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Extracellular Matrix Degrading Enzymes at the Prostasome Surface

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Extracellular Matrix Degrading Enzymes at the Prostasome Surface Prostate Cancer and Prostatic Diseases (2005) 8, 344–348 & 2005 Nature Publishing Group All rights reserved 1365-7852/05 $30.00 www.nature.com/pcan Extracellular matrix degrading enzymes at the prostasome surface I Bellezza1, MC Aisa2, R Palazzo1, E Costanzi1, E Mearini3 & A Minelli1* 1Dipartimento Medicina Sperimentale Scienze Biochimiche, Sezione Biochimica Cellulare, Perugia, Italy; 2Dipartimento Medicina Interna, Sezione Biochimica Applicata Scienze Nutrizionali, Perugia, Italy; and 3Diparimento Specialita` Chirurgiche, Sezione di Urologia, Universita` di Perugia, Perugia, Italia Prostasomes, prostatic secretory vesicles found in human ejaculates, were analyzed to verify the existence at their surfaces of enzymes involved in the degradation of the extracellular matrix. Findings were compared with those of prostasomes isolated from two human adenocarcinoma cell lines that reflect clinical features and molecular pathways of androgen-insensitive and hormone-responsive prostate cancer. Our aim was to determine whether neoplastic transformation is accompanied by changes of glycosidase and protease activities. Our results show that decreases of dipeptidyl peptidase IV and increases of urokinase plasminogen activator and cathepsin B are consistent with the clinical features of the cell lines, whereas increases of glycosidase activities seem to be of scarce biological significance. Prostate Cancer and Prostatic Diseases (2005) 8, 344–348. doi:10.1038/sj.pcan.4500828; published online 30 August 2005 Keywords: PC3; LNCaP; glycosidases; proteases Introduction istics and the presence of saturated and monounsatu- rated fatty acids make the prostasome membrane highly The human prostate gland has a remarkably high ordered and rigid.1 A proteomic analysis has shown the incidence of neoplastic disease and prostate cancer is existence at the prostasome surface of at least 139 one of the most commonly detected male cancers that proteins including enzymes (35%), transport/structural result in a high incidence of mortality. At present, proteins (19%), GTP proteins(14%), chaperone proteins prostate cancer is an enormous medical and socio- (6%), signal transduction proteins (17%) and novel economic problem that is going to worsen because of proteins (9%).3 Some of these components have already the aging of the population.1 been shown in the prostasomes.1 Prostasomes are present in human seminal plasma as Dipeptidyl peptidase IV (DPPIV), which has an prostate secretory vesicles, and an ordinary exocytotic extremely high specific activity in prostasomes,4 has event involving the fusion of the membranes that been identified as the antigen of a monoclonal anti- surround the storage vesicle and the prostatic secretory prostasome antibody, that is, mAb 78.5 In prostatic cancer cell is the probable mechanism by which they are tissues, there is an increased activity of DPPIV that may released. Discovered more than 20 years ago by Ronquist influence the metabolism and encourage cancer growth,6 and Brody,2 they are now biochemically well character- and in the highly invasive 1-LN human prostate tumor ized. Prostasomes are membrane-surrounded organelles cell line, DPPIV is connected with the activation of with an unusual membrane lipid composition and a plasminogen and the expression of matrix metallopro- striking cholesterol/phospholipid ratio. They contain a teinase (MMP)-97 followed by the degradation of large amount of sphingomyelin and a small amount of extracellular matrix components and cancer invasion. polyunsaturated phosphatidyl-choline. These character- The invasive behavior of prostate cancer involves the increased expression of enzymes that can decompose parts of the extracellular matrix, thus permitting passage *Correspondence: A Minelli, Dipartimento Medicina Sperimentale of malignant cells through basement membranes and Scienze Biochimiche, Sezione Biochimica Cellulare, Via del giochetto stromal barriers. Among these enzymes, glycosidases,8 06123 Perugia, Italia. E-mail: [email protected] 9 Received 23 June 2005; revised 27 July 2005; accepted 1 August 2005; cathepsin B, plasminogen activator (PA) and MMPs 10 published online 30 August 2005 play important roles. Malignant cells depend on these Prostasome glycosidases and proteases I Bellezza et al enzymes to disrupt basement membranes, invade Protein concentration was determined by the Bio-Rad 345 neighboring tissues and metastasize to different organs. Protein Assay kit (Bio-Rad Lab, Hercules, CA, USA). Since cells either of benign prostate tissue or of prostate Unless otherwise stated, all the reagents were purchased carcinoma show similarities in synthesis, storage and from Sigma (St Louis, MO, USA). release of prostasomes,11,12 it is possible that several prostasomal properties, although mainly involved in assisting the fertilization of sperm cells, can also Peptidoglycan hydrolyzing enzyme assays promote the transition from a normal to a neoplastic cell and help the neoplastic prostate cells to survive as Activities of N-acetyl- b -D-hexosaminidase (E.C.3.2.1.52), metastases, although it has recently been shown that b-D-galactosidase (E.C.3.2.1.23), a-D-mannosidase (E.C.3. 