1 Dipartimento di Medicina Sperimentale, Plesso Biotecnologico Integrato, Università di Parma, Via Volturno, 39-43100 Parma, Italy, 2 Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Via G. Campi 287-41100 Modena, Italy and 3 Ospedale EstenseS. Agostino, Divisione di Urologia, Via S. Agostino, 18-41100 Modena, Italy
4 To whom correspondence should be addressed Email: saverio.bettuzzi{at}unipr.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: CaP, prostate cancer; CLU, clusterin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Gas1, growth arrest-specific gene 1; GTC, green tea catechins; PIN, prostate intra-epithelial neoplasia; TRAMP, transgenic adenocarcinoma mouse prostate
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epidemiological studies on the incidence of CaP in some Asian countries, where green tea is consumed regularly (2), have suggested that green tea beverages might exert an effect in lowering the incidence of this malignancy. Most of the dry matter resulting from green tea infusion is represented by polyphenolic compounds known as catechins, the most common of which are ()-epigallocatechin-3-gallate (EGCG), ()-epigallocathechin, ()-epicatechin-3-gallate and ()-epicatechin. These compounds, and especially the most abundant of green tea polyphenols, EGCG, have been shown to exert an effective chemopreventive action in a vast number of animal models of induced carcinogenesis (3). This effect may be related to the complex endocrine changes occurring upon catechin administration (4) and/or to the inhibition of 5--reductase (the prostatic enzymes that transform testosterone into the prostate specific androgen, 5-
-dihydrotestosterone) by the polyphenols (5).
An autochthonous transgenic animal model of CaP, the transgenic adenocarcinoma mouse prostate (TRAMP) mouse model (6), was developed as a very important tool for understanding the earlier events and the progression of this kind of tumor. The TRAMP mouse model displays in situ and invasive carcinoma of the prostate, mimicking the whole spectrum of human CaP progression from prostate intra-epithelial neoplasia (PIN) to androgen-independent disease (7). TRAMP mice express SV40 T/t antigen under the control of the minimal rat probasin promoter, which is prostate specific. Recent laboratory studies have demonstrated that green tea polyphenol administration can actually prevent CaP development in this model (8).
Clusterin (CLU) (Apo J, SGP-2, TRPM-2, etc.) was found as one of the most potently over-expressed genes in the regressing rat prostate 210 days after androgen ablation, i.e. under conditions inducing massive cell death and cell atrophy (9). Different forms of CLU have been recognized (10), coded by a single gene. The data, together with some controversial reports about its action, pose the question of the different (even opposite) functions in which CLU appears to be involved. In fact, the secreted form of CLU (sCLU) has been shown to be over-expressed in cancer cells and may provide protection against cytotoxic agents that induce apoptosis (11). In contrast, the nuclear form of CLU (nCLU) has been implicated in pro-death processes (12).
We found previously that the level of expression of CLU (mRNA and protein) is lower in specimens of human prostate carcinoma than in the benign portion of the same gland (13,14). The data, and results indicating that CLU over-expression exerts an anti-proliferative effect in several SV40-immortalized cell lines (15), together with the fact that CLU is generally down-regulated when cell proliferation is induced (16), have suggested a tumor-suppressor function for this gene product. However, other authors have reported that CLU expression increases in CaP (17), while in colon adenocarcinoma a translocation of CLU from the nucleus to the cytoplasm appears to promote tumor progression (18).
A possible explanation of this discrepancy may rest on the existence of different CLU forms, and the possibility that they may undergo specific changes during the different phases of neoplastic transformation. In addition, sub-cellular localization of CLU might also be very important for interpretation of its biological effects.
Here we present the first data on the effect of the polyphenols of the green tea on SV40-immortalized human prostate epithelial cells, PNT1A, and androgen-independent human prostate cancer cells, PC-3, in comparison with normal human prostate epithelial cells in primary culture. Then we have studied the levels of expression of CLU in the prostate of TRAMP mice at different steps of tumor progression. Our data suggest the possible involvement of CLU in the chemopreventive action of green tea catechins (GTC).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of the IC50
Cells were seeded in 6-well plates to obtain 5060% confluence 24 h after plating, and then treated with different concentrations of EGCG (Sigma-Aldrich s.r.l.) for 24 h. Cells were then fixed with 3% formaldehyde in phosphate-buffered saline (PBS) and stained with 0.5% Crystal violet (Sigma-Aldrich s.r.l.). The stain was extracted with 0.1 mM sodium citrate/ethanol 2:1 and the absorbance was measured at 540 nm.
