a-Tomatine inactivates PI3K/Akt and ERK signaling pathways in human
lung adenocarcinoma A549 cells: Effect on metastasis
Yuan-Wei Shih a , Jiunn-Min Shieh b , Pei-Fen Wu c , Yi-Chieh Lee a , Yi-Zhi Chen a , Tai-An Chiang d, * a Department of Biological Science and Technology and Graduate Institute of Biomedical Science, Chung Hwa University of Medical Technology, Tainan 717, Taiwan b Department of Pulmonary Medicine, Chi Mei Medical Center, No. 901, Chung-Hua Road, Yong-Kang, Tainan 710, Taiwan c Department of Occupational Safety and Hygiene, Tajen University, Pingtung 907, Taiwan d Department of Medical Technology and Graduate Institute of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan 717, Taiwan a r t i c l e i n f o Article history: Received 6 January 2009 Accepted 12 May 2009 Keywords: a-Tomatine Invasion Migration PI3K/Akt ERK a b s t r a c t This study rst investigates the anti-metastastic effect of a-tomatine in the human lung adenocarcinoma cell line: A549. In this study, we rst noted a-tomatine inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed a-tomatine could inhibit phos- phorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase- type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase (JNK) and p38. Next, a-tomatine signicantly decreased the nuclear levels of nuclear factor kappa B (NF-jB), c-Fos, and c-Jun. Also, treating A549 cells with a-tomatine also leads to a dose-dependent inhi- bition on the binding abilities of NF-jB and activator protein-1 (AP-1). Further, the treatment of inhibitors specic for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2, MMP-9, and u-PA. These results showed a-tomatine could inhibit the metastatic ability of A549 cells by reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-jB or AP-1 binding activities. These ndings proved a-tomatine might be an anti-metastastic agent against human lung adenocarcinoma. Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved. 1. Introduction Lung cancer is the major cause of malignancy-related deaths worldwide, and its incidence is rising in many countries (Greenlee et al., 2001). About 40% of lung cancers are adenocarcinomas. Ade- nocarcinomas belonging to the subgroup of the non-small cell lung cancers are the most common type in US and Asia (Shivapurkar et al., 2003). Studies have shown lung cancer cases are caused by smoking, air pollution, environmental risk factors (for example, exposure to radiation, asbestos, heavy mental, and polycyclic aro- matic hydrocarbon) and oncogene (for example, slug gene) (Lee et al., 2005; Shih et al., 2005). Most diagnosed patients with lung adenocacinoma are in an advanced stage because of its highly met- astatic properties, and such patients are not candidates for surgical resection. One of the glycoalkaloids, a-tomatine, occurs naturally in toma- toes (Lycopersicon esculentum). Immature green tomatoes contain up to 500 mg a-tomatine/kg fresh fruit weight. The compound is partly degraded as the tomato ripens until at maturity levels in red tomatoes are about 5 mg/kg fresh fruit weight (Friedman and Levin, 1995). Fig. 1A shows a-tomatine is constructed of an aglycon moiety (tomatidine), and a tetrasaccharide moiety (b-lycotetrose) that contains two molecules of D-glucose and one each of D-galact- ose and D-xylose. Previous studies demonstrated a-tomatine has exhibited anti-proliferative and apoptotic effects on the growth of cancer cells originating from the human colon and liver (Lee et al., 2004). Although it was quite clear a-tomatine may inhibit the growth of various cancers by inducing cancer cells toward apoptosis and anti-proliferation, whereas the precise impact and related molecular mechanism of a-tomatine on metastasis of can- cer cells was still unclear. Metastasis is a multistep process involving overexpression of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and u-PA. MMP-2 and MMP-9 (also known as type IV collagenases or gelatinases) which can degrade most ECM components that forming the basal membrane (Bernhard et al., 1994). In addition, u-PA may initiate the activation of an enzymatics cascade and con- vert the zymogen plasminogen to plasmin. The activation of these 0278-6915/$ - see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.05.011 Abbreviations: MMPs, matrix metalloproteinases; u-PA, urokinase-type plas- minogen activator; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; ERK, extracellular signaling-regulating kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; NF-jB, nuclear factor kappa B; AP-1, activator protein-1, IjB, Inhibitor of NF-jB. * Corresponding author. Tel.: +886 6 2674567x450; fax: +886 6 2605598. E-mail address: giantful@mail.hwai.edu.tw (T.