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Carcinogenesis Advance Access originally published online on May 4, 2006
Carcinogenesis 2006 27(11):2180-2189; doi:10.1093/carcin/bgl054
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A novel anticancer effect of garlic derivatives: inhibition of cancer cell invasion through restoration of E-cadherin expression

Qingjun Chu, Ming-Tat Ling, Huichen Feng, Hiu Wing Cheung, Sai Wah Tsao, Xianghong Wang* and Yong Chuan Wong*

Cancer Biology Group, Department of Anatomy, Faculty of Medicine, The University of Hong Kong Hong Kong, China

*To whom correspondence should be addressed at: Department of Anatomy, The University of Hong Kong, 1/F, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong, China. Tel: +852 2819 2867; Fax: +852 2817 0857; Email: TUxhwang{at}hkucc.hku.hkUT and Uycwong{at}hkucc.hku.hk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metastatic cancer is one of the main causes of cancer-related death since they rarely respond to available treatments. Recently, certain compounds isolated from the dietary supplement, garlic, have shown anti-proliferation effect on cancer cells. The aim of this study was to investigate whether certain garlic derivatives had any effect on the potentially invasive androgen-independent prostate cancer (PCa) cells. Using colony-forming, wound-closure as well as matrigel-invasion assays, we found that two main water-soluble constituents of the garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), were able to suppress PCa cell proliferation and invasive abilities. This inhibitory effect was associated with induction of mesenchymal to epithelial transition. Most importantly, the SAC and SAMC treatment led to restoration of E-cadherin expression at transcription and protein levels. In contrast, the expression of E-cadherin repressor, Snail, was reduced in the SAC- and SAMC-treated cells. Furthermore, examination of cell lines from other types of cancer (ovarian, nasopharyngeal and esophageal carcinomas) also confirmed that the effect of SAC and SAMC on activation of E-cadherin might be a general effect on human cancer cells. Our results demonstrate a novel anticancer effect of garlic and suggest that certain garlic-derived compounds may be potential agents for suppression of invasive growth through restoration of E-cadherin expression in cancer cells.

Abbreviations: EMT, epithelial to mesenchymal transition; MET, mesenchymal to epithelial transition; PCa, prostate cancer; SAC, S-allylcysteine; SAMC, S-allylmercaptocysteine


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Garlic (Allium sativum), a widely used herbal vegetable, has been suggested as an anticancer agent for several decades in epidemiological studies (1). The most convincing evidence comes from studies on digestive tract tumors (i.e. esophageal, gastric and colorectal cancers) and prostate cancer (PCa). For example, a study on the association of gastric cancer and consumption of Allium vegetables showed that persons with high intake of total Allium vegetables (>24 kg/year) had 60% reduced risk of this cancer compared to those with low consumption (<11.5 kg/year) on 564 patients with stomach cancer and 1131 normal controls (2). A more significant population-based, case–control investigation on 238 histologically confirmed PCa patients and 471 normal male controls conducted in China reported that men with high intake of garlic (>2.14 g/day) had significantly lower risk of PCa than those with low or no garlic consumption (3). These results suggest that garlic may play a positive role in the prevention of certain human cancers.

Recently, several individual compounds have been isolated from garlic and two major groups of compounds that show active anticancer effects have been identified. One group is the lipid-soluble allyl sulfur compounds such as diallyl disulfide (DADS) and diallyl trisulfide (DATS), and the other one is the water-soluble compounds {gamma}-glutamyl S-allylcysteine group such as S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) (1,4). Both in vivo and in vitro studies have shown that these individual compounds are not only able to suppress the skin, esophageal, stomach, colon, liver, lung and breast cancer growth in animal models (1,4), but also directly inhibit proliferation of a variety of cancer cell lines derived from colon, lung, leukemia, skin, breast and PCas in vitro (4). For example, intraperitoneally injecting nude mice with DADS significantly inhibited the growth of colon cancer xenograft by 69% compared to the control group (P < 0.05) (5). In addition, DATS is able to inhibit proliferation of PCa cell lines by induction of apoptosis through downregulation of Bcl-2 protein and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways (6). Recently, SAMC has been shown to exert anti-proliferation effects on colon cancer cells through disrupting the microtubule assembly that triggers JNK1 and caspase 3 signaling pathways leading to apoptosis (7). These lines of evidence suggest that in addition to their cancer preventive effect, the garlic derivatives may also be used as effective agents in the treatment of human primary cancers.

