gastric carcinoma is the fourth most common gastrointestinal cancer and has the second highest mortality rate among all gastrointestinal cancers . three critical measures improving patient prognosis are early diagnosis , radical surgery , and endoscopic surgery . although radical resection has been widely promoted , the post - surgery recovery is still unfavorable due to tnm stages . the prognosis of gastric cancer is also affected by patient biological factors and clinical pathological conditions , necessitating the identification of novel bio - markers for diagnosis and treatment . a widely accepted opinion is that oxidative stress ( os ) participates in pathogenesis of gastric cancer because it can damage dna and cause mucous injuries . nuclear factor e2-related factor 2 ( nrf2 ) , as a basic redox - sensitive bzip transcription factor , plays a role in intracellular anti - oxidation and phase ii detoxification through the up - regulation of various antioxidant response elements ( are ) genes   via the keap1-cul3-dependent e3 ubiquitination pathway . under oxidative stress or chemical stimulus , nrf2 translocates into the nucleus , where it forms heterodimers with maf and binds onto the are sequence on the chromosome to activate the transcription of downstream genes , including antioxidants and phase ii detoxification enzymes [ 1214 ] . the importance of nrf2 resides in its protective roles against various human disease and pathological conditions , including cancer , neurodegenerative disease , cardiovascular dysfunction , inflammation , and pulmonary fibrosis , along with ameliorating aging [ 1519 ] , making nrf2 a  beneficial  transcriptional factor protecting the body from oxidative stress . in tumor tissues , therefore , nrf2 has a dual role in both preventing oncogenesis and protecting tumor cells from stress injury , making it a candidate factor for tumor growth and invasion . currently there is no available study regarding either expression or clinical significance of nrf2 in gastric carcinoma . this study therefore aimed to address this issue and further elucidate the clinical implication of anti - oxidative stress - related nrf2 expression . gastric cancer cells mkn74 , mnk45 , katoiii , and nugc4 ( cell institute of chinese academy of science , china ) were cultured in rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells collected by centrifugation were rinsed in phosphate - buffered saline ( pbs ) and extracted for total proteins by lysis buffer . cell nucleus / cytoplasmic fractions were separated using a test kit ( biovision , usa ) according to the manufacturer s instructions . denatured proteins were separated on 10% sds - page and transferred to pvdf membrane , which was blocked in 5% defatted milk powders overnight . mouse monoclonal anti - nrf2 antibody ( 1:500 , santa cruz , usa ) was used to incubate the membrane overnight . after rinsing in tris - buffered saline - tween 20 ( tbst ) , anti - mouse igg conjugated with horseradish peroxidase ( rd systems , us ) was used for a further 15-min incubation . the signal was finally developed using ecl - plus reagents and was visualized on x - ray films . the nucleus and cytoplasm fractions were labelled by anti - lamin b1 ( 1:1 000 , abcam , usa ) or anti- tubulin antibody ( 1:1000 , calbiochem , usa ) . all patients underwent surgical resection of stomach ( 57 cases of distal , 14 cases of proximal , 99 cases of total , and 5 cases of partial stomach resection ) with lymph node clearance between jan 2004 and dec 2013 in our hospital . there were 116 males and 59 females in the patient group , ages 3184 years ( average , 66 years ) . among all patients , 47 were at stage i when diagnosed , 33 were at stage ii , and 95 were at stage iii . histopathological typing showed 71 cases of differentiated ( papillary , high - differentiated , and moderate differentiated tubular adenocarcinoma ) tumors and 104 undifferentiated tumors ( including low differentiated adenocarcinoma , mucinous adenocarcinoma , and signet ring cell carcinoma ) . this study was pre - approved by the ethics committee of our hospital and we obtained written consent from all patients and their families . tumor tissues were first fixed in 10% formalin - pbs buffer , embedded in paraffin , sectioned ( 4-m thickness ) , and was mounted on glass slides . after de - waxing in xylene and re - hydration in gradient ethanol , endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol . after gentle washing in pbs , antigen retrieval was performed using heated citrate buffers for 10 min . non - specific binding sites were further blocked in 10% fbs . anti - nrf2 antibody ( 1:200 , santa cruz , usa ) further detection was accomplished by streptavidin - biotin peroxidase ( sp ) kit ( nichirei , japan ) and was visualized by diaminobenzidine incubation . two independent blinded observers evaluated staining images . in brief , 10 representative regions within the tumor samples were selected . in each region , the expression was observed in 100 cells under a high - magnification microscope ( 400 ) . the undifferentiated region was preferred to differentiate tissues on the same slide . averaged nrf2 index was calculated based on the number of positive cells in each region . the expressional profile was described from both the positive rate and staining intensity , the latter classified as 0 , 1 + , and 2 + . we compared the nrf2 expression level in patients who underwent 5-fluorouracil ( 5-fu ) treatment . as only stage ii / iii patients were qualified for chemotherapy