BACKGROUND
globally, gastric cancer is the second leading cause of cancer-related death, and is the most prevalent cancer in south korea.
in the past two decades, the mortality and incidence of gastric cancer has decreased gradually but it is still the second most common cancer in asia
<cit> .
most stomach cancers are an adenocarcinoma type, which accounts approximately 90%
<cit> .
the well-established risk factors are helicobacter pylori-infection and cigarette smoking, and the role of dietary factors has also been suggested
<cit> .
the most widely used treatments for stomach cancer are surgery, chemotherapy, and/or radiation therapy.
the available treatments are not effective and recuperation is also still problematic.
hence, there is an urgency to apply new therapeutic agents to increase survival rates of gastric cancer patients.
vitamin c  is an essential nutrient of most living tissues, and readily acts as a strong reducing agent
<cit> .
epidemiological studies have reported that vitamin c deficiency in humans are linked to more severe h. pylori-associated gastritis and a gastric cancer risk is also higher
<cit> .
the study reported that supplementation of vitamin c has been associated with reduced gastric cancer risk in humans.
in addition, a reduced risk for most types of cancer is associated with a high intake of fresh fruits and vegetables, which contain vitamin c
<cit> .
despite, the controversial cancer treatment history, the in-vitro studies reported that ascorbate induces cell cycle arrest and apoptosis in various tumor cells
<cit> .
however, the exact mechanism of vitamin c involved in cancer treatment is not fully elucidated.
a global proteomic approach is being extensively applied in cancer research
<cit> .
this approach uses a combination of two-dimensional gel electrophoresis , image analysis, matrix-assisted laser desorption/ ionization-time of flight  mass spectrometry , and bioinformatics analyses to comprehensively resolve, identify, and characterize proteins in the cells, tissues and animal models.
these high-throughput proteome techniques allow us to examine the changes in protein expression of ags cells in response to vitamin c. identification of differentially expressed proteins is important to understand the molecular events involved in vitamin c anti-cancer mechanism and protective effects, as well as brings new insights into ags carcinogenesis.
regarding gastric cancer, proteome analysis has been reported mainly in kato-iii and epg 85– <dig> human gastric cancer cell lines
<cit> .
2-de maps have also permitted to obtain an overview of the expressed proteins in the human stomach
<cit> .
also, proteome analysis has been carried out in  <dig> human gastric cancer samples to find the biomarkers of gastric cancer
<cit> .
however, no 2-de proteome of vitamin c-treated ags cells have hitherto been reported.
our previous study demonstrated that vitamin c induced apoptosis in human adenocarcinoma ags cells at pharmacological concentrations, and inhibited ags cells proliferation
<cit> .
in the present study, we perform a proteome analysis of ags cells treated with vitamin c at pharmacological concentrations  and the control , and  <dig> different expressed proteins were identified by maldi-tof/ms.
also, the expression of isoforms of 14-3- <dig> proteins was confirmed by immuno-blotting.
the cytotoxicity assay suggests that vitamin c inhibited ags cells growth and proteome results revealed that apoptosis related proteins were involved in promoting and regulating cell death of ags cells.
methods
chemical and reagents
rpmi  <dig> medium was purchased from hyclone .
fetal bovine serum  and antibiotics  were purchased from gibco .
materials and chemicals used for electrophoresis were obtained from biorad .
antibody to 14-3-3σ and β-actin were purchased from millipore .
14-3-3ϵ and 14-3-3δ were obtained from bioworld technology inc. ).
vitamin c was provided by animal resources research bank .
all other chemicals used in this study were purchased from amresco  and sigma-aldrich .
all the chemicals used were of the highest grade commercially available.
cell culture and treatments
ags human gastric cancer cell line was purchased from atcc .
cells were grown in rpmi  <dig> medium supplemented with 10% fbs and 1% penicillin/streptomycin , and grown in a humidified incubator with 5% co <dig> in air at 37°c.
experiments were performed when cell growth was approximately 80% confluent.
cytotoxicity assay
the 3-- <dig>  5-diphenyltetrazolium bromide -based assay was performed to determine the cytotoxicity of vitamin c on ags cells.
cells were seeded at  <dig> ×  <dig> cells/ml in a 12-well plate and incubated for  <dig> h. cells were treated with various concentrations of vitamin c  or only vehicle  and incubated for  <dig> h. after incubation,  <dig> μl of a mtt solution  was added to the wells and incubated for  <dig> h. then,  <dig> μl of dimethyl sulfoxide  was added to each well after the medium was removed completely to dissolve the cellular crystalline deposits.
the optical density was measured at  <dig> nm using an elisa plate reader.
protein extraction and two-dimensional gel electrophoresis
a total of 1× <dig> cells was plated onto 100ml plates and incubated overnight at 37°c in an atmosphere of 5% co <dig>  cells were treated with  <dig> μg/ml of vitamin c and 1x pbs used as the control.
after  <dig> h incubation, cells were trypsinized and washed twice with cold 1x pbs.
then, cells were lysed in a lysis buffer  chaps) on ice for  <dig> h. the lysates were centrifuged at  <dig> rpm for  <dig> min at 4°c, and the collected supernatant was stored at −80°c until analysis.
proteins in lysates were precipitated with equal volume  of 20% v/v trichloroacetic acid and dissolved in  <dig> m urea,  <dig> m thiourea, and 4%  chaps,  <dig> %  ipg buffer, and 1% dithiothreitol .
protein concentration was determined by the non-interfering™ protein assay kit , in accordance to the manufacturer’s protocol.
