tumor cells in the tumor mass interact with adjacent tumor cells through homotypic adherence molecules such as e - cadherin on epithelial tumor cells . it is widely known that the process of cancer metastasis is accompanied by changes in the adherence capacity of tumor cells . for instance , the loss in the capacity for homotypic adherence , which is caused by downregulation of e - cadherin , is often observed in highly metastatic tumor cells . loss of e - cadherin function is important in the acquisition of a more invasive phenotype to promote the dissemination of tumor cells from a tumor mass [ 1 , 2 ] . in contrast , loss of integrin expression , which weakens cell - matrix interactions , reportedly correlates with the metastatic capacity of breast cancer cells . additionally , it has been suggested that a reduction in the adherence capacity to the ecm induces formation of multicellular aggregates or spheroids of tumor cells , facilitating tumor cell dissemination [ 35 ] . the disseminated cell spheroids may cause emboli in blood vessels or lymph nodes [ 68 ] . although changes in the activities of e - cadherin and integrins in tumor cells are important for tumor metastasis , the factors governing adherence capacity remain unknown . neutrophils are thought to secrete a variety of factors , including proteases , cytotoxic factors , cytokines , and reactive oxygen species , that affect tumor growth and metastasis [ 12 , 13 ] . these factors can have both beneficial and harmful effects on the host . to determine whether neutrophils produce factor(s ) that alter(s ) tumor cell adherence , we previously examined the effect of the neutrophil lysate on the adherence capacity of mcf-7 mammary breast carcinoma cells . serine proteases , cathepsin g , and neutrophil elastase ( hereafter referred as to elastase ) were shown to induce homotypic cell - cell aggregation in vitro . the cell aggregates formed are e - cadherin - dependent three - dimensional ( 3d ) multicellular spheroids [ 14 , 15 ] . these observations indicate that neutrophil serine proteases affect tumor metastasis by modulating the adherence capacity of tumor cells . cathepsin g may attenuate binding between integrin and extracellular adhesion molecules such as fibronectin ( fn ) , resulting in e - cadherin - mediated homotypic cell - cell adhesion . cathepsin g is typically stored in the azurophilic granules of neutrophils and is released by a variety of stimuli , such as the presence of bacteria [ 16 , 17 ] . cathepsin g is not only involved in resistance against bacterial or fungal infection [ 1720 ] , but also has various physiological functions , such as induction of platelet activation [ 21 , 22 ] , induction of chemotaxis of leukocytes , activation of inflammatory cytokine release , induction of endothelium - dependent vascular relaxation , cardiomyocytes anoikis , and endothelial cell damage . we previously reported that the aggregation - inducing activity of cathepsin g against mcf-7 cells is dependent on enzyme activity , while the binding of cathepsin g to specific binding sites on cells is independent of enzyme activity . accordingly , we hypothesized that cathepsin g binds to a specific protein on mcf-7 cells and causes proteolytic cleavage either of the binding protein itself or putative adjacent proteins in order to signal a change in adherent capacity . cathepsin g is reportedly a ligand for protease - activated receptors ( pars ) [ 17 , 22 , 24 , 29 ] . pars are g - protein coupled receptors in which cleavage of tethered ligands of receptor molecules by specific proteases is required for par activation . however , our previous study showed that agonist peptides for each par did not induce mcf-7 aggregation , suggesting that this function of cathepsin g may involve molecules other than pars on the surface of mcf-7 cells . it is well known that cathepsin g and elastase degrade adhesion molecules of the ecm such as fn [ 3032 ] . serum contains adherent molecules such as fn and vitronectin ; the presence of fetal bovine serum ( fbs ) in the culture medium is indispensable for the adherence of mcf-7 cells to culture substrates . therefore , degradation of the adherence molecules by cathepsin g or elastase may reduce the adherence capacity of mcf-7 cells to adhesion molecules on culture substrates , inducing e - cadherin - dependent cell - cell adhesion , which is protease - resistant . in this report , we examined whether neutrophil proteases , specifically cathepsin g and elastase , induce mcf-7 cell aggregation by degrading adhesion molecules on culture substrates or if a cell - oriented mechanism exists . for this , we determined the adherence capacity of cells when adhesion molecules on the culture substrates were treated with proteases prior to cell seeding . cell aggregation occurred on the protease - treated substrate . to elucidate whether the