Cathepsin L,target in cancer treatment?

Cathepsin L, a cysteine protease, is considered to be a potential therapeutic target in cancer treatment. Proteases are involved in the development and progression of cancer. Inhibition of activity of specific proteases may slow down cancer progression. In this review, we evaluate recent studies on the inhibition of cathepsin L in cancer. The effects of cathepsin L inhibition as a monotherapy on apoptosis and angiogenesis in cancer are ambiguous. Cathepsin L inhibition seems to reduce invasion and metastasis, but there is concern that selective cathepsin L inhibition induces compensatory activity by other cathepsins. The combination of cathepsin L inhibition with conventional chemotherapy seems to be more promising and has yielded more consistent results. Future research should be focused on the mechanisms and effects of this combination therapy.     

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.     

Serum cathepsin H as a potential prognostic marker in patients with colorectal cancer

Cathepsin H is a lysosomal cysteine protease that may participate in tumor progression. In order to evaluate its potential as a prognostic marker, its protein levels were measured by ELISA in preoperative sera from 324 patients with colorectal cancer. The level of cathepsin H was significantly increased in patient sera, the median level was 8.4 ng/mL versus 2.1 ng/mL in 90 healthy blood donors (p < 0.0001). A weak association of cathepsin H levels was found with patient age (p = 0.02) but not with Dukes' stage, sex, or the level of carcinoembryonic antigen (CEA). In survival analysis a significant difference was found between the group of patients with low cathepsin H (first tertile) who had a poor prognosis and the remaining patients (p = 0.03). The risk of patients was further stratified when cathepsin H levels were combined with CEA. Patients with high CEA and low cathepsin H had the highest risk of death with a hazard ratio of 2.72 (95% CI 1.73-4.28), p < 0.0001. Our results show that the prognostic information of cathepsin H differs from that of the related cathepsins B and L and suggest different roles during the progression of malignant disease.     

Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S

Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied proteomic identification of protease cleavage sites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P3' due to an occluding loop blocking access to the primed subsites. In P1', cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P1' phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

Synthetic cyclohexenyl chalcone natural products possess cytotoxic activities against prostate cancer cells and inhibit cysteine cathepsins in vitro

A number of cyclohexenyl chalcone Diels-Alder natural products possess promising biological properties including strong cytotoxicity in various human cancer cells. Herein, we show that natural products in this class including panduratin A and nicolaioidesin C inhibit cysteine cathepsins as indicated by protease profiling assays and cell-free cathepsin L enzyme assays. Owing to the critical roles of cathepsins in the biology of human tumor progression, invasion, and metastasis, these findings should pave the way for development of novel antitumor agents for use in clinical settings.     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Elastin Degradation by Cathepsin V Requires Two Exosites

Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

Cysteine cathepsins B,X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.     

Cystatin C deficiency promotes epidermal dysplasia in K14-HPV16 transgenic mice

Background

Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.

Methods and Results

We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.

Conclusion

Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization.     

Digestive proteases in bodies and faeces of the two-spotted spider mite,Tetranychus urticae

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.     

Immunonanoparticles--an effective tool to impair harmful proteolysis in invasive breast tumor cells

Breast cancer cells exhibit excessive proteolysis, which is responsible for extensive extracellular matrix degradation, invasion and metastasis. Besides other proteases, lysosomal cysteine protease cathepsin B has been implicated in these processes and the impairment of its intracellular activity was suggested to reduce harmful proteolysis and hence diminish progression of breast tumors. Here, we present an effective system composed of poly(D,L-lactide-coglycolide) nanoparticles, a specific anti-cytokeratin monoclonal IgG and cystatin, a potent protease inhibitor, that can neutralize the excessive intracellular proteolytic activity as well as invasive potential of breast tumor cells. The delivery system distinguishes between breast and other cells due to the monoclonal antibody specifically recognizing cytokeratines on the membrane of breast tumor cells. Bound nanoparticles are rapidly internalized by means of endocytosis releasing the inhibitor cargo within the lysosomes. This enables intracellular cathepsin B proteolytic activity to be inhibited, reducing the invasive and metastatic potential of tumor cells without affecting proteolytic functions in normal cells and processes. This approach may be applied for treatment of breast and other tumors in which intracellular proteolytic activity is a part of the process of malignant progression.     

Tumor cell- and microenvironment-specific roles of cysteine cathepsins in mouse models of human cancers

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.     

Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.     

