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 degrading 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 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- (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- (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 product was obtained by using a solution of 30 mM Tris-HCl, 130 mM NaCl, pH 7.6 and stored at MCA-NH2 as reference. Substrates and standards of À801. Then, cells were thawed, sonicated (10–10 s bursts, tested proteinases were from Bachem (Switzerland). 5 s intervals) with Virsonic 60 (Virtis Co., Gardiner, NY, For all enzyme activities assayed under these condi- USA) and centrifuged at 1500 g for 30 min and at 6000 g tions, total substrate hydrolysis was always below 5%. for 20 min to remove cell debris. The supernatant was One milliunit of activity was defined as the amount of then conventionally treated.2 enzyme that releases 1 nmol of product per hour under

Prostate Cancer and Prostatic Diseases Prostasome glycosidases and proteases I Bellezza et al

346 specified conditions. In all measurements, blank sub- LNCaP than in prostasomes from healthy ejaculates. b- strates and blank samples were used, and all determina- Galactosidase specific activity, although significantly tions were made in duplicate. All activities (mU/ml) altered compared to the control, was slightly modified were normalized to protein concentration (mg/ml) of the in LNCaP, whereas it was increased 10-fold in PC3 cell sample used. line. a-Mannosidase was significantly increased in cancer prostasomes by a factor ranging between 200 and b Statistical analysis 320-fold, whereas -mannosidase activity was increased 20-fold in LNCaP and not significantly modified in All results were confirmed in at least five separate PC3. experiments and expressed as mean7s.d. Data were Whether these results can be attributed either to an analyzed for statistical significance by Student’s t-test. P- enrichement of the enzymes at the surface of neoplasia- values of less than 0.05 were considered significant. derived prostasomes or to a higher enzyme activity is still unclear. It has been reported that membranes of tumor cells undergo modifications of membrane fluid- Results and discussion ity22 and that modifications of membrane fluidity can alter the activity of enzymes localized in distinct To verify whether prostasomes present at their surface membrane microdomains.23 Therefore, it may be plau- enzymes involved in the degradation of the extracellular sible to suggest that prostasomes derived from neoplastic matrix components and whether the same enzymes are cell lines have a modified fluidity that could be present in prostasomes extracted from PC3 and LNCaP responsible for the observed specific activity variations. cell lines, we analyzed a series of enzymes involved in On the other hand, since the granular secretions of the degradation of the glycanic and protein components human prostate cancer cell lines are likely to reflect of the extracellular matrix. features and common molecular pathways of clinical Peptidoglycan hydrolyzing enzymes are glycoproteins androgen-independent and androgen-sensitive prostate located primarily in , but also in extracellular cancer, they may reflect cellular properties that promote fluids such as serum, urine and synovial and cerebrosp- the transition from a normal to a neoplastic cell and help inal fluid. These enzymes undergo cotranslational the neoplastic prostate cells to survive as metastases. glycosylation on selected asparagine residues in the However, we think that the reported variations cannot be endoplasmic reticulum and achieve compartmentaliza- regarded as prostate cancer hallmarks. If these enzymes tion through binding of their phosphomannosyl moiety reflected the invasive and aggressive behavior of prostate to the phosphomannosyl receptor and translocation of cancer, the highest specific activities of the enzymes the enzyme–receptor complex to the .20 These degrading the peptidoglycan components of the extra- enzymes have also been found at the surface of human cellular matrix should have been found in PC3 cells. spermatozoa, and increasing evidence indicates a role for Major biological significance can be attributed to sperm glycosidases in mammalian fertilization: a-man- results of proteolytic enzymes involved in the degrada- nosidase has been associated with the binding of tion of the protein component of the extracellular matrix spermatozoa to eggs, b-galactosidase has been impli- (Table 2). cated in sperm maturation during epididymal transit, We have found that normal prostasomes, that is, and a-N-acetylglucosaminidase has been shown to play a isolated from ejaculates of healthy donors, present role in the penetration of egg coats.21 Although these proteolytic enzymes at their surfaces and that these peptidoglycan hydrolyzing activities are involved in activities are significantly modified in protasomes many physiological and pathological processes,14 studies isolated from prostate cancer cell lines. to verify their presence at the surface of human Cathepsin B is a lysosomal cysteine protease that, prostasomes have not been carried out and, to our under normal conditions, participates in the early and knowledge, this is the first report showing the presence late stages of protein degradation. Its role in the process of these enzymes at the prostasome membranes (Table 1). of cancer invasion and metastasis has been under Prostasomes from healthy ejaculates are characterized by discussion for more than 10 y, and it is still unclear glycan hydrolyzing activities that appear significantly whether increased cathepsin B activity in body tissues modified in prostasomes extracted from human cancer and liquids could be used as a biochemical marker of cell lines. All the specific activities were increased. b- tumor progression.9 The fact that cathepsin B membrane Hexosaminidase was approximately 3- and 12-fold higher in prostasomes extracted from the PC3 and Table 2 Proteases on prostasomes from different sources

Table 1 Peptidoglycan hydrolyzing activities on prostasomes from Enzyme Healthy LNCaP PC3 cell different sources ejaculates cell line line

Enzyme Healthy LNCaP PC3 cell DPPIV 501751 267747* 239743* ejaculates cell line line Procathepsin B 3.670.9 7378.0* 96710*,f Latent activity 1472.5 85711* 136718*,f b-Hexosaminidase 4.971.4 63.3713* 14.573.0*,f Cathepsin B 9.171.0 3474.0* 397 5* b-Galactosidase 0.1670.09 0.3570.1* 1.370.8*,f Total PA 1.470.3 3076.0* 4178.0*,f a-Mannosidase 3.3 Â 10À370.01 1.170.4* 0.6170.28* UPA 0.270.1 8.072.0* 1573.0*,f b-Mannosidase 0.0370.01 0.6970.26* 0.0770.06f MMPs 0.670.2 1.970.6* 4.871.2*,f

Values, expressed as specific activity (mU/mg protein), are means7s.d. Values, expressed as specific activity (mU/mgprotein), are means7s.d. (n ¼ 5); *Po0.05 vs healthy ejaculated prostasomes; fPo0.05 vs LNCaP- (n ¼ 5); *Po0.05 vs healthy ejaculated prostasomes; f Po0.05 vs LNCaP- isolated prostasomes. isolated prostasomes.

