Anti-proliferative action of endogenous dehydroepiandrosterone metabolites on human cancer cell lines.

Article abstract of DOI:10.1016/S0039-128X(02)00117-4

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.

Full text of DOI:10.1016/S0039-128X(02)00117-4

Steroids 68 (2003) 73–83
Anti-proliferative action of endogenous dehydroepiandrosterone
metabolites on human cancer cell lines
a
a,b
a,∗
a
Shigemasa Yoshida , Akira Honda , Yasushi Matsuzaki , Sugano Fukushima ,
a
c
c
d
e,f
Naomi Tanaka , Aya Takagiwa , Yoshinori Fujimoto , Hiroshi Miyazaki , Gerald Salen
a
Department of Gastroenterology and Hepatology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
b
Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan
Department of Chemistry and Materials Science, Tokyo Institute of Technology, Tokyo 152-8551, Japan
Department of Drug Metabolism Disposition, Niigata College of Pharmacy, Niigata 950-2081, Japan
c
d
e
Gastrointestinal Division, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ 07103, USA
f
Veterans Affairs Medical Center, East Orange, NJ 07018, USA
Received 23 April 2002; received in revised form 8 July 2002; accepted 26 July 2002
Abstract
Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal
tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by
the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was
undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone,
epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is
responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl
tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all
DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by
DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r = −0.031,
n = 9, NS) or HMGR (r = 0.219, n = 9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not
overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by
the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that
is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or
anti-proliferative therapies.
©
2002 Elsevier Science Inc. All rights reserved.
Keywords: Dehydroepiandrosterone; Metabolites; Cancer; Anti-proliferation; Glucose-6-phosphate dehydrogenase; 3-Hydroxy-3-methylglutaryl CoA
reductase
1
. Introduction
and breast [13,14]. In addition, DHEA inhibits proliferation
of cancer [15–17] and non-cancer cells [18,19] in vitro and
in vivo studies.
Dehydroepiandrosterone (DHEA) is an endogenous
steroid synthesized in the adrenal cortex, gonads, brain, and
gastrointestinal tract [1,2]. Plasma concentrations of this
steroid reach maximum levels during adolescence and then
decline with age. Low plasma levels of DHEA have been
correlated with increased risks of age-associated tumori-
genesis [3,4]. DHEA prevented spontaneous tumors when
given to p53-knockout mice [5] and retarded chemically in-
duced carcinogenesis in liver [6–10], colon [11], lung [12],
Although the mechanisms of the anti-proliferative action
of DHEA have not yet been elucidated, there are two dif-
ferent explanations. First, DHEA is a potent uncompetitive
inhibitor of glucose-6-phosphate dehydrogenase (G6PD)
[20,21], the rate-limiting enzyme in the pentose phosphate
pathway. This pathway provides ribose phosphate, which
is necessary for RNA and DNA biosynthesis [8]. Indeed,
many types of cancers and cultured tumor cells exhibit large
increases in G6PD activity [22]. Second, DHEA may inhibit
cholesterol biosynthesis and the mitogen-activated protein
kinase (MAPK)-mediated signal transduction pathway for
∗
Corresponding author. Tel.: +81-298-53-3138; fax: +81-298-53-3138.
E-mail address: ymatsuzaki-gi@umin.ac.jp (Y. Matsuzaki).
0
039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00117-4

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S. Yoshida et al. / Steroids 68 (2003) 73–83
DNA synthesis by down-regulating 3-hydroxy-3-methyl-
glutaryl-CoA reductase (HMGR) [23,24], the rate-limiting
enzyme in cholesterol biosynthesis. Inhibition of HMGR de-
pletes endogenous pools of mevalonic acid (MVA), the im-
mediate product of HMGR. MVA is a precursor of farnesyl
diphosphate, which is necessary for the farnesylation of Ras
protein, an essential reaction in the MAPK cascade [25,26].