immobilized  <dig> cm linear ph gradient  strips, ph 4– <dig>  were rehydrated in a rehydration buffer  chaps,  <dig> % bromophenol blue).
for the first dimension,  <dig> μg protein was focused using the ettan ipg phor ii  at  <dig> v for  <dig> h, followed by  <dig> v for  <dig> h,  <dig> v for  <dig> min,  <dig> v for  <dig> min,  <dig> v for  <dig> h,  <dig> v for  <dig> h,  <dig> v for  <dig> h, and  <dig> v for  <dig> h. the focused strips were equilibrated twice,  <dig> min each time, first with  <dig> mg/ml dtt and then with  <dig> mg/ml iodoacetamide  prepared in equilibration buffer containing  <dig> mm tris–hcl ,  <dig> m urea, 30%  glycerol, 2%  sds, and  <dig> %  bromophenol blue.
the focused proteins were then separated in the second dimension by 12% linear gradient sds-page with a constant current of  <dig> ma/gel at 20°c.
gels were run until the bromophenol dye front reached the end of the gel.
protein detection, analysis, and in-gel digestion
the gels were stained with silver nitrate, similar to the method described by swain and ross
<cit>  with slight modifications.
three independent gels were performed in triplicate.
gels were scanned and image analysis was performed, using progenesis samespots software .
using this software, the differentially expressed spots were identified by automatic matching of the detected protein spots.
those spots differing significantly  in their intensities with a fold-change ≥ <dig> were used for further analysis.
selected protein spots were excised manually from the two-dimensional electrophoresis  gel and protein digestion was performed
<cit>  with slight modifications.
briefly, the excised gel pieces were washed with  <dig> μl of  <dig> mm nh4hco <dig> for  <dig> min, and then dehydrated in  <dig> μl of acetonitrile for  <dig> min.
after being dried in a lyophilizer , the gel pieces were rehydrated in 5– <dig> μl of  <dig> mm nh4hco <dig> containing  <dig> ng/μl trypsin  on ice.
after  <dig> min, the trypsin solution was removed and replaced with 10– <dig> μl of  <dig> mm nh4hco <dig> without trypsin, and digestion was carried out for a minimum of  <dig> h at 37°c.
these peptide mixtures were collected and analyzed by a mass spectrometry.
matrix-assisted laser desorption/ionization-time of flight mass spectrometry  mass spectrometry and database searching
tryptic peptides obtained as described above were subsequently extracted by an addition of  <dig> μl of the extraction buffer, followed by an addition of 10– <dig> μl of acetonitrile.
pooled extracts were dried in a lyophilizer  and the extracts were re-dissolved in  <dig> μl of extraction buffer and  <dig> μl of matrix solution  and targeted onto a maldi-tof plate.
after drying the samples completely onto the targeting plate, maldi-tof/ms was conducted using a voyager- de str mass spectrometer  equipped with delay ion extraction.
mass spectra were obtained over a mass range of 800– <dig>  da.
for identification of proteins, the peptide mass fingerprinting data were used to search against the swissprot database using the mascot program .
the following parameters were used for database searches: taxonomy, homo sapiens ; cleavage specificity, trypsin with one missed cleavage allowed; peptide tolerance of  <dig> ppm for the fragment ions; and allowed modifications, cys carbamidomethyl , and oxidation of met .
protein scores > <dig> were considered statistically significant .
western blot analysis
ags cells were cultured in 6-well plates and incubated with vitamin c at  <dig> μg/ml or pbs as the solvent control for  <dig> h. after incubation, cells were washed with ice-cold pbs and lysed with a lysis buffer , containing the protease inhibitor cocktail.
the cell debris was removed by centrifugation at  <dig>  rpm for  <dig> min and protein concentration was determined using a bradford assay .
proteins were separated by 10% sds-polyacrylamide gel electrophoresis  and transferred to a polyvinyldene fluoride  membrane  using the te  <dig> semi-dry transfer unit .
the membrane was blocked with 5% non-fat skim milk in tris-buffered saline containing 1% tween- <dig>  at room temperature for  <dig> h, and the blots were probed with rabbit monoclonal antibody to 14-3-3σ, 14-3-3ϵ and 14-3-3δ, and mouse monoclonal antibody for β-actin.
the proteins were visualized using an enhanced chemiluminescence kit  and western blotting detection reagents , and exposed to x-ray film .
each band was quantitatively determined using image j  software.
the densitometry readings of the bands were normalized to β-actin expression.
statistical analysis
the data represents the mean±standard deviation  of three independent experiments.
the statistical significance between the control and sample groups was calculated by the student’s t-test.
a p value < <dig>  was considered as significant.
RESULTS
growth inhibition of ags cells by vitamin c
to evaluate the effects of growth inhibition and survival of ags cells, the ags cells were cultured in the presence of various concentrations  of vitamin c for  <dig> h. vitamin c had a strong inhibitory effect on cell proliferation of ags cells in a dose-dependent manner when compared to the control, after  <dig> h treatment with vitamin c .
especially, vitamin c at  <dig>   <dig> and  <dig> μg/ml decreased the cell growth by approximately 50%, 36% and 27%, respectively.
therefore, the ic <dig>  of vitamin c was found to be approximately  <dig> μg/ml.
moreover, microscopic observations revealed morphological changes in ags cells, such as cell shrinkage and density compared with the control cells .
further, 2-de gel analysis was performed to study the protein expressions in ags cells due to inhibitory effects of vitamin c.
proteomic analysis to identify differentially expressed proteins in vitamin c-treated ags cells
we performed a proteomic approach to identify proteins that were differentially expressed in vitamin c treated  ags cells,  <dig> μg of total proteins were separated by ief on  <dig> cm ipg strips in the first dimension and 12% sds-page in the second dimension.
we observed a total of approximately  <dig> protein spots in silver stained gels.
control  and vitamin c treated gels were analyzed by using progenesis samespots software , and we found  <dig> statistically significant differentially expressed protein spots .
these  <dig> differentially expressed proteins spots were chosen for further analysis by maldi-tof/ms.
finally,  <dig> differentially expressed proteins were successfully identified by using the mascot search engine and the swissprot database .