Prostate Cancer and Prostatic Diseases Prostasome glycosidases and proteases I Bellezza et al activity has been detected in metastatic melanoma those of ejaculated prostasomes. We have found 347 and colon cancer cells and that the inhibition of an elevation of PA activity that ranges between 20- extracellular activity by oral administration of a soluble and 30-fold, the highest increase found in PC3-derived selective enzyme inhibitor causes significant reduction prostasomes. In ejaculated prostasomes, uPA represents in the number and dimension of colon cancer metastases about 10% of the total PA, whereas it rises to approxi- in rat liver lobes24 seems to confirm the participation mately 30% in PC3 and LNCaP. uPA has been shown of this enzyme in the process of metastasis. On the to correlate with human prostate cancer metastasis contrary, there are reports in which cathepsin B activity and an elevated concentration of uPA is a strong was found unchanged in tumors compared to non-tumor indicator of poor prognosis. uPA and its receptor tissues and increased serum cathepsin B activity was (u-PAR) contribute to prostate cancer metastases by found in patients without neoplastic diseases. Some promoting extracellular matrix degradation and growth reports do not show a clear correlation between factor activation.33,34 carcinoma progression and cathepsin B.25 Nevertheless, Our results show that PC3- and LNCaP-derived the marked increase of cathepsin B specific activity prostasomes are characterized by increases of proteolytic that we have found in prostasomes derived from activities, which may degrade the extracellular matrix prostate cancer cell lines indicates that higher levels and control cell adhesion and migration through the of cathepsin B activity characterize prostatic neoplastic reorganization of the actin cytoskeleton. These data are cells. It is also to note that some cancer cells are able in agreement with the hypothesis that proteolytic to produce the proenzyme, procathepsin B.26 Our results degradation is one of the processes responsible for show that procathepsin B is significantly increased in invasion and metastatsis. It is plausible to assume that both cell lines compared to normal prostasomes, and these differences mirror the different cellular environ- that, after pepsin activation, cathepsin B activity is ments of neoplastic cells. significantly increased in PC3 compared to LNCaP cell lines. DPPIV is a multifunctional cell surface protein that, as well as having dipeptidase activity, is the major binding Conclusions protein for adenosine deaminase and binds extracellular 16,27 By showing that normal prostasomes have extra- matrix proteins such as fibronectin and collagen. This cellular matrix degrading enzymes and that these serine protease, aberrantly expressed in many cancers, enzyme activities are modified in prostate cancer, functions as a tumor suppressor by regulating the we think that prostasomes can be tentatively proposed activities of mitogenic peptides implied in cancer as potential tools for a non-invasive diagnosis of prostate development and its decreased expression has been cancer. linked to increases in invasion and metastasis. Progres- sion of benign prostate cancer to malignant metastasis is linked to increased production of basic fibroblast growth factor (bFGF), a powerful mitogen whose signaling Acknowledgements pathway can be blocked by DPPIV thus inhibiting the malignant phenotype of prostate cancer cells.28 Our We thank Fondazione Cassa di Risparmio di Perugia for results show a remarkable reduction of DPPIV activity, funding the research. We are grateful to Dr M Kerrigan more marked in PC3. These findings are in agreement (MA, Cantab) for helpful linguistic suggestions. with the tumor-suppressing function of this enzyme. They are also in agreement with data from literature showing that PA and MMPs are involved in the References proteolytic degradation of extracellular matrix compo- 29 nents. We have found increased activities of PA and 1 Ronquist G, Nilsson BO. The Janus-faced nature of prostasomes: MMPs in cancer cell-derived prostasomes. In normal their pluripotency favours the normal reproductive process and tissue, these enzymes are highly regulated, whereas they malignant prostate growth. Prostate Cancer Prostatic Dis 2004; 7: are upregulated in cancer cells, in particular prostate 21–31. cancer cells, by a mechanism that is as yet unknown. 2 Ronquist G, Brody I. The prostasome: its secretion and function Significant increases in active MMP-2 are associated with in man. 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Histol Histopathol 2004; 19: 1345–1351. invasive potential in many tumor types.31 It has already 7 Gonzalez-Gronow M et al. Interaction of plasminogen with dipeptidyl peptidase IV initiates a signal transduction mechan- been reported that membrane vesicles shed by PC3 cells ism which regulates expression of matrix proteinase-9 by have uPA, an enzyme activity that can promote tumor 32 prostate cancer cells. Biochem J 2001; 355: 397–407. invasion. In this study, we have analyzed the variation 8 Nobuhisa T et al. Heparanase expression correlates with of PA and uPA at the surface of prostasomes isolated malignant potential in human colon cancer. J Cancer Res Clin from PC3 and LNCaP and compared these findings with Oncol 2005; 131: 229–237.

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