ViTOL 26, 301-314. 10. Loria,. R. M., Padgett,. D. A. (1992). Androstenediol regulates systemic re- sistance against lethal infections in mice. Areh. ViTOI. 127,.
Contrasting
effects
of a- and -androstenedioI
on oncogenic P. N. Huynh Department
and
College
The
of
in vitro
growth HL-60 RAW
RAW
cells were cells for 48
increasing
cell
clusion.
264.7,
At these
as
DNA
had
trypan
synthesis
such
basal
effect.
human
blue
ex-
with P388D1 and or above, 17aAED HL-60 as detected and the
These
data
that
Key
Words:
steroid
.
level
,pncroplmge
J.
pmlfration
.
Leu-
apoptosis.
.
factor
oncogenic
can
and
IL-3
studies the
the
of IL-4
synthesis
seems
gonadotropin 17aAED by
181.
Although
the
INTRODUCTION
has
not
of
17aAED
is
of
derived
that
from
DHEAS
occurs
peripheral have shown herpes faecalis
17aAED
mice
may, in part, ing released
[2-4J,
from
various
This
counteract the during infection
illness. It was proposed pends on its biotransformation to 5-androstene-33,73,1fl3
of [61,
infections,
virus
B4,
protective
the
efficacy
in the
triol
Enterococcus
effect
of 17AED
Journal
of
Leukocyte
Biology
of of
in
and
to
cells
or
stimulated
(5 jiM), 173AED of hydrocortisone on
response
IgE
to ConA
to regulate
in
vitro
the
TH2
can on immune
has
to 2:1
Low
in
levels
been
chorionic
to the
the
in the
in favor
ofthe
17IAED
in fetal
[19].
fluid
the
Further-
and
ratio
is approxi-
subsequently
re-
in adulthood
development
have
insufficiency in the late
211.
[20, been
associated
on the biological on myeloid oncogenic
the
The
fetus
and
level
peripheral
recently
pregnancies
and placental been discovered
higher
amniotic in
[17,
activity
pregnancies.
173AED 17aAED
in vitro
than
in the
of the
testes
tissue,
confirmed
favor
reported 17aAED
of
testis
of normal
of 17aAED
been
effects
epiIt is
dehydrogenase
vein
in the pathological
emia, diabetes, 17aAED had had
from
is present
17aAED
verses
human
of boar
steroid
circulation
9:1
by
fraction
spermatic
steroid
mately
is a natural (i7AED).
was found to be secreted testes. The secretion of
enhanced
detected
plasma
this
the
be
in the
skin
and
(3AET).
In
Volume
62,
in the
de-
im-
with
tox-
1221. Although 1960s, no studies significance cell lines.
androstene
sulfoxide;
to
versity,
Medical
Received 24,
1997.
Immunology
or the These
necrosis
College
M.
1997;
a;
factor
chorionic Loria,
Pathology,
of Virginia, 3,
DHEA,
concanavalin
deA; LPS,
17aAED,
gonadotropin;
5-
DMSO,
iodide.
Roger and
ConA,
human
propidium
Dr.
February
tumor HCG,
P1,
diol;
interleukin-2;
TNF-a, 3.3,17a-diol;
dimethyl
5-androstene-33,l73
IL-2,
lipopolysaccharide;
Microbiology,
the reFurther-
1997
173AED,
Correspondence:
brain
contrast
August
Abbreviations: hydroepiandrosterone;
besevere
of 17I3AED
3AET, 17AED in vitro is less effective in inducing lease of interleukin-2 (IL-2), IL-3 by splenocyte.
258
RAW
or
IL-i
including
and
elevation of glucocorticoid mediated stress and
that
by
of either
have also shown that 17fAED inhibitory effect of glucocorticoid
i7a-hydroxy
been
plicated
cortex The level as a result
release
prolif(ConA)
and DHEAS 171. We injection of 17I3AED
lethal
in vivo
natural
Synthesis
adrenal
[7, 81. tumors
from DHEA subcutaneous
2, Coxsackie
are
290.4. the
mammary tissues in primary breast
type
19-121.
