Effects of androgen on intracellular calcium of LNCaP cells

Vol. 179, No. 1, 1991 August 30, 1991

EFFECTS

BIOCHEMICAL

OF ANDROGEN

AND BIOPHYSICAL

ON INTRACELLULAR

Jaime Steinsapir,*

Robin

RESEARCH COMMUNICATIONS Pages 90-96

CALCIUM

OF LNCaP

CELLS

Socci and Peter Reinach

Department of Physiology and Endocrinology Medical College of Georgia Augusta, GA 30912

Received

July

8,

1991

Mibolerone

(dimethylnortestosterone) calcium (Ca,?2 of human prostate

intracellular treatment.

cancer

These effects were concentration-dependent

by preincubation mibolerone

with

hydroxyflutamide

(10e6M)-induced

1mM CaC12 introduced in Caf+

in LNCaP

membrane. hormone

in a Ca2+ -free media.

cells as a result

in target cells.

Steroid evidence

hormones

suggesting

and metabolism determine

B 1991

a critical

[3,4],

how steroids

groups which

Academic

indicate

hormones

in endometrial

known

about

LNCaP

cells are a widely

may control

used model

The main aims of this work

and growth

in target

in the initiation

and/or

0

I991

All

of reproduction

There

may trigger a rapid

by Academic

Press.

cells [1,2].

In view of of cell growth is essential

evidence

to

from several

plasma membrane-mediated

events in

(2.5 min) of the rate of calcium

Ca,2+ levels in normal

rats [l].

However,

and neoplastic

action [5] in responsive

target

little

is

cells.

cancer cells.

or mibolerone on Caf+ 2+ on Cai , in the presence or

a L-type

changes of Caf+ in LNCaP cells, in vitro.

irt cm? form

and growth,

responses to steroid

regulation

is accumulating

increase

to study androgen

should be addressed

$1.50

Copyright

in the plasma

as a second messenger

the effects of hydroxyflutamide

to whom correspondence

0006-291X/91

and mitogenic

and c) to assess the effects of verapamil,

[6], on the androgen-induced

channels

were: a) to study the effects of 5a-DHT

of LNCaP cells; b) to determine

*Author

L-type

elicit changes

of prostate cell division

cells isolated from uteri of ovariectomized

how androgens

absence of androgen

promote

that androgens

Inc.

these processes.

that steroid

and they were blocked

through

of its involvement

target cells [ 1,2], because estrogens exchange

Press,

role for calcium

control

( 10T6- lo-12M)

in metabolic

cell division

consideration

as early as 2 min after

in the regulation

is involved

control

increase

cells (LNCaP)

The results indicate

of Ca2+ influx

are involved

suggest that calcium

(DHT)

( 10W6M). Verapamil ( 10d6M) also suppressed the in Ca,2+ , in cells which were previously exposed to

increase

Since androgens

these findings

rights

or So-dihydrotestosterone

1~. reserved.

90

calcium

channel

blocker

Vol. 179, No. 1, 1991

Materials:

Mibolerone

Radioinert

BIOCHEMICAL

AND BIOPHYSICAL

MATERIALS

AND METHODS

(dimethylnortestosterone)

So-dihydrotestosterone

RESEARCH COMMUNICATIONS

was purchased

was obtained

from

Amersham,

from Sigma (St. Louis, MO).

Hydroxyflutamide

(2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoro-methyl)phenyl]propanamide, from Schering

Corp.,

(Eugene,

Media,

Grand

OR).

Bloomfield,

sera and antibiotics

Island Biological

Woodland,

CA.

Cell culture:

Co. (Grand

Cells were grown

were fed at 3 to 4-day

Plastics, Oxnard,

CA).

these studies,

levels: testosterone,

steroid

measurements:

(0.05%)

and EDTA

22 “C).

