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J. Agric. Food Chem. 2011, 59, 370–377 DOI:10.1021/jf102803a
Peptides from Lactobacillus Hydrolysates of Bovine Milk Caseins Inhibit Prolyl-peptidases of Human Colon Cells LUCIENNE JUILLERAT-JEANNERET,*,† MARIE-CLAUDE ROBERT,§ AND MARCEL A. JUILLERAT§ †
University Institute of Pathology, Centre Hospitalier Universitaire Vaudois (CHUV), and University of Lausanne (UNIL), Bugnon 25, CH-1011 Lausanne, Switzerland, and §Nestle´ Research Centre, Nestec Ltd., P.O. Box 44, CH-1000 Lausanne 26, Switzerland
Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from RS1-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-aminodipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects. KEYWORDS: Casein peptides; human colon cells; dipeptidyl prolyl-amino-peptidases; prolyl-oligopeptidases; matrix metalloproteinases
INTRODUCTION
Proteolytic post-translational modification is a major regulatory event for biologically active peptides. Proteolytic hydrolases (peptidases and proteases) are involved in several human diseases, such as cardiovascular, neoplasic, degenerative, or immune/ inflammatory disorders. Therefore, proteolytic activities are targets for the development of drugs aimed at treating human disorders, and selective inhibitors for these activities are in clinical use or under development. Inappropriate proteolytic activity and balance have been implicated in diseases of the human digestive tract and, in particular, the involvement of matrix metalloproteinases (MMPs) (1, 2) and serine proteases of the dipeptidyl peptidase IV (DPP IV)/prolyl oligopeptidase (POP) families (3-6). Both families of enzymes are able to hydrolyze prolyl-rich proteins or peptides, and their involvement in the development of diseases of the digestive tract, which are frequently associated with increased incidence of cancer, has been suggested. Many peptide hormones related to the functions of the digestive tract, including in type 2 diabetes for which DPP IV inhibitors are now in clinical use, have been suspected or demonstrated to be substrates of either DPP IV-like or POP-like peptidases. The involvement of prolyl-specific proteases in diseases other than diseases of the digestive tract has also been recognized (7-12). Many of the inhibitors developed for these *Address correspondence to this author at University Institute of Pathology, rue du Bugnon 25, CH-1011 Lausanne, Switzerland (phone þ41 21 314 7173; fax þ41 21 314 7115; e-mail lucienne.juillerat@ chuv.ch).
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Published on Web 12/02/2010
enzymes, either experimental for preclinical evaluation or now in clinical use for DPP IV inhibition in type 2 diabetes, have been developed on a prolyl scaffold (10-12). Several studies have demonstrated that bacteria-fermented milk can produce peptide inhibitors for proteolytic enzymes implied in human diseases, in particular for the metalloprotease angiotensin converting enzyme (ACE) involved in cardiovascular disorders (6, 13). Many of the casein-derived peptides discovered as ACE inhibitors, able to control hypertension in animal models, contain prolyl residues, including ours (13), and the first ACE inhibitors used in clinics, captopril and lisinopril, contain a prolyl scaffold. Epidemiological studies and experimental approaches have correlated ingestion of cultures of lactic bacteria or their fermented dairy products with a reduced risk of some cancers of the digestive tract (14-18). Fermented milk may act by inhibiting tumor growth directly (18) or by inhibiting the proteolytic activity of tumor-associated stromal cells, which have been shown to promote tumor progression (19, 20). In these previous studies the involvement of MMPs has mainly been investigated. Most of the active factors described in the literature were initially not present in the raw dairy materials but were produced de novo by lactic acid bacteria during milk fermentation (21-24). Only a few studies have identified and characterized the bioactive factor(s) (23-27). In the present study, we selected prolyl-containing caseinderived peptides form our previous library (13), initially defined as ACE inhibitors, and evaluated whether these peptides may also be inhibitors for MMPs or for prolyl-specific proteases. We show that synthetic peptides bearing such sequences selectively inhibited the enzymatic activities of several MMPs and prolyl-specific
