chemotherapeutic drugs , 
 alone or as an adjuvant therapy to surgery 
 and radiation , are a vital part in cancer treatment .
however , their 
 effectiveness is often offset by severe side effects caused by poor 
 selectivity and toxicity to normal cells . in recent years
, the rapid 
 advance in the fields of bioinformatics and genomics facilitated the 
 identification of numerous target molecules that are uniquely or overly 
 expressed in cancer cells , including certain receptors and enzymes .
these discoveries were successfully utilized for 
 the specific delivery of chemotherapeutic drugs to tumor cells , with 
 the double goal of increasing their therapeutic efficacy while decreasing 
 their toxicity to normal cells .
the enzyme dipeptidyl peptidase iv ( dppiv ) ( ec 3.4.14.5 ) , 
 also 
 known as cd26 ( t - cell surface activation antigen ) or adenosine deaminase 
 ( ada ) , was first identified in 1966 .
dppiv 
 is a member of the prolyl oligopeptidase ( pop ) family of serine proteases and specifically hydrolyzes the peptide bond 
 c - terminal to proline or alanine in the penultimate p1 position .
it is normally present on the apical membrane 
 surface of several cell types , including t cells and b cells , natural killer ( nk ) cells , epithelial cells of 
 kidney , intestine brush border membranes , plasma , and endothelial cells of blood vessels , and is involved in many diverse physiological processes .
in addition , it was recently discovered that dppiv is overly expressed 
 in human renal cell carcinoma tissues , and it is thought to play a role in the pathogenesis of other various 
 human cancers as well .
moreover , proline - containing substrates are hydrolyzed 
 exclusively by proline - specific enzymes and are less susceptible to 
 nonspecific peptidases and proteases .
thus , a dppiv - cleavable 
 prodrug containing a proline dipeptide conjugated to a cytotoxic chemotherapeutic 
 agent may limit nonspecific activation .
the purpose of this study 
 was to investigate the feasibility of 
 exploiting dppiv as a prodrug activating enzyme , to allow specific 
 targeting of chemotherapeutic agents to cancer cells . to that end
, 
 the expression profiles of dppiv in 60 cancer cell lines ( nci 60 ) 
 were obtained , and 7 cell lines , which represent the spectrum of dppiv 
 expression , were selected .
a candidate prodrug was then synthesized 
 by linking the cytotoxic drug melphalan ( figure 1a ) to a proline dipeptide , creating glycyl - prolyl - melphalan prodrug 
 ( gp - mel ; figure 1b ) with expected dppiv affinity . 

functional activity studies of dppiv with the prodrug , in the absence 
 vs presence of inhibitor , confirmed that the prodrug is a specific 
 dppiv substrate .
finally , hydrolysis and cell proliferation studies 
 were performed in high and low dppiv expression cancer cell lines . 

overall , this work indicates that dppiv may be exploited as a prodrug 
 activating enzyme for efficient chemotherapeutic drug targeting .
chemical 
 structures of ( a ) melphalan and ( b ) its gly - pro dipeptide 
 prodrug , gp - mel .
melphalan , porcine kidney 
 dipeptidyl peptidase ( porcine dppiv ) , glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalanyl - p - nitroanilide ( gf - pna ) , glycyl - arginyl - p - nitroanilide ( gr - pna ) , p - nitroaniline ( pna ) , diprotin a ( ile - pro - ile ) , xtt , 
 pms ( n - methyldibenzopyrazine methyl sulfate ) , and 
 ladder sybr green were purchased from sigma chemical co. ( st . louis , 
 mo ) .
n , n - dimethylformamide ( dmf ) , 
 piperidine , triisopropylsilane ( tis ) , triethanolamine ( tea ) , and trifluoroacetic 
 acid ( tfa ) were purchased from aldrich chemical co. ( milwaukee , wi ) . 

boc - l - gly - l - pro , 1-hydroxybenzotriazole anhydrous 
 ( hobt ) , 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium 
 hexafluorophosphate ( hbtu ) , triisopropylsilane ( tis ) , n-(9-fluorenylmethoxycarbonyloxy)succinimide ( fmoc - osu ) , and wang 
 resin ( 100200 mesh ) were obtained from calbiochem - novabiochem 
 ( san diego , ca ) .
access rt - pcr kit , dna blue / orange loading dye , 1 
 kb ladder and 100 bp ladder were obtained from promega ( madison , wi ) . 