Trypsin

reactions

cells were then washed serum-free

RPM1

Following

the cell pellets

Ca2+-free/buffer) cuvettes (2~10~ International vehicle

were

with

3pM

each experiment,

(pentetic performed

Calcium constructed different association

the fluorescence

Ca2+/EGTA constant,

were

ratios

expressed

as percentages [(Caf+after

corrected

of increase stimuli

for the percentage

of unloaded

added

(Photon of agonist

fluorescence

buffer

The fluorescence

on calibration

91

or

to 1 cm of

or antagonist. of 10m5M DTPA

emission ratios at Corrections

curves

nm) of solutions for

by Grynkiewicz

in intracellular

with

Technology

1.25mM

at 340 and 380nm.

calcium control)].

which

were

were

contained

The apparent is in agreement

et al [9,1 I]. concentration These percentage

to the vehicle alone.

that

which

pH 7.4 [9,10].

with Ca2+ was 222nM,

- Cai 2+ control)/Ca2+

B. 1640.

cells, in each experiment.

(340 nm/380

reported

in

(Fmax and Fmin)

10m5M ionomycin,

based

increase in Cai2+due

twice

any changes upon addition

the addition

excitation

interactions

the range of values (135 to 225nM)

experiment:

period,

MnC12,

The

(i.e. Ca2+ containing

was checked with the introduction

and the appropriate

Kd for Fura-

buffer

Washed cells were

before

calculated

ratios

1640.

used for cell loading

spectrofluorometer.

a control

of 0.15mM

fluorescence

concentrations

from

the same buffer

or 50mM CaC12 to cuvettes.

for the background

RPM1

pH 7.2 at

was assessed with Erythrosin

and the minimum

alternating

NaCl,

[7,8] at 34 “C for 45 min in RPM1

fluorescence

leakage, the maximum

following

0.125

10 min in trypsin

0.15M

at 600 xg for 6 min. and washed

in the appropriate

were determined

acid), 5.0 mM EGTA

17P-estradiol,

FBS-containing

and their viability

NJ). After

by the addition

of 5%

in 75-sq. cm.

analysis of FBS indicated

0.127nM;

at 37 “C for

with

of 1~10~ cells/ml.

the maximum

Any fura-

atmosphere

10 and 17 passages in vitro

(0.05 M KH2P04,

ml) in a Delta Scan-l

Inc., South Brunswick,

(50 mg/ml)

fresh medium

cells between

Fura -2/AM

were resuspended

ethanol)

with

by incubation

buffer

Cells were counted

510 nm were determined

with

harvested

at a cell density

determined

gentamicin

RPM1 1640 contains 0.42 mM Ca(N03)2x4H20.

in phosphate

cells/cuvette/2

(0.25% (v/v)

ionomycin.

with

0.28 nM; progesterone,

three washes at 600 xg for 6 min with

Fura-2,

After

intervals

three times in this medium

1640.

from

RPM1 1640 was from JR Scientific,

(GIBCO)

were stopped by 1:3 dilution

cells were loaded

Probes

cells were obtained

The results of radioimmunoassay

Cells were (0.02%)

was

Molecular

grade.

LNCaP

3.75 mM Ca2+ (GIBCO).

(Ca?+)

from

the LNCaP

Ca ++-free

in RPM1 1640 medium

were used to conduct

LNCaP

used to culture

were of analytical

the following

FBS contains

was purchased

fetal bovine serum (FBS), at 37 0C in a humidified

The cultures

plastic flasks (Falcon

nM.

SCH-16423)

Fura-

Island, NY).

All other chemicals

and 5% heat-inactivated CO2 in air.

NJ, U.S.A.

Searle.

Results were (nM),

in each

values were

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS Testosterone used to examine increased

stable,

14nM

because

reported

synthetic

to 88nM

neither

that DHT

mibolerone

androgen,

supplemented

with calcium

antiandrogens

is important

increase

LNCaP

cell growth

that hydroxyflutamide

Two increase

immediately increase in Cai2+ .

different

in Caf+.

studies.

It has been

[ 131. We used an 3 shows that

We observed with

in a medium

that this

1 mM which

alone

mibolerone lo6

were

to analyze

of mibolerone,

increased

106M before

[ 16,171 and androgens

it was of interest

or absence

types of experiments

CaC12, was not

increased

M mibolerone

performed

EGTA.

as well

as

the effects of

in this cell system.

intracellular

calcium

in a Ca2+

Caf+, the addition

effectively

to delineate

In the first type, the cells were bathed in a Ca 2+-free

presence or absence of 0.2mM

2).