1entry name, protein name and accession number from swiss-prot database identified by maldi-tof/ms.
2theoretical molecular weight  and pi from swiss-prot database; the experimental values of pi and mw for every isoelectric spot were calculated using progenesis samespots software .
3score is −10*log , where p is the probability that the observed match is a random event, protein scores greater than  <dig> are significant .
of these  <dig> proteins, six were up-regulated and fourteen were down-regulated in vitamin c-treated ags cells compared with the control.
down-regulated proteins involved in cell motility included tropomyosin alpha- <dig> chain  and tropomyosin alpha- <dig> chain , whereas xin actin-binding repeat-containing protein  <dig>  was up-regulated.
in addition, the peroxiredoxin- <dig>  and thioredoxin domain-containing protein  <dig>  were involved in antioxidant and detoxification, which was up-regulated.
while proteins participating in signal transduction were significantly down-regulated, including 14-3- <dig> protein sigma , 14-3- <dig> protein epsilon  and 14-3- <dig> protein zeta/delta , whereas tnfaip3-interacting protein  <dig>  was up-regulated.
proteins involved in protein metabolism eukaryotic translation initiation factor  <dig> subunit k , proteasome subunit alpha type- <dig>  and proteasome subunit beta type- <dig>  were down-regulated.
further, we showed the enlarged 2-de images of  <dig> important protein spots, one spot  was up-regulated and the other five  were down-regulated in the vitamin c-treated ags cells compared with the control .
validation of expression of 14-3- <dig> isoforms by immunoblotting
recent research on cancer targets have focused 14-3- <dig> proteins that are known to be involved in various biological processes like signal transduction, cell cycle control, apoptosis, cellular metabolism, proliferation, cytoskeletal regulation, transcription, and redox-regulation or stress response.
the ags cells were treated with vitamin c  and the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were examined by immuno-blotting.
quantification of the protein bands revealed that the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were decreased in the vitamin c-treated group compared to the vehicle-treated control group .
these data indicated that vitamin c decreased the expression of 14-3- <dig> isoforms  in ags cells.
discussion
apart from antioxidant activity, vitamin c plays an effective role of cancer prevention and treatment.
the numerous studies have reported that vitamin c prevents cell proliferation and metastasis of many human cancer cells
<cit> .
but, it’s exact molecular mechanisms still has not been fully elucidated.
in the previous study, we demonstrated that vitamin c at pharmacological concentration induced apoptosis in ags cells, mainly through the down-regulation of 14–3-3σ protein and dephosphorylation bad proteins via a mitochondrial dependent pathway
<cit> .
in the present study, we performed 2-de analysis coupled with maldi-tof/ms of ags cells treated with vitamin c at a pharmacological concentration  and the control , and  <dig> differentially expressed proteins were identified.
proteomic analysis revealed that the apoptosis related proteins were involved in promoting and regulating cell death of ags cells.
ascorbic acid is an excellent antioxidant and ascorbate caused toxicity to cancer cells, but had no effect on normal cells at the same concentration
<cit> .
in the present study, vitamin c had a strong inhibitory effect on cell proliferation of ags cells in a dose-dependent manner after  <dig> h treatment with vitamin c , and the ic <dig> of vitamin c was found approximately  <dig> μg/ml or  <dig>  mm/ml.
and also, morphological changes were observed in ags cells, such as cell shrinkage and density in vitamin c treated cells compared with the control cells .
this result revealed that vitamin c inhibited ags cell growth at pharmacological concentrations.
further, 2-de gel analysis was performed to study the protein expressions in ags cells due to inhibitory effects of vitamin c. the silver stained gels of control  and vitamin c treated gels were analyzed by using progenesis samespots software , and we found  <dig> statistically significant differentially expressed protein spots .
finally,  <dig> differentially expressed proteins were successfully identified by maldi-tof/ms analysis using the mascot search engine and the swissprot database .
among  <dig> proteins, six were up-regulated and fourteen were down-regulated in vitamin c-treated ags cells compared with the control.
these proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism.
vitamin c down-regulated proteins involved in the signal transduction, 14-3- <dig> isoforms
research on cancer targets have determined that 14-3- <dig> proteins are known to be involved in various biological processes like signal transduction, cell cycle control, apoptosis, cellular metabolism, proliferation, cytoskeletal regulation, transcription, and redox-regulation or stress response
<cit> .
among these differentially expressed proteins, three isoforms of 14-3- <dig> proteins, 14-3-3σ and 14-3-3ϵ and 14-3-3δ were down-regulated .
the bad protein, a proapoptotic family member, is one of the targets of 14-3- <dig> proteins
<cit> .
when bad disassociated from 14-3- <dig>  the bad is found localized to the mitochondria bound to bcl- <dig> and bcl-xl, and induced cell death
<cit> .
