gallbladder cancers ( gbcs ) are highly aggressive cancers with the highest mortality rate among gastrointestinal cancers . the highest prevalence rates of gbc have been observed in american indian , chilean , and japanese women [ 1 , 2 ] . early diagnosis of gbc is generally impossible due to a lack of specific signs or symptoms . over 90% of gbc patients are diagnosed at an advanced , inoperable stage with serious invasion and metastasis to other organs . a majority of gbcs encountered in the clinic are adenocarcinomas ( ac , 9095% ) , while rare subtypes such as squamous cell / adenosquamous carcinoma ( sc / asc ) , mucinous carcinoma , signet ring cell carcinoma , and undifferentiated carcinoma are hardly ever encountered [ 2 , 5 ] . at present , cholecystectomy is only viable for early stage gbc [ 68 ] , while patients with unresectable or metastatic gbc have extremely poor prognosis and few treatment options . generally , palliative chemotherapy and radiation therapy offer limited benefits to patients with gbc . therefore , there is an urgent need to identify biological markers for the diagnosis , prognosis , and target therapy of gallbladder cancers . numerous studies have observed elevated levels of oxidative stress during tumor growth , which is characterized by an increase in reactive oxygen species ( ros ) . ros serve as secondary messenger molecules to increase tumor cell proliferation , genetic mutations , and instability , which lead to subsequent invasion , angiogenesis , and drug resistance in cancer cells . most importantly , cancer cells can develop mechanisms to evade high ros - induced apoptosis [ 10 , 11 ] . the elevation of oxidative stress levels in tumor cells is generally thought to be associated with two factors : the increased generation of ros and the decreased capacity of the cell to eliminate ros . enhanced release of o2 and/or h2o2 from the mitochondria and activation of nadph oxidase ( nox ) systems increase cellular ros levels [ 13 , 14 ] . the increase in ros can also be caused by the suppression of antioxidant enzymes , such as mnsod and gpx . thus , the differential expression of antioxidant enzymes in cancer can serve as biomarkers of tumor initiation and/or progression . tumor suppressive roles of the superoxide dismutase ( sod ) family of proteins have been observed in a variety of cancers . glutathione peroxidase ( gpx ) has been revealed to modulate the tumor suppressive effect of mnsod in various tumor cells . a number of studies have demonstrated that gpx3 expression is downregulated in a variety of cancers . for example , gpx3 is strongly downregulated in prostate cancer , thyroid cancer , colorectal cancer , gastric cancer , and breast cancer [ 15 , 16 ] . intriguingly , a previous study has revealed that gpx3 expression is higher in clear cell epithelial ovarian carcinoma tissues compared to control tissues . since gpx3 is always highly expressed in healthy tissues , it has been suggested to be a tumor suppressor in many tumors . aldehyde dehydrogenases ( aldhs ) are a family of enzymes that metabolize both endogenous and exogenous aldehydes to carboxylic acids as well as other reactive compounds . for example , aldh1a1 and aldh3a1 function as ocular antioxidants that play unique roles in the defense of the eye against uv radiation and in the downstream effects of oxidative stress . some aldh isoforms function in retinoic acid ( ra ) signaling via ra production by oxidizing all - trans - retinal and 9-cis - retinal . recently , aldh1 activity has been employed as a marker of stem - like cells in many cancers , such as cervical and breast cancer . moreover , aldh1 activity was demonstrated to be significantly higher in metastatic breast tumor cells that escaped chemotherapy . furthermore , high activity of aldh1 is associated with poor prognosis in breast , bladder , and prostate cancer patients [ 24 , 25 ] . however , most aldh prognostic data is based on using aldh1a1 expression levels as an indicator for cancer patients ' outcome [ 26 , 27 ] . these studies also yielded varied results , and the correlation of aldh1a1 with prognosis may be dependent on cancer type . in contrast , aldh1a3 , but not aldh1a1 , has been shown to correlate consistently with tumor grade and metastasis of breast cancer patients , suggesting that aldh1a3 may be a better marker in some tumors than aldh1a1 . the association of other aldh subtypes with tumor progression and prognosis has not been established . our preliminary study using microarray assay showed a 24-fold increase in aldh1a3 level and 23-fold decrease in gpx3 in gbc - sd cells compared to normal gbc epithelial cells ( data not shown ) . in this study , the expression of aldh1a3 and gpx3 in surgically resected specimens , including ac and sc / asc , was examined by immunohistochemistry . the correlation of aldh1a3 and gpx3 expressions with the clinicopathological characteristics and prognosis of ac and sc / asc was comparatively evaluated . a total of 46 squamous cell / adenosquamous carcinomas ( sc / asc ) from gbc patients that underwent surgical resection or biopsy were collected from january 1995 to december 2009 . the percentage of sc / asc in various gallbladder cancers is 4.34% ( 46/1060 gbcs ) . 