In this study, using androgen-independent PCa cell lines, we demonstrated for the first time that garlic derivatives, SAC and SAMC, were able to suppress the invasion ability of androgen-independent PCa cells, indicating that they may be potential agents for the treatment of invasive PCa. In addition, our results also showed that the inhibitory effect on PCa cell invasion was mediated through restoration of E-cadherin expression, inactivation of which is one of the common characteristics of metastatic PCa (8–10). Furthermore, the garlic derivatives induced E-cadherin expression was also observed in additional three types of human cancer cell lines, suggesting that their anti-invasive effect may be a general effect on human cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines and cell culture conditions
Human androgen-independent PCa cell lines PC-3 and DU145, ovarian cancer cell line Skov-3 (obtained from ATCC, Rockville, MD), nasopharyngeal carcinoma cell line CNE-3 (11) and esophageal carcinoma cell line EC-109 (12) were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 2 mmol/l L-glutamine, 5% fetal calf serum (FCS) and 2% penicillin–streptomycin at 37°C in 5% CO2.

Garlic-derived compounds
SAC and SAMC are water-soluble compounds isolated from garlic and generously provided by Wakunaga Pharmaceutical (Hiroshima, Japan), and have been used in previous studies (7,13). Stock solutions of SAC (100 mM) and SAMC (5 mM) were prepared freshly in distilled water and phosphate-buffered saline (PBS), respectively, according to the manufacturer's suggestions.

Colony-forming assay
Detailed experimental procedures have been described previously (14). Briefly, single-cell suspension was produced and cultured in 12-well plates at a density of 200 cells per well. Twenty-four hours after plating, five concentrations of SAC or SAMC were added and the cells were incubated for 10–12 days. The cells were then fixed in 70% ethanol and stained with 1% Giemsa blue. The colonies that consisted of >50 cells were scored and compared with the vehicle-treated controls. Two wells were used for each concentration. Each experiment was repeated at least three times and each survival curve showed the means and standard deviations.

Cell-proliferation assay
Proliferation assay was determined as follows. Cells were plated in 12-well plates at a density of 1 x 103 cells per well and cultured for 24 h in RPMI 1640 medium. Then cells were treated with SAC or SAMC at different concentrations (IC50 or IC90). Cells were trypsinized and counted with Coulter Counter (Coulter Electronics, BEDS, England) every 24 h. Each experiment was repeated at least three times in triplicate wells and the survival curves showed the means and standard deviations.

Wound-closure assay
Cells (105) were seeded into 6-well culture plates and allowed to grow to ~90–95% confluence. Similar sized wounds were introduced to monolayer cells using a sterile yellow pipette tip. Wounded monolayer cells were washed three times by PBS to remove cell debris and then cultured with or without SAC or SAMC. The speed of wound closure was monitored and photographed every 8–12 h using a phase contrast microscope until complete wound closure was observed in the untreated control.

Matrigel-invasion assay
Matrigel-invasion assay was performed according to a previously published method with modifications (15). Briefly, cell culture plate inserts with 8 µm pore size (Millipore, Bedford, MA) were coated with 100 µl Matrigel/PBS solution (0.2 mg/ml, BD Bioscience, Bedford, MA) and dried in the culture hood at room temperature. All the cells were preincubated in media with or without SAC or SAMC at different time points. Two hundred microliters of 0.1% BSA-RPMI 1640 containing single cell suspension was added to the upper chamber insert and 600 µl of invasion buffer (0.1% BSA-RPMI 1640) containing fibronectin (10 µg/ml) and 600 µl of 5% FCS RPMI 1640 were added in the lower well as a chemoattractant. Then different cells were incubated for different time points (18 h for PC-3, 20 h for DU145, 24 h for Skov-3, 36 h for CNE-3 and 40 h for EC-109 cells) at 37°C in 5% CO2 humidified conditions. After different time points, the insert membranes were fixed for 10 min with 10% formaldehyde and then stained with Mayer's hematoxylin and 1% w/v aqueous eosin solution. The cells on the upper surface of the membrane were removed with cotton wool and discarded. After washing with double distilled water, membranes were cut from the chambers and mounted with Depex. The number of cells migrated to the lower surface of the membrane was counted using a PC-based image-analyzing system (Stereo Investigator, VT) attached to a Nikon microscope under x100 magnifications from 16 consecutive fields, representing ~60% total area of the membrane. Data presented are the means and standard deviations of two wells from three experiments.