in addition, vitamin c induced apoptosis by down-regulation of 14–3-3σ and dephosphorylation of bad via a mitochondrial dependent pathway in ags cells
<cit> .
moreover, the remarkable dissociation of bad from 14-3-3β is the apoptosis mechanism of vitamin c through the increasing of er stress and the translocation of bad to mitochondria after dissociation from 14-3-3β in human colon cancer cell line, hct-8
<cit> .
these findings suggest that bad dissociated from 14-3- <dig> is a key mediator in vitamin c-induced apoptosis through the disruption of mitochondrial membrane potential.
the down-regulation of 14-3-3σ protein has been reported in many types of cancer, including breast cancer
<cit> , and expression is frequently lost in other human epithelial carcinomas
<cit> .
moreover, other 14-3-3ϵ, ζ, γ, β, θ isoforms have also been identified in cancer
<cit> .
after dna-damage, 14-3-3σ down-regulated cells fail to maintain a g2/m arrest and undergo a mitotic catastrophe, which results in apoptosis
<cit> .
the reduction of 14-3-3ζ expression induced the g <dig> arrest, which leads to mitotic catastrophe and increase radio sensitivity
<cit> .
to verify the expressions of identified proteins, we performed an immuno-blotting analysis on 14-3-3σ, 14-3-3ϵ and 14-3-3δ, which have been suggested to be involved in various cancers.
quantification of the protein bands determined that the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were decreased in the vitamin c-treated group compared to the vehicle-treated control group .
these data indicated that vitamin c decreased the expression of 14-3- <dig> isoforms  in ags cells.
these data suggest that down-regulation of 14-3- <dig> could be a useful information in therapeutic targets of human gastric cancers.
vitamin c down-regulated the cytoskeleton and associated proteins, tropomyosin alpha- <dig> chain and tropomyosin alpha- <dig> chain proteins
tropomyosins are actin-binding proteins that can integrate cell mechanics and signaling essential for cellular migration and invasion.
proteomic studies showed that the expression of tropomyosin changes, which suggest an important role for tropomyosin in maintaining cell shape
<cit> .
in addition, tropomyosins increase filament stiffness, stabilize actin filaments by protecting them against the severing action of gelsolin and cofilin
<cit> .
in our  <dig> d gel system, the spots corresponding to tropomyosin  <dig> and tropomyosin  <dig> showed a decreased expression in the response to vitamin c .
these decreased changes in the tropomyosins expression might be coincidental with morphologic changes and migratory characteristics of ags cells in response to vitamin c.
vitamin c up-regulated the antioxidant proteins, peroxiredoxin- <dig> and thioredoxin domain-containing protein 5
generally, antioxidant proteins play a pivotal role in the antioxidant defense system and protect the cells from oxidative stress.
there are six peroxiredoxins found in mammalians, and peroxiredoxin  <dig>  is localized in the endoplasmic reticulum
<cit> .
in the present study, antioxidant and detoxification proteins, like peroxiredoxin- <dig> and thioredoxin domain-containing protein  <dig>  were over expressed in vitamin c treated ags cells .
since, vitamin c is an excellent antioxidant that increased antioxidant protein expressions in ags cells and protects from oxidative stress.
to our knowledge there has not been a reported study regarding peroxiredoxin- <dig> protein expression in human gastric cancer adenocarcinoma ags cells response to vitamin c. therefore, a detailed study is necessary regarding the effects of vitamin c on peroxiredoxin- <dig> protein expressions and the role of peroxiredoxin- <dig> protein in tumorigenesis of gastric cancer.
vitamin c altered the proteins involved in protein metabolism
also, we found that eif3k was down-regulated in vitamin c-treated ags cells.
a previous study has been reported that the down-regulation of eif3k attenuating apoptosis in simple epithelial cells
<cit> .
tumor necrosis factor  alpha-induced protein  <dig>  or tnfaip <dig> is a novel tumor suppressor protein and a key player in the negative feedback regulation of nf-kb signaling in response to multiple stimuli.
tnfaip <dig> also regulates tnf-induced apoptosis
<cit> .
moreover, tnfaip <dig> induces cell growth arrest and apoptosis, accompanied by down-regulation of nuclear factor-kappa b  activation
<cit> .
presently, tnfaip <dig> was up-regulated in vitamin c treated ags cells.
figure
<dig> represents the overview of the growth inhibition effect of vitamin c on ags cells and protein expression patterns.
these proteomic results reveal that vitamin c inhibited cell growth, and apoptosis related proteins were involved in promoting and regulating cell death in ags cells.
CONCLUSIONS
in summary, vitamin c showed strong inhibitory effect on ags cell growth at pharmacological concentrations , and  <dig> differentially expressed proteins were identified in ags cells after exposure to vitamin c by using 2-de and madli-tof analysis.
in particular, proteins involved in signal transduction 14-3-3σ, 14-3-3ϵ and 14-3-3δ, and cytoskeletal proteins tropomyosin alpha- <dig> chain and tropomyosin alpha- <dig> chain were down-regulated; peroxiredoxin- <dig> was up-regulated in vitamin c-treated ags cells compared with the control.
further, the expressions of 14-3- <dig> isoforms  were verified with a western blot analysis.
the findings of this study suggest that vitamin c could inhibit ags cell growth, alter the apoptosis related proteins, and might be helpful to understand the molecular mechanism of vitamin c ’s anti-tumor effect in ags cells.
competing interests
we declare that there are no conflicts of interests.
authors’ contributions
conceived and designed the experiments: an and gs-k; performed the experiments: an and hs-p; analyzed the data: an, hs-p and ki-p; statistical analysis: ja-k, ge-h, sr-k and jz; wrote the manuscript: an; drafting and critical review of manuscript for important intellectual content: eh-k, ws-l and ck-w. all authors read and approved the final manuscript.