aggregation observed on protease - treated substrates was solely due to the degradation of adhesion molecules on the culture substrates , protease - treated substrates were treated with phenylmethylsulfonyl fluoride ( pmsf ) or 1-antitrypsin , which are irreversible protease inhibitors , and cell aggregation was observed . for cathepsin g , the degree of cell adhesion was potently attenuated by treatment with protease inhibitors . among the proteases examined , the activity of cathepsin g induced mcf-7 cell aggregation , possibly involving a cell - oriented mechanism in addition to the degradation of adhesion molecules on the substrates . in contrast , induction of mcf-7 cell aggregation by elastase or other proteases , such as chymotrypsin and trypsin , appears to be dependent only on the degradation of adhesion molecules on culture substrates . cathepsin g purified from human neutrophils ( 95% purity ) was purchased from biocentrum ( krakw , poland ) . bovine pancreas chymotrypsin ( c7762 ) and bovine pancreas trypsin ( t1426 ) , 1-antitrypsin , pmsf , poly - l - lysine ( pll , mw ; 150300  kd ) , and fluorescein isothiocyanate ( fitc)-labeled pll ( pll - fitc , mwvis ; 68.3  kd , 0.0030.01  mol fitc per mol lysine monomer ) were from sigma ( st . louis , mo , usa ) . elastase purified from human neutrophils and chymostatin were from calbiochem ( san diego , ca ) . human fn ( fn , > 95% purity ) was from agc techno glass ( chiba , japan ) . rabbit anti - human fn polyclonal antibody ( ab1945 ) was obtained from millipore ( billerica , ma , usa ) and horseradish peroxidase- ( hrp ) conjugated secondary antibody was from santa cruz biotechnology ( santa cruz , ca , usa ) . the human breast cancer cell lines mcf-7 ( e - cadherin - positive ) and mda - mb-231 ( e - cadherin - negative ) were used . hiroshi kosano ( teikyo university , japan ) and mda - mb-231 cells were obtained from atcc ( manassas , va , usa ) . cells were maintained in rpmi 1640 medium supplemented with 10% heat - inactivated fetal bovine serum ( fbs ; mp biomedicals , solon , oh , usa ) and 80  g / ml kanamycin ( wako pure chemical , osaka , japan ) as described previously . cell motility was measured by chronological observation using a confocal laser microscope with a built - in co2 incubator fv10i ( olympus , tokyo , japan ) . cells were inoculated on a glass - bottomed dish ( agc techno glass ) in rpmi 1640 supplemented with 5% fbs on the day before the measurement . after changing the medium to the cathepsin g - containing rpmi 1640 supplemented with 1% bovine serum albumin ( bsa ) and nucred live 647 ( life technologies , carlsbad , ca , usa ) , which is a fluorescent probe used to visualize cell nuclei , cells were chronologically observed using confocal laser microscopy at 1-hour intervals . the incubator was maintained at 37c in a humidified atmosphere of 5% co2 in air . cell motilities were quantified by drawing lines to connect between the center positions of the nuclei of the cell over 2 hours . the lines for 10 randomly selected cells in a visual field were quantified using software imagej . to quantitatively assess the degree of cell aggregation or multicellular spheroid formation , we quantified the mcf-7 human breast cancer cells that were tightly attached to the culture plate after staining with crystal violet as previously described . briefly , mcf-7 cells ( 1  10 cells / well ) were seeded in 96-multiwell plates ( without precoating ; iwaki glass inc . , tokyo , japan ) and cultured in rpmi 1640 medium containing 5% fbs for 24  h. after adherent cells were washed with serum - free rpmi 1640 medium , the medium was replaced with rpmi 1640 medium containing 1% bsa ( 1% bsa - medium ) and cultivated with cathepsin g or other proteases , respectively . after cultivation for 24  h , the plate was vigorously tapped on paper towels 10 times to remove loosely attached cell aggregates and spheroids . remaining cells were stained with 0.1% crystal violet in phosphate - buffered saline ( pbs ) for 10  min . after extensive washing with tap water , the plate was dried , and residual crystal violet in the cells was lysed with 100  l of 0.5% sodium dodecyl sulfate ( sds ) . optical density at 595  nm ( od595 ) was measured , and the aggregation index was calculated as follows : 
