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Departments of Surgery and Molecular Biology, Division of Urology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Address all correspondence and requests for reprints to: Natasha Kyprianou, Ph.D., Division of Urology, Department Surgery, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201. E-mail: nkyprianou{at}smail.umaryland.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the normal prostate gland, homeostasis is maintained by a unique balance between the rates of proliferation and apoptosis, such that neither overgrowth nor involution of the gland occurs (2). Normal prostatic development and cell proliferation require the presence of androgens and certain growth factors. The expression of several growth factors and their cognate receptors, such as transforming growth factor-ß (TGF-ß) and TGF-ß receptors I (TßRI) and II (TßRII), is controlled by androgens, suggesting that androgens mediate proliferation through growth factor signaling pathways (3, 4, 5). Additionally, removal of androgens by hormone ablation therapy causes the secretory cells of the glandular epithelium to undergo apoptosis with TGF-ß being an important mediator in this process (2, 4, 6).
The loss of function or expression of TßRI and/or TßRII may contribute to the absence of TGF-ß-mediated growth inhibition and apoptosis during tumorigenesis. In several different tumor cell lines, TßRII inactivation is associated with decreased TGF-ß growth inhibition (7, 8, 9). Furthermore, mutations or loss of expression of TßRII have been demonstrated in breast cancer, T-cell lymphomas, colon cancers, and head and neck squamous carcinomas (9, 10, 11, 12). In prostate cancer, evidence from several groups has demonstrated a correlation of down-regulation of TßRI and TßRII expression with tumor progression (3, 13, 14). Previous studies in this laboratory demonstrated that expression of TGF-ß RII in the normally TGF-ß insensitive LNCaP prostatic carcinoma cell line, restores sensitivity to TGF-ß, which results in growth arrest and apoptosis induction (15). In vivo, these LNCaP TßRII cells exhibit decreased tumorigenicity through decreased bcl-2 expression and increased caspase-1 expression, documenting that bcl-2 and caspases are involved in TGF-ß-mediated apoptosis (16).
Considering that increased expression of both TGF-ß and its receptors is associated with androgen-ablation-induced apoptosis and that deregulated TGF-ß signaling is implicated in prostate tumorigenesis, in this study we examine the effects of androgens on TGF-ß-mediated apoptosis in prostate cancer cells with restored TGF-ß signaling pathways. Our results demonstrate that dihydrotestosterone (DHT) enhances (instead of protecting against) TGF-ß-mediated apoptosis and cell cycle arrest in LNCaP TßRII prostate cancer cells through the modulation of key apoptotic and cell cycle regulatory proteins. These findings suggest that DHT may have potential therapeutic benefits by sensitizing prostate cancer cells to physiological apoptotic inducing agents.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell viability
LNCaP Neo and TßRII clone 61 transfectant cells were plated at
a density of 1 x 105 cells per well in
6-well plates. Forty-eight hours after plating, the cells were washed
twice with PBS and the media was changed to DMEM (without phenol red)
supplemented with 10% charcoal-stripped serum (CSS), 100 U penicillin,
100 mg/ml streptomycin, and/or 1 nM DHT, 0.5 ng/ml TGF-ß,
and/or 5.0 ng/ml TGF-ß. Five days after treatment, cell viability was
assessed by trypan blue exclusion as previously described
(15) and the mean values (from triplicate wells) for each
treatment were determined.
[3H]Thymidine incorporation assay
Cells were plated at 5 x 104 cells
per well in 24-well plates, and after 48 h, the cells were washed
and treated with DHT and/or TGF-ß as described above. Five days
following treatment, cells were pulsed with 7 µCi/ml
[3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) at 37 C for 3 h as previously
described (15). DNA was precipitated with 10% (wt/vol)
trichloroacetic acid, and the amount of
[3H]thymidine incorporated was determined by
liquid scintillation counting. The mean values from triplicate wells
for each treatment were determined (experiments performed in
triplicate).
