AMPK-dependent signaling modulates the suppression of invasion and
migration by fenofibrate in CAL 27 oral cancer cells through NF-κB
pathway
Shih-Chang Tsai1, Ming-Hsui Tsai2, Chang-Fang Chiu3, Chi-Cheng Lu4, Sheng-Chu Kuo5, Nai-Wen Chang6 and Jai-Sing Yang7
1Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
2Department of Otolaryngology, China Medical University Hospital, Taichung 404, Taiwan 3Department of Hematology and Oncology, China Medical University Hospital, Taichung 404, Taiwan
4Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 402, Taiwan
5Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 404, Taiwan
6Department of Biochemistry, China Medical University, Taichung 404, Taiwan 7Bracco Pharmaceutical Corp. Ltd., Taipei 104, Taiwan
Running title: FENOFIBRATE STIMULATES ANTIMETASTATIC EFFECTS ON ORAL CANCER CELLS
Correspondence to: Dr. N.-W. Chang; e-mail: [email protected] and Dr. J.-S. Yang; e-mail: [email protected]
Key words: Fenofibrate, invasion, migration, AMPK, MMPs, oral cancer CAL 27 cells
Abstract. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist and lipid-lowering agent, has been used worldwide for the treatment of hyperlipidemia. The clinical trials demonstrated that fenofibrate possess multiple pharmacological activities, including antitumor effects. However, the precise mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we investigated the anticancer effects of fenofibrate on the migration and invasion of human oral cancer CAL 27 cells. Fenofibrate inhibited the cell migration and invasion of CAL 27 cells in vitro by the wound healing and Boyden chamber transwell assays, respectively. In addition, fenofibrate reduced the protein expressions of MMP-1, MMP-2, MMP-7 and MMP-9 by Western blotting and inhibited enzyme activities of MMP-2/-9 using gelatin zymography assays in CAL 27 cells. Moreover, the proteins of p-LKB1 (Ser428), LKB1, p-AMPKα (Thr172), p-AMPKα1/α2 (Ser425/Ser491), p-AMPKβ1 (Ser108), and AMPKγ1 were up-regulated by fenofibrate; the levels of p-IKKα/β (Ser176) and p-IκBα were reduced in fenofibrate-treated CAL 27 cells,
which were determined utilizing Western blotting analysis. Also, fenofibrate suppressed the expressions of nuclear NF-κB p65 and p50 by immunoblotting and NF-κB DNA binding activity by EMSA assay in CAL 27 cells. The anti-invasive effect of fenofibrate was attenuated by compound C (an AMPK inhibitor) or dominant negative form of AMPK (DN-AMPKα1). The results suggest that signaling pathways for fenofibrate-inhibited migration and invasion of CAL 27 cells might be mediated through blocking NF-κB signaling, resulting in the inhibition of MMPs. We found that fenofibrate considerably inhibited the migration, invasion and MMPs activities of CAL 27 cells, and these effects were AMPK-dependent rather than PPARα signal. Our findings provide a molecular rationale, whereby fenofibrate exerts anticancer effects and additional beneficial effects for the treatment of cancer patients. Introduction
Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer with poor prognosis due to frequent lymph node metastasis and local invasion (1). The addiction to betel, tobacco and alcohol are found to be highly correlated with the risk of OSCC in Taiwan (2). Inhibition of the metastatic and invasive process is important for treating cancer (3). It has been shown that elevated expression of matrix metalloproteinases (MMPs) plays a crucial role in the development of several human malignancies, including OSCC (4, 5). MMPs, proteolytic enzymes that degrade extracellular matrix proteins (EMPs), show an important role in tumor progression, including invasion, metastasis, growth and angiogenesis in OSCC (5-7). MMPs could cleave one or more of the components of the extracellular matrix (ECM) (8). MMP-1 has the capability to cleave interstitial collagens I, II and III at a specific site relative to the N terminus (9). Gelatinases (MMP-2 and MMP-9) are able to degrade collagen I, III and IV from basement membranes and extracellular matrix. MMP-9, the most prevalent form of MMPs expressed by monocytes/macrophages, is shown to be critically involved in the infiltration of monocytes into atherosclerotic lesions (10, 11). Suppression of MMP-9 activity was considered to exert anti-atherosclerotic effects mediated by decreased monocyte infiltration (12).
