor volume reached an average volume of approximately 200 mm 3 , the mice were randomly divided into two groups: 5 mice received intraperitoneal injection of normal saline as control (n = 5) and 5 received intraperitoneal administration of Antroquinonol (0.082 mg/kg/ daily, n = 5) for 10 days. The mice were sacrificed and tumors were excised and weighed. Major organs such as liver, kidney, spleen, and tumor were subjected to histological analysis.

Statistical analysis
The results are presented as the mean ± SD. Data were analyzed with a one-way ANOVA Tukey test for multiple-group comparisons. The significant differences ( Ã p < 0.05), ( ÃÃ p < 0.01), and ( ÃÃÃ p < 0.001) between the means of control and the treatment groups were analyzed.

Cell viability
Fig 1A illustrates the incubation of cells with the desired concentrations of antroquinonol for 24 h to determine the cytotoxic effects of antroquinonol on both the C6 and N18 cell lines, and an MTT assay was performed to analyze cell viability. After 24 h of treatment, the cell viability was significantly decreased with an increasing concentration of antroquinonol. Antroquinonol reached the IC 50 value at a concentration of 10 μM. Hence, this concentration was applied for the subsequent experiments. The cytotoxic effect was further validated on BEAS-2B Human bronchial epithelium normal cell lines for 12 and 24 h, intriguingly antroquinonol treatment did not exhibit any toxicity (0 to 25 μM) concentration ( Fig 1B). This result implies that antroquinonol was more potent cytotoxicity to cancer cells compared with the normal cells.

Cell cycle (TUNEL assay)
The cell-cycle distribution was investigated by flow cytometry analysis. Cells exposed to antroquinonol for 24 h showed a cell cycle arrest at the sub-G1 phase in a dose-dependent manner compared with the untreated cells (Table 1; Fig 2A). Antroquinonol-induced apoptosis in C6 and N18 cells was further determined with a TUNEL assay. Antroquinonol increased the accumulation of green fluorescent apoptotic cells after 24 h treatment ( Fig 2B). This evidence reveals that antroquinonol caused the apoptosis of C6 and N18 cells.

Alterations of pro-apoptotic and anti-apoptotic proteins
The effects of antroquinonol on the expression of apoptotic proteins in N18 and C6 cell lines were determined. The expression of Bad, Bax, Bak, and p53 were significantly increased while the expression of Bcl-2 was decreased after 24 h of antroquinonol treatment (Fig 2C). The effect of antroquinonol on the expression of caspase and PARP, two apoptosis associated proteins were further analyzed. Unexpectly, antroquinonol did not cause any change in the cleavage of caspase and PARP proteins in both cell lines. These results demonstrate that antroquinonol induced apoptosis through the down-regulation of Bcl-2.
Effects of FAK/Src signaling pathway FAK, Src, Cdc42, and Rac1 are involved in the mediation of actin cytoskeleton remodeling, invasion, and cell migration. The expression of these proteins was measured by Western blot analysis. The protein levels of FAK, pFAK, Src, pSrc, Rac1, and Cdc42 were decreased after antroquinonol treatment for 24 h (Fig 3A). The results also showed that treatment with antroquinonol significantly decreased the phosphorylation of Y397 in a dose-dependent manner when compared with the control in both N18 and C6 cells (Fig 3Bi and 3Bii). These data illustrate that antroquinonol might regulate pFAK. The PF 431396 (FAK inhibitor) was applied as a reference control to verify that antroquinonol acted as a specific pFAK (Y397) inhibitor, and it effectively blocked the autophosphorylation of pFAK (Y397) (Fig 3C). These data illustrate that antroquinonol might regulate pFAK.

Molecular docking and validation of FAK
To verify that antroquinonol acted as a pFAK (Y397) inhibitor, we computed the molecular docking to investigate the interaction of antroquinonol and FAK and furthermore examine the effect of antroquinonol on pFAK (Y397) with a pFAK (Y397) activity assay. The binding modes in the active sites were investigated with the Discovery Studio software (Fig 4A). From the interaction mode of the antroquinonol with the predicated active site, it has been noted that antroquinonol has a hydrogen bond donor interaction with Arg 86. Additionally, the oxygen of the carbonyl group of the cyclohexenone ring (antroquinonol) is responsible for the hydrogen bonding interaction with the components of the binding pocket ( Fig 4B). These data confirm that antroquinonol indeed acted as a specific pFAK (Y397) inhibitor.

