    the main cause of treatment failure and death in cancer patients is metastasis  the formation of secondary tumors in organs distant from the original neoplastic cell tissue .
adjuvant therapy of proven efficacy is not currently available for cancer patients ; therefore , the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases . during metastasis ,
it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer .
the receptor for advanced glycation end - products ( rage ) , a multiligand member of the immunoglobulin superfamily of cell surface molecules , interacts with distinct molecules implicated in homeostasis , development , and inflammation .
rage binding by ligands such as advanced glycation end - products ( ages ) triggers the activation of key cell signaling pathways , thereby reprogramming cellular properties .
in addition , several reports have suggested that rage is associated with cancer malignancy [ 4 , 5 ] .     
the advanced stage of the glycation process ( one of the posttranslational modifications of proteins ) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients , aging , and neurodegenerative diseases [ 69 ] .
a growing body of evidence suggests that the interaction of glyceraldehyde - derived ages ( glycer - ages ) , but not glucose - derived ages ( glc - ages ) , with rage elicits oxidative stress generation in numerous types of cells ( vascular wall cells , mesangial cells , schwann cells , and cortical neurons ) , all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer 's disease [ 1013 ] .
we have recently found that glycer - ages stimulated the growth and migration of cultured human melanoma cells and that anti - rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice .
however , the effects of glycer - ages on other cancer cells remains poorly understood , and the molecular mechanisms behind their effects have not been clarified .      in the present study , we examined the effects of glycer - ages on cultured human lung adenocarcinoma a549 cells and showed that glycer - ages enhanced their malignancy rather than their proliferation .
n - acetyl - l - cysteine ( nac ) was purchased from sigma - aldrich ( st . louis , mo , usa ) .
briefly , 25  mg / ml of bsa ( a0281 , sigma - aldrich ) were incubated at 37c for 7 days under sterile conditions with 0.1  m glyceraldehyde ( ga ; nakalai tesque , kyoto , japan ) and 5  mm diethylenetriamine - pentaacetic acid ( dojindo , kumamoto , japan ) in 0.2  m phosphate buffer ( ph 7.4 ) .
the modified albumin was then purified by pd-10 column ( ge healthcare , buckinghamshire , england ) chromatography and dialysis against phosphate - buffered saline ( pbs ) .
control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control .
protein concentrations were determined with the dc protein assay reagent ( bio - rad , richmond , ca , usa ) using bsa as a standard .
the preparations were tested for endotoxin using the pyrotell - t test ( seikagaku bio - business , tokyo , japan ) , but no endotoxin was detected .
human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco 's modified eagle 's medium ( dmem ; sigma - aldrich ) supplemented with 10% fetal bovine serum ( fbs ; equitech - bio , kerrville , tx , usa ) under standard cell culture conditions ( humidified atmosphere , 5% co2 , 37c ) .
yamagishi , and its mock vector - transfected hep3b cells were maintained in 10% fbs / dmem in the presence of 700  g / ml g418 ( roche , mannheim , germany ) .
the cells were washed with ice - cold ca and mg free pbs ( pbs ( - ) ) and subjected to lysis buffer ( 1% tritonx-100/nonidet p-40 , 10  mm sodium fluoride , 1  mm sodium orthovanadate , 5  mm sodium pyrophosphate , 2  mm egta , 5  mm edta , and 1 protease inhibitor cocktail ( complete , mini ; roche ) ) .
subsequently , the cell lysates were incubated on ice for 5 min and centrifuged at 10,000  g for 10  min at 4c .
their protein concentrations were then measured using the bradford assay ( bio - rad ) .
cell lysates ( 30  g of proteins / lane ) dissolved in sds sample buffer ( 62.5  mm tris - hcl ( ph 6.8 ) , 2% sds , 10% glycerol , and 0.01% bromophenol blue ) containing 5% 2-mercaptoethanol ( 2-me ) were boiled for 3 min at 95c , separated by sds - page , and then electrotransferred onto pvdf membranes ( millipore , billerica , ma , usa )
. biotinylated markers ( cell signaling , beverly , ma , usa ) were used as molecular weight markers .
the membranes were blocked for 1  h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate ( pbs - t ) . after being washed twice with pbs - t
, the membranes were incubated with goat anti - rage antibody ( n-16 ) or mouse anti--actin antibody ( santa cruz , santa cruz , ca , usa ) for 1.5  h. subsequently , the membranes were washed twice with pbs - t and incubated with anti - goat igg antibody ( santa cruz ) or anti - mouse ig 's antibody ( biosource , camarillo , ca , usa ) and anti - biotin antibody ( cell signaling ) for 1  h. after being washed a further two times with pbs - t , the immunoreactive proteins were detected with ecl plus western blotting detection reagents ( ge healthcare ) using a luminescent image analyzer ( las-1000uvmini ; fujifilm , tokyo , japan ) .
the density of the bands was analyzed using a multi gauge version 3.0 ( fujifilm ) .
the cells ( 710  cells / cm ) were seeded in various plates or culture dishes ( bd biosciences , franklin lakes , nj , usa ) and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in 0.1% fbs / dmem for 48 and 72  h , and cell viability was determined by the wst-1 assay . after removing the medium , 100  l / well of 10% fbs / dmem and 10  l / well of wst-1 solution ( 5  mm wst-1 , 0.2  mm 1-methoxy-5-methylphenazinium , and 20  mm hepes buffer ( ph 7.4 ) ) ( dojindo ) were added , and the cells were incubated for 2  h. absorbance was measured at 450  nm and 650  nm using a microplate reader ( labsystems multiskan ascent , model no .
