Inhibition of Matrix Metalloproteinase Activities and Tightening of Tight Junctions by Diallyl Disulfide in AGS Human Gastric Carcinoma Cells Hyun Soo Park, Gi-Young Kim, Il-Whan Choi, Nam Deuk Kim, Hye Jin Hwang, Young-Whan Choi, and Yung Hyun Choi
Abstract: The effect of diallyl disulfide (DADS), a major component of an oil-soluble allyl sulfide garlic (Allium sativum)
derivative, on the correlation between anti-invasive activity and tightening of tight junctions (TJs) was investigated in human gastric adenocarcinoma AGS cells. Our data indicated that the inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance. Activities of matrix metalloprotease (MMP)-2 and -9 in AGS cells were dosedependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins; however, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 mRNA levels and proteins were increased. Additionally, immunoblotting results indicated that DADS repressed the levels of claudin proteins (claudin-2, -3, and -4), major components of TJs that play key roles in control and selectivity of paracellular transport. Although further studies are needed, these results suggest that DADS treatment may inhibit tumor cell motility and invasion and, therefore, act as a dietary source to decrease the risk of cancer metastasis.
Introduction Garlic (Allium sativum) has been used as a vegetable spice and medicinal herb for many centuries. Several epidemiologic or preclinical researches have demonstrated that consumption of garlic and other allium vegetables may be associated with low incidence of certain cancer types by way of suppressing the inflammatory processes that lead to transformation, hyperproliferation, and initiation of carcinogenesis (Hsing and others 2002; Aggarwal and Shishodia 2006; Iciek and others 2009). Diallyl disulfide (DADS), a compound derived from organosulfur products’ breakdown of allin (S-allylcysteine sulfoxide), has been considered as a potential molecular target against human cancer (Dwivedi and others 1992; Dwivedi and others 1998). Although the roles and mechanisms of DADS as a potential chemopreventive and/or chemotherapeutic agent have not been completely elucidated, several recent studies have indicated that DADS inhibits growth of cancer cells. This inhibition of cancer cell growth is associated with cell cycle arrest at the G2/M phase in a p53-independent manner and stimulation of apoptosis though generation of reactive oxygen species; increase in endoplasmic reticulum stress; and mitochondrial dys-
MS 20101190 Submitted 10/20/2010, Accepted 1/1/2010. Authors Park and N.D. Kim are with Dept. of Pharmacy, Pusan Natl. Univ., Busan 609-735, Republic of Korea. Author G.Y. Kim is with Dept. of Marine Life Sciences, Jeju Natl. Univ., Jeju 690-756, Republic of Korea. Author I.W. Choi is with Dept. of Microbiology, Inje Univ. College of Medicine, Busan 633-165, Republic of Korea. Author Hwang is with Dept. of Food and Nutrition, College of Human Ecology, Dongeui Univ., Busan 614-714, Republic of Korea. Author Y.W. Choi is with Dept. of Horticultural Bioscience, Pusan Natl. Univ., Miryang 627-706, Republic of Korea. Author Y.H. Choi is with Dept. of Biochemistry, College of Oriental Medicine and Research Inst. of Oriental Medicine, Dept. of Biomaterial Control, Graduate School and Blue-Bio Industry Regional Innovation Center, Dongeui Univ., Busan 614-052, Republic of Korea. Direct inquiries to author Choi (E-mail:
[email protected]).
