Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

ARTICLE IN PRESS European Journal of Cell Biology 86 (2007) 405–415 www.elsevier.de/ejcb Cholesterol regulates prostasome release from secretory lys...

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ARTICLE IN PRESS

European Journal of Cell Biology 86 (2007) 405–415 www.elsevier.de/ejcb

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells Alicia Llorentea,, Bo van Deursb, Kirsten Sandviga,c a Centre for Cancer Biomedicine, Faculty Division The Norwegian Radium Hospital, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Centre, N-0310 Oslo, Norway b Department of Cellular and Molecular Medicine, The Panum Building, University of Copenhagen, DK-2200 Copenhagen N, Denmark c Department of Molecular Biosciences, University of Oslo, N-0316 Oslo, Norway

Received 4 May 2007; received in revised form 25 May 2007; accepted 25 May 2007

Abstract Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same sizedistribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-bcyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane. r 2007 Elsevier GmbH. All rights reserved. Keywords: Prostasomes; PC-3 cells; Secretion; Cholesterol; Secretory lysosomes; Prostate cancer

Introduction Prostasomes are vesicles produced and secreted by epithelial cells of the prostate gland (Ronquist and Nilsson, 2004; Ronquist and Brody, 1985). Prostasomes are secreted with prostatic fluid and mixed with semen Corresponding author. Tel.: +47 2293 4282; fax: +47 2250 8692.

E-mail address: [email protected] (A. Llorente). 0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.05.001

during ejaculation, and have several functions related to human male reproduction and fertility (Burden et al., 2006). Interestingly, recent studies suggest that prostasomes may also be implicated in prostate cancer (Ronquist and Nilsson, 2004; Delves et al., 2006; Carlsson et al., 2000), and that they have the potential to be used as prognostic indicators for tumour progression (Larsson et al., 2006). Furthermore, it has been suggested that prostasomes of non-malignant origin and

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prostasomes from metastatic prostate cancer cell lines have opposite effects on angiogenesis (Delves et al., 2006). Prostasome membranes contain high amounts of cholesterol and sphingomyelin (Arvidson et al., 1989; Arienti et al., 1998), molecules that are enriched in lipid rafts (Simons and Ikonen, 1997; Simons and Toomre, 2000). Moreover, a proteomic analysis has revealed the presence of 139 proteins in human seminal prostasomes (Utleg et al., 2003). However, there are differences between the protein composition of prostasomes isolated from seminal fluid of healthy donors and from malignant prostate cells, i.e. caveolin-1 (cav-1), a protein that is overexpressed in human metastatic prostate cancer cells (Yang et al., 1998), has only been found in prostasomes isolated from prostate cancer cell lines (Llorente et al., 2004). Intracellularly, prostasomes are apparently localised to the interior of large storage vesicles (Sahlen et al., 2002). So far it is not clear how prostasomes are released by prostate cells, but several mechanisms have been proposed. It has been suggested that the storage vesicles originate from Golgi membranes, and that prostasomes are released after fusion of the storage vesicles with the plasma membrane (Sahlen et al., 2002; Ronquist and Brody, 1985). However, the release of vesicles in prostate cancer PC-3 cells continues when the constitutive and the regulated secretory pathways are inhibited (Llorente et al., 2004). Furthermore, prostasomes could be released by apocrine secretion, i.e., through protrusions or blebs that detach from the cell surface (Aumuller et al., 1997, 1999). Finally, it has been suggested that prostasomes may be secreted in a way similar to exosomes, after fusion of multivesicular bodies (MVBs)/late endosomes with the plasma membrane (van Niel et al., 2006; Johnstone, 2006). Remarkably, exosomes have both morphological and molecular similarities with prostasomes (Llorente et al., 2004). For instance, both organelles have a membrane enriched in cholesterol and sphingomyelin and several proteins in common (Utleg et al., 2003; Johnstone, 2006). Clearly, more studies on the mechanism and regulation of prostasome release are needed. To better characterise prostasomes and their secretion by prostate epithelial cells we have chosen the PC-3 cell line, a cell line isolated from a bone metastasis of a human prostatic adenocarcinoma (Kaighn et al., 1979). We have investigated whether several proteins involved in the endocytic pathway are found in vesicles released by PC-3 cells. Our results show that prostasomes contain proteins that are also found associated with the plasma membrane, endosomes, MVBs/late endosomes, and lysosomes. This is the first time that most of these proteins have been found in prostasomes. Moreover, our data suggest that prostasomes are released from characteristic, multivesicular body-like