420% tbe gels , trizol reagent , and custom - ordered dppiv sense 
 and antisense primers were from invitrogen life technologies ( carlsbad , 
 ca ) .
the cancer cell lines igrovi ( ovarian ) , pc-3 ( prostate ) , 786 - 0 
 ( renal ) , sk - mel-5 ( melanoma ) , sk - ov-3 ( ovarian ) , caco-2 ( colon ) , and 
 hepg2 ( liver ) were obtained from national cancer institute or atcc . 

dulbecco s modified eagle s medium ( dmem ) , rpmi-1640 , 
 fetal bovine serum ( fbs ) , fetal calf serum ( fcs ) , phosphate buffered 
 saline ( pbs ) , and trypsin - edta were purchased from gibco brl ( grand 
 island , ny ) . minimal essential medium ( mem )
was obtained from atcc . 
 all hplc grade solvents ( acetonitrile , dmf , ethyl acetate , and piperidine ) 
 used for peptide synthesis or hplc analysis
trifluoroacetic acid for hplc analysis was 
 obtained from pierce ( rockville , il ) .
the solid - phase synthesis of the l - glycyl - l - prolyl prodrug of melphalan was carried out in a stepwise 
 fashion with wang resin as described below and is summarized in the 
 schematic shown in figure 2 .
fmoc - l - melphalan synthesis : 
 fmoc - osu ( 300 mg , 0.89 mmol ) was added to an ice - cold solution of 
 melphalan , 1 ( 200 mg , 0.82 mmol ) , in a mixture of acetone 
 ( 20 ml ) , distilled water ( 10 ml ) , and nahco3 ( 300 mg ) . 