-A. Chiang). Food and Chemical Toxicology 47 (2009) 19851995 Contents lists available at ScienceDirect Food and Chemical Toxicology j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchemt ox enzymes enable the degradation of extracellular matrix (ECM) by tumor cells, allowing their access to the vasculature, migration and invasion into the target organ and development of tumor metastasis (Duffy and Duggan, 2004; Itoh and Nagase, 2002). As well as MMPs and u-PA, the mitogen-activated protein ki- nases family members (MAPK) are also known to mediate metasta- sis. The MAPK serine/threonine kinase superfamily is activated by numerous extracellular stimuli and is involved in signal transduc- tion cascades playing an important regulatory role in cell growth, differentiation, apoptosis, and metastasis (Chan-Hui and Weaver, 1998). Three major mammalian MAP kinases have been described: ERK1/2 or p44/42 MAPK, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAPK. The diverse MAP kinase members are activated in response to different extracellular stimuli and have distinct downstream targets, thus serving different roles in cellular responses. ERK1/2, p38 MAPK, and JNK/SAPK play a cen- tral role in regulating the expression of MMPs and u-PA (Chen et al., 2005; Kwon et al., 2008; Lee et al., 2008). In addition, PI3K/Akt sig- nal transduction pathway regulates the cell metastasis of non-small cell lung cancer (NSCLC) and is closely associated with the develop- ment and progression of various tumors. Overexpression of PI3K and low expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are closely correlated with the develop- ment, invasion and metastasis of NSCLC (Liao et al., 2006). NF-jB is a multisubunit transcription factor, which is involved in immune response, inammation and malignant transformation. The active NF-jB consists of p50, p52, p65 (RelA), Rel B, and c-Rel. under normal condition, NF-jB is maintained in the cytoplasm through interactions with an inhibitor of NF-jB (IjB), but upon dissociation, moves into the nucleus and promotes cancer cells proliferation, angiogenesis and metastasis. AP-1 is a nuclear tran- scription, which is involved in cell proliferation, differentiation, apoptosis and neoplastic transformation. AP-1 consists of homodi- mers and heterodimers of members from Fos (c-Fos, Fos B, Fra-1, and Fra-2) and Jun (c-Jun, Jun B, and Jun D) families (Karin and Ben-Neriah 2000; Lee et al., 2007). Previous papers have showed the MMP-2, MMP-9, and u-PA promoters are coordinately regu- lated by both NF-jB and AP-1 (Aguirre Ghiso et al., 1999; Ma et al., 2004; Westermarck and Kahari, 1999). As with NF-jB and AP-1 regulates the expression of matrix metalloproteinase and u- PA, consistent with a role for this protein in regulating metastasis. Further, to establish anti-metastastic mechanism of a-tomatine, the objective of this work was to examine the inhibitory effects and the related signaling pathways of a-tomatine on the inva- sion/migration of human lung adenocarcinoma A549 cells in vitro. 2. Materials and methods 2.1. Chemicals and reagents a-tomatine (purity >97%), DMSO, TrisHCl, EDTA, SDS, phenylmethylsulfonyl uoride, bovine serum albumin (BSA), gelatin, casein, plasminogen, leupeptin, Non- idet P-40, deoxycholic acid, sodium orthovanadate, wortmannin, and U0126 were purchased from SigmaAldrich (St. Louis, MO); the protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA). Dulbeccos phosphate buffer solution (PBS), tryp- sin-EDTA, and powdered Dulbeccos modied Eagles medium (DMEM) were pur- chased from Gibco/BRL (Gaithersburg, MD). Matrigel was from BD Biosciences (Bedford, MA). Antibody against Akt, MAPK/ERK1/2, JNK/SAPK and p38 MAPK, pro- teins and phosphorylated proteins were purchased from Cell Signaling Tech. (Bev- erly, MA). PI3K (p85), NF-jB (p65), c-Fos, c-Jun, b-actin, and C23 antibodies were from BD Transduction Laboratories (San Diego, CA). The enhanced chemilumines- cence (ECL) kit was purchased from Amersham Life Science (Amersham, UK). 2.2. Cell culture and a-tomatine treatment A549, a human lung adenocarcinoma cell line, was obtained from BCRC (Food Industry Research and Development Institute in Hsin-Chu, Taiwan). Cells were cul- tured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ ml of penicillin, 100 mg/ml streptomycin mixed antibiotics and 1 mM sodium pyru- vate. All cell cultures were maintained at 37 C in a humidied atmosphere of 5% CO 2 95% air. The culture medium was renewed every 23 days. Adherent cells were detached by incubation with trypsin. For a-tomatine treatment, the stock solution of a-tomatine was dissolved in dimethyl sulfoxide (DMSO) and sterilized by ltra- tion through 0.2 lm disc lters. Suitable amounts of stock solution (1 mg/ml in DMSO) of a-tomatine were added into the cultured medium to achieve the indi- cated concentrations (Final DMSO concentration was less than 0.2%) and then incu- bated with cells for the indicated time periods. 2.3. Analysis of cell viability (MTT assay) To evaluate the cytotoxicity of a-tomatine, an MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-tetrazolium bromide] assay was performed to determine cell via- bility (Mosmann, 1983). Briey, cells were seeded at a density of 4 10 4 cells/ml in a 24-well plate for 24 h. Then, the cells were treated with a-tomatine at various concentrations (0, 1, 1.5, 2, 2.5, 3, and 3.5 lM) for various periods of time (24 and 48 h). Each concentration was repeated three times. After the exposure period, the medium was removed that was followed by washing the cells with PBS. Then, the medium was changed and incubated with MTT solution (5 mg/ml)/well for 4 h. The medium was removed, and formazan was solubilized in isopropanol and mea- sured spectrophotometrically at 563 nm. The percentage of viable cells was esti- mated by comparing with untreated control cells. 