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The Prostate 38:268–277 (1999)
Early Castration-Induced Upregulation of
Transforming Growth Factor ␤1 and Its
Receptors Is Associated With Tumor Cell
Apoptosis and a Major Decline in Serum
Prostate-Specific Antigen in Prostate
Cancer Patients
Pernilla Wikström,1 Patrick Westin,2 Pär Stattin,1 Jan-Erik Damber,1* and
Anders Bergh2
1
2
Department of Urology and Andrology, Umeå, Sweden
Department of Pathology, Umeå University, Umeå, Sweden
BACKGROUND. The mechanism behind castration-induced apoptosis in prostate cells is
unknown, but data from other species suggest that transforming growth factor ␤1 (TGF-␤1)
may be involved.
METHODS. By using quantitative RT-PCR and immunohistochemistry, expression of TGF␤1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of core
biopsies taken before and 2–11 days after castration therapy. The TGF-␤ responses were
related to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA).
RESULTS. In normal prostate tissue, apoptosis was generally increased by castration, and
apoptosis was accompanied by an increase in TGF-␤1 and RII mRNA levels (P < 0.05). In
tumors, apoptosis was seen only in 44% of the cases and in these, but not in the others,
TGF-␤1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing a
prognostically favorable PSA response (nadir PSA <5 ng/ml), but not in the others, RI and RII
mRNA levels were significantly upregulated (P < 0.05).
CONCLUSIONS. Short-term upregulation of TGF-␤1 and its receptors is associated with
apoptosis in human prostate and prostate cancer, and possibly with a favorable clinical
outcome after castration therapy. Prostate 38:268–277, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
TGF-␤1; apoptosis; prostate cancer; castration therapy
INTRODUCTION
Castration treatment has remained the first-line
therapy for metastatic prostate cancer over the past 50
years [1], since it initially relieves clinical symptoms
for most patients. However, almost all tumors relapse
to an androgen-independent stage within a few
months or up to several years later. This wide timespan in progression-free survival indicates tumor differences at the cellular level. At present, the best way
to predict clinical outcome after castration is to measure the serum level of prostate-specific antigen (PSA)
3–6 months after therapy initiation [2,3]. Patients with
© 1999 Wiley-Liss, Inc.
normalized PSA values at that time (PSA-responders)
have longer predicted progression-free survival times
than non-PSA-responders. However, a recent study
showed that it may be possible to predict clinical reGrant sponsor: Swedish Cancer Society; Grant number: Project 1760;
Grant sponsor: University Hospital of Northern Sweden; Grant
sponsor: Maud and Birger Gustavsson Foundation; Grant sponsor:
Lions Cancer Research Foundation; Grant sponsor: Umeå University; Grant sponsor: Swedish Society for Medical Research.
*Correspondence to: J.-E. Damber, Department of Urology and Andrology, Umeå University, 901 85 Umeå, Sweden.
Received 27 March 1998; Accepted 30 July 1998
TGF-␤1 and Apoptosis in Prostate Cancer
sponse at an earlier time point by examining shortterm cellular effects induced by castration therapy [4].
In the rat ventral prostate (VP), androgen deprivation causes massive cell death within days due to induction of apoptosis [5,6]. Castration-induced apoptosis in the prostate is believed to be mediated by different intermediate factors. It has been suggested that
transforming growth factor ␤1 (TGF-␤1) is a crucial
factor for the apoptotic process in the VP [7]. TGF-␤1
is a multifunctional growth factor with a number of
effects in the development, differentiation, and
growth of epithelial tissues [8]. The expression of TGF␤1 and its receptors, TGF-␤ receptor type I and type II
(RI and RII), is negatively regulated by androgens and
increases during castration-induced apoptosis in the
rat VP [9–11]. Moreover, TGF-␤1 has been shown to
induce apoptosis in normal prostatic epithelial cells
both in vivo and in vitro [12,13]. In the rat prostatic
Dunning R3327 PAP adenocarcinoma, on the other
hand, neither apoptosis nor TGF-␤1 or RII expression
increase shortly after castration treatment [11,14,15].