 ( 1)aggregation  index  ( % )  = od595  without  sampleod595  with  test  samplesod595  without  sample  100 . although some of the calculated aggregation index values were slightly below zero , microscopic observation revealed that the cells were morphologically similar to nonaggregated control cells , and , therefore , the negative values are expressed as zero in the figures to avoid confusion . the effect of proteases against mda - mb-231 human breast cancer cells was also examined using the same assay . the effect of protease treatments on this cell type was expressed as the  detachment index  because cell aggregation did not occur . to prepare fbs - coated plates , 96-multiwell plastic plates were incubated in 5% fbs - containing rpmi 1640 medium at 37c overnight and were washed with serum - free medium before use . to prepare fn - coated plates , plates were treated with 10  g / ml fn in pbs at 4c overnight , blocked in 3% bsa / pbs for 1 hour , washed once with serum - free medium , and immediately used . for pll - coating , the multiwell plates were incubated with pll solution ( 0.01% , w / v in pbs ) at 37c for 1 hour , washed with sterile purified water three times , dried , and rewashed once with serum - free medium before use . to examine the effect of protease treatment on cell aggregation in fbs- , fn- , or pll - coated plates , proteases in 1% bsa - medium were added to the coated plates , and the plates were incubated at 37c for 24 hours . plates were then washed once with serum - free medium and mcf-7 cells ( 1  10 cells / well ) were seeded . after 24 hours of culture , the aggregation index was evaluated as described above . in some experiments , to inhibit proteases bound to the wells , the irreversible protease inhibitors pmsf or 1-antitrypsin were used . briefly , 1% bsa - medium solution containing 4  mm pmsf or 200  g / ml 1-antitrypsin was added to protease - treated plates , which were incubated at 37c for 1 hour . for pmsf , 1% dimethyl sulfoxide ( dmso ) in 1% bsa - medium was used as a vehicle control . after the treatment with protease inhibitors , the plates were washed once with serum - free medium before adding the cell suspension . the enzymatic activity of cathepsin g bound to the culture plates was measured using n - succinyl - ala - ala - pro - phe p - nitroanilide ( sigma ) as a substrate ; 200  l of the substrate ( 1.2  mg / ml ) in hepes buffer ( ph 7.5 ) containing 10% dmso was added to the wells and incubated at 37c for 90  min . after incubation , liberated p - nitroanilide was evaluated by measuring changes in the optical density at 405  nm ( od405 ) . to measure elastase in the wells , elastase substrate i ( calbiochem ) was used as a substrate , and the levels of liberated p - nitroanilide were measured after 10  min . the amount of fn that remained adhered to the culture wells after protease treatment was analyzed by western blotting . each protease - treated culture well was washed once with pbs , after which 20  l laemmli 's sample buffer containing 2-mercaptoethanol was added . after extensive scraping of the wells with micropipette tips , the solutions from three wells were pooled , and the samples were separated by sds - page . western blotting was performed using an anti - fn rabbit polyclonal antibody and an hrp - labeled secondary antibody as previously described . pll - fitc ( 100  g / ml in pbs ) was mixed with each protease , and the 1  ml reaction mixtures were incubated at 37c for 4 hours . next , the solutions were applied to a millipore ultrafiltration system ( membrane cut - off of 10  kda ) and the fractions ( < 10,000  fr . ) that passed through the ultrafiltration unit were diluted 10-fold with pbs . each 200  l aliquot was added into a 96-well microplate ( nunclon delta surface , nunc , roskilde , denmark ) , and the fluorescence intensity of fitc was measured using a multimode plate reader ( 485  nm excitation wavelength , 535  nm emission wavelength ; arvo x3 , perkinelmer , waltham , ma , usa ) . for statistical analysis of the data data are expressed as the mean  standard deviation ( sd ) , unless otherwise indicated . we previously observed that when cathepsin g was added to adherent mcf-7 cells , the cells moved to contact each other and form cell aggregates , eventually forming 3d - sheroidal shapes when adherence to substrates is reduced . to characterize the early phase of this reaction , we first quantified the degree of cell movement when cells were cultured with cathepsin g.   figure 1(a ) shows that only in the presence of cathepsin g did the cells generally touch each other at 0.5 hours and maintain their cell - cell adhesions during subsequent culturing , finally forming spheroids . quantitative analysis was performed with mcf-7 cells cultured on a glass - based dish at lower concentration ( 0.25 or 1.25  nm ) of cathepsin g ( figures 1(b ) and 1(c ) ) . notably , mcf-7 cells cultured on a glass - based dish ( figure 1(b ) ) were more sensitive to cathepsin g than cells cultured on a plastic plate ( figure 1(a ) ) , presumably because adhesion properties are weaker on a glass - based dish . in fact , mcf-7 cells treated with 40  nm cathepsin g formed complete spheroids on a glass - based dish even after 1 hour ( data not shown ) , although the cathepsin g - treated cells completely formed spheroids on a plastic plate after 4 hours ( figure 1(a ) ) . the results showed that the degree of cell movement was significantly