cancer cells are characterised by uncontrolled proliferation and accelerated cellular metabolism . as a consequence , in addition , a state where cancer cells inherently favour aerobic glycolysis rather than aerobic respiration has been widely described 1 . this results in an increased demand for atp because in glycolysis , atp production from glucose is considerably lower than that produced during oxidative phosphorylation in aerobic respiration . the first rate - limiting step for atp production through glucose metabolism is the transport of glucose across the plasma membrane of the cell via the facilitative sugar transporters ( gluts ) . the glut / slc2a family of transporters is comprised by 14 isoforms , which are divided into 3 different classes according to their sequence homology 2 . the diverse glut isoforms are distributed within the body according to their distinct functional properties to allow the appropriate distribution of the whole - body glucose 2 . due to high glucose demand , most cancer cells overexpress one or more of the gluts . these gluts may or may not be present in the respective tissue of origin under non - cancer conditions . altered expression of gluts 1 - 5 has been reported in many tumours such as hepatic ( glut1 , -2 , -5 ) 3 - 5 , pancreatic ( glut1 ) 3 , breast ( glut1 , -2 , -4 ) 3 - 5 , lung ( glut5 ) 4 , digestive ( glut2 , -4 , -5 ) 4 , 6 , 7 , reproductive tract ( glut5 ) 4 and lymphomas ( glut5 ) 5 . glut1 expression , in particular , has been shown to have a close relationship with tumour development and with unfavourable prognosis 4 , 7 . glut1 and glut3 are also known to be up regulated in tumours and normal tissues by hypoxia 4 , 8 , 9 . it was cloned from the human breast adenocarcinoma cell line mcf-7 by its homology with glut4 10 . glut12 transports 2-deoxy - glucose glucose , fructose and galactose , and it has been proposed that could work as a proton coupled symporter 10 . glut12 protein is expressed in adipose tissue and skeletal muscle , where seems to work as an insulin sensitive transporter , and it is also found in small intestine and placenta 10 . in tumour tissues , glut12 has been found in human breast tumours 10 , human prostate carcinoma and the prostate adenocarcinoma cell line lncap 10 , oligodendrogliomas , oligoastrocytomas and astrocytomas 11 . interestingly , a recent study has shown that p53 transcription factor , a tumour glycolytic metabolism inhibitor , specifically represses glut12 and glut1 expression , suggesting that both transporters could be the main glucose transporters involved in the glycolytic metabolism characteristic of human cancer 12 . firstly , to determine the expression of glut12 in a range of tumour cell lines in which expression in the corresponding non - malignant tissue has not been reported . secondly , to investigate whether glut12 expression might , like glut1 , be induced under hypoxic conditions . human lung adenocarcinoma ( a549 ) , colorectal adenocarcinoma ( ht29 ) , paediatric rhabdomyosarcoma ( rh-36 ) , neuroblastoma ( nb - eb ) and breast adenocarcinoma ( mcf-7 ) cell lines were cultured in rpmi 1640 medium supplemented with 10% foetal bovine serum ( fbs ) . ht29 cells were seeded into t75 flasks , previously coated with agarose gel ( 0.5% w / v ) . cells were then cultured under normoxic conditions for approximately 10 days until cell spheroids were becoming visible to the naked eye . spheroids of approximately equal sizes were selected and cultured on for a further 10 days in a magnetic spinner flask in rpmi-1640 ( hepes modification ) supplemented with 10% fbs until fully formed . cell monolayers were harvested with trypsin / edta solution followed by centrifugation ( 2000 g , 4c , 10 minutes ) . cell pellets were washed with phosphate buffered saline ( pbs ) before fixation overnight in 4% formalin buffered saline ( 4c ) . after fixation , the cells and spheroids were processed through to wax and sectioned ( 5 m ) then mounted onto glass slides . for immunohistochemical detection of glut-12 , sections were de - waxed and rehydrated . endogenous peroxidase