173AED
weight
skin
conversion that a single
virus
and
molecular
in the
the testis, and 173AED increases
protects
Both
with
173AED
diol (173AED) (DHEA) and
dehydroepiandrosterone
[1, 7, 8J.
C19i5-steroid
5-androstene-33,173
the
a (TNF-a)
microsomal
fetal-placental
known
A
(HCG) stimulation [15, 161. Subsequently, been shown to be synthesized from pregneno-
has
lone
venous
been
and
production
1121. In vivo counterbalance
more,
It has
on splenocyte
LPS [131. At high concentrations counteract the inhibitory effect
with
17aAED
of myelopoiesis. 1997.
overall 258-267;
(LPS),
necrosis
effect concanavalin
a precursor of testosterone that in the spermatic vein of human
determine
the 62:
tumor
if any either
1141. 5-Androstene-3,i7a-diol (17aAED) meric steroid of 5-androstene-3.3,17-dio1
anti-proliferative effect of effects of 173AED may
Biol.
little by
response
TUNEL isomer
suggest
has
stimulated
IL-2
as measured
with
173AED
lipopolysaccharide
the balance between the 17aAED and the proliferative Iwc.
University,
eration
was suppressed by as effect was timeand on removal of the ste-
electron microscopy treating cells
no
and
by
roid. Similar results were obtained human HL-60 cell lines. At 50 nM induced apoptosis in RAW cells and
173AED
Commonwealth
more,
isomer
the
17aAED treatment of total cell number without
incorporalion P < 0.05. This and reversible
by transmission assays. By contrast,
on
P388D1,
detected
doses,
by [3lljthymidine much as 65%, dose-dependent
Virginia
the
(173AED)
investigated. h reduced death
Immunology,
Richrrwnd
of 17aAED,
diol
of murine
and
Virginia,
effects
of 5-androstene-33,17f3
cell lines in vitro
R. M. Loria
of Microbiology
Medical
Abstract:
myeloid
revised
Professor, Virginia
Richmond, April
Department Commonwealth
VA 23,
of Uni-
23298-09678.
1997;
accepted
April
findings
prompted
us to investigate
ural structural epimer biological activity.
whether
of 173AED,
i7aAED,
has
similar
a nat-
or different
were
washed
were
then
twice
harvested studies,
HL-60
METHODS
were
Reagents The
steroid
MO)
and
stock
was were
final
obtained
the
effect
on
Steraloid
and
before of the
use and
as
Inc.
(St.
(Wilton,
equal
this
in the
concentration
determined
in
no signifi-
blue
exclusion.
ptotic
macrophage-like
promyelocytic MD)
HL-60
were
fetal
U/mL
These
in
calf
lines
were
Type
200
and
RAW
264.7
cells in the
2.5
control in 25-cm2 vehicle
flasks
in the
as control.
Cells
washed,
and
hyde
fixed
before
cells
106 vehicle
control
RAW
or
with
were
were
washed
twice
tiation
of culture.
buffer
or
absence
(1:1
v/v)
viable
cells
or
at 48
h,
2% glutaralde-
x l0
(1
replaced
steroid
removal
media
steroid.
alone
by trypan
blue
on
day
0.
blue
experiments plates
After
48
were h, cells
for an additional
exclusion
x or
by trypan
in 24-well
of the
(1.0
steroids
of 1 mL
cells/well)
with
determined
plates
determined
For
72
5 days
h.
mi-
after
assays
To determine
the
P388D1, were
or
effects
HL-60
performed.
pared
Briefly,
These
and
cells
plates
at a density
night.