augmented by cathepsin g , indicating that cathepsin g actively induced spheroidal aggregate formation of mcf-7 cells by altering cell motility ( figures 1(b ) and 1(c ) ) . in contrast , elastase ( 0.2510  nm ) did not increase the motility of the cells ( see supplementary figure 1 in supplementary materials available online at http://dx.doi.org/10.1155/2014/971409 ) . cathepsin g shows more potent aggregation - inducing activity against mcf-7 cells than does elastase   or chymotrypsin . since cathepsin g has chymotrypsin - like and trypsin - like substrate specificities , we first compared the activity of cathepsin g with elastase , chymotrypsin , and trypsin . cathepsin g induced mcf-7 aggregation at low concentrations ; cathepsin g induced aggregation in a dose - dependent manner beginning at a concentration of 2.5  nm , while the aggregation - inducing activity of elastase was observed beginning at approximately 10  nm in an all - or - none manner ( figure 2(a ) ) . in contrast , higher concentrations ( greater than 40  nm ) of chymotrypsin and trypsin were required to induce aggregation . thus , among these proteases , cathepsin g was the most potent inducer of mcf-7 spheroidal aggregation . since aggregation induction of mcf-7 cells by cathepsin g is e - cadherin - dependent [ 14 , 15 ] , we next examined how these proteases affect the morphology of e - cadherin - negative mda - mb-231 cells . when left overnight in the presence of each protease , the cells lost the spindle morphology and detached easily during the washing step of crystal violet staining ( figure 2(b ) ) . however , in contrast to the cadherin - positive mcf-7 cells , aggregation of mda - mb-231 cells was not induced by any of the proteases . the dose - response relationships of these proteases in inducing the detachment of mda - mb-231 cells were similar to those that induced aggregation of mcf-7 cells ; the effects of cathepsin g and elastase were observed beginning at concentrations of 2.5  nm and 10  nm , respectively , while the effects of chymotrypsin and trypsin were observed beginning at 40  nm ( figure 2(a ) ) . these findings suggest that these proteases affect interactions between cells and culture substrates and that these changes occur regardless of e - cadherin expression . evaluation of the adherence of mcf-7 cells to the culture substrate requires the presence of adhesion molecules in the substrates because the cells only adhere to plastic plates when fbs is in the medium . in the experiments shown in figure 2 , culture plates were not precoated with adhesion molecules before cell seeding ; it was thought that adhesion factors such as fn and vitronectin in fbs became bound to the plates during the preculture of mcf-7 cells , maintaining the adhesion of these cells . to understand the implication of adhesion molecule degradation by proteases and the reduction of substrate adherence of mcf-7 cells , we examined cell aggregation on fbs - coated wells that had been treated overnight with each protease prior to adding cells . as shown in figure 3(a ) , cell aggregation was observed with all protease - treated substrates . the effect of cathepsin g pretreatment was clear at lower concentration ranges compared to that of elastase , and chymotrypsin and trypsin required much higher concentrations to take effect than cathepsin g and elastase . thus , overall dose - response relationships showed similar trends as those shown in figure 2(a ) , which reflect results obtained when the proteases were added to adhered mcf-7 cells . because fbs contains multiple adhesion molecules , including fn and vitronectin , as well as inhibitory factors such as 1-antitrypsin and 1-antichymotrypsin against serine proteases , we performed similar experiments using human fn rather than fbs to coat the culture substrates . figure 3(b ) shows that protease treatments of fn - coated plates induced aggregation of mcf-7 cells with a dose - response relationship similar to that observed for fbs - coated plates . cathepsin g morphologically induced cell aggregation even at 0.32~0.63  ng / ml ( not shown ) , although the deviation of the aggregation index at 0.63  ng / ml was very high . the effect of elastase on fn - coated plates was observed at lower concentration ranges than that on fbs - coated plates . the reason for this is unclear , but elastase might exert more potent effect in fn - coated plates due to the lack of the fbs - derived protease inhibitors . to examine whether the extent of cell aggregation on protease - treated fn was correlated with the amounts of fn remaining in the culture after treatment , residual fn as shown in figure 4 , cathepsin g and elastase digested fn ( molecular weight under reduced conditions was 220  kd ) much more effectively than trypsin and chymotrypsin ; a reduction in residual fn was observed from 2.5  nm cathepsin g and elastase