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Carcinogenesis Advance Access originally published online on December 12, 2005
Carcinogenesis 2006 27(6):1222-1231; doi:10.1093/carcin/bgi306
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A proteomic investigation into a human gastric cancer cell line BGC823 treated with diallyl trisulfide

Na Li 1, 2, 4, {dagger}, Ruifang Guo 3, {dagger}, Wenmei Li 3,, Jianmin Shao 1, 2, Shuting Li 1, 2, Kang Zhao 1, 2, Xishu Chen 1, 2, Ningzhi Xu 1, 2, Siqi Liu 1, 2 and Youyong Lu 1, 2, 3, *

1 Beijing Genomics Institute, Chinese Academy of Sciences and 2 Beijing Proteomics Institute, Beijing Airport Industrial Zone B-6, Shunyi, Beijing 101300, China, 3 Peking University School of Oncology, Beijing Institute for Cancer Research, Beijing 100034, China and 4 Graduate School of Chinese Academy of Sciences, Beijing 100049, China

* To whom correspondence should be addressed. Tel: +86 10 6616 3061; Fax: +86 10 6617 5832; Email: yongylu{at}public.bta.net.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Garlic is generally used as a therapeutic reagent against various diseases worldwide. Although a great effort is made to understand the pharmaceutical mechanisms of garlic and its derivatives, there are many mysteries to be uncovered. Using proteomic means, herein we have systematically studied the responses of protein expression in BGC823 cells, a gastric cancer cell line, induced by diallyl trisulfide (DATS), a major component of garlic derivatives. A total of 41 unique proteins in BGC823 were detected with significant changes in their expression levels corresponding with DATS administration. Of these proteins, five typical ones, glutathione S-transferase-pi (GST-pi), voltage-dependent anion channel-1 (VDAC-1), Annexin I, Galectin and S100A11, were further examined by Western blotting, resulting in coincident data with the proteomic evidence. Moreover quantitative real-time RT–PCR experiments offered dynamic data of mRNA expression, indicating the responses of Annexin I and GST-pi expression within a short period after DATS treatment. Interestingly, ~50% of DATS-sensitive proteins (19/41) in BGC823 are tightly associated with apoptotic pathways. These proteomic results presented, therefore, provide additional support to the hypothesis that garlic is a strong inducer of apoptosis in tumor cells.

Abbreviations: 2DE, two-dimensional electrophoresis; DADS, diallyl disulfide; DATS, diallyl trisulfide; GST-pi, glutathione S-transferase-pi; HGC, Human gastric cancer; LC-MS/MS, tandem mass spectrometry; MALDI-TOF MS, matrix assisted laser desorption ionization time-of-flight mass spectrometry; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Historically numerous scientific data have evidenced the therapeutic effects of garlic for various ailments of heart, arthrosis, pulmo and uterus (1). Recent epidemiological studies have continued to support the premise that increased consumption of garlic is associated with decreased risk of various types of human cancer (2). Furthermore these extensive investigations have suggested that the chemical constituents of garlic are able to exert their inhibitory effects on carcinogenesis using in vitro and in vivo models. For instance, diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, was reported to have anticancer properties against both hormone-dependent and hormone-independent breast cancers through the interactions between DADS and polyunsaturated fatty acids, which were known as modulators of breast cancer cell growth (3). Other recent studies have also indicated that garlic derivatives, such as diallyl trisulfide (DATS), could inhibit proliferation of cancer cells from the human stomach, prostate, colon, lung and skin (4).

The correlation of garlic consumption and gastric cancer has been drawn the attention of many. On the basis of epidemiological data reported by You et al. (5), a low mortality of gastric cancer (3.45/100 000) was observed in Cangshan county, China, where the residents consumed a large amount of garlic (~20 g/day); conversely, the neighboring county, Linqu county with lower consumption of garlic, had high death rates for gastric cancer (40/100 000). In animal models, the different derivatives of garlic were examined for their inhibitions to tumor formation in the mouse stomach induced by benzo[a]pyrene (BP) (6). The human gastric cancer (HGC) cell line is sensitive to treatment of garlic (7). The exposure to 125 µM DATS led to significant morphological changes and a slow growth rate in HGC cells. Now the challenge is how to elucidate the pharmaceutic mechanisms of garlic, especially for anticarcinogenic functions, which will greatly benefit the development of garlic-related medicines for prevention and therapy of gastric cancer.

