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Proc. West. Pharmacol. Soc. 46: 153-155 (2003)
153
Effect of β-sitosterol as Inhibitor of 5α-reductase in Hamster Prostate
Marisa Cabeza
1*
, Eugene Bratoeff
2
, Ivonne Heuze
1
, Elena Ramírez
2
, Mauricio Sánchez
1
and Eugenio Flores
2
Department of Biological Systems & Animal Production Metropolitan University-Xochimilco
1
, Mexico D. F., Mexico, Department
of Pharmacy, Faculty of Chemistry
2
of National University of Mexico, Mexico D. F.
*email: marisa@cueyatl.uam.mx
It has been reported that β-sitosterol (U) obtained
from plants inhibits the growth and migration of one
type of prostate cancer cell and to slow the growth of
prostate tumors in laboratory mice [1]. These data
suggest that an androgenic mechanism of action
could be involved, since growth of most prostate
cancers is androgen-dependent.
On the other hand β-sitosterol like other drugs derived
from plants have a long tradition in the medical
treatment of benign prostate hyperplasia (BPH) in
Europe. At the present time no mechanism of action
or precise classification of the active compounds for
many of these drugs has been established, although
substantial symptomatic improvement has been
reported. Recently it has been reported that β-
sitosterol significantly improves the symptoms and
urinary flow parameters present in BPH [2].
The purpose of this study was to determine if β-
sitosterol has an effect on the weight of hamster
prostate. Also it was of interest to know if this steroid
could act an inhibitor of 5α-reductase, the enzyme
that converts testosterone (T) to his more active form,
dihydrotestosterone (DHT)[3]. Furthermore it was of
interest to determine if this compound binds to the
androgen receptor.
METHODS: Animal treatment: Adult male golden hamsters (150-
200 g) were obtained from Metropolitan University-Xochimilco of
Mexico. Gonadectomies were performed under light ether
anesthesia 30 d before the experiments.
The biological activity of the pure compound (U), obtained from
Sigma-Aldrich, was determined in gonadectomized male
hamsters divided in several groups. Daily subcutaneous
injections of 400 and 800 µg of U dissolved in 200 µl of sesame
oil were administered for 6 d together with 200 µg of T. Two
groups of animals were kept as a control; one was injected with
200 µl of sesame oil, the second with 200 µg of T for 6 days.
After treatment, animals were sacrificed by ether anesthesia and
the prostate dissected and weighed
In vitro metabolic studies with prostates: Prostates from
gonadectomized animals were removed, blotted and weighed
prior to use. Tissues were homogenized in 3 volumes of TEDAM
buffer (20 mM tris-HCl, 1.5 mM EDTA and 1 0mM sodium
molybdate) at pH 7.4 (4ºC). Homogenates were centrifuged at
140,000 x g for 60 min [8] in a SW 60 Ti rotor (Beckman
instruments, Palo Alto, CA). Pellets were separated, washed with
3 tissue volumes of medium A [20 mM potassium phosphate, pH
7 containing 0.32 M sucrose, 0.1 mM dithiothreitol (Sigma-Aldrich,
Inc)] and centrifuged two additional times at 440 x g at 0ºC for 10
min [4]. Washed pellets were suspended in medium A and stored
at -70ºC. The suspension (6.8 mg protein/ml determined by the
Bradford method [5]) was used as source of 5α-reductase.
Inhibitory effect of finasteride and U on hamster prostatic 5∝-
reductase: In order to calculate IC
50
(the concentration of the
steroid required to inhibit 5α-reductase activity by 50%), two
series of tubes containing different concentrations of finasteride
(100 pM-100 nM) or U (20 nM-10 µM) were incubated in
duplicate, in the presence of: 1 mM of dithiothreitol, 40 mM
sodium phosphate buffer pH of 7, 2 mM NADPH, 2 nM [1,2,6,7-
H
3
]T (specific activity 95 Ci/mmol) and 250 µg of protein in a final
volume of 1 ml. The reaction was carried out in duplicate at 37ºC
for 60 min and stopped by mixing with 1 ml of dichloromethane.
The dichlorometane fraction was separated and the extraction
was repeated x4. The extract was evaporated to dryness under a
nitrogen stream and suspended in 50 µl of methanol spotted on
HPTLC Keiselgel 60 F
254
plates. T and DHT were used as
carriers and the plate was developed in chloroform:acetone=9:1.
Plates were air-dried and the chromatography was repeated x2.
T standard was visualized under UV lights [254 nm] and DHT was
detected using phosphomolibdic acid reagent (8% in methanol)
and heating of the plate. DHT containing areas were cut out and
the strips soaked in 5 ml in Ultima Gold [Packard] and the
radioactivity measured by scintillation.
