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Tumor Biology |
Departments of Immunology [C. L., H. H., T. E.] and Chemistry [C. S., K. J.], The Scripps Research Institute, La Jolla, California 92037-1092
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we present evidence that legumain is overexpressed in the majority of human solid tumors. We demonstrate that legumain promotes cell migration, and overexpression is associated with enhanced tissue invasion and metastasis. Its unique functional properties and high-level expression in many human tumors lends itself as a potential candidate enzymatic target for prodrug activation and tumor eradicative therapy.
The integrity of the amino group of doxorubicin is essential for function. It has been shown that doxorubicin tolerates the addition of a leucine residue at this site; however, incorporation of additional amino acids abolishes cytotoxic activity (6 , 7) . A prototype prodrug was synthesized by addition to doxorubicin through a peptide bond of an asparaginyl endopeptidase substrate peptide. Upon exposure to legumain, the compound was converted to an active cytotoxic leucine-doxorubicin molecule. This compound had markedly reduced toxicity compared with doxorubicin and was effectively tumoricidal in a murine colon carcinoma model. It is proposed that legumain represents a new functional target for tumoricidal prodrug development and therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rapid Isolation of Tumor Endothelial Cells and mRNA Extraction.
CD31 antibody-coated Dynabeads were prepared by mixing 300 µl of bead suspension with 500 µl of PBSA (PBS, 1% BSA). Biotinylated antimouse CD31 antibody (20 µg) was added to the suspension, and association of antibody to beads was for 20 min at 4°C. The beads were washed three times with PBS to remove unbound antibody. CT26 tumors grown to 
1.5-cm greatest diameter were surgically removed cooled to 4°C for following steps, and the tumor minced into 1-mm3 bits with sterile scissors. The minced tumor was gently pressed through metal meshes and filtered through a 40-µm Falcon cell strainer (Becton Dickinson, Franklin Lakes, NJ) to rapidly recover the single cell suspensions. Strepavidin-conjugated paramagnetic Dynabeads (Dynal, Lake Success, NY) coated with biotinylated antimouse CD31 antibody (Mec 13.3; PharMingen, La Jolla, CA) were immediately added to the single cell suspensions. Capture by beads of CD31-positive cells was conducted at 4°C for 20 min with gentle agitation. Beads with bound CD31-positive cells were recovered with a magnetic trap column and washed three times with cold PBS. Unbound CD31-negative cells were collected separately and were recovered by centrifugation at 1000 rpm for 3 min. Both CD31-positive and CD31-negative cells were used for mRNA extraction (Qiagene mRNA direct kit). The concentration of mRNA was quantitated with RiboGreen RNA quantitation reagents (Molecular Probes, Eugene, OR).
Differential Gene Expression Profiling Using Restriction Fragment Differential Display.
Five hundred ng of mRNA were used for differential profiling using the displayPROFILE method (Display Systems Biotech, Vista, CA). The mRNA was first used to synthesize double-stranded cDNA. The resultant double-stranded DNA was digested with Taq I, and adaptors were ligated onto the fragment ends. Display primer was used to PCR amplify the gene fragment profiles, which were then displayed on a 6% sequencing gel. Differentially displayed bands were cut from the sequencing gel and extracted with 50 µl of water for 15 min in a boiling water bath. The fragments were reamplified with the same set of primers and then electrophoresed on 4% agarose gels. The amplified fragments were recovered from the gels and cloned into a pCRII vector by the Topo cloning method (Invitrogen). The vectors were then sequenced and BLAST searches performed with National Center for Biotechnology Information database to identify genes.
Histological and Immunohistochemical Analysis.