DHEA is often referred to as an adrenal androgen because
it can be converted into testosterone and dihydrotestosterone
in the peripheral tissues. Although DHEA is a naturally
occurring steroid and seems to be less cytotoxic than other
anti-neoplastic drugs, administration for extended periods
increases circulating testosterone and dihydrotestosterone
manifold above normal levels, especially in women [27], and
may cause masculinization. Endogenous DHEA metabo-
lites (Fig. 1), i.e. 7␣-hydroxy-DHEA (7␣-OH-DHEA),
7␤-hydroxy-DHEA (7␤-OH-DHEA), 7-oxo-DHEA, an-
drosterone, epiandrosterone, and etiocholanolone, cannot be
converted to testosterone. Although 7-oxygenated DHEA
may be converted to 7-oxygenated testosterone, this steroid
seems to have no androgenic activity [28]. Therefore, these
DHEA metabolites may be more useful therapeutic agents
than DHEA. However, the effects of the DHEA metabolites
on cell proliferation have not been studied.
The present study was undertaken to investigate whether
DHEA metabolites also have anti-proliferative effects on
Fig. 1. Biosynthetic pathway of DHEA and related steroids from cholesterol. Solid arrows represent known enzymatic reactions whereas broken arrows
represent putative reactions that have yet to be confirmed.

S. Yoshida et al. / Steroids 68 (2003) 73–83
75
cancer cells and to clarify which pathway, the pentose phos-
phate pathway or mevalonate pathway, is responsible for
growth inhibition. We used a ras-activated human hepatoma
cell line, Hep G2 [29], and human colonic adenocarcinoma
cell lines, Caco-2 [30] and HT-29 [31], and systemati-
cally studied the effects of DHEA and its metabolites on
cell growth and G6PD and HMGR activities. The results
showed that not only DHEA, but DHEA metabolites also
had an anti-proliferative effect. However, tumor growth in-
hibition was not explained by the inhibition of either G6PD
or HMGR activities.
cultures were grown and maintained in Eagle’s minimum es-
sential medium (Life Technologies, Inc., Grand Island, NY)
supplemented with 1 mM pyruvate, 0.1 mM non-essential
amino acids, and 10% fetal bovine serum (FBS). The cul-
tures were incubated at 37 C in a humidified incubator con-
taining 5% CO2, 95% air.
◦
2.4. Growth inhibition assays
2.4.1. MTT assay
The effects of DHEA and related steroids on cell growth
were tested by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl
tetrazolium bromide (MTT) assay [34]. On day 0, cells were
4
seeded in 96-well plates at a density of 1.0 × 10 cells per
2
. Experimental
well. On day 1, the original growth medium was removed
and replaced with fresh medium containing various concen-
trations of steroids (1–100 ␮M) dissolved in 1% ethanol.
Although 1% ethanol in the medium had no detectable
effects on cell growth, the same concentration of ethanol
was also added to the controls. On days 2–4, the medium
was discarded, MTT solution (0.5 mg/ml) was added to
2
.1. Chemicals
DHEA, DHEA-sulfate, pregnenolone, androsterone, epi-
androsterone, and etiocholanolone were purchased from
Sigma Chemical Co. (St. Louis, MO) and purified by recrys-
3
tallization. RS-5-[ H]mevalonolactone (MVL), (888.0 GBq/
◦
all wells, and the cells were incubated at 37 C for 4 h.
mmol) was obtained from NEN (Boston, MA). 3-Hydroxy-
1
4
Formazan products were dissolved with dimethylsulfoxide
(DMSO), and the absorption at 540 nm was measured by
Microplatereader M-Emax (Wako, Japan).