Lack of TGF-␤ induction after castration has also been
demonstrated in other Dunning tumor sublines [16].
Taken together, these results suggest that castrationinduced apoptosis in androgen-dependent prostatic
tissues involves a TGF-␤1 response that might be lost
in androgen-independent prostatic tumors.
In earlier studies, we found that human prostate
tumors respond to castration in a highly heterogeneous way. Only approximately one third of tumors,
mainly belonging to PSA-responding patients,
showed increased apoptotic index 1 week after treatment [4,17]. This could be compared to a sevenfold
increase in apoptotic cells in adjacent normal prostatic
glands [17]. However, it is not known if castrationinduced apoptosis in human prostate and prostate tumors involves a TGF-␤ response. Neither is it known
if the lack of short-term apoptosis induction in some
prostate tumors after androgen withdrawal could be
due to some failure in mediating such a TGF-␤ response. Human prostate tumors often express high
levels of TGF-␤1 [18,19], and overproduction of TGF␤1 has been shown to be associated with angiogenesis,
metastasis, and short survival in prostate cancer [20].
This may be due to possible tumor-stimulating properties of TGF-␤1 such as inhibition of immune responses [21] and stimulation of angiogenesis and cell
motility [22,23]. Furthermore, low expression of RI
and RII has been associated with short prostate cancer-specific survival [20,24], which may indicate some
failure of prostate tumor epithelial cells in responding
to TGF-␤1 growth-inhibiting and/or apoptotic signals.
The aim of this study was to examine if short-term
269
effects on TGF-␤1, RI, and RII expression are associated with apoptosis and clinical response after castration therapy in patients with advanced prostate cancer.
MATERIALS AND METHODS
Tissue Collection and Processing
Several ultrasound-guided core biopsies were taken
shortly before and 2–11 days after castration therapy
in a series of patients with advanced prostate cancer.
Local tumor stage was evaluated by digital rectal examination, according to the 1992 UICC classification
[25], and radionuclide bone scan was performed for
metastasis staging at the time of diagnosis (Table I).
Biopsies were immediately frozen in liquid nitrogen
or fixed in phosphate-buffered formalin, before being
embedded in TissueTek (Miles, Inc., Elkhart, IN) and
paraffin, respectively. The biopsies were cut into
4-␮m-thick sections and stained with hematoxylin and
eosin (HE). The tumors were classified into high (G1),
moderate (G2), and low (G3) differentiation by evaluating the HE-stained slides from the fixed biopsies,
according to the World Health Organization (WHO)
classification system (Table I) [26]. The HE-stained
cryosections were used for localizations of normal areas (biopsy areas where no malignant cells were detected) and tumor areas (Fig. 1). Biopsy parts corresponding to these areas were microdissected from the
frozen biopsies, by using a sterile scalpel blade, and
further processed for total RNA extraction. By using
this procedure, total RNA from normal and tumor
prostate tissue were isolated from 9 and 18 pairs of
frozen pre- and posttherapy biopsies, respectively.
Clinical Classification
Response to castration therapy was defined, as previously described [4], as a serum level of PSA of 艋5
ng/ml 3–6 months posttreatment (PSA-responders),
and nonresponse as a nadir PSA 艌10 ng/ml (nonPSA-responders, Table I).
Determination of Apoptotic Indexes
Apoptotic indexes (number of apoptotic cells per
1,000 cells) in the HE-stained sections from the frozen
tumors were determined by counting approximately
250–1,000 and 300–2,500 normal and tumor cells, respectively, at 400× magnification. Apoptotic cells were
defined as single rounded cells or fragments with
270
Wikström et al.