Immunohistochemical staining was performed on both formalin-fixed and -unfixed frozen 5-µm thick sections on poly-L-lysine slides. For endothelial identification, biotinylated rat antimouse CD31 monoclonal antibody (MEC 13.3) was used with fluorescein-conjugated strepavidin as the secondary reporting reagent. Rabbit anti-legumain antisera was prepared by immunization with purified human legumain produced in Escherichia coli (8)
. This antisera recognizes both mouse and rat legumain in frozen sections, as well as human legumain in formalin-fixed sections. For staining of legumain in both frozen and formalin-fixed sections, rabbit polyclonal anti-legumain antisera was used at 1:500 dilution, followed by biotinylated antirabbit IgG as the second antibody. The reaction was visualized with Texas-red conjugated strepavidin, and the slides were analyzed by laser scanning confocal microscope (Bio-Rad, Hercules, CA). For chromogenic staining, the rabbit polyclonal anti-legumain antibody was followed by a biotinylated goat antirabbit antisera (Vector, Burlingame, CA). Strepavidin-conjugated peroxidase was used and developed with the substrate BAD (Vector).
Western Blot.
Proteins were dissolved in 2x SDS sample buffer for SDS-PAGE analysis using gradient Tris-glycine gels (8–16%). After electrophoresis, the proteins were transferred to nitrocellulose membranes and blocked with nonfat milk. The anti-legumain antisera was used as the first antibody and was incubated with membrane for 1 h (1:1,000 dilution). The blot was washed three time with PBS, incubated with strepavidin-peroxidase for 15 min, and developed by the enhanced chemiluminescence method (Sigma, St. Louis, MO).
Zymogram.
Control 293 cells and legumain +293 cells were plated onto 96-well plates at 4000 cells/well. The cells were allowed to attach overnight, then were serum starved for 4 h. Zymogen forms of metalloproteinase 2 or 9 (Chemicon, Temecula, CA) were added at concentration of 0.1 µg/well with 50 µl of reaction buffer [39.5 mM citric acid, 121 mM Na2HPO4 (pH 5.8), 1 mM EDTA, and 0.8% Na2Cl], and the reactions were continued for 10 min. The reactants were collected and mixed with an equal volume of SDS sample buffer and held at room temperature for 10 min then applied to a zymogram gel (10% Tris-glycine gel with 0.1% gelatin substrate). After electrophoresis, the gel was washed briefly and incubated with 2.5% (v/v) Triton X-100 at room temperature for 30 min with gentle agitation. Digestion of the incorporated gelatin by activated collagenase was conducted in buffer [50 nM Tris (pH 7.25), 200 mM NaCl, 10 nM CaCl2, 0.05% Brij-35, and 0.02% NaN3] overnight. The gel was stained with Coomassie Blue R250 (Novex, San Diego, CA), and the presence of a protease was readily observed as a clear band.
Cell Invasion and Mobility Assays.
Cell migration and invasion assays were performed as described with modifications (9)
. Stock solutions (15 mg/ml) of Matrigel basement membrane matrix (Becton Dickinson, Bedford, MA) were stored at -80°C in 100-µl aliquots. After thawing on ice, the stock was diluted 1:50 with cold serum-free culture media and immediately applied to each membrane insert (8-µm pore) that formed the upper chambers of the multiwell invasion assay plate. The Matrigel was incubated overnight in a sterile laminar tissue culture hood. The membranes were hydrated for 2 h with 250 µl of serum-free medium, and excess medium was removed by aspiration. Medium containing 10% FBS was added to the bottom of each well. A suspension of 105 cells in 150 µl of serum-free medium was added to the upper chamber and incubated for 12 h at 37°C, 5% CO2. At the indicated times, the membrane inserts were removed from the plate, and the noninvading cells were removed from the upper surface. Membrane-associated cells were stained with 0.09% crystal violet for 30 min and washed twice with PBS. The invading cells were counted microscopically. Cell mobility assays were performed in a similar manner, except the membrane inserts were not coated with Matrigel, and duration was shortened. In some assays, protease inhibitors were added to the invasion chamber at the beginning of the incubation.
Prodrug Synthesis.