Morphological evaluation of apoptosis
Cells were plated at 1 x 105 cells
per well in 6-well plates and treated with DHT and TGF-ß for 5 days
as described above. Cells were subsequently fixed with 4%
paraformaldehyde (Sigma, St. Louis, MO) and stained with
10 µg/ml Hoechst 33342 (B2261; Sigma) in the presence of
0.1% Triton X-100 (Sigma) overnight in the dark at 4 C as
previously described (17). Cells were visualized using a
Carl Zeiss Axiovert 10 fluorescent microscope
(Thornwood, NY) with a UV filter (365 nm) and those cells containing
fragmented nuclei were designated as apoptotic (x32 magnification).
The apoptotic index was assessed by counting 3 random fields in
duplicate wells per treatment group (from experiments performed in
triplicate).
Ribonuclease protection assays
LNCaP TBRII receptor transfectant clone 61 (TßRII) cells were
plated at 1 x 105 cells per well on
100-mm plates and after 24 h, cells were washed twice in
PBS and the media was changed to DMEM containing 10% CSS, no phenol
red, and supplemented or not with the following: 5.0 ng/ml TGF-ß,
and/or 1 nM DHT. After 72 h, cells were harvested and
total RNA was isolated using the TRIzol Reagent (Life Technologies, Inc.). Total RNA (20 µg) isolated from the LNCaP
TßRII cells was coprecipitated with 4.2 x
105 cpm of purified antisense
32P-labeled RNA probes generated from the hAPO-1c
template set for caspases 1–10a and the housekeeping genes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32 (45607P;
PharMingen, San Diego, CA). The subsequent assay was
performed according to the protocol outlined for HybSpeed RNase
protection assay (Ambion, Inc.). Briefly, the
32P-labeled probe was coprecipitated with 20 µg
total RNA, resuspended in 10 µl hybridization buffer, and solubilized
at 95 C for 3 min. Hybridization occurred at 68 C for 10 min followed
by digestion of unprotected probes by RnaseA/T1 mix at 1:100 dilution
for 45 min at 37 C. Protected RNA fragments were electrophoresed on 5%
polyacrylamide/8 M urea gels, and radiolabeled fragments
were visualized by autoradiography. Densitometric analysis was
performed using the Scion Image program and bands were normalized to
GAPDH and L32 fragments. Results were quantitatively expressed as fold-
induction or fold-reduction of the control nontreated samples
(CSS).
Western blot analyses
Cells were plated at 1 x 105 cells
per well in 6-well plates and exposed to DHT and TGF-ß treatment for
5 days as described above. Total cellular protein was extracted from
the cell pellets by homogenization in RIPA buffer [150
mM NaCl, 50 mM Tris (pH 8.0), 1% Nonidet P40,
0.5% deoxycholate sodium salt, 1 µM PMSF, and 2 mg/ml
aprotonin] for 1 h at 4 C. Aliquots of protein (20–40 µg) were
loaded on 4%/12% SDS stacked polyacrylamide gels and subjected to
electrophoretic analysis. After electrophoresis, proteins were
electrotransferred onto Immobilon-P membranes (Amersham Pharmacia Biotech). Membranes were blocked for 1 h at room
temperature with 5% dry milk in PBS containing 0.05% Tween 20 then
incubated with the primary antibody overnight at 4 C. Expression of
bcl-2 (clone 124; DAKO Corp., Carpintera, CA), bax
(sc-493; Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
p21 (OP64; Calbiochem, San Diego, CA), p27 (sc-528;
Santa Cruz Biotechnology, Inc.), caspase-1 (sc-515;
Santa Cruz Biotechnology, Inc.), caspase-3 (65906E;
PharMingen, San Diego, CA), caspase-9 (9502; New England Biolabs, Inc., Beverly, MA), and 
-actin (CP01;
Calbiochem) proteins was assessed using the appropriate
primary antibodies. Following incubation with the primary antibody, the
membranes were washed and incubated with species specific horse-radish
peroxidase (HRP)-labeled secondary antibodies (1 h at room
temperature). Membranes were subsequently incubated with the enhanced
chemiluminescence system (ECL, RPN 2108; Amersham Pharmacia Biotech) and autoradiographed using x-ray film (Amersham Pharmacia Biotech). Densitometric analysis was performed using
the Scion Image program (Scion Corp., Frederick, MD) and all bands were
normalized to actin and shown as fold-change of the control nontreated
samples.
