Novel Action of Botulinum Toxin on the Stromal

and Epithelial Components of the Prostate Gland

Yao-Chi Chuang,* Chao-Cheng Huang, Hong-Yo Kang, Po-Hui Chiang, Fernando Demiguel,
Naoki Yoshimura and Michael B. Chancellor*,†
From the Departments of Urology (YCC, PHC) and Pathology (CCH) and Center for Menopause and Reproductive Medicine Research
(HYK), Chang Gung Memorial Hospital, Kaohsiung, Taiwan, Republic of China, and Department of Urology, University of Pittsburgh
School of Medicine (FD, NY, MBC), Pittsburgh, Pennsylvania

Purpose: Intraprostatic injection of BTX-A has demonstrated clinical improvement in men with bladder outlet obstruction.
We investigated the mechanisms of action of BTX-A on the prostate.
Materials and Methods: Adult male Sprague-Dawley rats were injected with varying doses of BTX-A into the prostate and
the prostates were harvested after 1 or 2 weeks. The effects of BTX-A on prostate histology, and the proliferative and
apoptotic indexes were determined using hematoxylin and eosin staining, proliferative cell nuclear antigen staining and
TUNEL staining, respectively. Changes in ␣1A adrenergic receptor and androgen receptor were evaluated by Western
blotting.
Results: One week after BTX-A injection generalized prostate atrophy was observed. There was a significant increase in
apoptotic cells (12, 16 and 22-fold), and decrease in proliferative cells (38%, 77% and 80%) and ␣1A adrenergic receptor (13%,
80% and 81%) for 5, 10 and 20 U, respectively. There was no significant change in androgen receptors. The effects were
decreased 2 weeks after BTX-A treatment.
Conclusions: BTX-A injection into the prostate alters cellular dynamics by inducing apoptosis, inhibiting proliferation and
down-regulating ␣1A adrenergic receptors. BTX-A may potentially be the drug that has dual actions on the static and dynamic
components of benign prostatic hyperplasia.

Key Words: prostate; botulinum toxin type A; rats, Sprague-Dawley; prostatic hyperplasia; receptors, adrenergic

PH is nonmalignant enlargement of the prostate that presynaptic nerve terminal.4 – 6 BTX-A has been used to

B is regarded as a major cause of voiding dysfunction in

aging men.1,2 Excessive growth (static component)
and contraction (dynamic component) are the 2 main BPH
components. The 2 classes of pharmacotherapy for BPH
treatment are ␣1-adrenergic antagonists, which relax pros-
tate smooth muscle (dynamic component), and 5␣-reductase
inhibitors, which shrink the prostate (static component) by
blocking formation of the main intracellular androgen, dihy-
drotestosterone.1–3 These drugs are not all always effective
and they have potential side effects, including postural hy-
potension, retrograde ejaculation and impotence. To our
knowledge there are no agents today with efficacy for the
dynamic and static components of BPH or that may be
effective when ␣-blocker and 5␣-reductase fail to be clini-
cally effective.
BTX-A is known to block the release of neurotransmit-
ters, including acetylcholine, norepinephrine, calcitonin
gene-related peptide, substance P and glutamate, from the
treat diseases related to muscular spasticity by inhibiting
acetylcholine release at the neuromuscular junction. Injec-
tion of BTX-A into the urethra or bladder is also used in
urology for external sphincter hyperactivity and overactive
bladder.4,7A previous study using rat models has provided
direct evidence that intraprostatic injection of BTX-A in-
duces selective denervation, and subsequent apoptosis and
atrophy of the glands.8 Furthermore, a recent clinical study
revealed a marked reduction in prostate size and improve-
ment in lower urinary tract symptoms after BTX-A injection
into the human prostate.9
Prostate function is under the influence of acetylcholine
as well as norepinephrine and testosterone.10,11 Thus, addi-
tional mechanisms might be involved in BTX-A effects on
the prostate. Toward this goal we examined the immuno-
blotting of ␣1A-adrenergic receptor and androgen receptor,
and cellular dynamic in terms of proliferation and apoptosis
in the rat prostate after BTX-A injection.