activity was inhibited with 3% h2o2 in tris - buffered saline ( tbs ) ( 20 minutes ) before antigen retrieval was carried out ( 10 mm citric acid , 0.5% tween-20 in tbs ( tbs - t ) ( ph6.0 ) at 100c for 20 minutes ) . sections were blocked with 5% bovine serum albumin ( bsa ) in tbs - t ( 20 minutes ) . sections were then incubated in rabbit anti - glut12 polyclonal antibody ( 1:200 ) , ( bs-2540r , bioss , woburn , ma , usa ) 13 or glut1 ( 1:100 ) ( gt12-a , adi , san antonio , tx , usa ) overnight at 4c or incubated in tbs - t as a negative control . sections were then washed in tbs - t before 2ab treatment ( anti - rabbit igg - hrp ( dako , carpinteria , usa ) room temperature for 1 hour ) . the sections were again washed then peroxidase activity was detected using 3-3-diaminobenzidine tetrahydrochloride before counter - staining with haematoxylin . human lung adenocarcinoma ( a549 ) , colorectal adenocarcinoma ( ht29 ) , paediatric rhabdomyosarcoma ( rh-36 ) , neuroblastoma ( nb - eb ) and breast adenocarcinoma ( mcf-7 ) cell lines were cultured in rpmi 1640 medium supplemented with 10% foetal bovine serum ( fbs ) . ht29 cells were seeded into t75 flasks , previously coated with agarose gel ( 0.5% w / v ) . cells were then cultured under normoxic conditions for approximately 10 days until cell spheroids were becoming visible to the naked eye . spheroids of approximately equal sizes were selected and cultured on for a further 10 days in a magnetic spinner flask in rpmi-1640 ( hepes modification ) supplemented with 10% fbs until fully formed . cell monolayers were harvested with trypsin / edta solution followed by centrifugation ( 2000 g , 4c , 10 minutes ) . cell pellets were washed with phosphate buffered saline ( pbs ) before fixation overnight in 4% formalin buffered saline ( 4c ) . after fixation , the cells and spheroids were processed through to wax and sectioned ( 5 m ) then mounted onto glass slides . for immunohistochemical detection of glut-12 , sections were de - waxed and rehydrated . endogenous peroxidase activity was inhibited with 3% h2o2 in tris - buffered saline ( tbs ) ( 20 minutes ) before antigen retrieval was carried out ( 10 mm citric acid , 0.5% tween-20 in tbs ( tbs - t ) ( ph6.0 ) at 100c for 20 minutes ) . sections were blocked with 5% bovine serum albumin ( bsa ) in tbs - t ( 20 minutes ) . sections were then incubated in rabbit anti - glut12 polyclonal antibody ( 1:200 ) , ( bs-2540r , bioss , woburn , ma , usa ) 13 or glut1 ( 1:100 ) ( gt12-a , adi , san antonio , tx , usa ) overnight at 4c or incubated in tbs - t as a negative control . sections were then washed in tbs - t before 2ab treatment ( anti - rabbit igg - hrp ( dako , carpinteria , usa ) room temperature for 1 hour ) . the sections were again washed then peroxidase activity was detected using 3-3-diaminobenzidine tetrahydrochloride before counter - staining with haematoxylin . in non - malignant tissue , glut12 protein is detectable under normal physiological conditions in human skeletal muscle , adipose tissue and small intestine , but not in brain , liver or kidney . glut12 mrna has been found in the myocardium , skeletal muscle and prostate , and at low levels in the brain and kidney , but glut12 mrna has not been detected in the lung or colon 14 . rogers et al ( 2002 ) also reported breast cancer cell line mcf-7 to be positive for glut12 . our work confirms this and the specificity of the antibody by the intense glut12 cytoplasmic and nuclear staining in the mcf-7 cell pellet sections ( figure 1 ) . interestingly , expression of glut12 mrna was detected in different subtypes of human renal tumours 15 , human colon carcinoma ( hct116 ) , osteosarcoma ( u20s and saos-2 ) and non - small cell lung carcinoma ( h1299 ) cell lines 12 . based on these data , we decided to investigate glut12 expression in a range of human cancer cell lines derived from tissues that have not been reported to express glut12 protein in their non - malignant state . figure 1 shows that glut12 was highly expressed in the lung adenocarcinoma ( a549 ) and paediatric rhabdomyosarcoma ( rh-36 ) cell lines , whereas its expression was relatively low in the colorectal adenocarcinoma ( ht29 ) cell line and undetectable in the neuroblastoma ( nb - eb ) cell line . altered protein expression has been widely observed in tumours and this has been demonstrated with other members of the glut family . for example , glut3 protein is expressed in lung , ovarian , and gastric cancers but not in the corresponding normal tissues 16 . similarly , glut5 expression has been reported in human breast cancer cells , but not in normal breast tissue 17 . glut12 is widely expressed in rat foetus , human placenta and in foetal human term membrane 18 - 20 , and foetal tissue , like tumour tissue , has high glucose requirements . the fact that glut12 is found in foetal tissues and in some cancer cell lines , but not in the corresponding normal tissues , suggests a specific role for glut12 in response to particular metabolic needs . ( 2011 ) that glut12 is a major glucose transporter involved in glycolytic metabolism of cancer cells . we did not detect glut12 expression in the neuroblastoma ( nb - eb ) cell line , indicating that glut12 may not necessarily be expressed in all cancer cell lines or tumours . since glut12 expression has been related to foetal tissues and the nb - eb cell line is derived from embryological cancer , glut12 expression could have been down regulated during tumour genesis . however , further work is necessary to address this question . cell spheroids are spherically symmetric aggregates of cells , which have been widely used as in vitro model for the study of tumour response to therapy 21 . in the peripheral layers of the spheroid , cells are proliferating ; deeper cells are quiescent and characterised by hypoxia , low glucose and atp production , lactate accumulation , and induction of cellular apoptosis / necrosis 22 . according to our results , low expression of glut12 was found in ht29 cell monolayers ( figure 1 , q - t ) and high expression in the hypoxic centre of the ht29 spheroids ( figure 2a ) . interestingly , we also found that glut1 showed a similar pattern of expression in ht29 spheroids ( figure 2b ) . it might be hypothesised that increased glut12 expression , like glut1 , is involved in cancer cell glycolysis and inducible by hypoxia . the p53 transcription factor , a tumour glycolytic metabolism inhibitor , specifically represses glut12 and glut1 expression 12 . further , p53 expression has been found to be low in mcf-7 , a549 23 and rh-36 cell lines 24 , but high in neuroblastoma and ht-29 cell lines 23 , 25 . our results offer further evidence that in cell lines with low levels of p53 , there would be a correlation with high expression levels of glut12 and vice versa . this observation excepts ht-29 cells , which we found to express glut12 despite reports of high levels of p53 . however , this cell line , in contrast to the other cell lines analysed , expresses mutant p53 , which will be inactive and abolish any suppressive effect on glut-12 expression 23 . in summary , glut12 expression in certain tissues seems to be related to cancer development and hypoxia as well as to the characteristic glycolytic metabolism observed in malignant cells . this effect may be mediated via a p53-dependent mechanism , where down - regulation of glut-12 could be an important tumour suppressor . this warrants further studies to validate glut12 as a therapeutic target for novel anticancer treatment .
background : recent studies proposed glut12 to be a major glucose transporter involved in the glycolytic metabolism of cancer cells.methods : glut12 expression was determined by immunohistochemistry in a selection of cancer cell lines and a tumour spheroid model.results : glut12 expression was high in a549 and rh-36 ; low in ht29 ; and absent in nb - eb cancer cell lines . 
 glut12 expression was located in the necrotic centre of ht29 spheroids , which is characterised by anaerobic metabolism.conclusion : the data supports the involvement of glut12 in the glycolytic metabolism of cancer cells and therefore , its potential as a novel therapeutic target for cancer treatment .