3
[
-methyl-3-[ C]glutaryl CoA (2.15 GBq/mmol) and ␥-
3
2
P]ATP (110 TBq/mmol) were obtained from Amersham
(Aylesbury, UK). All other chemicals were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
2.4.2. BrdU assay
Inhibition of cell proliferation by the steroids was also
evaluated by bromodeoxyuridine (BrdU) incorporation as-
2
.2. Syntheses of 7-oxygenated DHEA derivatives
␣-OH-DHEA, 7␤-OH-DHEA, and 7-oxo-DHEA were
4
say. On day 0, 96-well plates were seeded with 1.0 × 10
7
cells per well. On day 1, the original growth medium was re-
moved and replaced with fresh medium containing 100 ␮M
of steroids dissolved in 1% ethanol. After 48 h of incubation,
BrdU incorporation was measured by using the cell prolifer-
ation ELISA system, version 2 (Amersham). Briefly, BrdU
synthesized from 3␤-(t-butyldimethylsilyloxy)-17,17-ethy-
lenedioxyandrost-5-en-7-one, which was obtained from
DHEA in three steps (ketalization, silyl ether formation, and
allylic oxidation with tert-butyl hydroperoxide using ruthe-
nium chloride as a catalyst [32]). Deprotection of the enone
under acidic conditions (2N HCl in methanol tetrahydro-
furan) gave 7-oxo-DHEA. 7␤-OH-DHEA was prepared by
reduction of the enone with NaBH4 in the presence of CeCl3
labeling solution was added to each well and incubated for
◦
2
h at 37 C. After fixation and blocking, peroxidase-labelled
anti-BrdU solution (100 ␮l per well) was added, and the
mixture was incubated for 90 min at room temperature. At
the end of the assay, tetramethylbenzidine (TMB) was added
into each well as a peroxidase substrate (100 ␮l per well).
After 5 min of incubation at room temperature, the reaction
was stopped, and the absorption at 450 nm was measured
by Microplatereader M-Emax. Results were expressed as a
percentage of the control absorbance.
[33] followed by removal of the protecting groups under the
acidic conditions as described above. 7␣-OH-DHEA was
synthesized from the enone in four steps, i.e. reduction with
lithium tri-sec-butylborohydride in tetrahydrofuran [33],
acetylation of the resulting 7␣-alcohol with Ac2O-pyridine,
removal of the silyl and ethylenedeoxy groups in the
same manner as described above, and final removal of the
acetyl group by 5% KOH in methanol tetrahydrofuran.
The purities of these compounds were checked by gas–
liquid chromatography (GLC), and each gave only a single
peak.
2
.5. Assay of HMGR activity
HMGR activities in Hep G2 cells were measured ac-
cording to the method described by Goldstein et al. [35]
with minor modifications. Cells were seeded at a density of
5
2
2
.3. Cell cultures
5×10 /12.5 cm tissue culture flask. After 3 days, when the
cells were about 50% confluent, the medium was replaced
with fresh medium containing 50 ␮M of DHEA or related
steroids dissolved in 1% ethanol. After 24 h of incubation,
the medium from each flask was discarded, and the attached
Hep G2 cells were supplied by Riken Cell Bank (Tsukuba,
Japan), and Caco-2 and HT-29 cells were obtained from
American Type Culture Collection (Rockville, MD). Stock

7
6
S. Yoshida et al. / Steroids 68 (2003) 73–83
cells were rinsed twice with phosphate-buffered saline
PBS). Cells were then harvested by use of a cell scraper
nitrotetrazolium blue (NO2-TB), and 1.8 U/ml diaphorase,
and the mixture was incubated at 37 C for 10 min. LDH
◦
(
and centrifugation at 10,000×g for 1 min. Cell-free extracts
were prepared by adding 65 ␮l of phosphate buffer contain-
ing 0.25% (v/v) Brij 96 to the cell pellets, and aliquots were
used for determination of protein concentration [36]. The
cell extract (50 ␮l) was incubated in a final volume of 150 ␮l
of 100 mM potassium phosphate buffer (pH 7.4) contain-
ing a NADPH generating system. The reaction was started
activity was determined by measuring the absorbance at
560 nm.
2.8. Screening for apoptosis by DNA-binding
fluorochromes
6
2
Hep G2 cells were seeded at a density of 2 × 10 /10 cm
14
with the addition of 15 ␮l of [ C]HMG-CoA and stopped
tissue culture dish. Medium was replaced with fresh medium
containing each steroid (100 ␮M) dissolved in 1% ethanol.
Both attached and floating cells were harvested after 72 h.