Fenofibrate is a potent and effective lipid-lowering reagent which is widely used to treat hyperlipidemia due to its actions of lowering the levels of triglycerides and low-density lipoprotein cholesterol, and raising high-density lipoprotein levels (13-17). The lipid-lowering effect of fenofibrate is dependent on the stimulation of nuclear receptor peroxisome proliferator-activated receptor α (PPARα) (18-20). PPARs are members of the nuclear hormone receptor superfamily and regulate gene expression in response to ligand binding (21). Three isoforms of PPARs (α, β and γ) have been found and revealed a tissue specific distribution (22). PPARα seems to mediate the hypotriglyceridemic effect of fibrates by inducing high rates of mitochondrial and peroxisomal β-oxidation in liver, kidney, heart, and muscle and by decreasing the plasma concentration of triacylglycerol-rich lipoproteins
triacylglycerol (23) . Moreover, PPARα is known to regulate fatty acid metabolism by controlling fatty acid oxidation (24-26). PPARα and AMP-activated protein kinase (adenosine 5’-monophosphate-activated protein kinase; AMPK) are central regulators of fatty acid oxidation. It is well known that activation of AMPK inhibits PPARα transcriptional activity in human hepatoma cells (27). AMPK is an energy sensor and a serine/threonine protein kinase that mediates systemic glucose homeostasis (28) and that ameliorates the pathogenesis of metabolic disorders, including diabetes, by controlling the expression and activation of various downstream molecules (29). Cells are in ATP-depleting conditions such as calorie restriction and physical exercise, and AMPK is activated and subsequently phosphorylates key metabolic enzymes resulting in an increase in generating adenosine triphosphate (ATP) (30, 31). Several studies have reported that activation of AMPK inhibits mammalian target of rapamycin (mTOR) signaling and NF-κB activation (32, 33). Additionally, stimulation of AMPK by anti-tumor agents inhibits the metastatic potential of cancer cells (34-36).
Our previous study demonstrated that fenofibrate reduced the tumor frequency and suppressed the tumor progression, which might be linked to the EGFR and COX-2 pathways in an oral-specific 4-nitroquinoline 1-oxide/arecoline mouse model in vivo (14). However, the molecular mechanism underlying the anti-tumor ability of fenofibrate in OSCC remains to be elucidated in vitro. Herein, we report the first evidence that the connection between fenofibrate and the AMPK pathway for the inhibition of tumor cell invasion in OSCC. Our results demonstrate that fenofibrate inhibits OSCC cell migration and this inhibitory action of fenofibrate is associated with the attenuation of the AMPK signaling pathway. MMPs (MMP-1, -2, -7 and -9) expression was expected to be diminished in fenofibrate-treated CAL 27 cells. These findings might suggest a new therapeutic approach for the treatment of OSCC. Materials and Methods
Chemicals and reagents
Dulbecco’s Modified Eagle medium (DMEM), L-glutamine, fetal bovine serum (FBS), and Trypsin-EDTA were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). Fenofibrate, dimethyl sulfoxide (DMSO), anti-β-actin and Triton X-100 were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). These primary antibodies (MMP-1, anti-MMP-2, anti-MMP-7, anti-p-IKKα/β (Ser176), anti-IKK, anti-p-IκBα, anti-IκB, anti-NF-κB (p50), anti-NF-κB (p65), α-tubulin and PCNA) and second antibodies for Western blotting were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-MMP-9 (Cat. AB19016), Immobilon-P transfer membrane (Cat. IPVH00010) and Immobilon Western Chemiluminescent HRP substrate (Cat. WBKLS0500) were bought from Merck Millipore (Billerica, MA, USA). Primary antibodies to p-LKB1 (Ser428), LKB1, p-AMPKα (Thr172), p-AMPKα1/α2 (Ser425/Ser491), p-AMPKβ1 (Ser108), AMPKβ1/2 and AMPKγ1 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell culture and treatment
Human oral cancer cell line (CAL 27) was obtained from Dr. Pei-Jung Lu (Graduate Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan). Cells were grown in DMEM medium supplemented with 10% FBS, 100 Units/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere incubator. Cells were treated with fenofibrate for concentrations indicated as in each experiment and equivalent volume of 0.1% DMSO was used as vehicle (37-39).