Western blotting
Detailed protocols have been described previously (14). Briefly, cell lysates were prepared by suspending cell pellets in lysis buffer [50 mmol/l Tris–HCl (pH 8.0), 150 mmol/l NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin and 1 mmol/l phenylmethylsulfonyl fluoride]. Protein concentration was measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA). Equal amount of protein (10–30 µg) was loaded onto a SDS polyacrylamide gel for electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Amersham, Piscataway, NJ). The membrane was then incubated with primary antibodies for 1 h at room temperature against E-cadherin (BD Biosciences, Bedford, MA), {alpha}-catenin, ß-catenin, {gamma}-catenin (Santa Cruz Biotechnology, CA), vimentin (Chemicon, International, Temecula, CA) and {alpha}-Smooth Muscle Actin ({alpha}-SMA, Sigma, St Louis, MO). After incubation with appropriate secondary antibodies, signals were visualized by ECL western blotting system (Amersham, Piscataway, NJ). Expression of actin was assessed as an internal loading control.

RT–PCR analysis
Total RNA (2 µg) was reverse transcribed using a SuperScript II RT kit (Invitrogen, Carlsbad, CA) primed with oligo(dT). E-cadherin and Snail expression was examined by PCR and GAPDH expression was used as an internal reference. The primer sequences and expected sizes of the PCR products were as follows. Human E-cadherin: forward, 5'-GTAACCGATCAGAATGAC-3' and reverse, 5'-CGTGGTGGGATTGAAGAT-3' (380 bp) (11); human Snail: forward, 5'-AATCGGAAGCCTAACTACAG-3' and reverse, 5'-GGAAGAGGCTGAAGTAGAG-3' (320 bp); human GAPDH: forward, 5'-CTCAGACACCATGGGG-3' and reverse, 5'-ATGATCTTGAGGCTGTTGTCATA-3' (450 bp) (16). Preliminary experiments were conducted to ensure that the PCR conditions were at the logarithmic phase of the PCR reaction for each set of primers. PCR amplification were performed for 40 cycles of 94°C for 30 s, 54°C for 1 min, 72°C for 1 min for E-cadherin; 33 cycles of 94°C for 30 s, 58°C for 1 min, 72°C for 1 min for Snail; 25 cycles of 94°C for 30 s, 58°C for 1 min, 72°C for 1 min for GAPDH. PCR products were subjected to electrophoresis on 1.5% agarose gels containing ethidium bromide and photographed using a gel documentation system (Ultra-Violet Product Limited, CA).

Quantitative real-time PCR
To confirm the results of RT–PCR, mRNA expression was also analyzed using an iCycler iQ Multi-Color Real Time PCR Detection System (Bio-Rad, Hercules, CA) with SYBR Green (Molecular Probe, Eugene, OR). The primer sequences used for real-time PCR and expected products size were as follows. Human E-cadherin: forward, 5'-TGAAGGTGACAGAGCCTCTGGAT-3' and reverse, 5'-TGGGTGAATTCGGGCTTGTT-3' (151 bp); human Snail: forward, 5'-GGATCTCCAGGCTCGAAAGG-3' and reverse, 5'-GACATTCGGGAGAAGGTCCG-3' (309 bp); human GAPDH: forward, 5'-TGCACCACCAACTGCTTAGC-3' and reverse, 5'-GGCATGGACTGTGGTCATGAG-3' (86 bp). In brief, 1 µl of cDNA was added in a 24 µl reaction mixture containing 0.5x SYBR Green, 1x PCR buffer, 0.6 µM MgCl2, 0.4 µM dNTP, 0.5 µM primer sets and 0.625 U Amplitaq gold DNA polymerase (Applied Biosystems, Foster City, CA). The cycling conditions for all genes were as follows: preincubation at 50°C for 2 min, 95°C for 4 min, followed by 55 cycles of denaturation at 95°C for 20 s, annealing at 57°C for 30 s and extension at 72°C for 30 s. At the completion of cycling, melting curve analysis was performed to establish the specificity of the PCR product. The expression level of cDNA of each candidate gene was internally normalized using GAPDH. The relative quantitative value was expressed by the 2–{Delta}{Delta}CT method (17), representing the amount of candidate gene expression with the same calibrators. Each experiment was performed in duplicates and repeated three times.