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Article metalloproteinases and serine peptidases, without displaying cytotoxic or cytostatic effects, suggesting that casein-derived fragments may control proteolytic balance in the digestive tract at several levels of proteolytic activity. MATERIALS AND METHODS Casein Peptides. Casein digestion by lactic acid bacteria and the separation of the peptides in the hydrolysate by chromatrography, their characterization by mass spectrometry, their selection based on ACE inhibition, and their chemical synthesis have been previously described in detail (13). MMP-2, MMP-7, and MMP-9 Activity Determination. MMP-2 and MMP-7 were obtained from Calbiochem, La Jolla, CA; MMP-9 was obtained from Oncogene, SanDiego, CA; and DQ-gelatin was obtained from Molecular Probes, Leiden, Germany. One microliter of enzyme stock solution (MMP-2, 0.05 μg/μL; MMP-7, 1 U/μL; and MMP-9, 0.05 μg/μL) and increasing amounts of casein peptides in PBS for a final volume of 200 μL per well of a 96-well plate were incubated for 5 min at room temperature, and then 5 μL of substrate (DQ-gelatin, 1 mg/mL PBS, final concentration = 25 mM) solution was added and the increase in fluorescence was recorded for 120 min at 37 °C in a multiwell plate reader (CytoFluor Series 4000, PerSeptive Biosystems, Framingham, MA; λex = 485 nm, λem = 530 nm). Means and standard deviation were calculated. Graphs of enzyme inhibition versus inhibitor concentration were constructed, and IC50 values were determined graphically as the inhibitor concentration able to inhibit 50% of the enzyme activity. Cell Culture Conditions, Cell Proliferation, and Survival. Human HT-29 and SW480 colon carcinoma cells were obtained from ATCC (American Tissue Culture Collection, Manassas, VA) and were grown in DMEM medium, 4.5 g/L glucose, 10% FCS, and antibiotics (all from Gibco, Invitrogen, Basel, Switzerland). Two days before experiments, cells were seeded in 48-well plates (Costar, Corning, NY) and grown in complete culture medium. Cell proliferation was determined by quantifying DNA synthesis using [3H]-thymidine incorporation, essentially as previously described (28). Briefly, following treatments, 1 μCi/mL [3H]-thymidine (Amersham Pharmacia, D€ubendorf, Switzerland) was added for the last 2 h, and incorporation was quantified in a β-counter (Rackbeta, LKB) after precipitation with 10% trichloroacetic acid and solubilization in 0.1 M NaOH. Cell survival was determined using the AlamarBlue test. Briefly, following treatment, cells were exposed to 10% AlamarBlue (Serotec, D€usseldorf, Germany) added to the cell culture medium without medium change for 15 min, and then fluorescence increase was recorded for 30 min at 37 °C in a thermostated multiwell fluorescence reader (Cytofluor Series 4000, PerSeptive BioSystems) at λex/λem = 530/580 nm, respectively. Experiments were performed at least three times in triplicate wells, and means ( SD were calculated. Post-Pro-Cleaving Activities. To prepare cell extracts, the culture medium was removed and the cell layers were dissolved directly in the culture wells with PBS-0.1% Triton X-100 (Fluka, Buchs, Switzerland), then casein-derived peptides or synthetic inhibitors, then substrates were added to the extracts, and enzyme activity and inhibition were determined by increase of fluorescence. For nonextracted cells, after removal of the cell culture medium, casein-derived peptides or synthetic inhibitors, then substrates in PBS, were added directly on the cell layers and the increase of fluorescence was analyzed in intact nonhydrolyzed cells. In detail, for enzyme activity determination, culture medium was aspirated, then 200 μL per well of a 48-well plate of either PBS (for determination of enzyme activities in intact cells) or 0.1% Triton X-100 (Fluka) in PBS (for determination of enzyme activities in cell extracts) was added on the cell layer, and the enzymatic activities were determined as described below. Alternatively, for cell extracts, cells were grown to confluence in 10 cm Petri dishes (Falcon, Franklin Lakes, NJ) in complete culture medium, medium was aspirated, the cell layer was extracted in Triton-PBS, and the enzymatic activities were determined in the cell extracts. Peptidase activities were determined using Pro-AMC (prolyl-aminopeptidase activities, final concentration = 29 μM), Gly-Pro-AMC (prolylexopeptidase activities, final concentration = 24 μM), and Z-Gly-ProAMC (prolyl-endopeptidase activities, final concentration = 15 μM) fluorogenic substrates (all from Bachem, Bubendorf, Switzerland) either in