secretory lysosomes. Importantly, we have also investigated whether cholesterol plays a role in prostasome release. Cholesterol is an abundant and essential component of mammalian cell membranes that is enriched in rafts, cell membrane microdomains that retain or exclude cellular proteins (Simons and Ikonen, 1997; Simons and Toomre, 2000). Cholesterol and cholesterol-enriched domains have been implicated in the regulation of several membrane trafficking events along the secretory and the endocytic pathway (Helms and Zurzolo, 2004; Ikonen, 2001). It has been shown that cholesterol accumulates in prostate cancer cells due to a deficient regulation of cholesterol metabolism (Hager et al., 2006). In the present study cholesterol has been depleted in the prostate cancer cell line PC-3 to investigate whether this lipid plays a role in prostasome release. Interestingly, our results show that cholesterol regulates prostasome release by PC-3 cells.

Materials and methods Reagents and antibodies Methyl-b-cyclodextrin (MBCD; average degree of substitution: 10.5–14.7 methyl groups per molecule), filipin complex, lovastatin and mevalonate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid protein assay kit and Western blotting detection reagents were from Pierce (Rockford, IL, USA). EasyTag L-[35S]methionine was purchased from Perkin-Elmer (Wellesley, MA, USA). Antibodies to cav-1, annexin II, EEA1, LAMP-2, and calnexin were from BD Biosciences (San Diego, CA, USA). The mouse monoclonal antibody to dynamin was from Upstate (Charlottesville). The antibody to clathrin heavy chain was from Research Diagnostics (Concord, MA, USA). The antibody against PDI was from Biosite (San Diego, CA, USA). The antibody against Hrs was kindly donated by Harald Stenmark. The antibody against LAMP-1 and CD63/LAMP-3 was from DHB (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The antibody against Tsg101 was from GeneTex (San Antonio, TX, USA). The antibody against Rab11 was from Zymed (San Francisco, CA, USA).

Cell culture The androgen-independent epithelial human prostate cancer cell line PC-3 has been isolated from a bone metastasis of a prostatic carcinoma (Kaighn et al., 1979). It was obtained from the American Type Culture Collection. Cells were maintained in a 1:1 mixture of

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Ham’s F12 medium and Dulbecco’s modified Eagle’s medium supplemented with 7% foetal calf serum (FCS), 100 units/ml penicillin and 100 mg/ml streptomycin at 37 1C in an atmosphere of 5% CO2/95% air.

Prostasome isolation Prostasomes were isolated from the serum-free cell culture medium of PC-3 cells as previously described (Llorente et al., 2004). The supernatant was centrifuged to remove cell debris first at 400g for 10 min and then at 10,000g for 30 min. Prostasomes were then collected by ultracentrifugation at 100,000g for 2 h in a SW40 or SW28 rotor, washed with phosphate-buffered saline (PBS), and then concentrated by ultracentrifugation at 100,000g for 2 h in a SW40 rotor first, and then in a TLA 120.1 rotor. As mentioned above, the experiments were done in the absence of serum. We chose to do the experiments under these conditions since PC-3 cells have a greatly reduced serum dependence for growth (Kaighn et al., 1979), and serum-derived vesicles (Wubbolts et al., 2003) or cholesterol could influence our results.

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Cholesterol determination PC-3 cells were washed with PBS and lysed for 5 min at 37 1C with a lysis buffer containing 0.1% (w/v) SDS, 1 mM EDTA and 0.1 M Tris–HCl, pH 7.4. Subsequently, the lysates were homogenised by using a 19-gauge needle. Cholesterol levels were then determined using the Infinity Cholesterol Liquid Stable reagent (ThermoDMA, Louisville, CO, USA).