2.4. Analysis of MMP-2, MMP-9 and u-PA activity (zymography assay) The activities of MMP-2 and MMP-9 were assayed by gelatin zymography as de- scribed previously (Chu et al., 2004). Briey, conditioned media from cells cultured in the absence of serum for 24 h were collected. Samples were mixed with loading buffer and electrophoresed on 8% SDSpolyacrylamide gel containing 0.1% gelatin. Electrophoresis was performed at 140 V and 110 V for 3 h. Gels were then washed twice in zymography washing buffer (2.5% Triton X-100 in double-distilled H 2 O) at room temperature to remove SDS, followed by incubation at 37 C for 1216 h in zymography reaction buffer (40 mM TrisHCl (pH 8.0), 10 mM CaCl 2 , 0.02% NaN 3 ), stained with Coomassie blue R-250 (0.125% Comassie blue R-250, 0.1% ami- no black, 50% methanol, 10% acetic acid) for 1 h and destained with destaining solu- tion (20% methanol, 10% acetic acid, 70% double-distilled H 2 O). Non-staining bands representing the levels of the latent form of MMP-2 and MMP-9 were quantied by densitometer measurement using a digital imaging analysis system. Fig. 1. Effect of a-tomatine on the viability in A549 cells. (A) Chemical structure of glycoalkaloid a-tomatine isolated from the leaves and fruits of tomato (Lycopersicon esculentum). (B) Cells (4 10 4 cells/ml) were treated with various concentrations (0, 1, 1.5, 2, 2.5, 3, and 3.5 lM) of a-tomatine for 24 and 48 h. Cell viability was determined by MTT assay. The cell viability was directly proportional to the production of formazan, which was measured spectrophotometrically at 563 nm. Values are expressed as mean SD of three independent experiments. **p < 0.01, ***p < 0.001 compared with the untreated control (dose 0). 1986 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 Visualization of u-PA activity was performed by casein-plasminogen zymogra- phy. Briey, 2% casein and 20 lg/ml plasminogen were added to 8% SDSPAGE gel. Samples with a total protein of about 20 lg were then loaded onto the gels. The u-PA activity of cells treated or untreated with a-tomatine was measured as de- scribed in the gelatin zymography section. 2.5. Wound-healing assay For cell motility determination, A549 cells (1 10 5 cells/ml) were plated in six- well tissue culture plate and grown to 8090% conuence. After aspirating the med- ium, the centers of the cell monolayers were scraped with a sterile micropipette tip to create a denuded zone (gap) of constant width. Subsequently, cellular debris was washed with PBS, and A549 cells were exposed to various concentrations of a-tom- atine (0, 1, 1.5, and 2 lM). The wound closure was monitored and photographed at 0, 12, 24, 36, and 48 h with an Olympus CKX-41 inverted microscope and an Olym- pus E-410 camera. To quantify migrated cells, pictures of the initial wounded mon- olayers were compared with the corresponding pictures of cells at the end of the incubation. Articial lines tting the cutting edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures following incubation. Mi- grated cells across the white lines were counted in six random elds from each trip- licate treatment, and the data were presented as mean SD. 2.6. Boyden chamber invasion and migration assay The ability of A549 cells to pass through Matrigel-coated lters was measured by the Boyden chamber invasion assay (Ochi et al., 1993). Matrigel was diluted to 200 lg/ml with cold ltered distilled water and applied to the top side of the 8- lm pore polycarbonate lter. Briey, A549 cells were treated with various con- centrations of a-tomatine. After 48 h, cells were detached by trypsin and resus- pended in serum-free medium. Medium containing 10% FBS-medium was applied to the lower chamber as chemoattractant, and then the cells were seeded on the upper chamber at a density of 1 10 5 cells/well in 50 ll of serum-free medium. The chamber was incubated for 8 h at 37 C. At the end of incubation, the cells in the upper surface of the membrane were carefully removed with a cotton swab and cells invading across the Matrigel to the lower surface of the membrane were xed with methanol and stained with 5% Giemsa solution. The invasive cells on the lower surface of the membrane lter were counted with a light microscope. The data are presented as the average number of cells attached to the bottom surface from randomly chosen elds. Each experiment was carried out in triplicate. To measure the ability of A549 cells on migration, cells were seeded into a Boy- den chamber with 8 lm pore polycarbonate lters which were not coated with Matrigel. The migration of cells was treated with various concentrations of a-tom- atine. The migration assay was measured as described in the invasion assay. 