Assessment of the Anti-invasion Potential and Mechanism of Select

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Assessment of the Anti-invasion Potential and Mechanism of Select Cinnamic Acid Derivatives on Human Lung Adenocarcinoma Cells Chiung-Man Tsai,†,‡ Gow-Chin Yen,§ Fang-Ming Sun,∥ Shun-Fa Yang,*,†,⊥ and Chia-Jui Weng*,#,∇ †

Institute of Medicine, Chung Shan Medical University, No. 110, Sec.1, Jianguo N. Rd., Taichung 40256, Taiwan Department of Health, Tainan Hospital, Executive Yuan, No. 125, Zhongshan Rd., Tainan City 70043, Taiwan § Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan ∥ Department of Health and Nutrition, ChiaNai University of Pharmacy and Science, 60, Sec. 1, Erh-jen Rd., Jen-te District, Tainan City 71710, Taiwan ⊥ Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan # Graduate Institute of Applied Living Science, Tainan University of Technology, 529 Zhongzheng Road, Yongkang District, Tainan City 71002, Taiwan ∇ Research & Development Center of Environment and Life Science, Tainan University of Technology, 529 Zhongzheng Road, Yongkang District, Tainan City 71002, Taiwan ‡

S Supporting Information *

ABSTRACT: Patients with lung adenocarcinoma are often diagnosed with metastasizing symptoms and die of early and distal metastasis. Metastasis is made up of a cascade of interrelated and sequential steps, including cell adhesion, extracellular matrix degradation, cell movement, and invasion. Hence, substances carrying the ability to stop one of the metastasis-associated steps could be a potential candidate for preventing tumor cells from metastasizing and prolonging the life of cancer patients. Cinnamic acid (CA) was demonstrated to be such a candidate for human lung adenocarcinoma cells. Nevertheless, the effectiveness of CA derivatives on invasion of lung cancer cells is still unclear. The aims of this study were to explore the mechanisms underlying several select CA derivatives against invasion of human lung adenocarcinoma A549 cells. The results revealed that caffeic acid (CAA), chlorogenic acid (CHA), and ferulic acid (FA) can inhibit phorbol-12-myristate-13-acetate (PMA)-stimulated invasion of A549 cells at a concentration of ≥100 μM. The MMP-9 activity was suppressed by these compounds through regulating urokinase-type plasminogen activator (uPA), tissue inhibitor of metalloproteinase (TIMP)-1, plasminogen activator inhibitor (PAI)-1, and PAI-2; the cell-matrix adhesion was decreased by CAA only. The proposed molecular mechanism involved not only decreasing the signaling of MAPK and PI3K/Akt but also inactivating NF-κB, AP-1, and STAT3. In the present study, we selected CAA, CHA, and FA as potential inhibitors for invasive behaviors of human lung adenocarcinoma cells and disclosed the possible mechanisms. The association between structural features and anti-invasive activity of these compounds cannot be determined here and needs to be further verified. KEYWORDS: caffeic acid, chlorogenic acid, cinnamic acid derivatives, ferulic acid, invasion, lung adenocarcinoma cells

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INTRODUCTION Lung cancer is responsible for approximately 20% of tumorrelated deaths worldwide and is attributed to major cancerrelated death for both males and females.1 Lung cancer is broadly categorized into two groups: small cell lung carcinoma and nonsmall cell lung cancer (NSCLC).2 The NSCLC group is further classified into several subgroups such as squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Of these, adenocarcinoma is the most common type and accounts for approximately 75−85% of lung cancers. Patients with lung © 2013 American Chemical Society

adenocarcinoma are often diagnosed with metastasizing symptoms, suffer from metastatic diseases, and die of early and distal metastasis.4 It is estimated that metastasis causes approximately 90% of death from human cancers.3 Accordingly, the strategy to effectively inhibit cell invasion and limit tumor Received: Revised: Accepted: Published: 1890

November 20, 2012 April 3, 2013 April 5, 2013 April 5, 2013 dx.doi.org/10.1021/mp3006648 | Mol. Pharmaceutics 2013, 10, 1890−1900