It is well known that cancer development is associated with increased cell proliferation and decreased apoptosis (8). Using a flow cytometric analysis, we have observed that DATS was able to induce cell cycle arrest and to enhance apoptosis in BGC823 cells, a gastric cancer cell line. Some apoptotic proteins in BGC823 were susceptible to garlic treatment, in which Bcl-2 and cyclin D1 were downregulated but caspase-3 and p27kip1 were upregulated (9,10). Garlic and its organic sulfur components have been also reported to induce apoptosis in several other cancer cell lines. Recently, DATS-induced cell cycle arrest and apoptosis have been documented in human prostate tumor cells, which are associated with a decline of Cdc25 protein (11,12). The apoptotic process functions as a network, in which many proteins and multiple steps are involved. Though a few apoptotic biomarkers have been identified so far corresponding to garlic treatment, this limited information is lacking for all features of proteomic responses. An investigation is virtually required to globally profile the molecular responses triggered by garlic or its derivatives.

The proteomic strategy has paved the access to profile the cellular responses at protein level under stress. Currently it has been successfully employed in the area of cancer research, particularly in biomarker studies (13). Proteomic analysis of the primary tumors or cell lines of the stomach has been reported by some investigators (14–16). In this communication, we present proteomic data based upon a systematic analysis, combining several techniques of proteomics, such as two-dimensional electrophoresis (2DE), mass spectrometry and western blotting, to study the protein alterations in BGC823 cells induced by DATS treatment. Our data revealed that DATS indeed induced apoptosis in BGC823 cells; subsequently, several apoptotic-associated proteins were expressed responding to this event. The information provided here, therefore, will deepen our knowledge on how garlic is involved in the apoptosis in cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemical reagents
DATS (98% purity) with commercial name as Allitridi, was obtained from Shanghai Hefeng Pharmacy Company (Shanghai, China). IPG strips were purchased from Bio-Rad Laboratories (Hercules, CA). All chemicals employed for electrophoresis were from Amersham Biosciences (Uppsala, Sweden). All chemicals of analytical grade were from Sigma (St Louis, MO). Modified trypsin (sequence grade) was acquired from Promega (Madison, WI). All HPLC grade solvents were from J.T. Baker (Phillipsburg, NJ).

Cell culture and DATS treatment
The HGC cell line, BGC823, was established in People's Hospital of Peking University, China. The cells were cultured in complete DMEM (Dulbecco's modified Eagle's medium) containing 5% (v/v) fetal bovine serum (FBS)made by Hyclone (Logan, UT), penicilin (100 kU/l) and streptomycin (100 mg/l) at 37°C in a humidified atmosphere containing 5% CO2. When cell confluence reached 40%, the cells were treated with 25 or 50 µM DATS. To avoid degradation of this compound, the culture medium with DATS was refreshed daily. Considering that DATS dissolved in 0.2% Tween-80, the cells cultured at the same condition with 0.2% Tween-80 were referred to as a mock.

MTT assay
The inhibition of cellular proliferation was measured by the modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, based on the ability of live cells to converting thiazolyl blue to dark blue formazan. Approximately 2.5 x 103 cells were seeded into 96-well culture plates and treated with or without DATS at 24, 48, 72 and 96 h, respectively. After treatment, 100 µl MTT (5 g/l) was added into the wells and incubation continued at 37°C for 4 h, and 100 µl DMSO was pipetted to solubilize the formazan product for 30 min at room temperature. The absorbency at 570 nm was measured using Bio-Rad micro-plate reader.

The sample preparation for 2DE
The harvested cells were washed three times using washing buffer (250 mM D-Sorbitol and 10 mM Tris, pH 7.0), subsequently lysed with lysis buffer containing 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 40 mM dithiothreitol (DTT), 2% pharmalyte (pH 3–10NL), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylene diamine tetra-acetic acid (EDTA). The lysed cells were sonicated with a probe sonicator for 5 min followed by a centrifuge at 40 000 g for 30 min. After quantification of proteins by the Bradford method, the supernatants were stored at –20°C until use for electrophoresis.