the mixture was stirred for 1 h at 0 c and then reacted at room 
 temperature for 16 h. after the reaction was complete , the mixture 
 was concentrated .
15 ml of ethyl acetate and 15 ml of distilled water 
 were added . the aqueous phase was then extracted with ethyl acetate 
 ( 3  15 ml ) .
the combined organic phases were washed with distilled 
 water and brine and dried over mgso4 .
the filtrate was 
 concentrated under vacuum to yield fmoc - l - melphalan , 2 ( 330 mg , yield 98% ) .
fmoc - l - melphalan ( 330 
 mg ) and wang resin ( 0.1 mmol ) were added into a 100 ml round reaction 
 bottle with 20 ml of dmf .
hobt ( 0.3 mmol ) , hbtu ( 0.3 mmol ) , and tea 
 ( 0.3 mmol ) were then added to the resin mixture and stirred overnight 
 at room temperature .
the wang resin , 3 , was washed 3 
 times with 10 ml of dmf after filtration and then washed 3 times for 
 10 min each in 10 ml of 20% piperidine in dmf .
boc - l - glycyl - l - proline ( 0.3 mmol ) and wang resin 3 were added to 20 
 ml of dmf in a 100 ml round reaction bottle .
hobt ( 0.3 mmol ) , hbtu 
 ( 0.3 mmol ) , and tea ( 0.3 mmol ) were added and stirred overnight at 
 room temperature .
the resultant wang resin , 4 , was washed 
 3 times with 10 ml of dmf after filtration and then washed 3 times 
 for 10 min each in 10 ml of 20% piperidine in dmf .
wang resin 4 was then added to 10 ml of cleavage buffer ( tfa : tis : h2o , 95:2.5:2.5 ) and stirred for 1 h at room temperature . after 
 filtration , the solution was concentrated and cold ether ( 10 ml ) was 
 added to precipitate out the prodrug 5 . after removing 
 ether , the residues were reconstituted with 5 ml of distilled water 
 and lyophilized .
the purity of the melphalan prodrug 
 was determined by hplc to be 
 95% .
electrospray ionization mass spectra ( esi - ms ) were obtained on 
 a thermoquest lcq electrospray ionization mass spectrometer .
the observed 
 molecular weight of the prodrug was found to be consistent with that 
 required by its structure .
the structural identity of the prodrug 
 was then confirmed using proton nuclear magnetic resonance spectra 
 ( h nmr ) .
schematic of solid - phase synthesis of l - glycyl - l - prolyl - melphalan prodrug .
l - glycyl - l - prolyl - melphalan : 
 yield 13% ; percent 
 purity 95% ; h nmr ( dmso - d6 )  1.952.20 ( m , 4h , ch2 on proline ) , 3.053.35 
 ( m , 12h , ch2 ) , 4.444.80 ( m , 5h , ch , ch2 ) , 6.506.90 ( m , 4h , phenol h ) ; esi - ms 459.4 ( m + h ) .
the microarray data and the u95av2 affymetrix 
 data were downloaded from the dtp database ( https://wiki.nci.nih.gov/display/ncidtpdata/molecular+target+data ) .
the programming language perl was used to sift through more than 
 26,000 genes to find all enzymes with names that end with the suffix 
  ase  .
the enzyme text file was then used to separate 
 the enzymes into different classes , such as hydrolases , peptidases , 
 and esterases as described earlier .
visual basic 
 was then used to arrange the data retrieved so that the expression 
 of each enzyme gene in the 60 cancer cell lines could be easily visualized 
 using visual tools such as cluster and treeview programs .
the arranged 
 data from peptidases was normalized using a pool of 12 cell lines 
 ( nci - h226 , colo 205 , mcf-7 , hs 578 t , ovcar-3 , ovcar-4 , k-562 , hl-60 
 ( tb ) , caki-1 , lox imvi , pc-3 , snb-19 ) as reference and clustered according 
 to the origin of the cell lines .
the sorted expression data for the 
 60 cancer cell lines was clustered using hierarchical clustering , 
 and a file was created ( .cdt ) such that the expression of the genes 
 in 60 cancer cell lines could be visualized using treeview .
the potential 
 enzyme target , dppiv , was selected on the basis of the differential 
 expression in various cell lines or tissues with emphasis on high 
 expression in a particular tissue as compared to other tissues and 
 high substrate specificity of the enzyme .
candidate cancer cell 
 lines were selected from the nci 60 cell 
 lines based on the expression levels of dppiv , doubling time , and 
 growth requirements .
thus , the cancer cell lines selected , igrovi , 
 pc-3 , 786 - 0 , sk - ov-3 , and sk - mel-5 , represent cells with high , medium , 
 and low expression of dppiv , reasonably short doubling times , and 
 standard growth requirements .
in addition , caco-2 as well as hepg2 
 cell lines were also used in the studies .
total rna was lysed and purified 
 from each cancer cell line with 
 trizol reagent according to the manufacturer s protocol .