In vitro invasion assay by boyden chamber assay
Cell invasion was assessed using 24-well transwell inserts with 8 μm pore polycarbonate membranes (Cat. PIEP12R48, Merck Millipore) pre-coated with 30 μg Matrigel (BD Biosciences, Bedford, MA, USA) as described previously (40, 41). Briefly, CAL 27 cells were cultured for 24 h in serum-free medium and then cells were placed in the upper chambers of the transwell inserts (2 × 104 cells/0.4 ml medium) and treated with 0.1% DMSO (as a vehicle control) or fenofibrate (12.5, 25 and 50 μM) in presence and absence of compound C (an AMPK inhibitor) for 24 h. The medium containing 10% FBS was then placed in the lower chambers. After that, the inserts were washed with PBS, and the non-migratory cells were wiped out with cotton swabs; the filters were stained and fixed with 4% formaldehyde as well as 2% crystal violet crystal mixture for 20 min. The blue-stained cells were counted under a light microscope at 200× magnification. Each sample was analyzed triplicates of each treatment including the control and treated conditions and three independent experiments were performed.
Wound closure assay
CAL 27 cells were seeded in 12-well plates at confluence a wound was made with a 200-μl pipette tip followed by washing with serum-free medium. Cells were then incubated with or without fenofibrate (12.5, 25 and 50 μM) at 37°C and 5% CO2 as described previously (42, 43). Following cells were allowed to migrate into the wound area for up to 24 h and gently washed with PBS. Cells in the wound area (denuded zone) were photographed and measured at 200× magnification. The experiments were performed in triplicate.
Gelatin zymography assay
Determination of the enzyme activities of MMP-2 and -9 in CAL 27 cells were measured after exposure to fenofibrate. CAL 27 cells (1 × 106 cells/well) were plated in 12-well tissue culture plates and then were incubated in serum-free DMEM in the presence of 0, 12.5, 25 and 50 μM of fenofibrate for 24 h. The conditioned medium was then collected and was separated by electrophoresis on 10% SDS–PAGE containing 0.2% gelatin (Sigma-Aldrich
Corp.). At the end of electrophoresis, the gels were soaked in 2.5% Triton X-100 in dH2O twice for a total of 60 min at 25°C, then were incubated in substrate buffer (50 mM Tris HCl, 5 mM CaCl2, 0.02% NaN3 and 1% triton X-100, pH 8.0) at 37 °C for 18 h. Bands corresponding to activity of MMP-2 and -9 were visualized by negative staining using 0.2% Coomassie blue in 50% methanol and 10% acetic acid as described elsewhere (44, 45). The NIH ImageJ software was applied to quantify these bands.
Whole cell protein extraction and nuclear protein preparation
Fenofibrate-treated and -untreated cells were suspended in lysis buffer (PRO-PREP protein extraction solution, iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea) as described elsewhere (46). Protein concentrations of the extracts were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Nuclear proteins were extracted according to the instructions of the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA).
Western blotting analysis
Western blot assays were done as described elsewhere (41, 44). In brief, equal amounts (40 μg) of cell lysates were uploaded and separated by 12% SDS-polyacrylamide gel electrophoresis and electro-transferred to Immobilon-P transfer membrane (Merck Millipore). The transferred blots were blocked in TBST (10 mM Tris, 150 mM NaCl and 0.1% Tween 20) containing 5% nonfat dry milk for 1 h and incubated with primary antibodies at 4°C overnight. Membranes were washed three times with TBST for 10 min and incubated with secondary HRP-conjugated antibody. The blots were detected by using an enhanced cheniluminescence (ECL) kit (Merck Millipore) and Kodak Bio-MAX MR film (Eastman Kodak, Rochester, NY, USA). The same membrane was incubated again with a specific primary antibody for β-actin, α-Tubulin or PCNA. To quantitate Western blot results, densitometric analysis was done by the NIH ImageJ software. All Western blot experiments were repeated at least three times with a different cell preparation.
EMSA for NF-κB p65 DNA-binding activity
NFκB DNA binding activity was determined by use of a commercial LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. In brief, 5 μg of nuclear extract from treated or control CAL 27 cells was incubated with biotin-labeled NF-κB consensus oligonucleotide (5’-AGTTGAGGGGACTTTCCCAGGC-3’) in the reaction buffer at room temperature for 20 min and then loaded onto the 6% polyacrylamide gel and transferred to nylon membrane. The membrane was blocked and incubated with streptavidin-horseradish peroxidase conjugate for 15 min. The membrane was incubated with substrate solution and exposed to Kodak film
(Eastman Kodak).