Submitted for publication April 27, 2005.

Study received Institutional Animal Care Committee approval.
Supported by National Institutes of Health DK06695 and
DK066138, and National Science Council Taiwan NSC 93-2314-B-
182A-195.
* Correspondence: Suite 700, Kaufmann Building, 3471 Fifth
Ave., Pittsburgh, Pennsylvania 15213 (telephone: 412-692-4096;
FAX: 412-692-4101; e-mail: chancellormb@msx.upmc.edu).
† Financial interest and/or other relationship with Allergan.
See Editorial on page 817.
MATERIALS AND METHODS
BTX-A injection. All experimental procedures were per-
formed in male Sprague-Dawley rats weighing 450 to 550
gm, and reviewed and approved by the Institutional Animal
Care Committee before the study began. With the rat under
halothane anesthesia a lower midline abdominal incision
was made and the bladder and prostate were carefully ex-

0022-5347/06/1753-1158/0 1158 Vol. 175, 1158-1163, March 2006

THE JOURNAL OF UROLOGY® Printed in U.S.A.
Copyright © 2006 by AMERICAN UROLOGICAL ASSOCIATION DOI:10.1016/S0022-5347(05)00318-6
 BOTULINUM TOXIN INJECTION INTO RAT PROSTATE 1159

TABLE 1. Effects of saline and BTX-A on ventral prostate weight, and PCNA and TUNEL staining at 1 and 2 weeks
Mean Prostate Wt/100 Gm Mean PCNA ⫾ SE Mean TUNEL ⫾ SE
Body Wt ⫾ SE (500 cells/4 high power fields) (500 cells/4 high power fields)

1 Wk:
A. Saline 0.166 ⫾ 0.010 214.8 ⫾ 5.9 3.7 ⫾ 0.8
B. 5 U 0.125 ⫾ 0.012 132.8 ⫾ 5.8 45.0 ⫾ 4.2
C. 10 U 0.115 ⫾ 0.009 50.0 ⫾ 2.2 57.7 ⫾ 3.3
D. 20 U 0.075 ⫾ 0.009 44.0 ⫾ 4.4 82.7 ⫾ 4.3
2 Wks:
E. Saline 0.167 ⫾ 0.009 244.8 ⫾ 21.2 3.0 ⫾ 0.6
F. 5 U 0.146 ⫾ 0.007 231.7 ⫾ 20.2 2.8 ⫾ 0.5
G. 10 U 0.139 ⫾ 0.008 193.5 ⫾ 15.7 4.0 ⫾ 0.6
H. 20 U 0.119 ⫾ 0.007 163.2 ⫾ 13.2 4.8 ⫾ 0.5
Six preparations per group.