2D electrophoresis and image analysis
Isoelectric focusing electrophoresis was carried out with 18 cm (pH 3–10NL) IPG strips at 20°C according to the manufacturer's instructions. Approximately 1 mg protein was loaded onto each gel and triplicate gels for each sample were run to achieve reproducible 2DE results. Briefly, the strips were rehydrated without voltage for 4 h and with 50 V for 8 h. The isoelectric focusing was programmed at a gradient mode, which was first focused for 3 h at the different voltages, 500, 1000 and 8000 V, respectively, then continued at 8000 V until a total of 50 kVh. The focused strips were equilibrated in buffer with 6 M urea, 50 mM Tris–HCl, 30% glycerol, 2% SDS and trace bromophenol blue, and were subsequently treated by the reduction of DTT and alkylation of iodoacetamide. The treated strips were transferred onto 12% uniform SDS–polyacrylamide gels running in 2.5 W each gel for 30 min and 15 W each gel until the bromophenol blue dye reached the bottom of the gel. The gels were stained by Coomassie brilliant blue.

The gels were scanned by Amersham Imagescanner and the image analysis was conducted with ImageMaster 2D Platinum licensed by Amersham Biosciences. The threshold as the significant change in 2DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the treated and control groups.

Mass spectrometry for protein identification
The gel spots verified as the significant changes in spot volume were separately excised by spot picker P2D1.5 from the gel company (San Francisco, USA) and transferred into Eppendorf tubes. The particles were treated by reduction of DTT and alkylation of iodoacetamide followed by a thorough process of washing and drying. Finally, the treated gel particles were incubated with 20 µl of 25 mM NH4HCO3 containing 0.05 µg/µl trypsin at 37°C overnight. After a centrifuge, the resulted supernatants were mixed with 2 µl of 5% trifluoroacetic acid (TFA) and delivered to mass spectrometry.

The tryptic digests were desalted by POROS R2 from Applied Biosystems (Foster City, CA), then co-crystallized with a matrix of {alpha}-cyna-4-hydroxycinnamic acid spotted on the target wells. Then the dried matrixes were subjected to a Bruker autoflex matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bremen, Germany). The mass spectrometer was operated under 19 kV accelerating voltage in the reflectron mode and the m/z range was from 600 to 4000. All PMFs were externally calibrated using standard peptide mixtures and internally calibrated using the masses of trypsin autolysis products. The monoisotopic peptide masses obtained from MALDI-TOF MS were analyzed by m/z software, and interpreted utilizing Mascot produced by Matrix Science (Boston, MA) against the NCBInr database, in which one incomplete cleavage was allowed and alkylation of cysteine by carbamidomethylation, oxidation of methionine and pyroGlu formation of N-terminal Gln were considered as possible modifications.

Some digestive products from 2DE spots were carried out by liquid chromatography ion-trap tandem mass spectrometry (LC-MS/MS) using a LCQ DecaXP ion-trap mass spectrometer from Thermo Finnigan (Ringoes, NJ) for further confirmation of the amino acid sequences. After capillary reverse phase HPLC, the separated peptides were subjected to ion-trap mass spectrometer with 3.2 kV of spray voltage and 150°C at the heated desolvation capillary. The m/z range from 400 to 2000 was scanned in 1.2 s, and the ions were detected with a high energy conversion dynode detector. The LC-MS/MS data were converted into DTA-format files, which were further searched for proteins with Sequest.