Dead,
RAW or vehicle
jiL
0. At the
on day
to each
before
cubation vester
(Cambridge
a KLB Likewise,
the
withdrawal
were
treated
P388D1
cells
same
counter.
with
increasing
time
point, then glass
RAW
samples
by trypan
carried
cells
(1
concentrations
by aspiration
the
cells
(1:1
in a volume
x
were
the
last
filter
using
a PHD
It was
then
out
with
104/well) of steroids.
or
were
and
fixed
to the analysis.
each
sample.
For
incubated jig
stages
of apoptotic
iodide
(P1)
apoptosis,
were
with
of
and
positive
of camptothecin,
respectively.
cells
in parallel
were
without
After
48
h,
events
control
ape-
collected
apoptosis,
or in the
Furthermore, by
Mann-
were
of
transferase
determined
or
(Boehringer
10,000
of terminal
(1 x
cells
with
isothiocyanate-labeled
instructions
Minimums
absence
cultures.
fluorescein
manufacturer’s
RAW
incubated
in duplicate
stained
negative
in the
of 2.5
106)
vehicle
according FACS
undergoing
x
cells presence
necrotic
the
uptake
and
late
of propidium
cultures.
analysis
Differences
between
by unpaired
experimental Student’s
groups
t-test
and
and
analysis
vehicle
control
were
evalu-
P < 0.05
of variance;
was
significant.
Morphological presence
changes
dish 25J. studied
by using
treating diminished
HAW
day.
with
By
48
cells were
h,
vehicle
were viable effect
microscopy.
cells with spreading
cell
50
(Fig.
density
ib)
vehicle
or
the
or medium markedly
medium
control
(Fig.
cells
with
some
The remaining blue exclusion
cells and
steroid.
In
is required
to observe
the
such
At con-
there was no noticecompared with either
control.
173AED,
ic).
and
of the id),
is la),
significantly surface com-
reduced
on removal nM (Fig. morphology
[23-
in (Fig.
of 17aAED the plastic
the plate. by trypan
reversible
centrations below 12.5 able difference on cell
high density cell morphology
at
As shown
nM onto
was
detached from as determined was
up HAW
contrast,
structural
treating
analogue
morphological
of
morphothe spa17 deterpresence
changes.
was
6 h of incell counted
17(3AED.
For
in a 96-well
Huynh
cell
light
rounding on the
17aAED at doses up to 50 nM, was without gross logical effect as shown in Figure 1, e and f. Thus cial configuration of the hydroxyl group at carbon mines the effect of 17aAED and its continuous
of 200
48
cell line, HAW 264.7, grew onto a glass or plastic culture
blue
haron
in quadruplicate.
After
macrophage-like cells spreading
at low density and The effect 17aAED
pared
of RAW 264.7 in the
of 17aAED
HAW
exposed
f3Hjthymidmne
MA).
assayed
173AED,
and
the
overnext
HL-60
for
and
by TUNEL
pre-
microtiter
to adhere
v/v)
RAW,
was
96-well
1 jiCi
were
were
cells
allowed
pulsed
Watertown,
procedures studies,
or
of
incorporations
of RAW
and
cells,
onto a All
growth
in flat-bottom removed
were
harvest
basal
determined
DMSO:ETOH
indicated
their
was
cells/well were
Technology,
scintillation
roid
seeded
10
the
I3Hjthymidmne
suspension
then cells
The
cell viability
control
well.
on 6-h
cell
x
cells,
to 17aAED
added
were
of 2.0
nonadherent
Subsequently,
17aAED
standard a single
by trypsinization
exclusion.
of
cells,
of cells (1
heim)
this
Proliferation
cells
The murine as adherent
indicated
in a volume
6 h of incubation
grown
173AED,
or 24-well
of the
cells were
concentrations
and
were
in 12-
for 48 h. Cells
jiM)
last
RESULTS
were
were
policeman
containing
the
con-
or absence
106
and viability
a hemocytometer.