and from 20  nm of chymotrypsin and trypsin . at lower concentrations of cathepsin g ( 0.63 and 1.25  nm ) , some fn remained . however , cell aggregation was induced by cathepsin g at 1.25  nm and below ( figure 3(b ) ) , indicating that cathepsin g induces cell aggregation through an additional mechanism other than substrate adhesion molecule degradation . in contrast , elastase caused potent decomposition of fn at about 2  nm , and it was entirely decomposed at approximately 5  nm . since the potent cell aggregation induced by elastase at approximately 2  nm plateaued when concentrations reached 5  nm , the increase in the aggregation index was correlated with the degree of fn degradation . for trypsin and chymotrypsin , cell aggregation was observed at around 40  nm ( figure 3(b ) ) ; at this concentration , fn was almost entirely decomposed . at 20  nm of concentration for both enzymes , cell aggregation was either weakly induced or not induced at all ( figure 3(b ) ) , although extensive fn decomposition was observed . it is likely that the residual fn fragment may have been able to sustain cell adherence , although further experiments are necessary to determine the validity of this hypothesis . to determine whether cathepsin g or elastase induces cell aggregation when being bound to culture substrates , we next measured the enzymatic activities remaining in the culture substrates after fn - coated plates were treated with proteases and washed out . supplementary figure 2 shows that approximately one - third of the enzymatic activities of the added cathepsin g and elastase remained . therefore , cathepsin g or elastase bound to the bottom of the wells may induce cell aggregation through their enzymatic activity . consequently , we examined whether cell aggregation on the cathepsin g- or elastase - treated fn - coated wells is attenuated by treatment with pmsf against substrate - bound enzymes . if the cell aggregation was induced by adhesion molecule digestion on the culture substrates by the proteases , aggregation intensity should not be influenced by pmsf treatment . the enzymatic activities of cathepsin g and elastase in the bottom of the plate wells were nearly completely or extensively inhibited by pmsf treatment ( supplementary figure 2 ) , suggesting that pmsf is effective against proteases bound to the culture substrates . as shown in figures 5(b ) and 5(c ) , the cell aggregation - inducing effect of 2.5 and 5  nm cathepsin g treatments against fn - precoated plates were markedly attenuated by subsequent treatment with pmsf , while the aggregation caused by 10  nm treatment of cathepsin g was suppressed by pmsf to a lesser degree . in contrast , the aggregation - inhibiting effects of elastase ( 2.28.6  nm ) were not disturbed by pmsf . these results suggest that cathepsin g induces cell aggregation by not only degrading adhesion molecules , but also through putative cell - oriented mechanisms , and that at the higher concentration ( 10  nm ) , extensive decomposition of fn on the substrates is the primary cause of aggregation . similar results were obtained using fbs - coated plates rather than fn - precoated plates ( supplementary figure 3 ) ; the cell aggregation - inducing activity of treatment with 0.63  nm cathepsin g against fbs - precoated plates was completely suppressed by subsequent treatment with pmsf , whereas those of 2.5 and 10  nm cathepsin g were only weakly reduced . similar results were also obtained using 1-antitrypsin rather than pmsf as an irreversible protease inhibitor ( data not shown ) . these results support the notion that cathepsin g induces cell aggregation through a cell - oriented mechanism that is dependent on enzymatic activity . however , at higher concentrations , the degradation effect of cathepsin g against fn may mask cell - orientation activity . to further support that mcf-7 cell aggregation by cathepsin g is not solely caused by the degradation of adhesion molecules , we next examined the effect of the proteases on poly - l - lysine- ( pll- ) coated plates because pll was not expected to be a good substrate for cathepsin g. to measure the hydrolytic activity of each protease towards pll , we mixed pll - fitc with each protease for 37c for 1 hour and then measured fitc fluorescence in fractions that had been ultrafiltered ( 10  kd cut - off ) . supplementary figure 4 shows that cathepsin g and elastase treatments did not augment fluorescence in fractions < 10  kd , whereas chymotrypsin and trypsin treatment revealed small and large increases , respectively . the fluorescence intensity of degraded pll - fitc may be higher than that of the intact molecule because fluorescence of the < 10  kd fraction of the trypsin - treated pll - fitc was higher than that of the untreated control . these results indicate that , in contrast to chymotrypsin and trypsin , cathepsin g and