Western blotting analysis
Equal amounts of protein (10 µg) of each sample, quantified by Bradford method, were electrophoresed on 12% SDS–PAGE and electrotransferred onto PVDF membranes (350 mA for 1 h) using Bio-Rad Mini PROTEAN 3 system (Hercules, CA) according to the standard protocol. The PVDF membranes were blocked with PBS containing 5% milk powder and 0.1% Tween-20 at room temperature for 2 h, and incubated with 1:200 anti-Bcl-2 from Santa Cruz (Santa Cruz, CA), 1:1000 anti-ß-actin from Santa Cruz, 1:1000 anti-GST-pi from BML (Kawagoe, Saitama, Japan), 1:1000 anti-VDAC-1 from Calbiochem (Cambridge, MA), 1:5000 anti-Annexin I from BD Biosciences (San Jose, CA), 1:5000 anti-Galectin from Santa Cruz, and 1:300 anti-S100 A11—provided by Dr Richard—at 4°C overnight, respectively. The antibody against rabbit, goat or mouse IgG conjugated with horseradish peroxidase (HRP) was adopted as secondary antibody corresponding to the primary antibody. Peroxidase activity was visualized with the ECL kit produced by Amersham Biosciences according to the manufacturer's instructions.

Quantitative real-time RT–PCR
Quantification of gene expression of Annexin I and glutathione S-transferase-pi (GST-pi) was performed via quantitative real-time RT–PCR using an ABI PRISM 7300 system (Foster City, CA). The cDNA libraries were generated from the total RNA preparations of BGC823 cells treated with or without 25 µM DATS at different time spots of 2, 8, 12, 24, 48 and 72 h. The primers were designed to span the exon–intron junctions of these genes as follows, Annexin I: 5'-gcgtcaacagatcaaagcagcata-3' (forward) and 5'-gcagcacgaagttcatcagcatc-3' (reverse); GST-pi: 5'-gcccgggcaactgaagc-3' (forward) and 5'-gcactgaggcgccccacata-3' (reverse); ß-actin: 5'-caaggccaaccgcgagaaga-3' (forward) and 5'-ggatagcacagcctggatag-3' (reverse).

Each sample was normalized on the basis of ß-actin expression. Data were analyzed according to relative gene expression by 2–{Delta}{Delta}Ct method. Moreover, melting curves for each PCR reaction were analyzed carefully to ensure the purity of the amplification product.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
DATS treatment caused apoptosis in BGC823 cells
In our previous observation, the growth of gastric cancer cell line BGC823 was inhibited dramatically by 125 µM DATS. To avoid any toxic effect generated from DATS, the BGC823 cells were seeded at low density and treated with the relatively low doses of DATS, 25 and 50 µM, respectively. The rates of cell growth were monitored by MTT assay as shown in Figure 1A, in which 50 µM DATS exerted inhibition to BGC823 growth in 48 h incubation with increased extent of inhibition during prolonged incubation with DATS. However, a significant inhibition of BGC823 growth was observed until 72 h incubation with 25 µM DATS. To examine whether the cell growth slowed down was caused by apoptosis, an antibody against Bcl-2, one of apoptotic markers, was employed to immunoreact with the proteins extracted from the BGC823 cells treated with or without DATS. The results of western blotting (Figure 1B) demonstrated that Bcl-2 protein expression was significantly inhibited corresponding to DATS treatment, suggesting that suppression of BGC823 growth induced by DATS might be partially attributed to apoptosis in the cells. In addition, the DATS-treated BGC823 cells were examined by Hochest 33342 staining, flow cytometry and DNA fragmentation assay (data not shown). All the results from these experiments substantiated that DATS indeed caused apoptosis in BGC823 cells.


Figure 1
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  		Fig. 1. DATS caused growth suppression and apoptosis in BGC823 cells. (A) The BGC823 cells were treated with 0, 25 and 50 µM of DATS at different incubation periods, 0, 24, 48, 72 and 96 h. In Tween experiments, the cells were treated with 0.2% Tween-80. The treated cells were conducted for MTT assay as described in Materials and methods. Asterisks indicate the significant inhibition of cell growth (P < 0.01). (B) Western blotting to measure the Bcl-2 expression in BGC823 cells treated with or without DATS.