2.7. Preparation of whole-cell lysates and nuclear extracts The cells were lysed with iced-cold RIPA buffer (1% NP-40, 50 mM Tris base, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5) and then the fol- lowing were added phenylmethylsulfonyl uoride (10 mg/ml), leupeptin (17 mg/ ml), and sodium orthovanadate (10 mg/ml). After vortexing for 30 min on ice, the samples were centrifuged at 12,000 g for 10 min, and then the superna- tants were collected, denatured, and subjected to SDSPAGE and Western blot- ting. Nuclear extracts were prepared as previously described (Hoppe-Seyler et al., 1991) and then used for NF-jB, c-Fos, c-Jun, and AP-1 detection. Each nu- clear pellet was resuspended in nuclear extract buffer (1.5 mM MgCl 2 , 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsul- fonyl uoride, 25% glycerol, and 420 mM NaCl). The nuclear suspension was incubated on ice for 20 min and then centrifuged at 14,000 g for 5 min. The supernatant (corresponding to the soluble nuclear fraction) was saved, and the remaining pellet was solubilized by sonication in PBS. The protein content was determined with Bio-Rad protein assay reagent using bovine serum albu- min as a standard. 2.8. Western blotting assay To analyze the migration-related proteins, Western blotting was performed as follows. The denatured samples (50 lg puried protein) were resolved on 1012% SDSPAGE gels. Proteins were then transferred onto nitrocellulose membranes. Non-specic binding of the membranes was blocked with Tris-buffered saline (TBS) containing 1% (w/v) non-fat dry milk and 0.1% (v/v) Tween-20 (TBST) for more than 2 h. Membranes were washed with TBST three times for 10 min and incubated with a suitable dilution of specic primary antibodies in TBST overnight at 4 C. Subsequently, the membranes were washed with TBST and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti- mouse or antirabbit IgG) for 1 h. After washing the membrane three times for 10 min in TBST, the band detection was revealed by enhanced chemiluminescence using ECL Western blotting detection reagents and exposed ECL hyperlm in a UVP Luminescent image analyzer. 2.9. Analysis of NF-jB and AP-1 binding assay (electrophoretic mobility shift assay) Cell nuclear proteins were extracted with a nuclear extract buffer and measured by an electrophoretic mobility shift assay (EMSA) (Ma et al., 2001). Cells (1 10 5 / ml) were collected in PBS buffer (pH 7.4) and centrifuged at 2000 g for 5 min at 4 C. Cells were lysed with buffer A (10 mM HEPES, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF (pH 7.9) containing 5% NP-40) for 10 min on ice, and this was followed by vortexing to shear the cytoplasmic membranes. The ly- sates were centrifuged at 2000 g for 10 min at 4 C. The pellet containing the nu- clei was extracted with high salt buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, and 25% glycerol) for 15 min on ice. The lysates were claried by centrifuge at 13,000 g for 10 min at 4 C. The supernatant containing the nuclear proteins was collected and frozen at 80 C un- til use. The protein content of nuclear fractions was determined with Bio-Rad pro- tein assay. Five microgram aliquot of nuclear proteins were mixed with either biotin-labeled NF-jB or AP-1 oligonucleotide probes for 15 min at room tempera- ture or with oligonucleotides containing (sense of NF-jB, 5 0 -AGTTGAGGGGACTTTCC CAGGC-3 0 , antisense of NF-jB, 3 0 -TCAACTCCCCTGAAAGGGTCCG-5 0 ; sense of AP-1, 5 0 -CG CTTGATGACTCAGCCGGAA-3 0 , antisense of AP-1, 3 0 -GCGAACTACT- GAGTCGGCCTT). DNA probes were added to 10 ll binding reactions containing dou- ble-distilled H 2 O, 5 lg nuclear protein, 1 ll poly (dI-dC), 1 ll biotin-labeled double- stranded NF-jB or AP-1 oliginucleotides and 2 ll of 10-fold binding buffer into a microcentrifuge tube and were incubated for 15 min at room temperature. Specic competition binding assays were performed by adding 200-fold excess of unlabeled probe as a specic competitor. Following protein-DNA complexes formation, sam- ples were loaded on a 6% non-denaturing polyacrylamide gel in 0.5 TBE buffer and were then transferred to positively charged nitrocellulose membranes (Mili- pore, Bedford, MA) by a transfer blotting apparatus and cross-linked in a Stratagene cross-linker. Gel shifts were visualized with streptavidin-horseradish peroxidase followed by chemiluminescent detection. 2.10. Statistical analysis Data were expressed as mean SD of three independent experiments and ana- lyzed by Students t-test (Sigmaplot 2001). Signicant differences were established at p 6 0.05. 3. Results 3.1. Cytotoxicity of a-tomatine in A549 cells We rst assayed the cytotoxicity of a-tomatine by treating A549 cells with a-tomatine at various concentrations (0, 1, 1.5, 2, 2.5, 3, and 3.5 lM) for 24 and 48 h followed by MTT assay. As shown in Fig. 1B, a-tomatine showed a dose- and time-dependent inhibitory effect on the growth of A549 cells. Compared to 0 lM (DMSO was treated alone, data not shown), after 24 h and 48 h treatment with a-tomatine at a concentration between 0 to 2 lM was not signicantly altered, indicating that a-tomatine was not toxic to A549 cells at these dosages. When cells were treated with 2.53.5 lM a-tomatine for 24 and 48 h, cell viability was signi- cantly decreased. These results demonstrated the treatment of a- tomatine with doses higher than 2 lM for 24 and 48 h resulted in dose- and time-dependent loss of cell viability in A549 cells, but doses lower than 2 lM for 24 and 48 h did not cause cytotox- icity. In the following experiments, these doses below 2 lM of a- tomatine were applied in all subsequent experiments. 