posed from the surrounding tissues. Saline or various doses amount of ␤-tubulin was also detected as the internal con-
of BTX-A (Botox®) (5, 10 or 20 U in 0.2 cc saline) were then trol. Quantitative analysis was done using LabWorks™ im-
injected slowly into the ventral lobes of the prostate using a age acquisition and analysis software.
30 gauge needle at 0.1 cc per lobe. After injection the wound
was closed in layers. Statistical analysis. Quantitative data are expressed as
the mean ⫾ SE. Statistical analyses were performed using
Tissue sectioning and analysis. The animals were sacri- ANOVA with the Scheffe post-test when applicable with
ficed at intervals of 1 or 2 weeks by an overdose of pento- p ⬍0.05 considered significant.
barbital sodium (200 mg/kg intraperitoneally). The prostate
was harvested, weighted and divided into 2 parts. One part RESULTS
was fixed in 10% buffered formaldehyde for 24 to 48 hours
and embedded in paraffin. It was then stained with hema- Prostate volume. We observed a reduction in prostate size
toxylin and eosin or analyzed immunohistochemically for in BTX-A injected animals. Table 1 shows that mean ventral
markers of cell proliferation using antibodies to PCNA prostate weight was decreased in animals 1 week after the
(Dako, Carpinteria, California) or apoptosis with TUNEL injection of 5, 10 and 20 U BTX-A in a dose dependent
staining using a commercially available kit (Detection Kit manner (24.7%, 30.7% and 54.8% decrease, respectively).
POD, Boehringer Mannheim, Mannheim, Germany).8,12,13 BTX-A effects on prostate volume were less significant at 2
The remainder was frozen in liquid nitrogen and stored until weeks (table 2).
use. The proportion of PCNA positive or TUNEL positive
nuclei per 500 nuclei was quantified at 400⫻ magnification Histology. BTX-A induced a diffuse atrophy of prostatic
using a 10 ⫻ 10 grid in the eyepiece in 4 fields. glands and flattened epithelial cells in a dose dependent
manner 1 week after injection (fig. 1). The effect was de-
Western blot analysis for ␣1A-adrenergic receptor and creased at 2 weeks (fig. 2). There was no inflammatory
androgen receptor. Western blot analysis for ␣1A-adreno- reaction after BTX-A injection. TUNEL staining of sections
ceptor and androgen receptor was done according to the of control animals revealed few apoptotic cells, which were
standard protocol (Amersham Biosciences, Piscataway, New
Jersey).13 Samples were homogenized with protein extrac-
tion solution (iNtRON Biotechnology, Kyungki-Do, Korea)
prior to sonication and purification. The amount of total TABLE 2. Saline vs 5, 10 and 20 U BTX-A
protein was measured with the Bradford protein assay p Value (Scheffe post-test)
method (Bio-Rad Laboratories, Hercules, California). So- Wt PCNA TUNEL
dium dodecyl sulfate-polyacrylamide gel electrophoresis was
1 Wk
performed using the Laemmli buffer system. An aliquot of
the extracts equivalent to 50 ␮g protein was loaded onto 8% Saline vs:
5U 0.192 0.018 0.000
polyacrylamide gel, electrophoresed at a constant voltage of 10 U 0.046 0.000 0.000
100 V for 1 hour and transferred to Hybond-P polyvinylidene 5 U vs:
fluoride membrane (Amersham Biosciences). The membrane 10 U 0.999 0.016 0.097
20 U 0.060 0.007 0.000
was immunoblotted using a 1:500 dilution of androgen 10 vs 20 U 0.236 1.000 0.000
receptor rabbit polyclonal antibody or 1:500 dilution of
␣1A-adrenoceptor goat polyclonal antibody (Santa Cruz Bio- 2 Wks
technology, Santa Cruz, California). Primary antibody was Saline vs:
applied overnight at 4C and secondary antibody was applied 5U 0.912 0.999 1.000
for 2 hours at room temperature. Protein were detected 10 U 0.688 0.391 1.000
20 U 0.076 0.019 1.000
using horseradish peroxidase linked anti-rabbit or anti-goat 5 U vs:
IgG. Primary and secondary antibody incubations were per- 10 U 1.000 0.746 1.000
20 U 0.708 0.085 1.000
formed in 5% defatted milk powder in tris-buffered saline. 10 vs 20 U 0.922 0.906 1.000
Western blots were visualized by an enhanced chemilumi-
Saline vs 20 U BTX-A at 1 week p ⫽ 0.000.
nescence detection system (Amersham Biosciences). The
FIG. 1. Photomicrographs of prostate sections after saline (A), and 5 (B), 10 (C) and 20 U (D) BTX-A treatment at 1 week. Significant
glandular proliferation with papillary infolding in lumen was seen in saline injected rat. Atrophy change in glandular component with
flattening of lining epithelium was seen in BTX-A treated rat. Reduced from ⫻200.

FIG. 2. Photomicrographs of prostate sections after saline (A), and 5 (B), 10 (C) and 20 U (D) BTX-A treatment at 2 weeks. Significant
glandular proliferation with papillary infolding in lumen was seen in saline injected rat. Atrophy change in glandular component with
flattening of lining epithelium was seen in BTX-A treated rat, which was less significant than in figure 1, B to D. Reduced from ⫻200.
FIG. 3. TUNEL staining of prostate sections of saline (A), and 5 (B), 10 (C) and 20 U (D) BTX-A injected preparations at 1 week. Few apoptotic
nuclei are recognizable in saline injected animal. Increasing amounts of apoptotic nuclei (arrow) were seen in BTX-A treated animal at 1 week.
Reduced from ⫻200.