elastase show no cleavage activity towards pll . additionally , we examined cell aggregation in pll - coated plates that had been treated with cathepsin g and elastase . figure 6(a ) shows that cathepsin g treatment against the pll substrate induced mcf-7 cell aggregation , whereas elastase pretreatment did not induce aggregation . we confirmed that the amounts of cathepsin g bound to the bottoms of the pll - precoated plates were similar to those bound to fn - coated plates and that the activity of substrate - bound cathepsin g was nearly completely inhibited by pmsf treatment ( data not shown ) . figure 6(b ) shows that cell aggregation was not observed when substrate - bound cathepsin g was inhibited by pmsf treatment prior to cell seeding . these data support that cathepsin g , but not elastase , induces cell aggregation through an enzyme activity - dependent , cell - oriented mechanism . in this study , we found that cathepsin g induces the formation of multicellular aggregates in low metastatic and e - cadherin - positive mcf-7 cells , while reducing the adherent capacity of highly metastatic and e - cadherin - negative mda mb-231 cells to culture substrates . because the dose - response curves of cathepsin g inducing changes in the adherence capacity of mcf-7 and mda mb-231 cells are very similar , cathepsin g may have a common molecular mechanism in the induction of reduced cell - substrate interactions among these two cell types . we previously reported that cathepsin g - induced cell aggregation is e - cadherin - dependent [ 14 , 15 ] . this hypothesis was supported in the current study because cathepsin g did not induce aggregation of e - cadherin - negative mda mb-231 cells . two mechanisms have been proposed for the dissemination mechanism of tumor cells from the tumor mass . first , cancer cells gain the capacity to infiltrate the ecm by augmentation of the integrin - mediated adhesive activity to adhesion molecules in the ecm while also increasing the expression of metalloproteinases . such highly metastatic cells reportedly lose cadherin - dependent adhesive activity between tumor cells [ 1 , 2 ] . second , it was proposed that tumor cells lose the capacity to adhere to the ecm [ 35 ] and that they form homotypic aggregates , disseminate into small vessels , and become trapped as tumor emboli [ 68 ] . cathepsin g secreted by neutrophils infiltrating intratumor environments is a candidate factor that influences the metastatic capacity of tumor cells . cathepsin g may act on e - cadherin - positive tumor cells to induce detachment from the ecm to facilitate the dissemination of tumor cell aggregates . in contrast , cathepsin g - caused suppression of the adhesion capacity between highly metastatic e - cadherin - negative tumor cells and the ecm may inhibit metastasis . we observed that in the presence of cathepsin g , mcf-7 cells move to make contact with adjacent cells . the quantification assay of cell motility showed that cathepsin g augments the motility of mcf-7 cells , suggesting that cell aggregation is not passively caused by the loss of adhesion to the substrates , but is caused by increased cell motility . in addition to mcf-7 cells , a comparison to other proteases of the effect of cathepsin g on the motility of other tumor cells , including highly metastatic mda mb-231 cells , should be further examined . on the other hand , elastase did not increase the motility , suggesting that the reaction mode of cathepsin g in induction of cellular spheroids is different from that of elastase . the aim of the present study was to determine whether the induction of cell aggregation of cathepsin g and other proteases is cell - oriented or is the result of adhesion molecule degradation on the culture substrates . we first examined the effect of protease treatment on a culture substrate precoated with fbs or fn . protease treatments against fbs - coated plates induced cell aggregation of mcf-7 cells with a similar overall dose - response relationship as the results obtained when the proteases were added to adherent cells . cathepsin g induced aggregation at the lowest concentration ranges in both assay systems , elastase exerted its effect at higher concentrations than cathepsin g , and chymotrypsin and trypsin only induced the aggregation at much higher concentrations . a similar order of the dose - response relationship was observed when fn - coated plates were treated with proteases . since 10 neutrophils reportedly secrete approximately 1  g of cathepsin g   and the concentration of cathepsin g that induces the cell aggregation is less than 40  nm ( 1  g / ml ) , the concentrations are physiologically relevant in environments containing relatively low amounts of inhibitory factors , such as serpines . to understand whether the activity of cathepsin g and other proteases depends only on the digestion of adhesion