Figure

of Cap.

was supplemented

cell growth

[18,19],

Although

10m3M hydroxyflutamide androgen-induced

increase

(Figure

stable for as long as 5 min, in the presence of 106M

in the presence

manner.

1 shows that DHT was concentration-

cells, in vitro

in subsequent

for prostate

Figure

dependent

Ca:+

levels than those observed

on Caf+

concentration

Figure

was

3).

hydroxyflutamide 4 indicates

increased

the cell medium

at higher (Figure

cells.

this increase

by LNCaP

[14,15]

that remained when

Cap

Since calcium

DHT

a concentration-dependent

Furthermore, increased

Furthermore,

metabolized

mibolerone

DHT in prostate cells [12] which

on Ca,& in LNCaP min.

10e9 nor 10-12M

response reached a “plateau” mibolerone.

in l-2

can be partially

also induced

mibolerone

to its active metabolite

the effects of androgen

from

dependent

is metabolized

blocked

the source

medium,

either

of the

of an in the

In the second type, the cells were bathed instead in the

100

150

TimeCseconde)

Figure 1. Effect of So-dihydrotestosterone (DHT) on the intracellular calcium concentration (nM) of LNCaP cells. Cells (1 x lo6 cells/ml) were exposed to 10e6M DHT or vehicle (ethanol, final concentration = 0.25% v/v). The fura- fluorescence ratio (340/380) was measured and intracellular calcium quantitated as indicated in “Materials and Methods”.

92

Vol.

179,

No.

1, 1991

BIOCHEMICAL

-9

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-12

10

DHT

AND

10

03

[M]

MIBOLERONE

[M]

Figure 2. Effects of different concentrations of DHT on Cai2+ of LNCaP cells in the presence of RPM1 1640. Cai2+ was measured as indicated in “Materials and Methods”. Experimental values were corrected for vehicle values shown in Figure 1. The same effect of 106M mibolerone was observed in three different experiments. Figure 3. Effects of different mibolerone concentrations on Cai2+ of LNCaP cells, in the presence of a phosphate-saline buffer (50 mM KH2P04, 0.15 M NaCl, pH 7.2 at 22°C) supplemented or not with 1 mM Ca Cl2 ( n : - Ca C12, ma:+ Ca CI2). Cai2+ was measured as indicated in “Material and Methods”. Each percentage value is shown after subtraction for the vehicle effect (25.5% over basal Cai’+).

Ca*+-containing 5 indicate bath.

medium

which

that mibolerone

Mibolerone

also contained

did not increase

was also ineffective

EGTA.

The requirement

external

Caf+ because after subsequent

Caf+was

restored

occurs through

(Figure

5).

That

dependent

shown by the effect of preincubation Ca*+-free increase

system, mibolerone in Caf+ following

bath through

in the Ca2+ -free

for mibolerone

a voltage

failed

exposure

a voltage dependent

10m6M Verapamil.

The results shown in Figure

Ca?+ .m the absence of calcium

to elicit addition

medium

in the external

supplemented

an increase in Cat+ is clearly

with

0.2mM

dependent

of 1mM CaC12 the mibolerone-induced

on

rise in

the Ca2+ influx elicited by mibolerone in LNCaP cells Ca 2+ channel pathway in the plasma membrane is

L-type

of the cells with to elicit

10e6M

a significant

to mibolerone

verapamil

increase

(Figure

5). As in the

in Caf+ . Therefore,

stems from Ca*+ influx

from

the

the external

Ca*+ channel (L-type).

DISCUSSION Little

is known

about the mechanisms

“early” responses in responsive hormones

have been previously

by which

tissues and cells although described

in target

93

steroid

hormones

numerous

tissues [20,21].

induce

the so-called

“early” responses to steroid For example,

estrogens

Vol.