FIG. 4. PCNA immunostaining of prostate sections of saline (A), and 5 (B), 10 (C) and 20 U (D) BTX-A injected preparations at 1 week. Most
glandular epithelial cell nuclei were positive for PCNA in saline injected animal. Marked decrease in number of PCNA positive cells (arrow)
was seen in 5, 10 and 20 U BTX-A treated animals at 1 week. Difference in number of PCNA positive cells between 10 and 20 U BTX-A treated
animals at 1 week was not conspicuous (C and D). Reduced from ⫻200.
1162 BOTULINUM TOXIN INJECTION INTO RAT PROSTATE
FIG. 5. Western blot for detecting ␣1A-adrenoceptor (AR) and andro-
gen receptor (AndR). Protein amount of ␣1A-adrenoceptor was de-
creased by BTX-A treatment at 1 week and effect was decreased at
2 weeks (A). There was no significant difference in amount of an-
drogen receptor in saline and BTX-A treated animals (B).
significantly increased in 5, 10 and 20 U BTX-A injected
animals at 1 week (12, 16 and 22-fold, respectively, table 1
and fig. 3). However, the number of apoptotic cells recovered
to baseline at 2 weeks. PCNA staining demonstrated signif-
icantly decreased cellular proliferation 1 week after 5, 10
and 20 U BTX-A injection (38%, 77% and 80% reduction,
respectively, table 1 and fig. 4). The effects were decreased at
2 weeks. Taken together these findings suggest that BTX-A
causes decreased cell survival and proliferation in the rat
prostate and the effects recovered at 2 weeks.
Effects on ␣1A-adrenergic receptor and androgen re-
ceptor. Figure 5 shows that 5, 10 and 20 U BTX-A de-
creased ␣1A-adrenergic receptor in a dose dependent manner
(13%, 80% and 81% reduction, respectively) at 1 week. How-
ever, at 2 weeks the BTX-A effect was decreased. There was
no significant change in androgen receptor by BTX-A injec-
tion. These findings suggest that BTX-A might cause a vol-
ume reduction and down-regulation of ␣1A-adrenergic
receptor, which is independent of androgen receptor.
DISCUSSION
By inhibiting the release of acetylcholine on the nerve ter-
minal BTX-A can suppress the secretomotor function and
trophic effects of acetylcholine on the prostate.11,14 We found
that intraprostatic injection of BTX-A blocks the effects of
acetylcholine on the prostate and induces subsequent cellu-
lar apoptosis, similar to the findings of Doggweiler et al.8 In
addition, our study revealed that BTX-A caused decreased
cellular proliferation and ␣1A-adrenoceptor down-regula-
tion. These new findings suggest that BTX-A has effects the
negative balance of cellular dynamics and might regulate
the static and dynamic components of BPH.
It has been suggested that an increase in the rate of cell
replication or decrease in the rate of cell death can disturb
the balance of cell cycles and result in the net accumulation
of cells and prostatic hyperplasia.10 The expression of spe-
cific prostate apoptosis related genes, such as bcl2 and trans-
forming growth factor-␤, has been implicated in BPH
pathogenesis.15 As the prostate epithelium receives cholin-
ergic innervation, cholinoceptor activation leads to the
growth of prostate epithelial cells and stimulates exocrine
secretion of epidermal growth factor.11 Furthermore, there
is evidence that acetylcholine can act as a cellular modula-
tor, regulating various intracellular functions such as mito-
sis, proliferation and differentiation.14 Thus, the increase in
cellular apoptosis and decrease in cellular proliferation after
BTX-A treatment are likely to be related to the inhibition of
acetylcholine effects and the subsequent decreased neuro-
trophic influence on the gland. Further study to investigate
cholinergic receptors after BTX-A treatment would be inter-
esting for a future report.
The prostate gland is androgen dependent, requiring a
source of testosterone for its growth and function. Treatment
with androgen ablation or 5 ␣-reductase inhibitor decreases
the testosterone or intraprostatic dihydrotestosterone level,
followed by proliferation inhibition and an increase in cell
death in the prostate.1–3 However, 5␣-reductase treatment is
not effective in all patients and it has potential side effects,