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penicillin/streptomycin (PS) antibiotic solution, sodium pyruvate, nonessential amino acids (NEAA), trypsin-EDTA (TE) solution, bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), Bradford reagent, type I collagen, type IV gelatin, casein, and plasminogen were purchased from Sigma Chemical Co. (St. Louis, MO). Dimethylsulfoxide (DMSO) and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Merck Chemical Inc. (Gibbstown, NJ). Acrylamide, Coomassie Brilliant blue R-250, and bromophenol blue were purchased from BIOVOVAS Co. (Toronto, Ontatio, Canada). Matrigel was purchased from Biocoat Inc. (Horsham, PA). Polyvinylidene fluoride (PVDF) membrane was purchased from Pall Co. (Port Washington, NY). Horseradish peroxidase (HRP) substrate and anti-β-actin antibody were purchased from Millipore Co. (Bedford, MA). Anti-TIMP-1 and anti-PAI-2 antibodies were purchased from GeneTex Inc. (San Antonio, TX). Transwell insert (8 μm pore size), anti-PAI-1, antilamin B, total and phosphorylated antibodies of anti-ERK, antip38, anti-JNK, anti-Akt, and anti-PI3K were purchased from BD Biosciences (San Jose, CA). The phosphorylated antibodies of antiNF-kB (p65), anti-AP-1 (c-Jun), and anti-STAT3 were purchased from Cell Signaling Technology (Beverly, MA). Cell Culture. Human lung adenocarcinoma A549 cells (BCRC No 60074) were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsin Chu, Taiwan). Cells were grown in RPMI1640 medium, supplemented with 10% (v/v) FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.37% (w/v) NaHCO3, 0.1 mM NEAA, and 1 mM sodium pyruvate at 37 °C, in a humidified atmosphere of 95% air and 5% CO2. In the invasive and metastatic experiments, cells were cultured in a serum-free medium. Cell Viability Assay. Cell viability was determined with the MTT assay. A549 cells were seeded onto 96-well plates at a number of 1 × 104 cells/well in RPMI-1640 medium without FBS. After 24 h of incubation, the cells were treated with various concentrations (0−200 μM) of compounds dissolved in 0.1% DMSO and incubated for further 24 h. The controls were treated with 0.1% DMSO alone. The dye solution (10 μL; 5 mg/mL phosphate buffered saline, PBS) was added to each well for an additional 2 h of incubation at 37 °C. After the addition of DMSO (100 μL/well), the reaction solution was incubated 30 min in the dark. The absorbances at 570 and 630 nm (reference) were recorded with a Fluostar Galaxy plate reader (BMG LabTechologies, GmbH, Offenburg, Germany). The percent viability of the treated cells was calculated as follows:

spread is urgently required for increasing the survival of patients with lung cancer. Metastasis is a fundamental property of malignant cancer cells and is made up of a cascade of interrelated and sequential steps. To start the metastasizing process, adhesions between cells and surrounding tissues must be weakened to possibly facilitate cell migration; proteolytic enzymes are secreted to degrade environmental barriers [e.g., extracellular matrix (ECM) and basement membrane] for promoting cell movement and invasion.5 In these enzymes, matrix metalloproteinase (MMP)-2 and MMP-9 are highly expressed in malignant tumors and correlated to the invasive behaviors of numerous types of cancer cells.6,7 In general, several endogenous activators and inhibitors are mutually cooperated to regulate MMP activity in a balance status. Once the balance is interrupted, the MMPs activity might therefore shift. Urokinasetype plasminogen activator (uPA) and tissue inhibitor of metalloproteinases (TIMPs) are common activators and inhibitors, respectively, for MMPs. The uPA is initially secreted as an inactive pro-uPA form, and the pro-uPA is activated by binding with its receptor, uPAR, to catalyze MMP-9/-2 activation. The uPA activity can be negatively modulated by plasminogen activator inhibitor (PAI)-1 and PAI-2, and the activity of MMP-9 and MMP-2 is able to directly inhibit by TIMP-1 and TIMP-2, respectively.8 Hence, a compound with the capability to inhibit uPA and/or MMPs and enhance TIMPs and/or PAIs might abolish the hydrolytic activity of a cell and could be used to prohibit tumors from invading and metastasizing. Cinnamic acid (CA) and its derivatives are naturally occurring phenolic compounds. In them, caffeic acid (CAA) is a 3,4dihydroxycinnamic acid that widely presents in agricultural products such as fruits, vegetables, wine, olive oil, and coffee.9 Chlorogenic acid (CHA) is an ester of CAA and quinic acid that exists in sweet potatoes, burdock roots, prunes, fruits (peaches, apples, and pears), and vegetables (corn salad, eggplant, and artichoke). Ferulic acid (FA) is a 4-hydroxy-3-methoxycinnamic acid in natural extracts of medicinal plants, spices, chocolate, and coffee.10,11 Sinapic acid (or sinapinic acid, SA) and m-coumaric acid (m-CA) are also CA derivatives from botanical foodstuffs. Natural plant-derived phenolic phytochemicals were widely revealed to be effective preventative for cancer metastasis.12 Although CA was demonstrated to be an inhibitor for the invasion of human lung adenocarcinoma cells,13 the effectiveness of CA derivatives on invasion of lung cancer cells is still unclear. Cell metastasis can be induced and magnified by environmental stimulators. Phorbol-12-myristate-13-acetate (PMA) is such a common inducer, which uses to simulate an environmental chemical, to provoke cancer cells into metastasis. In the present study, five CA derivatives, CAA, CHA, FA, m-CA, and SA (structures shown in Figure 1A), were employed to treat the PMA-exposed human lung adenocarcinoma A549 cells, and several invasive behaviors of the cells were assayed. The aims of this study were to select the CA derivatives with anti-invasive potential on human lung adenocarcinoma cell line and further explore the underlying molecular mechanisms.