N-(-t-Butoxycarbonyl-L-alanyl-L-alanyl-L-asparaginyl-L-leucyl)doxorubicin was synthesized as follows. To cold (0°C) solution of -t-butoxycarbonyl-L-alanyl-L-alanyl-L-asparaginyl-L-leucine (43 mg, 95 µmol) and 4-methylmorpholine (20 µl, 200 µmol) in 5 ml of dimethylformamide was added O-benzotriazol-1-N,N,N',N'-tetramethyluronium hexafluorophosphate (54 mg, 142.5 µmol). After 10 min, doxorubicin hydrochloride (50 mg, 86 µmol) was added, and the mixture was stirred for 2 h at room temperature in the dark. The solution was diluted with 30 ml of EtOAc and washed with water. The solvent was evaporated, and solids were dried over MgSO4 and purified over silica gel using CHCl3/methanol (90/10) while protected from light to yield 65 mg of compound 1 (75% yield). 1H NMR (600 MHz, CD3OD, 
): 0.82 (d, [3H], J = 6.1), 0.88 (d, [3H], J = 6.6), 1.28–1.35 (m, 9H), 1.43 (s, 9H), 1.59–1.74 (m, 4H), 2.05 (m, 1H), 2.17 (m, 1H), 2.36 (d, 1H, J = 14.5), 2.67 (m, 1H), 2.79 (m, 1H), 2.91 (d, 1H, J = 18.0), 3.04 (d, 1H, J = 18.0), 3.62 (m, 1H), 4.01–4.04 (m, 4H), 4.11 (m, 1H), 4.22–4.32 (m, [3H]), 4.59 (dd, 1H, J = 5.9, 7.2), 4.74 (d, 2H, J = 4.4), 5.08 (s, 1H), 5.39 (d, 1H, J = 3.1), 7.51 (d, 1H, J = 8.8), 7.78 (dd, 1H, J = 7.9, 7.9), 7.86 (d, 1H, J = 7.5). Preparation high-resolution mass spectrometry (matrix-assisted desorption ionization) calculated for C48H64N6O18 [M + Na]+ is 1035.4169 and found is 1035.4234. The compounds were purified by semipreparative high-performance liquid chromatography.
Cytotoxic Assays.
The WST-13
cell proliferation reagent (Roche Molecular Biochemicals, Indianapolis, IN) was used to determine cell proliferation by quantitation of cellular metabolic activity. Control 293 cells and legumain+ 293 cells were cultivated in microtiter plates (5 x 103 cells/well in 100 µl) and were incubated with serial concentrations of legubicin or doxorubicin for 48 h. Subsequently, 10 µl of WST-1 solution (1 mg/ml WST-1, 25 µM methyldibenzopyrazine methyl sulfate) were added/well, and mixtures were incubated for an additional 4 h. The tetrazolium salt WST-1 was cleaved by the mitochondrial succinate-tetrazolium-reductase system to formazan in cells, which directly correlates to the number of metabolically viable cells in the culture. The amount of formazan salt was quantified in three replicates by absorbance at 450 nm using a microplate reader (Molecular Devices, Palo Alto, CA). All results were derived from replicate experiments with similar results.
Animals Models.
The CT26 syngeneic murine colon carcinoma model was generated and maintained in The Scripps Research Institute animal facility. Balb/C mice ages 4–6 weeks from the breeding colony were inoculated with 500,000 syngeneic CT26 tumor cells/site s.c. on the back. Treatment was initiated when the tumors reached 4 mm in diameter through bolus i.p. injection of the indicated reagents. Treatment was repeated at 2-day intervals. The human 293 tumor models were generated in WEHI nude mice (The Scripps Research Institute breeding colony). Either legumain+ 293 cells or control 293 cells (106 cells/site) were inoculated s.q. on the back. Tumor growth and physical signs were monitored daily, including any gross evidence of tumor necrosis, local tumor ulceration, as well as evidence of toxicity, including mobility, response to stimulus, eating, and weight of each animal. These procedures have been reviewed and approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute. The work was conducted in The Scripps Research Institute facilities, which are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. The Scripps Research Institute maintains an assurance with the Public Health Service, is registered with the United States Department of Agriculture, and is in compliance with all regulations relating to animal care and welfare.
Statistical Analysis.
Statistical significance of data were determined by the two-tailed Student’s t test.