Filipin staining To study the cellular distribution of cholesterol PC-3 cells grown on coverslips were fixed in a 10% formalin solution (approximately 4% formaldehyde) for 1 h at room temperature. The cells were then incubated with 50 mM NH4Cl in PBS for 10 min and with PBS containing 0.05% filipin complex and 5% FCS for 30 min at room temperature. After filipin staining the coverslips were mounted in Mowiol. Images were taken at  100 magnification using a Zeiss (Hallbergmoos, Germany) fluorescence microscope with the AxioVision system. Images were prepared with AdobePhotoshop 7.0.

SDS-PAGE and Western blot analysis

Lactate dehydrogenase (LDH) release

Prostasome pellets isolated from 1 to 3 100-mm diameter tissue culture dishes were solubilised in loading buffer. Whole cell lysates were solubilised in lysis buffer (25 mM Tris–HCl, 125 mM NaCl, 5 mM EDTA, 1% Triton X-100, SDS 0.1%, deoxycholate 2 g/l, pH 7.4) in the presence of a protease inhibitor mixture. Sample buffer was added to the cell lysates after removal of insoluble material. Prostasome and lysate samples were then subjected to SDS-PAGE in 12% or 4–20% gradient polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were then blocked with 5% nonfat dry milk and 0.1% Tween 20 in PBS and incubated with the indicated primary antibody. The membranes were then extensively washed with 0.1% Tween 20 in PBS, and then incubated with secondary antibodies coupled to horseradish peroxidase. Finally, the membranes were washed and developed using an enhanced chemiluminescence detection kit.

To measure LDH release, culture medium was centrifuged to remove prostasomes and then concentrated with an Amicon centrifugal filter device (pore 10,000 MWCO) (Millipore, Billerica, MA, USA). The sample was subjected to SDS-PAGE and then analysed by Western blotting using a goat polyclonal antibody raised against human LDH-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Protein determination PC-3 cells and isolated prostasomes were lysed in lysis buffer (25 mM Tris–HCl, 125 mM NaCl, 5 mM EDTA, 1% Triton X-100, SDS 0.1%, deoxycholate 2 g/l, pH 7.4) in the presence of a protease inhibitor mixture. Subsequently, the protein content was determined using a bicinchoninic acid protein assay kit according to the manufacturer’s instructions (Pierce).

Metabolic labelling PC-3 cells were starved in methionine- and serumfree DMEM for 30 min and labelled with EasyTag L-[35S]methionine for 90 min. The radiolabel-containing medium was removed, and the cells were washed twice in serum-free medium. Culture medium with or without MBCD (1.25 mM) was then added, and prostasomes and cells were collected after 16 h. Cells were lysed and prostasomes isolated as previously described, and the radioactivity was measured. In addition, proteins in prostasomes and cells lysates were separated by SDS-PAGE and transferred to Immobilon-P membranes that were exposed to X-ray films.

Electron microscopy PC-3 cells were washed in PBS and fixed with 0.1% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.2, followed by dehydration

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and embedding in Epon. Alternatively, the fixed cells were processed for ultracryosectioning and the sections labelled with mouse monoclonal anti-LAMP-1, anti-LAMP-2 or anti-CD63/LAMP-3 followed by gold-conjugated goat-anti mouse antibody. Isolated prostasomes were negatively stained on grids with phosphotungsten acid.

Results Isolation of prostasomes released from PC-3 cells Prostasomes released to the culture medium of PC-3 cells were in principle isolated following the protocol established for human seminal fluid prostasomes (Carlsson et al., 2003; Ronquist and Brody, 1985). It has previously been shown that these vesicles contain cav-1 (Llorente et al., 2004), and we have used this protein as well as other markers (see below) to characterise the extent of prostasome release in Western blot experiments. As shown in Fig. 1A, the amount of cav-1 associated with vesicles released to culture supernatants increased with time. In the experiments described in this paper prostasomes were isolated from the medium of PC-3 cells grown for at least 16 h.