[(A570nm − A 630nm )sample /(A570nm − A 630nm )control ] × 100

Gelatin and Casein Zymography. A549 cells were incubated in 200 nM PMA-containing serum-free RPMI-1640 medium with or without compounds (in DMSO) for a given time; then, the conditioned media were collected as samples. The unboiled samples were separated by electrophoresis on 8% sodium dodecyl sulfate (SDS)/polyacrylamide gels containing enzyme substrate (0.1% gelatin in gelatin zymography; 2% casein and 20 μg/mL plasminogen in casein zymography). After electrophoresis, the gels were washed twice in washing buffer (2.5% Triton X-100 in dH2O) for 30 min at room temperature and were then incubated in reaction buffer (10 mM CaCl2, 0.01% NaN3, and 40 mM Tris-HCl, pH 8.0) at 37 °C for 12 h. Bands corresponding to activity were visualized by negative staining using Coomassie Brilliant blue R-250 (Bio-Rad Laboratories, Richmond, CA), and molecular weights were estimated by reference to prestained SDS-PAGE markers.

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MATERIALS AND METHODS Materials and Chemicals. The 99.5% purity of caffeic acid (3,4-dihydroxycinnamic acid, CAA), chlorogenic acid [3-(3,4dihydroxycinnamoyl) quinic acid, CHA], ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA), m-coumaric acid (3-hydroxycinnamic acid, m-CA), and sinapic acid (or sinapinic acid; 3,5-dimethoxy-4-hydroxycinnamic acid, SA), PMA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI-1640 medium, fetal bovine serum (FBS), 1891

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Figure 1. (A) Chemical structures of several cinnamic acid derivatives. (B) Effects of caffeic acid (CAA), chlorogenic acid (CHA), and ferulic acid (FA) on the viability of A549 cells. Cells were incubated in a serum-free medium with 0, 50, 100, 150, and 200 μM compounds for 24 h. Cell in a serum-free medium with 0.1% DMSO was used as the control. (C) Concentration-dependent inhibitory effects of CAA, CHA, and FA on the invasion of PMAtreated A549 cells. Photography (100×) represents the cells invading through Matrigel-coated membrane. The bar graphs represent the invasive cell number that was treated with various concentrations of compounds in the presence of 200 nM PMA for 24 h. Values are reported as means ± SD, n = 3. *, indicated p < 0.05 compared with the PMA treatment only.

Cell−Matrix Adhesion Assay. After treating with various concentrations (0−200 μM) of compounds in the presence of 200 nM PMA for 24 h, the cells (1 × 105 cells/well) were transferred to a 24-well Transwell plate that was coated with 20 μg/well type I collagen and were then cultured for 30 min. Nonspecific binding was blocked by incubation with 2% BSA (in PBS) for the next 2 h at room temperature. The cells were washed twice in PBS at room temperature to remove nonadherent cells. The adherent cells on the plate were measured by MTT assay. Wound-Healing Assay. A549 cells were grown to 90% confluence in a 6-well plate at 37 °C, 5% CO2 incubator. A wound was created by scratching cells with a sterile 200 μL pipet tip; cells were washed twice with PBS to remove floating cells and

then added to a serum-free medium with 200 nM PMA and various concentrations (0−200 μM) of compounds. Photos of the wound were taken each 12 h under ×100 magnitude microscope. The percent migration of the treated cells was calculated as follows: [(cell free width 0h − cell free width48h)sample ] /(cell free width 0h − cell free width48h)control ] × 100

Cell Migration and Invasion Assay. Boyden chamber assay was employed to determine the cell invasive and migratory activities. A549 cells were detached from the tissue culture plates, washed with PBS buffer and resuspended in a serum-free RPMI1640 medium (5 × 104 cells/200 μL) with 200 nM PMA and 1892