80 adenocarcinomas ( ac ) samples for patients that underwent surgical resection or biopsy were collected from january 2005 to december 2009 . a gallbladder cancer sample was diagnosed as sc when most malignant cells are squamous cells and less than 10% are adenocarcinoma cells . a gallbladder cancer sample was diagnosed as asc when the tumor contains both squamous cells and adenocarcinoma cells , but the tumor must contain at least 10% adenocarcinoma or squamous cell carcinoma cells . among the 46 sc / asc patients , 19 patients were male and 27 patients were female with an age variation from 35 to 82 ( 55.8  9.6 ) years . among the 80 ac patients , 26 patients were male and 54 patients were female with an age variation from 33 to 80 ( 53.8  9.9 ) years . the differentiation ( refer to squamous cells ) of the 46 sc / ascs includes 16 well - differentiated , 24 moderately differentiated , and 6 poorly differentiated carcinomas . among the 80 acs , 27 were well differentiated , 25 were moderately differentiated , and 28 were poorly differentiated . among the 46 sc / asc patients , invasion of gallbladder , surrounding tissues and organs , was found in 30 patients , while 29 patients had regional lymph node metastasis . among the 80 ac patients , invasion of gallbladder surrounding tissues and organs was found in 49 patients , while 50 patients had regional lymph node metastasis . surgery included radical resection for 14 sc / ascs and 26 acs , palliative surgery for 18 sc / ascs and 28 acs , and no operation for 14 sc / ascs and 26 acs with only biopsy . the 2-year survival information of 80 ac and 46 sc / asc patients was obtained through letters and phone calls . this study was preapproved by the ethics committee for human research , central south university . four - micrometer - thick sections were cut from routinely paraffin - embedded tissues of ac and sc / asc . staining was conducted with the peroxidase - based envision detection kit ( dako laboratories , ca , usa ) by following the user manual and the rabbit anti - aldh1a3 ( abgent company , california , usa ) and rabbit anti - gpx3 ( novus biologicals , littleton , usa ) antibodies which specifically stain human aldh1a3 and human gpx3 . briefly , the sections were deparaffinized and then incubated with 3% h2o2 for 15 minutes . after being soaked with phosphate - buffered saline ( pbs ) for 3  5 minutes , the sections were incubated with primary antibody for 1  hr at room temperature . after rinsing sections with pbs for 3 times , solution a was added , and the sections were incubated for 30 minutes . the substrate dab was added followed by hematoxylin counterstaining . the slides were dehydrated with different concentrations ( 70%100% ) of alcohol and soaked in xylene for 3  5 minutes . the positive control was the positive biopsy provided by beijing zhongshan biotechnology company ( beijing , china ) , while the negative control was designed by replacing the primary antibody with 5% fetal bovine serum . by considering that heterogeneity of staining pattern exists , the percentage of positive cells was calculated from 500 tumor cells in 10 random fields . the staining strength was graded as previously described : a score of 1 was given for no positive staining or a uncertainly weak staining ; a score of 2 was given for weak to moderate staining ; a score of 3 was given for moderate to strong staining . for gpx3 , cases with both positive cells  25% and scores  2 were considered positive . a few cases with 10% to 25% positive cells and 3 scores in staining cases with both positive cells  10% and scores  2 were considered positive . the slides were systematically scanned , and 7 - 8 representative digital images were acquired from each slide . digital images were quantified by image analysis with adobe photoshop , version 7.0 ( adobe systems , san jose , ca ) , and the extent of staining was assessed as the percentage of positively stained area per field under 100 magnification . data was analyzed using the statistical package for the social sciences version 13.0 ( spss 13.0 ) . the interrelationship of aldh1a3 or gpx3 expressions with histological or clinical factors was analyzed using  , fisher 's exact test , or student 's t - test . kaplan - meier and time series tests ( log - rank test ) were used for univariate survival analysis . cox proportional hazards model was used for multivariate analysis and to determine the 95% confidence interval . the percentage of cases with age older than 45 years and tumor mass > 3  cm was significantly higher in sc / ascs than in acs ( p < 0.05 ) . the percentage of cases with poorly differentiated tumor was significantly lower in sc / ascs than in acs . no significant differences in other clinicopathological characteristics were observed between sc / asc and ac patients . envision immunohistochemistry revealed that aldh1a3 and gpx3 positive reaction was mainly localized in the cytoplasm