Proteomic Identification of Cysteine Cathepsin Substrates Shed from the Surface of Cancer Cells
2015; Elsevier BV; Volume: 14; Issue: 8 Linguagem: Inglês
10.1074/mcp.m114.044628
ISSN1535-9484
AutoresBarbara Sobotič, Matej Vizovišek, Robert Vidmar, Petra Van Damme, Vasilena Gocheva, Johanna A. Joyce, Kris Gevaert, Vito Türk, Boris Turk, Marko Fonovič,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoExtracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192. Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192. Cysteine cathepsins, a family of cysteine proteases normally confined to the endolysosomal system, emerged as major players in cancer progression (1.Mohamed M.M. Sloane B.F. Cysteine cathepsins: multifunctional enzymes in cancer.Nat. Rev. Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1009) Google Scholar, 2.Palermo C. Joyce J.A. Cysteine cathepsin proteases as pharmacological targets in cancer.Trends Pharmacol. Sci. 2008; 29: 22-28Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 3.Vasiljeva O. Reinheckel T. Peters C. Turk D. Turk V. Turk B. Emerging roles of cysteine cathepsins in disease and their potential as drug targets.Curr. Pharm. Des. 2007; 13: 387-403Crossref PubMed Scopus (199) Google Scholar). Genetic ablation of several cathepsins, including cathepsins B, L, and S, significantly slowed down cancer growth and metastatic spread in several mouse cancer models including mammary gland tumors and pancreatic islet cancer (3.Vasiljeva O. Reinheckel T. Peters C. Turk D. Turk V. Turk B. Emerging roles of cysteine cathepsins in disease and their potential as drug targets.Curr. Pharm. Des. 2007; 13: 387-403Crossref PubMed Scopus (199) Google Scholar, 4.Gocheva V. Zeng W. Ke D. Klimstra D. Reinheckel T. Peters C. Hanahan D. Joyce J.A. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis.Genes Develop. 2006; 20: 543-556Crossref PubMed Scopus (440) Google Scholar, 5.Sevenich L. Schurigt U. Sachse K. Gajda M. Werner F. Muller S. Vasiljeva O. Schwinde A. Klemm N. Deussing J. Peters C. Reinheckel T. Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2497-2502Crossref PubMed Scopus (140) Google Scholar, 6.Vasiljeva O. Turk B. Dual contrasting roles of cysteine cathepsins in cancer progression: apoptosis versus tumour invasion.Biochimie. 2008; 90: 380-386Crossref PubMed Scopus (110) Google Scholar). 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In cancer, cathepsins S and L are secreted into the tumor microenvironment by tumor cells, fibroblasts, endothelial cells, and infiltrating immune cells (13.Mason S.D. Joyce J.A. Proteolytic networks in cancer.Trends Cell Biol. 2011; 21: 228-237Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Among the immune cells, macrophages are a major source of tumor-associated cathepsins (14.Gocheva V. Wang H.W. Gadea B.B. Shree T. Hunter K.E. Garfall A.L. Berman T. Joyce J.A. IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion.Genes Develop. 2010; 24: 241-255Crossref PubMed Scopus (519) Google Scholar). Secreted cathepsins were found to be involved in several processes that contribute to carcinogenesis, including extracellular matrix (ECM)1 degradation, activation of proteases such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), and in E-cadherin cleavage (2.Palermo C. Joyce J.A. Cysteine cathepsin proteases as pharmacological targets in cancer.Trends Pharmacol. Sci. 2008; 29: 22-28Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). However, this evidence comes predominantly from in vitro studies and little is known about the in vivo substrates of these enzymes. Identification of the substrates of secreted cathepsins is therefore key to understanding their biological functions in cancer (15.Turk B. Turk D. Turk V. Protease signalling: the cutting edge.EMBO J. 2012; 31: 1630-1643Crossref PubMed Scopus (221) Google Scholar). Membrane-anchored proteins, including receptors, growth factors, cytokines, and adhesion proteins, have a major role in cancer progression. A general mechanism for their functional regulation is the release of their extracellular domains through limited proteolysis, also known as ectodomain shedding (16.Garton K.J. Gough P.J. Raines E.W. Emerging roles for ectodomain shedding in the regulation of inflammatory responses.J. 