3.2. a-Tomatine inhibits the activation of MMP-2, MMP-9, and u-PA in A549 cells For the cell migration and invasion processes, pointing to the inevitable involvement of matrix-degrading proteinases, the ef- fects of a-tomatine on MMP-2, MMP-9, and u-PA activities were investigated by gelatin and casein zymography. The conditioned media were collected, concentrated, and the inhibition of metasta- sis was measured after A549 cells were treated for 24 h by a-tom- atine. As shown in Fig. 2A, dose-dependent and markedly reduced MMP-2 and MMP-9 activities were noted in the serum-free med- ium treated with 2 lMa-tomatine for 24 h. Similarly, u-PA activity was also inhibited in a dose-dependent manner by a-tomatine treatment (Fig. 2B). These results suggested the anti-metastatic Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1987 effect of a-tomatine was related to the inhibition of the enzymat- ically degradative processes of tumor metastasis. This study gives a rst glimpse to demonstrate a-tomatine reduced the metastasis in human lung adenocarcinoma cells. The activities of MMP-2, MMP- 9, and u-PA have been shown to play a critical role in degrading the basement membrane in cancer invasion and migration. 3.3. a-Tomatine inhibits the migration and invasion in A549 cells To investigate the inhibitory effect of a-tomatine on A549 cells migration and invasion process, a wound-healing assay and a Boy- den chamber assay were used. In wound-healing assay, the conu- ent monolayer was scraped with a sterile micropipette tip to create a scratch wound. After incubation with 1.5 and 2 lM of a-tomatine for 24 and 48 h, the cells migrated to the denuded zone, and they were counted. The results demonstrated a-tomatine dose-depen- dently suppressed A549 cell migration to the denuded zone. According to a quantitative assessment, treatment with 1.5 and 2 lM of a-tomatine inhibited 60% and 69% of cell migration after 24 h, respectively; and such doses of a-tomatine inhibited 45% and 55% of cell migration at 48 h, respectively. Also, the cells were treated with various concentrations of a-tomatine for 0, 12, 24, 36, and 48 h. The results showed 2 lM of a-tomatine exhibited the most inhibiting effect on cell motility after 48 h incubation. Espe- cially, compared with the untreated cells, the level of A549 cells number decreased almost 2.2-fold with the treatment of 2 lM a- tomatine for 48 h (Fig. 3A). Although the cells were not treated by a-tomatine, the number of cells in migration property was increased with increasing time (Because A549 cells is under non- cytotoxic concentrations). Also, the cells were treated with non- a-tomatine for 24, 36 and 48 h compared with the untreated control (dose 0), the results was presented signicantly different and dened as constituting statistical signicance. These results revealed that a-tomatine signicantly inhibited the motility of A549 cells. One important characteristic of metastasis is the migratory and invasive ability of tumor cells. We used Boyden chamber assay to quantify the migratory and invasive potential of A549 cells. The re- sults showed a-tomatine induced a dose-dependent decrease in migration with increasing concentrations of a-tomatine (Fig. 3B). At 1.5 lM, the migration was reduced to 54.9% and at 2 lM the migration was reduced to less than 43%. Subsequently, a-tomatine also induced a dose-dependent decrease in invasion with increas- ing concentrations of a-tomatine (Fig. 3C). At 1.5 lM the invasion was reduced to 68.1% and at 2 lM the invasion was reduced to less than 51%. The results demonstrated a-tomatine signicantly inhib- ited the migration and invasion of A549 cells. 3.4. a-Tomatine inhibits phosphorylation of ERK and Akt Since we have shown treatment of A549 cells with a-tomatine inhibited the cell metastasis and activities of MMP-2, MMP-9, and u-PA, the underlying mechanisms were further investigated. Sev- eral studies have indicated the transcription factors (for example, NF-kB, c-Fos, and c-Jun), JNK1/2, ERK1/2, p38 MAPK, and Akt that are involved in activity of MMP-2, MMP-9, and u-PA on different cell types (Chen et al., 2005; Turner et al., 2007). To assess whether a-tomatine mediates and/or inhibits phosphorylation of JNK1/2, ERK1/2, p38 MAPK, Akt, and the protein level of PI3K, we investi- gated the effect of a-tomatine on the phosphorylated status of MAPK family members (JNK1/2, ERK1/2, and p38 MAPK) and Akt in A549 cells which were treated with various concentrations of a-tomatine for 3 h and 2 lM of a-tomatine for various periods of time (0, 1, 2, 3, and 6 h). Fig. 4A and B showed a-tomatine signi- cantly inhibited the activation of ERK1 and ERK2 as shown by decreasing the phosphorylation of ERK1 and ERK2. In contrast, Fig. 2. Effect of a-tomatine on MMP-2/MMP-9 and u-PA activities in A549 cells. Cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 24 h. The conditioned media were collected, and then (A) MMP-2/MMP-9 and (B) u-PA activities were determined by gelatin zymography or casein zymography. MMP-2/MMP-9 and u-PA activities were quantied by densitometric analysis. The densitometric data were expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control (dose 0). 