 
Proteomic comparison of BGC823 cells treated with and without DATS
In this study the fundamental judgement is based upon the comparison of different intact proteins expressed in BGC823 cells treated with or without DATS; hence, 2DE is a proper technique to serve the purpose. Since DATS used for cell culture was dissolved in Tween-80, the experiments of quality control were always conducted to ensure whether low concentration of Tween-80 contributed any side-effect to the protein expression in BGC823. The image analysis of 2DE revealed that the patterns of eletrophoretic spots were comparable in the two media with and without 0.2% Tween-80 (data not shown) and the total spots detected by Coomassie blue were quite close, 358 ± 3 (n = 3, control) and 363 ± 6 (n = 3, 0.2% Tween-80), respectively. So this implies that 0.2% Tween-80 does not exhibit an obviously regulatory effect to the protein expression of BGC823, either in the distribution patterns of spots or in the total spots stained.

As illustrated in Figure 2, the proteins extracted from BGC823 cells were well resolved on 2DE. Upon 2DE images, the proteins in BGC823 cells either with or without DATS treatment behaved electrophoretically in similar modes along molecular mass and pI. The electrophoretic spots were biased toward the specific pH regions on the IPG strips, whereas they were evenly distributed over the range of molecular mass from 10 to 100 kDa on 12% SDS–polyacrylamide gel. The images of 2DE were further analyzed by combination of software, ImageMaster and manual check. The total spots counted were 358 ± 3 (n = 3) and 369 ± 6 (n = 3) from the control and DATS-treated groups, respectively. As the variation of 2DE quality is not easily controlled, 3-folds of change in spot volume, gel by gel, were set as the threshold to define the significant differences among the images. A total of 51 spots were defined as garlic-sensitive due to DATS treatment in BGC823 cells, 20 downregulated including 10 spots only found in control group, and 31 upregulated including 6 spots only detected in DATS-treated group. The typical comparisons for these different spots and the changes in spot volume are represented in Figure 3.


Figure 2
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  		Fig. 2. The images of 2DE for the protein extractions of BGC823 cells. The BGC823 cells were treated with or without 25 µM DATS in culture media for 72 h, respectively, followed by 2DE analysis. Triplicate gels for each sample were run.

 

Figure 3
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  		Fig. 3. Image analysis and comparison of spot volume for two typical DATS-sensitive 2DE spots. (A) The closed-up images of 2DE for the Spot 9 identified as GST-pi by mass spectrometry; (B) closed-up images of 2DE for the Spot 30 identified as Annexin I by mass spectrometry. (C) and (D) represent the comparison of the Spots 9 and 30, which spot volumes changed after DATS treatment, respectively. Each sample was parallelly examined three times.

 
Identification of the DATS-sensitive 2DE spots was carried out mainly by MALDI-TOF MS
Mass spectra were internally calibrated with the masses of two trypsin autolysis products at m/z = 842.509 and m/z = 2211.105 to reach a typical mass measurement accuracy of 100 ppm. Stringent criteria were adopted to ensure the accuracy of protein identification: (i) the identified protein must rank at the top two hits with at least five matched sequences and (ii) the total coverage must be >10%. If the quantity of the digestive products was available, double examinations, using two approaches of mass spectrometry, MALDI-TOF MS and LC-MS/MS, were conducted for the same gel spot to achieve more reliable mass data for protein identification. The Spot 9 in Table I was identified using two methods of mass spectrometry. Figure 4 illustrates the verification result for this spot using LC-MS/MS. By MALDI-TOF MS (mass spectrum not shown), eleven unique peptide fragments matched to the protein of GST-pi (accession no. gi:598158); whereas, the spectrum of LC-MS/MS detected eight unique partial amino acid sequences of GST-pi. The protein identification with two different measurements of mass techniques strengthens the confirmation of the proteomic data and reduces the errors generated from different mass machines as well as peptide search engines.


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  		Table I. Downregulated proteins of BGC823 cells treated with DATS

 

Figure 4
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  		Fig. 4. Identification of GST-pi by LC-MS/MS. (A) Peptide base peak ion chromatogram of digestive peptides from Spot 9; (B) an MS survey scan at time 28.33 min during LC-MS analysis; and (C) the MS/MS spectra for parent ion 678.2. The amino acid sequence, AFLASPEYVNLPINGNGKQ, was confirmed by analyzing b-ions and y-ions derived from the peptide ion.