Luciferase assay
Same number of cells was plated into 12-well plates until grown to ~30% confluence. After 24 h, the medium was changed to serum free medium (SFM). pGL2-Basic-Ecad108, pGL2-Basic-Ecad368 and GL2-Basic-Ecad1359 (luciferase reporter containing different length of E-cadherin promoter, kindly provided by Dr Karen M Hajra and Dr Amy S Woodard, Departments of Internal Medicine, Human Genetics and Pathology, University of Michigan Medical School, USA) (18,19) and pRL-CMV-Luc (internal control) were cotransfected into PC-3 cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN, USA). Cells were treated with SAC or SAMC 24 h after transfection and were lysed after 24 or 48 h of treatment. Luciferase activity was assayed using the dual-luciferase reporter assay system with procedure described by the manufacturer (Promega, WI, USA). The percentage increase in luciferase activity of the treated cells was calculated as relative to that of the untreated controls. Each experiment was repeated at least three times in duplicate wells and each data point represented the mean and standard deviation.

Immunofluorescence staining
Cells were grown on chamber slides and incubated in culture medium with or without SAC, SAMC. Forty-eight hours later, cells were fixed in 100% methanol solution for 10 min. The fixed cells were washed three times with PBS and blocked in 1% bovine serum albumin (BSA) for 30 min. After washing with PBS, cells were incubated with anti-E-cadherin antibody (1:100) at 4°C overnight and then washed three times with PBS. They were then incubated with fluorescein isothiocyanate (FITC)-conjugated second antibody (1:40 DAKO, Carpinteria, CA) for 1 h and washed again with PBS. At last the chamber slides were mounted. Images of the cells with fluorescent signal were captured by a laser confocal microscope (Carl Zeiss, 07740 Jena, Germany).

Statistical analysis
The results were analyzed by two-tailed student t-test SD using SPSS 11.0 (Aspire Software International, Leesburg, VA) and P-values were calculated. The difference was considered significant between two samples if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Inhibitory effect of SAC and SAMC on PCa cell growth
To investigate if SAC and SAMC exerted any effect on the growth of PCa cells, colony-forming assay was performed on two androgen-independent human PCa cell lines PC-3 and DU145. As shown in Figure 1 (left panels), the clonogenic survival of these cells was significantly inhibited after exposure to SAC and SAMC in both cell lines in a dose-dependent manner. Comparison of the IC50 and IC90 (Table I) revealed that PC-3 cells were more sensitive (by ~2-fold) than DU145 to both compounds. To confirm these results, proliferation assay was performed using IC50 and IC90 concentrations at different time points. As shown in Figure 1 (right panels), SAC and SAMC treatment suppressed cell proliferation in a time- and dose-dependent manner. This effect became much more significant after ≥3 days of treatment. These results indicate that SAC and SAMC are able to inhibit both clonogenecity and growth of androgen-independent PCa cells with SAMC more effective than SAC.


Figure 1
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  		Fig. 1 Evidence of SAC- and SAMC-induced growth suppression in PCa cells. Left panels: Effect of SAC (A) and SAMC (B) on clonogenecity of PC-3 and DU145 cell lines examined by colony-forming assay. Right panels: Effect of SAC (A) and SAMC (B) on cell growth rate (*P < 0.05). Note that both SAC and SAMC are able to inhibit clonogenecity and proliferation of androgen-independent PCa cells.

 

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  		Table I IC50 and IC90 concentrations of SAC and SAMC determined by colony-forming assay

 
Inhibitory effect of SAC and SAMC on invasion ability of PCa cells
Since androgen-independent stage of PCa is the main obstacle in the treatment of PCa, we next examined whether SAC and SAMC could suppress the invasive ability of androgen-independent PCa cells. As shown in Figure 2A and B, using matrigel-invasion assay, we found that SAC and SAMC (IC90 concentrations) treated PC-3 and DU145 cells showed much lower invasion ability compared to the untreated control as evidenced by decreased number of cells migrated through the matrigel. Using wound-closure assay, however, we found that the suppressive effect of SAC and SAMC treatment on PCa cell migration was not as obvious as their effect on invasion ability (Figure 3). After exposure to IC90 concentrations of SAC and SAMC, there was little difference in migration rate of both cell lines compared with the untreated control (Figure 3, arrows). The reason for this difference is unknown at this stage. As migration and invasion are two different phenomena during the process of cancer metastasis, one possible explanation may be that the two garlic derivatives are more effective in inhibiting invasion than migration of PCa cells. This inhibitory effect on cell invasion was not the result of cell growth inhibition induced by these compounds as there was no significant difference in cell growth rate between the treated and control cells up to 48 h post-exposure time (Figure 1, right panels). These results indicate that SAC and SAMC are able to inhibit invasion ability of androgen-independent PCa cells, independent to their toxic effects.