The cells were fixed with 4% paraformaldehyde solution
for 24 h, and DNA was stained with 0.2 mM bisbenzimide
H33258 solution (Wako). The bisbenzimide H33258 solu-
tion at 1 mM in PBS was freshly prepared before each ex-
periment. Fluorescent DNA-dye complexes were observed
under a fluorescence microscope with a 340/380 nm excita-
tion filter. About 200–300 cells of attached cells and about
30 cells of floating cells were counted at a magnification of
◦
after 30 min at 37 C with the addition of 20 ␮l of 6N HCl.
After adding unlabeled MVL (0.5 mg) as a marker for vi-
3
sualization on thin-layer plates, [ H] MVL (50,000 dpm)
as an internal recovery standard, 200 ␮l of ethanol, 0.2 g of
anhydrous Na2SO4, and 50 ␮l of water, MVL was extracted
twice with 1 ml of diethyl ether, separated by thin-layer
chromatography, and measured by dual-label liquid scintil-
lation counting. The result was expressed as a percentage of
the control radioactivity measured with the cells incubated
in the presence of 1% ethanol.
400× [38].
2
.6. Assay of G6PD activity
2.9. Statistics
Pooled Hep G2 cell-free extracts were used for mea-
suring G6PD activity. Cells were seeded at a density of
Data are reported here as the mean ± S.D. The statistical
6
2
1
× 10 /25 cm tissue culture flask. After 4 days, when the
significance of differences between the results in the dif-
ferent groups was evaluated with the Student’s two-tailed
t-test. We tested correlation by calculating Pearson’s corre-
lation coefficient, r. For all comparisons, significance was
accepted at the level of P < 0.05.
cells were about 70% confluent, the medium from each flask
was discarded, and the attached cells were rinsed twice with
PBS. Cells were then harvested by use of a cell scraper and
centrifugation at 10,000 × g for 1 min. Cell-free extracts
were prepared by the same method as in the HMG-CoA re-
◦
ductase assay and stored at −80 C. The reaction was started
by the addition of the cell extracts (20 ␮l) to 980 ␮l of 0.1 M
Tris–HCl buffer (pH 8.1) containing 1 M MgCl2, 10 mM
NADP, 200 mM glucose-6-phosphate, and various concen-
trations of steroids (1–100 ␮M) dissolved in 1% ethanol,
3. Results
3.1. Effects on growth inhibition
◦
and the mixture was incubated at 25 C. The initial rate of
When 10–100 ␮M of DHEA and related steroids were
added to Hep G2 cell cultures and incubated for 24–72 h,
cell growth evaluated by MTT assay was significantly inhib-
ited in a dose- (Fig. 2) and time-dependent manner (Fig. 3).
However, each steroid revealed various efficacies of inhibi-
tion. The growth inhibition rates at 100 ␮M after 72 h incu-
bation in order of inhibition efficacy were: etiocholanolone
NADPH formation was determined by measuring the in-
crease of absorption at 340 nm [37]. The result was ex-
pressed as a percentage of the control activity measured with
the cell extract incubated in the presence of 1% ethanol.
2
.7. Determination of LDH activity in cells and medium
(
79%) > pregnenolone (70%) > epiandrosterone (67%) >
Cytotoxicity of DHEA and related steroids was assayed
DHEA (57%) > androsterone (55%) > 7␣-OH-DHEA
(48%) > 7-oxo-DHEA (44%) > DHEA-sulfate (35%) >
7␤-OH DHEA (33%). Growth inhibition of Hep G2 cells
by these steroids was also evaluated by BrdU assay, which
determines DNA synthesis. The results obtained by the two
different assay methods were similar, and a significant cor-
relation was observed between the data of both assays (r =
0.808, n = 9, P < 0.05).