TABLE 1. Clinical Characteristics of Patients With Advanced Prostate Cancer Treated With Castration Therapy and
Included in the mRNA Experiments
Tumor stagec
T1–T2
T3–T4
Tumor graded
G1
G2
G3
Metastasis (bone scan)
PSA before therapye
PSA nadir
AI before therapy
AI after therapy
Time for biopsyf
Ap-responders
(n = 8)a
Non-Ap-responders
(n = 10)
PSA-responders
(n = 10)b
Non-PSA-responders
(n = 7)
1
7
2
8
2
8
1
6
1
4
3
7
1,400 (1,000)
26 (21)
8.8 (1.5)
19 (3.6)
6.4 (1.1)
5
5
8
550 (210)
25 (11)
17 (2.0)
13 (1.8)
6.3 (0.47)
1
5
4
7
1,100 (850)
2.3 (0.44)
12 (1.5)
14 (1.9)
5.6 (0.92)
3
4
7
720 (230)
61 (23)
14 (3.4)
16 (4.4)
7.3 (0.29)
a
Apoptotic (Ap) response defined as increased apoptotic index (AI) in post- compared to pretherapy biopsy. Indexes calculated as
number of apoptotic cells per 1,000 cells in HE-stained sections.
b
Response defined as serum prostate-specific antigen (PSA) 艋5 ng/ml, and nonresponse as PSA 艌10 ng/ml 3–6 months after therapy.
One patient had a nadir PSA value of 8 ng/ml.
c
According to UICC [25].
d
According to WHO [26].
e
Values expressed as means and SEM (in parentheses).
f
Days between therapy and posttherapy biopsy.
Total RNA Preparation and Competitive Reverse
Transcription-Polymerase Chain Reaction
(RT-PCR)
Fig. 1. Identification of normal (N) and tumor (T) areas in HEstained cryosection from a pretherapy prostate cancer core biopsy (×200).
densely aggregated chromatin and condensed cytoplasm, often lying in ‘‘halos’’ of extra cellular space
(Fig. 2) [27]. If more than one apoptotic body was seen
per ‘‘halo,’’ these were considered to originate from
the same cell and were counted as one.
Apoptotic response to castration therapy was defined as increased apoptotic index in the posttherapy
biopsy compared to the pretherapy biopsy (Apresponders), and apoptotic nonresponse as unchanged
or decreased apoptotic index after treatment (non-Apresponders, Table I).
Total RNA was isolated from the frozen prostatic
tissues by the TRIzol extraction method (Life Technologies AB, Täby, Sweden). The mRNA levels for
TGF-␤1, RI, RII, and the housekeeping gene cyclophilin [28] were quantified, by using competitive
RT-PCR and PCR primers as previously described
[11,15]. Briefly, 50 ng of total RNA were reverse transcribed together with appropriate amounts of internal
RNA standards (IS) [11,15] for TGF-␤1, RI, RII, and
cyclophilin. Each RNA sample was titrated with three
amounts (double samples) of IS, in ranges of 8.5–170,
0.52–10, 2.3–46, and 34–680 amol for TGF-␤1, RI, RII,
and cyclophilin, respectively. After RT completion,
samples were divided into four PCR tubes, in order to
amplify cDNA for TGF-␤1 (248 bp), RI (177 bp), RII
(215 bp), and cyclophilin (362 bp) separately. During
32 cycles of PCR (95°C, 30 sec; 63°C, 30 sec; and 72°C,
45 sec), the TGF-␤ templates were competitively amplified with cDNA for their corresponding IS (266,
163, 199, and 315 bp, respectively). Resulting PCR
products were analyzed in an automatic laser fluorescence system (ABI PRISM™ 377 DNA sequencer, Perkin Elmer, Askim, Sweden). The data were processed
by the ABI PRISM™ GeneScan software (Perkin
Elmer), and RNA levels were calculated from tem-
TGF-␤1 and Apoptosis in Prostate Cancer
271
TABLE II. Clinical Characteristics of Patients With
Advanced Prostate Cancer Treated With Castration
Therapy and Included in the
Immunohistochemistry Experiments
Tumor stageb
T1–T2
T3–T4
Tumor gradec
G1
G2
G3
Metastasis (bone scan)
PSA before therapyd
PSA nadir
Time for biopsye
PSAresponders
(n = 12)a
Non-PSAresponders
(n = 9)
4
8
1
8
3
7
2
6
770 (720)
2.0 (0.38)
7.0 (0.64)
2
4
3
8
1,600 (460)
84 (22)
6.8 (0.40)
a
Response defined as serum prostate-specific antigen (PSA) 艋5
ng/ml, and nonresponse as PSA 艌10 ng/ml 3–6 months after
therapy. One patient had a nadir PSA value of 8 ng/ml.
b
According to UICC [25].
c
According to WHO [26].
d
Values expressed as means and SEM (in parentheses).
e
Days between therapy and posttherapy biopsy.