Transgenic animals and GTC chemoprevention
Tramp mice, heterozygous for the PB-Tag transgene, were maintained in a C57BL/6 background. Transgenic males for the studies were routinely obtained as [TRAMP x C57BL/6]F offspring. Purification of mouse-tail DNA and PCR screening were performed routinely as described previously (6). GTC were extracted from green tea leaves as reported previously (5) and content and purity were assessed by HPLC (EGC 5.5%; EC 12.2%; EGCG 51.9%; ECG 6.1%; total GTCs 75.7%, caffeine <1.0%) (20). Male TRAMP mice and their non-transgenic littermate were divided into four groups of 60 mice each. One group of TRAMP mice and one group of non-transgenic ones were given freshly prepared 0.3% GTC solution in tap water every Monday, Wednesday and Friday, starting at the age of 8 weeks. Another group of TRAMP mice and one group of non-transgenic ones were given tap water alone (controls). The animals had access to laboratory chow ad libitum. At 12, 17 and 24 weeks of age, 20 animals for each group were killed by cervical dislocation and the prostate glands were quickly removed and used for northern and western blot and immunohistochemistry experiments.
Northern blot hybridization analysis
Total RNA was extracted from frozen TRAMP mice prostate as described previously (13). Ten microgram aliquots were electrophoresed and blotted onto nylon membranes. Mouse CLU cDNA was amplified by RTPCR from wild mouse prostate total RNA using the following primers: forward primer 5'-GCCGCAGAGCGGCCGCCAGATTCC-3'; reverse primer 5'-CAGAATCCAAGAGAAGGGCAGGCTGC-3'.
Mouse CLU cDNA amplification product was then purified and used as probe for northern blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), growth arrest-specific gene 1 (Gas1) and histone H3 full-length cDNA were purified, labeled and used for northern blot hybridization as described previously (13).
Western blot analysis and developing of anti-human -375 CLU antibody
Affinity-purified, polyclonal -375 anti-human CLU antibodies were produced by injecting the following peptide into rabbits: NH2-LTQGEDQYYLRVTT (position 375387 on the human CLU sequence GenBank accession number P10909). The
-375 antibody against the 14-aa peptide injected as an immunogen was used in the TRAMP mice model because it recognized a region of the mouse protein between positions 375 and 387 (GenBank accession number Q06890; sequence comparison: identities = 12/14, 85%) that share high homology with human CLU. Polyclonal
-375 was affinity-purified on a peptide-armed CNBr-Sepharose column before use. Antibody specificity was then checked by western blot and immunohistochemistry analysis in strict comparison with commercially available polyclonal anti-mouse CLU antibody from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-human CLU from clone 41D Upstate Biotechnology, Lake Placid, NJ, for validation in human and mouse samples as part of an ongoing research project aimed at developing new antibodies against CLU (data not shown). Total cell extract and frozen mice prostates were homogenized in RIPA buffer (TrisHCl 50 mM, pH 7.4; NaCl 150 mM, Nadeoxycholate 0.25%, NP-40 1%, DNase 50 µg/ml, RNase 50 µg/ml), added with CompleteTM protease inhibitor according to manufacturer's instruction (Roche Diagnostics, Milan, Italy) and heated at 100°C for 10 min in SDSPAGE loading buffer. The equivalent of 50 µg of total protein was loaded on each lane and resolved by electrophoresis on 10% polyacrylamide gel. Immunoreactive bands were detected with the Chemiluminescence Blotting Substrate (POD) (Roche Diagnostics) using affinity-purified polyclonal anti-human CLU
-375, anti-human CLU (clone 41D Upstate Biotechnology), anti-caspase 9, anti-caspase 8, anti-caspase-3 (Santa Cruz Biotechnology) and anti-SV40 large T antigen (BD Pharmigen, Hilderberg, Germany) antibodies.