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various concentrations (0−200 μM) of compounds. The cells were then seeded onto the upper chambers of Matrigel-coated filter inserts. A serum-containing RPMI-1640 medium (500 μL) was added to the lower chambers. After 24 h of incubation, filter inserts were removed from the wells. The cells on the upper surface of the filter were wiped with a cotton swab. Filters were fixed with methanol for 10 min and stained with crystal violet for 1 h. The cells that invaded the lower surface of the filter were counted under a microscope. The migration assay was performed as described for the invasion assay, but without the coating of Matrigel. Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA was prepared from A549 cells using the Trizol reagent (Invitrogen Co., Carlsbad, CA) and performed by following the manufacturer’s instructions. RT-PCR was performed using One Step Ex Taq qRT-PCR kit (TaKaRa Biomedicals, Shiga, Japan). A sample of 30 ng of total cellular RNA was used as template in a 20 μL reaction solution which contained 2× One Step RT-PCR Buffer 10 μL, TaKaRa Ex Taq HS (5U/μL) 0.4 μL, RTase Enzyme Mix 0.4 μL, PCR forward primer (20 μM) 0.4 μL, PCR reverse primer (20 μM) 0.4 μL, and RNase free dH2O 6.4 μL. The reaction was performed with the following primers: MMP-9 (94 bp), 5′-GGGCTTAGATCATTCCTCAGTG-3′ (sense) and 5′-GCCATTCACGTCGTCCTTTAT-3′ (antisense); MMP-2 (108 bp) 5′- ATCCTGGCTTTCCCAAGCTC-3′ (sense) and 5′- CACCCTTGAAGAAGTAGCTGTG-3′ (antisense); G6PD (glucose-6-phosphate dehydrogenase; 110 bp), 5′-ATCGACCACTACCTGGGCAA-3′ (sense) and 5′-AGGATAACGCAGGCGATGT-3′ (antisense). RT-PCR amplification was performed under the following conditions: 42 °C for 5 min and 95 °C for 10 s (stage 1); 35 cycles of 95 °C for 5 s, 60 °C for 20 s followed by a final incubation at 13 °C. Preparation of Cell Lysates and Nuclear Fractions. Cell lysates and nuclear fractions were prepared using a Nuclear Extraction Kit (Signosis Co., Sunnyvale, CA). Briefly, cells (1 × 107 cells/10 cm plate) were treated with the indicated concentrations of compound at 37 °C for 24 h. Then, the harvested cells were washed twice with 5 mL cold 1× PBS. A portion of 1 mL of buffer I (containing: 1X buffer I, 10 μL of DTT, 10 μL protease inhibitor/1 mL) solution was added to each plate. The plate was transferred to an ice bucket on a rocking platform at 200 rpm for 10 min. The treated cells suspension was transferred to a sterile Eppendorf tube and centrifuged at 10 000 × g for 5 min at 4 °C. The supernatant (cytosolic fraction) was removed, and the pellet was kept on ice. A portion of 0.25 mL of buffer II (containing: 1X buffer II, 10 μL DTT, 10 μL protease inhibitor/1 mL) solution was added to each pellet. Pellets were vortexed at maximum speed for 10 s, and the Eppendorf tubes were kept on ice and shook on a rocking platform at 200 rpm for 2 h. After centrifugation at 12 000 × g for 5 min at 4 °C, the supernatant was nuclear extract and was stored at −80 °C until use. Western Blotting. The 10 μg samples of total cell lysates or nuclear fractions were separated by SDS-PAGE on 10% polyacrylamide gels and transferred onto a PVDF membrane using the Maxi Electrophoresis and Blotting System (Major Science; Saratoga, CA). The blot was subsequently blocked with 5% skim milk in PBST (phosphate buffered saline Tween-20) for 1 h and probed with antibodies to total and phosphorylated MAPK/ERK, p38MAPK, SAPK/JNK, PI3K, and Akt. β-actin, NF-κB (p65), AP-1 (c-Jun), STAT3, and lamin B were detected with their respective specific antibodies overnight at 4 °C. Detection was performed with an appropriate peroxidase-conjugated secondary

antibody at room temperature for 1 h. Intensive PBS washing was performed after each incubation. After the final PBS wash, the signal was visualized by the ECL (enhanced chemiluminescence) detection system and Kodak X-OMAT Blue Autoradiography Film. Protein Content Determination. The protein content was determined according to the method described by Bradford14 using BSA as a standard. Statistical Analysis. Data are presented as the mean ± SD of three independent measurements. Differences between variants were analyzed using the Student’s t-test for unpaired data. Values of p < 0.05 (*) were regarded as statistically significant.