Fig. 2. Cryosections showing castration-induced apoptosis (arrows) in tumor epithelial cells of a prostate cancer core biopsy
(×400). A: Tumor cells before therapy. B: Tumor cells after
therapy.
plate- to IS-cDNA ratios, as previously described [15].
The TGF-␤1, RI, and RII levels were corrected for the
corresponding cyclophilin levels in each RNA sample
and expressed as relative levels (amol/amol cyclophilin mRNA) in the resulting figures.
Immunohistochemistry
In addition to quantify the mRNA levels for TGF-␤1
and its receptors before and after castration, we
wanted to use immunohistochemistry (IHC) to examine TGF-␤1, RI, and RII protein expression in response
to castration therapy. Unfortunately, the biopsy material used in the mRNA experiment was not sufficient
for these studies, and we had to use core biopsies from
another series of patients. The two series of patients
were similar according to tumor and metastasis classification and PSA levels before and after therapy, and
the biopsies were collected and processed in the same
way (Tables I and II).
Four-micron sections from formalin-fixed tumor biopsies were deparaffinated and rehydrated according
to standard procedures, washed with phosphatebuffered saline (PBS), and heated in a microwave oven
at 600 W for 2 × 7.5 min and 1 × 5 min in 0.01 M citrate
buffer, pH 6.0, as earlier described [29]. To quench
endogenous peroxidase activity, slides were immersed in 3% H2O2 in methanol for 20 min. Slides
were incubated overnight, at 4°C, with the following
antibodies: anti-TGF-␤1 (10 ␮g/ml, anti-TGF-␤1 neutralizing antibody, R&D Systems, Oxon, UK), anti-RI
(0.25 ␮g/ml, V-22, Santa Cruz Biotechnology, Santa
Cruz, CA), and anti-RII (1 ␮g/ml, anti-human TGF-␤
RII neutralizing antibody, R&D Systems). For detection, the ABC technique was used, with aminoethylcarbazole as chromogen, according to the manufacturer’s instructions (Vector Laboratories, Burlingame,
CA). Sections were counterstained with Mayer’s hematoxylin solution. Specificity of TGF-␤ immunoreactions was examined by preincubation of the primary
antibodies with a 25-fold (w/w) excess of the corresponding control peptides (recombinant human TGF␤1 and TGF-␤1 soluble receptor II, R&D Systems, and
V-22P, Santa Cruz Biotechnology).
The immunoreactivity in tumor and normal areas
272
Wikström et al.
TABLE III. Short-Term Effects Induced by Castration Therapy on Apoptosis
and TGF-␤1, RI, and RII mRNA Levels in Patients With Advanced
Prostate Cancer
Normal tissue (n = 9)
AIa
TGF-␤1b
RI
RII
Time for biopsyc
Tumors (n = 18)
Untreated
mean (SEM)
Castrated
mean (SEM)
Untreated
mean (SEM)
Castrated
mean (SEM)
5.0 (0.64)
0.12 (0.017)
0.022 (0.0034)
0.23 (0.085)
14.0 (2.2)*
0.37 (0.15)*
0.060 (0.032)
0.71 (0.36)
7.0 (0.65)
13.0 (1.6)
0.11 (0.012)
0.014 (0.0023)
0.094 (0.014)
16.0 (2.0)
0.21 (0.045)*
0.020 (0.0025)
0.17 (0.032)**
6.3 (0.55)
a
Apoptotic indexes (AI) calculated as number of apoptotic cells per 1,000 cells in HEstained sections.
b
Relative mRNA levels expressed as amol/amol cyclophilin mRNA (see Materials and
Methods).
c
Days between therapy and post-therapy biopsy.
*P < 0.05, significant increase after castration therapy.