Immunofluorescence and co-localization of proteins
CAL 27 cells (5×104 cells/well) on 4-well chamber slides were treated with 50 μM fenofibrate for 6 h. Cells were then fixed in 3% formaldehyde (Sigma-Aldrich Corp.) in PBS for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 30 min and stained with primary antibodies, including anti-PPARα, anti-pAMPKα and NF-κB (p65) at 4°C overnight, followed utilizing FITC-conjugated secondary antibodies (green fluorescence, Invitrogen/Life Technologies) at 1:100 dilutions for 1 h at room temperature as previously described (47, 48). Cells were stained with PI (red fluorescence) for nucleus and coverslips were mounted before photomicrographs were subjected to fluorescent microscope (Leica Microsystems, Heidelberg, Mannheim, Germany)
Stably expressing a dominant negative AMPK CAL 27 cells
The vector pcDNA-Zeo (Invitrogen, Carlsbad, CA, USA) and DN-AMPK were gifts from Jill Suttles (Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USA). CAL 27 cells were transfected plasmids with Arrest-In transfection reagent (Open Biosystems, Lafayette, CO, USA) and followed the manufacturer’s instructions. To establish for the stable cell lines, CAL 27 cells were split after 48hr transfection. The following day, cells were selected with media containing 50 μg/mL Zeocin. Medium were changed every three days until stable clones showed up. Clones were trypsinized and transferred into a well in the 6-well plate. All clones were performed and checked AMPK expression by Western blotting. Also, in vitro cell invasion in fenofibrate-treated pcDNA-Zeo and DN-AMPKα1 CAL 27 cells as described above.
Statistical analysis
All the statistical results were expressed as the mean ± standard deviation (S.D.) of triplicate samples. Statistical significance of the differences between groups was done using one-way ANOVA followed by Student’s t-test. A p-value less than 0.05 was considered to be statistically significant.
Results
Febofibrate suppresses invasion and migration of CAL 27 cells
The abilities to cell migration and invasion are the hallmarks of malignant transformation (5, 6). To assess whether febofibrate influences the migration and invasion of CAL 27 cells, the Matrigel invasion assay was performed. Results displayed that CAL 27 cells moved from the upper chamber to the lower chamber in the absence of febofibrate (control group) (Fig. 1). Moreover, the penetration of the Matrigel-coated filter by CAL 27 cells was inhibited in the
presence of febofibrate. Data showed that the inhibition rate was at 12.6% to 60.4% when cells were incubated with febofibrate at 12.5-50 μM for a 24-h treatment (Fig. 1). Also, mean number cells in the denuded zone decreased (14.3% to 75.3%) at concentrations between 12.5 and 50 μM in a concentration-dependent manner (Fig. 2) when cells were exposed to febofibrate for 24 h exposure. The migration assay showed that febofibrate had a significant inhibitory effect on cell migration as shown in Figure 2. We found that fenofibrate treatment led to impaired cell invasion and migration (Figs. 1 and 2). The next experiments were characterized whether specific cell signaling pathways account for these changes after fenofibrate treatment.
Fenofibrate decreases the MMPs proteins expression and MMP-2/-9 enzyme activities in CAL 27 cells
To explore the inhibitory mechanism of CAL 27 cell invasion by fenofibrate, we investigated the effects of fenofibrate on the levels of protein expression associated with migration and invasion. The previous study has shown that MMPs play vital roles in tumor cell invasion (4, 5). Western blot analysis showed that treatment of CAL 27 cells with fenofibrtae clearly reduced protein expression of MMP-1, -2, -7, and -9 as seen in Figure 2A. Gelatin zymography was used to analyze the effects of 0, 12.5, 25 and 50 μM of febofibrate-treated CAL 27 cells on MMP-2 and -9 activities for 24 h. Data in Figure 2B indicate that febofibrate at 12.5-40 μM dramatically reduced activities of both MMP-2 and -9 levels in CAL 27 cells (Fig. 3B). Based on these findings, we suggest that febofibrate-inhibited CAL 27 cell invasion and migration was mediated through suppressing MMPs signaling.