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RESULTS CAA, CHA, and FA in Five CA Derivatives Display AntiInvasive Potential on PMA-Stimulated A549 Cells under Nontoxic Concentration. We first used the MTT assay to determine cytotoxicity of the candidate CA derivatives on A549 cells. Cells were treated with each CA derivative by 0, 50, 100, 150, and 200 μM for 24 h. All of the CA derivatives were nontoxic to the cells, and the cell viabilities remaining at least 88% compared to the controls after treating with CAA, CHA, and FA (Figure 1B). The MMPs secretion was indistinct in A549 cells incubated for 24 h (data not shown). We hereby used 200 nM PMA, which was able to induce MMP-9 secretion and invasive activity of cancer cells,15 to stimulate A549 cells before applying nontoxic compound for the following experiments. By Boyden chamber assay, the cell invasion significantly (p < 0.05) inhibited by ≥100 μM of CAA, CHA, and FA was observed (Figure 1C). Nevertheless, the inhibition was unobservable by a treatment of m-CA and SA (data not shown). Hence, CAA, CHA, and FA were selected to study the mechanism underlying the antiinvasion activity of these compounds on A549 cells. CAA, CHA, and FA Inhibit MMP-9 Activity through Regulating uPA, TIMP-1, PAI-1, and PAI-2. To clarify whether the activities of MMP-2 and MMP-9 are involved in the CAA-, CHA-, and FA-inhibited invasion, we treated the PMAinduced A549 cells with each compound for 24 h and analyzed the conditioned media by gelatin zymography. Figure 2A showed that MMP-9 activity was significantly reduced to 83, 35, and 22% of the PMA treatment only group by 150 μM of CAA, CHA, and FA, respectively; the MMP-2 activity was significantly decreased only under high dose (200 μM) treatment. It was supposed that such a reduction in MMP-2 activity might result from the depression of cell viability (as Figure 1B shows). Because MMP-9 activity is highly associated to the levels of uPA and TIMPs (especially TIMP-1), we further employed casein zymography and Western blots to determine the activity of uPA and the protein levels of TIMP-1/PAI-1/PAI-2, respectively. The data indicated that uPA activity was significantly (p < 0.05) inhibited by ≥150 μM of CAA and ≥100 μM of FA (Figure 2B). Moreover, CAA and CHA increased both PAI-1 and PAI-2, but FA increased only PAI-2; TIMP-1 was increased by CHA only (Figure 2C). Gathering these results, we concluded that CAAand FA-inhibited MMP-9 were via depressing PAIs-dependent uPA activity and CHA-inhibited MMP-9 was directly through increasing TIMP-1. Only CAA Decreases Cell-Matrix Adhesion. Cell-matrix interaction is one of the crucial steps for cell invasion. Here, cellmatrix adhesion assay was performed to examine the effect of CAA, CHA, and FA on the adhesive capability of A549 cells. Compared to the PMA treatment only, CAA (100, 150, and 200 μM) decreased the adhesive capability of the PMA-treated A549 cells in a dose-dependent manner, but CHA and FA did 1893

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Figure 2. Effects of CAA, CHA, and FA on the activities of MMP-9/-2 (A) and uPA (B) and the protein levels of TIMP-1, PAI-1, and PAI-2 (C) in PMAtreated A549 cells. Cells were incubated in a serum-free medium with 0, 50, 100, 150, and 200 μM compounds in the presence of 200 nM PMA for 24 h. The activity of MMP and uPA was determined by gelatin zymography and casein zymography, respectively; the level of TIMP-1, PAI-1, and PAI-2 was determined by Western blotting. β-actin was used as the internal control of Western blots. The density of each band was subsequently quantified by densitometric analyses with that of PMA treatment only set to 100% or 1.0. *, indicated p < 0.05 compared with the PMA treatment only.

CAA, CHA, and FA Inhibit the Transcriptional Level of the PMA-Stimulated MMP-9 and Block the Phosphorylations of MAPK and PI3K/Akt. Using RT-PCR, we demonstrated that CAA, CHA, and FA inhibited MMP-9 of PMA-treated A549 cells at the transcriptional level (Figure 5A). The mitogen activated protein kinase (MAPK) and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) are well-known transduction signaling pathways associate with the MMPs expression. To clarify the transcriptional mechanism, the impact of CAA, CHA, and FA on these signaling was further investigated. The signaling expression was evaluated by a timedependent test, and 6 h treatment was determined to be the appropriate time for detection (Figure S1). The densitometric analyses of Western blots indicated that the phosphorylations of PI3K/Akt and MAPK signaling were respectively depressed by ≥100 μM and ≥150 μM of CAA, CHA, and FA (Figure 5B). The CHA- and FA-inhibited MMP-9 expression through a p38- and ERK-independent, respectively, manner was also shown.