R 2011 Institute of Food Technologists doi: 10.1111/j.1750-3841.2011.02114.x
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regulation, and activation of cysteine proteases (Lu and others 2004; Druesne-Pecollo and others 2006; Das and others 2007; Jo and others 2008; Yang and others 2009). Because DADS is relatively nontoxic to normal cells, an important implication of these findings is that this agent might play a useful role in treatment of cancer. However, the biochemical mechanisms underlying DADS-induced anti-invasiveness and antimetastasis have not yet been clarified. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, process a broad spectrum of cell surface molecules and function in several important biological processes. MMPs are capable of cleaving virtually all extracellular matrix (ECM) substrates, and degradation of matrix is a key event in progression, invasion, and metastasis of potentially malignant and malignant lesions (Duffy and others 2002; Vihinen and others 2005). Since MMPs have many physiological functions in metastasis, inhibition of the activity of MMPs holds great promise for prevention or inhibition of metastasis. Among various MMPs, MMP-2 and -9 (gelatinases A and B) seem to play an important role in tumor invasion and metastasis and are highly expressed in epithelial cancer cells (Matrisian 1992; Mook and others 2004). MMP-2 is preferentially secreted from fibroblasts and various epithelial cells, while MMP-9 is preferentially expressed by inflammatory cells (Gibbs and others 1999). Both have been frequently associated with the invasive metastatic potential of tumor cells. Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the catalytic activity of MMPs by binding to activated MMPs and control breakdown of ECM (Uzui and others 2002). TIMPs can also inhibit proliferation, invasion, and metastasis of malignant cells. Disturbance in balance of MMPs and TIMPs is found in various pathologic conditions, including cancer (Lambert and others 2004). Thus, the balance between MMPs and TIMPs plays a vital role in maintaining the integrity of
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Keywords: AGS, DADS, invasion, matrix metalloproteinases, tight junctions
Anti-invasive activity of DADS. . . healthy tissues; and MMP inhibitors, as well as TIMP activators, are expected to be useful chemotherapeutic agents for treatment of malignant cancer. Since cell adhesion between neighboring epithelial cells is a crucial and tightly controlled process, several specialized structures, including tight junctions (TJs), are responsible for establishment of contact between neighboring cells. TJs form the primary barrier to paracellular transport of solutes across cells and play a critical role in the establishment and maintenance of epithelial cell polarity (Soler and others 1999; Schneeberger and Lynch 2004). However, in precancerous and cancerous epithelia, TJ strands become disorganized or lost altogether and TJs become “leaky,” as indicated by decreased resistance to electrical current (transepithelial electrical resistance; TER) and increased paracellular permeability of markers (Soler and others 1999; Schneeberger and Lynch 2004). The claudin family proteins are key integral membrane proteins that form the backbone of TJs. The proteins can form homodimers or heterodimers to produce paired strands between adjacent cells and act as a barrier to paracellular flux of water, solutions, and transmigration of other cells, thereby determining the characteristic permeability properties of different epithelial tissues (Morin 2005). Recent studies have provided evidence that claudins are aberrantly expressed in various cancers and are associated with development and progression of cancer, which suggests that they have key cellular functions that are distinct from their roles in TJcomplexes. For example, claudin-3 and -4 are overexpressed in many types of cancer and in metastatic cells in particular. These cells show a greater tendency to spread to other sites (Rangel and others 2003; Soini 2004; Agarwal and others 2005; Kominsky 2006). Conversely, inhibition of claudin-3 and -4 in ovarian cancer cells reduced their invasive potential (Van Deun and others 2008). In addition, overexpression of claudin-1 is correlated with an advanced stage of disease in oral squamous cell carcinoma and with angiolymphatic and perineural invasion (Van Deun and others 2008; Dos Reis and others 2008). However, the exact role of claudin overexpression and the functional importance of these proteins in development of cancers remain unclear. Therefore, in this study, we investigated the effects of DADS on invasive parameters, including matrigel membrane invasion, zymography for MMP activities, and gene expression for TJassociated proteins, using human gastric adenocarcinoma AGS cells.
Materials and Methods
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Cell culture and MTT assay The human gastric adenocarcinoma cell line AGS was purchased from the American Type Culture Collection (Rockville, Md., U.S.A.), and maintained at 37 ◦ C in a humidified 95% air and 5% CO2 in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. DADS (C6 H10 S2 ) (Sigma Chemical Co., St. Louis, Mo., U.S.A.) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at a 100 mM concentration and was stored in aliquots at –20 ◦ C. For the cell viability study, AGS cells were grown to 70% confluence and treated with DADS. Control cells were supplemented with complete media containing 0.1% DMSO (vehicle control). Following treatment, cell viability was determined by MTT [3-(4,5-dimethyl-2 thiazolyl)-2,5-diphnyl2H-tetrazolium bromide, MTT, Sigma] assay (Jang and others 2010).