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Here we show that extracellular cathepsins can act as sheddases and release protein ectodomains from the surface of cancer cells. Among the identified substrates are cell adhesion proteins and membrane receptors. We confirmed cathepsin-mediated shedding of these substrates in cell based models as well as in vivo in a mouse model of pancreatic cancer. Collectively, this work has identified possible molecular mechanisms by which cysteine cathepsins may regulate cancer progression. Human cathepsin B was expressed in E. coli and purified as described in (21.Rozman J. Stojan J. Kuhelj R. Turk V. Turk B. Autocatalytic processing of recombinant human procathepsin B is a bimolecular process.FEBS Lett. 1999; 459: 358-362Crossref PubMed Scopus (104) Google Scholar). Human cathepsins S and L were expressed in the methylotrophic yeast P. pastoris and purified as described in (22.Mihelic M. Dobersek A. Guncar G. Turk D. Inhibitory fragment from the p41 form of invariant chain can regulate activity of cysteine cathepsins in antigen presentation.J. Biol. Chem. 2008; 283: 14453-14460Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Cancer cell lines MDA-MB-231, MCF-7, PANC-1, HT-144, and T98-G were grown to confluence in Dulbecco's modified Eagles media supplemented with 10% fetal bovine serum (FBS), 1% glutamine and penicillin/streptomycin (Lonza, Verviers, Belgium). U937 cells were grown in RPMI (Roswell Park Memorial Institute, Buffalo, NY) media supplemented with 10% FBS, 1% glutamine and 1% penicillin/streptomycin (Lonza). U937 cells were plated in a 12-well culture plate (7 × 105 cells per well) and differentiated into macrophages with 30 nm phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO) for 48 h, followed by 24 h of recovery without PMA in the completed RPMI media. For a coculture experiment, 1.4 × 106 of detached MDA-MB-231 cells were resuspended in PBS buffer (Lonza) (pH 6.0, 0.5 mm dithiothreitol (DTT) (Fluka Biochemica)) and plated in 12-well cell culture dish containing differentiated U937 cells (0.7 × 106 cells per well). Cells were detached using an enzyme-free cell dissociation solution (Millipore, Darmstadt, Germany). Per condition, thirty million cells were incubated in parallel in 500 μl of PBS (Lonza) (pH 6.0, containing 0.5 mm DTT (Fluka Biochemica, Steinheim, Germany)), with human recombinant cathepsin L, S, or B (1 μm and 0.2 μm) or with E-64-inhibited cathepsin (1 μm cathepsin L, S, or B incubated in PBS containing 20 μm broad spectrum cysteine cathepsin inhibitor E-64 (Peptide Institute, Osaka, Japan) for 1 h at 37 °C) serving as a negative control for 1 h at 37 °C, followed by collection of the supernatant (sample was centrifuged for 5 min at 500 × g, supernatant was removed and centrifuged again for 5 min at full speed). Residual cathepsin activity was blocked by the addition of E-64 to each sample (20 μm final concentration). In the microscopy experiments, MDA-MB-231 cells were first grown to confluence, thereby establishing contacts with neighboring cells, in contrast to most other experiments herein that were performed on detached cells. Similar protease concentrations were used in vitro in recent degradomic studies to identify putative substrates of various matrix metalloproteases, caspase-3, and aspartic cathepsins D and E (23.Impens F. Colaert N. Helsens K. Ghesquiere B. Timmerman E. De Bock P.J. Chain B.M. Vandekerckhove J. Gevaert K. A quantitative proteomics design for systematic identification of protease cleavage events.Mol. Cell. Proteomics. 2010; 9: 2327-2333Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24.Plasman K. Van Damme P. Kaiserman D. Impens F. Demeyer K. Helsens K. Goethals M. Bird P.I. Vandekerckhove J. Gevaert K. Probing the efficiency of proteolytic events by positional proteomics.Mol. Cell. Proteomics. 2011; 10M110.003301Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 25.Prudova A. auf dem Keller U. Butler G.S. Overall C.M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.Mol. Cell. Proteomics. 2010; 9: 894-911Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 26.Starr A.E. Bellac C.L. Dufour A. Goebeler V. Overall C.M. Biochemical characterization and N-terminomics analysis of leukolysin, the membrane-type 6 matrix metalloprotease (MMP25): chemokine and vimentin cleavages enhance cell migration and macrophage phagocytic activities.J. Biol. Chem. 2012; 287: 13382-13395Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The obtained protein supernatants were separated on a 12.5% SDS-PAGE gel (Lonza). The gel was stained with Comassie Brilliant Blue and each protein lane was cut into six bands (six samples), which were destained using destaining solution (25 mm NH4HCO3 (Fluka Biochemica), 50% acetonitrile (JT Baker, Deventer, The Netherlands)). The gel pieces were washed with acetonitrile and vacuum dried before rehydrating the gel pieces in reducing solution (10 mm DTT (Fluka Biochemica), 25 mm NH4HCO3) followed by an incubation at 56 °C for 45 min before exchanging to an alkylating solution (55 mm iodoacetamide (Amersham Biosciences, Little Chalfont, UK), 25 mm NH4HCO3). The reaction was allowed to proceed in the dark for 30 min before washing the gel pieces with 25 mm NH4HCO3 using intense vortexing. Afterward, the gel pieces were washed with acetonitrile and vacuum dried before rehydrating in 80 μl of trypsinization buffer (25 mm NH4HCO3) containing 1 μg of sequencing-grade modified porcine trypsin (Promega, Madison, WI) per sample. The gel pieces were allowed to rehydrate on ice for 15 min before adding more trypsinization buffer to cover the gel pieces completely. Trypsin was then left to digest overnight at 37 °C. Next day, the trypsin solution was collected and remaining peptides were extracted from the gel pieces using extraction solution (50% acetonitrile, 5% formic acid (JT Baker)). Trypsin solution was added to extraction solution and concentrated by vacuum drying to a final volume of about 20 μl. Liquid chromatography tandem MS (LC-MS/MS) analyses were performed with an EASY-nanoLC II HPLC unit (Thermo Scientific, San Jose, CA) coupled to an Orbitrap LTQ Velos mass spectrometer (Thermo Scientific). The peptide sample was first loaded on a C18 trapping column (Proxeon EASY-ColumnTM, 2 cm (length), 100 μm internal diameter, 5 μm 120Å, C18-A1 beads) and then separated on a 10 cm long C18 PicoFritTM AQUASIL analytical column, (75 μm internal diameter, 5 μm 100 Å, C18 beads) (New Objective, Woburn, MA) using forward flushing. Peptides were eluted with a 90 min linear gradient of 5–50% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–2000 m/z and 30,000 resolution. MS/MS spectra were obtained by higher-energy collision dissociation fragmentation (normalized collision energy at 35) of the nine most intense precursor ions from the full MS scan. Dynamic exclusion was enabled with repeat count of 2 and 120 s exclusion time. The database search and quantification by spectral counting were performed using the MaxQuant proteomics software (version 2.0.18), with imbedded Andromeda search engine (27.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9210) Google Scholar, 28.