Fig. 1. Western Blot analysis of PC-3 cell lysates and released prostasomes: (A) prostasomes were isolated from PC-3 cell culture supernatants after different periods of time. The vesicles were then subjected to SDS-PAGE and Western blot. The figure shows a representative Western blot of cav-1 bands detected using an enhanced chemiluminescence detection kit. (B, C) Prostasomes were isolated from PC-3 cells culture supernatants after 24 h. Similar amounts of protein from cell lysates and isolated prostasomes (vesicles) were separated by 4–20% SDS-PAGE and analysed by immunoblotting using antibodies to clathrin, Tsg101, Hrs, LAMP-1, annexin II (annex II), Rab5, Rab11 (B) and EEA1, dynamin, calnexin, PDI (C). The figure shows representative Western blots.

Proteins of the endocytic pathway are found in prostasomes released by PC-3 cells It has been reported that prostasome-containing granules morphologically resemble MVBs (Aumuller et al., 1997). Moreover, it has been suggested that prostasomes may be secreted in a way similar to exosomes, by an endocytic secretory pathway in which MVBs fuse with the plasma membrane (Llorente et al., 2004). We have here investigated by Western blotting whether prostasomes released by PC-3 cells contain proteins involved in the endocytic pathway. Clathrin plays an important role in the endocytic pathway since it is involved in the formation of clathrin-coated transport vesicles, in the recycling of internalised material and in the endosomal sorting of cargo into the degradative pathway (Brodsky et al., 2001; Raiborg et al., 2006). Interestingly, clathrin-heavy chain was detected in PC-3 released prostasomes (Fig. 1B). Furthermore, the tumour-susceptibility gene product 101 (Tsg101) and the hepatocyte-growth-factor-regulated tyrosine kinase substrate (Hrs), both proteins involved in endosomal sorting of ubiquitinated membrane proteins at the endosomal membrane (Gruenberg and Stenmark, 2004), were found on prostasomes released by PC-3 cells (Fig. 1B). It has previously been reported that the lysosomal tetraspanin protein CD63 (LIMP-I, LAMP3) is found in vesicles released by PC-3 cells (Llorente et al., 2004), and also other lysosomal proteins, the lysosome-associated membrane protein type 1 (LAMP1) (Fig. 1B) and LAMP-2 (data not shown) are found in vesicles released by PC-3 cells. In addition, members of the Rab family of small GTPases that regulate the endocytic pathway were found on prostasomes. Rab11 and Rab5 have been found to be involved in recycling to the plasma membrane and in endocytosis, respectively (Stenmark and Olkkonen, 2001). As shown in Fig. 1B, both Rab5 and Rab11 were detected in prostasomes. We also tested whether annexin II was released in association with prostasomes. Several members of the annexin family of proteins play important roles in the endocytic pathway, and annexins may be implicated in the generation, localisation, or fusion of endocytic compartments (Rescher and Gerke, 2004). As shown in Fig. 1B, annexin II was detected in PC-3-derived prostasomes. However, two other proteins functioning in the endocytic pathway were not found in prostasomes. As shown in Fig. 1C, the early endosome antigen 1 (EEA1) and the large GTPase dynamin were not found in the vesicles, although both proteins were detected in PC-3 cell lysates (Fig. 1C). Furthermore, as shown in Fig. 1C, the prostasomes were not contaminated with ERresident proteins since both calnexin, an integral ERmembrane protein, and the protein disulphide isomerase (PDI), a protein primarily located in the ER lumen, were excluded from prostasomes. These experiments

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reveal that endocytic proteins are well represented in prostasomes released by PC-3 cells, and support the idea that prostasomes may originate from endocytic organelles.

Origin of prostasomes To further investigate the cellular origin of prostasomes and how they are released, PC-3 cells and prostasomes released by PC-3 cells were examined by electron microscopy. Both control cells and cells treated with MBCD to extract cholesterol (see below) were included. Isolated, negatively stained prostasomes are shown in Fig. 2A. In agreement with previous studies (Burden et al., 2006; Sahlen et al., 2002) most prostasomes had a diameter of 30–90 nm, but larger ones (including about 10% larger than 150 nm) also occurred (data not shown). No differences in appearance and size distribution were observed between prostasomes from control cells and cholesterol-depleted cells. Electron microscopy of PC-3 cells revealed the presence of several large MVBs, often localised close to Golgi cisterns (Fig. 2B) or the cell surface (Fig. 2C). The vesicular content of these MVBs was highly heterogeneous, both with respect to size and density.