by measuring the activity of lactate dehydrogenase (LDH)
in the cells and the medium. Hep G2 cells were seeded at
5
2
a density of 5 × 10 /10 cm dish. After 24 h, the medium
was replaced with fresh medium containing various kinds
of steroids (10–100 ␮M) dissolved in 1% ethanol, and the
cells were incubated for 48 h. LDH activities in the medium
and in the cells were measured by using a LDH CII kit
(
(
Wako). Briefly, a 10 ␮l aliquot of cell culture medium
released sample) or cell lysate (non-released sample)
was added to 100 ␮l of 0.1 M Tris–HCl buffer (pH 8.4)
containing 0.1 M lithium lactate, 2.6 mM NAD, 0.49 mM
DHEA and its metabolites, except for 7␤-OH-DHEA, also
significantly inhibited the proliferation of other cancer cell
lines, Caco-2 and HT-29 (Fig. 4). In particular, the growth
inhibition due to etiocholanolone was significantly greater

S. Yoshida et al. / Steroids 68 (2003) 73–83
77
Fig. 2. Effects of steroid concentrations on cell growth. Hep G2 cells were incubated for 72 h with each steroid at concentrations of 0–100 ␮M. Effects
of steroid concentrations on cell growth were determined by MTT assay. Each data point and error bar represent the mean ± S.D. from quadruplicate
determinations. Where S.D. bars are not apparent, the S.D. was smaller than the symbol.
than that caused by DHEA at the same concentration and
incubation time in all cell lines.
DNA-binding fluorochromes. After addition of each steroid
at a concentration of 100 ␮M, a few floating cells were ob-
served. However, no apoptotic cells were detected in both
attached and floating cells.
3
.2. Evaluation of cytotoxicity
3
.3. Effects of deoxyribonucleosides (DN), ribonucleosides
The effects of treatment with DHEA and related steroids
(RN), or MVL on DHEA or etiocholanolone-mediated
for 48 h on cytotoxicity were studied by measuring LDH
activity in medium and cells. Cell cultures exposed to each
steroid at concentrations up to 50 ␮M showed neither a
significant increase of LDH activity in the medium nor a
significant decrease of the activity in cells. However, a sig-
nificant increase of LDH activity in medium and a decrease
of the activity in cells were observed when the cells were
exposed to 100 ␮M of etiocholanolone (Fig. 5). To screen
whether each steroid causes apoptosis to cells, we employed
growth inhibition
Hep G2 cells were treated with 50 ␮M of DHEA or
etiocholanolone in combination with 20 ␮M of DN (thymi-
dine, deoxycytidine, deoxyadenosine, and deoxyguanosine),
30 ␮M of RN (uridine, cytidine, adenosine, and guano-
sine), or 2 mM of MVL to test their ability to prevent the
growth-inhibitory effects of DHEA and etiocholanolone.
Fig. 3. Effects of incubation time on cell growth. Hep G2 cells were incubated for 0–72 h with each steroid at a concentration of 100 ␮M. Effects
of incubation period on cell growth were determined by MTT assay. Each data point and error bar represent the mean and S.D. from quadruplicate
determinations. Where S.D. bars are not apparent, the S.D. was smaller than the symbol.

7
8
S. Yoshida et al. / Steroids 68 (2003) 73–83
Fig. 4. Effects of DHEA and its metabolites on proliferation of different cell lines. Hep G2, Caco-2, and HT-29 cells were incubated for 72 h with each
steroid at a concentration of 100 ␮M. Cell growth was evaluated by MTT assay. Each column and error bar represent the mean and S.D. from quadruplicate
∗
∗∗
†
determinations. P < 0.001, significantly different from control; P < 0.0001, significantly different from control; P < 0.0001, significantly different
from DHEA.

S. Yoshida et al. / Steroids 68 (2003) 73–83
79
Fig. 5. Effects of DHEA and related steroids on the percentage of total activity of LDH in the Hep G2 cells and in the medium. Cells were incubated with
∗
each steroid for 48 h. Each column and error bar represent the mean and S.D. from quadruplicate determinations. P < 0.0001, significantly different
from control.
The addition of DN or MVL to the medium did not prevent
growth inhibition while the addition of RN partially over-
came the growth inhibition induced by DHEA (Fig. 6A). In
contrast, the growth inhibition by etiocholanolone was not
prevented by the addition of DN, RN, or MVL (Fig. 6B).