354 ; thermo fisher scientific , kanagawa , japan ) , and the net difference ( a450  a650 ) was used to express cell viability .
the migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers ( bd biosciences ) , respectively . in both assays ,
the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8  m .
ten percent fbs was used as a chemoattractant in the lower chamber compartments . before starting the invasion assay ,
the matrigel matrix of the chambers was reconstituted by adding serum - free dmem for 2  h. in the migration assay , 1    10 cells in serum - free dmem with control unglycated bsa or glycer - ages ( 100  g / ml ) were added into each of the upper chambers for 20  h. in the invasion assay , 2    10 cells in serum - free dmem with control unglycated bsa or glycer - ages were added to each of the upper chambers for 48  h. then , the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs .
the cells that migrated to or invaded the opposite side of the chamber were counted .
the active form of rac1 was detected with the ez - detect rac1 activation kit ( pierce , rockford , il , usa ) .
this assay uses the ability of a glutathione s - transferase ( gst ) fusion protein corresponding to the p21-binding domain ( pbd ) of human p21 activated protein kinase 1 ( pak1 ) to specifically bind to the active gtp - bound and not the inactive gdp - bound forms of rac1 and cdc42 .
cell lysates were incubated with gst - pak1-pbd and swellgel immobilized glutathione discs for 1  h at 4c , leaving an aliquot for measuring the levels of total rac1 .
the bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5  min at 95c and characterized by western blotting using monoclonal anti - rac1 antibody .
rac1 activity was determined by densitometric quantification of the pulled - down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates .
total rna was isolated from the cells with isogen ( nippon gene , tokyo , japan ) , and 50  ng of rna were reverse transcribed into cdna with primescript rt reagent kit ( takara , shiga , japan ) using a geneamp pcr system 9700 ( perkin - elmer applied biosystems , foster city , ca , usa ) .
real - time pcr was performed with sybr premix ex taq using a smart cycler ii system ( takara ) .
the reaction mixture ( 25  l ) contained 1 sybr premix ex taq , 0.2  m pcr forward primers , 0.2  m pcr reverse primers , and 10  ng of cdna as a template .
the primers used were as follows : tgf- : 5- gcg tgc taa tgg tgg aaa cc   -3 and 5- cgg agc tct gat gtg ttg aag a -3 , mmp-2 : 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3 , and -actin : 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer 's instructions .
-actin was used as an endogenous control gene in order to normalize target gene expression values .
the cells were seeded and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in serum - free dmem for 48  h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system ( ge healthcare ) .
all experiments were performed in duplicate and repeated at least two to three times , with each experiment yielding essentially identical results .
the significance of differences between group means was determined by one - way analysis of variance .
to investigate whether rage proteins are present in human lung adenocarcinoma a549 cells , we carried out western blot analysis using anti - rage antibody ( n-16 ) .
rage proteins of different molecular weights were detected in a549 cells ( figure 1 ) . in full length rage
cdna - transfected human hepatocellular carcinoma hep3b cells , the major band ( 57  kda ) ( indicated by an arrow in figure 1 ) represents the full - length rage protein .
likewise , the full - length rage protein was also detected in a549 and mock transfected hep3b cells .
no bands were detected in a neutralization experiment using blocking peptide ( data not shown ) so the other bands may represent deglycosylated rage proteins .
we examined the effect of glycer - ages on the cell viability of a549 cells .
cell viability was determined after 48  h incubation with control unglycated bsa or glycer - ages .
twenty , 50 , and 100  g / ml of glycer - ages significantly decreased cell viability to 83 , 64 , and 54% , respectively , ( figure 2(a ) ) .
in contrast , control unglycated bsa had no effect . furthermore , glycer - ages significantly attenuated cell proliferation ( figure 2(b ) ) ; however , no cell death was induced by glycer - ages according to cell death detection elisa ( data not shown ) .
we examined the effect of glycer - ages on cell migration and invasion . in the migration assay ,
the glycer - ages - treated cells showed significantly ( 2.7 times higher ) greater migration than the control unglycated bsa - treated cells ( figure 3(a ) ) , and in the invasion assay , the glycer - ages - treated cells showed significantly ( 3.3 times higher ) greater invasion than the control unglycated bsa - treated cells ( figure 3(b ) ) . when glycer - ages were added to the lower chamber
, there was no effect on the results of the invasion assay ( data not shown ) .
these results suggest that glycer - ages - rage interaction is necessary for cell migration and invasion .
rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility .
interestingly , rage signaling has been shown to result in the activation of rac1 [ 1720 ] , and the activation of rage by ages has been shown to induce the generation of reactive oxygen species ( ros ) .