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Importantly, the size distribution of the internal vesicles and that of isolated secreted prostasomes (see above) was largely similar, strongly suggesting that the prostasomes derive from these MVBs. Moreover, occasionally profiles were found suggesting fusion of MVBs with the plasma membrane and subsequent release of the internal vesicles (Fig. 2C, inset). Because the lysosome-associated membrane protein LAMP-3/CD63 has previously been found in prostasomes released by PC-3 cells (Llorente et al., 2004), we next localised this protein in PC-3 cells by ultrastructural immunogold labelling. As shown in Fig. 2D, LAMP-3/CD63 was localised to the MVBs, mainly to the heterogeneous population of inner vesicles, and labelling of other structures was not seen. Also the lysosomal proteins LAMP-1 and LAMP-2 were localised to the MVBs, although with a lower level of labelling than LAMP-3/CD63 (not shown). Interestingly, even LAMP-1, which is normally associated with the limiting membrane of lysosomes, was also seen associated with the inner vesicles. These findings, together with the morphological observations presented above, strongly suggests that the characteristic MVBs of PC-3 cells are secretory lysosomes releasing prostasomes by fusing with the plasma membrane.

Fig. 2. Electron microscopy of isolated PC-3 prostasomes and intact PC-3 cells: (A) isolated, negatively stained prostasomes. (B, C) Multivesicular body-like secretory lysosomes in PC-3 cells (arrows). Note the varying density and size of the inner vesicles. Go, Golgi cisterns. Inset in (C) shows an apparent release of small vesicles from a secretory lysosome fused with the plasma membrane. (D) Ultracryosection of a PC-3 cell showing MVB-like secretory lysosomes (arrows) immunogold-labelled for LAMP-3/CD63. Bars, 500 nm.

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Cholesterol distribution in human prostate cancer PC-3 cells Cholesterol accumulates in prostate cancer (Freeman and Solomon, 2004; Hager et al., 2006), and it has recently been reported that the human prostate cancer cell line PC-3 contains more cholesterol than normal prostate epithelial cells (Li et al., 2006). To visualise the distribution of free cholesterol in PC-3 cells, cells were fixed and stained with the UV-light fluorescent sterolbinding antibiotic filipin. As shown in Fig. 3, the plasma membrane, and in particular the perinuclear region of the cell, were strongly labelled in many cells, probably due to the association of cholesterol with the Golgi apparatus and the perinuclear recycling compartment (Maxfield and Wustner, 2002).

Effect of cholesterol depletion on prostasome release: MBCD treatment To investigate whether cholesterol plays a role in the secretion of prostasomes, PC-3 cells were incubated with the membrane-impermeable, cholesterol-extracting drug MBCD (Christian et al., 1997). As shown in Fig. 4A, treatment with MBCD (1.25 mM) reduced the cholesterol content of PC-3 cells by nearly 40% after 16–18 h, and control experiments showed that the long incubation time with MBCD (1.25 mM) did not affect cell growth (Fig. 4B). Prostasomes were isolated by centrifugation as described in Materials and methods, and electron microscopy showed that prostasomes isolated from MBCD-treated cells were similar in morphology and size to prostasomes isolated from untreated cells (see above). The amount of released prostasomes under the different conditions was then measured by quantifying the amount of prostasome proteins in Western blots. Interestingly, in the presence of MBCD the amount of cav-1 in the prostasome fraction was increased by 10-fold. A representative experiment and a densitometric