HL-60
cells
0. Cells
cells
a rubber
for
increasing
presence
or vehicle
of 17aAED,
with
seeded
presence
increasing
Viable
absence
M cacodylate
time point, using
cells
treated
or
x
(1.0
on day
Alternatively,
number
considered
studies
plates
with
in the
DNA fragmentation
the
Statistical
at 37#{176}C.
steroids
of 1 mL
point.
collected
DMSO:ETOH
exclusion
12-well
treated (250
I3Hlthymidine
dUTP
ated
indicated
and
anti-proliferation
heat-
bicarbonate, CO2
microscopic
in a volume
presence were
Rockville, 10%
weekly.
of the
time
cells
in the
At the indicated dye
sodium in 5%
of cell number
or HL-60
cells/well)
L-glutamine.
human
microscopy.
Determination RAW
v/v)
in 0.1
electron
Collection, containing
in flat-bottom
indicated
and
streptomycin
twice
or absence (1:1
at the
jiM
seeded
DMSO:ETOH
photographed
Culture
electron
presence
P388D1,
medium
jig/mL
passaged
were
264.7,
1640
Light and transmission cells/well)
RAW
RPMI
serum,
penicillin,
cell
lines,
(American
maintained
inactivated 2.5
cell
h. Cells
above.
HL-60
cells
were
culture
Coil
or
17aAED,
0.15%
had
by trypan
10)
media.
than
were or vehicle
2’,5’-dideoxyadenosine with
72
6 h of incubation
cAMP-mediated
105/well)
1713AED,
described
To quantify
solutions
culture
to or less
For
last
The
(DMSO:
for
Murine
as
for an additional the
assays and flow cytometry
Louis,
NH).
at 4#{176}C.Working
by diluting was
studies
Co.
sulfoxide:ethanol
stored
vehicle
cells
Chemical
dimethyl
sterilized,
throughout
Sigma
from in
immediately
cytotoxic
from
dissolved
concentration
all samples cant
obtained
filtered,
prepared
The
was
17aAED 1:1 v/v),
were
x
(1
Oligonucleosomal 173AED
solutions
ETOH,
cells
pulsed
harvested
media for
above.
inhibitor,
then
with
3Hthymidmne
of 17aAED,
of a cAMP
AND
replaced
with
described
as
centrations
MATERIALS
and
pulsed
ste. plate
h, cells
and Loria
Assessment To assess number
of RAW whether
and
viability
cell viability
17aAED were
induced
eter and trypan blue dye exclusion. treating HAW cells with 17aAED
17aAED
and
173AED
effects
cell
quantified
on
death,
using
total
cell
a hemocytom-
As shown in (Fig. 2a), ranging from 12.5 to 50
macrophage
proliferation
259
Fig.
1.
(a-f)
concentrations
of 17aAED
of the
(c) medium cells
Effects control;
compared
and
indicated (d)
with
5 nM
over
17aAED;
(e)
17AED-treated
(P < 0.01).
portional shown).
to the At 6.25
reduced manner
This
ity or on total cell number There was also no significant cell
number
shown).
260
between
By comparison,
Journal
of
Leukocyte
5 nM
vehicle
and
treating
Biology
morphology.
were
173AED;
vehicle
cell
had compared effect
cell
Cells
cells
number
no effect with on cell
(1) 50
Volume
was
nM
control cells
62,
pro-
(data on cell
vehicle viability
medium RAW
RAW
photographed
cells
at the 173AED.
(1 x
106)
indicated
Note
the
were time
grown
in the
point.
absence
(a)
50
of spreading
absence nM in
or presence
17aAED;
(b)
17aAED
(50
of increasing vehicle
nM)
control;
treated
RAW
control.
in viable
in total
17aAED
h.
the number of viable compared with vehicle
decrease
decrease nM,
on RAW 48
or the
nM for 48 h significantly cells in a dose-dependent control
173AED
steroid
with
August
not
viabilcontrol. or total
(data 17AED
1997
not
for
did
48 not
ment
h at the have with
same
an affect 173AED
range
of concentrations
on cell (50
viability.