 
A total of 51 spots corresponding to the BGC823 cells treated with DATS were picked up and digested by trypsin. On the basis of the data of mass spectrometry, 44 spots matched with the proteins (86.3% of identification rate), in which 41 were assigned as unique proteins including 17 downregulated and 24 upregulated, which are involved in many biological functions, such as cell growth and differentiation, cell structure and adhesion, basic metabolism and protein degradation. All information of the proteins is listed in Tables I and II.


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  		Table II. Upregulated proteins of BGC823 cells treated with DATS

 
Confirmation of the DATS-sensitive proteins associated with apoptotic pathways
A number of documents have provided solid data to support our observation that garlic derivatives are able to induce apoptosis in cancer cells. As described above, apoptosis induced by DATS was also observed in BGC823 cells. These facts provoked us to further seek the relationship of DATS-sensitive proteins and apoptosis in BGC823 cells. Analyzing the data listed in Tables I and II, and referring the literatures published, 19 DATS-sensitive proteins in BGC823 identified in this study are closely or partially involved in apoptotic pathways. In Table III, there is the basic information on these proteins particularly on how they participate in apoptotic pathway in cancer cells. In order to validate the proteomic data, two proteins downregulated by DATS, GST-pi and voltage-dependent anion channel-1 (VDAC-1), and three proteins upregulated by DATS, Annexin I, Galectin and S100A11, were selected for further conformation using western blotting with the proper antibodies. As depicted in Figure 5, the five proteins were specifically recognized using these antibodies, and the patterns of band intensities developed with enhanced chemiluminescence (ECL) were comparable with the image analysis based upon 2DE. For instance, the decreased folds of GST-pi and VDAC-1 in western blotting were 0.08 and 0.34, corresponding with the changes of spot volume of 0.13 and 0.18, respectively; whereas, the increased folds of Annexin I, Galectin and S100A11 in western blotting were 1.58, 2.71 and infinite, corresponding with the changes of spot volume of 4.38, 3.00 and infinite, respectively. It is obvious that the western blotting results match well with the proteomic observation.


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  		Table III. The garlic-sensitive proteins in BGC823 cells may be involved in apoptotic pathways

 

Figure 5
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  		Fig. 5. The DATS-sensitive proteins in BGC823 cells identified by western blotting analysis. After 72 h treatment the BGC823 cells were collected followed by protein extraction, and the proteins were resolved by 12% SDS–PAGE followed by western blotting using specific antibodies as described in Materials and methods. ß-Actin was used as an internal control.

 
Furthermore, the protein translocations responding to DATS induction were determined. After preparation of cellular organelles from BGC 823, Annexin I and GST-pi were analyzed in the different fractions using western blotting. After DATS treatment, Annexin I protein appeared significant increase in cytosol and nucleus fractions and no change in mitochondria and membrane fractions. In contrast to Annexin I, GST-pi was detected the attenuated expression in both nucleus and mitochondria and constant expression in cytosol (data not shown).

The expression of the DATS-sensitive genes measured by quantitative real-time RT–PCR
In spite of the solid proteomic data, there are still two questions to be addressed. First, proteomic technique only offers semi-quantitative data. Second, the proteomic results do not reflect the acute responses of gene expression induced by DATS. Therefore, quantitative real-time RT–PCR experiments were introduced to accurately measure and monitor gene expression within a short induction. As shown in Figure 6, both genes, Annexin I and GST-pi, take fast responses after incubation with DATS. Within 2 h, their mRNA levels were significantly enhanced or attenuated, with 1.37 or 0.72-folds, respectively. The changes reached to maximum at 12 h administration with 2.73-folds for Annexin I and 0.71-folds for GST-pi, and eventually reduced the expression extents. Interestingly, the mRNA changes of both genes were minimized after 72 h administration with 1.27 and 1.10-folds, respectively; whereas, at this time point both protein expressions were indeed found significant alternations as described above. Taking the observations together, the trends of gene expression induced by DATS, either in mRNA or in protein, are consistent for the two genes, Annexin I and GST-pi. Furthermore, the fact that DATS triggered the fast response of mRNA expression leads to a deduction that this chemical can regulate transcriptional processes somehow.