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RESULTS |
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. Legumain overexpression in mouse tumors was confirmed by Western blot analysis of a panel of mouse tumors but is also expressed by some normal mouse tissues (Fig. 1B)
. Legumain expression is not detected in the CT26 cell line in culture that was used to generate the syngeneic mouse colon carcinoma model and is negative in other tumor cell lines in culture that we have tested. The remarkable up-regulation during tumor development in vivo infers an in vivo environmental response. Legumain appears to be a stress-responsive gene, although not detectable in cultured cells under typical tissue culture conditions, its expression was markedly elevated in cells subjected to environmental stress such as serum starvation or in vivo growth.
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. Whereas, in normal tissues legumain expression is sparse, highest in the kidney, presence in liver and spleen was detectable, agreeing with published results (1
, 14)
. Notably, legumain was highly expressed in the majority of human tumor tissue panels analyzed, which encompassed a wide variety of solid tumors (Table 1)
. Expression was highest for prostate carcinomas and positive for most specimens from breast and colon carcinomas, and all central nervous system malignancies were positive.
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, consistent with the proposed function as a lysosomal protease. The delivery of membranous vesicles containing proteases, adhesion molecules, and actin-binding proteins toward the leading edge of migratory cells has been implicated in cell locomotion (15
, 16)
. The legumain positive membranous vesicles were often concentrated at the invadopodia of tumor cells (Fig. 2B)
. However, we also observed it in apparent association with cell surfaces (Fig. 2C)
, as well as with the actin cortex (Fig. 2D)
. Double staining of legumain+ 293 cells with antibody against integrin ß1 and anti-legumain antibody disclosed the presence of legumain inside cells in a granular organelle resembling aggregated lysosomes and also on the cell surface colocalized with ß1 integrins (Fig. 2E)
. The potential binding of legumain to ß1 integrins is provocative considering the Arg-Gly-Asp sequence motif present in legumain, a motif that might facilitate legumain association with cell surface integrins.
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. Recently, activation of zymogen gelatinase A by legumain has been reported (18)
. No effect of legumain on zymogen gelatinase B was detected (Fig. 2F)
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Legumain Expression Promotes Cell Migration and Invasion.
We evaluated the effect of legumain expression on cell migration and invasion. In an in vitro migration assay, legumain+ 293 cells exhibited an increased migration in comparison with wild-type 293 cells, and the enhanced migration was inhibited by cystatin, a known inhibitor of legumain protease function, weakly by TIMP-2 protein but not by E64 (Fig. 3A)
. Next, we evaluated control 293 cells and legumain+ 293 cells in a modified Boyden chamber invasion assay. Legumain+ cells exhibited increased invasion of extracellular matrix, which was inhibited by cystatin (Fig. 3B)
but to only a limited extent by TIMP-2. Again E64 was without effect. These experiments were repeated with a 293 cell line in which the transcription of legumain was conditionally regulated by tetracycline with comparable results (data not shown).
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, predominantly lung and liver, whereas none were observed for control 293 tumors. The increased invasion and metastasis associated with legumain overexpression is consistent with legumain-facilitated tumor metastasis and progression. The more invasive legumain+ 293 tumors frequently invaded muscles and frequently lacked the well-defined pseudoencapsulation observed with control 293 tumors (Fig. 4, B and C)
. This more invasive tumor behavior was evident in early as well as later stage tumors.
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. The effect of doxorubicin on both 293 cell types was similar, with legumain+ cells only slightly more resistant to doxorubicin. In contrast, the cytotoxic effect of legubicin on control 293 cells was <1% of that of doxorubicin, indicating peptide conjugation had abolished the cytotoxic effect of the doxorubicin. The dose-responsive curve of legubicin on control 293 cells was parallel to that of doxorubicin on both 293 cells, suggesting that the residual cytotoxicity may result from slight (1%) doxorubicin contamination. In contrast, a profound cytotoxic effect of legubicin was observed for legumain+ 293 cells. The dose-response curve of legumain+ cells challenged with legubicin differed from that for these cells exposed to doxorubicin.
Tumoricidal Effect of Legubicin in Vivo.