Competition binding studies: Tubes containing 3.15 nM
[H
3
]DHT (Specific activity 110 Ci/mmol) plus a range of increasing
concentrations (10
-8
-10
-4
M) of nonradioactive DHT and U were
prepared [6]. Aliquots of 200 µl of prostatic cytosol (2.4 mg
protein, determined by the Bradford method [5]) were added and
incubated (by duplicate) for 18 h at 4º C. After this time, 800 µl of
dextran-coated charcoal in TEDAM buffer (containing dithiotreitol)
was added and the mixture incubated for 40 min at 4°C. To
prepare the dextran-coated charcoal mixture, dextran was
agitated for 30 min before adding the charcoal to the mixture.
The tubes were vortexed and immediately centrifuged at 800 g for
10 min and 200 µl aliquots counted by scintillation.
RESULTS: After gonadectomy the weight of the
prostate decreased significantly (p<0.005). Treatment
with vehicle alone (control) did not change this
whereas subcutaneous injection of 200 µg of T for 6 d
significantly increased (p<0.005) the weight of the
prostate in gonadectomized male hamsters (Table I).
When T and U were injected together at two different
doses, the weight of seminal vesicles decreased in a
dose dependent manner suggesting an inhibitory
effect of 5α-reductase. Duplicate experiments were
carried out in quadruplicate. The mean of weights are
shown in Table I.
154
Table I. Effect of β-sitosterol (U) on prostatic weight in
gonadectomized hamsters treated with T. The weight of the
prostate is given in mg ± standard deviation. Significant
differences were observed between treated with T (p<0.009) and
treated with T+U at different doses.
Treatment
Prostate Weight
(mg)
Control
47.2 ± 9.9
T
93.3 ± 26.9
T+U (400 µg) 72.1 ± 14.7
T+U (800 µg) 60.8 ± 7.2
5α-reductase activity inhibition: Since the weight
of the prostate depends on the 5α-reduced T [7], it
was important to determine the effect of U on the in
vitro activity of 5α-reductase. The results (Fig. 1)
obtained from two separate experiments performed in
duplicate demonstrated that β-sitosterol (U) inhibited
5α-reductase activity. The concentration of U
necessary to inhibit 50% of the enzymatic activity
(IC
50
) was of 2.7 µM at pH of 7, whereas that for
finasteride, a type 2 selective 5α-reductase inhibitor,
at pH of 7 was 10.12 nM (see: Cabeza et al. this
volume).
Figure 1. Inhibitory effect of β-sitosterol on hamster prostatic 5α-
reductase activity. Results are the average of two experiments.
IC
50
represents the β-sitosterol concentration necessary to inhibit
50% enzymatic activity.
β-sitosterol competition of androgen receptor
binding. The effect of increasing concentrations of
non-radioactive steroids upon [H
3
]DHT binding to
androgen receptors from male hamster prostate in
two different experiments is shown in Fig. 2. U was a
poor competitor and did not exhibit any apparent
affinity for the androgen receptor.
Figure 2. Binding Specificity for the androgen receptor.
Compound U (β-sitosterol) was a poor competitor at the androgen
receptor, whereas cold DHT competed for [H
3
]DHT binding to the
androgen receptor.
DISCUSSION:
This study describes the 5α-reductase inhibitory
activity of the β-sitosterol. This compound
significantly inhibits growth of the gonadectomized
hamster prostate treated with T at two different doses
thus indicating that β-sitosterol has a pharmacological
activity from subcutaneous application in this model.
In humans treated orally with β-sitosterol, no relevant
reduction of prostatic volume was observed [2]. This
difference between humans and hamsters may be
attributable to the doses chosen or the purity of the
compounds utilized [2]; we employed pure β-sitosterol
purchased from Sigma-Aldrich, whereas Berges et al.
[2] used a mixture of phytosterols.
On the other hand β-sitosterol inhibited 5α -reductase
activity with a value of IC
50
2.7 µM, as compared to
that of finasteride of 10.12 nM. These data indicate
that β-sitosterol is less potent than finasteride in the
inhibition of 5α-reductase. However, U did not exhibit
any affinity to the androgen receptor thus indicating
that the effect observed is not due to the binding of β-
sitosterol to the androgen receptor.
ACKNOWLEDGEMENTS: We gratefully acknowledge the
financial support of CONACYT for the project G-33450-M.
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Compound U
0
25
50
75
100
0246810
mM concentration of U
% of activity of 5a-reductase
HO
IC
50
= 2.7 mM
0
10
20
30
40
50
60
70
80
90
100
0 200 400 600 800 1000 1200
[uM]
% Binding
155
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