Further prevention of growth inhibition was not observed
even when higher concentrations of DN, RN, or MVL were
added to the medium.
3.4. Effects on G6PD activity
Table 1 shows the effects of adding increasing amounts
of DHEA and related steroids on G6PD activity in Hep G2
cell extracts. When any steroid was added to the incubation
mixture, G6PD activity was suppressed in a dose-dependent,
variable manner. Epiandrosterone and DHEA were the
most potent inhibitors of G6PD, while DHEA-sulfate and
etiocholanolone had little effect. The inhibition rates of a
5
0 ␮M concentration of the steroids and the relative order of
inhibition efficacy were: epiandrosterone (86%) > DHEA
85%) > 7␣-OH-DHEA (76%) > 7␤-OH-DHEA (57%) >
(
Table 1
Effects of each steroid on G6PD activity in Hep G2 cells
Steroid added to
incubation mixturea
G6PD activityb (% of controlc)
1
␮M
10 ␮M
100 ␮M
DHEA
DHEA-sulfate
Pregnenolone
82 ± 9
98 ± 3
75 ± 6
92 ± 4
97 ± 4
91 ± 13
99 ± 1
82 ± 12
38 ± 3
100 ± 5
61 ± 2
53 ± 4
80 ± 1
78 ± 4
80 ± 1
27 ± 6
99 ± 7
7.4 ± 1.0
92 ± 8
51 ± 5
14 ± 4
31 ± 1
62 ± 6
55 ± 7
9.6 ± 3.2
98 ± 9
7
7
7
␣-OH-DHEA
␤-OH-DHEA
-Oxo-DHEA
Androsterone
Epiandrosterone
Etiocholanolone
104 ± 7
Fig. 6. Effects of DN, RN, or MVL on DHEA- or etiocholanolone-induced
growth inhibition. Hep G2 cells were incubated for 48 h with 50 ␮M
of DHEA (D), etiocholanolone (E) and DN (20 ␮M each), RN (30 ␮M
each), or MVL (2 mM). Effects on cell growth were determined by MTT
assay. Each column and error bar represent the mean and S.D. from
quadruplicate determinations.
a
Pooled Hep G2 cell-free extracts were incubated with each steroid at
concentrations of 1, 10 and 100 ␮M, and G6PD activities were measured.
b
Data represent the mean ± S.D. (n = 4).
c
The absolute G6PD activity in the absence of steroid was 178 ±
14 nmol/min/mg protein (n = 4).

8
0
S. Yoshida et al. / Steroids 68 (2003) 73–83
Table 2
Effects of each steroid on HMGR activity in Hep G2 cells
Steroid added
to medium
HMGR activity (pmol/min/mg protein)
a
b
100%d
38%
88%
43%
47%
42%
97%
32%
52%
72%
Control
DHEA
DHEA-sulfate
Pregnenolone
137 ± 16
52 (42, 63)c
121 (104, 138)
59 (63, 56)
64 (66, 63)
58 (55, 62)
133 (126, 140)
44 (51, 37)
71 (69, 75)
99 (93, 105)
7␣-OH-DHEA
7␤-OH-DHEA
7-Oxo-DHEA
Androsterone
Epiandrosterone
Etiocholanolone
a
Hep G2 cells were exposed for 24 h to 50 ␮M of each steroid. The
cells were then harvested for determination of HMGR activity.
b
Mean ± S.D. (n = 5).
c
Average of two assays with individual values in parentheses.
d
Relative enzyme activities.
pregnenolone (47%) > androsterone (39%) > 7-oxo-
DHEA (34%) > DHEA-sulfate (6%) > etiocholanolone
(1%).
3
.5. Effects on HMGR activity
The effects of 50 ␮M steroid treatments for 24 h on
HMGR activity in Hep G2 cells are summarized in Table 2.
HMGR activity was obviously inhibited by DHEA and re-
lated steroids except for 7-oxo-DHEA and DHEA-sulfate.