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3467) Google Scholar). Search was performed against the human IPI protein database v.385 (89,952 sequences, 36,291,020 residues), using the trypsin cleavage specificity with maximum two missed cleavages. Carbamidomethylation of cysteines was set as static, whereas methionine oxidation and N-terminal acetylation were set as dynamic modifications. Precursor and fragment mass tolerances were set at 6 and 20 ppm. Reversed database search was performed and false discovery rate (FDR), was set at 1% for peptide and protein identifications. In MaxQuant, identified peptides are assigned to protein groups rather than proteins and groups with at least two identified peptides were considered as positive identifications. Relative quantification of identified proteins was performed by spectral counting of their razor and unique peptides. This approach is known to reliably detect differences in protein abundance if at least a twofold difference in spectral count is observed (29.Liu H. Sadygov R.G. Yates 3rd, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2069) Google Scholar). To minimize the number of false positives especially in the case of proteins with lower spectral counts (5–10 counts per protein), only proteins with at least threefold spectral count change were considered to have significantly altered abundance (30.Old W.M. Meyer-Arendt K. Aveline-Wolf L. Pierce K.G. Mendoza A. Sevinsky J.R. Resing K.A. Ahn N.G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics.Mol. Cell. Proteomics. 2005; 4: 1487-1502Abstract Full Text Full Text PDF PubMed Scopus (1018) Google Scholar). Protein spectral count ratios (SCRs) were computed by the division of spectral counts in cathepsin treated sample with spectral counts in negative control (sample treated with inhibited cathepsin). Spectral counts of all proteins identified in compared data sets were increased by 1 in order to avoid division by zero. Gene Ontology analysis was performed by g:Profiler web interface (31.Reimand J. Kull M. Peterson H. Hansen J. Vilo J. g:Profiler–a web-based toolset for functional profiling of gene lists from large-scale experiments.Nucleic Acids Res. 2007; 35: W193-200Crossref PubMed Scopus (747) Google Scholar). MDA-MB-231 cells were grown to confluence and detached with enzyme-free cell dissociation solution (Milipore). A total of 7.2 million cells per parallel were incubated in 120 μl PBS (Lonza) (pH 6.0, with 0.5 mm DTT (Fluka Biochemica)), with added human recombinant cathepsin L (0.2 or 1 μm) or inhibited cathepsin (1 μm cathepsin 1 h preincubated with 20 μm E-64 (Peptide Institute)) as a negative control. After 1 h of incubation at 37 °C the cells were washed with PBS. Annexin V-PE and PI were used to determine the phosphatidylserine exposure and the loss of membrane integrity according to the manufacturer's instructions (BD PharmigenTM, Erembodegem, Belgium). Analysis was made with a FACSCalibur flow cytometer (Becton Dickinson) and the CellQuest software. The supernatants used in the sample preparation for mass spectrometry were also analyzed on 12% SDS-PAGE gels followed by immunoblotting. For the detection of the shed proteins in the supernatant, antibodies against neuropilin 1 (R&D Systems, Minneapolis, MN) (sheep polyclonal, dilution 1:500), transferrin receptor protein 1 (R&D Systems, goat polyclonal, dilution 1:500), CD44 (R&D Systems, mouse monoclonal, dilution 1:500), ALCAM (R&D Systems, goat polyclonal, dilution 1:500), L1CAM (R&D Systems, goat polyclonal, dilution 1:500), plexin B2 (R&D Systems, sheep polyclonal, dilution 1:200), MUC18 (R&D Systems, goat polyclonal, dilution 1:500), and ephrin type A receptor 2 (Novus Biologicals, Abingdon, UK) (rabbit polyclonal, dilution 1:5000) were used. Cathepsins B and S in the RIP1-Tag2 tumors were detected by antibodies against cathepsin B (R&D Systems, goat polyclonal, dilution 1:2000) and cathepsin S (R&D Systems, goat