Caspase inhibitor assays
LNCaP TßRII cells were plated at 1x105
cells per well in 6-well plates. After 48 h, the cells were washed
twice with PBS and treated with 1 nM DHT and/or 5 ng/ml
TGF-ß in DMEM containing 10% CSS, Pen/Strep, L-glutamine
in the presence of either the vehicle (DMSO), or either caspase-1
inhibitor z-YVAD-FMK (20 µM, FK-013; Enzyme Systems,
Livermore, CA), or caspase-3 inhibitor z-DQMD-FMK (20 µM,
FK-025; Enzyme Systems). Five days following treatment the cells were
fixed in 4% paraformaldehyde and stained with Hoechst 33342 and the
incidence of apoptosis was determined as described above from duplicate
wells (in experiments performed in triplicate).
Statistical analyses
Statistical analyses of the numerical data were performed using
ANOVA in the Graph Pad Prism program. All data are represented
as average values ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As shown in Fig. 1A, a further
significant decrease in cell proliferation was observed in cells
treated with both TGF-ß and DHT, compared with TGF-ß alone
(P = 0.023). A similar trend was observed in the loss
of cell viability on the basis of trypan blue exclusion although
statistical significance was not achieved (Fig. 1B).
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), the expression levels
of the cell cycle regulators p21 and p27 proteins were examined.
Exposure of the LNCaP TßRII cells to the combined treatment of
TGF-ß and DHT resulted in a 1.5-fold induction of p21 protein levels
24 h following treatment (Fig. 2A, lane 10). This induction was significantly higher than that observed in
cells grown in CSS or DHT alone. Additionally, levels of p27 protein
actually decreased 50% by day 3 following combined TGF-ß and DHT
treatment (Fig. 2B, lane 13). Treatment with DHT and TGF-ß did not
lead to increased p27 protein expression levels over those cells grown
in CSS. However, growth in CSS did increase p27 protein levels over
cells grown in FBS (Fig. 2B, lane 1).
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, combined treatment with
5.0 ng/ml TGF-ß and DHT resulted in an increased number of apoptotic
cells, over that induced by TGF-ß alone. Quantitative analysis
revealed that 15% of the cells treated with both TGF-ß and DHT were
undergoing apoptosis, whereas only 7% of the TGF-ß-treated cells
exhibited apoptotic morphology (Fig. 3B). The apoptotic response
occurred in a TGF-ß dose-dependent fashion, which reached statistical
significance with 5.0 ng/ml TGF-ß and DHT (P =
0.023). Minimal levels of apoptosis induction were observed in cells
grown in CSS or DHT alone (Fig. 3B). In these cases, dividing cells
could also be identified (Fig. 3A).
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), the potential modulations in
apoptotic regulatory proteins were subsequently assessed. As shown in
Fig. 4, Western blot analysis revealed
that there was a significant decrease (approximately 2-fold) in bcl-2
protein following TGF-ß treatment (5.0 ng/ml), and a further
considerable decrease (to almost undetectable levels) following the
combined TGF-ß (5.0 ng/ml) and DHT treatment over cells grown in CSS
control. Although treatment with TGF-ß alone at the low dose (0.5
ng/ml) had no significant effect on bcl-2 expression, combination with
DHT resulted in a moderate reduction in bcl-2 levels (Fig. 4). No
significant alterations in the expression profile of the
pro-apoptotic protein, bax were observed (Fig. 4).
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. A
moderate increase in caspase-1 mRNA expression was detected in
TGF-ß-treated cells over CSS cells (Fig. 5). All other caspases
demonstrated only slight increases following TGF-ß treatment (Fig. 5). DHT treatment resulted in a slight decrease in caspases 1, 5, 6, 7,
and 10a with more significant decreases in caspase-2 (Fig. 5). The
combined treatment with TGF-ß and DHT did not alter the expression of
any of the caspase mRNA over those observed in the control (CSS) (Fig. 5).