isolated 
 rna ( 0.5 g ) was reverse transcribed to cdna with amv reverse 
 transcriptase , oligo ( dt ) , and access rt - pcr kit following the standard 
 protocol .
each of the 20 cycles of amplification consisted of denaturation 
 ( 94 c for 25 s ) , annealing ( 50 c for 25 s ) , and extension 
 ( 68 c for 40 s ) .
the primer set used for dppiv amplification 
 was the sense primer ( 5-ccttctactctgatgagtcactgc-3 ) 
 and the antisense primer ( 5-gtgccactaagcagttccatcttc-3 ) .
pcr product was identified by electrophoresis 
 in 420% tbe gels followed by sybr green staining .
the gel 
 was then visualized with uv light , and the relative intensities of 
 the rt - pcr product bands were measured using metamorph software .
porcine kidney dppiv 
 was used in the studies due to its commercial availability and its 
 close alignment with human dppiv ( 88% homology ) .
stock solutions of porcine dppiv were prepared by reconstituting 
 lyophilized powder ( 0.87 unit , 23 units / mg ) in 1 ml of 0.1 m tris - hcl 
 ( ph 8.0 ) and stored at 80 c .
solutions of the various 
 substrates glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalaninyl - p - nitroanilide 
 ( gf - pna ) , or glycyl - argininyl - p - nitroanilide 
 ( gr - pna ) at 0.4 mm were mixed with appropriate amounts 
 of diluted dppiv solution ( final dppiv concentration in mixture , 0.473 
 g / ml ) and incubated at 37 c for 1530 min in flat - bottom 
 96-well plates .
the hydrolysis of the substrates 
 was monitored by measurement of absorbance at 405 nm ( generation of p - nitroanilide ) using a precision microplate reader ( emax , 
 molecular devices ) .
the competitive inhibition of hydrolysis of the 
 substrates by porcine kidney dppiv in the presence of the inhibitor 
 diprotin a was determined by including 0.4 mm diprotin a in the diluted 
 dppiv mixture prior to incubation .
initial hydrolysis rates were determined 
 in all hydrolysis experiments by assaying the amounts of pna released .
dppiv activity in the 
 selected cancer cells was determined using the standard substrate 
 gp - pna .
briefly , cells were grown to 90% confluence 
 in 150 mm culture plates , washed carefully with 0.15 m nacl , and collected 
 by scraping .
the cells were resuspended in 0.15 m nacl and then centrifuged 
 at 3,000 rpm for 5 min .
the cell pellet was resuspended in phosphate 
 buffered saline ( pbs , ph 7.4 ) , and sonicated for 5 s two times on 
 ice .
the sonicated cell suspension was centrifuged at 18000 g for 30 min at 4 c .
the supernatant was then used 
 in hydrolysis studies , and to determine protein content ( bio - rad dc 
 protein assay ) .
the protein content was adjusted to approximately 
 1000 g / ml by appropriate dilutions before use in hydrolysis 
 studies .
hydrolysis studies were conducted in a 96-well microplate 
 containing the cell homogenate suspensions at 37 c .
the reactions 
 were initiated by the addition of gp - pna ( final concentration 
 1 mm ) to the suspensions .
the absorbance was monitored every minute 
 for a total of 3060 min using a precision microplate reader 
 ( emax , molecular devices ) at 405 nm . in the competitive inhibition 
 studies , diprotin a at 1 mm was included along with 1 mm gp - pna , and the mixture was added to the cell homogenate suspensions 
 and absorbance monitored as described above .
dppiv activity was expressed 
 as the amount of pna ( micromoles ) released per minute , 
 normalized to the amount of protein .
hydrolysis of the melphalan 
 prodrug by porcine kidney dppiv and 
 the effect of the competitive inhibitor diprotin a were assayed using 
 96-well microplates .
porcine dppiv enzyme solution ( 230 l at 
 a final concentration 4.4 g / ml ) was placed in triplicate wells 
 maintained at 37 c .
the reactions were initiated by the addition 
 of gp - mel ( 1 mm final concentration in mixture ) , and 40 l aliquots 
 were removed at predetermined time points and added to two volumes 
 of 10% ice - cold tfa to quench the reaction and precipitate protein . 
 in the inhibition studies , diprotin a and gp - mel were both added ( 1 
 mm final concentration ) to the enzyme solution , incubated at 37 c , 
 and sampled as described above .
the quenched precipitated samples were then filtered through 
 a 0.45 m filter plate and centrifuged at 1800 g and 4 c for 20 min .
the extent of hydrolysis of gp - mel in caco-2 
 and sk - mel-5 cell homogenates was determined as follows .
caco-2 and 
 sk - mel-5 cells and cell homogenates were prepared as described earlier . 