**P < 0.01, significant increase after castration therapy.
was evaluated without knowledge of any patient data.
Immunoreactivities for TGF-␤1, RI, and RII were classified as negative (−), moderate (+), or intense (++),
and assessed as either increased or unchanged by castration therapy.
Statistics
Groups were compared by the Mann-Whitney Utest and paired observations by the Wilcoxon paired
test. To test correlations, the Spearman rank sum test
was applied. P 艋 0.05 was considered statistically significant.
RESULTS
Effects of Castration Therapy on Apoptosis, and
on TGF-␤1, RI, and RII Expression
Apoptotic indexes were determined and TGF-␤1,
RI, and RII mRNA levels were quantified in normal
and tumor parts of prostate cancer biopsies taken before and 2–11 days after castration therapy (Table III).
Furthermore, TGF-␤1 and TGF-␤ receptor protein expression was examined in pre- and posttherapy biopsies by using IHC (Table IV).
In normal prostatic tissue, apoptotic indexes were
increased after castration in 8 of 9 cases (89%), and
TGF-␤1, RI, and RII mRNA levels were increased in 7
(78%), 5 (56%), and 7 (78%) cases, respectively. In the
one normal case where no induction of apoptosis was
seen after treatment, there was also no increase in
TGF-␤1 or TGF-␤ receptor mRNA levels. The average
TGF-␤1 mRNA levels and apoptotic indexes were significantly higher in the post- than in the pretherapy
biopsies (3.1- and 2.8-fold; P = 0.028 and 0.011, respectively), while the increase in RI and RII mRNA levels
were nonsignificant (P = 0.21 and 0.14, Table III).
In the tumor tissue, apoptotic response to castration
was more heterogeneous than in normal tissue, with
only 8 of 18 cases (44%) showing increased apoptotic
indexes after therapy. For the whole group, castration
did not induce a significant increase in apoptosis. The
TGF-␤1 and RI mRNA levels were increased by castration in 12 (67%, P = 0.031 and 0.102, respectively),
and the RII mRNA levels in 13 (72%, P = 0.006), of the
tumors (Table III).
Immunoreactivity for TGF-␤1, RI, and RII was
found mainly in normal and tumor epithelial cells
(Fig. 3), which is in line with recent results demonstrating protein expression of TGF-␤1 and its receptors, and mRNA expression of TGF-␤1, preferentially
in prostate epithelial cells [20]. The epithelial immunostaining in the pretherapy biopsies was classified as
negative (−), moderate (+), or intense (++), and was
found to be unchanged or intensified (Fig. 3) in the
posttherapy biopsies (Table III). In accordance with
the mRNA results (Table III), protein levels of TGF-␤1,
RI, and RII seemed to be heterogeneously upregulated
by androgen ablation in the prostate tissues (Table IV).
TGF-␤1 and TGF-␤ receptor protein induction were
more frequently seen in normal than in tumor tissue
(Table IV). The specificity of the immunoreactions has
previously been determined [20], and control slides
incubated with preblocked antibodies showed no
staining (results not shown).
TGF-␤1 and Apoptosis in Prostate Cancer
273
TABLE IV. Short-Term Effects Induced by Castration Therapy on TGF-␤1, RI,
and RII Immunoreactivity in Patients With Advanced Prostate Cancer
Increased immunoreactivity
after therapy
TGF-␤
RI
RII
No. of cases
(normal/tumor)
Time for biopsy
(normal/tumor)a
Normal tissue
no. (%)
Tumor
no. (%)
13/21
13/21
16/22
6.5 (0.27)/6.8 (0.44)
7.3 (0.36)/6.7 (0.42)
7.3 (0.55)/7.0 (0.33)
11 (85)
9 (69)
10 (63)
9 (43)
11 (52)
12 (55)
a
Days between therapy and posttherapy biopsy. Values expressed as means and SEM (in
parentheses).
TGF-␤1, RI, and RII mRNA Expression in Relation
to Castration-Induced Apoptosis
Normal tissue with increased apoptotic index after
castration showed three- and fivefold inductions of
TGF-␤1 and RII mRNA, respectively (P = 0.025, Fig.