The inhibition rates and the order of inhibition efficacy were:
androsterone (68%) > DHEA (62%) > 7␤-OH-DHEA
Fig. 7. Relationship between growth inhibition and inhibition of G6PD
A) and HMGR (B) activities in Hep G2 cells treated with DHEA and
(58%) > pregnenolone (57%) > 7␣-OH-DHEA (53%) >
(
epiandrosterone (48%) > etiocholanolone (28%). To eval-
uate whether the inhibition of HMGR activity depends on a
direct interaction of the steroids with the enzyme, cell-free
extracts were also incubated in the presence of 50 ␮M of
each steroid. However, no significant inhibition of HMGR
activities occurred.
related steroids. G6PD activities were measured by incubating cell-free
extracts with each steroid at concentrations of 50 ␮M. HMGR activities
were determined by use of the cells exposed to 50 ␮M of each steroid for
24 h. Growth inhibition was determined by MTT assay (50 ␮M of each
steroid, 72-h incubation).
these steroids on cell growth have not been tested. There are
clinical reports that plasma 7␣-OH-DHEA concentration
[39] and urinary etiocholanolone and androsterone excre-
tion [40,41] in breast cancer patients were lower than the
concentrations in control women. In the present study, we
demonstrated that many endogenous DHEA metabolites, in-
cluding 7␣-OH-DHEA, etiocholanolone, and androsterone,
had anti-proliferative effects. In addition, we investigated
whether the pentose phosphate pathway (G6PD) or the
mevalonate pathway (HMGR) is more responsible for the
growth inhibition.
It has been reported that ketonization of the C-17 or C-20
position and 3␤-hydroxylation are required for the inhibi-
tion of G6PD activity [42]. The mechanism is uncompetitive
with respect to both NADP and glucose-6-phosphate and is
probably due to the binding of the steroids to the ternary
enzyme–coenzyme–substrate complex(es) [21]. Since the
inhibition was not caused by decreased expression of G6PD
protein [19], we directly added each steroid to the G6PD
3
.6. Relationship between growth inhibition and
G6PD or HMGR activities
Fig. 7A shows the relationship between growth inhibition
evaluated by MTT assay and inhibition of G6PD activity.
There was no significant correlation between inhibition of
cell growth and inhibition of G6PD activities by DHEA and
related steroids (r = −0.031, n = 9, NS). The relationship
between inhibition of cell growth and inhibition of HMGR
activity is shown in Fig. 7B, and no significant correlation
was observed (r = 0.219, n = 9, NS).
4
. Discussion
It has been reported that not only DHEA but also cer-
tain endogenous DHEA metabolites inhibit the activities
of G6PD [20,21] and HMGR [23]. However, the effects of

S. Yoshida et al. / Steroids 68 (2003) 73–83
81
assay mixture and measured enzyme activity in vitro. If the
anti-proliferative effect of DHEA-related steroids depends
on the inhibition of G6PD activity, there ought to be a sig-
nificant relationship between growth suppression and the
inhibition of G6PD activity. However, the activity was not
correlated with growth inhibition at all (Fig. 7A), suggest-
ing that the anti-proliferative effect of these steroids is not
explained by the inhibition of G6PD alone. A recent report
demonstrated that DHEA had growth inhibitory effects in
G6PD deficient cells (89 cells) [43], which lends support
to our results that the inhibition of G6PD is but one of the
anti-proliferative mechanisms of DHEA-related steroids.
HMGR is the rate-limiting enzyme in the biosynthesis of
farnesyl diphosphate, which is a precursor of cholesterol and
is also necessary for the farnesylation of Ras protein. In the
MAPK mediated cell proliferation signaling pathway, acti-
vated Ras (farnesylated and bound GTP) switches on Raf1
was not affected by the addition of RN. It may be mentioned
here that G6PD is the rate-limiting enzyme in the pentose
phosphate pathway, which provides ribose-5-phosphate.