1988 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 Fig. 3. Effect of a-tomatine on the migration and invasion in A549 cells. (A) In wound-healing assay, A549 cell monolayers were scraped by a sterile micropipette tip and the cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 0, 12, 24, 36, and 48 h. The number of cells in the denuded zone was quantitated after indicated times (0, 12, 24, 36, and 48 h) by inverted microscopy. White lines indicate the wound edge. Pictures only were presented 24 and 48 h. Migrated cells across the white lines were counted in six random elds from each treatment. (B) In Boyden chamber migration assay, cells were treated with various concentrations of setin for 48 h, then cell migration were measured by Boyden chamber for 6 h with polycarbonate lters (pore size, 8 lm); (C) In Boyden chamber invasion assay, cells were treated with various concentrations of setin for 48 h, then cell invasion were measured by Boyden chamber for 8 h; polycarbonate lters (pore size, 8 lm) were precoated with Matrigel. Migration and invasion ability of A549 cells were quantied by counting the number of cells that invaded to the underside of the porous polycarbonate membrane under microscopy. Values are expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control (dose 0); ## p < 0.01, ### p < 0.001 compared with the 0-h treated time. Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1989 a-tomatine did not signicantly affect phospho-JNK1/2 and phos- pho-p38 activity (Fig. 4CF). In addition, a-tomatine inhibited the protein level of PI3K and phosphorylation of Akt in a dose- and time-dependent manner (Fig. 4G and H). To further investigate whether the inhibition of a-tomatine was mainly occurred through the inhibition of ERK1/2 or PI3K/Akt sig- naling pathway, A549 cells were pretreated with a PI3K inhibitor (Wortmannin; 5 or 10 lM) or ERK inhibitor (U0126; 10 or 20 lM) for 1 h and then incubated in the present or absence of a-tomatine (1 lM) for 24 h. Results of gelatin zymography assay has shown that a sole treatment of Wortmannin (5 or 10 lM) and U0126 (10 or 20 lM) or a-tomatine separately reduced the expressions of MMP-9 or MMP-2 or by 8.7%, 28.3%, 14%, 34.3% and 12.3% or 4.7%, 14.2%, 7.3%, 30% and 3.3%, respectively, and the combination treatment could even dramatically reduced the secretions of MMP-9 or MMP-2 by 50.7% or 25% (5 lM Wortman- nin + 1 lM a-tomatine), 63.3% or 40% (10 lM U0126 + 1 lM a- tomatine) and 79.2% or 62.5% (5 lM Wortmannin + 10 lM U0126 + 1 lM a-tomatine) (Fig. 5A). Similarly, in a casein zymog- raphy assay, a sole treatment of Wortmannin (5 or 10 lM) and U0126 (10 or 20 lM) or a-tomatine reduced the expression of u- PA by 12%, 27.5%, 14.5%, 32% and 15%, respectively, and the com- bination treatment could further reduce the secretion of u-PA by 67% (5 lM Wortmannin + 10 lM U0126 + 1 lM a-tomatine) (Fig. 5B). The data nding revealed the inhibition of the expres- sions of MMP-2 and MMP-9 by a-tomatine on A549 cells could partly occur through ERK1/2 and Akt inactivation, while the inhi- bition of the expression of u-PA could partly occur through ERK1/2 inactivation. 3.5. a-Tomatine inhibits the DNA binding activities of NF-jB, c-Fos, and c-Jun NF-jB and AP-1 family of transcriptional factors have been known to translocate to the nucleus and regulate the expression of multiple genes involved in MMPs or u-PA secretion. To clarify Fig. 4. Dose- and time-dependent effect of a-tomatine on the phosphorylation of ERK, JNK, p38, Akt, and the protein expression level of PI3K. In dose-dependent assay (A, C, E, and G), A549 cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 3 h. In time-dependent assay (B, D, F, and H), A549 cells were treated with 2 lM of a-tomatine for 0, 1, 2, 3, and 6 h. Activities of ERK phosphorylation, ERK, JNK phosphorylation, JNK, p38 phosphorylation, p38, Akt phosphorylation, Akt, and the expression of PI3K were analyzed by Western blotting. b-Actin was used as a loading control. Values are expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control (dose 0). 1990 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 the involvement of NF-jB and AP-1 proteins in the mechanism of a-tomatines action, the effect of a-tomatine on the DNA binding activities of NF-jB and AP-1 in A549 cells was explored by EMSA. As shown in Fig. 6A, A549 cells were treated with 02 lM of a- tomatine for 24 h, and a-tomatine inhibited NF-jB and AP-1 tran- scriptional activities in a dose-dependent manner. Especially, the binding activities of NF-jB and AP-1 were strongly inhibited by treating with 2 lM a-tomatine. Further, the expressions of NF- jB, c-Fos, and c-Jun in nuclear extracts were analyzed by Western blotting to assess the possible inhibitory effect of a-tomatine on NF-jB, c-Fos, and c-Jun. As explained in Fig. 6B, the nuclear levels of NF-jB, c-Fos, and c-Jun were gradually diminished by a-toma- tine in a dose-dependent manner. Especially, data was shown to be strongly inhibited by treating with 2 lM a-tomatine. 