molecules on the culture substrate , we used western blotting analysis to first estimate fn decomposition after cathepsin g treatment of fn - coated plates . in contrast to other proteases examined , cathepsin g induced cell aggregation at low concentrations , at which a substantial amount of fn remained . therefore , cathepsin g may exert its effect through a mechanism other than digestion of adhesion molecules . this hypothesis was supported by the observation that pmsf attenuated the degree of cell aggregation on cathepsin g - treated fn plates . the inhibitory activity of pmsf against aggregation was clearly observed at relatively low concentrations ( 2.55  nm ) of cathepsin g. in contrast , the effect of pmsf against 10  nm cathepsin g - treated fn plates was very weak , suggesting that cathepsin g - induced degradation of fn induces cell aggregation at higher concentrations . accordingly , cathepsin g bound to the culture substrates may exert cell - oriented cell aggregation - inducing activity ; however , the cell - oriented activity may be masked by the degradation effect of cathepsin g against fn at higher concentrations of cathepsin g. in contrast to cathepsin g , the activities of elastase , chymotrypsin , and trypsin appeared to depend only on adhesion molecule degradation on the culture substrates because pmsf did not suppress the induction of cell aggregation in fn- or fbs - coated plates treated with any of these enzymes . treatment with cathepsin g , but not elastase , induced cell aggregation on pll substrates , although cathepsin g did not decompose pll . moreover , the activity of cathepsin g on pll was nearly completely inhibited by subsequent pmsf treatment . these results strongly support that cathepsin g affects the character of cell adherence of mcf-7 through a possible cell - oriented mechanism , although this mechanism remains unknown . we previously reported that cathepsin g binds to a specific binding site on mcf-7 cells . recently , zen et al . reported that the neutrophil serine proteases cathepsin g , elastase , and proteinase-3 cleave 2 integrin cd11b / cd18 to inhibit neutrophil chemotaxis , although another report indicated that cathepsin g modulates integrin cd11b clustering without proteolytical procession of integrin molecules . one possibility is that cathepsin g alters the character of adherence by cleaving the integrins of tumor cells . the results of this study are that cathepsin g induced cell aggregation not only on fn - coated substrates but also on pll - coated surfaces and cell adhesion to pll is not dependent on integrins . because cathepsin g loses extended mcf-7 morphology , it is thought that cathepsin g decreases focal adhesion of the cells . we are planning to examine whether cathepsin g modulates formation of focal adhesion complex . in summary , in contrast to elastase , cathepsin g may induce cell aggregation of mcf-7 cells through a cell - oriented mechanism in addition to degrading substrate adhesion molecules . further studies are needed to identify the binding molecule(s ) of cathepsin g in cells to elucidate the mechanism underlining this nonoverlapping activity of cathepsin g - induced alterations in the adherence capacity of tumor cells . these results contribute to the understanding of tumor invasion and metastasis and the role of neutrophils .
we previously found that a neutrophil serine protease , cathepsin g , weakens adherence to culture substrates and induces e - cadherin - dependent aggregation of mcf-7 human breast cancer cells through its protease activity . in this study , we examined whether aggregation is caused by degradation of adhesion molecules on the culture substrates or through an unidentified mechanism . 
 we compared the effect of treatment with cathepsin g and other proteases , including neutrophil elastase against fibronectin- ( fn- ) coated substrates . 
 cathepsin g and elastase potently degraded fn on the substrates and induced aggregation of mcf-7 cells that had been subsequently seeded onto the substrate . 
 however , 
 substrate - bound cathepsin g and elastase may have caused cell aggregation . after inhibiting the proteases on the culture substrates using the irreversible inhibitor phenylmethylsulfonyl fluoride ( pmsf ) , we examined whether aggregation of mcf-7 cells was suppressed . 
 pmsf attenuated cell aggregation on cathepsin g - treated substrates , but the effect was weak in cells pretreated with high concentrations of cathepsin g. in contrast , pmsf did not suppress cell aggregation on elastase - treated fn . 
 moreover , cathepsin g , but not elastase , induced aggregation on poly - l - lysine substrates which are not decomposed by these enzymes , and the action of cathepsin g was nearly completely attenuated by pmsf . 
 these results suggest that cathepsin g induces mcf-7 aggregation through a cell - oriented mechanism .