polyclonal, dilution 1:500). K-ras was detected using sheep polyclonal antibodies (R&D Systems, dilution 1:200). Secondary antibodies were used at 1:5000 dilutions and ECL kit was used for detection (GE Healthcare). The cleavage of the fluorogenic substrate Z-Phe-Arg-AMC (Bachem, Bubendorf, Switzerland) was used to determine cathepsin activity in the coculture supernatants. Fifty microliters of each sample were mixed in a 96-well plate with buffer (100 mm phosphate buffer, 1 mm EDTA, 1 mm dithiothreitol, and 0.1% (w/v) polyethyleneglycol, pH 6.0) to the final volume of 90 μl. After 15 min incubation at 37 °C, substrate was added to a final concentration of 10 μm and its hydrolysis continuously measured in a 96-well plate reader (Tecan Safire, Mannedorf, Switzerland) at excitation and emission wavelengths of 370 and 460 nm, respectively. In an additional experiment, the active concentration of the cathepsins in the supernatants was determined by active site titration with E-64 (32.Rozman-Pungercar J. Kopitar-Jerala N. Bogyo M. Turk D. Vasiljeva O. Stefe I. Vandenabeele P. Bromme D. Puizdar V. Fonovic M. Trstenjak-Prebanda M. Dolenc I. Turk V. Turk B. Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.Cell Death Differ. 2003; 10: 881-888Crossref PubMed Scopus (181) Google Scholar, 33.Turk B. Krizaj I. Kralj B. Dolenc I. Popovic T. Bieth J.G. Turk V. Bovine stefin C, a new member of the stefin family.J. Biol. Chem. 1993; 268: 7323-7329Abstract Full Text PDF PubMed Google Scholar). For activity probe labeling, differentiated U937 cells (7 × 105 cells per well) and MDA-MB-231 cells (1.4 × 106 cells per well) were grown separately and in a coculture on a 12-well culture plate. Cells were incubated for 2 h at 37 °C in PBS buffer (Lonza) (pH 6.0, 0.5 mm DTT (Fluka Biochemica)) in the presence of 10 μm DCG-04 probe. After the incubation, the PBS buffer was collected and centrifuged for 5 min at 500 × g and 30 min at maximum speed. The supernatant was then used for immunological detection of biotinylated cathepsins. MDA-MB-231 and differentiated U937 cells were plated in a coculture as described in the cell culture section. After 2 h incubation at 37 °C, the PBS buffer was collected and centrifuged once for 5 min at 500 × g and once for 5 min at the maximum speed. The buffer was then used for imunodetection of the shed ectodomains by Western blotting. As a negative control, differentiated U937 and MDA-MB-231 cells were pretreated with E-64 (25 μm) (Peptide Institute), GM6001 (10 μm) (Calbiochem, San Diego, CA) or batimastat (10 μm) (Tocris Bioscience, Bristol, UK). Cells were pretreated with inhibitor for 2 h before the coculturing and same inhibitor concentration was also present during the coculture Additionally, differentiated U937 and MDA-MB-231 cells were also incubated with PBS buffer (Lonza) for 2 h (pH 6.0, 0.5 mm DTT (Fluka Biochemica)) in the presence and absence of 50 μm E-64, 10 μm GM6001, and batimastat. The cathepsin activity in the coculture and macrophage supernatant was monitored as described in the Cathepsin activity assay. MDA-MB-231 cells were grown to confluence followed by starvation in FBS-free media for 48 h with 1% nutridoma (Roche, Basel, Switzerland) added. Cells were subsequently detached with a cell dissociation solution. A total of 4.2 × 104 cells per experimental setup were incubated in 100 μl of PBS buffer (Lonza) (pH 6.0 or 7.4, with 0.5 mm DTT (Fluka Biochemica)) with cathepsin B, S, or L (0.2 μm and 0.02 μm) or inhibited cathepsins (0.2 μm cathepsin, 1 h preincubated with 20 μm E-64 (Peptide Institute)) as a negative control. After 10 min at 37 °C the buffer with cathepsin was removed and the cells were put into a 24-well cell culture insert (BD Falcon Cell culture inserts, 8 μm pore size) to assay migration. 