4. Discussion Lung cancer is the most common neoplasm in humans in both developed and developing countries (Erridge et al., 2007; Gajra et al., 2003; McCracken et al., 2007). This research has conrmed a-tomatine can inhibit the invasion and migration of A549 human adenocarcinoma cells in vitro model. We found a-tomatine can suppress cancer cell invasion and migration possibly occurs through inactivation of PI3K/Akt or ERK signaling pathways, exert- ing inhibitory effects on NF-jB, c-Fos, and c-Jun transcriptional fac- tors, inhibiting NF-kB and AP-1 DNA binding activities, decreasing MMP-2, MMP-9, and u-PA activities, and then having an anti-met- astatic effect. Our results strengthen the potential of a-tomatine as a new strategy for anti-cancer therapy. Fig. 4. (continued) Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1991 a-Tomatine, a tomato glycoalkaloid, may also have benecial ef- fects. Glycoalkaloids are reported to inactivate the Herpes simplex and Herpes zoster viruses in humans (Chataing et al., 1997), to en- hance the duration of action of anesthetics, which act by inhibiting acetylcholinesterase (McGehee et al., 2000), and to potentiate the immune response of vaccines in mice (Rajananthanan et al., 2000). a-Tomatine may benet cancer chemotherapy by inhibiting multidrug resistance in human cancer cells (Lavie et al., 2001). As part of an effort designed to improve food safety through identica- tion and reduction of the content of the most toxic alkaloids in plant foods using safety evaluation. In previous study has demonstrated a-tomatine dose not appear to be toxic when consumed orally in moderate amount, and observation that the absence of a 5, 6-dou- ble bond in the B-ring of tomatidine results in a much less toxic molecule in mice (Friedman et al., 2000). Wilson et al. studied the pharmacology and toxicology of a-tomatine. In mice, a-tomatine appears to be non-toxic following oral consumption, presumably because of poor absorption from the digestive tract into the blood- stream due to formation of an insoluble complex with dietary and endogenous cholesterol which is then eliminated in the faeces (Cayen, 1971; Roddick, 1979). Nevertheless, further studies need to be done in order to investigate the anti-metastatic effect of a- tomatine in humans. Malignant tumors invade the tissue, involving three indepen- dent processes: the degradation of the extracellular matrix (ECM), cell metastasis and proliferation. Metastasis has been found to be accompanied by various physiological alterations involved in degrading ECM, such as the overexpression of proteolytic enzyme activity as in MMPs or u-PA, as well as the migration and invasion of tumor cells into the bloodstream or lymphatic system to spread to other tissues or organs (Kleiner and Stetler-Stevenson, 1999). More specically, the ability to penetrate the basement membrane (BM) is related to an increased potential for metastasis. Basement membranes are thin extracellular matrices underlying cells in vivo. Matrigel Basement Membrane Matrix is a solubilized basement membrane preparation extracted from the Englebreth-Holm Swarm (EHS) mouse tumor. It major component is lamin, collagen I, entactin, heparin sulfate proteoglycan (perlecan), growth factors, and so on. A number of methods have been developed using Matri- gel Matrix to investigate the invasion of the basement membrane matrix by tumor in vitro. The invasive ability of A549 cells to pass through Matrigel-coated lters was measured by the Boyden chamber invasion assay. So, our study demonstrated treatment with a-tomatine at a non-cytotoxic concentration below 2 lM for 24 h exerted an inhibitory effect in a dose- and time-dependent manner on the migration and invasion of the highly metastatic A549 cells. In recent years, attention has been drawn to the phys- iological relevance of MMPs and u-PA markers related to the met- astatic ability and malignancy of tumor cells (Chen et al., 2005; Shih et al., 2007). Thus, many studies have shown proteinases re- lated to degradation of matrix are required for tumor cell metasta- sis and heightened production of MMPs and u-PA correlates with the invasion, migration and angiogenesis of the tumors (Mackay et al., 1990). To further explore the exact expression of a-toma- tine-induced inhibition on migration and invasion, we performed gelatin or casein-plasminogen zymographic assays, to detect activ- ities of MMP-2, MMP-9, and u-PA. The result showed a-tomatine noticeably downregulated the activities of MMP-2, MMP-9, and u-PA. These results suggested the anti-metastastic effect of a-tomatine was associated with the inhibition of the enzymatically degradative processes of tumor metastasis. One important charac- teristic of metastasis is the migratory and invasive ability of tumor cells. Further, we used wound-healing assay and Boyden chamber assay to quantify the migratory potential of A549 cells. The results demonstated a-tomatine signicantly inhibited the migration and invasion of A549 cells. A major mechanism through which signals from extracellular stimuli are transmitted to the nucleus involves the activation of ki- nases. These kinases, serine/threonine kinases related to the mito- gen-activated proteins kinase (MAPK) superfamily, mediate signals from cell membrane receptors triggered by growth factors, cyto- kines, and cell-matrix interactions. MAPKs are intricately involved in the expression of the components involved in MMPs or u-PA promoters induction by NF-kB, AP-1, and its association with c- Fos and c-Jun. At least three subgroups of MAPK family members have been