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). In the
case of procaspase-1, minimal basal expression is observed in cells
grown in 10% CSS and 10% FBS (Fig. 6A, lanes 1 and 7). Treatment with
either DHT or TGF-ß (0.5 ng/ml and 5.0 ng/ml) alone resulted in an
approximately 2-fold increase in procaspase-1 protein (Fig. 6A, lanes
2–4). A further significant increase in procaspase-1 expression
(2.2-fold) was observed following the combined treatment of DHT and
TGF-ß (5.0 ng/ml) (Fig. 6A, lane 6). As shown in Fig. 6B there were
no significant changes in procaspase-3 protein levels following any of
the treatment regimes. There was a moderate increase in procaspase-9
expression levels in response to the combined DHT and TGF-ß (5.0
ng/ml) treatment, which was similar to the increase observed in cells
grown in 10% FBS (approximately 1.3-fold) (Fig. 6C, lanes 6 and 7,
respectively).
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).
Interestingly, apoptosis induced by TGF-ß alone or combined TGF-ß
and DHT treatment was suppressed in a comparable manner by the
caspase-1 inhibitor, implicating the involvement of caspase-1 in the
initiation of apoptosis in response to both treatments (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study focused on exploring the effects of androgens on TGF-ß-mediated apoptosis as an initial approach to understand the apoptotic process in hormone-sensitive prostate cancer cells. The documented ability of DHT to enhance the apoptotic effect of TGF-ß against prostate cancer cells (rather than protect them from it) surprisingly does not resonate with the above dogma. Although it has been previously shown that superphysiological concentrations of DHT have suppressive effects on prostate cancer cell proliferation, physiological concentrations (1 nM) are known to be growth permissive (23). Additionally, a recent review by Prehn (24) discussed the correlation between decreasing androgens and increasing incidence of prostate cancer with age and states that subphysiological concentrations of androgens also yield negative effects (24). This review provided a convincing argument that androgen treatment instead of androgen ablation may lower the incidence of prostate cancer. In accord with this provocative concept, our study demonstrates that DHT sensitizes prostate cancer cells to undergo apoptosis following receipt of an apoptotic signal by TGF-ß. If one considers that most human prostate cancers arise in aging men with relatively low and declining androgen levels, a potential priming of prostate cancer cells (by androgens) to the apoptotic effects of TGF-ß might not be fully reached. Attractive as this argument might be, one also has to consider that evidence in rat prostate cell types (in contrast with our data) indicates that DHT inhibits the negative growth of TGF-ß signaling by down-regulation of the TGF-ß and its receptors (25).
Increased bcl-2 expression correlates with development of androgen-independent prostate cancer, an apoptotic defect that renders these tumors resistant to therapy (20, 21, 22). Previous reports demonstrated that both DHT and TGF-ß can independently down-regulate bcl-2 expression (16, 20, 26). In accordance, our observations indicate that the combined treatment of DHT and TGF-ß enhanced these effects. Although the mechanisms by which DHT and TGF-ß regulate the expression of bcl-2 remain unclear, a recent report suggested that bcl-2 regulation by DHT occurred through an indirect mechanism (27). Because androgens also regulate the expression of TGF-ß and its receptors (6), it is tempting to propose that the DHT effects against prostate growth can be potentially mediated by intracellular effectors of TGF-ß signaling.