Figure 2
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  		Fig. 2 Effect of SAC and SAMC on invasion ability of PCa cells. Cells were pretreated with IC90 doses of SAC (A), SAMC (B) or vehicle (open bars) for indicated time points and then tested for invasion ability using matrigel-invasion assay. Cell invasion ability was calculated as percentage of cells migrated through plate chamber filter to the vehicle-treated controls (set up as 100%). Data represent the mean ± SE of at least three independent experiments. Note that the SAC- and SAMC-treated cells showed much lower invasion ability than the controls.

 

Figure 3
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  		Fig. 3 Effect of SAC and SAMC on PCa cell migration. Differential cell migration rate was examined using wound-closure assay as described in Materials and methods. Note that treatment of SAC (A) and SAMC (B) leads to suppression of cell migration rate in PC-3 and DU145 cells.

 
Effect of SAC and SAMC on mesenchymal to epithelial transition (MET)
It has been reported that ‘epithelial to mesenchymal transition’ (EMT), transdifferentiation from epithelial type to mesenchymal phenotype, is one of the major events during the acquisition of the invasive phenotype in tumors of epithelial origin (20,21). This process is often accompanied by expression of mesenchymal markers and loss of epithelial markers, especially E-cadherin, which is one of the most commonly observed changes in invasive and metastatic carcinomas (22–24). We therefore studied whether the inhibitory effect of the garlic derivatives on PCa cell invasion was mediated through MET. We found that the morphology of SAC- or SAMC-treated cells changed from a more elongated fibroblast-like morphology to a round and packed appearance of epithelial cells (Figure 4A, arrows). In addition, the expression of epithelial markers, such as E-cadherin and {gamma}-catenin, was increased in both cell lines treated with both compounds at IC90 concentrations in a time-dependent manner (Figure 4B and C). Owing to a deletion of the {alpha}-catenin gene in PC-3 cells (25), there was no {alpha}-catenin expression detected throughout. However, there was no change in ß-catenin levels in PC-3 and both {alpha}- and ß-catenin in DU145 cells after treatment by the two garlic derivatives. In contrast, expression of mesenchymal markers, such as {alpha}-smooth muscle actin ({alpha}-SMA) and vimentin, was reduced (Figure 4B and C). Most importantly, we found that intense immunofluorescent staining of E-cadherin was observed in the SAC- and SAMC-treated PC-3 and DU145 cells compared to the controls (Figure 4D). The membrane localization of the E-cadherin, which has been indicated as activation of this protein (26,27), was found in the treated cells while negative or weak positive E-cadherin staining was mainly observed in cytoplasm of the untreated controls. Taken together, both the morphological and molecular changes indicate that the inhibitory effect of SAC and SAMC on PCa cell invasion is associated with MET.


Figure 4
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  		Fig. 4 SAC- and SAMC-induced mesenchymal–epithelial transition (MET) in PCa cells. (A). Cells were treated with SAC or SAMC for 48 h and photos were taken under x400 magnifications on a phase contrast microscope. Note that SAC and SAMC treatment results in morphological changes associated with MET (B and C). Expression of epithelial markers E-cadherin, {gamma}-catenin and mesenchymal markers {alpha}-SMA and vimentin in PC-3 and DU145 cells before and after SAC (IC90 dose, B) and SAMC (IC90 dose, C) treatment. (D) Representative immunofluorescent images of PC-3 and DU145 cells stained for E-cadherin in the control and SAC- and SAMC-treated cells under a confocal microscope at x400 magnifications. Note that increased expression of epithelial markers and decreased mesenchymal markers are observed after exposure to SAC and SAMC in a time-dependent manner in both cell lines. Note also there is a distinctive absence of {alpha}-catenin signal in PC-3 cells due to reported deletion of {alpha}-catenin gene. ß-catenin levels in both PC-3 and DU145 and {alpha}-catenin in DU145 are unchanged.