implicated: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38MAPK (Robinson and Cobb, 1997). Also, the PI3K and Akt signal pathways also play a critical role in MMP-9 gene regulation (Yao et al., 2001). Fig. 5. Effect of PI3K inhibitor (Wortmannin), ERK inhibitor (U0126), and a- tomatine on the activities of MMP-2, MMP-9, and u-PA. Cells were plated in six-well and pre-treated with Wortmannin (5 or 10 lM) or U0126 (10 or 20 lM) for 1 h and then incubated in the presence or absence of a-tomatine (1 lM) for 24 h. Afterwards, the culture medium was subjected to gelatin and casein zymography to analyze the activities of (A) MMP-2/MMP-9 and (B) u-PA. Determined activities of these proteins were subsequently quantied by densitometric analysis with that of control being 100% as shown just below the gel data. Data represented the mean SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). 1992 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 Indeed, we have demonstrated treatment of a-tomatine inhib- ited phosphorylation of ERK1/2 and Akt. In contrast, a-tomatine did not signicantly affect phospho-ERK1/2 and phospho-p38 activities. The involvement of the ERK and PI3K/Akt pathways was further supported by using the ERK and PI3K inhibitors in our experimental model. Treatment with an inhibitors specic for ERK could inhibit the MMP-2, MMP-9, and u-PA secretion. Also, the PI3K/Akt signaling pathway also played a crucial role in MMP- 2, MMP-9, and u-PA gene regulation, cell survival and tumor cell metastasis (Kim et al., 2001; Rao, 2003; Westermarck and Kahari, Fig. 6. Effects of a-tomatine on the DNA binding activities of NF-jB and AP-1 in A549 cells. Cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 24 h. Cell nuclear extracts were prepared and analyzed for (A) NF-jB and AP-1 DNA binding activities using biotin-labeled consensus NF-jB and AP-1 specic oligonucleotide, then EMSA assay were performed as described in materials and methods. Lane 1: nuclear extracts incubated with 100-fold excess unlabeled consensus oligonucleotide (comp.) to conrm the specicity of binding. Excess free probe is indicated at the bottom. (B) Nuclear extracts were also analyzed by Western blotting with anti-NF-jB (p65), c-Fos, and c-Jun antibodies. C23 was a nucleus protein loading control. Determined the protein expressions of NF-jB, c-Fos, and c-Jun were subsequently quantied by densitometric analysis with that of control being onefold. The densitometric results are expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control. Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1993 1999). Our ndings suggested a-tomatine-caused decreased MMP- 2 and MMP-9 activities in the culture media could possibly occur through suppression of phospho-ERK1/2 or phospho-Akt, while the reduction in u-PA activity may only be associated with a sup- pression of phospho-ERK1/2. Here, it was demonstrated a-toma- tine clearly decreased MMP-2, MMP-9, and u-PA activities through inactivating the PI3K/Akt or ERK signaling pathways, and such inhibitory effect on proteinases expression may contribute to the capability of a-tomatine for the inhibition of cell metastasis. In this study, the activity of u-PA, an upstream activating en- zyme of MMP-2 and MMP-9 involved in invasion and migration, was also shown to be inhibited in a dose-dependent manner by a-tomatine treatment. The transcription of MMPs and u-PA gene is regulated by upstream regulatory sequences, including NF-jB, AP-1, and Ets-1 binding sites (Nagase and Woessner, 1999; Roth- hammer et al., 2004; Sliva, 2004; Westermarck and Kahari, 1999). Therefore, this study provides insight into how a-tomatine suppresses the ERK1/2 and Akt signaling pathways and reduces NF-kB and AP-1 transcriptional activities in A549 lung adenocarci- noma cells. Indeed, one or more of these binding sites have been implicated in mediating the effects of a diverse set of agents. Here, we have also found the treatment of a-tomatine to A549 cells re- sults in an inhibition of NF-kB and AP-1 DNA binding activities, which was accompanied by the inhibition of nuclear translocation of these factors. Thus, the inhibitory effect of a-tomatine on the migration and invasion of non-small lung cancer cells probably oc- curs by abating the expressions of NF-kB, c-Fos, and c-Jun, then reducing the activities of MMP-2, MMP-9, and u-PA. In addition, Chishma et al. (1997) indicated the tumor host-or- gan chimeric histoculture system developed in the present study with GFP uorescing tumor cells has signicantly advanced the ability to understand and treat human metastatic cancer. The re- sults provide an invaluable new tool for understanding the most important steps in tumor host-organ interaction, tumor progres- sion, and metastasis. Because the metastatic colony expansion takes place in vitro, it offers a unique opportunity for developing agents for intervention. We will carry out this histoculture exper- iment in the further. Finally, the involvement of ERK and PI3K/Akt signaling path- ways in cell metastasis were further supported by experiments with ERK and PI3K/Akt inhibitors, showing treatment with inhibi- tors of ERK and PI3K/Akt to A549 cells inhibited the cell metastasis. 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