Figure 6
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  		Fig. 6. The alternations of mRNA expression of Annexin I and GST-pi in BGC823 measured by quantitative real-time RT–PCR. The BGC823 cells treated with or without 25 µM DATS were harvested at intervals, 2, 8, 12, 24, 48 and 72 h followed by total RNA preparation and reverse transcription. Gene expression of Annexin I and GST-pi was quantitatively analyzed by quantitative real-time RT–PCR. The Ct of both genes was normalized by Ct value of ß-actin. Data were analyzed according to relative gene expression by 2–{Delta}{Delta}Ct method.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In an effort to determine the effects of garlic on the protein expression in HGC, we have examined the protein profiles of BGC823 cells treated with or without DATS, using proteomic means of 2DE and mass spectrometry. The present study reveals that ~14% of gel spots that were stained with Coomassie blue significantly changed their spot volumes after treatment with DATS. A total of 44 spots were verified to be proteins with 41 unique ones, in which almost a half of these proteins (19/41) are apoptosis-related. Furthermore, the results of western blotting were in agreement with the proteomic observations. The data obtained from real-time RT–PCR experiments also supported that garlic derivatives are able to evoke a widely apoptotic response in BGC823 cells.

In the last decade, a great effort has been taken for manipulation of apoptosis as a promising strategy in cancer studies. Three biological processes, mitochondrial signals, calcium homeostasis and oxidative stress, are typical events that result in or from apoptosis (17). Apoptosis induction by garlic derivatives has been drawn attention in many mammary cancer cells. For instance, several components of apoptotic pathways have been widely investigated under garlic-induction, such as upregulation of bax, downregulation of Bcl-2, stimulation of cytochrome c release and activation of caspase-3 (18–20). Corresponding to these alterations, naturally a network response related with apoptosis can be expected, in particular the three typical events discussed briefly below.

Several models have been proposed that apoptosis might closely associate with permeability changes in the mitochondrial out membrane. Shimizu et al. (21) hypothesized that the apoptogenic proteins were released through the VDAC–Bax channels. The attenuated expression of VDAC in BGC823 cells treated with DATS appears in agreement with the apoptotic process in these cells. However, VDAC-dependent apoptosis is regulated by a number of Bcl-2 proteins, some anti-apoptotic and some pre-apoptotic. The molecular mechanism remains to be elucidated in future studies how VDAC expressions and functions are mediated by DATS. Interestingly, Galectin was found a significant increase of protein expression after DATS administration. Following apoptotic stimuli, Galectin was able to translocate to the perinuclear mitochondrial membranes, and the translocation was regulated through the interactions of Galectin and the proteins on mitochondrial membrane, one identified as Synexin (22). Downregulation of Synexin abolished the anti-apoptotic activity of Galectin, suggesting that translocation to mitochondrial membrane was a key step for its functions. It merits note that Synexin is a member of the Annexin family and binds to phospholipids on mitochondrial membrane. In this study, Annexin I expression positively correlated with the elevated expression of Galectin in BGC823 cells treated with DATS. The phenomenon leaves a question whether Annexin I locates on mitochondrial membrane and interacts with Galectin during apoptosis in BGC823.

Both Ca2+ signaling and Ca2+ homeostasis provide critical environment for apoptosis, either through intra-mitochondria or extra-mitochondria. Garlic derivatives have been observed to disturb Ca2+ homeostasis, and activate Ca2+-dependent endonucleases and apoptosis. When HCT cells were treated with DADS (50–500 µM), an increase in intracellular Ca2+ concentration was well correlated with cell arrest and apoptosis induction (23). Similar effects have been reported in lung cancer cells due to exposed to DATS (24). In the proteomic survey to the BGC823 cells treated with DATS, several garlic-sensitive proteins are believed to participate in regulation of Ca2+, such as Annexin I and S100A11. Some Annexins have been demonstrated to form the Ca2+ channels with lipid bilayers (25). A S100 protein was characterized by the presence of two different Ca2+ binding motifs, and by the formation of functional dimers. Impressively the interactions between S100A11 and Annexin I have been deeply investigated recently. Using a proteolytically truncated Annexin I derivative as well as a number of N-terminal Annexin I peptides, Seemann et al. (26) mapped the S100A11-binding site to the N-terminal 13 residues of Annexin I, and found that the residues D91 to I94 in C-terminal extension of S100A11 were indispensable for Annexin I binding. Furthermore, the complex of S100A11 and Annexin I induced the apoptosis in human carcinoma cell lines; however, this induction was apparently independent from p53, p21WAF1/CIP1 and caspase activity. Makino et al. (27)therefore proposed that the interactions of S100A11 and Annexin I resulted in partial translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. Taken together, the formation of Annexin I and S100A11 complex is a plausible model to explain this apoptotic process induced by DATS in BGC823 cells.