nM)
as
17aAED,
Furthermore,
resulted
in
treat-
a significantly
different total cell number than cells treated with 17aAED (1.14 x 106 vs. 0.49 x 106), respectively. Thus, treatments with 17aAED for 48 h result in both cell killing from the initial inoculum and in the inhibition of the proliferation
of
173AED
has
the no
surviving effect
cells, on
RAW
whereas cell
viability.
treatment
with
18
or
greater
viable 16
fewer
cells
decrease
after
48
the
vehicle
had
no effect
in the
h. This
control.
was not affected
12
10
number
resulted
of
in 91.7%
At a concentration
on cell
viability.
by vehicle
the continuous presence of 17aAED nfficantly reduced the mean number well compared with vehicle control
0
-.,
well,
than
17aAED
of RAW
a marked
per
cells
5 nM, 14
caused
cells
The
of viability
treatment.
By 72 h,
(50-100 nM) sigof viable cells per by 98.2 and 81.2%,
respectively.
(I)
C)
Comparison of 17aAED on RAW cell proliferation
.0
4 2
the
17aAED
h (Fig.
(5-100 in the
manner
&,#
I).
compared
50
(5-50
Sc
for 48
the
50
nM
for 48
vehicle
control
i7aAED,
the
hibited
thymidine
vehicle
control.
RAW
At
proliferation
of
by
with
i73AED
inhibitory
at 10 times nM) treatment
uptake
This antiin RAW had no antiwith vehicle
cells
no significant
uptake. However, i73AED (500
3) re-
(P < 0.001).
treatment compared
treating
h, had
of by of
in a dose-dependent
h. At 24 h, 17aAED effect on RAW cells
nM)
thymidine as 17aAED, 30
with
By comparison,
control.
cells
growth in vitro Addition
was significantly decreased by 65%. effect of 17aAED is first observed
cells at 48 proliferative
40
to RAW
of proliferation
of
RAW cells proliferative
f
nM)
inhibition
a concentration I,
effects
The effects of 17aAED and i73AED on macrophage-like cells were further evaluated measuring tritiated thymidine incorporation. sulted
0
and 17AED
RAW
the
cells
effect
on
concentration significantly
in-
relative
the
to
(I)
C) a,
14
:
20
5 12 10 10
8
0 0
12
24
36
48
60
72
Hour Fig. 2.
(a)
24-well
Viability
plates
Viable
cells
addition
different t-test, (b)
Effects
in RPM!
or different were represent cultures.
vehicle
containing
concentrations
control are
mean
indicated
number
as
on
RAW
cell
viability.
FCS
were
treated
of viable
48,
by trypan cells
per
the
and
sig-
RAW
blue
ex-
cells the
h. Viable
SD
cul-
0
vehicle
exclusion. ±
2
unpaired
separate
with
or 72 well
4
after
by
6
in
steroid.
at 48 h
of five
10%
for 24,
105/well)
cultures
determined
representative
interval
x
of the
blue exclusion
of 17aAED
at the
the
Data
(1
of quadruplicate
SD
of 17aAED
1640
determined
±
Cells
concentrations
by trypan
from
cells.
increasing
* Mean
P < 0.01.
C)
of RAW
with
determined steroid.
nificantly periments.
treated
were
of the
Student’s tured
assessment
were
a.
cells
,‘
of triplicate
P < 0.05.
Fig.
3.
ation
over
trations
Time
are
shown
17aAED
courses
for
in Figure over
72
the effect 2b. When
h, 17aAED
of 17aAED RAW cells
on cell viability were treated with
at a concentration
f31t
Data
of 50
Huynh
and
nM
Loria
with mean
Comparison 48 of
17aAED,
(3Hthymidmne cpm
±
independent
17aAED
of
h. Cells
SD
17aAED
x
and
10)
were
173AED, for
the
or last
of quadruplicate
experiments.
and
(2
173AED
vehicle 6
h of
cultures *P
effects
c::
10
2
.
17AED < 0.05
p
.. P