A decrease in the liver content of ribulose-5-phosphate,
the immediate precursor of ribose-5-phosphate, was re-
ported in rats treated with DHEA [55]. DN is synthesized
from RN while RN is not synthesized from DN, but from
ribose-5-phosphate, which may be the reason why the addi-
tion of DN did not diminish the inhibitory action of DHEA
(Fig. 6).
A previous report showed that DHEA had a cytostatic
effect and arrested cells in the G1 phase of the cell cycle [15].
In fact, except for etiocholanolone, the steroids we tested
did not cause cytotoxicity or apoptosis at a concentration of
100 ␮M. However, 100 ␮M of etiocholanolone significantly
increased LDH activity in the medium and decreased the
LDH activity in cells (Fig. 5), which suggests that, in contrast
to DHEA and other DHEA metabolites, etiocholanolone has
a cytotoxic effect at a concentration of 100 ␮M. It should
be noted that to determine HMGR activity and to test the
ability of DN, RN, and MVL to prevent growth inhibition
by the steroids, we exposed cells to 50 ␮M of each steroid.
This concentration of etiocholanolone did not change LDH
activity in neither medium nor cells, which shows that 50 ␮M
of etiocholanolone was not cytotoxic.
In summary, this study demonstrated that endoge-
nous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated
DHEA derivatives, androsterone, epiandrosterone, and eti-
ocholanolone, produced anti-proliferative effects on cancer
cell lines. Since the growth inhibitory effects were not cor-
related with the inhibition of G6PD nor HMGR activity,
some other mechanisms of the growth inhibition may ex-
ist. However, we can not exclude the possibility that the
anti-proliferative effects of any one of the individual steroids
may have been mediated via the inhibition of G6PD and/or
HMGR. These non-androgenic DHEA metabolites may
serve as chemopreventive or anti-proliferative therapies.
kinase [44], which phosphorylates MEK1 (MAP kinase ki-
MAPK
nase) [45], and in turn, phosphorylates p44
p42
(ERK1) and
MAPK
(ERK2) [46,47]. These MAPKs can phosphorylate
a variety of substrates, including Fos and Jun [48]. Fos and
Jun are protein products of c-fos and c-jun proto-oncogenes,
respectively, and are components of the transcription fac-
tor, activator protein-1 (AP-1) [49]. Binding of AP-1 to the
DNA regulatory sites activates transcription of a variety of
target genes that leads to initiation of DNA synthesis and
eventually, to mitosis [50]. A recent study using cultured
airway smooth muscle cells showed that DHEA decreased
DNA binding of AP-1 [51]. Although the exact mechanism
has not been elucidated, one possibility is that it may be due
to inhibition of a kinase, which mediates AP-1 phosphoryla-
tion events that are necessary for DNA binding [50,52,53].
In our studies, however, HMGR activity was not correlated
with growth inhibition (Fig. 7B), and the addition of MVL to
the medium did not mitigate the growth inhibition induced
by DHEA (Fig. 6). Therefore, the anti-proliferative action
of DHEA is not explained by the inhibition of cholesterol
biosynthesis or the MAPK signal transduction pathway due
to suppression of HMGR activity. Direct interference with
the MAPK pathway may be another possible mechanism for
the growth inhibition by DHEA.
Acknowledgments
In the liver, DHEA is metabolized to multiple prod-
ucts, including 7-oxygenated derivatives [54], androsterone,
epiandrosterone, and etiocholanolone (Fig. 1). Although
many unique biological effects of DHEA have been re-
ported, some may be due to metabolism of DHEA to more
biologically active products. In the present study, all DHEA
metabolites we tested showed anti-proliferative actions. In
particular, the growth inhibition due to etiocholanolone was
significantly greater than that caused by DHEA (Fig. 4).
However, it is possible that the inhibitory action due to
etiocholanolone and DHEA are caused by different mech-
anisms. DHEA markedly inhibited G6PD activity, and the
anti-proliferative effects of DHEA were diminished by the
addition of RN. In contrast, etiocholanolone did not inhibit
G6PD activity, and the growth inhibition by etiocholanolone
This work was supported in part by a grant (B)10470130
from the Ministry of Education, Science, and Culture of
Japan.
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