Fenofibrate activates AMPK in CAL 27 cells
There are two different isoforms of the catalytic α AMPK subunit (α1 and α2) that are differentially expressed in different tissues (28, 29). Cells express both α subunits and different groups report the predominance of different isoforms, a finding that may explain the inconsistent dependence on changes in ADP/ATP for stimulation (30, 31). CAL 27 cells were treated with 0, 12.5, 25 and 50 μM of fenofibrate for 6 h and resulted in a concentration-dependent activation of AMPK signals, as monitored by phosphorylation of AMPKs (Fig. 4). The protein levels of p-LKB1 (Ser428), LKB-1, p-AMPKα (Thr172), p-AMPKα1/α2 (Ser425/Ser491), p-AMPKβ1 (Ser108) and AMPKγ1 were increased using Western blotting analysis compared with the internal β-actin control as can be seen in Figure 4. Thus, we found that fenofibrate modulates AMPK pathway in human oral cancer CAL 27 cells.
Fenofibrate inhibits phosphorylated IKKα/β and IκBα, and NF-κB activation in CAL 27 cells
activation in different cell types (32, 33). NF-κB is bound to IκB, and once phosphorylation and subsequent degradation of IκB after NF-κB translocates into nucleus and becomes activated (34, 36). To determine if fenofibrate reduces IκBα phosphorylation and inhibits NF-κB activation in CAL 27 cells, Western blot analysis was performed. As shown in Figure 5A, febofibrate increased protein level of IκB and decreased that of IKKα/β (Ser176) and p-IκBα. In addition, nuclear NF-κB p50 and p65 expression levels were suppressed in fenofibrate-treated CAL 27 cells (Fig. 5B). DNA binding activity of NF-κB protein was blocked by fenofibrate in CAL 27 cells (Fig. 5C). Therefore, NF-κB activation signaling is involved in fenofibrate-affected CAL 27 cells in vitro.
Fenofibrate-suppressed NF-κB activation is mediated through AMPK signaling but not PPARα in CAL 27 cells
Fenofibrate has been reported to stimulate the nuclear receptor PPARα (18-20). However, we found that nuclear fraction of PPARα was not altered (data not shown) and the trafficking of PPARα into nuclei was not observed (Fig. 6A) in CAL 27 cells after fenofibrate treatment for 6 h. We further investigated if fenofibrate influences p-MAPKα (Thr172) and NF-κB (p65) in CAL 27 cells. The results showed that inductions of p-MAPKα (Thr172) and reduction of NF-κB (p65) translocations occurred in fenofibrate-treated CAL 27 cells. We suggest that p-MAPKα is likely to decrease NF-κB (p65) activity by fenofibrate in CAL 27 cells.
Fenofibrate-inhibited cell invasion was reversed by compound C and a dominant negative AMPK mutant in CAL 27 cells
Our results indicated that fenofibrate inhibited cell invasion (Fig. 1). We further explored that cells were pre-treated with compound C (an AMPK inhibitor), and the fining revealed that effects of fenofibrate on cell invasion was enhanced in comparison to fenofibrate-treated only cells (Fig. 7A). It has a similar result that cells expressing a dominant negative-AMPK (DN-AMPK) attenuated the effects of fenofibrate on cell invasion CAL 27 cells after fenofibrate exposure (Fig. 7B). Based on this finding, AMPK signaling correlated with fenofibrate-inhibited cell invasion in CAL 27 cells.
Discussion
Fenofibrate has beneficial effects in amelioration of metabolic syndromes through regulating multiple target genes in a variety of human tumors including colorectal carcinoma (49), melanoma (50), hepatoma (51) and breast carcinoma (52). The present study demonstrated that fenofibrate exhibited anticancer abilities in CAL 27 cells with: i) blunted MMPs expression and activities (Figure. 3); ii) increased AMPK phosphorylation (Figure. 4); and iii) decreased NF-κB p65 and its DNA binding activity (Figure 5). Metastasis is one of cancer hallmarks and a multistep process. Invasion and migration are thought to be two of the critical
metastatic steps from benign to malignant tumors (4, 5). Malignant tumor cells have the ability to degrade components of the extracellular matrix (ECM) proteins (8). The basement membrane (BM) is a thin layer of basal lamina consisting of various type IV collagens. The production of MMPs and the degradation of BM are enhanced in the process of cancer invasion and metastasis. MMPs are Zn2+-dependent enzymes, which are classified into four
groups based on their substrate specificities. The four groups are collagenases (such as MMP-1, -8, and -13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3 and MMP-10) and metalloelastases (such as MMP-12) (9-11). Our data showed that fenofibrate-treated CAL 27 cells dramatically decreased MMP-1, -2, -7 and -9 expressions (Figure 3A). The proteolytic activity of MMP-2 becomes active during tumor angiogenesis and subsequently degrades the ECM proteins (11, 53). We demonstrated that there is also a significant decrease for MMP-2 and MMP-9 enzyme activities in CAL 27 cells after fenofibrate treatment (Figure 3B). It is implicated that MMP-2 and MMP-9 play a vital role in CAL 27 progression and metastasis.
AMPK is a heterotrimeric protein comprising a catalytic α subunit and regulatory β and γ subunits. AMPK α1 and α2 are differentially expressed in various tissues. AMPK α1 predominates in adipose tissue, while AMPKα2 is expressed higher in skeletal muscle and cardiomyocytes (54). AMPK exists in the cytoplasm without activation and translocates into the nucleus after phosphorylation of α subunit induced by metabolic stresses. Our current finding is the first to show significant activation of p-AMPK expression in fenofibrate-treated CAL 27 cells (Figure 4). It is consistent with previously reported activation of AMPK in vascular endothelial cells by fenofibrate (33). It is reported that MMPs expression is regulated by modulating the activation of NF-κB in different cell types through the AMPK pathway (55). NF-κB is a transcription factor and is bound to IκB in the cytoplasm. Once phosphorylation and subsequent degradation of IκB, NF-κB trnaslocates into nucleus, becomes active and regulates various pro-inflammatory gene expressions (32, 33). Our data demonstrated that phosphorylation of IKK and IκB were reduced in fenofibrate-treated CAL 27 cells (Figure 5A). Moreover, fenofibrate suppressed nuclear NF-κB p65 and p50 expressions (Figure 5B) as well as a decrease of NF-κB DNA binding activity were observed (Figure 5C) in CAL 27 cells.
Interestingly, fenofibrate has been reported to activate PPARα (a PPARα agonists) and use to treat dyslipidemia in clinic (18-20). However, our results showed that fenofibrate did not stimulate expressions of nuclear fraction of PPARα (data not shown) and significantly alter the trafficking of PPARα into nuclei (Fig. 6A) in CAL 27 cells. Thus, we suggest that fenofibrate-affected CAL 27 cells might be through PPARα-independent signaling. Okayasu
et al. reported that NF-κB activation is involved in alterations of AMPK (33), and which was
activated during metastatic potential of cancer cells (34-36). In the present study, we agree these reports and found that fenofibrate induced p-MAPKα (Thr172) trafficking but reduced NF-κB (p65) translocations in treated CAL 27 cells (Figure 6). It is reported that presence of
AMPK inhibited MMP-9 expression through suppressing NF-κB signal during the invasion and metastatic effects in tumor cells (56). Our data showed that attenuation of AMPK signal by an AMPK inhibitor (compound C) and a dominant negative-AMPK (DN-AMPK) contributed to cell invasion in CAL 27 cells (Figure 7C). Correctively, based on these observations, we suggest that fenofibrate-suppressed metastatic effect (invasion) is mainly correlated with AMPK-modulated NF-κB pawhway, but not PPARα in CAL 27 cells.
In summary, our results provide first evidence to understand the mechanisms that fenofibrate inhibits the invasion and migration of CAL 27 cells by suppressing MMPs expression through the AMPK and NF-κB signaling pathway (Figure 8). It is probable that we are still far from unveiling the complete mechanisms underlying fenofibrate inhibitory effect on tumor cell metastasis. Our work supports a theoretical basis which is therefore worthy of further research for the therapeutic use of fenofibrate to treat CAL 27 cells.
Acknowledgements
This study was supported by research grant from the China Medical University ( CMU100-TS-01 and CMU101-S-27) and the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH101-TD-C-111-005). This work was also supported in part by a Grant from the National Science Council of Taiwan (NSC 97-2320-B-039-004-MY3).