the hydrolysis reactions were carried out in 96-well plates ( corning , 
 corning , ny ) .
230 l of the cell suspensions ( 1000 g / ml 
 protein ) were placed in triplicate wells and the reactions initiated 
 by the addition of gp - mel ( final concentration 1 mm in mixture ) and 
 incubated at 37 c . at predetermined time
points 40 l 
 aliquots were removed and added to two volumes of 10% ice - cold tfa 
 to quench the reaction and precipitate protein . in the inhibition 
 studies , diprotin a and gp - mel were both added ( 1 mm final concentration ) 
 to the cell suspensions , incubated at 37 c , and sampled as described 
 above .
the quenched 
 precipitated samples were then filtered through a 0.45 m filter 
 plate and centrifuged at 1800 g and 4 c for 
 20 min .
dppiv activity was expressed as the amount ( picomoles ) of melphalan 
 released per minute , normalized to the amount of protein .
the concentrations 
 of gp - mel and melphalan were determined on a waters hplc system ( waters 
 inc . ,
the hplc system consisted of two waters pumps 
 ( model 515 ) , a waters autosampler ( wisp model 712 ) , and a waters uv 
 detector ( 996 photodiode array detector ) . the system was controlled 
 by waters millennium 32 software ( version 3.0.1 ) .
samples were injected 
 onto a waters xterra c18 reversed phase column ( 5 m , 
 4.6  250 mm ) equipped with a guard column .
the flow rate was 
 1 ml / min , and the mobile phase was 70:30 ( %
standard curves generated 
 for prodrug and parent drug were utilized for quantitation of integrated 
 area under peaks .
cell proliferation 
 assays were conducted to determine the cytotoxic activities of the 
 prodrug gp - mel and the parent melphalan .
the assays were carried out 
 with caco-2 and sk - mel-5 cells since the expression of dppiv was found 
 to be highest and lowest , respectively , in these cells , based on rt - pcr 
 expression results .
caco-2 and skmel-5 cells were plated overnight 
 in a 96-well cell culture plate at a density of 5,000 cells / well per 
 0.1 ml .
stock solutions ( 1 mm ) of gp - mel and melphalan were prepared 
 in rpmi-1640 phenol red free medium supplemented with fbs .
stock solutions 
 were serially diluted to obtain a total of six drug concentrations , 
 1 mm , 0.5 mm , 0.25 mm , 0.125 mm , 0.0625 mm , and 0.03125 mm , for cell 
 proliferation studies .
after 24 h , the medium in the 96-well plate 
 was aspirated and replaced with drug solutions in the medium .
the cells were then incubated at 
 37 c and 5% co2 for 48 h. after 48 h , 50 l 
 of xtt labeling mixture ( 5 ml of 1 mg / ml xtt in rpmi-1640 phenol red 
 free medium mixed with 100 l of 0.383 mg / ml pms in phosphate 
 buffered saline ) was added to each well .
the color development , due 
 to formation of formazan dye by metabolically active cells , was monitored 
 for 4 h , after which the plates were read at 490 nm ( 805 nm as the 
 reference wavelength ) with a precision microplate reader ( emax , molecular 
 devices ) .
the percent cell viability , at different drug and prodrug 
 concentrations , relative to control was then plotted as a function 
 of drug / prodrug concentration to compute the gi50 values .
after the genes had been sorted as hydrolases , 
 peptidases , and esterases , they were clustered and their expression 
 in the 60 cancer cell lines was visualized as described previously .
the expression patterns of proline - specific 
 peptidases such as prolinases / peptidase  , prolyl carboxypeptidase , 
 prolyl oligopeptidase / prolyl endopeptidase , dppiv / cd26 , aminopeptidase 
 p , and prolidase / peptidase d , were selected for further assessment . 