quantitation of the bands obtained from several experiments are shown in Fig. 4C. The increased cav-1 levels in prostasomes released by MBCD-treated PC-3 cells were not due to increased cellular levels of cav-1 (Fig. 4D). In addition, MBCD also caused an increase in the amount of other prostasome-associated proteins such as LAMP-1 (Fig. 4D). To control that the increase in prostasome release by MBCD was not due to a cytotoxic effect of the drug, the extracellular levels of the enzyme LDH were measured. LDH is a stable cytosolic protein often used as a sensitive marker for cell death or membrane permeabilisation since it is released to the cell culture medium upon cell lysis or membrane damage. When prostasome-free cell culture medium was concentrated and submitted to SDS-PAGE and Western blot analysis, similar amounts of LDH were found in control and MBCD-treated cells (Fig. 4D), thus indicating that the effect of MBCD on prostasome release is not due to leakage of cellular material. Furthermore, to investigate whether the total protein composition of PC-3 cells and prostasomes was altered by MBCD, PC-3 cells were labelled with L-[35S]methionine in some experiments. Similar amounts of radioactively labelled proteins of either cell lysates or prostasomes were analysed by SDSPAGE and autoradiography. As shown in Fig. 5, the protein profiles of both cell lysates and prostasomes were quite similar in control cells and in MBCD-treated cells, although some quantitative differences were observed in prostasomes (Fig. 5, asterisks). Finally, we found more radioactivity associated with prostasomes isolated from MBCD-treated cells than from control cells (data not shown), thus in agreement with previous results showing that MBCD increases prostasome release (Fig. 4).

Effect of cholesterol depletion on prostasome release: Lov/Mev treatment MBCD has been the most frequently employed drug to assess the effects of cholesterol depletion and the

Fig. 3. Cholesterol distribution in PC-3 cells. (A, B) Cholesterol distribution in human prostate cancer PC-3 cells as visualised by staining with the UV-light fluorescent sterol-binding compound filipin. Images were taken using a Zeiss fluorescence microscope with the AxioVision system. Bars, 10 mm.

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Fig. 4. MBCD treatment increases prostasome release. PC-3 cells were incubated without or with MBCD (1.25 mM) for 16–18 h. Cholesterol levels were then measured (A), and cells were counted (B). Data represent the average of 6 and 5 experiments, respectively, expressed as the percentage of control cells, and error bars show the standard deviation. (C) Prostasomes (vesicles) released to cell culture supernatants of control cells and cells treated with MBCD (1.25 mM) for 16–18 h were isolated as described in Materials and methods. Samples of prostasomes were subjected to SDS-PAGE, and analysed by Western blotting using antibodies to cav-1. Quantitative analyses were performed with a computing densitometer. The figure shows a representative Western blot, and a densitometric quantitation of cav-1 bands detected using an enhanced chemiluminescence detection kit. Data represent the average of 4 experiments expressed as the percentage of control cells, and the error bar shows the standard deviation. (D) Samples of lysates and prostasomes from control PC-3 cells and cells treated with MBCD (1.25 mM) for 16–18 h were subjected to SDS-PAGE and Western blotting for cav-1 and LAMP-1. Representative Western blots of bands detected using an enhanced chemiluminescence detection kit are shown. In addition, culture medium of cells treated without or with MBCD (1.25 mM) for 16–18 h was centrifuged to remove prostasomes, and then concentrated with an Amicon centrifugal filter device. The concentrated medium was subjected to SDS-PAGE and Western blotting using a goat polyclonal antibody raised against human LDH-A. The figure shows a representative Western blot of LDH-A bands detected using an enhanced chemiluminescence detection kit.

subsequent disruption of cholesterol-dependent domains on biological processes (Christian et al., 1997). However, recent studies have indicated that MBCD may induce membrane alterations that are not due to reduced cholesterol levels only (Shvartsman et al., 2006; Goodwin et al., 2005). As shown above, treatment with MBCD increases the release of vesicles by PC-3 cells. To confirm that the effect of MBCD on prostasome release is due to cholesterol depletion, cellular cholesterol levels were reduced by an alternative mechanism. De novo synthesis of cholesterol in the ER was inhibited by

incubating PC-3 cells for 2 days with 5 mM lovastatin (Lov), a drug that inhibits the enzyme that catalyses the rate-limiting step (HMG-CoA to mevalonate) in the biosynthesis of cholesterol (Alberts et al., 1980). The experiments were done in the presence of 0.25 mM mevalonate (Mev) to allow the synthesis of isoprenoid compounds and avoid additional effects of lovostatin (Keller and Simons, 1998). As shown in Fig. 6A, treatment of PC-3 cells with Lov/Mev for 2 days reduced cellular cholesterol levels by 20–25%. This treatment did not affect cell growth (Fig. 6B). Prostasomes were then