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Cell motility assay AGS cells were grown to confluency on 35-mm cell culture dishes coated with 1mL of rat-tail collagen (20 μg/mL, BD Biosciences, Bedford, Mass., U.S.A.). Confluent cells were wounded by scraping with a pipette tip. After washing with phosphatebuffered saline (PBS), serum-free media (to prevent cell proliferation) containing either vehicle (DMSO) or various concentrations of DADS was added for the indicated times. Wound closure of cells was observed and photographed under the microscope at 40× magnification (Kim and others 2009). Measurement of TER TER was measured using an EVOM Epithelial Tissue Voltohmmeter (World Precision Instruments, Fla., U.S.A.) equipped with a pair of STX-2 chopstick electrodes. Briefly, AGS cells were seeded into the 8.0 μm pore size insert (upper chamber) of a TranswellR (Corning Costar Corp., N.Y., U.S.A.) and allowed to reach full confluence, after which, fresh medium was replaced for further experiments. Inserts without cells, inserts with cells in medium, and inserts with cells with DADS were treated for 48 h. Electrodes were placed at the upper and lower chambers, and resistance was measured with the voltohmmeter. In vitro invasiveness assay Matrigel invasion assays were used to assess the ability of AGS cells to penetrate ECM in the presence or absence of DADS, as previously described (Kim and others 2009). Briefly, the chamber membrane was coated with 50 μL of matrigel (BD Biosciences) diluted at 10:1 in ice-cold serum-free medium, and then cells were exposed to DADS for 6 h, and treated cells (50000) were then plated onto the apical side of the Matrigel-coated filters in serum-free medium containing either DMSO or DADS. Medium containing 20% FBS was placed in the basolateral chamber to function as a chemo-attractant. After 48 h, cells on the apical side were wiped off with a Q-tip. Cells on the bottom of the filter were stained with hematoxylin and Eosin Y (Sigma) and counted (3 fields of each triplicate filter) with an inverted microscope. RNA extraction and reverse transcription-polymerase chain reaction (PCR) Total RNA was prepared according to the manufacturer’s instructions using an RNeasy kit (Qiagen, La Jolla, Calif., U.S.A.) and primed with random hexamers for synthesis of complementary DNA through avian myeloblastosis virus reverse transcriptase (Amersham Corp., Arlington Heights, Ill., U.S.A.). A PCR was carried out in a Mastercycler (Eppendorf, Hamburg, Germany) using the primers indicated in Table 1. Conditions for PCR reactions were 1 × (94 ◦ C for 3 min), 35 × (94 ◦ C for 45 s; 58 ◦ C for 45 s; and 72 ◦ C for 1 min), and 1 × (72 ◦ C for 10 min). Amplification products obtained by PCR were electrophoretically separated on 1% agarose gel and visualized by ethidium bromide (EtBr) staining. Protein extraction and Western blot analysis The cells were harvested, lysed, and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad Lab., Hercules, Calif., U.S.A.), according to the procedure reported by the manufacturer (Jang and others 2010). For Western blot analysis, an equal amount of protein was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide
Anti-invasive activity of DADS. . . gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, N.H., U.S.A.) by electroblotting. The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies and visualized by enhanced chemiluminescence (ECL) according to the recommended procedure (Amersham). Primary antibodies against MMP-2, -9, TIMP-1, and -2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., U.S.A.), and claudin-2, -3, and -4 were obtained from Invitrogen Corp. (Carlsbad, Calif., U.S.A.). Antibody against actin was purchased from Sigma. All primary antibodies except for actin (1:2000) were diluted at 1:500 in phosphate buffer saline tween-20 buffer (PBST). Peroxidase-labeled donkey antirabbit immunoglobulin and peroxidase-labeled sheep antimouse immunoglobulin were purchased from Amersham.
supernatant was mixed with 2× sample buffer (Invitrogen) and zymography was performed using precast gels (10% polyacrylamide and 0.1% gelatin). Following electrophoresis, gels were washed twice at room temperature for 30 min in 2.5% Triton X-100, and subsequently washed in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2 , 1 μM ZnCl2 , and 0.02% NaN3 at pH 7.5, and incubated in this buffer at 37 ◦ C for 24 h. Thereafter, gels were stained with 0.5% (w/v) Coomassie brilliant blue G250 (Bio-Rad) for 1 h, then lightly destained in methanol:aceticacid:water (3:1:6). Clear bands appeared on the Coomassie stained blue background in the areas of gelatinolytic activity. Gels were scanned and images were processed for extraction of the blue channel signal, which converted it to black and white, and inverted it for quantification of gelatinolytic activities from the integrated optical density (Lee and others 2009).