Immunohistochemistry
Tissues were fixed in 10% formalin for 24 h, transferred to 70% ethanol and then embedded in paraffin. Sections (5 µm) were cut and mounted on slides. Slides were hydrated through xylene and graded alcohol and equilibrated in PBS. Antigen retrieval was performed with sodium citrate 10 mM pH 6, using a microwave for 10 min at 400 W. Endogenous peroxidase was quenched with 3% H2O2 in methanol. Non-specific binding was blocked with normal serum (Pierce, Rockford, IL). The same polyclonal antibody -375, exhibiting specific binding in western blot, was used for immunohistochemical staining of prostate gland sections at dilutions 1:200. All the slides were then washed several times in PBS and incubated with biotinylated goat anti-rabbit IgG (Amersham Bioscience Europe GmbH, Milan, Italy) at dilutions 1:200, followed by peroxidase-labeled streptavidin (Amersham Bioscience Europe GmbH). The antigen was visualized by a 10-min incubation with diaminobenzidine tetrahydrochloride (Sigma Chemical, St Louis, MO). Negative controls, made by excluding monoclonal antibodies from the reaction, showed no specific staining. Counterstaining was performed with hematoxylin (Sigma Chemical, St Louis, MO) and cover slips were mounted with Eukitt (O. Kindler GmbH, Freiburg, Germany).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A recent study has shown that EGCG induced caspase activation and apoptosis in different cell lines (21). Consistent with the latter results, under our conditions, EGCG administration dramatically reduced pro-caspase 8 and 3 concentrations in PC-3 and PNT1A cell lines, suggesting that both caspases were cleaved, leading to activation of the caspase cascade. Caspase activity in normal prostate epithelial cells was unaffected by EGCG treatment. The same inhibitory effects on the proliferation activity of cultured cells were obtained when a GTC extract containing the same amounts of EGCG indicated above were used (data not shown). Therefore, the experimentation with TRAMP mice was continued using GTC.
In order to evaluate a possible GTC chemopreventive effect, TRAMP and non-transgenic (wild) mice of 8 weeks of age were fed a solution of 0.3% GTC in tap water or tap water alone (controls) as drinking fluid. At 17 weeks, none of the 20 TRAMP mice drinking GTC and 1 out of 20 TRAMP mice drinking water exhibited a palpable tumor that was confirmed by histology (Table I). At 24 weeks, 100% of water-fed TRAMP mice developed CaP, but only 4 out of 20 GTC-treated TRAMP mice developed the disease (Table I). All tumors were confirmed by histology. Thus, GTC chemopreventive activity was evident in as many as 80% of the transgenic animals. These results fully confirm the data obtained previously by other authors (8).
|
|
Consistently with increased cell proliferation induced by SV40 expression that sustains CaP progression, histone H3 transcript accumulated at very high levels in the prostate of tumor-bearing TRAMP mice drinking water (Figure 2A, lanes 5 and 7) or GTC (Figure 2B, lane 14), but was almost undetectable in tumor-free specimens. In this regard, it may be worth noticing the inverse relationship between the abundance of this transcript and that of CLU, again supporting the anti-proliferative role of the latter. It was expected that the pattern of the transcript coding for Gas1 was reciprocal to that of histone H3; however, this was not always so. In water-fed TRAMP mice of 12 weeks of age and in those of 17 weeks that did not show signs of tumor, the abundance of this transcript was maintained at about the same levels as wild counterparts (Figure 2A, lanes 14). However, in the one mouse of 17 weeks exhibiting the tumor burden, the level of Gas1 mRNA dropped dramatically (lane 5) with respect to wild controls (lane 3) and a similar decrease of the transcript occurred in the prostate of 24-week-old TRAMP mice (all with tumors, lane 7) as compared with wild controls (lane 6). Also in the prostate of mice receiving GTC (Figure 2B), the abundance of Gas1 transcript did not change very much between wild and TRAMP mice of 12 and 17 weeks (lanes 811). At 24 weeks, Gas1 transcript accumulated at higher levels in the TRAMP mice that remained tumor-free (lane 13) with respect to wild controls (lane 12). This was expected, considering that CLU was very high and histone H3 was undetectable under the same conditions. However, in TRAMP mice of the same age that developed the tumor in spite of receiving GTC, and exhibited undetectable CLU mRNA and high levels of histone H3 transcript, unexpectedly Gas1 accumulated as high as in those not developing the neoplasm (lane 14).
In the same specimens, the hybridization signal of the transcript coding for the housekeeping glycolytic enzyme GAPDH was also studied for comparison. The results obtained indicated a modest increase in the accumulation of its mRNA in prostate tumor of water-fed and GTC-administered TRAMP mice.