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Fig. 5. Protein profiles of PC-3 cells and prostasomes with and without MBCD treatment. PC-3 cells were labelled with L-[35S]methionine for 90 min. The radiolabel-containing medium was removed and medium with or without MBCD (1.25 mM) was added. After 16 h prostasomes were isolated and cells were lysed. Proteins in prostasomes and cells lysates were separated by SDS-PAGE and exposed to X-ray films. Asterisks point to proteins enriched in prostasomes isolated from MBCD-treated cells.

isolated from culture supernatants and the amount of released prostasomes in control cells and in cells treated with Lov/Mev was analysed by measuring the levels of cav-1 in Western blots. Interestingly, as shown in Fig. 6C, the amount of cav-1 in the prostasome fraction was also increased after treatment of PC-3 cells with Lov/Mev. Finally, as shown in Fig. 6D, control experiments showed that PC-3 cells treated with Lov/ Mev did not contain higher amounts of cav-1. The fact that depletion of cholesterol by two different methods causes an increase in prostasome release by PC-3 cells supports the idea that a reduction in the cholesterol levels, and not cholesterol-independent effects, are responsible for the observed effect.

Discussion An important finding in this study is that cholesterol plays an important role in the release of prostasomes from the human prostate cancer cell line PC-3. Furthermore, biochemical characterisation of prostasomes released by PC-3 cells revealed the presence of several proteins involved in the endocytic pathway that may be targeted in future studies to unravel the mechanism of prostasome release.

Prostasomes isolated from human seminal plasma have been best characterised so far (Ronquist and Brody, 1985; Utleg et al., 2003), but prostasomes can also be isolated from other sources such as prostate tissue (Carlsson et al., 2003) and prostate cancer cell lines (Llorente et al., 2004; Babiker et al., 2005). In this study the human prostate cancer cell line PC-3 has been used in order to better characterise prostasomes in prostate cancer cell lines. This work provides new important information about the biochemical composition of prostasomes isolated from PC-3 cells and, in particular, about the presence of proteins normally associated with organelles of the endocytic pathway. Our results show that clathrin, Tsg101, Hrs, LAMP-1, LAMP-2, LAMP-3/CD63, Rab5, Rab11, and annexin II, but not proteins associated with the ER, are found in prostasomes. Interestingly, with exception of annexin II, these proteins have not been found in seminal prostasomes isolated from healthy donors. It is possible that these proteins were not found in seminal prostasomes due to limitations in the proteomic methodology that was used (Utleg et al., 2003). However, some of these proteins could selectively be overexpressed in prostate cancer cells and their presence could be relevant for the different behaviour of prostasomes isolated from normal cells and from prostate cancer cells. In fact, it has been shown that proteins such as the glycosylphosphatidylinositol-anchored complement regulatory protein CD59 (Babiker et al., 2005), the protein kinases A, C, and casein kinase II (Babiker et al., 2006) are expressed to a higher level in prostasomes isolated from prostate cancer cell lines than in prostasomes isolated from seminal plasma. It is interesting to note that most of the proteins identified in this study have previously been detected in exosomes (van Niel et al., 2006), and that Rab11 has been shown to play a role in exosome release (Savina et al., 2002). In conclusion, our results reveal biochemical similarities between exosomes and the vesicles released by the prostate cancer cell line PC-3 and, together with the electron microcopy analysis, are consistent with the idea that prostasomes can have an endosomal/lysosomal origin and be released by secretory lysosomes. This paper clearly shows that cholesterol can regulate the release of prostasomes from the human prostate cancer cell line PC-3. This conclusion was reached after observing that cholesterol-depleting drugs increase prostasome secretion as measured by Western blot analysis of specific proteins found in prostasomes and by radioactive labelling of prostasomes. Both reduction of cholesterol levels metabolically and by the cholesterol-sequestering agent MBCD caused an increase in prostasome release. The fact that in our experimental conditions MBCD did not cause an increase in the release of LDH or inhibit cell growth indicates that the effect of MBCD on prostasome release was not due to a cytotoxic effect of the drug. Cholesterol plays an important role in cell signalling and cell survival in prostate cancer cells. Simvastatin, an