the glycer - ages - induced rac1 activity was about 2 times higher than that of the control unglycated bsa , and it was suppressed by pretreatment with n - acetyl - l - cysteine ( nac ) , an ros scavenger ( figure 4(a ) ) .
likewise , the glycer - ages - enhanced migration capacity was also significantly suppressed by pre - treatment with nac ( figure 4(b ) ) .
thus , glycer - ages - rage interaction enhances migration capacity by activating rac1 via ros generation .
members of the matrix metalloproteinase ( mmp ) family are widely accepted to be key regulators of tumor invasion [ 2224 ] .
in particular , mmp-2 and -9 , which are called gelatinases , are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis .
we examined the expression of transforming growth factor- ( tgf- ) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system .
neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer - ages at 24  h ( figures 5(a ) and 5(b ) ) .
pro - mmp-2 activity was also not significantly altered by the addition of glycer - ages at 48  h ( figure 5(c ) )
. however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% ( figure 5(d ) ) .
    rage , which is a multiligand receptor , affects diseases such as cancer and diabetes through its ligands .
several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [ 26 , 27 ] .
ages have been implicated in the development and progression of diabetic angiopathies , including skin dermopathy .
however , it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells .
we have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages - modified proteins and peptides that circulate in the sera of diabetic patients .
recently , we found that glycer - ages perform a diverse range of biological activities on vascular wall cells , mesangial cells , schwann cells , and cortical neurons [ 1013 ] .
we also found that glycer - ages , but not glucose - derived ages ( glc - ages ) , significantly stimulated the growth and migration of human melanoma cells .
furthermore , the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti - rage antibody . in tumor bearing - mice ,
survival rates were prolonged , and spontaneous pulmonary metastases were inhibited by treatment using anti - rage antibody . in addition , glycer - ages were present in the beds of human melanoma tumors , whereas they were hardly detected in normal skin .
these results suggest that glycer - ages are involved in the growth and invasion of malignant melanoma through their interactions with rage .
recently , we found that glycer - ages have the strongest binding affinity for rage [ 28 , 29 ] and subsequently elicit oxidative stress generation and evoke vascular inflammation , thereby implicating them in accelerated atherosclerosis in diabetes [ 10 , 11 ] .
taken together , glycer - ages - rage interactions play an important role in the progression of melanoma cells to malignancy .
however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind these effects have not been clarified .   in the present study
rage includes full - length , c - truncated , and n - truncated rage . the rage antibody ( n-16 ) used in this study , which recognizes the n - terminus of rage , detects full - length and c - truncated rage .
indeed , full - length rage ( 57  kda ) was detected in a549 cells .
the other bands may have represented the de - glycosylated form or c - truncated rage .
glycer - ages are associated with cell cytotoxicity , but glc - ages are not [ 21 , 31 , 32 ] . in mesangial cells , intracellular ros produced by glycer - ages were found to induce apoptotic cell death . however , no a549 cell death induced by glycer - ages was observed in this study .
it is thought that the cytotoxicity of glycer - ages depends on the sensitivity of their target cells to ros .
furthermore , glycer - ages enhanced the migration and invasion activity of the a549 cells , both of which are prominent features of cancer malignancy .
glycer - ages induced the invasion of a549 cells across matrigel , indicating that matrix degradation and migration mechanisms had been stimulated in these cells . at the molecular level ,
glycer - ages - induced phenomena resemble the effects of long - term oxidative stress or tgf-. both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [ 3335 ] .
tgf- strongly induces mmp-2 expression in a549 cells ; however , mrna expression of tgf- was not induced by glycer - ages , and the mrna levels of mmp-2 , which is produced by tgf- , did not change . in long - term oxidative stress , mori et al .
showed that intracellularly produced ros activated rac1   and enhanced the invasion capacity of tumor cells by activating mmp [ 33 , 3739 ] .
a recent report showed that sustained exposure of cells to h2o2 , but not one time exposure to h2o2 , increased pro - mmp-2 activation through the induction of membrane type 1-mmp ( mt1-mmp ) expression .
indeed , our results also showed that glycer - ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2 .
however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% .
it is suggested that the activation of other mmp such as mmp-13 may also participate in the above - mentioned changes , and future studies are necessary to clarify this matter . in conclusion
, we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation .
these results suggest that glycer - ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention .
 the receptor for advanced glycation end - products ( rages ) is associated with the malignancy of cancer . 
 a recent study has suggested that glyceraldehyde - derived ages ( glycer - ages ) enhanced the malignancy of melanoma cells , but glucose - derived ages did not . however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind the above - mentioned effect have not been clarified . the present paper aimed to examine the effect of glycer - ages on cultured lung cancer a549 cells . 
 rage was expressed in a549 cells . 
 glycer - ages significantly attenuated cell proliferation . 
 furthermore , glycer - ages enhanced the migration capacity of the cells by activating rac1 via ros and also increased their invasion capacity . 
 we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation . 
 these results suggest that glycer - ages play a critical role in the malignancy of cancer rather than its proliferation and are potential targets for therapeutic intervention . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion