Abstract
Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles in the form of prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. The aim of the present investigation was to conduct a proteomic analysis of metastasis-derived prostasomes. Materials and Methods: We investigated prostasomes from vertebral metastases of prostate cancer by 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) protein characterization. Results: Twenty-five unique protein spots were identified by MALDI-TOF and another five proteins were determined by mass spectrometry (MS)/MS. Annexins A1, A3 and A5, as well as dimethylarginine dimethylaminohydrolase 1 were among the identified proteins. The annexins and dimethylarginine dimethylaminohydrolase 1 found in cancer-derived prostasomes can act, among others, as angiogenic factors and can increase the vascular development in the neighbourhood of the tumour. Conclusion: Cancer-derived prostasomes may play an important role in the interaction between tumour cells and their environment.
Epithelial cells lining the prostate acini release in a defined fashion organellar structures named prostasomes. Accordingly, membrane-surrounded prostasomes can exist extracellularly in vivo (1-3), albeit their authenticity was initially questioned. Similar extracellularly occurring organelles named exosomes were later described in other cell systems (4-6), giving further support to the idea of an in vivo existence of membrane-surrounded organelles that could be released into an extracellular fluid. Prostasomes as components of the seminal fluid raised the justified question about their relevance to reproductive health. The impact of prostasomes in a physiological context was highlighted by the finding that prostasomes were able to interact with spermatozoa (4). This fundamental extracellular reaction between a cell (spermatozoon) and organelles (prostasomes) was subsequently confirmed in different ways (8-10). Herewith, a membrane-to-membrane contact can be established between sperm cells and prostasomes in an orderly fashion. The corollary of such a contact is that a prostatic epithelial cell, from which the prostasomes originate, is able to mediate different abilities to many sperm cells that are of importance for their survival in the female genital tract and for their capability to reach and penetrate the zona pellucida for fertilization of the ovum. It should be kept in mind that on a stoichiometric basis the number of prostasomes in an ejaculate is manyfold compared to that of spermatozoa (11). Prostasomes promote forward motility of sperm cells and evoke hyperactive sperm cells (12, 13) and they are influential on the capacitation process and acrosome reaction (14-16). Furthermore, they are immunosuppressive (17, 18) and mitigate the effects of the complement system (19, 20), thus protecting the sperm cells in the female genital tract from an immunological attack.
Prostasomes play a role as an antioxidant factor in semen by interaction with polymorphonuclear neutrophils and inhibition of their NADPH oxidase activity (21, 22). Finally, prostasomes can act as antibacterial agents (23).
Not only normal prostate acinar secretory cells but also neoplastic prostate cells have the capacity to synthesize and export prostasomes to the extracellular environment (24, 25). Even poorly differentiated cells of prostate cancer metastases are able to produce and secrete prostasomes to the interstitial space surrounding the metastatic cell (26). It is a long known principle that tumour cells tend to exploit the host's physiological systems in order to gain support in terms of, for example, nutrition, growth and metastasis. It seems that several prostasomal abilities, which are developed to assist the fertilizing sperm cells, can also be promotive in the transition from a normal to a neoplastic cell and help the prostasome-producing, poorly differentiated cancer cells to survive as metastases.
The control of cell proliferation, differentiation and signal transduction pathways are generally mediated by protein kinases and phosphatases (27, 28) whose actions are modified by hormones, growth factors and mitogens (29). Such phosphorylation/dephosphorylation cycles of certain proteins have been recognized to play a pivotal role in regulating cell proliferation and malignant transformation. Recent work from our laboratory showed an up-regulation of protein kinase activities of prostasomes of malignant cell origin compared to those of prostasomes of normal cell origin (30). Other proteins and enzymes also exist that are related to malignant transformation and proliferation on the prostasome surface. They are up-regulated in prostasomes of malignant cell origin as protectin (31), tissue factor (32) and matrix metalloproteinase (33).
The aim of the present study was to investigate by 2D-electrophoresis and by ensuing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) or electrospray ionization (ESI) MS/MS the identity of different protein spots derived from prostasomes originating from cells of vertebral metastases of prostate cancer.
Materials and Methods
Samples and patients. Skeletal metastases were surgically removed from 12 patients with clinically established metastatic prostate cancer at Uppsala University Hospital, Uppsala, Sweden. This metastatic material was directly frozen (−20°C) in phosphate-buffered saline (PBS) containing protease inhibitor (Sigma-Aldrich, Hamburg, Germany).
Written consents were obtained from the patients and the study was approved by the local Ethics Committee.