not (Figure 3). The result infers that the cell-matrix interaction was involved in the CAA-inhibited invasion, but the interaction was not highly implicated in the CHA- and FA-reduced invasion. CAA, CHA, and FA Suppress the Motility of PMATreated A549 Cells. Cell invasion is mostly dependent on motility available. Wound healing and Boyden chamber-based cell migration assays are common methods to evaluate the motile ability of a cell on the surface of tissue culture plate and through the biological membrane, respectively. We observed that the migratory distance of the PMA-induced A549 cells was significantly (p < 0.05) decreased by CAA, CHA, and FA at a concentration of ≥100 μM in a wound healing assay (Figure 4A). Additionally, the migratory ability of the cells reduced to 58, 60, and 69% of the PMA treatment only by treating with 100 μM of CAA, CHA, and FA, respectively, was also obtained in Boyden chamber assay (Figure 4B). Based on the data, we suggest that the anti-invasive activity of CAA, CHA, and FA should be partly attributed to the inhibition of cell migration. 1894

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cancer.19 Generally, MMP-2 is overexpressed but constitutive in highly metastatic tumors, whereas MMP-9 can be stimulated by cytokines, growth factors, and phorbol esters through the activation of different intracellular signaling pathways.20,21 PMA is a tumor promoter and selective activator of protein kinase C (PKC).22 Shih et al.23 and Shieh et al.4 revealed that PMA can be used to induce the activities of MMP-9/MMP-2 and the abilities of adhesion, migration, and invasion of A549 cells. Combining the results of Figures 1−4, the efficacy of CAA, CHA, and FA on the inhibition of invasive behaviors of PMA-induced A549 cells was clear. It was disclosed that the PMA-induced invasion, MMP-9, and migration were suppressed by each CA derivative, but only CAA attenuated the cell-matrix adhesion. A successful metastasis has to overcome several barriers and accomplish a series of sequential steps. Hence, substance carry the ability to inhibit one of the metastasisrelated steps could be a potential candidate for antimetastasis and for survival of cancer patients. Anti-invasive/antimetastatic substance is considered that might play a role in either oxidative stress release or signaling molecule inhibition. PMA is able to stimulate an increasing rate of oxidative stress levels,24 and the MMP-9 expression in A549 cells can be induced by PMA. Yu et al.25 have reported that the reduction in oxidative stress might result in the decrease in MMP-9 expression. Since CA derivatives possess antioxidant properties, the observable antimetastatic properties of these compounds on PMA-treated cells are therefore doubted that might derive from the reduction of oxidative stress. However, we had found that SA and m-CA, two CA derivatives with antioxidant activity, did not exert anti-invasive effect on A549 cell line in a Boyden chamber-based cell invasion assay. The thought of CAA, CHA, and FA against invasion of A549 cells simply originate from their antioxidant activity was hence exclusive. We therefore focused the investigation on the modulation of invasion-related intracellular signaling molecules by inhibitors but not on the reduction of oxidative stress by antioxidants. In some cases, a specific chemical structure might account for the function of a compound. In other cases, the compounds with similar structure might display quite dissimilar biological characteristic and pharmacokinetic profile. 6-Shogaol and 6-gingerol, two active compounds in ginger, are chemically with a double bond on the carbon side chain forming α,βunsaturated ketone moiety and a hydroxyl moiety, respectively. Accordingly, they are functionally exerted different intensity of power on migration and invasion of hepatoma cells.26 Chemically, the functionality and efficacy of cinnamic acid (CA) is probably offered by three main reactive sites in 3-phenyl acrylic acid: substitution at the phenyl ring, addition at the α- and β- unsaturation, and the action of carboxylic acid.27 The structural difference between two structure-related CA isomer, cis-CA and trans-CA, is only in the out-plane protons on α and β carbons of double bond. Cis-CA has showed a better bioactivity than trans-CA in certain functionality, such as a nearly 10-fold power on inhibiting the root growth of Arabidopsis thaliana and an approximately 120-fold activity on antituberculosis.28,29 Nevertheless, the anti-invasive activity of cis-CA and trans-CA on A549 cells is only a moderate extent difference.13 The CA derivative, CAA, can suppress the invasion of AH109A and PC3 cancer cells and inhibit PMA-induced MMP-9 expression of HepG2 cells.30−32 Another CA derivative, CHA, can be a strong MMP-9 inhibitor and anti-invasive agent in Hep3B and AH109A cells, respectively, and can be used to inhibit sphingosine-1phosphate induced or noninduced MMP-2 secretion and migration of U-87 glioma.30,33,34 The isolated molecule CA has been

Figure 3. Effects of CAA, CHA, and FA on cell-matrix adhesion of PMAtreated A549 cells. Cells were treated with 0, 50, 100, 150, and 200 μM compounds in the presence of 200 nM PMA for 24 h and were subjected to analyze for cell-matrix adhesion as described in Materials and Methods. Photos represented the adherent cells on the plate which were measured by MTT assay. Data are represented as mean ± SD of three independent experiments. *, indicated p < 0.05 compared with the PMA treatment only.

CAA, CHA, and FA Inhibit the Activations of NF-κB, AP-1, and STAT3. Nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1) are transcription factors localized downstream of the MAPK and PI3K/Akt pathways, and the signal transducer and activator of transcription 3 (STAT3) is a transcription factor directly activated by receptor-associated kinase. All of them are implicated in the regulation of MMP-9 expression. The influences of CAA, CHA, and FA on the protein levels of nuclear NF-κB, AP-1, and STAT3 in PMA-treated A549 cells were examined. Cells were treated with various concentrations (0, 50, 100, 150, and 200 μM) of CA derivatives in the presence of 200 nM PMA for 6 h according to the time dependent test in Figure S1. The nuclear protein was extracted and applied to Western blotting. The levels of nuclear NF-κB, AP-1, and STAT3 were dose-dependently decreased by CA derivatives compared to the PMA treatment only (Figure 6). Accordingly, the inactivation of nuclear NF-κB, AP-1, and STAT3 might be associated with the inhibition of MMP-9 and invasion by CAA, CHA, and FA in PMA-treated A549 cells.

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DISCUSSION MMPs expression is highly associated to the invasive capability of cancer cells. Suppression of MMPs is therefore suggested to be an effective strategy for preventing tumor from metastasizing.16 MMP-2 secretion is correlated to the invasion of highly metastatic human lung adenocarcinoma cells,17,18 and MMP-9 oversecretion is shown in the invasive lung, colon, and breast 1895

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Figure 4. (A) Effects of CAA, CHA, and FA on PMA-induced wound healing of A549 cells. Cells were treated with 0, 50, 100, 150, and 200 μM compounds in the presence of 200 nM PMA for 48 h. Photos of the wound were taken each 12 h under a ×100 magnitude microscope. The bar graph was drawn in according to the equation described in Materials and Methods. (B) Effects of CAA, CHA, and FA on motility of PMA-treated A549 cells. Photography (100X) represents the cells migrating through PCF membrane. The bar graphs represent the migratory cell number that was treated with 0, 50, 100, 150, and 200 μM compounds in the presence of 200 nM PMA for 24 h. Values are reported as means ± SD, n = 3. *, indicated p < 0.05 compared with the PMA treatment only.

evidenced that having selective inhibitory activity against invasion and MMP-9 of A549 cells13,35 but the activity of CA derivatives on lung cancer cells was still unknown. This study provides the first demonstration that CAA, CHA, and FA are capable of inhibiting invasive behaviors, including invasion, migration, adhesion, and MMP-9, in lung adenocarcinoma cells. The relationship between chemical structure and anti-invasive behavior of a compound has also been proposed in numerous studies. CAA is a 3,4-dihydroxycinnamic acid, CHA is an ester of

CAA and quinic acid, and FA is a 4-hydroxy-3-methoxycinnamic acid. These compounds are CA derivatives that have a similar structural backbone but specific functional group. Yagasaki et al.30 have compared the anti-invasive activity among CAA (with dihydroxy group), CA (without hydroxy group), and p-coumaric acid (4-hydroxycinnamic acid, with 4-hydroxy group) on hepatoma cells, and only CAA significantly suppressed the invasion of AH109A cells. The in vitro invasive activity of hepatoma (AH130) cells potentiates by superoxide radicals and suppresses by superoxide 1896

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Figure 5. (A) Effects of CAA, CHA, and FA on mRNA expression of MMP-9 and MMP-2 in cytosol of PMA-treated A549 cells. Cells were incubated in a serum-free medium with 0, 50, 100, 150, and 200 μM compounds in the presence of 200 nM PMA for 24 h. Semiquantitative RT-PCR was used to determine the mRNA expression. (B) Effects of CAA, CHA, and FA on the signaling of MAPK and PI3K/Akt pathways in cytosol of PMA-treated A549 cells. Cells were treated with 0, 50, 100, 150, and 200 μM compound in the presence of 200 nM PMA for 6 h, and the cell lysates were subjected to SDS-PAGE followed by Western blots with the indicated total and phosphorylated antibodies. Determined levels of these proteins were quantified by densitometric analyses with that of PMA treatment only being 1.0. *, indicated p < 0.05 compared with the PMA treatment only.