 globally, gastric cancer is the second leading cause of cancer-related death, and is the most prevalent cancer in south korea. in the past two decades, the mortality and incidence of gastric cancer has decreased gradually but it is still the second most common cancer in asia. most stomach cancers are an adenocarcinoma type, which accounts approximately 90%. the well-established risk factors are helicobacter pylori-infection and cigarette smoking, and the role of dietary factors has also been suggested. the most widely used treatments for stomach cancer are surgery, chemotherapy, and/or radiation therapy. the available treatments are not effective and recuperation is also still problematic. hence, there is an urgency to apply new therapeutic agents to increase survival rates of gastric cancer patients. vitamin c is an essential nutrient of most living tissues, and readily acts as a strong reducing agent. epidemiological studies have reported that vitamin c deficiency in humans are linked to more severe h. pylori-associated gastritis and a gastric cancer risk is also higher. the study reported that supplementation of vitamin c has been associated with reduced gastric cancer risk in humans. in addition, a reduced risk for most types of cancer is associated with a high intake of fresh fruits and vegetables, which contain vitamin c. despite, the controversial cancer treatment history, the in-vitro studies reported that ascorbate induces cell cycle arrest and apoptosis in various tumor cells. however, the exact mechanism of vitamin c involved in cancer treatment is not fully elucidated. a global proteomic approach is being extensively applied in cancer research. this approach uses a combination of two-dimensional gel electrophoresis, image analysis, matrix-assisted laser desorption/ ionization-time of flight mass spectrometry, and bioinformatics analyses to comprehensively resolve, identify, and characterize proteins in the cells, tissues and animal models. these high-throughput proteome techniques allow us to examine the changes in protein expression of ags cells in response to vitamin c. identification of differentially expressed proteins is important to understand the molecular events involved in vitamin c anti-cancer mechanism and protective effects, as well as brings new insights into ags carcinogenesis. regarding gastric cancer, proteome analysis has been reported mainly in kato-iii and epg 85– human gastric cancer cell lines. 2-de maps have also permitted to obtain an overview of the expressed proteins in the human stomach. also, proteome analysis has been carried out in human gastric cancer samples to find the biomarkers of gastric cancer. however, no 2-de proteome of vitamin c-treated ags cells have hitherto been reported. our previous study demonstrated that vitamin c induced apoptosis in human adenocarcinoma ags cells at pharmacological concentrations, and inhibited ags cells proliferation. in the present study, we perform a proteome analysis of ags cells treated with vitamin c at pharmacological concentrations and the control, and different expressed proteins were identified by maldi-tof/ms. also, the expression of isoforms of 14-3- proteins was confirmed by immuno-blotting. the cytotoxicity assay suggests that vitamin c inhibited ags cells growth and proteome results revealed that apoptosis related proteins were involved in promoting and regulating cell death of ags cells. methods chemical and reagents rpmi medium was purchased from hyclone. fetal bovine serum and antibiotics were purchased from gibco. materials and chemicals used for electrophoresis were obtained from biorad. antibody to 14-3-3σ and β-actin were purchased from millipore. 14-3-3ϵ and 14-3-3δ were obtained from bioworld technology inc. ). vitamin c was provided by animal resources research bank. all other chemicals used in this study were purchased from amresco and sigma-aldrich. all the chemicals used were of the highest grade commercially available. cell culture and treatments ags human gastric cancer cell line was purchased from atcc. cells were grown in rpmi medium supplemented with 10% fbs and 1% penicillin/streptomycin, and grown in a humidified incubator with 5% co in air at 37°c. experiments were performed when cell growth was approximately 80% confluent. cytotoxicity assay the 3-- 5-diphenyltetrazolium bromide -based assay was performed to determine the cytotoxicity of vitamin c on ags cells. cells were seeded at × cells/ml in a 12-well plate and incubated for h. cells were treated with various concentrations of vitamin c or only vehicle and incubated for h. after incubation, μl of a mtt solution was added to the wells and incubated for h. then, μl of dimethyl sulfoxide was added to each well after the medium was removed completely to dissolve the cellular crystalline deposits. the optical density was measured at nm using an elisa plate reader. protein extraction and two-dimensional gel electrophoresis a total of 1× cells was plated onto 100ml plates and incubated overnight at 37°c in an atmosphere of 5% co cells were treated with μg/ml of vitamin c and 1x pbs used as the control. after h incubation, cells were trypsinized and washed twice with cold 1x pbs. then, cells were lysed in a lysis buffer chaps) on ice for h. the lysates were centrifuged at rpm for min at 4°c, and the collected supernatant was stored at −80°c until analysis. proteins in lysates were precipitated with equal volume of 20% v/v trichloroacetic acid and dissolved in m urea, m thiourea, and 4% chaps, % ipg buffer, and 1% dithiothreitol. protein concentration was determined by the non-interfering™ protein assay kit, in accordance to the manufacturer’s protocol. immobilized cm linear ph gradient strips, ph 4– were rehydrated in a rehydration buffer chaps, % bromophenol blue). for the first dimension, μg protein was focused using the ettan ipg phor ii at v for h, followed by v for h, v for min, v for min, v for h, v for h, v for h, and v for h. the focused strips were equilibrated twice, min each time, first with mg/ml dtt and then with mg/ml iodoacetamide prepared in equilibration buffer containing mm tris–hcl, m urea, 30% glycerol, 2% sds, and % bromophenol blue. the focused proteins were then separated in the second dimension