4A). The RI mRNA level was increased twofold, but
this was not statistically significant (P = 0.123, Fig. 4A).
In the tumors, the relative changes in TGF-␤1, RI,
and RII mRNA levels observed after castration
therapy were correlated to the corresponding changes
in apoptosis indexes (rs = 0.60, 0.63, and 0.61; P = 0.016,
0.005, and 0.008, respectively). TGF-␤1, RI, and RII
mRNA levels were increased in the Ap-responding
tumors after castration (2.5-, 1.8-, and 2.3-fold; P =
0.036, 0.036, and 0.017, respectively, Fig. 4B), while
neither TGF-␤1 nor the TGF-␤ receptor mRNA levels
were significantly increased in the non-Ap-responding tumors (Fig. 4C).
TGF-␤1, RI, and RII mRNA Expression in Relation
to PSA Response After Castration Therapy
There was no significant correlation found between
the relative changes in tumor TGF-␤1, RI, or RII
mRNA levels and the corresponding changes in patient PSA levels after castration therapy. In accordance
with the Ap-responding patients, however, the PSAresponding patients showed increased RI and RII
mRNA levels after treatment (1.7- and 1.5-fold, P =
0.047 and 0.022, respectively, Fig. 5A). There was also
a tendency for TGF-␤1 induction in this group, although the increase was nonsignificant (P = 0.114, Fig.
5A). Neither TGF-␤1, nor the TGF-␤ receptor mRNA
levels, were significantly increased in the non-PSAresponding tumors after therapy (Fig. 5B).
DISCUSSION
Androgen ablative therapy today, due to its ability
to initially relieve clinical symptoms for most patients,
is the most frequently used method in the treatment of
advanced prostate cancer. The beneficial therapy responses are believed to be achieved by castrationinduced apoptosis and decreased cell proliferation
among androgen-dependent epithelial cells [5,6,30].
Only a minority of human prostate tumors, however,
respond with increase in apoptosis. Recent studies
have shown increased, unchanged, or even decreased
apoptosis in human prostate cancer 1 week after castration [17], and this heterogeneity in short-term apoptotic response was demonstrated to be differential for
subsequent clinical outcome [4]. The molecular
mechanism for castration-induced apoptosis in prostate epithelial cells is not clear, but it has been thought
to involve a TGF-␤1-dependent pathway (see Introduction).
Several studies have demonstrated a relationship
between TGF-␤1 and castration-induced apoptosis in
animal models [9,10,30,31]. However, the present
study is the first to report short-term effects on TGF␤1, RI, and RII expression in the human prostate and
advanced prostate cancer after castration. Induction of
TGF-␤1, RI, and RII expression was found to be related to the apoptotic response in tumor cells after
castration therapy. The TGF-␤1, RI, and RII mRNA
levels were significantly increased in the Apresponding, but not in the non-Ap-responding tumors
after treatment. This is in line with previous results
showing increased expression of TGF-␤1 and its receptors in apoptosis-responding model systems, such
as the rat VP and the human PC-82 tumor [9,10,30,31],
but not in apoptosis-nonresponding Dunning tumors
after castration [11,15,16]. Taken together, these results
suggest that TGF-␤1 is involved in mediating castration-induced apoptosis in the prostate, and furthermore, that androgen-independent tumors may evade
castration-induced apoptosis due to defects in their
TGF-␤1 response to androgen ablation.
Defects in TGF-␤ receptor expression have been associated with TGF-␤1 insensitivity in many systems,
274
Wikström et al.