Nano-morphological changes by AFM
Furthermore, to better understand how the compound may alter the ultra-structural cell morphology of cancer cells, atomic force microscopy (AFM) was used to measure the effect of    antroquinonol on the morphology of C6 and N18 cells treated with 5 and 10 μM of antroquinonol. From the deflection image of the AFM (Fig 5), we observed that the compound increased the surface roughness of the cells and pore formations. Additionally, 10 μM of antroquinonol diminished the formation of filopodia when compared with the control cells. It seems that the anti-tumorigenic activity of antroquinonol abrogates the morphological structure of cancer cells.

Effects of EMT markers
Next, the effect of antroquinonol on epithelial-to-mesenchymal transition (EMT) markers was examined. After a 24 h treatment, antroquinonol increased the expression of E-cadherin in N18 and markedly decreased the protein levels of NF-kB, Smad2, and Smad3 in both N18 and C6 cells when compared with the untreated controls. Furthermore, antroquinonol treatment did not show any significant difference in protein levels of β-catenin and vimentin in either N18 or C6 cells (Fig 6A).

Alterations of MMP expression
The effects of antroquinonol on the MMP family were examined. The cells were treated with various concentrations of antroquinonol for 24 h. The supernatant was collected and analyzed by gelatin zymography. After antroquinonol treatment, MMP2 and MMP9 activities were reduced in N18 cells, but no significant change in MMP9 activity was seen in the C6 cell line (Fig 6B).

Antroquinonol inhibited tumor growth in nude mice
Subcutaneous C6 rat glioma cells were induced in nude mice as described in the methods section to test the in vivo efficacy of antroquinonol. Antroquinonol treatment significantly decreased the tumor volume when compared to the controls (Fig 7A) at day 10 of drug treatment. Furthermore, no difference in the body weight was found after antroquinonol treatment (Fig 7B). Major organs such as the liver, kidneys, and spleen, as well as tumors were stained with hematoxylin and eosin (H&E) and analyzed histologically. No tissue damage in the liver, kidneys, or spleen was detected after antroquinonol treatment. However, antroquinonoltreated tumor micro-sections revealed large areas of cell death when compared to the controls (Fig 7C). Biochemical analysis showed no significant differences in creatinine, amino transferase (ALT), and aspirate amino transferase (AST) levels after antroquinonol treatment (data not shown). These data indicate that antroquinonol could suppress tumor growth and that these doses of antroquinonol treatment were safe and non-toxic.