including impotence or the loss of libido. Therefore, a new
treatment with local tissue effects that decrease prostate vol-
ume without altering androgen function is highly desirable to
decrease the adverse effects of androgen ablation. Because the
stability of androgen receptor is mainly regulated by androgen,
BTX-A causes a decrease in prostate volume and, as long as it
does not significantly alter the androgen level in prostate
glands, it may not affect the turnover rate of androgen receptor
protein. Our study demonstrates that BTX-A induction of cel-
lular apoptosis and inhibition of cellular proliferation may re-
store the cellular dynamics of BPH. This may emerge as an
alternative for the management of BPH.
It has been observed that there is an overall 6-fold in-
crease in ␣1-adrenoceptor expression and a 9-fold increase in
␣1A-adrenoceptor in BPH compared with normal prostate.16
In addition, ␣1-adrenoceptor antagonists have been success-
fully used for the treatment of benign prostatic obstruction
to relieve lower urinary tract symptoms. However, overall
improvement in BPH symptoms decreased significantly with
time and long-term use of ␣1-adrenoceptor antagonists can-
not prevent BPH progression.17,18 It is conceivable that
chronic administration of ␣1-adrenoceptor antagonists may
result in an alteration of ␣1-adrenoceptor properties. Fur-
thermore, a recent study in a rat model suggested that
long-term use of doxazosin, an ␣1-adrenoceptor antagonist,
resulted in up-regulation of ␣1-adrenoceptor and increased
prostate weight.19 Thus, a drug that can down-regulate ␣1-
adrenoceptor may help relieve newly diagnosed patients
with symptomatic BPH as well as those whose condition is
refractory to ␣1-adrenoceptor antagonists. Our current
study demonstrates that BTX-A down-regulates ␣1A-adreno-
ceptor, which might provide a new rationale for using intra-
prostatic BTX-A injection for the treatment of patients with
BPH, even those whose condition is refractory to ␣-blockers.
The effects of denervation provided by the BTX-A wears
off as new axons sprout and new synaptic contacts form.4
The time necessary to recover function after BTX-A injection
might depend on the different characteristics of targeted
tissue, the therapeutic dose and the interval. The process
requires approximately 2 to 6 months at the mammalian
neuromuscular junction and 17.3 months for sweat glands in
gustatory sweating.20 In the current study BTX-A effects on
the rat prostate were prominent at 1 week but decreased at
2 weeks. As proliferated cells increase and receptor protein
synthesis recovers, the effects of BTX-A on the prostate
 BOTULINUM TOXIN INJECTION INTO RAT PROSTATE
decrease. Effects on the rat prostate were shorter than that
found in the human prostate. We can speculate that prostate
tissue in the rat may have a higher turnover rate than that
in humans. Rat prostate is primarily epithelium, whereas
the human prostate is primarily stroma. Thus, the current
results in the rat model cannot be completely applied to the
human therapeutic arena. Furthermore, various doses of
BTX-A have been injected into the urethra (30 U), bladder
(25 U) and prostate (2.5 to 12 U) to evaluate the impact of
BTX-A on lower urinary tract function in rat models.5,6,8 All
doses used in rat models are larger than those used in
humans, in comparison to the body weight of rats and hu-
mans. Since species differences result in different responses
to BTX-A treatment, we selected the doses of BTX-A (5, 10 or
20 U) in accordance with previous studies. Further clinical
research is needed to assess the true mechanisms of BTX-A
in the human prostate.
CONCLUSIONS
The current study demonstrates that intraprostatic BTX-A
injection down-regulates ␣1-adrenoceptor as well as cellular
dynamics. Thus, BTX-A appears unique in having dual ac-
tions on the dynamic and static components of BPH.7,12
ACKNOWLEDGMENTS
Moya Wu assisted with the study.
Abbreviations and Acronyms
BPH ⫽ benign prostatic hyperplasia
BTX-A ⫽ botulinum toxin type A
PCNA ⫽ proliferating cell nuclear antigen
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