by 12% linear gradient sds-page with a constant current of ma/gel at 20°c. gels were run until the bromophenol dye front reached the end of the gel. protein detection, analysis, and in-gel digestion the gels were stained with silver nitrate, similar to the method described by swain and ross with slight modifications. three independent gels were performed in triplicate. gels were scanned and image analysis was performed, using progenesis samespots software. using this software, the differentially expressed spots were identified by automatic matching of the detected protein spots. those spots differing significantly in their intensities with a fold-change ≥ were used for further analysis. selected protein spots were excised manually from the two-dimensional electrophoresis gel and protein digestion was performed with slight modifications. briefly, the excised gel pieces were washed with μl of mm nh4hco for min, and then dehydrated in μl of acetonitrile for min. after being dried in a lyophilizer, the gel pieces were rehydrated in 5– μl of mm nh4hco containing ng/μl trypsin on ice. after min, the trypsin solution was removed and replaced with 10– μl of mm nh4hco without trypsin, and digestion was carried out for a minimum of h at 37°c. these peptide mixtures were collected and analyzed by a mass spectrometry. matrix-assisted laser desorption/ionization-time of flight mass spectrometry mass spectrometry and database searching tryptic peptides obtained as described above were subsequently extracted by an addition of μl of the extraction buffer, followed by an addition of 10– μl of acetonitrile. pooled extracts were dried in a lyophilizer and the extracts were re-dissolved in μl of extraction buffer and μl of matrix solution and targeted onto a maldi-tof plate. after drying the samples completely onto the targeting plate, maldi-tof/ms was conducted using a voyager- de str mass spectrometer equipped with delay ion extraction. mass spectra were obtained over a mass range of 800– da. for identification of proteins, the peptide mass fingerprinting data were used to search against the swissprot database using the mascot program. the following parameters were used for database searches: taxonomy, homo sapiens; cleavage specificity, trypsin with one missed cleavage allowed; peptide tolerance of ppm for the fragment ions; and allowed modifications, cys carbamidomethyl, and oxidation of met. protein scores > were considered statistically significant. western blot analysis ags cells were cultured in 6-well plates and incubated with vitamin c at μg/ml or pbs as the solvent control for h. after incubation, cells were washed with ice-cold pbs and lysed with a lysis buffer, containing the protease inhibitor cocktail. the cell debris was removed by centrifugation at rpm for min and protein concentration was determined using a bradford assay. proteins were separated by 10% sds-polyacrylamide gel electrophoresis and transferred to a polyvinyldene fluoride membrane using the te semi-dry transfer unit. the membrane was blocked with 5% non-fat skim milk in tris-buffered saline containing 1% tween- at room temperature for h, and the blots were probed with rabbit monoclonal antibody to 14-3-3σ, 14-3-3ϵ and 14-3-3δ, and mouse monoclonal antibody for β-actin. the proteins were visualized using an enhanced chemiluminescence kit and western blotting detection reagents, and exposed to x-ray film. each band was quantitatively determined using image j software. the densitometry readings of the bands were normalized to β-actin expression. statistical analysis the data represents the mean±standard deviation of three independent experiments. the statistical significance between the control and sample groups was calculated by the student’s t-test. a p value < was considered as significant.  growth inhibition of ags cells by vitamin c to evaluate the effects of growth inhibition and survival of ags cells, the ags cells were cultured in the presence of various concentrations of vitamin c for h. vitamin c had a strong inhibitory effect on cell proliferation of ags cells in a dose-dependent manner when compared to the control, after h treatment with vitamin c. especially, vitamin c at and μg/ml decreased the cell growth by approximately 50%, 36% and 27%, respectively. therefore, the ic of vitamin c was found to be approximately μg/ml. moreover, microscopic observations revealed morphological changes in ags cells, such as cell shrinkage and density compared with the control cells. further, 2-de gel analysis was performed to study the protein expressions in ags cells due to inhibitory effects of vitamin c. proteomic analysis to identify differentially expressed proteins in vitamin c-treated ags cells we performed a proteomic approach to identify proteins that were differentially expressed in vitamin c treated ags cells, μg of total proteins were separated by ief on cm ipg strips in the first dimension and 12% sds-page in the second dimension. we observed a total of approximately protein spots in silver stained gels. control and vitamin c treated gels were analyzed by using progenesis samespots software, and we found statistically significant differentially expressed protein spots. these differentially expressed proteins spots were chosen for further analysis by maldi-tof/ms. finally, differentially expressed proteins were successfully identified by using the mascot search engine and the swissprot database. 1entry name, protein name and accession number from swiss-prot database identified by maldi-tof/ms. 2theoretical molecular weight and pi from swiss-prot database; the experimental values of pi and mw for every isoelectric spot were calculated using progenesis samespots software. 