The in vivo effects of legubicin on normal and tumor-bearing hosts, and efficacy in tumor eradication was investigated using the CT26 murine syngeneic colon carcinoma model. Legubicin was very well tolerated in mice with much reduced toxicity compared with doxorubicin. i.p. injection of legubicin at 5 mg/kg three times at 2-day intervals induced complete growth arrest of the tumors (Fig. 6A
, top and middle panel) with little evidence of toxicity, as most readily evidenced by the absence of weight loss (Fig. 6A
, bottom panel). In contrast, doxorubicin failed to produce similar antitumor efficacy at doses approaching its maximum-tolerable dose. When doxorubicin was administered by the same protocol and dosage as for legubicin toxicity was fatal (Fig. 6A
, top and middle panel).
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. TUNEL assay analysis of tumor tissues revealed a higher apoptotic index for legubicin than for doxorubicin treatment (Fig. 6, D and E)
. Surprisingly, in organs that do express legumain such as kidney and liver, no injury was evident (data not shown). These observation indicate that legubicin has significantly improved safety and therapeutic indices compared with doxorubicin. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we present evidence for, not only atypical expression, but also the participation of legumain in effector functions and as an apparent regulator of cellular behavior in migration and tissue invasion. Cells that highly express legumain-exhibited enhanced migratory and invasive properties. A correlation between tumor invasion and metastasis with the presence of cysteine endopeptidases (particularly cathepsins B and L) has been observed (21) . Hydrolysis of asparaginyl bonds is prominent in the posttranslational processing of cathepsin B, D, and H (1 , 14 , 22) . Legumain might activate local cysteine protease zymogens to their active two-chain protease forms. In addition to the established plasminogen/plasmin system and the metalloproteinase system, a cysteine protease cascade may represent an additional tumor invasion/metastasis cascade. We as well as others have provided evidence that legumain activates the gelatinase A zymogen, an important mediator of extracellular matrix degradation. The activation mechanism of gelatinase A by legumain differs from that involved with the membrane type matrix metalloproteinases (23) . This may be important for tumor cell adaptation to a more invasive and metastatic phenotype. Legumain-promoted cell migration and invasion can be partially inhibited by cystatin and TIMP-2, which suggests that there may be a set of derivative events and downstream effector enzymes. The inhibition of mammalian legumain by cystatin is attributable to a novel second reactive site (24) . Another cysteine inhibitor E64 has no affect on legumain and excludes typical papain family cysteine proteases, which are characteristically susceptible to E64 inhibition. Analysis by site-directed mutagenesis of the catalytic residues of mammalian legumain implicates a catalytic dyad with the motif His-Gly-spacer-Ala-Cys. The presence of this motif is also found in the catalytic sites of the caspases, the aspartate-specific endopeptidases central to the process of apoptosis in animal cells, as well as in the families of clostripain and gingipain which are arginyl/lysyl endopeptidases of pathogenic bacteria. Therefore, these four families of proteases may be evolutionarily related and share similar protein folding. In this respect, legumain is notably distinct from other lysosomal cysteine proteases, and its catalytic activity is rather unique. It is the only asparaginyl endopeptidase identified to date, its conservation through evolution and unique enzymatic activity support selection for a significant biological function.
Animal tumor models generated with cells overexpressing legumain demonstrated an in vivo behavior that is vigorous with more invasive growth and metastasis. This phenotype is proposed to result from the proteolytic function of legumain to activate other protease zymogens. The inhibitory effect of cystatins on tumor cells (25 , 26) is consistent with the involvement of legumain and perhaps other cysteine proteases in tumor invasion and metastasis. Whether the tumor arrest effect is mediated through inhibition of legumain catalytic activity or other cysteine proteases is presently unknown. Tumor invasion and metastasis are critical determinants of cancer lethality, linked to 90% of human cancer deaths (27) . Invasion and metastasis are considered as associated properties of tumor cells because they use similar processes involving physical attachment of cells to their environment through cell adhesion molecules and activation of extracellular proteases (20) . Increased expression of proteases and down-regulation of protease inhibitors is commonly observed in tumors (28 , 29) . Notably, cell surface proteases are often associated with invasive and metastatic tumor cells (19) . Some proteases are linked to other properties of tumors such as angiogenesis (30) and growth signaling (31) as perhaps with legumain.