and patients with severe adverse effects were excluded , only 72 patients were recruited for the analysis of nrf2 expression and tumor chemoresistance . between - group comparisons were performed by chi - square , fisher s exact , or mann - whitney u test . the correlation between nrf2 expression and clinical indexes was analyzed by kaplan - meier curve , univariate / multivariate analysis , and log - rank test . gastric cancer cells mkn74 , mnk45 , katoiii , and nugc4 ( cell institute of chinese academy of science , china ) were cultured in rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells collected by centrifugation were rinsed in phosphate - buffered saline ( pbs ) and extracted for total proteins by lysis buffer . cell nucleus / cytoplasmic fractions were separated using a test kit ( biovision , usa ) according to the manufacturer s instructions . denatured proteins were separated on 10% sds - page and transferred to pvdf membrane , which was blocked in 5% defatted milk powders overnight . mouse monoclonal anti - nrf2 antibody ( 1:500 , santa cruz , usa ) was used to incubate the membrane overnight . after rinsing in tris - buffered saline - tween 20 ( tbst ) , anti - mouse igg conjugated with horseradish peroxidase ( rd systems , us ) was used for a further 15-min incubation . the signal was finally developed using ecl - plus reagents and was visualized on x - ray films . the nucleus and cytoplasm fractions were labelled by anti - lamin b1 ( 1:1 000 , abcam , usa ) or anti- tubulin antibody ( 1:1000 , calbiochem , usa ) . all patients underwent surgical resection of stomach ( 57 cases of distal , 14 cases of proximal , 99 cases of total , and 5 cases of partial stomach resection ) with lymph node clearance between jan 2004 and dec 2013 in our hospital . there were 116 males and 59 females in the patient group , ages 3184 years ( average , 66 years ) . among all patients , 47 were at stage i when diagnosed , 33 were at stage ii , and 95 were at stage iii . histopathological typing showed 71 cases of differentiated ( papillary , high - differentiated , and moderate differentiated tubular adenocarcinoma ) tumors and 104 undifferentiated tumors ( including low differentiated adenocarcinoma , mucinous adenocarcinoma , and signet ring cell carcinoma ) . this study was pre - approved by the ethics committee of our hospital and we obtained written consent from all patients and their families . tumor tissues were first fixed in 10% formalin - pbs buffer , embedded in paraffin , sectioned ( 4-m thickness ) , and was mounted on glass slides . after de - waxing in xylene and re - hydration in gradient ethanol , after gentle washing in pbs , antigen retrieval was performed using heated citrate buffers for 10 min . non - specific binding sites were further blocked in 10% fbs . anti - nrf2 antibody ( 1:200 , santa cruz , usa ) further detection was accomplished by streptavidin - biotin peroxidase ( sp ) kit ( nichirei , japan ) and was visualized by diaminobenzidine incubation . two independent blinded observers evaluated staining images . in brief , 10 representative regions within the tumor samples were selected . in each region , the expression was observed in 100 cells under a high - magnification microscope ( 400 ) . the undifferentiated region was preferred to differentiate tissues on the same slide . averaged nrf2 index was calculated based on the number of positive cells in each region . the expressional profile was described from both the positive rate and staining intensity , the latter classified as 0 , 1 + , and 2 + . we compared the nrf2 expression level in patients who underwent 5-fluorouracil ( 5-fu ) treatment . as only stage ii / iii patients were qualified for chemotherapy and patients with severe adverse effects were excluded , only 72 patients were recruited for the analysis of nrf2 expression and tumor chemoresistance . between - group comparisons were performed by chi - square , fisher s exact , or mann - whitney u test . the correlation between nrf2 expression and clinical indexes was analyzed by kaplan - meier curve , univariate / multivariate analysis , and log - rank test . as shown by western blotting in both nucleus and cytosolic fractions , nrf2 protein is mainly expressed in the nucleus , with minimal expression in the cytoplasm ( figure 1 ) . ihc staining obtained results consistent with those in the abovementioned western blotting , as the nrf2 signal localizes in the nucleus ( figure 2 ) . we calculated the staining scored as described in the methods section , and classified those with scores higher than 100 into the nrf2-positive group . in all 175 patients , 108 were classified into the positive group ( 61.7% ) and the other 67 were classified into the negative group ( 38.3% ) . further correlation analysis between nrf2 expression and clinical indexes significant correlations were discovered between nrf2-positive expression and patient sex , tumor volume , infiltrative depth , lymph node metastasis , lymph tube invasion , and tnm staging . among all 72 patients who underwent 5-fu therapy , there were 59 who were nrf2-positive and 13 who were nrf2-negative . among those 59 nrf2-positive patients , a total of 43 patients ( 72.9% ) developed 5-fu resistance , which occurred in only 5 of the 13 nrf2-negative patients ( 38.5% ) . using kaplan - meier method , we found that the overall survival rate in nrf2-positive patients was significantly lower than that in nrf2-negative patients ( 61% vs. 79% , p<0.01 , figure 3 ) . significant correlation factors of