3score is −10*log, where p is the probability that the observed match is a random event, protein scores greater than are significant. of these proteins, six were up-regulated and fourteen were down-regulated in vitamin c-treated ags cells compared with the control. down-regulated proteins involved in cell motility included tropomyosin alpha- chain and tropomyosin alpha- chain, whereas xin actin-binding repeat-containing protein was up-regulated. in addition, the peroxiredoxin- and thioredoxin domain-containing protein were involved in antioxidant and detoxification, which was up-regulated. while proteins participating in signal transduction were significantly down-regulated, including 14-3- protein sigma, 14-3- protein epsilon and 14-3- protein zeta/delta, whereas tnfaip3-interacting protein was up-regulated. proteins involved in protein metabolism eukaryotic translation initiation factor subunit k, proteasome subunit alpha type- and proteasome subunit beta type- were down-regulated. further, we showed the enlarged 2-de images of important protein spots, one spot was up-regulated and the other five were down-regulated in the vitamin c-treated ags cells compared with the control. validation of expression of 14-3- isoforms by immunoblotting recent research on cancer targets have focused 14-3- proteins that are known to be involved in various biological processes like signal transduction, cell cycle control, apoptosis, cellular metabolism, proliferation, cytoskeletal regulation, transcription, and redox-regulation or stress response. the ags cells were treated with vitamin c and the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were examined by immuno-blotting. quantification of the protein bands revealed that the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were decreased in the vitamin c-treated group compared to the vehicle-treated control group. these data indicated that vitamin c decreased the expression of 14-3- isoforms in ags cells. discussion apart from antioxidant activity, vitamin c plays an effective role of cancer prevention and treatment. the numerous studies have reported that vitamin c prevents cell proliferation and metastasis of many human cancer cells. but, it’s exact molecular mechanisms still has not been fully elucidated. in the previous study, we demonstrated that vitamin c at pharmacological concentration induced apoptosis in ags cells, mainly through the down-regulation of 14–3-3σ protein and dephosphorylation bad proteins via a mitochondrial dependent pathway. in the present study, we performed 2-de analysis coupled with maldi-tof/ms of ags cells treated with vitamin c at a pharmacological concentration and the control, and differentially expressed proteins were identified. proteomic analysis revealed that the apoptosis related proteins were involved in promoting and regulating cell death of ags cells. ascorbic acid is an excellent antioxidant and ascorbate caused toxicity to cancer cells, but had no effect on normal cells at the same concentration. in the present study, vitamin c had a strong inhibitory effect on cell proliferation of ags cells in a dose-dependent manner after h treatment with vitamin c, and the ic of vitamin c was found approximately μg/ml or mm/ml. and also, morphological changes were observed in ags cells, such as cell shrinkage and density in vitamin c treated cells compared with the control cells. this result revealed that vitamin c inhibited ags cell growth at pharmacological concentrations. further, 2-de gel analysis was performed to study the protein expressions in ags cells due to inhibitory effects of vitamin c. the silver stained gels of control and vitamin c treated gels were analyzed by using progenesis samespots software, and we found statistically significant differentially expressed protein spots. finally, differentially expressed proteins were successfully identified by maldi-tof/ms analysis using the mascot search engine and the swissprot database. among proteins, six were up-regulated and fourteen were down-regulated in vitamin c-treated ags cells compared with the control. these proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism. vitamin c down-regulated proteins involved in the signal transduction, 14-3- isoforms research on cancer targets have determined that 14-3- proteins are known to be involved in various biological processes like signal transduction, cell cycle control, apoptosis, cellular metabolism, proliferation, cytoskeletal regulation, transcription, and redox-regulation or stress response. among these differentially expressed proteins, three isoforms of 14-3- proteins, 14-3-3σ and 14-3-3ϵ and 14-3-3δ were down-regulated. the bad protein, a proapoptotic family member, is one of the targets of 14-3- proteins. when bad disassociated from 14-3- the bad is found localized to the mitochondria bound to bcl- and bcl-xl, and induced cell death. in addition, vitamin c induced apoptosis by down-regulation of 14–3-3σ and dephosphorylation of bad via a mitochondrial dependent pathway in ags cells. moreover, the remarkable dissociation of bad from 14-3-3β is the apoptosis mechanism of vitamin c through the increasing of er stress and the translocation of bad to mitochondria after dissociation from 14-3-3β in human colon cancer cell line, hct-8. these findings suggest that bad dissociated from 14-3- is a key mediator in vitamin c-induced apoptosis through the disruption of mitochondrial membrane potential. the down-regulation of 14-3-3σ protein has been reported in many types of cancer, including breast cancer, and expression is frequently lost in other human epithelial carcinomas. moreover, other 14-3-3ϵ, ζ, γ, β, θ isoforms have also been identified in cancer. after dna-damage, 14-3-3σ down-regulated cells fail to maintain a g2/m arrest and undergo a mitotic catastrophe, which results in apoptosis. the reduction of 14-3-3ζ expression induced the g arrest, which leads to mitotic catastrophe and increase radio sensitivity. to verify the expressions of identified proteins, we performed an immuno-blotting analysis on 14-3-3σ, 14-3-3ϵ and 14-3-3δ, which have been suggested to be involved in various cancers. quantification of the protein bands determined that the expression of 14-3-3σ, 14-3-3ϵ and 14-3-3δ were decreased in the vitamin c-treated group compared to the vehicle-treated control group. these data indicated that vitamin c decreased the expression of 14-3- isoforms in ags cells. these data suggest that down-regulation of 14-3- could be a useful information in therapeutic targets of human gastric cancers. vitamin c down-regulated the cytoskeleton and associated proteins, tropomyosin alpha- chain and tropomyosin alpha- chain proteins tropomyosins are actin-binding proteins that can integrate cell mechanics and signaling essential for