Discussion
In this study, we investigated the effect of antroquinonol on cancer cells (mainly focused on C6 glioma cells), and subsequently tested antroquinonol on N18 neuroblastoma cells to confirm its anti-cancer properties and possible molecular mechanisms of action. The results demonstrated that antroquinonol was cytotoxic in both cell lines with IC50 values of 10 μM. Antroquinonol shows a significant inhibition of cell viability in both the N18 and C6 cell lines, which exhibited cell cycles in the sub-G1 phase suggesting an induction of apoptosis. Apoptosis is a major mechanism of cell death in response to various cancer therapies and is characterized by morphological events such as cell shrinking, DNA fragmentation, and fragmentation into membrane bound apoptotic bodies [29]. Antroquinonol treatment induced a significant accumulation of an apoptotic-portion sub-G1 peak as well as an increase of green TUNEL-positive apoptotic bodies in both C6 and N18 cells. Furthermore, AFM was used to measure the cells treated with antroquinonol to have a better understanding of how the compound altered the ultra-structural cell morphology of cancer cells. Cells were treated with various concentrations of antroquinonol and showed signs of apoptosis. Cells also displayed shrinking, disruption of the cytoskeleton, nucleus fragmentation, membrane blebbing, or slightly and peripheral chromatin condensation. This result is consistent with our previous study in which Kumar et al. observed structural and morphological changes in A549 cells after antroquinonol treatment [23]. Lamellipodia and filopodia are cytoskeletal actin structures that are involved in cell mobility. Filopodia formation is activated by Cdc42 while Rac1 promotes the formation of lamellipodia [30]. FAK/Src complex activates several pathways that lead to protruding activity via Rac and Cdc42 GTPases at sites of integrin ligation. Our data showed that antroquinonol treatment greatly suppressed the activity of FAK, pFAK, Src, and pSrc proteins. Molecular docking is a computation for molecular recognition and produces a fast prediction of the structures of the protein-ligand complex, particularly for structure-based drug designs [31]. Our direct predicted-binding model showed that antroquinonol docked in the Y397 site of FAK, which suggests a decrease in the phosphorylation level and some degree of specificity of antroquinonol towards this enzyme. Furthermore, Rac1 and Cdc42, which are the key regulators of cell adhesion and spreading, were substantially inhibited by antroquinonol in this study. Bcl-2 family proteins have a demonstrated role in the process of apoptosis. Two different types of Bcl-2 family proteins have been identified: (1) pro-apoptotic proteins such as Bax, Bak, and Bcl-Xs, and (2) anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1. Previous studies have indicated that an increase in pro-apoptotic Bcl-2 family proteins and a decrease in antiapoptotic Bcl-2 family proteins participate in apoptosis [32,33]. In addition, the over-expression of anti-apoptotic Bcl-2 proteins can protect cells from stimulant-induced apoptosis [34]. p53 is a tumor suppressor protein and transcription factor that plays an important role in apoptosis and it can induce apoptosis by activating the pro-apoptotic protein Bax [35]. Antroquinonol treatment increased pro-apoptotic proteins and decreased Bcl-2, without showing any changes in caspase levels. Antroquinonol induced apoptotic cell death in both N18 and C6 cells through a caspase-independent mechanism and was associated with a reduction in the Bcl-2. This demonstrates that antroquinonol-induced apoptosis is mediated by a p53-and caspase-independent pathway. Our findings differ from a previous report [23] that suggest antroquinonol induces a caspase-dependent apoptosis. FAK has been shown to be important for survival signaling, angiogenesis, motility, and metastasis and has been shown to be overexpressed in a number of tumor cells [36]. Moreover, FAK has been proposed as a new potential therapeutic target for cancer [37,38]. Recently, deletion of FAK promotes p53-mediated DNA damage in advanced squamous cancer cells [39] and the localization of apoptotic inducing factor (AIF) from mitochondria to nucleus leads to DNA fragmentation and regulates the caspase-independent apoptotic pathway [40]. We speculate that antroquinonol could induce apoptosis in both N18 neuroblastoma and C6 glioma cells by the nuclear translocation of AIF rather than through caspase activation; however, further investigation is necessary.
Losses of expression of epithelial adherens junction protein (E-cadherin) with a concomitant gain of mesenchymal marker expression (vimentin) are distinctive events in EMT and are common in metastatic carcinomas [41,42]. FAK and Src play a critical role in tumors associated with EMTs promoting intracellular signaling pathways that lead to the induction of E-cadherin repressors and to the subsequent down-regulation of E-cadherin to allow tumor cell migration and invasion [43]. Src and FAK also induce cytoskeletal reorganization, causing the dissociation of E-cadherin from the membrane, loss of epithelial morphology, and increased cell motility [44]. Here, we demonstrate that antroquinonol treatment elicits E-cadherin induction and suppresses the expression of NF-kb, Smad2, and Smad3. Matrix metalloproteases (MMPs) play an important role in degrading ECM components. FAK siRNA dramatically decreased MMP9 and MMP2 at both the mRNA and protein levels in SMMC7721 and SK-hep1 cells [45]. FAK plays a pivotal role in the regulation of MMP2 and/or MMP9, which are considered to be critical for cancer metastasis and invasion [46]. The present results have shown that antroquinonol also decreases MMP2 and MMP9 protein levels, suggesting that antroquinonol possesses anti-invasive and anti-migratory properties.
In vitro studies have shown that antroquinonol is more potent in C6 and N18 cell lines. We also confirmed the anti-tumorigenic activity of antroquinonol in vivo. In mouse xenograft models with C6 glioma cells, antroquinonol significantly inhibited tumor growth and prolonged the doubling time of the tumor. Furthermore, antroquinonol treatment showed no significant body weight loss or tissue damage. These data indicate that antroquinonol has no toxic effects and possesses an anti-tumorigenic activity against glioma in both in vitro and in vivo assays.
In conclusion, this study demonstrated that antroquinonol has a potential effect on cell viability and induces apoptosis. Antroquinonol also suppresses FAK/Src complex formation, which subsequently inhibits Rac1 and Cdc42 activation. In addition, antroquinonol alters the expressions of EMT proteins and effectively reduces tumor volume in a xenograft mouse model. Thus, antroquinonol suppresses tumor proliferation through FAK inhibition, which suggests that antroquinonol could be a promising anti-tumor drug.