Oxidative stress, generated through a serious imbalance between reactive oxygen species (ROS) and antioxidant systems, is a crucial factor to induce and execute apoptosis. With the neuroblastoma cell line SH-SY5Y, Filomeni et al. (28) observed that ROS production occurred during the first 15 min with DADS treatment and reached a peak value at 30 min. Several end products of lipid peroxidation, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), concomitantly increased with massive generation of ROS after 1 h treatment of 50 µM DADS. Generally, GST-pi is a detoxification enzyme and has been found to have a tight association with c-Jun N-terminal kinase (JNK), the c-Jun upstream mitogen-activated protein (MAP) kinase (29). Overexpression of GST-pi was detected in many cancer cells, resulting in high intrinsic JNK inhibitory activity to apoptosis. Importantly, garlic derivatives enable to regulate the dissociation of JNK–GST-pi complex leading to JNK activation and apoptosis stimulation. The proteomic data presented in this study demonstrated a significantly decreased GST-pi in BGC823 cells after treatment with DATS for 72 h; and the data of real-time PCR provided further evidence that GST-pi transcription was attenuated within 12 h after incubation of DATS. Thus, the decreased GST-pi expression seems inversely correlated with the increased of BGC823 arrest responding to DATS treatment. The phenomenon could be explained by the ROS model mediated by garlic. Accordingly, administration of DATS is able to enhance oxidative stress in BGC823 cells, subsequently ROS inhibit GST-pi expression and dissociate JNK–GST-pi complex. These events accelerate the apoptotic process in BGC823 cells.

It merits note that the expression of mRNA and protein, Annexin I and GST-pi, were not synchronous, the mRNA expression responding earlier to DATS than the protein expression. First of all, upon the central principle, translation events are expected to occur after transcription, quickly or slowly, depending upon the regulatory mechanism for transcription and translation. Secondly, the half-life of either mRNA or protein is controlled by many factors. Sachs (30) pointed out that some eukaryotic mRNAs were degraded with a half-life of 20 min while other mRNAs remained intact for up to 24 h. For instance, the half-life of GST-pi in Vcrems cells was ~4–5 h after treatment with actinomycin D. In contrast, the GST-pi mRNA levels in EJ cells remained unchanged for 6 h after treatment with actinomycin D. As a control, levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were demonstrated to be unaffected in both cell lines (31). The turnover rates of protein may be variable due to the different regulations as well.

In summary, the results of the present study shed light on a massive response of protein expression in BGC823 cells induced by DATS, and demonstrate that numerous DATS-sensitive proteins in this cell line are related with apoptosis. Therefore, it seems reasonable to conclude that garlic derivatives exert multiple stimuli to several biological pathways; moreover, the diverse molecular responses merge into apoptotic pathways that could strengthen the anticarcinogenic effects. The proteomic investigation into BGC823 cells treated with DATS contributes additional effort to understand the molecular mechanisms of the garlic-enriched diet in cancer prevention.


   Notes
 
{dagger} These authors contributed equally to the work Back


   Acknowledgments
 
We thank Dr Richard L. Eckert (Case Western Reserve University School of Medicine, Cleveland, Ohio, USA) and Dr Hong Tang (Institute of Biophysics, CAS, China) for providing the antibody. This work was supported by grant of 863 program in China (2002BA711A11) and science and technology project in Beijing (H010210440111) and by State Key Basic Research Program, No. 2004CB518708.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 18, 2005; revised September 30, 2005; accepted December 6, 2005.


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