Millimaggi 27_7

Although it has been shown that the cross-talk between osteoblasts and tumor cells stimulates proliferation and invasion of prostate carcinoma (PCa) cells, the molecular mechanisms underlying this event are largely unknown. In this study, we demonstrated that the PCa cells, PC3, derived from bone metastasis, undergo changes of their invasive capability if grown in the presence of osteoblast-derived conditioned media (OBCM). Specifically, they were able to organize tridimensional structures in Matrigel, such as large branching colonies, tube-like structures and clusters of proliferating cells, after treatment. At the ultrastructural level, we observed that PC3 cells grown in the presence of OBCM presented an increment of membrane activity with a blast of shed membrane vesicles from the cell surface. After 6 h of incubation, protein content was approximately 5-fold more elevated in vesicles isolated from PC3 cells cultured in OBCM than in unstimulated cultures. Gelatin zymography of vesicles collected from OBCM-treated PC3 cells showed an increment of lytic bands of MMP family members identified as proenzymatic and active forms of gelatinase A (MMP-2) and gelatinase B (MMP-9). By casein-plasminogen zymography, this latter culture also presented an elevated level of highmolecular weight urokinase plasminogen activator (HMWuPA). Purified vesicles from OBCM-treated PC3 cells incubated with Matrigel cleaved its components more efficiently than vesicles from untreated PC3 cells. Collectively, these findings indicate that osteoblasts produce factor/s able to modify the invasive capability of prostate cancer cells, increasing the amount of shed vesicles and of their associated lytic enzymes. Introduction Prostate Cancer (PCa) metastasizes to bone with high frequency producing osteoblastic lesions. Several authors have demonstrated that PCa metastatization to bone is mainly dependent on the peculiar characteristics of the target organ. Following haematogenous dissemination, PCa cells are able to form a synergistic relationship with bone environment, creating favorable conditions for their development and growth (1). Since bone is continuously remodelled through the action of osteoblasts and osteoclasts, its microenvironment is particularly rich in growth factors (2). Many of these factors, such as insulin-like growth factors, have a proliferative effect on PCa cells, driving the initial phase of tumor implantation. At the same time, tumors reaching a critical mass, may release additional factors that determine an aberrant response by bone cells. Increasing evidence suggests that the modulation of protease secretion by extracellular signals may play a key role in determining the ability of tumor cells to ‘seed’ in a particular target organ. Among the principal classes of matrix degrading proteases, metalloproteases (MMPs), a family of multidomain zinc-containing neutral endopeptidases, and plasminogen activators are involved in PCa progression. Specifically, PCa progression is associated with the expression of elevated levels of MMP-2 and MMP-9 (3,4). Moreover, the MMP-2 and MMP-9 detected in plasma from PCa patients could be important for diagnosis and prognosis of malignant progression (5). A causal role of MMPs in PCa metastasis formation is clearly demonstrated by the effective action of MMP inhibitors in reducing experimental bone metastasis in vivo (6). The plasmin cascade driven by urokinase (uPA) and tissue plasminogen activator (tPA) was shown also to be associated with extracellular matrix (ECM) degradation (7). In detail, uPA, through its protease activity, converts plasminogen to plasmin, a serine protease capable of activating a cascade of extracellular proteases at focal sites adjacent to the cell surface, thus determining the local degradation of the ECM. The uPA expression and uPA-mediated extracellular matrix degradation by PCa correlates with their metastatic behaviour (8), and elevated uPA levels in the serum from PCa patients correlate with increased numbers of bone metastases (9). INTERNATIONAL JOURNAL OF ONCOLOGY 28: 909-914, 2006 909 Osteoblast-conditioned media stimulate membrane vesicle shedding in prostate cancer cells DANILO MILLIMAGGI1, CLAUDIO FESTUCCIA1, ADRIANO ANGELUCCI2, SANDRA D'ASCENZO1, NADIA RUCCI1, SILVIO FLATI1, MAURO BOLOGNA3, ANNA TETI1, ANTONIO PAVAN1 and VINCENZA DOLO1 Departments of 1Experimental Medicine, 2Surgery, and 3Basic and Applied Biology, University of L'Aquila, 67100 L'Aquila, Italy Received July 27, 2005; Accepted September 1, 2005 _________________________________________ Correspondence to: Professor Vincenza Dolo, Dipartimento di Medicina Sperimentale, Università di L'Aquila, Via Vetoio-Coppito 2, 67100 L'Aquila, Italy E-mail: [email protected]