Gelatin zymographic analysis of secreted MMPs Following incubation with DADS, cell culture supernatants Statistical analysis All data were derived from at least 3 independent experiments. were collected and centrifuged at 400 × g for 5 min. Cell-free The blots were visualized with the Chemi-Smart 2000 (Vilber Table 1–Sequences of the primer pairs employed in the RT-PCR Lourmat, Marine, Cedex, France). Images were captured usreactions. ing Chemi-Capt software and imported into Adobe Photoshop (Adobe Systems, San Jose, Calif., U.S.A.). Scion Imaging softName Sequence of primers ware (http://www.scioncorp.com) was used to quantify the RT TIMP-1 Sense 5 -TGG-GGA-CAC-CAG-AAG-TCA-AC-3 PCR, Western blots, and zymography. Statistical analyses were Antisence 5 -TTT-TCA-GAG-CCT-TGG-AGG-AG-3 TIMP-2 Sense 5 -GTC-AGT-GAG-AAG-GAA-GTG-GAC-TCT-3 conducted using SigmaPlot software (Aspire Software Intl., Ashburn, Va., U.S.A.), and values are presented as mean ± standard Antisence 5 -ATG-TTC-TTC-TCT-GTG-ACC-CAG-TC-3 MMP-2 Sense 5 -GGC-CCT-GTC-ACT-CCT-GAG-AT-3 deviation (SD). Student’s t-test was used for comparisons between Antisence 5 -GGC-ATC-CAG-GTT-ATC-GGG-GA-3 2 experiments. A value of ∗ P < 0.05 was considered statistically MMP-9 Sense 5 -CGG-AGC-ACG-GAG-ACG-GGT-AT-3 significant. Antisence 5 -TGA-AGG-GGA-AGA-CGC-ACA-GC-3 5 -ACA-CAC-AGC-ACA-GGC-ATC-AC-3 5 -TCT-CCA-ATC-TCA-AAT-TTC-ATG-C-3 5 -AAG-GCC-AAG-ATC-ACC-ATC-GTG-3 5 -AGA-CGT-AGT-CCT-TGC-GGT-CGT-3 5 -TGG-ATG-AAC-TGC-GTG-GTG-CAG-3 5 -GAG-GCG-GCC-CAG-CCG-ACG-TA-3 5 -CGG-AGT-CAA-CGG-ATT-TGG-TCG-TAT-3 5 -AGC-CTT-CTC-CAT-GGT-GGT-GAA-GAC-3
Results DADS inhibited cell growth and migration of AGS cells To investigate the effects of DADS on cell viability, AGS cells were treated with various concentrations of DADS for 48 h and subjected to MTT assay. We found that DADS had inhibitory effects on cell viability in a dose-dependent manner (Figure 1A). Figure 1–Inhibition of cell growth and motility of AGS cells by DADS. (A) AGS cells were treated with the indicated concentrations of DADS for 48 h, and cell viability was estimated by MTT assay. Each point represents the mean ± SD of 3 independent experiments. Significance was determined by the Student’s t-test (∗ P < 0.05 versus untreated control). (B) Cells were grown to confluency on 30-mm cell culture dishes; a scratch was then made through the cell layer using a pipette tip. After washing with PBS, serum-free media (to prevent cell proliferation), containing either vehicle or DADS, was added for 48 h. Photographs of the wounded area were taken for evaluation of cell movement into the wounded area.
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Claudin-2 Sense Antisence Claudin-3 Sense Antisence Claudin-4 Sense Antisence GAPDH Sence Antisence
Anti-invasive activity of DADS. . . When compared with the controls, treatment with 100 μM and 150 μM of DADS resulted in approximately 25% and 40% inhibition of cell growth, respectively. To determine whether or not DADS inhibits motility of AGS cells, wound-healing experiments were performed. Results demonstrated that 70 μM of DADS, which was not cytotoxic, as shown by MTT assay, timedependently delayed the motility of AGS cells compared to that of control cells (Figure 1B).
DADS decreased cell invasion and down-regulated activities and expression of MMPs in AGS cells Using a Boyden chamber invasion assay, we next examined the question of whether or not DADS decreases the activity of cell invasion. As shown in Figure 2, treatment of cells with 70 μM of DADS markedly reduced cell invasion through the Matrigel chamber. Because cell migration plays an important role in the metastasis process, and since invasion of the basement membrane is primarily mediated by gelatinase MMPs (Sternlicht and Werb 2001; Egeblad and Werb 2002), we tested the effects of DADS on TIMPs and MMPs mRNA levels by reverse transcriptionpolymerase chain reaction (RT-PCR). As shown in Figure 3A, DADS treatment increased TIMP-1 and -2 mRNA and protein levels in a concentration-dependent manner. We next investigated the effects of DADS on the levels and activities of MMPs using RT-PCR, Western blot, and gelatin zymography. Data indicated that activities of MMP-2 and -9 in AGS cells were significantly decreased by DADS treatment, which was connected with a concurrent down-regulation of their mRNA and protein levels (Figure 3B and C). These results suggest that the anti-invasive effect of DADS is associated with increased TIMP-1 and -2 levels, as well
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Figure 2–Inhibition of invasion by DADS in AGS cells. Cells pretreated with DADS for 6 h were plated onto the apical side of matrigel-coated filters in serum-free medium containing either vehicle or DADS. Medium containing 20% FBS was placed in the basolateral chamber to act as a chemo-attractant. After 48 h, cells on the apical side were wiped off using a Q-tip. Next, cells on the bottom of the filter were stained using hematoxylin and Eosin Y, and then counted. Data are shown as the mean ± SD of triplicate samples and represent invasive cell numbers compared with those of control cells. Significance was determined using a Student’s t-test (∗ P < 0.05 versus untreated control).