2,3-Dihydroquinazolin-4(1H)-one derivatives as potential non-peptidyl inhibitors of cathepsins B and H

A direct correlation between cathepsin expression–cancer progression and elevated levels of cathepsins due to an imbalance in cellular inhibitors-cathepsins ratio in inflammatory diseases necessitates the work on the identification of potential inhibitors to cathepsins. In the present work we report the synthesis of some 2,3-dihydroquinazolin-4(1H)-ones followed by their evaluation as cysteine protease inhibitors in general and cathepsin B and cathepsin H inhibitors in particular. 2,3-Dihydroquinazolin-4(1H)-ones, synthesized by the condensation of anthranilamide and carbonyl compound in presence of PPA-SiO₂ catalyst, were characterized by spectral analysis. The designed compounds were screened as inhibitors to proteolysis on endogenous protein substrates. Further, a distinct differential pattern of inhibition was obtained for cathepsins B and H. The inhibition was more to cathepsin B with K_i values in nanomolar range. However, cathepsin H was inhibited at micromolar concentration. Maximum inhibition was shown by compounds, 1e and 1f for cathepsin B and compounds 1c and 1f for cathepsin H. The synthesized compounds were established as reversible inhibitors of cathepsins B and H. The results were also compared with the energy of interaction between enzyme active site and compounds using iGemdock software.     

Characterization of mouse cathepsin R, a new member of a family of placentally expressed cysteine proteases

A new mouse cysteine protease, termed cathepsin R, has been identified. The complete nucleotide sequence of this gene was derived from a set of cDNAs generated from 15.5-day mouse placenta. Sequence analysis revealed an open reading frame encoding a 334 amino acid long polypeptide closely related to placentally expressed cathepsins P, Q, and M. RT-PCR analysis indicated that cathepsin R is only expressed in placenta and thus is a new member of the emerging family of cathepsins whose expression is regulated during mouse embryonic development. Modeling and structural analysis suggests that cathepsin R will have a restricted substrate specificity when compared to that of cathepsin L.     

Synthesis and cathepsin D inhibition of peptide-hydroxyethyl amine isosteres with cyclic tertiary amines

Summary Cathepsin D, a lysosomal aspartic protease, has been suggested to play a role in the metastatic potential of several types of cancer. A high activated cathepsin D level in breast tumor tissue has been associated with an increased incidence of relapse and metastasis. High levels of active cathepsin D have also been found in colon cancer, prostate cancer, uterine cancer and ovarian cancer. Hydroxyethyl isosteres with cyclic tertiary amine have proven to be clinically useful as inhibitors of aspartyl proteases similar to cathepsin D in activity, such as the HIV-1 aspartyl protease. The design and the synthesis of (hydroxyethyl)amine isostere inhibitors with cyclic tertiary amines is described. The IC₅₀ and K_i(app) values for the six cathepsin D inhibitors and pepstatin are reported. Compounds7b,3(S)-[Acetyl-L-valyl-L-phenylalanylamino]-4-phenyl-1-N-piperidine-2(S)-butanol, and7c, 3(S)-[Acetyl-L-leucyl-L-phenylalanylamino]-4-phenyl-1-N-piperidine-2(S)-butanol, showed the most potent inhibition of cathepsin D hydrolysis of hemoglobin with IC₅₀ values of 3.5 nM and 4.5 nM, respectively.     

Cathepsins as effector proteases in hepatocyte apoptosis

Conclusion Cathepsins B and D appear to act as part of the effector protease cascade in hepatocyte apoptosis, both in bile salt-induced apoptosis and CPT-induced apoptosis of hepatocellular cancer cell lines. It is important to note that these proteases do not appear to participate in many models of apoptosis studied to date; in fact, cathepsin inhibitors have been used as negative controls to show that enzymes other than caspases are not involved in apoptosis. In particular, it has been shown that cathepsin B inhibitors do not prevent many models of apoptosis in lymphocytes (43). Further, our experiments have shown that not all models of hepatocyte apoptosis are mediated by cathepsins. For example, staurosporine-induced apoptosis is not inhibited by cathepsin B inhibitors in primary hepatocytes or in cell lines stably transfected with the cathepsin B antisense construct. Although the signaling pathways leading to activation of cathepsins B and D in hepatocyte apoptosis are not completely understood, we hypothesize that a caspase 8-like protein may be involved proximal to cathepsins D and B (Fig. 6). The precise mechanism by which cathepsin B is translocated from lysosomes to “apoptotic targets” is currently under investigation in our laboratory. Because of the relative promiscuity of cathepsin B as protease, it is likely that it is involved in nonspecific protein degradation in apoptotic bodies; however, cathepsin B has also been shown to degrade certain specific proteins, such as histones, which may be directly relevant to the apoptotic process. Further evaluation of the role of cathepsins B and D in apoptosis should include the determination of specific proteolytic targets that result in the biochemical and morphologic manifestations of apoptosis.     

Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.