13 prostasomes inhibit in vivo angiogenesis. Human 2.1.24), b-D-mannosidase (E.C.3.2.1.25) were measured prostate cancer cell lines also express a granular according to Costanzi et al15 using the appropriate 11 secretion with properties similar to prostasomes. fluorigenic substrates at concentrations of 1 mM for Both PC3 cells, a line that reflects critical features N-acetyl-b-D-hexosaminidase and 3 mM for the other and common molecular pathways of clinical andro- enzymes. Intensity of fluorescence was measured at gen-insensitive prostate cancer, and LNCaP cells, a line 446 nm (excitation at 360 nm). The concentration of that represents the hormone-responsive tumor model, product was obtained by reference to a solution of contain prostasome-like organelles. Although prosta- 4-methyl-umbelliferone. For all enzyme activities as- somes are the secretory product of the normal prostate sayed under these conditions, total substrate hydrolysis gland, of prostate cancer of different grading and of was always below 5%. In all measurements, blank human prostate cancer cell lines, and although it has substrates and blank samples were used, and all recently been hypothesized that lysosomal and prosta- determinations were made in duplicate. One milliunit somal hydrolytic enzymes and redox processes can of activity is the amount of enzyme that hydrolyzes initiate prostate cancer,14 so far no studies have been 1 nmol of substrate per minute at 371C. All activities carried out to investigate whether enzymes involved in (mU/ml) were normalized to protein concentration (mg/ the degradation of the extracellular matrix are associated ml) of the sample used. with these organules. In this study, we have shown the presence of such activities on prostasomes from healthy donors and compared the findings with those obtained Proteolytic enzyme assays with prostasomes isolated from PC3 and LNCaP cell lines to find out whether neoplastic transformation is DPPIV (E.C.3.4.14.5) activity was assayed using Gly- followed by enzyme activity changes at the surface of Pro-p-NA as substrate as already described.16 prostasomes. Cathepsin B (E.C.3.4.22.1) activity and PA (E.C.3.4.21. 68/73) activities were measured according to Aisa et al17 using Z-Arg-Arg-NH-Mec (Bachem, Switzerland) and Z- Gly-Gly-Arg-MEC (Bachem, Switzerland), as specific Materials and methods synthetic substrates. Proform of cathepsin B, activated in vitro by treatment with pepsin,18 was determined after Materials a 60 min incubation at 371C with 20 mg/ml pepsin in Ejaculates were obtained after a 3-day abstinence from 50 mM sodium formiate, pH 3.0, as latent activity that healthy donors. The parental prostate carcinoma cell represents the difference between native activity mea- lines PC3 and LNCaP were purchased from American sured in the absence of activation and the total activity Type Culture Collection (ATCC, Manasas, VA, USA) and measured subsequent to activation with pepsin. In were cultured in RPMI medium (GIBCO, Grand Island, preliminary experiments, pepsin did not hydrolize the NY, USA) supplemented with 10% fetal bovine serum synthetic substrate under the above-mentioned assay and 1% penicillin/streptomycin. Cells were maintained conditions. Fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of in 5% humidified CO2 chamber, subculture 1:6 split once per week by trypsinization. In each experiment, cells 460 nm for cathepsin B activity and at an excitation were seeded at 7.8 Â 103/cm2. wavelength of 383 nm and an emission wavelength of 455 nm for PA activity. Bz Arg-NH2 HCl, a competitive inhibitor of urokinase plasminogen activator (uPA), was Prostasome preparation used at 6 mM to distinguish between tPA and uPA. The product concentration was obtained by reference to a Prostasomes were isolated from pooled ejaculates ac- solution of 7-amino-4-methylcoumarin. cording to conventional procedure.2 Metalloproteinase (E.C.3.4.24.7) activity was assayed Prostasomes from PC3 and LNCaP cell cultures were with Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 according obtained by following the conventional procedure with to Knight et al.19 The intensity of fluorescence was slight modifications. Briefly, cells were recovered at measured at 393 nm (excitation at 328 nm). The concen- confluence with 0.25% trypsin-EDTA, washed with tration of the
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