 
Effect of SAC and SAMC on E-cadherin transcription
Downregulation of E-cadherin expression is one of the most frequently reported characteristics of metastatic cancers (28), and restoration of E-cadherin in cancer cells leads to suppression of invasive and metastatic ability of cancer (29–31). In PCa, downregulation of E-cadherin expression is correlated with high-grade tumors and poor prognosis (8–10), indicating its role in the progression of this cancer. To further characterize the effect of SAC and SAMC on E-cadherin expression, we performed RT–PCR and quantitative real-time PCR as well as luciferase assay to study whether the SAC- and SAMC-induced E-cadherin expression occurred at transcription level. Since the untreated PC-3 cell line showed undetectable levels of E-cadherin protein expression, we decided to focus on this cell line for the following experiments. As shown in Figure 5A, exposure to IC90 concentrations of SAC and SAMC led to upregulation of E-cadherin mRNA expression in a time-dependent manner. At the same time, the expression of its transcriptional suppressor, Snail (32,33), was reduced (Figure 5A). We further confirmed these results using quantitative real-time PCR, which showed that mRNA expression of E-cadherin was increased by SAC and SAMC for up to 2.2- and 4-folds, respectively, in PC-3 cells after 48 h treatment. Concurrently, the Snail mRNA expression was decreased by SAC and SAMC for 88 and 84%, respectively (Figure 5B). In addition, we transfected E-cadherin promoter reporter constructs containing 108 bp (E-Cad108), 368 bp (E-Cad368) and 1359 bp (E-Cad1359) of the 5' flanking region of the E-cadherin promoter into PC-3 cells, respectively, and then treated them with SAC or SAMC. We found that SAC and SAMC were able to activate all three E-cadherin promoter constructs, with the highest induction level in the E-Cad1359 transfectants (up to 8-fold higher) (Figure 5C). These results indicate that in addition to the well-characterized E-boxes located within the 108 bp of E-cadherin promoter, additional elements located between –1359 and –108 bp of the E-cadherin 5' flank region may also play a role in the SAC- and SAMC-induced E-cadherin activation.


Figure 5
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  		Fig. 5 Effect of SAC and SAMC on E-cadherin transcription in PC-3 cells. (A) RT–PCR analysis of E-cadherin and Snail expression in PC-3 cells treated by SAC and SAMC (IC90 concentrations) at indicated time points. (B) Quantitative real-time PCR was used to confirm the above results. Quantification of E-cadherin and Snail expression was calculated according to method described in Materials and methods. The mean fold expression of each candidate gene in PC-3 cell line was normalized by GAPDH. (C) Relative luciferase activity in PC-3 cells treated with SAC and SAMC. PC-3 cells were transiently transfected with E-cadherin promoter reporter constructs containing E-Cad108, E-Cad368 and E-Cad1359, respectively, and luciferase activity was measured by the dual-luciferase reporter assay system (Promega, WI). The percentage increase in luciferase activity of the treated cells was calculated as relative to that of the untreated controls (*P < 0.05). Note that SAC and SAMC treatment leads to increased E-cadherin gene expression and promoter activity in PC-3 cells.

 
Evidence of SAC- and SAMC-induced E-cadherin expression in ovarian, nasopharyngeal and esophageal cancer cell lines
To investigate whether the SAC- and SAMC-induced E-cadherin expression was a specific phenomenon on PCa cells or a common effect on human cancer cells, we then treated ovarian (Skov-3), nasopharyngeal (CNE-3) and esophageal (EC-109) cancer cell lines, which have been reported to have low or undetectable levels of the E-cadherin protein (11,12), with SAC (10 mM) and SAMC (250 µM), respectively, and studied E-cadherin expression at both protein and transcriptional levels. As shown in Figure 6, we found that both compounds were able to restore the expression of E-cadherin at both protein (Figure 6A) and mRNA levels (Figure 6B and C) in a time-dependent manner in all three cancer cell types. However, treatment with SAC and SAMC reduced the expression of Snail gene expression (Figure 6B and C). We next treated these three cancer cell lines with two garlic derivatives to further confirm whether SAC and SAMC could suppress migration and invasion ability of these cancer cell lines. As shown in Figure 6D using wound-closure assay, we found SAC and SAMC could suppress the migration of Skov-3 and EC-109 cancer cell lines, but not as obvious in CNE-3 cells. Moreover, using matrigel-invasion assay, SAC and SAMC treatment inhibited the number of cells migrated through the Matrigel in a time-dependent manner (Figure 6E). These results showed that the two garlic derivatives were more effective in suppressing the invasion ability of these cancer cell lines but had a more varied effect on migration of cancer cells. Taken together, our results suggest that the SAC- and SAMC-induced E-cadherin expression may not be a specific effect on PCa cells but a more general effect on invasive cancer cells.