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cell extracts of HT-29 and SW480 cells or in intact cells. Casein-derived synthetic peptides (stock solutions of 10 mg/mL H2O), at the indicated concentrations, were added 5 min before the respective substrates. The increase in fluorescence was continuously recorded for 30 min at 37 °C in a fluorescence multiwell plate reader (CytoFluor; λex = 360 nm, λem = 460 nm). The synthetic prolyl-oligopeptidase inhibitor Y29794 and the dipeptidylaminopeptidase inhibitor P32/98 were purchased from Tocris Biosciences, Bristol, U.K., and were prepared as 20 μM stock solutions in DMSO and 25 mM stock solution in H2O, respectively. Each experiment was repeated at least three times. Means and standard deviation were calculated. Graphs of enzyme inhibition versus inhibitor concentration were constructed, and IC50 values were determined graphically as the inhibitor concentration able to inhibit 50% of the enzyme activity. Western Blotting and Immunohistochemistry of DPP IV/CD26 and FAP-r/Seprase. Human colon tissues were retrospectively selected from surgical samples of the University Institute of Pathology tissue bank, according to an accepted protocol, either fixed in 4% buffered paraformaldehyde and embedded in paraffin or frozen in liquid nitrogen and stored at -80 °C. For immunohistochemistry, paraffin-embedded samples were used. For Western blotting, fragments (15-25 mg of tissue) were retrieved from frozen samples. The frozen or fixed samples were retrieved at different distant locations of the samples representing the tumor tissue and the associated nontumoral tissue of the same patients. For immunohistochemistry, paraffin-embedded sections (5 μm) of surgical samples were deparaffinized in xylene and ethanol, antigen was retrieved by boiling for 4 min at pH 9 in the presence of EDTA, and endogenous peroxidase was inactivated in 3% hydrogen peroxide in methanol. Sections were incubated overnight at 4 °C with a murine monoclonal antihuman DPP IV/CD26 antibody (clone 2A6, eBiosciences, CBI Medical Products, Baar, Switzerland, catalog no. 14-0269-82, diluted 1:150) or a rabbit polyclonal antihuman FAP-R/seprase antibody (Abcam, Cambridge, U.K., catalog no. ab53066, diluted 1:150) and subsequently exposed to anti-mouse or anti-rabbit polymer peroxidase complex (EnVision, Dako, Baar, Switzerland), according to the manufacturer’s instructions. Peroxidase activity was visualized using 0.035% diaminobenzidine (Fluka) as a chromogen, and slides were counterstained with hematoxylin. Reactions performed without the primary antibody were used as controls for nonspecific reactions (not shown). For Western blotting, proteins were extracted from tissue samples in 50 mM Tris-HCl. pH 7.2, 150 mM NaCl. 2 mM EDTA, 0.5% Triton X-100, 2 mM vanadate, and 50 mM NaF, and the protein content was quantified using the BCA kit (Pierce, Socochim, Le Mont, Switzerland) using bovine albumin as standard. The tissue extracts were submitted to electrophoresis performed either following boiling in SDS-β-mercapthethanol (DPP IV/ CD26) or without boiling and without β-mercaptoethanol for FAP-R/ Seprase, and the proteins were determined by Western blotting following transfer using the same murine monoclonal antihuman DPP IV/CD26 antibody (dilution 1:1000) as used for immunohistochemistry or with a rabbit polyclonal anti-human FAP-R/Seprase antibody (ABR, Affinity BioReagents, Golden, CO, catalog no. PA1-8467, dilution 1:3000) and peroxidase-labeled anti-mouse or anti-rabbit immunoglobulins (Sigma, Buchs, Switzerland), respectively, and ECL detection kit (Amersham, Otelfingen, Switzerland). To control for identical protein loading, the membranes were probed with anti-human β-actin antibody (results not shown). RESULTS
The previous disclosure of ACE inhibitors from casein hydrolysates and their chromatographic fractionation produced families of peptides of molecular weight