Western blot analysis, using the affinity-purified polyclonal anti-human CLU antibody specifically cross-reacting with mouse CLU that we have developed, showed constant levels of CLU protein in the prostate of water-fed wild-type mice between 12 and 24 weeks of age (Figure 3A, lanes 13). In the prostate of 12-week-old TRAMP mice (lane 4), CLU protein accumulated at about the same level as non-transgenic counterparts (lane 1). But in 17-week-old TRAMP mice (lane 5), CLU precursor form (60 kDa) was at much lower level than controls (lane 2), while the 40-kDa molecular weight form increased substantially (lane 5). All the isoforms of CLU totally disappeared in the tumor-bearing prostate of 24-week-old mice (lane 6). Figure 3B shows that also in GTC-fed wild-type mice the levels of CLU did not change substantially between 12 and 24 weeks of age (lanes 79). CLU protein accumulation in the prostate of 12- and 17-week-old TRAMP mice was similar to that of non-transgenic mice (lanes 10 and 11), except that, in 17-week-old mice, a strong signal for the low molecular form of the protein appeared (lane 11). The same was true for 24-week-old TRAMP mice that did not develop CaP (lane 12). At variance, in those animals escaping the chemopreventive action of GTC and developing CaP, CLU was undetectable. Thus, the preventative effect of GTC for CaP was strictly correlated with CLU expression in this experimental model.
|
Immunohistochemistry analysis (Figure 4) was performed using the same antibody used previously for western blot analysis. The prostates of non-transgenic mice (Figure 4A) were composed of a single layer of columnar secretory epithelium and CLU was present prevalently as secreted protein in the lumen of the glands. In poorly differentiated adenocarcinoma composed of sheets of anaplastic tumor cells, CLU was generally undetectable (Figure 4B). The prostate glands of GTC-fed TRAMP mice at 24 weeks of age (Figure 4D) displayed high grade PIN, similar to that described at an earlier age (1016 weeks) in TRAMP mice (22). This was associated with extensive infolding of the epithelial cell layer into the glandular lumen. Under these conditions, CLU immunostaining appeared confined mostly to the cytoplasm of epithelial cells and in a few nuclei. Interestingly, Figure 4C shows a field taken from prostate glands of GTC-fed TRAMP mice at 17 weeks of age, where a large number of epithelial cells, that appeared normal rather than transformed, showed nuclear localization of CLU. Thus, nuclear localization of CLU seems to be an early response to GTC administration, preceding the late stage of CaP development.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As shown by others (23) with different cell lines, EGCG is relatively ineffective on the proliferative activity of normal human prostate epithelial cells in primary culture, but it inhibits growth of both well differentiated, immortalized, non-tumorigenic PNT1A cells and tumorigenic, androgen-independent, highly malignant PC-3 cells. Sensitivity to the polyphenolic compound appears to be much higher with SV40-immortalized, well-differentiated cells (PNT1A, IC50 83.6 ± 1.3 µg/ml) than with truly malignant ones (PC-3, IC50 202.3 ± 3.2 µg/ml). This may show a relation to the preventive, rather than curative, action proposed for catechins (3), suggesting that EGCG would exert its maximal effect at the initial stages of loss of cell cycle control.
CLU appeared undetectable by western blot analysis in normal cells, but it was present at basal levels both in PNT1A and PC-3 cells, where it was markedly induced by EGCG treatment. In both cell lines the increase in CLU accumulation was accompanied by the cleavage of both pro-caspase 8 and 3, which suggest that CLU induction might affect caspase cascade activation.
Experiments of northern and western analyses conducted with TRAMP mice prostate glands during CaP development fully confirmed the down-regulation of CLU mRNA and protein in poorly differentiated tumors. This finding agrees nicely with the data already reported for human CaP specimens, demonstrating that CLU mRNA and protein down-regulation indeed accompany human CaP onset and progression (13,14). Interestingly enough, further confirmation came very recently from another laboratory using micro-array analysis (24) (please search for CLU in the online database at http://microarray-pubs.stanford.edu/cgi-bin/gx?n=prostate1&rx=5).
The anti-proliferative action of CLU that we have reported many times with different biological systems (1316) is corroborated, under the present conditions, by the pattern of expression of histone H3 and Gas1 mRNAs (markers of cell proliferation and cell quiescence, respectively). In fact, the former resulted undetectable in all specimens except cancer tissue, but the levels of expression of Gas1 mRNA exhibited reciprocal changes with respect to histone H3 (Figure 2A, lanes 5 and 7) and paralleled the pattern of CLU (Figure 2B, lane 13), with the exception represented by GTC-fed 24-week-old TRAMP mice developing tumors (Figure 2B, lane 14). The coexistence, in these animals, of high levels of Gas1 and histone H3 transcripts may be envisioned as the consequence of the heterogeneity of the cell population within the tumor, which, at specific phases of its growth, may include actively proliferating malignant cells that have escaped the inhibitory action of catechins and cells that are still under the inhibitory effects exerted by the polyphenols on the cell proliferation (25). It seems that changes in the levels of histone H3 or Gas1 do not necessarily correlate with tumor progression, rather changes in CLU levels appears to be more consistently involved in this process. In this regard, it is noteworthy that the level of CLU mRNA increased dramatically in the prostate of 24-week-old transgenic mice GTC-chemoprevented (Figure 2B, lane 13) with respect to non-transgenic controls (Figure 2B, lane 12), which prevented CLU protein expression from being abrogated, as it occurred in 24-week-old TRAMP mice water-fed (Figure 3A, lane 6) or in those escaping chemoprevention (Figure 3B, lane 13), thus maintaining a CLU protein level similar to that of non-transgenic mice (Figure 3A, lane 3) or tumor-free GTC-treated TRAMP animals (Figure 3B, lane 12).