Figure 6
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  		Fig. 6 Effect of SAC and SAMC on E-cadherin and Snail expression in ovarian, nasopharyngeal and esophageal cancer cells. (A, left panels) Western blotting analysis of E-cadherin expression bands in Skov-3, CNE-3 and EC-109 cells treated by SAC and SAMC at 24 and 48 h. (A, right panels) The density of the E-cadherin bands was quantified by densitometry analysis. Data are presented after normalization with the ß-actin bands. (B) RT–PCR analysis of E-cadherin and Snail expression in Skov-3, CNE-3 and EC-109 cells treated by SAC and SAMC. (C) Quantitative real-time PCR was used to confirm the RT–PCR results. Quantification of E-cadherin and Snail expression was calculated according to the method described in Materials and methods. The mean fold expression of each candidate gene in each cell line was normalized by GAPDH. (D) Differential cell migration rate was examined using wound-closure assay as described in Materials and methods after SAC and SAMC treatment. (E) Cells were pretreated with 10 mM of SAC, 250 µM of SAMC or vehicle (open bars) for indicated time points and then tested for invasion ability using matrigel-invasion assay. Cell invasion ability was calculated as percentage of cells migrated through plate chamber filter to the vehicle-treated controls (set up as 100%). Note that SAC and SAMC treatment leads to induction of E-cadherin at both transcriptional and protein levels in all three types of cancer cells. Note also that SAC- and SAMC-treated cells showed much lower migration and invasion ability than the controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, we have demonstrated a novel anticancer effect of garlic-derived compounds, SAC and SAMC, and provided possible mechanisms responsible for their anti-invasive effect. Several new points are generated from the current study. First, we demonstrated that SAC and SAMC were able to suppress the invasion ability of androgen-independent PCa cells and other three cancer cell lines (Figures 2 and 6E). Second, we showed that this effect was associated with their ability to promote MET (Figure 4). Third, we found that restoration of E-cadherin through transcriptional activation of the E-cadherin gene may be accounted for its inhibitory effect on cancer cell invasion (Figure 5). Most importantly, the SAC- and SAMC-induced E-cadherin expression was also confirmed in additional three types of cancer cell lines (Figure 6A–C). Although the chemoprevention effect as well as anti-proliferation effect of garlic have been well documented in many types of human cancers (1–7), our results provide first evidence to suggest that they may also be potential agents for the treatment of invasive cancer.

Downregulation of E-cadherin is one of the most frequently reported phenomena in metastatic cancers (26–28). It is suggested that loss of E-cadherin expression is able to promote EMT, which plays a key role in the progression of cancer cells to metastatic stage (20,21). Although the precise mechanism responsible for E-cadherin inactivation in cancer cells is not clear, alterations at transcriptional level seem to be one of the mechanisms responsible for its decreased expression in several cancer types (18,19,34–37). In this study, we found that the SAC- and SAMC-treated PCa cells showed increased E-cadherin expression (Figure 4B and C), which was associated with reduced invasion ability (Figure 2), as well as cell morphological changes from mesenchymal to epithelial phenotype (Figure 4A). In contrast, the expression of an E-cadherin suppressor, Snail, was decreased (Figure 5A and B). Recently, Snail has been identified which binds to the E-boxes located in the promoter region leading to suppression of E-cadherin gene transcription (32). Since it has been suggested that upregulation of Snail expression promotes EMT and cancer metastasis through downregulation of E-cadherin (33), our results suggest that the SAC- and SAMC-induced E-cadherin expression may be a result of downregulation of Snail which in turn promotes MET, leading to inhibition of cancer cell invasion. The effect of SAC and SAMC on E-cadherin and Snail expression was further confirmed on additional cancer cell lines derived from other cancer types including ovarian (Skov-3), nasopharyngeal (CNE-3) and esophageal cancers (EC-109) (Figure 6), suggesting that it may be a more general effect on cancer cells.