dppiv was overexpressed in most renal carcinoma cell lines ( uo-31 , 
 tk-10 , sn12c , rxf 393 , caki-1 , achn , a498 , and 786 - 0 ) compared to 
 cancer cell lines derived from other tissues .
the relatively higher 
 dppiv expression in 786 - 0 renal carcinoma cell line compared to its 
 expression in sk - ov-3 ovarian carcinoma or sk - mel-5 melanoma cell 
 lines was consistent with affymetrix expression data .
thus , in addition 
 to the selection of igrovi ( ovarian cancer cell line ) , pc-3 ( prostate 
 cancer cell line ) , 786 - 0 ( renal cancer cell line ) , sk - ov-3 ( ovarian 
 cancer cell line ) , and sk - mel-5 ( melanoma ) based on the u95av2 affymetrix 
 gene expression of dppiv ( table 1 ) , hepg2 and 
 caco-2 cells were also selected for characterization of dppiv activity 
 with standard substrates and rt - pcr determinations of dppiv expression .
rt - pcr of extracellular mrna in the seven selected 
 human cancer cell lines was performed with primers specific for dppiv . 

the expression profiles of dppiv in the seven cancer cell lines evidenced 
 by the band at 315 bp are shown in figure 3 .
the results of semiquantitative rt - pcr analysis ( table 1 ) indicated that dppiv was expressed at high levels 
 in caco-2 and hepg2 cells , and at very low levels in sk - mel-5 cells . 