In water-fed TRAMP mice at 17 weeks of age (Figure 2A, lane 4), CLU mRNA accumulated at higher levels than in non-transgenic counterparts (Figure 2A, lane 3). Under the same conditions, the level of the 60-kDa precursor of CLU protein decreased substantially (Figure 3A, lane 5). This apparent lack of correlation between CLU mRNA and protein levels may indicate translational regulation of CLU expression, suggesting that changes in CLU proteomic pattern are early events during CaP progression in the TRAMP model.
Loss of caspase activities during human CaP progression has been reported previously (26). Detection of caspase 9 in TRAMP mice receiving water suggests that this protease is involved in this model of tumor progression; in fact, it markedly decreased at 17 weeks and became undetectable at 24 weeks (Figure 3A, lanes 5 and 6, respectively). Caspase 9 appeared also to respond dramatically to treatment with GTC and to be associated to the chemopreventive action of the latter, in that, while disappearing in TRAMP mice escaping chemoprevention (Figure 3B, lane 13), it increased markedly in tumor-free TRAMP mice at 24 weeks of age (Figure 3B, lane 12).
Immunohistochemistry data (Figure 4A) show that in non-transgenic mice at 24 weeks of age, CLU is present in huge amounts in the lumen of the glands while practically undetectable in poorly differentiated TRAMP tumors (Figure 4B), confirming the results of Figures 2 and 3. It has been reported (12) that nuclear CLU is associated with a high rate of apoptotic activity, thus since the epithelial cells in GTC-fed TRAMP mice at 17 weeks of age (Figure 4C) appeared normal and non-transformed, induction of nuclear CLU and apoptotic activity might be the result of GTC chemopreventive effects. The picture shown in Figure 4D, obtained from GTC-fed TRAMP mice at 24 weeks of age, indicates that CLU is actively expressed in epithelial cells at this stage of chemoprevention, mostly in the cytoplasm, but also in the nuclei of a limited number of cells. The presence of CLU in the cytoplasm of epithelial cells in GTC-fed TRAMP mice at 24 weeks of age may indicate that, in the prostate of these animals, normal cells protected from apoptosis still coexist with cells doomed to cell death, marked by nuclear CLU. In non-treated mice at this age, the tumor has yet progressed to poorly differentiated grade (22), but in these GTC-fed TRAMP mice only PIN lesions can be appreciated, showing a marked delay in tumor development which may be the major effect of GTC chemoprevention. Preliminary experiments (data not shown) suggest, by immunohistochemistry, that SV40 is still expressed following GTC administration even at 24 weeks of age in TRAMP animals. Thus, GTC administration does not abrogate SV40 expression in TRAMP mice. In this regard, the sustained expression of CLU, which we have already shown capable of inhibiting the cell growth of SV40-immortalized cells (15), and in particular its nuclear translocation well evident in 17-week-old TRAMP, GCT-treated (Figure 4, panel C) is very likely a key event in the inhibition of CaP progression in this experimental model.
The data presented here, altogether, suggest that CLU might participate in the chemopreventive action of GTC. Considering the anti-proliferative activity shown by CLU in many biological systems (1316) and, particularly, the action exerted by nuclear CLU in this regard as reported by us more recently (27,28), a possible role for CLU as a novel tumor-suppressor gene in the prostate may perhaps be postulated. Two relevant papers, one by Hastak et al. (29) showing that EGCG induction of apoptosis involves NF-B inactivation, the other by Santilli et al. (30) showing that CLU is required for the regulation of NF-
B activity, appeared very recently. The data lend further support to the hypothesis of a role for CLU in mediating the chemopreventive action of catechins. The validity of this hypothesis is currently being investigated in our laboratory.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|