cellular migration and invasion. proteomic studies showed that the expression of tropomyosin changes, which suggest an important role for tropomyosin in maintaining cell shape. in addition, tropomyosins increase filament stiffness, stabilize actin filaments by protecting them against the severing action of gelsolin and cofilin. in our d gel system, the spots corresponding to tropomyosin and tropomyosin showed a decreased expression in the response to vitamin c. these decreased changes in the tropomyosins expression might be coincidental with morphologic changes and migratory characteristics of ags cells in response to vitamin c. vitamin c up-regulated the antioxidant proteins, peroxiredoxin- and thioredoxin domain-containing protein 5 generally, antioxidant proteins play a pivotal role in the antioxidant defense system and protect the cells from oxidative stress. there are six peroxiredoxins found in mammalians, and peroxiredoxin is localized in the endoplasmic reticulum. in the present study, antioxidant and detoxification proteins, like peroxiredoxin- and thioredoxin domain-containing protein were over expressed in vitamin c treated ags cells. since, vitamin c is an excellent antioxidant that increased antioxidant protein expressions in ags cells and protects from oxidative stress. to our knowledge there has not been a reported study regarding peroxiredoxin- protein expression in human gastric cancer adenocarcinoma ags cells response to vitamin c. therefore, a detailed study is necessary regarding the effects of vitamin c on peroxiredoxin- protein expressions and the role of peroxiredoxin- protein in tumorigenesis of gastric cancer. vitamin c altered the proteins involved in protein metabolism also, we found that eif3k was down-regulated in vitamin c-treated ags cells. a previous study has been reported that the down-regulation of eif3k attenuating apoptosis in simple epithelial cells. tumor necrosis factor alpha-induced protein or tnfaip is a novel tumor suppressor protein and a key player in the negative feedback regulation of nf-kb signaling in response to multiple stimuli. tnfaip also regulates tnf-induced apoptosis. moreover, tnfaip induces cell growth arrest and apoptosis, accompanied by down-regulation of nuclear factor-kappa b activation. presently, tnfaip was up-regulated in vitamin c treated ags cells. figure  represents the overview of the growth inhibition effect of vitamin c on ags cells and protein expression patterns. these proteomic results reveal that vitamin c inhibited cell growth, and apoptosis related proteins were involved in promoting and regulating cell death in ags cells.  in summary, vitamin c showed strong inhibitory effect on ags cell growth at pharmacological concentrations, and differentially expressed proteins were identified in ags cells after exposure to vitamin c by using 2-de and madli-tof analysis. in particular, proteins involved in signal transduction 14-3-3σ, 14-3-3ϵ and 14-3-3δ, and cytoskeletal proteins tropomyosin alpha- chain and tropomyosin alpha- chain were down-regulated; peroxiredoxin- was up-regulated in vitamin c-treated ags cells compared with the control. further, the expressions of 14-3- isoforms were verified with a western blot analysis. the findings of this study suggest that vitamin c could inhibit ags cell growth, alter the apoptosis related proteins, and might be helpful to understand the molecular mechanism of vitamin c ’s anti-tumor effect in ags cells. competing interests we declare that there are no conflicts of interests. authors’ contributions conceived and designed the experiments: an and gs-k; performed the experiments: an and hs-p; analyzed the data: an, hs-p and ki-p; statistical analysis: ja-k, ge-h, sr-k and jz; wrote the manuscript: an; drafting and critical review of manuscript for important intellectual content: eh-k, ws-l and ck-w. all authors read and approved the final manuscript. 
the identified proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism. our proteome results suggest that the apoptosis related proteins were involved in promoting and regulating cell death of ags cells, and might be helpful to understand the molecular mechanism of vitamin c on ags cell growth inhibition. hence, the purpose of this study was to investigate the molecular mechanism of the inhibitory effect of vitamin c on ags cell growth, and protein profiles in ags cells after exposure to vitamin c treatment, by using proteomic tools. vitamin c is an essential nutrient of most living tissues that readily acts as a strong reducing agent, which is abundant in fruits and vegetables. vitamin c showed a cytotoxic effect on ags cells and, differentially expressed proteins were successfully identified by assisted laser desorption/ ionization-time of flight/mass spectrometry. vitamin cgastric cancerags cellsproteome analysis14-3- isoforms
 vitamin c is an essential nutrient of most living tissues that readily acts as a strong reducing agent, which is abundant in fruits and vegetables. although, it inhibits cell growth in many human cancer cells in vitro, treatment in cancer is still controversial. hence, the purpose of this study was to investigate the molecular mechanism of the inhibitory effect of vitamin c on ags cell growth, and protein profiles in ags cells after exposure to vitamin c treatment, by using proteomic tools.  vitamin c showed a cytotoxic effect on ags cells and, differentially expressed proteins were successfully identified by assisted laser desorption/ ionization-time of flight/mass spectrometry. of the proteins, six were up-regulated and fourteen were down-regulated. specifically, 14-3-3σ, 14-3-3ϵ, 14-3-3δ, tropomyosin alpha- chain and tropomyosin alpha- chain were down-regulated and peroxiredoxin- and thioredoxin domain-containing proteins were up-regulated. the identified proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism. further, the expressions of 14-3- isoforms were verified with immuno-blotting analysis.  our proteome results suggest that the apoptosis related proteins were involved in promoting and regulating cell death of ags cells, and might be helpful to understand the molecular mechanism of vitamin c on ags cell growth inhibition. vitamin cgastric cancerags cellsproteome analysis14-3- isoforms 