Protease zymogens are dependent on limited proteolytic activation for conversion to the functional state. Protease cascades are characteristic of many biological pathways such as the coagulation, apoptosis, and complement cascades. Similar cascades appear to be involved in tumor invasion and metastasis. Characterization of the later is complicated by the diversity of neoplasms. However, comprehensive profiling of protease expression and function may advance understanding of tumor invasion and metastasis.
Some metalloproteinase inhibitors have demonstrated tumor stasis in animal models. Similarly, legumain may represent a target for inhibition of growth and metastasis based on its up-regulation associated with tumor growth and unique restricted specificity. Legumain functions both extracellular and intracellular, therefore, a cell permeable inhibitor might extend the efficacy observed with cystatin because the latter is cell impermeable and has shown limited inhibition of in vitro cell migration and invasion.
We observed that tumor cells with higher legumain levels appear more resistant to apoptosis. Although the precise molecular pathway has yet to be defined for this effect, lysosomal proteases are known to participate as effector enzymes in apoptosis (32 , 33) . In another context, others have been observed to inhibit apoptosis (34) . Thus, the subcellular localization of legumain may determine its targets and thereby its effects on the apoptosis cascades.
The high level of legumain expression by tumor cells coupled with its unusual and highly specific substrate requirement for catalytic function makes it an attractive candidate for prodrug conversion in a therapeutic mode. Current cancer chemotherapeutic agents have significant undesirable cytotoxicity. A promising approach to increase selectivity is to exploit enzymes more highly expressed by tumor cells to achieve local prodrug activation to the active compound. Peptide conjugates of doxorubicin designed for activation by plasmin (35 , 36) and cathepsins (37, 38, 39) have been described. However, they are relatively deficient in target selectivity because plasmin generation is not tumor selective. The prodrug example in the present study was synthesized by incorporating a peptide extension to the amino group of doxorubicin. This compound, designated legubicin, was analyzed for cytotoxicity on cells not expressing legumain where it was <1% as toxic as doxorubicin. However, on cells expressing legumain, it was profoundly cytotoxic consistent with conversion to leucine-doxorubicin. i.p. administration of legubicin at 5 mg/kg resulted in complete arrest of tumor growth without identifiable toxicity such as weight loss in contrast to doxorubicin-treated mice. Legubicin administration produced profound tumor cell apoptosis as indicated by TUNEL assay. To our surprise, in organs containing cells that normally express legumain such as kidney and liver, no injury was evident. These data indicated that legumain activation of this prodrug may require conditions not present in normal tissue. First, prodrug activation may be carried out by secreted or cell surface-associated legumain, whereas legumain appears to be localized in lysosomal vesicles in normal tissues. Second, legumain requires an acidic environment for optimal catalytic activity, which may not be present in normal tissues. Our data suggest legubicin has an improved therapeutic index compared with its parent doxorubicin. Whereas clinical use of doxorubicin is limited by its toxicity, a prodrug that preserves activity but with reduced toxicity is an attractive alternative.
In summary, we have demonstrated unsuspected high-level expression of legumain, a novel asparaginyl endopeptidase, in a wide variety of tumors, murine and human. Legumain expression appears to be associated with increased tumor invasion and metastasis, possibly through increased extracellular matrix degradation, resulting from activation of zymogens such as progelatinase A and also to reduce tumor cell apoptosis. The legumain-activated prodrug legubicin, exhibited reduced toxicity relative to doxorubicin, and was effectively tumoricidal in vivo. This study testifies to the strength of functional genomics in guiding rational drug design.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work is supported by the NIH Grant P01 HL-16411. 
2 To whom requests for reprints should be addressed, at E-mail: chengliu{at}scripps.edu or tse{at}scripps.edu
3 The abbreviations used are: WST-1, (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disfonate); TIMP-2, tissue inhibitor of metalloproteinase 2; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 2/10/03. Accepted 3/28/03.
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