Purification of prostasomes from metastatic material. After thawing, the excised skeletal metastatic material was washed and homogenized in PBS, containing protease inhibitors, for 30 s at 4°C (34). Tissues from metastases were obtained from 12 patients with bone metastases of prostate cancer. Tissue homogenizations were performed in isotonic Tris-HCl buffer using an Ultra-turrax homogenisator (Axel Kistner, Stockholm, Sweden) with a rotating pestle (30 s). The homogenates were centrifuged three times for 15 min at 3,000 ×g, then twice for 15 min at 10,000 ×g. The supernatants were then ultracentrifuged for 2 h at 100,000 ×g. The pellets were resuspended in isotonic Tris-HCl buffer and applied to a Superdex column (Amersham Bioscience, Uppsala, Sweden) to separate prostasomes from amorphous material. Fractions were collected displaying an elevated absorbance both at 260 nm (due to nucleic acid content of prostasomes) and at 280 nm. The pooled fractions were again ultracentrifuged at 100,000 ×g for 2 h and the resuspended prostasome protein concentration was adjusted to 2 mg/ml using a Protein Assay ESL method (Roche Diagnostic, Mannheim, Germany).
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The purified prostasomes were precipitated with a solution containing 8 mol/l urea, 4% CHAPS, 40 mmol/l Tris, 0.2% (w/v) immobilized pH gradients (IPG) buffer 3-10 for 1 h at 4°C and ultrasonicated on ice for 1.5 min at 40 kHz. The prostasome extract was then ultracentrifuged at 100,000 ×g and 4°C for 2 h. The supernatant intended for prostasomal 2D-protein analysis was saved and the protein concentration was determined. Prostasomal 2D-proteins (40 μg of soluble proteins) were rehydrated with a buffer consisting of 8 mol/l urea, 2% CHAPS (w/v), 50 mmol/l dithiotreitol (DTT) and 0.2 % IPG buffer 3-10. Isoelectric focusing, using IPG, was carried out by loading 300 μl onto the 17-cm strip. Focusing was performed at 20°C up to a total of 40 kVh using an IPG-phor unit (Bio-Rad, Hercules, CA, USA). The focused strips were equilibrated for 10 min, each in Bio-Rad Ready Prep 2D-starter kit equilibration buffers I and II.
The second dimension was carried out in an 8-16% SDS gel (1 mm thick, 17 cm IPG well) and protein visualisation was achieved by colloidal blue staining (InVitrogen, Carlsbad, CA, USA).
Mass spectrometry. In-gel digestion was carried out with trypsin (32) and digests were desalted using Zip Tip (Millipore, Billerica, MA, USA). Peptides were eluted in 70% acetonitrile/5% formic acid. The eluate was mixed 1:1 (v/v) with a saturated matrix solution of α-cyano-4-hydroxycinnamic acid in 30% acetonitrile/0.1% trifluoroacetic acid. Mass mapping of tryptic peptides was performed with an Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using alpha-cyano-4-hydroxy-trans-cinnamic acid as matrix.
Trypsin fragments of masses 842.50 Da and 2211.10 Da were used as internal standards for spectrum calibration. Data generated were screened in data bases, using a mass tolerance ≤20 ppm.
Results
2D-PAGE. Separated proteins appeared on the 2D gel (Figure 1) and identifiable spots within the range of possibility were punched out and subjected to trypsin digestion and peptide mass mapping by MALDI-TOF MS as previously described (35), followed by tandem mass spectrometry (MS/MS) when necessary.
Mass spectrometry. A total of 104 spots were punched out for identification by MALDI-TOF. Twenty unique protein spots had a MALDI-TOF score above 60, 5 others had a score of 49-57 and another 5 proteins were determined by MS/MS (Table I). The remaining 74 spots were either identical to already determined proteins or had no reliable score in the MALDI-TOF investigation.
Discussion
Prostasomes have several similarities with exosomes. Hence, prostasomes as well as exosomes are small microvesicles that are released from late endosomal compartments, storage vesicles, or multivesicular bodies (MVB) (36, 37). Exosomes have been the subject of much attention in recent years. They were originally described as a mechanism for the removal of receptor proteins in reticulocytes (38). Subsequently, pioneering work of several laboratories showed the importance of exosomes for general cell biology and in particular for the immune system (5). Exosomes have been demonstrated to play a role in the control of tumour growth (39) and the release of exosomes from tumour cells may be a novel mechanism for chemoresistance (40).
In fast-growing tumours, there is a constant struggle for oxygen and nutrients. Simultaneously, these tumours are bombarded by the body's defence system. Escaping tumour cells do not confront more pleasurable environments and very few of them will survive to create a metastatic outgrowth. The tumour cells must mount a battery of preventative measures to survive the harsh environments. A change of the proteome profile is accomplished by up-regulation or down-regulation of genes but also by degradation in late endosomes/lysosomes and secretion of unwanted proteins. Unwanted proteins might also be secreted by exocytosed vesicles from the late endosome and among these proteins are those that slow down the fast growth of the tumour cells. Hence, proteins hindering the fast dividing cancerous cells might be turned against fast dividing immune cells, if a transfer of these proteins is possible.
A curiosity in prostate cancer cells and metastasizing prostate cancer cells is their continued production and secretion of prostasomes (26). This prostasomal production and secretion demands a high energy investment and the continued prostasome production in malignant epithelial cells probably benefits the wellbeing and survival of the developing prostate tumour. Moreover, when identifying proteins, we noticed high score numbers for annexins A1 and A3, and especially for annexin A5, in prostasomes derived from prostate cancer metastases. In mammals, the annexin family consists of a group of at least 12 calcium ion-dependent phospholipid-binding proteins that are implicated in cell differentiation, immunomodulation and migration (41, 42). Annexin A1 was first identified as a mediator of the anti-inflammatory activity of glucocorticoids (43). However, identification of other annexins followed, including annexin A3 and A5. It has been suggested that annexin A3 promotes tumour growth by being an angiogenic factor that induces vascular endothelial growth factor (VEGF) production (44). Furthermore, a relationship exists between annexin A3 and prostate cancer (45). Moreover, annexin A3 immunostaining (positive versus negative) discerned two prognostic groups in the large ‘intermediate’ group between clearly identifiable low-risk and high-risk groups categorized by preoperative prostate-specific antigen, Gleason grade, and pT stage (45). Hence, a down-regulation of annexin A3 was apparent in prostate cancer (45) and this down-regulation could be a co-phenomenon in a context of autoimmunity in prostate cancer with elevated antibody titres against prostasomes in connection with prostate cancer (46). In contrast, annexin A3 was significantly up-regulated in primary lung adenocarcinoma with lymph node metastasis (47). Annexin A5 is associated with inhibition of phospholipase A2, a prostasome membrane-bound enzyme (48) and suppression of blood coagulation (49, 50). It binds phosphatidylserine which is transferred from the inner to the outer surface of the plasma membrane upon activation of thrombin and it acts as an anticoagulant in the microvillus space (49, 51, 52). Annexins are made up of a highly alpha-helical core domain that binds calcium ions, allowing them to interact with phospholipid membranes. In addition, annexins have effects on membrane organization, membrane trafficking, membrane-cytoskeleton linkage (53) and, especially annexin A5, stabilization of membranes (54). These properties qualify annexin A5 to be involved in intracellular signaling in at least some cell types (55-57). S100 calcium-binding proteins were also identified (Table I). Interactions between the two proteins S100 and annexin have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein (58). Hence, prerequisites are present for perfectly satisfactory interactions between prostate cancer cell messengers, i.e. prostasomes, and any neighboring cell in favor of prostate cancer growth and development.
We also observed the existence of dimethylarginine dimethylaminohydrolase 1 (DDAH-1) in prostasomes derived from prostate cancer metastasis. DDAH-1 is a zinc-containing enzyme that, through hydrolysis of side-chain methylated L-arginines, regulates the activity of nitric-oxide (NO) synthase (59) and, accordingly, DDAH-1 plays an important role in NO metabolism. There is strong evidence that NO, produced from arginine by nitric oxide synthase (NOS), is a crucial molecule with signaling functions and is also a regulator of angiogenesis (60). NO favours vascular permeability, induces extracellular matrix degradation, endothelial cell proliferation and migration (61, 62). Furthermore, NO also stimulates the expression of VEGF (63). A rat C6 glioma cell line was manipulated to overexpress the rat gene for DDAH-1 (64). Enhanced expression of DDAH-1 increased NO synthesis, expression and secretion of VEGF and induced angiogenesis in vitro and tumours derived from these cells grew more rapidly in vivo than cells with normal DDAH-1 expression (64). It was also claimed that human brain tumours express particularly high levels of DDAH-1 activity (64). This gives further credence to the view that prostasomes from prostate cancer cells are involved in the sustenance of prostate cancer cell growth and development (65).
Acknowledgements
This study was supported by a grant from the Swedish Cancer Society.
Footnotes
- Received September 3, 2009.
- Revision received January 22, 2010.
- Accepted January 25, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved