Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation
Bone metastasis is often occurs in patients with prostate cancer. There is a vicious cycle for bone metastases involving prostate cancer cells, osteoblasts, and osteoclasts. Acting among those cells during the process of metastasis are several molecules such as bone morphogenetic proteins, platelet-derived growth factor, endothelin-1, matrix metalloproteases, vascular endothelial growth factor, transforming growth factor-β, and insulin-like growth factors. Cell-derived microvesicles are endogenous carriers transporting proteins, mRNAs and miRNAs between cells, which is a candidate for participation in the bone metastasis of these cells. Here, we demonstrated that prostate cancer cells in vitro released microvesicles into the culture medium (PCa-MVs), which was shown by electron microscopic study and nanoparticle tracking analysis. In this study, we found for the first time that these PCa-MVs enhanced osteoblast differentiation mainly through the delivery of PCa cell-derived v-ets erythroblastosis virus E26 oncogene homolog 1, which is an osteoblast differentiation related-transcriptional factor.
KeywordsProstate cancer Microvesicles Osteoblast differentiation Ets1 Osteoblastic bone metastasis
In many cell types, microvesicles (MVs) including shedding microvesicles (SMVs) and exosomes (EXOs) are released into the extracellular environment as a cell-to-cell communication tool (Bastida et al. 1984; Mack et al. 2000; Morel et al. 2004; Tesse et al. 2005; Martinez et al. 2006; Wysoczynski and Ratajczak 2009). In this study, we defined that MVs include SMVs and EXOs. These MVs contain receptor proteins, proteolytic enzymes, miRNAs, and mRNAs which are transferred into the target cell, and then affect various cell functions (Ratajczak et al. 2006; Baj-Krzyworzeka et al. 2006). In tumor cells, α-disintegrin and metalloproteinase (ADAM) and matrix metalloprotease (MMP) in MVs enhance the matrix digestion, which action facilitates the migration and metastasis of tumor cells (Gutwein et al. 2003; Mochizuki and Okada 2007). Moreover, anti-cancer drugs such as a doxorubicin decrease the levels of SMVs (Shedden et al. 2003). Thus, the MV transfer system is one of the important systems for tumor cell proliferation and progression. Osteoblastic bone metastasis in prostate cancer (PCa) patients is frequently observed as the disease progresses, and is related to high patient mortality and morbidity (Coleman 1997; Bubendoef et al. 2000; Roudier et al. 2003). In osteoblastic metastasis, a vicious cycle is established between the PCa cells and bone cells, i.e., osteoblasts and osteoclasts. PCa cells supply osteoblastic factors (e.g., bone morphogenetic proteins [BMPs], platelet-derived growth factor [PDGF], endothelin-1 [ET1]) and osteolytic factors (e.g., MMPs and vascular endothelial growth factor [VEGF]) to osteoblasts and osteoclasts, respectively, thereby allowing these cells to elaborate bone-derived growth factors (e.g., transforming growth factor-β [TGF-β], Insulin-like growth factors [IGFs]) for cell growth (Casimiro et al. 2009; Morrissey et al. 2010; Ibrahim et al. 2010). Zhang et al. (2009) showed in a recent report that heterotypic cell-to-cell contact between cells of the human prostate cancer cell line PC3 and bone marrow stromal cells (BMSC) proportionally up-regulates urokinase plasminogen activator (uPA) gene expression, which is associated with PC3 cell invasion. On the other hand, osteoblast-conditioned medium stimulates releasing of MVs from PCa cells (Festuccia et al. 1999; Millimaggi et al. 2006). Therefore, many signal exchanges are performed by direct or indirect contact between PCa cell and osteoblast during the process of bone metastasis. However, the effect of PCa-MVs on osteoblast function is not yet understood. In this study, we present evidence that PCa-MVs enhanced osteoblast differentiation mainly through the delivery of PCa-derived v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1), which is an osteoblast differentiation-related transcriptional factor.
Materials and methods
Reagents and materials
The 25× Complete® mixture of protease inhibitors purchased from Roche (Penzberg, Germany); and Phosphatase Inhibitor Cocktail® 1 and 2 from Sigma (St. Louis, MO, USA). Antibodies against human TSG101, CD9, CD81, PTHrP, Ets1, GAPDH, and mouse Ets1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against β-actin as an internal standard was purchased from Sigma. Anti-rabbit and anti-mouse antibodies conjugated with horseradish peroxidase and the chemiluminescence (ECL) kit was obtained from GE Health Science (GE Healthcare UK Ltd., Amersham Place, Buckinghamshire, UK).