of tumor cells in sc / asc ( figure 1 ) and ac ( figure 2 ) , but a small amount of inflammatory reaction cells and fibroblasts was also stained . western blot with aldh1a3 antibody showed one main band of 56  kda and a weak band of 22  kda , while western blot with gpx3 antibody showed one band of 25  kda ( data not shown ) . no significant difference in the percentage of cases with positive aldh1a3 and gpx3 expressions was observed between sc / asc and ac patients ( data not shown ) . as shown in table 1 and figure 3(a ) , the percentage of cases with positive aldh1a3 expression was significantly higher in tumor tissues from sc / asc patients with high tnm stage and lymph node metastasis compared to tumor tissues from cases with low tnm stage and no lymph metastasis ( p < 0.05 or  p < 0.01 ) . the gpx3 expression was significantly lower in cases with higher tnm stage , lymph node metastasis , and invasion ( p < 0.05 or  p < 0.01 ) . aldh1a3 and gpx3 exhibited no significant association with other clinicopathological characteristics of sc / asc . the percentage of cases with positive aldh1a3 expression and negative gpx3 expression in ac tumors was significantly higher in cases with poor differentiation , large tumor mass , high tnm stage with lymph node metastasis , and invasion to the gallbladder surrounding tissues and organs compared to the cases with well - differentiated tumor , small tumor mass , lower tnm stage , no lymph metastasis , and no invasion ( p < 0.05 , p < 0.01 , or  p < 0.001 ) ( table 1 , figure 3(b ) ) . survival information of 46 sc / asc patients was obtained through letters and phone calls . the followup time was 2 years , and patients that survived longer than 2 years were included in the analysis as censored cases . thirty - three of the 46 sc / asc patients survived less than 1 year and 13 patients survived over 1 year ( 4 cases survived longer than 2 years ) with an average survival time of 10.07  0.78 months . fifty - seven of the 80 ac patients survived less than 1 year and 23 patients survived over 1 year ( 9 cases survived longer than 2 years ) with an average survival time of 10.34  0.63 months . the kaplan - meier survival analysis in sc / asc patients revealed that the differentiation , tumor size , tnm stage , lymph node metastasis , and invasion ( p < the average survival time of aldh1a3 positive patients was significantly lower than patients with negative aldh1a3 expression ( p = 0.005 ) , but the average survival time of gpx3 positive patients was significantly higher than patients with negative gpx3 expression ( p = 0.002 ) ( table 2 , figure 4 ) . cox multivariate analysis showed that the differentiation , tumor size ( 3  cm ) , tnm stage , invasion , and operative procedure as well as aldh1a3-positive expression or gpx3-negative expression were negatively correlated with postoperative survival , suggesting that aldh1a3 positivity is a risk factor and gpx3 has a strong impact on prognosis of scs / ascs ( table 3 ) . the kaplan - meier survival analysis in ac patients revealed that the differentiation , tumor size , tnm stage , lymph node metastasis , invasion , and operative procedure ( p < 0.001 ) were significantly associated with the average survival time ( table 2 ) . the average survival time of aldh1a3-positive ac patients was significantly lower than patients with negative aldh1a3 expression ( p < 0.001 ) , but the average survival time of gpx3 positive patients was significantly higher than patients with negative gpx3 expression ( p < 0.001 ) ( table 2 , figure 5 ) . cox multivariate analysis showed that the differentiation , tumor size ( 3  cm ) , tnm stage , lymph node metastasis , invasion , and operative procedure as well as aldh1a3-positive expression or gpx3-negative expression were negatively correlated with poor survival of ac patients ( table 3 ) . the clinical and pathological characteristics of squamous cell / adenosquamous carcinomas ( sc / asc ) of the gallbladder have not yet been widely recognized because these subtypes are rather rare . moreover , no study so far has systemically compared the clinical , pathological , and molecular differences between the rare sc / asc subtype and the common ac subtype of gallbladder cancers ( gbc ) . in this study , no significant differences in differentiation , metastasis , invasion , tnm stage , and prognosis were observed between these two subtypes . the clinicopathological presentations of sc / asc did not seem to be different from ordinary ac though more sc / asc patients exhibited larger tumor mass ( > 3  cm ) than ac patients . this suggested that squamous differentiation is as aggressive as glandular differentiation in the gallbladder , which is inconsistent with previous observations [ 31 , 32 ] . importantly , we found no differences in aldh1a3 and gpx3 expressions between sc / asc and ac patients , suggesting that they may have similar biological characteristics . numerous studies have observed an increase in oxidative stress during tumor growth , including gbc . therefore , the expression of aldh1a3 and gpx3 , two