Several key E-cadherin transcription regulation elements are suggested to be located within 108 bp of the 5' region of the E-cadherin promoter (18,19,34–37), and the repressing effect of Snail on E-cadherin gene transcription is suggested to be mediated through this region (32). In this study, we found that indeed the luciferase activity of the E-Cad108 transfectants was increased after SAC and SAMC treatment (Figure 5C). However, we also observed that luciferase activity was much higher in the E-Cad1359 and –368 transfectants. It is therefore possible that the SAC- and SAMC-induced E-cadherin expression may be also regulated through Snail-independent mechanisms. It has been reported that hypermethylation of E-cadherin promoter is another common mechanism responsible for E-cadherin inactivation in many types of metastatic cancers (11,12,37). Previously, complete hypermethylation of the E-cadherin promoter was reported in CNE-3 and EC-109 cell lines that were correlated with undetectable levels of E-cadherin expression in these cells (11,12). In the present study, we found that SAC and SAMC treatment was able to induce E-cadherin gene expression at both transcriptional and protein levels in these cell lines (Figure 6). It is possible that in addition to their direct effect on Snail, the SAC- and SAMC-induced restoration of E-cadherin may be also be mediated through demethylation of other regions of the E-cadherin promoter. However, further studies are necessary to confirm this hypothesis.

Catenins ({alpha}, ß and {gamma}), a family of cytoplasmic cadherin binding proteins, link E-cadherin to the actin cytoskeleton and are thought to be essential for normal E-cadherin function. Basically, two distinct E-cadherin–catenin adhesion complexes exist in the same cell. One complex is composed of E-cadherin, {alpha}- and ß-catenin, the other of E-cadherin, {alpha}- and {gamma}-catenin (23,27). In our study, we found that the two garlic derivatives only upregulated the expression of E-cadherin and {gamma}-catenin, but not {alpha}- and ß-catenin. For PC-3 cells, as reported earlier that {alpha}-catenin gene was defective (25), it is therefore not surprising that {alpha}-catenin protein expression was undetectable throughout in this cell line. However, the reason for the static level of ß-catenin in PC-3 and both {alpha}- and ß-catenin proteins in DU145 remains unknown. As it is known that {gamma}-catenin is structurally related to ß-catenin with similar functions, such as cell adhesion (38). We speculate that the garlic derivatives induced MET may be mediated through E-cadherin–{gamma}-catenin interaction. On the other hand, besides linking E-cadherin to {alpha}-catenin in the process of cell adhesion, ß-catenin also plays an important role in Wnt signaling pathway. For example, interaction between ß-catenin and APC (adenomatous polyposis coli) tumor suppressor is essential for Wnt-controlled stabilization of ß-catenin and transcriptional activation (39,40). In addition, E-cadherin and APC are known to compete for the same binding region on ß-catenin (41,42). Hence, it is possible that SAC and SAMC increase the E-cadherin expression without an increase in the ß-catenin level. In addition, our study showed the increased immunoflluoresence staining of E-cadherin protein in the SAC- and SAMC-treated PC-3 and DU145 cells compared to the untreated cells (Figure 4D). Although PC-3 cells do not express {alpha}-catenin, a key molecule for functional E-cadherin expression, SAC and SAMC might restore the function of E-cadherin through other molecules such as vinculin, which has been reported to play a role in the establishment of the E-cadherin-based cell adhesion complex (43,44).

In the recent years, use of garlic as food or dietary supplement for the chemoprevention or treatment of digestive tract cancers has shown remarkable results (1,2,4). In addition, the fact that certain garlic derivatives such as SAMC preferentially suppress cancer cell growth over non-malignant cells (45) further suggests that they may be potential alternative agents for the treatment of primary cancers. In this study, we have demonstrated that the garlic-derived compounds, SAC and SAMC, are able to restore E-cadherin expression in invasive cancer cells, suggesting that they may also be effective agents for the treatment of invasive cancers.


   Acknowledgments
 
This work was supported by grants to X.H.W. (HKU7478/03M, American Institute for Cancer Research 05A006-Rev2) and Y.C.W. (HKU 7314/01M and HKU7490/03M, 7470/04M, NSFC/RGC/03, HKU Foundation 2003). We thank Wakunaga Pharmaceutical Co., Ltd. for the supply of SAC and SAMC compounds.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 25, 2005; revised March 17, 2006; accepted April 13, 2006.


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