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as inhibition of MMP-2 and -9 mRNA, protein expression, and activity in AGS cells.
DADS increased TER values and decreased the expression levels of claudins in AGS cells TER (a measure of TJ formation) values were next measured for examination of the relationship between tightening of TJs and invasive activity of AGS cells treated with DADS. As shown in Figure 4, incubation of AGS cells with DADS increased their TER levels, suggesting that DADS increased TJ function in AGS cells (approximately 1.3-fold by 70 μM of DADS). To elucidate the question of whether or not DADS reduces levels of TJ components, AGS cells were treated with DADS and levels of claudins were examined using RT-PCR and Western blot analyses. As shown in Figure 5, DADS treatment dose-dependently decreased levels of claudin mRNA and proteins (claudin-2, -3, and -4), the most important components of the TJ (Morin 2005; Hewitt and others 2006), suggesting that this modulation contributed to TJ tightening, and up-regulation of TER by DADS was associated with inhibition of cell invasion in AGS cells.
Discussion Because cell migration and invasion are critical steps in metastasis, inhibition of tumor cell migration and invasion are important mechanisms in the antimetastatic effects of anticancer drugs. Many recent studies have shown that chemopreventive and/or chemotherapeutic agents can inhibit tumor cell invasion and metastasis. Although several recent investigations have indicated that DADS, a major oil-soluble allyl sulfide of garlic, induced G2/M arrest of the cell cycle; and induced apoptosis in various tumor cells, including human lung carcinoma A549, human leukemic HL-60, breast carcinoma MCF-7, and prostate carcinoma PC3 cells (Lu and others 2004; Druesne-Pecollo and others 2006; Das and others 2007; Jo and others 2008; Yang and others 2009), the molecular mechanisms of its antimetastasis and antiinvasive activities are not well known. Thus, in the present study, we investigated the effects of DADS on cell migration and invasion in human gastric adenocarcinoma AGS cells. We found that DADS significantly inhibited cell motility and invasive activity by decreasing MMP activity and tightening TJs. MMPs are important proteolytic enzymes during organ development and tissue regeneration; however, they also play important roles in cancer invasion and metastasis. In particular, MMP-2 and -9 play important roles in tumor invasion and angiogenesis; therefore, tumor metastasis can be inhibited by blocking MMPs synthesis and activity (Sternlicht and Werb 2001; Egeblad and Werb 2002). MMPs activity is tightly controlled by transcriptional activation, by a complex proteolytic activation cascade, and by an endogenous system of TIMPs. TIMPs inhibit MMPs by formation of 1:1 stoichiometric complexes for regulation of matrix turnover (Mannello and others 2005; Malemud 2006). As shown in Figure 3, DADS markedly inhibited the levels of mRNA, protein, and activity of MMP-2 and -9; however, the levels of TIMP1- and -2 mRNA as well as protein were concentrationdependently increased. The data suggest that the anti-invasive activity of DADS in AGS cells was associated with inhibition of MMP-2 and -9 activities through elevation of TIMPs expression, and increased TIMPs/MMPs ratio is a key factor in regulation of the antimetastatic process. Changes in permeability properties and loss of cell polarity are hallmarks of epithelial cell tumorigenesis. Modulation of TJs, which are structures critical for maintenance of these functions in
Anti-invasive activity of DADS. . . cellular permeability (Kominsky 2006; Hewitt and others 2006). Emerging evidence indicates that TJ disruption and dysregulation of TJ protein are early events in cancer cell invasion and metastasis (Yokoyama and others 2001; de Oliveira and others 2005; Van Itallie and Anderson 2006). These observations also indicate that claudins are dysregulated in many types of cancers and that the nature of the dysregulation is highly type-specific for
Figure 4–Increase of TER values by DADS in AGS cells. Cells were treated with 70 μM of DADS for 48 h, and TER values were measured. Results are shown as the mean ± SD of 3 independent experiments. Significance was determined using a Student’s t-test (∗ P < 0.05 versus untreated control).