thus , the relative rt - pcr expression ( expression in sk - mel-5 set at 
 unity ) in the seven cell lines listed in table 1 were caco-2 ( 13.49 ) , hepg2 ( 12.71 ) , igrovi ( 7.92 ) , pc-3 ( 5.51 ) , 
 786 - 0 ( 2.81 ) , sk - ov-3 ( 1.89 ) , and sk - mel-5 cells ( 1.0 ) .
the relative 
 rt - pcr expression in the cancer cell lines exhibited an excellent 
 linear correlation ( r = 0.94 ) with u95av2 
 affymetrix microarray data that was available for five of the seven 
 cell lines ( figure 4 ) .
expression profiles of 
 dppiv ( 315 bp product ) in 7 cancer cell 
 lines determined by semiquantitative rt - pcr : 100 base pair dna ladder 
 ( lane 1 ) , caco-2 ( lane 2 ) , hepg2 ( lane 3 ) , igrovi ( lane 4 ) , pc-3 ( lane 
 5 ) , 786 - 0 ( lane 6 ) , sk - ov-3 ( lane 7 ) , and sk - mel-5 ( lane 8) .
linear correlation of affymetrix genechip expression 
 of dppiv with 
 dppiv expression determined using rt - pcr in 5 cancer cell lines .
the specific activity of a standard substrate gp - pna for porcine dppiv was determined by the release of p - nitroanilide and the resultant effect on the clarity of 
 the solution .
the baseline activity was 14.02  0.90 nmol / min/g 
 dppiv . in the presence of the specific inhibitor diprotin a , it decreased 
 substantially to 0.15 nmol / min/g dppiv .
the kinetic parameters km and vmax obtained 
 from a plot of v0 versus [ s ] ( figure 5 ) were 231  33 m and 0.650  0.037 
 mol / min / mu dppiv , respectively ( fit estimate  sd ; n = 3 ) .
the kcat value determined 
 from vmax and dppiv concentration ( e0 ) was 36 s. in contrast , 
 gf - pna or gr - pna produced no detectable 
 release of p - nitroanilide ( negligible absorbance 
 at 405 nm ) , indicating that these two molecules were resistant to 
 cleavage by porcine dppiv .
gp - pna hydrolysis kinetic profile with porcine 
 dppiv ( mean  sd , n = 3 ) .
the activity 
 of dppiv in various cancer cell homogenates was assessed using the 
 standard substrate gp - pna .
the results shown in figure 6 indicate that dppiv activity was in the order hepg2 
  caco-2  igrovi  pc-3  786 - 0  
 sk - ov-3  sk - mel-5 cells .
caco-2 and hepg2 cells exhibited 
 high dppiv activity ( around 20 pmol / min/g protein ) whereas 
 sk - mel-5 cells showed the lowest activity ( 1.3 pmol / min/g protein ) 
 among the cells tested ( figure 6 ) .
these results 
 are in excellent correlation ( r = 0.96 ) 
 with the relative rt - pcr expression of dppiv in the cell lines ( figure 7 ) .
the hydrolysis of gp - pna in 
 the presence of diprotin a was significantly inhibited in all cancer 
 cell homogenates examined ( figure 6 ) , with 
 an average inhibition of 95% ( range : 85100% ) .
specific activity of 
 dppiv against gp - pna ( pmol / min/g 
 protein ) in 7 cell homogenates , in the absence vs presence of diprotin 
 a ( mean  sd ,
n = 3 ) . linear correlation of specific activity of dppiv against gp - pna ( pmol / min/g protein ) in 7 cancer cell homogenates 
 with dppiv expression determined using rt - pcr .
the specific activity of 
 pure porcine enzyme against gp - mel was 1.2  0.1 nmol / min/g 
 protein .
the activity was substantially lowered to 100 pmol / min/g 
 protein , a 93% inhibition , in the presence of diprotin a. dppiv activity 
 against the prodrug determined in caco-2 and sk - mel-5 homogenates 
 was 5.14  0.01 and 0.68  0.03 pmol / min/g protein , 
 respectively , which was significantly inhibited by an average of 85% 
 in the presence of diprotin a ( figure 8) .
specific 
 activity of dppiv against gp - mel ( pmol / min/g protein ) 
 in caco-2 and sk - mel-5 cell homogenates , in the absence vs presence 
 of diprotin a ( mean  sd , n = 3 )
. the antiproliferative 
 activity of gp - mel was determined in caco-2 and sk - mel-5 cancer cells 
 and compared with that obtained with the parent drug melphalan .
the 
 percent cell viability profiles as a function of drug or prodrug concentration 
 are shown in figure 9 .
the profiles indicate 
 that the antiproliferative action of melphalan was similar in caco-2 
 and sk - mel-5 cells with gi50 values of 34.9 m and 
 56.6 m , respectively . the gi50 value for gp - mel 
 in caco-2 cells ( 261.3 m ) , however , was significantly lower 
 than that obtained with sk - mel-5 cells ( 806.7 m ) .
the cytotoxic 
 activity of gp - mel in caco-2 and sk - mel-5 cells was consistent with 
 the expression levels of dppiv in these cells .
cell proliferation assay 
 of melphalan and gp - mel after 48 h incubation 
 with caco-2 and sk - mel-5 cells .
data are expressed as mean  
 sd ; n = 3 for each treatment at each concentration .
in the past three decades , the science of molecular biology was 
 revolutionized by the rapid advancement of the complementing fields 
 of genomics and bioinformatics .
recently , 
 a number of enzymes from the peptidase / protease class were found to 
 be highly expressed in several types of tumors , and to play an important 
 role in the pathophysiology of tumor cells .
dppiv has been previously associated with the onset and progression 
 of several cancer types ; inamoto et al .
demonstrated that the blockage 
 of ddpiv reduced several cancer - related processes in the human renal 
 carcinoma cell line caki-2 .
it also reduced the tumor size and increased 
 the survival of mice in a xenograft model .
furthermore , in patients with clear cell renal cell carcinoma ( ccrcc ) , 
 higher dppiv activity was linked with a significant decrease in patients 
 5 year survival rates .
dppiv was also 
 shown to be overexpressed in several human colon cancer tissues and 
 in human colon cancer cell lines , and its inhibition reduced carcinogenesis in a rat 
 model . in the prostate , dppiv activity 
 in cancerous versus benign prostatic hyperplasia was increased 2-fold .
an elevation of dppiv activity was also found in the prostatic secretions 
 and the peripheral zone of the prostate , where most prostate cancers 
 arise .
higher dppiv levels in cancerous 
 versus normal prostate tissue was correlated with psa level , tumor 
 residue , cancer stage , and tumor size in humans .
in addition to its expression 
 and involvement in cancer , dppiv 
 is one of the few proline - specific proteases that is able to cleave 
 proline - associated peptide bonds , as the unique cyclic structure of 
 proline serves as a structure regulation element which limits the 
 susceptibility for nonspecific enzymatic degradation .
thus , a high level of expression in cancer cells , combined with 
 high substrate specificity , indicated that dppiv may be a potential 
 target molecule for the delivery of chemotherapeutic drugs , and sparked 
 our interest in developing a dppiv - cleavable anticancer prodrug .
the functional activity of pure porcine dppiv was assessed against 
 three gly - x dipeptide chromogenic compounds , gp - pna , a well - known standard dppiv substrate , as well as gf - pna and gr - pna , 
 two dipeptide analogues that do not contain proline in the p1 position . 