BIOCHEMICAL

179, No. 1, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MIBOLERW

VEFWAMlLMlBOLERONE VE!W+ MIEOL

Figure 4. Effects of hydroxyflutamide (10S3M) and/or mibolerone (10m6M) on Cai’+ of LNCaP cells, in the presence of a phosphate-saline buffer (indicated in legend of figure 3) supplemented with 1 mM (m.) or 2 mM (0) Ca C12. Cytosolic calcium concentrations were measured as indicated in “Materials and Methods”. Each percentage value is shown after subtraction for the vehicle effect (26.1% over basal Cai2+). The bar shows the range of values of two different determinations obtained in two different cuvettes from the same batch of cells. The same effects of hydroxyflutamide were observed in two different experiments. Figure 5. Effects of verapamil (10e6M)

and mibolerone (10m6M) on Cai2+ of LNCaP cells. Fura- loaded cells were suspended in Ca ++-free RPM1 1640 and exposed to mibolerone or vehicle (ethanol, 0.25% v/v). After addition of 1 mM CaC12, cells were treated with verapamil, water (vehicle of verapamil), mibolerone (10e6M) again or verapamil (10m6M) immediately before 10m6M mibolerone. Cai’+ was measured in each condition as indicated in previous experiments. Values were corrected for the vehicle (ethanol) effect (25.0% over basal Cai2+). The bar shows the range of values of two different determinations obtained in two different cuvettes from the same batch of cells. The same effect of verapamil was observed in two different experiments.

induce early changes in the content of water and divalent is no information Moreover, trigger

although

cell division

available

we know

LNCaP

[3,4], which

if calcium

The findings

content

in cellular

shortly

calcium

is one end-point

in the uterus [l] but there after androgen

accumulation

of androgen

here that androgen to characterize

acutely

increases

the processes

precede

calcium

in metabolic

of and

responses to the hormone.

The LNCaP cells were selected as a model to study early events in androgen that the growth

androgen

responsiveness

with

receptor

in these cells [22], make them the most promising

androgen

and/or

action in prostatic cytosolic

involved

we know

human

stimulation.

action in responsive

serves as a second messenger for androgen

reported

cells are of significance

mitogenic

calcium

that increments

and growth

cells, we do not know cancer cells.

on the prostatic

electrolytes

(AR)

of these cells is androgen-dependent

prostate cancer currently

time in culture available.

[5].

These properties,

as well as the presence of

Prostate cell death and proliferation 94

action since

and that they do not lose in vitro

model of

have been both

Vol.

179,

No.

1, 1991

associated with calcium

alterations

channel

induction

blocking

of c-fos

calcium

BIOCHEMICAL

concentration.

calcium

levels [17].

drugs

a delay in rat prostate

levels [ 171. The finding

it is likely

binding

to mediate

[20].

effects

transport

distinct

on intracellular

that AR is involved

changes

interact

in this

with plasma

in intracellular

calcium that steroid

calcium

channels hormones

These calcium

channels

from the classical

which

are

may change

Several reports have described

sites for steroid hormones 123,241. However,

in the hand of others [25].

for androgen

rats with

in vivo, as well as

and antiandrogen

since it is known

state of target cell membranes

been reproducible mechanism

regression

cells may have voltage-dependent

and antiandrogen,

of castrated

was able to block mibolerone-induced

that both androgen cell

COMMUNICATIONS

Treatment

that androgen

hydroxyflutamide

of the LNCaP

The LNCaP

of plasma membrane

RESEARCH

cells are fast (2 min.) makes it unlikely

calcium,

by androgen

the polarization

induced

Although

components

controlled

in intracellular

response.

rises in cytosolic membrane

BIOPHYSICAL

mRNA

levels of LNCaP

acute androgen

AND

the existence

these results have not provide

an alternative

passive diffusion

mechanism

of

steroid entry into target cells [26]. Receptor membrane that

sites for androgens

components

androgens

other than plasma membranes

increase the cytosolic

the presence of extracellular Ca++ at intracellular that SaDHT,

phosphoinositide

RU23908,

(PI) turnover

responses in PI turnover in neoplastic

[27]. calcium

Furthermore,

cells reported

the results

in this neoplastic together,

upon

redistribution

of

androgen-induced

sources. It has been also are able to increase

of LNCaP cells [27].