PC3 and DU145 hormone-refractory human prostate cancer cells, and hormone-sensitive LNCaP cells were purchased from ATCC and cultured in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml streptomycin. MVs in FBS were excluded by ultra-centrifugation (250,000×g, 3 h) and filtration (0.45 μm). PrEC cells were used as normal human prostate epithelial cells. Murine pre-osteoblast cell line MC3T3-E1 was obtained from RIKEN Cell Bank (Tsukuba, Ibaraki, Japan) and cultured in phenol-red free α-MEM supplemented with 10 % heat-inactivated FBS, 100 U/ml of penicillin, and 100 μg/ml streptomycin. These cells were cultured in a humidified atmosphere of 5 % CO2 at 37 °C.
Electron microscopic observation
The PC3 and DU145 cells were harvested and rinsed with PBS, after which they were fixed for 30 min in 4 % paraformaldehyde and 1 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.4, PB), rinsed in PB, and postfixed in 1 % osmium tetraoxide for 30 min. After having been washed with PB, the cells were progressively dehydrated by passage through a 10 % graded series of 50–100 % ethanol and then cleared in QY-1 (Nissin EM, Tokyo, Japan). They were then embedded in Epon 812 resin (TAAB Laboratories Equipment, Reading, UK); subsequently, thin sections (70 nm thickness) were cut, stained with uranyl acetate and lead citrate, and then examined by transmission electron microscopy using an Hitachi-7650 (Hitachi, Tokyo, Japan).
Isolation of microvesicles from medium of PC3 or DU145 cell cultures
For preparation of MVs from PC3 or DU145 cells, the medium from either source was centrifuged at 1,500×g for 10 min to remove cells and other debris. These supernatants were then centrifuged at 250,000×g for 3 h at 4 °C. The centrifuged-microvesicles were resuspended in serum-free α-MEM and then filtered (0.45 μm). The filtered samples were quantified based on the protein levels by using the method of Bradford (BioRad, Hercules, CA, USA).
Nanoparticle tracking analysis (NTA)
Microvesicles were purified from the medium of PC3 cell cultures, as described above. The microvesicle samples after passage through the 1st filter (0.22 μm) of an ExoMir kit (Bioo Scientific, Austin, TX) were used for analysis. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd, UK) equipped with a blue laser (638 nm) illumination was used for real-time characterization of the vesicles. The results were presented at the average value of 2 independent experiments.
MC3T3-E1 cells were inoculated into 96-well plates (1 × 105 cells/ml, 100 μl/well; Nunc, Roskilde, Denmark) and cultured with or without PCa-MVs (2/100 μl of MEMα/well, equivalent protein conc. 20 μg/μl) for 3 days. After incubation, the treated cells were washed twice with PBS, and then fixed with EtOH for 10 min. The ALP activity was estimated by the using a TRAP & ALP double-staining kit (Takara Bio Inc. Ohtsu, Japan) according to the manufacturer’s protocol. As a positive control, MC3T3-E1 cells were treated with 100 ng/ml of BMP-2 (R&D Systems, Minneapolis, MN, USA).
Western blot analysis
Microvesicles was lysed with RIPA buffer containing the Complete protease inhibitor cocktail® (Roche, Penzberg, Germany). Samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes. The membranes were incubated with a primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunolabeled proteins were detected by using an ECL chemiluminescence kit (GE Healthcare, Piscataway, NJ, USA) and an LAS-4000 lumino-image analyzer (Fuji film, Tokyo, Japan).
The cells were washed twice with PBS and then fixed with 4 % paraformaldehyde for 15 min at room temperature. Fixed cells were washed twice with PBS containing 10 mM glycine (PBS-G) and then treated for 5 min at room temperature with PBS containing 0.1 % Triton X-100 (Sigma) (PBS-T). Subsequently, the cells were blocked with 3 % BSA for 10 min at room temperature. After incubation, the treated cells were incubated with primary antibody (anti-human Ets1) that had been diluted with PBS-G for 1 h at room temperature. After having been washed with PBS(−) containing 0.1 % BSA, the cells were incubated with secondary antibody (Alexa Fluor-488 Rabbit IgG, Invitrogen, Carisbad, CA, USA) for 30 min at room temperature. The nuclei and cell membranes of the treated cells were further stained with Hoechst33342 (Invitrogen) and Cell Mask Orange plasma membrane stain solution (Invitrogen) for 30 min. The cells were then mounted with a drop of mounting medium (Dako cytometion fluorescent mounting medium, Dako, CA, USA) and sealed with a coverslip. Photomicrographs of mounted cells were taken with a camera-equipped fluorescence microscope (Olympus BX-50, Tokyo, Japan).
Results and discussion
In summary, Ets1-containing MVs from hormone-refractory PCa cells were transferred into osteoblasts, and the Ets1 discharged into the cytoplasm functioned to induce differentiation. Our findings thus suggest that PCa-MVs acted as a cell-to-cell communication tool in osteoblastic metastasis.
The work was supported by Grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Ms. Ayako Irie (Quantum Design Japan, Inc.) for the analysis of NTA.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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