10% FBS (Lonza) was used as chemoattractant. Cells that migrated through the pores of the insert were counted after 40 h. To check whether the difference between the samples' arithmetic means were statistically significant, we used a homoscedastic (two samples with equal variance) Student's t-test with two-tailed distribution. If the probability associated with the Student's t-test (p value) was lower than 0.05, the sample was considered statistically significantly different from the control and was marked with an asterisk (*). The same statistical approach was applied also for the invasion assay below. MDA-MB-231 cells were grown to confluence, followed by starvation in the FBS-free medium containing 1% nutridoma (Roche) for 48 h. The cells were detached with a cell dissociation solution. A total of 4.2 × 104 cells per experimental condition was incubated in 100 μl of PBS buffer (Lonza) (pH 6.0 or 7.4, with 0.5 mm DTT (Fluka Biochemica)) with cathepsin B, S, or L (0.2 μm and 0.02 μm) or inhibited cathepsin (0.2 μm cathepsin B, S, or L pre-incubated with 20 μm E-64 (Peptide Institute) for 1 h) as a negative control. After 10 min at 37 °C the buffer was removed and the cells were put into a 24-well cell culture insert with an 8 μm pore size polycarbonate membrane coated with ECMatrixTM (Milipore QCMTM cell invasion assay). Ten percent FBS (Lonza) was used as a chemoattractant. The invaded cells that migrated through the ECM layer were dissociated from the membrane and detected by CyQuant GR® dye using a fluorescence plate reader. Tumors were prepared from wild-type, cathepsin B-deficient and cathepsin S- deficient RIP1-Tag2 mice as described previously (4.Gocheva V. Zeng W. Ke D. Klimstra D. Reinheckel T. Peters C. Hanahan D. Joyce J.A. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis.Genes Develop. 2006; 20: 543-556Crossref PubMed Scopus (440) Google Scholar). Three wild-type tumors (28 mm3, 33 mm3, and 65 mm3) and three tumors from each cathepsin knockout mouse (1.2 mm3, 4.2 mm3, and 6.5 mm3, respectively, from cathepsin B-deficient mice, and 1.2 mm3, 1.8 mm3, and 2.4 mm3, respectively, from cathepsin S-deficient mice) were used in the study, each originating from a different mouse. In agreement with previous results, tumors from cathepsin-deficient mice were considerably smaller than wild-type tumors (4.Gocheva V. Zeng W. Ke D. Klimstra D. Reinheckel T. Peters C. Hanahan D. Joyce J.A. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis.Genes Develop. 2006; 20: 543-556Crossref PubMed Scopus (440) Google Scholar). Samples were weighed and dounce homogenized on ice in an ice-cold PBS buffer (Lonza) containing 0.5 mm EDTA (Serva). The buffer volume was adjusted according to the tumor mass (700 μl buffer per 1 mg tumor mass). The homogenate was centrifuged at 4 °C for 30 min at the maximum speed. The supernatant was used for the immuno-detection of substrate ectodomains by Western blotting. Actin was used as a loading control. MDA-MB-231 cells were grown to confluence in a 24-well plate. They were washed twice with PBS. In each well the PBS was then replaced with 500 μl PBS (pH 7.4) with 0.5 mm DTT and active cathepsin B, L, or S at the final concentration of 1 μm. No cathepsins were added to the control wells. The cells were observed under the light microscope (Olympus IX81, 100× magnification). The pictures were taken immediately after addition of the cathepsins (t = 0), 3 (t = 3 min), and 10 min (t = 10 min) after the incubation. MDA-MB-231 cells were grown to confluence in Dulbecco's modified Eagles media. They were detached with enzyme-free cell dissociation solution (Milipore). Cells (30,000 cells per setup) were incubated in 300 μl PBS (Lonza) (pH 6.0, with 0.5 mm DTT (Fluka Biochemica)), with added human recombinant cathepsin L or S (0.05 μm) or inhibited cathepsin (0.05 μm cathepsin 1 h preincubated with 20 μm E
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