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Fig. 6. Lov/Mev treatment increases prostasome release. PC-3 cells were incubated without or with Lov/Mev for 2 days. Cholesterol levels were then measured (A), and cells were counted (B). Data represent the average of 4 experiments expressed as the percentage of control cells, and error bars show the standard deviation. (C) Prostasomes (vesicles) released to cell culture supernatants of control cells and cells treated with Lov/Mev for 2 days were isolated as described in Materials and methods. Samples of prostasomes were subjected to SDS-PAGE and Western blotting using antibodies to cav-1. Quantitative analyses were performed with a computing densitometer. The figure shows a representative experiment, and a densitometric quantitation of the cav-1 bands obtained from several experiments. Data represent the average of 4 experiments expressed as a percentage of control cells, and the error bar shows the standard deviation. (D) Samples of lysates from control PC-3 cells and cells treated with Lov/Mev for 16–18 h were subjected to SDS-PAGE and Western blotting for cav-1. A representative Western blot of cav-1 bands detected using an enhanced chemiluminescence detection kit is shown.

inhibitor of the synthesis of cholesterol, impairs Akt signalling and induces apoptosis in the human prostate cancer cell line LNCaP cells (Zhuang et al., 2005). Furthermore, it has recently been shown that MBCD inhibits cell proliferation and causes apoptosis in serumstarved PC-3 cells after 24 h, but not at the concentration used in this study (Li et al., 2006). Moreover, the fact that we did not observe the characteristic ultrastructural alterations of apoptosis supports the idea that induction of apoptosis and shedding of blebs are not responsible for the increase in prostasome release observed in the cholesterol-depleted PC-3 cells.

Remarkably, 63% of all cholesterol detected in the endocytic pathway is found in MVBs (Mobius et al., 2003), mostly in the internal vesicles from which exosomes originate. It would be interesting to know whether cholesterol also regulates exosome release. This is not clear so far. It has recently been shown that cholesterol is required for the IFN-g induced release of Hsp72 in K562 cells (Bausero et al., 2005), but not for the release of the heat shock protein HSP70 from human peripheral blood mononuclear cells (Lancaster and Febbraio, 2005). Cholesterol-enriched domains play a critical role in membrane protein sorting and signalling and could

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control prostasome release in several ways. Cholesterol depletion may alter different signalling pathways by disrupting the interaction of signalling proteins with cholesterol-enriched domains (Simons and Toomre, 2000). For example, it has been shown that cholesterol depletion activates protein kinase A and induces transcytosis in MDCK cells (Burgos et al., 2004). Therefore, it is possible that in PC-3 cells cholesterol depletion induces the activity of a kinase that regulates prostasome release. Furthermore, other molecules that may facilitate prostasome release could be retained in cholesterol-enriched domains and be accessible after disruption of these domains. Moreover, the levels of cholesterol in an organelle can affect the function of molecules involved in trafficking, for example Rab4 function is inhibited by an excess of cholesterol in early endosomes in Niemann-Pick cells, and recycling is impaired (Choudhury et al., 2004). It is possible then that a reduction in the cholesterol levels of the organelles involved in prostasome release can activate a molecule important for the secretion of these vesicles. Finally, cholesterol depletion may change the properties of the membrane, and the permeability of relevant molecules for prostasome release could be affected. MBCD increases the permeability of the basal Ca2+ concentration in rat basophilic leukaemia cells (Kato et al., 2003), and it has been shown that an increase in cytosolic calcium can stimulate exosome release (Savina et al., 2005). We cannot exclude that this may also be the case for prostasome release. Several biological functions have been assigned to prostasomes in fertilisation, and the potential role of prostasomes in prostate diseases is just starting to be studied. However, there are still many open questions about the mechanism and the regulation of prostasome release. Our results suggesting that cholesterol can regulate prostasome release, and the characterisation of proteins associated with these vesicles contribute to the understanding of the biology of prostasomes.

Acknowledgements We thank Ulla Hjortenberg and Mette Ohlsen for their excellent technical assistance. The present study was supported by The Norwegian Cancer Society, The Research Council of Norway, The Functional Genomics (FUGE) programme from The Research Council of Norway, The Danish Cancer Society, The Danish Medical Research Council, the Jahre foundation, and Jeanette and Søren Bothner’s legacy.

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