Figure 3–Effect of DADS on TIMPs and MMPs in AGS cells. (A) Cells were treated with the indicated concentrations of DADS for 48 h. Total RNAs were isolated and reverse transcribed with the indicated primers. Resulting cDNAs were then subjected to PCR and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as an internal control. (B) Cells were lysed, and proteins were separated by electrophoresis on SDS-polyacrylamide gels. Western blotting was then performed using anti-MMP-2 and anti-MMP-9 antibodies, and an ECL detection system. Actin was used as an internal control. (C) After incubation with DADS under the same conditions as those of A, medium was collected, and the activities of MMP-2 and -9 were measured by zymography. The quantitative analysis was based on densitometry of each band and appeared the fold versus control as described in Materials and Methods.
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epithelial cells, in a number of epithelial cancers, including gastric adenocarcinoma, has been demonstrated (Morin 2005; Mullin and others 2005; Ouban and Ahmed 2010). Thus, TJ disruption and dysregulation of its composite proteins play critical roles in cancer progression, invasion, and metastasis, particularly epithelial cancers (Van Itallie and Anderson 2006). For example, Soler and others (1999) first demonstrated that TER of colon carcinoma tissue was significantly lower than that of normal colon tissues but showed higher transepithelial paracellular permeability, which confirmed the loss of TJs. Other studies also have shown that many anticancer drugs are inhibitory to motility and invasiveness and that they act by enhancement of transepithelial paracellular permeability (Gitter and others 2000; Verghese and others 2006; Van Deun and others 2008; Choi and others 2009; Kim and others 2009). These observations indicated that TJ leakiness was associated with cancer progression and TJ tightening might have anticancer activity (Morin 2005; Mullin and others 2005). In this study, DADS treatment increased the TER of AGS cells, which was associated with lower cell motility and invasiveness (Figure 1 and 2), indicating that the anti-invasive activity of DADS may be due, in part, to its ability to enhance TJ activity. Several components of TJs have recently been identified. Among these, the members of the claudin family interact with other claudins associated with adjacent cells for regulation of para-
Anti-invasive activity of DADS. . . associated with the marked inhibition of claudins, major components of TJs. Although, the relation between DADS-induced inhibition of cell migration and invasion is still unclear, the results provide novel information on the possible mechanisms for the anticancer activity of DADS.
Acknowledgment This work was supported by a grant (Code nr 7–19-42) from Rural Development Administration, Republic of Korea.
References
Figure 5–Effect of DADS on expression of claudins in AGS cells. (A) Cells were treated with the indicated concentrations of DADS for 48 h. Total RNAs were isolated and reverse-transcribed with the indicated primers. Resulting cDNAs were then subjected to PCR and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as an internal control. (B) After incubation with DADS under the same conditions as those of A, cells were lysed, and proteins were separated by electrophoresis on SDS-polyacrylamide gels. Western blotting was then performed using the indicated antibodies, and an ECL detection system. Actin was used as an internal control. The quantitative analysis was based on densitometry of each band and appeared the fold versus control as described in Materials and Methods.
given cancers. In addition, these data suggest that claudin proteins may prove to be useful biomarkers for detection and diagnosis of certain cancers. In the present study, DADS treatment markedly altered the levels of claudins (-2, -3, and -4), indicating that downregulation of claudin expression by DADS relates to increased TJ tightening. Although this study will require validation, we tentatively suggest that DADS inhibits cell migration and invasion in AGS cells while concurrently repressing the MMPs activities, as well as tightening of TJs through inhibition of the levels of claudin expression. Taken together, the data indicate that DADS may be a promising new dietary source for decreasing the risk of cancer cell metastasis.
Conclusions
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In this study, we have suggested the mechanism of DADSinduced inhibition of cell motility and invasiveness in human gastric carcinoma AGS cells through the inactivation of MMPs along with the tightening of TJs. DADS exposure concomitantly caused inactivation of MMP-2 and -9 by the down-regulation of mRNA and protein levels and the up-regulation of TIMP-1 and TIMP-2 expression. The increased TJ tightening by DADS treatment was T110 Journal of Food Science r Vol. 76, Nr. 4, 2011
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