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Figure legend
Figure 1. Fenofibrate suppresses invasion of CAL 27 cells. Cells were treated with 0, 12.5, 25 and 50 μM of fenofibrate for 24 h. Cell invasion was examined in a Boyden chamber was pre-coated with Matrigel. Cells that had invaded to the lower face of the matrigel-coated membrane were stained with 2% crystal violet and then examined by light microscopy (at 200× magnification). The number of invaded cells was determined by counting the stained cells. Invasion abilities of CAL 27 cells were quantified by counting the number of cells under microscopy at 100× magnification and represented the average of three independent experiments. The values are presented the mean± S.D. from three independent experiments. The asterisk (*) indicates as p<0.05 significantly different from vehicle-treated cells.
Figure 2. Fenofibrate inhibits migration of CAL 27 cells. Cells were treated with 0, 12.5, 25 and 50 μM of fenofibrate for 24 h. Cell migration was examined by Wound closure assay as described in Materials and Methods. Migration abilities of CAL 27 cells in denuded zone were measured by under microscopy at 100× magnification and represented the average of three independent experiments. The results performed the mean± S.D. from three independent experiments. The asterisk (*) shows as p<0.05 significantly different from vehicle-treated cells.
Figure 3. Febofibrate inhibits MMPs proteins expression and enzyme activities in CAL 27 cells. Cells were incubated with 0, 12.5, 25 and 50 μM of fenofibrate for 24 h. (A) Cells were harvested, and cells lysates were prepared to subject to immunoblotting as described in Materials and Methods. The levels of MMP-1, MMP-2, MMP-7, and MMP-9 were examined by SDS–PAGE and Western blotting. (B) The supernatant was harvested from examined cells and were processed for zymography analysis. The images of MMP-2 and MMP-9 activities were quantitated by the NIH ImageJ software. Data are performed with representative at least three independent experiments with similar results.
Figure 4. AMPK-associated proteins expressions were increased by fenofibrate in CAL 27 cells. Cells were exposed to 0, 12.5 and 50 μM of fenofibrate for 24 h. The total proteins were collected and the proteins levels were measured using Western blotting analysis. The levels of p-LKB1 (Ser428), LKB1, p-AMPKα (Thr172), p-AMPKα1/α2 (Ser425/Ser491), AMPKα, p-AMPKβ1 (Ser108), AMPKβ1/2 and AMPK1 were examined by SDS–PAGE and Western blotting. β-actin was used as an internal control. Results were shown with similar results of at least three independent experiments.
Figure 5. Fenofibrate inhibits NF-κB expression and its related protein levels in CAL 27 cells. (A) Western blot assay of p-IKKα/β (Ser176), IKK, p-IBα and IκB protein expression.
α-Tubulin shows as an internal control. (B) Western blot analysis of nuclear NF-κB p65 and p50 protein levels. PCNA is a nuclear internal control. (C) Effect of fenofibrate on DNA-binding activity of nuclear NF-κB in CAL 27 cells using electrophoretic mobility shift assay (EMSA). A gel from three independent experiments is presented. The arrow indicates a specific NFκB DNA probe-protein complex. Our data were performed at least three independent experiments with similar results.
Figure 6. Fenofibrate alters the effects of PPARα, p-AMPKα and NF-κB (p65) nuclear translocations in CAL 27 cells. Cells were incubated with 50 μM fenofibrate for 6 h. Cells were fixed and stained with anti-PPARα, anti-p-AMPKα and anti-NF-κB (p65), respectively. The trafficking of PPARα, p-AMPKα and NF-κB (p65) were examined, following to be labeled secondary antibodies-FITC (green fluorescence) and the proteins were detected by a fluorescent microscope. The nuclei were stained by PI (red fluorescence). Our results were obtained at least three independent experiments with similar results.
Figure 7. Fenofibrate-inhibited CAL 27cell invasion is mediated via the AMPK signaling pathway. (A) CAL 27 cells were pretreated with 10 μM compound C for 1 h and then incubated with 50 μM fenofibrate for 24 h. (B) CAL 27 cells expressing a vector control (pcDNA-Zeo) or a dominant negative-AMPK (DN-AMPK) were incubated with 50 μM fenofibrate for 24 h. The ability of cell invasion (%) from three independent experiments are presented as mean values ± SD (the asterisk (*) indicates as p < 0.05 versus the fenofibrate treated only cells).
Figure 8. The proposed a schematic presentation of the mechanisms of fenofibrate-inhibited invasion and migration of CAL 27 cells. See text for details.