dismutase and catalase was demonstrated.36−38 The specific structure for scavenging free radical in CAA and CHA may contribute to their anti-invasive action. Our result in Figure 1C also showed that CAA and CHA were effective on inhibiting the invasion of lung cancer cells. Moreover, Zhang et al.39 and de Melo40 have evaluated cinnamoyl pyrrolidine derivatives as potent gelatinase inhibitors; the data indicated that compound substituted with trimethoxy group and hydroxy group shows respectively low and high activity for gelatinase inhibition. Nevertheless, these rules do not match the findings in Figure 2A which exhibiting a higher inhibitory activity on MMP-9 in FA (with a methoxy and a hydroxy group) and CHA (with a quinic acid and two hydroxy groups) compared to CAA (with two hydroxy groups). The MAPK and PI3K/Akt pathways transduce the signal sending by chemokines, growth factors, and cytokines to the downstream

transcription factors, NF-κB and STAT3, for modulating numerous reactions in cells.41 NF-κB is a well-known carcinogenesis-related transcription factor, and the hyperactivation of STAT3 is found in many types of malignancies.42,43 Several studies have clearly declared the importance of MAPK, PI3K/Akt, NF-κB, and STAT3 signaling on the regulation of uPA and MMPs expressions in invasive and metastasized cancer cells.6,7,44 The MMPs, especially MMP-9, expression and invasiveness induced by PMA via MAPK pathway and NF-kB and AP-1 in some types of cancer cells were also documented.45−47 The studies mentioned above strongly support our findings which indicating MAPK and PI3K/Akt pathways and NF-κB, AP-1, and STAT3 transcription factors were the molecular signaling involved to the inhibition of MMP-9 and invasion by the select CA derivatives (Figures 5 and 6). 1897

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Figure 6. Effects of CAA, CHA, and FA on protein levels of NF-κB, AP-1, and STAT3 transcription factor in nuclear of PMA-treated A549 cells. Cells were treated with 0, 50, 100, 150, and 200 μM compound in the presence of 200 nM PMA for 6 h, and the nucleus extracts were subjected to SDS-PAGE followed by Western blots with the indicated antibodies. Determined levels of these proteins were quantified by densitometric analyses with that of PMA treatment only being 1.0. *, indicated p < 0.05 compared with the PMA treatment only.

Figure 7. Schematic representation of the signaling pathways and invasive behaviors involved in the inhibition of invasion of PMA-treated human adenocarcinoma cells by CAA, CHA, and FA. ⊖ indicates inhibition. ⊕ indicates induction. The dotted line (----) indicates indirect pathway.

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In conclusion, CAA, CHA, and FA are potential inhibitors on various invasive behaviors, including MMPs expression, adhesion, and migration, of human lung adenocarcinoma cells. The proposed molecular mechanism involved not only decreasing the signaling of MAPK and PI3K/Akt but also inactivating NF-κB, AP-1, and STAT3. The overall signaling pathways and behaviors involved in the inhibition of invasion of PMA-treated human adenocarcinoma cells by these CA derivatives were represented schematically in Figure 7. As for the association between structural feature and antiinvasive activity of these compounds cannot be determined in this study and needs to be further verified.

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AUTHOR INFORMATION

Corresponding Author

*S.-F.Y.: Tel.: 886-4-24739595, #34253; fax: 886-4-24723229; e-mail: [email protected]. C.-J.W.: Tel.: 886-6-2532106, #256 or #5129; fax: 886-6-2433837; e-mail: [email protected]. edu.tw. Author Contributions

C.-M.T. and G.-C.Y. contributed equally to this article. Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

ACKNOWLEDGMENTS

This research work was supported by a research grant from National Science Council (NSC99-2313-B-165-001-MY3), Taiwan, Republic of China.

Time-dependent effect of PMA stimulation on the protein levels of signaling molecules in A549 cells. This material is available free of charge via the Internet at http://pubs.acs.org. 1898

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ABBREVIATIONS Akt, protein kinase B; AP-1, activator protein 1; CA, cinnamic acid; CAA, caffeic acid; CHA, chlorogenic acid; DMSO, dimethylsulfoxide; ECL, enhanced chemiluminescence; ECM, extracellular matrix; FA, ferulic acid; G6PD, glucose-6-phosphate dehydrogenase; HRP, horseradish peroxidase; MAPK, mitogen activated protein kinase; m-CA, m-coumaric acid; MMP, matrix metalloproteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor-kappa B; NSCLC, nonsmall cell lung cancer; PAI, plasminogen activator inhibitor; PBS, phosphate buffered saline; PBST, phosphate buffered saline Tween-20; PI3K, phosphoinositide-3 kinase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PVDF, polyvinylidene fluoride; RT-PCR, reverse transcriptionpolymerase chain reaction; SA, sinapic acid or sinapinic acid; STAT3, signal transducer and activator of transcription 3; TIMP, tissue inhibitor of metalloproteinase; uPA, urokinase-type plasminogen activator

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