the kinetic parameters obtained in this study were consistent with 
 those previously reported for gp - pna with porcine 
 dppiv .
the negligible activity observed with gf - pna and gr - pna is consistent with dppiv 
 substrate requirements , and eliminates the possibility of nonspecific 
 degradation .
similarly , in the seven investigated cancer cell 
 lines , gp - pna exhibited an excellent linear correlation 
 ( r = 0.96 ) with rt - pcr dppiv expression 
 ( figure 7 ) , and dppiv activity was significantly 
 inhibited in the presence of the specific inhibitor diprotin a ( average 
 inhibition 95% ) .
the similarity of the extent of inhibition 
 in cell homogenates with that observed with porcine dppiv underscores 
 the similarity of human and porcine dppiv .
melphalan , phenylalanine 
 mustard ( l - pam ) , is an established anticancer 
 agent that was originally approved for the treatment of multiple myeloma . 

recently it has been suggested as a therapeutic agent in the treatment 
 of a variety of cancers such as ovarian cancer , breast cancer , colorectal 
 cancer , and melanoma .
however , its use has been limited due to severe side effects 
 such as bone marrow suppression , leukopenia , and thrombocytopenia , largely due to lack of selectivity for cancer cells , and so it was 
 selected as the model drug for investigating our approach .
a proline 
 dipeptide prodrug of melphalan was designed to follow dppiv substrate 
 specificity requirements .
additionally , 
 the peptide bond between p1 and p1 requires a trans - configuration .
the release of melphalan 
 from the gp - mel prodrug by pure porcine 
 dppiv was extensive , and was 93% reduced in the presence of diprotin 
 a , indicating that the activation of gp - mel was dppiv - dependent .
this 
 was further corroborated in caco-2 and sk - mel-5 homogenates when the 
 prodrug activation was significantly inhibited by diprotin a ( figure 8) .
the 8-fold greater activation of the prodrug 
 in caco-2 compared to sk - mel-5 homogenates ( figure 8) was consistent with the 14-fold higher rt - pcr dppiv expression 
 levels , and the 15-fold higher dppiv activity against gp - pna in caco-2 cells compared to sk - mel-5 cells ( figure 7 ) . as a preliminary assessment of gp - mel potential to 
 serve as an 
 anticancer prodrug , we have investigated the antiproliferative activity 
 of gp - mel in caco-2 and sk - mel-5 cell lines .
the antiproliferative 
 activity of the prodrug was shown to be dependent on dppiv expression 
 level in the cells ; the cytotoxicity ( represented by gi50 ) of gp - mel in caco-2 cells was 3-fold higher ( 260 m ) than 
 that in sk - mel-5 cells ( 800 m ) ( figure 9 ) .
in contrast , for the free parent drug melphalan , similar gi50 values were obtained in caco-2 and in sk - mel-5 cells ( 35 
 and 44 m , respectively ) , demonstrating the nonselective cytotoxic 
 action of melphalan ( figure 9 ) .
the cytotoxic 
 effect of gp - mel on both cell lines was significantly lower in comparison 
 to melphalan ; the prodrug failed to show significant cytotoxic effect 
 in concentrations equimolar to those required for maximal growth inhibition 
 by melphalan .
this result may indicate that the gp - mel prodrug is 
 not likely to be cytotoxic by itself , and further validates the key 
 role of dppiv in the activation of the gp - mel prodrug . on the other 
 hand
, it may indicate insufficient activation , resulting in too low 
 free drug levels .
overall , our results demonstrate the potential to 
 exploit dppiv as a prodrug activating enzyme for efficient chemotherapeutic 
 drug targeting .
in conclusion , 
 dppiv was identified as a potential prodrug target 
 due to its differential expression levels in tumor and normal tissues 
 and relatively strict substrate specificity .
a gly - pro dipeptide prodrug 
 of melphalan , gp - mel , was designed and synthesized , based on the highly 
 specific substrate requirements of dppiv .
the finding that the activation 
 and antiproliferative activity of gp - mel in cells were highly dependent 
 on dppiv expression levels confirmed our hypothesis that dppiv is 
 a feasible functional prodrug target for effective and selective chemotherapeutic 
 action .
 the efficacy of chemotherapeutic 
 drugs is often offset by severe 
 side effects attributable to poor selectivity and toxicity to normal 
 cells . 
 recently , the enzyme dipeptidyl peptidase iv ( dppiv ) was considered 
 as a potential target for the delivery of chemotherapeutic drugs . 
 
 the purpose of this study was to investigate the feasibility of targeting 
 chemotherapeutic drugs to dppiv as a strategy to enhance their specificity . 
 
 the expression profile of dppiv was obtained for seven cancer cell 
 lines using dna microarray data from the dtp database , and was validated 
 by rt - pcr . 
 a prodrug was then synthesized by linking the cytotoxic 
 drug melphalan to a proline - glycine dipeptide moiety , followed by 
 hydrolysis studies in the seven cell lines with a standard substrate , 
 as well as the glycyl - prolyl - melphalan ( gp - mel ) . 
 lastly , cell proliferation 
 studies were carried out to demonstrate enzyme - dependent activation 
 of the candidate prodrug . 
 the relative rt - pcr expression levels of 
 dppiv in the cancer cell lines exhibited linear correlation with u95av2 
 affymetrix data ( r2 = 0.94 ) , and with 
 specific activity of a standard substrate , glycine - proline - p - nitroanilide ( r2 = 0.96 ) . 
 
 the significantly higher antiproliferative activity of gp - mel in caco-2 
 cells ( gi50 = 261 m ) compared to that in sk - mel-5 
 cells ( gi50 = 807 m ) was consistent with the 9-fold 
 higher specific activity of the prodrug in caco-2 cells ( 5.14 pmol / min/g 
 protein ) compared to sk - mel-5 cells ( 0.68 pmol / min/g protein ) 
 and with dppiv expression levels in these cells . 
 our results demonstrate 
 the great potential to exploit dppiv as a prodrug activating enzyme 
 for efficient chemotherapeutic drug targeting . 
Introduction
Materials
and Methods
Results
Discussion
Conclusions