oxygen stress - related molecules , was investigated in this study . previous studies have demonstrated that gpx3 is downregulated in a variety of tumors , such as prostate cancer , thyroid cancer , colorectal cancer , gastric cancer , and breast cancer [ 15 , 16 , 18 ] . however , the predictive role of gpx3 in the prognosis of these cancer patients has not been observed . moreover , no molecular markers related to the aggressive biological characteristics or prognosis of sc / asc have currently been identified . in this study , we demonstrated that loss of gpx3 expression was significantly associated with metastasis , invasion , tnm stage , and poor prognosis in both sc / asc and ac patients . these observed correlations may possibly indicate that gpx3 is a tumor suppressor in gallbladder cancers . increases in reactive oxygen species levels have been associated with tumor cell proliferation , genetic instability , and subsequent increases in invasion , angiogenesis , and drug resistance [ 9 , 10 ] . although this study provided no evidence on whether the expression and/or activity of other antioxidant enzymes were decreased and whether the suppression of these enzymes plays a key role in the increased ros levels in gbcs , the observed decrease in gpx3 expression suggested its involvement in oxidative stress . aldehyde dehydrogenases are known to modulate several cell functions , including proliferation , differentiation , and survival , as well as cellular response to oxidative stress . in addition , cancer cell - acquired drug resistance has been demonstrated to be associated with the transcriptional activation of aldh1 expression . in contrast , aldh1a1 and aldh3a1 have been demonstrated to function as ocular antioxidants . in this study , positive aldh1a3 expression in ac patients was associated with poor differentiation , large tumor size , high tnm stage , lymph node metastasis , and invasion . in sc / asc patients , positive aldh1a3 expression was also associated with high tnm stage , invasion , and lymph node metastasis . this suggests that aldh1a3 in gbc plays a role in the proliferation , differentiation , and survival of tumor cells . in addition , both the ac and sc / asc patients with positive aldh1a3 expression exhibited poor survival . recently , aldh1 activity has been employed successfully as a stem cell marker in various cancers . in addition , aldh activity detected in tumor cells may actually be an indication of stem cells , because they are resistant to various chemotherapeutic drugs and linked to poor prognosis [ 2225 ] . we acknowledge that although positive aldh1a3 expression or negative gpx3 expression significantly correlated the shorter survival in both sc / asc and ac patients , their regression coefficients or relative risk showed that the impact of positive aldh1a3 expression or negative gpx3 expression on survival was small when compared with differences in tumor size , invasion , and differentiation . therefore , aldh1a3/gpx3 may be more effective as markers for proliferation and differentiation of gallbladder carcinoma than as tools for predicting overall survival . however , the real role of aldh1a3/gpx3 on tumor growth and metastasis of gallbladder cancer should be investigated further in animal models using inhibitory agents of aldh1 , such as sirna and/or overexpression of gpx3 gene . the data from this study also raised questions on why do sc / asc and ac share very similar gene expression profiles for aldh1a3 and gpx3 , and why the expression profiles of aldh1a3 and gpx3 , both molecules associated with oxidative stress , are closely related to tnm staging and metastasis . in conclusion , the lowered expression of gpx3 and elevated expression of aldh1a3 in ac and sc / asc subtypes of gbc are important biological markers for the metastasis , invasion , and maybe prognosis of gallbladder cancer .
background . 
 gallbladder cancers ( gbcs ) are highly aggressive cancers with high mortality . 
 however , biological markers for the progression and prognosis of gbc are currently unavailable in the clinic . 
 objective . to identify biomarkers for predicting gbc metastasis and prognosis 
 . methods . 
 we examined aldh1a3 and gpx3 expressions in 46 squamous cell / adenosquamous carcinomas ( sc / asc ) and 80 adenocarcinomas ( ac ) by using immunohistochemistry . results . 
 positive aldh1a3 and negative gpx3 expressions were significantly associated with lymph node metastasis and invasion of sc / ascs and acs . 
 univariate kaplan - meier analysis showed that either positive aldh1a3 ( p < 0.001 ) or negative gpx3 ( p < 0.001 ) expression significantly correlated with decreased overall survival in both sc / asc and ac patients . 
 multivariate cox regression analysis showed that positive aldh1a3 expression or negative gpx3 expression was an independent poor - prognostic predictor in both sc / asc and ac patients . 
 conclusions . 
 our study suggested that positive aldh1a3 and negative gpx3 expressions are closely associated with clinical pathological behaviors and poor prognosis of gallbladder cancer .