patient survival rate include invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , by univariate analysis . a further multivariate analysis indicated lymph node metastasis as an independent prognostic index ( table 3 ) . as shown by western blotting in both nucleus and cytosolic fractions , nrf2 protein is mainly expressed in the nucleus , with minimal expression in the cytoplasm ( figure 1 ) . ihc staining obtained results consistent with those in the abovementioned western blotting , as the nrf2 signal localizes in the nucleus ( figure 2 ) . we calculated the staining scored as described in the methods section , and classified those with scores higher than 100 into the nrf2-positive group . in all 175 patients , 108 were classified into the positive group ( 61.7% ) and the other 67 were classified into the negative group ( 38.3% ) . further correlation analysis between nrf2 expression and clinical indexes significant correlations were discovered between nrf2-positive expression and patient sex , tumor volume , infiltrative depth , lymph node metastasis , lymph tube invasion , and tnm staging . among all 72 patients who underwent 5-fu therapy , there were 59 who were nrf2-positive and 13 who were nrf2-negative . among those 59 nrf2-positive patients , a total of 43 patients ( 72.9% ) developed 5-fu resistance , which occurred in only 5 of the 13 nrf2-negative patients ( 38.5% ) . using kaplan - meier method , we found that the overall survival rate in nrf2-positive patients was significantly lower than that in nrf2-negative patients ( 61% vs. 79% , p<0.01 , figure 3 ) . significant correlation factors of patient survival rate include invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , by univariate analysis . a further multivariate analysis indicated lymph node metastasis as an independent prognostic index ( table 3 ) . as an antioxidant , nrf2 can protect normal cells from oxidative stress injuries , thereby forming a self - protective mechanism . the over - expression of nrf2 and its downstream genes in various tumors , however , raised the potency of nrf2 in facilitating survival and proliferation of cancer cells [ 2026 ] . our study for the first time reports that a close relationship exists between nrf2 expression in gastric carcinoma cell nucleus and patient clinical features . the prominent expression of nrf2 in nucleus of gastric cancer cells from both in vivo and in vitro samples suggests a persistent expression of nrf2 may cause production of antioxidants , which further endow cancer cells with elevated anti - reactive oxygen species ( ros ) activity . this proposed mechanism has been reported by ma et al . , who found higher nuclear nrf2 levels and downstream anti - oxidative proteins in more advanced cervical cancer tissues . therefore , it is likely that gastric cancer cell nuclear nrf2 level is related with tumor malignancy . ihc staining results showed elevated nrf2 expression in gastric cancer tissues . among all patients , 61.7% were nrf2-positive , higher than those in non - small cell lung cancer ( nsclc ) ( 26% ) or gall bladder cancer ( 23% ) . the survival rate - related factors include tumor invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , in agreement with a gall bladder cancer study . recent study in gastric carcinoma found consistent effects of nrf2 expression on the prognosis , but only with immune reactivity in cytoplasm rather than the nucleus , probably due to the use of different antibodies . various studies have confirmed the importance of cytoplasmic - nuclear translocation of nrf2 for exerting its anti - oxidative activity . persistent over - expression of nrf2 may protect cancer cells from ros injuries because it can work as an anti - oxidant . therefore , it is necessary to quantify nuclear but not cytoplasmic nrf2 proteins in gastric cancer tissues . although nrf2-positive is not included in multivariate analysis as an independent prognostic evaluating factor , our results showed that lymph node metastasis can significantly affect nrf2-positive rates as a marker for unfavorable prognosis . previous studies in nsclc and gall bladder cancer all supported existence of relationships between nrf2 positive expression and unfavorable prognosis , suggesting the utility of nrf2 as a prognostic index for evaluating the postoperative survival rate of patients . various studies have shown that nrf2 can decrease the survival rate via its facilitation on tumor cell resistance against radio-/chemo - therapy [ 2934 ] . this study found a higher rate of 5-fu resistance in nrf2-positive tumors ; therefore , the evaluation of nrf2 in gastric cancer patients may help to optimize the chemotherapy plan . the genetic silencing or functional inhibition can suppress activity of nrf2-modulated oxidase , including glutathione , thioredoxin , and mercaptan sulfur , leading to the recovery of tumor cell sensitivity to chemo-/radio - therapy . there have been clinical trials supporting this mechanism , such as the sensitization of cancer cells against alkylation drugs by nrf2 inhibition   or the inhibition of tumor growth by the combination of nrf2 gene knockdown and cisplatin application . therefore , the next generation of chemotherapy plans involving modulating nrf2-related anti - oxidative functions may provide more satisfactory treatment efficacy and improve patient survival and prognosis . this study demonstrated a positive relationship between nrf2 expression and invasiveness of gastric carcinoma , suggesting the utility of nrf2 as a prognostic index .