Fig. 3. Sections from Ap-responding tumors before (A, C, E) and after (B, D, F) castration therapy, showing increased immunoreactivity
for TGF-␤1 (A, B), RI (C, D), and RII (E, F) after castration (×400).
including the prostate [32–36], and loss of epithelial
expression of RI and RII has been shown with human
prostate cancer progression [37,38]. Lack of RI or RII
expression in the epithelial cells would be one obvious
explanation for the absence of castration-induced
TGF-␤1 response in advanced prostate cancer. In this
study, however, TGF-␤ receptor expression in the intact tumors was not predictable for the apoptotic re-
sponse to castration. All untreated tumors expressed
similar amounts of RI and RII mRNA, and the Apresponding group contained intact tumors with negative as well as intense receptor immunoreactivity. Our
results suggest that the ability of androgen-independent tumors to avoid castration-induced apoptosis is
not simply due to loss of RI or RII expression in the
tumor cells, but may be influenced by the inability of
TGF-␤1 and Apoptosis in Prostate Cancer
275
Fig. 5. Competitive RT-PCR results, showing relative TGF-␤1,
RI, and RII mRNA levels before (open columns) and after (shaded
columns) castration therapy in PSA-responding (A) and non-PSAresponding (B) prostate tumors. RI and RII mRNA levels were
increased by castration in the PSA-responding tumors (*P < 0.05).
No significant induction of TGF-␤1, RI, or RII was seen in the
non-PSA-responding tumors after castration.
Fig. 4. Competitive RT-PCR results, showing relative TGF-␤1,
RI, and RII mRNA levels before (open columns) and after (shaded
columns) castration therapy in Ap-responding normal prostate
areas (A), and in Ap-responding (B) and non-Ap-responding (C)
prostate tumors. TGF-␤1 and RII mRNA levels were increased by
castration in the Ap-responding normal areas, and the TGF-␤1, RI,
and RII mRNA levels were increased in the Ap-responding tumors
(*P < 0.05). No significant induction of TGF-␤1, RI, or RII was seen
in the non-Ap-responding tumors after castration.
some tumors to upregulate the expression of these receptors in response to castration. The TGF-␤1 signaling pathway in prostate cancer cells therefore needs to
be thoroughly investigated.
In addition to being associated with apoptosis, the
tumor TGF-␤ response to castration therapy seemed to
be predictable for the subsequent PSA-response. The
RI and RII mRNA levels were significantly increased
by castration in the group of PSA-responding tumors,
but not in the group of non-PSA-responding tumors.
These results are consistent with results by Stattin et
al. [4], indicating the possibility of predicting clinical
outcome at an early time-point by studying short-term
effects of castration therapy. There was, however, no
significant induction of TGF-␤1 in the PSAresponding tumors after castration, although an overall induction of TGF-␤1 was seen in 67% of the tumor
cases. Speculatively, induction of TGF-␤1 in tumor
cells that do not go into apoptosis after castration may
be nonbeneficial to the patient, due to tumorpromoting effects of TGF-␤1 such as inhibition of immune responses [21] and stimulation of angiogenesis
[20,22], cell motility [23], and metastasis [20].
Apoptosis was more frequently seen in normal
prostate tissue than in tumors after castration. Only
one normal case showed lack of castration-induced
apoptosis, and in this tissue there were also no increases in TGF-␤1 or TGF-␤ receptor expression. The
average increase of TGF-␤1, RI, and RII mRNA in the
normal Ap-responding tissue was more pronounced
276
Wikström et al.
than the increase in the Ap-responding tumors, although the increase in RI mRNA was not statistically
significant. Lack of statistical confirmation of RI induction in this group, as well as the big standard deviations seen for TGF-␤1, RI, and RII in normal tissue,
suggest that normal, nonmalignant prostate tissue
could be rather heterogeneous. Possible reasons for
this are premalignant cellular defects in normallooking epithelial cells, but also regional differences in
normal epithelial cell function and/or functional
changes related to the proximity of cancer tissue.
11.
12.
13.
14.
CONCLUSIONS
The present study suggests that increased expression of TGF-␤1, RI, and RII is associated with castration-induced apoptosis in the human prostate and in
advanced prostate cancer. Moreover, short-term effects on RI and RII seem to be predictable for PSA
response and thus probably also for the long-term
clinical outcome after castration therapy.
ACKNOWLEDGMENTS
Mrs. Birgitta Ekblom, Mrs. Elisabeth Dahlberg, and
Ms. Pernilla Andersson have skillfully contributed to
this paper by their technical assistance.
15.
16.
17.
18.
19.
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