Smads are the transcriptional regulators of the TGF-ß family of growth factors that function to transmit the growth factor signal from the cell membrane to the nucleus (28). Known TGF-ß regulated genes, such as plasminogen activator inhibitor type-1, possess Smad binding elements (SBE) within the promoter sequences, which allow for Smad binding and transcriptional activation following TGF-ß treatment (29). In the present study, the presence of SBEs and TGF-ß-inducible elements were examined in the bcl-2 promoter region. However, following analysis, no SBEs of TGF-ß- inducible elements were identified suggesting that other factors aside from DHT, TGF-ß, and Smads directly regulate bcl-2 gene expression (data not shown). Interestingly, a recent study demonstrated that the glucocorticoid receptor could actually repress TGF-ß signaling by interacting with Smad 3 (30). Therefore, one may propose that a similar active interplay occurs between the androgen receptor and the TGF-ß signaling pathways. Because expression of both Smad 2 and Smad 4 is regulated by androgens in the rat ventral prostate (31), a dynamic cross-talk may exist between androgens and TGF-ß signaling with a potential interaction with the Smad intracellular substrates for TGF-ß signaling. Studies are currently in progress to investigate such interactions in our model system.
Several lines of evidence implicate caspase involvement in the execution of apoptosis in both androgen-sensitive and androgen-insensitive prostate cancer cell lines in response to diverse stimuli (32, 33, 34, 35). In the case of hormone-ablation therapy, the caspase inhibitor CrmA suppresses apoptosis induction in the LNCaP cell line (35). Additionally, we previously demonstrated that increases in caspase-1 expression occur following TGF-ß treatment (16). In the present study only slight increases in caspase-1 mRNA expression were observed following TGF-ß treatment. This could result from the differences between growth in 10% FBS and 10% CSS. Because treatment of serum with charcoal removes all of the steroid components including DHT, the possibility exists that TGF-ß-mediated up-regulation of caspase-1 requires an additional factor present in FBS that is removed from CSS. As expected, DHT treatment alone provided only moderate decreases in caspases mRNA. No significant changes were detected in the mRNA expression for the other caspases. The increased protein expression of procaspase-1 and procaspase-9 in response to the combined treatment could be due to increased protein stabilization and/or translation. The fact that the active fragments of either caspase-1 or caspase-9 were not detected, may be due to the relatively low levels of cells undergoing apoptosis in our system. Although the presence of the active caspase-1 protein fragment was not detected, the functional role of activated caspase-1 is documented by the ability of caspase-1 specific peptide inhibitor z-YVAD to significantly suppress apoptosis following combined DHT and TGF-ß treatment. No alterations in procaspase-3 expression were observed consistent with the functional assay, indicating that caspase-3 inhibitor z-DQMD did not change the apoptotic outcome of the cells. Together, these data demonstrate a functional role for activated caspase-1 (but not caspase-3) in apoptosis initiation in response to combined DHT and TGF-ß treatment.
Typically, caspase-1 is regarded as the cytokine-mediated caspase due
to its critical role in the cleavage and subsequent activation of
interleukin-1ß (36). Studies with caspase-1 knockout
mice indicated that apoptosis occurred following 
-radiation but not
following lipopolysaccharide treatment, clearly delineating its
role in cytokine-mediated apoptosis but not in response to other
stimuli (37). Considering this evidence and the knowledge
that TGF-ß functions as a cytokine promoting or inhibiting an immune
response following trauma and/or injury (38), it is
possible to hypothesize that in prostate cancer, an immune response
following hormone ablation, may involve the up-regulation of TGF-ß
and subsequent caspase-1 induction. Indirect support for this concept
stems from evidence that the expression of proinflammatory genes,
including interleukin-1ß, increase following estrogen- induced
immune response in the rat prostate, which documents the effects of
hormone manipulation on prostatic immune responses
(39).
In conclusion, our data provides a basis for the molecular interactions between TGF-ß and androgen signaling pathways in apoptosis induction in prostate cancer cells. In vivo studies are currently in progress to assess the interactions between the TGF-ß and androgen signaling pathways in prostate tumor apoptosis following castration. A further understanding of this cross-talk will facilitate the design of new mechanistic apoptosis-driven therapeutic modalities to effectively treat androgen-dependent and independent prostatic tumors.
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Acknowledgments |
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Footnotes |
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2 Recipient of an American Foundation for Urologic Research Disease
Research Fellowship.
Received December 27, 2000.
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