Phospholipase

cells [27].

the previously

Although

shown

metabolism here

with

and antagonistic

cell system, in agreement

on phosphatidylinositol these findings

action and its regulation

The acute (2 min) androgen

here may provide

in phosphoinositide

has both agonistic

hydroxyflutamide Taken

prostate cancer cells.

of LNCaP

hours) changes observed “antiandrogen”

to mediate calcium

here indicate

are usually secondary to a rise in cytosolic calcium from an . it is also possible that Ca 2+ influx from extracellular sources may increase

origin, calcium

be important

of LNCaP

microsomal

dependent

could also induce

12-24 hours after stimulation

described PI turnover

cells by mechanisms

acetate and hydroxyflutamide

treatment

with

The results reported

upon intracellular

cyproterone

also increases after 5aDHT

cytosolic

[25].

androgens

AR could

dependent

to be associated

of LNCaP

However,

Microsomal

calcium

C activity intracellular

calcium

calcium.

sites.

increases in intracellular reported

have been described

reported

[27] and LNCaP

a role for calcium

of LNCaP cell growth

mentioned

indicate

in the control

with previously

rises in

for the late (12

cells previously

hydroxyflutamide

metabolism

may indicate

of LNCaP

properties

- induced

an explanation

that

this

of intracellular

agonistic

effects of

cell growth

[18,19].

in the mechanisms

of androgen

and metabolism.

ACKNOWLEDGMENTS This work was supported J.S.) a Biomedical NIH grant EY04795

Research

by Grant No. IN-174-A

Support

Grant

from the American

from the Medical

(to P.R.). 95

College

Cancer Society (to

of Georgia

(to J.S.) and

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 21.

Pietras, R.J., Szego, C.M. (1975) Nature 253, 357-359. Brann, D.W., Mahesh, V.B. (1991) Frontiers in Neuroendocrinology 12:165-207. Wasserman, R.H., Corradino, R.A. (1973) Vitamins and Hormones, 31, 43-103. Whitfield, J.F., Rixon, R.H., Mac Manus, J.P., Balk, S.D. (1973) In Vitro 8, 257-278. Horoszewicz, J.S., Leong, S.S. Kaminski, E., Karr, J.P., Rosenthal, H., Chu, T.M., Mirand, E.A., Murphy, G.P. (1983) Cancer Res. 43:1809-1818. Tsien, R.W., Tsien, R.Y. (1990) Annu. Rev. Cell Biol. 6:715-760. Roe, M.W., Lemasters, J.J., Herman B. (1990) Cell calcium 11:63-73. Williams, D.A., Fay, F.S. (1990) Cell calcium 11:75-83. Grynkiewicz, G., Poenie, M., Tsien, R.Y. (1985) Biochemistry 260:3440-3450. Godt, R.E., Nosek, T.M. (1989) J. Physiol. 412155-180. Tsien, R.Y. (1980) Biochemistry 19:2396-2404 Motta, M., Zoppi, S., Martini, L. (1986) J Steroid Biochem. 25:897-903. Berns, E.M.J.J., Boer, W., Mulder, E. (1986) The Prostate 9:247-259. Liao, S., Liang, T., Fang, E., Castaneda, E., Shao, T.S. (1973) J. Biol. Chem. 248:61546162. Traish, A.M., Muller, R.E., Wotiz, H.H. (1984) Endocrinology 118:1327-1333. Chaproniere, D.M., McKeehan, W.L. (1986) Cancer Res. 46:819-824. Connor, J., Sawczuk, I.S. Benson, MC., Tomashefsky, P., O’Toole, K.M., Olsson, C.A., Buttyan, R. (1988) The Prostate 13:119-130. Wilding, G., Chen, M., Gelmann, E.P. (1989) The Prostate 14:103-l 15. Olea, N., Sakabe, K., Soto, A.M., Sonnenschein, C. (1990). Endocrinology 126:1457-1463. Szego, C.M., Pietras, R.J. (1984) Int. Rev. Cytol. 88:1-302. Szego, C.M., Sjostrand, B.M., Seeler, B.J., Baumer, J.W., Sjostrand, F.S. (1988). Am. J. Physiol. 254:E775-E785. Quarmby, V.E., Yarbrough, W.G., Lubahn, D.B., French, F.S., Wilson, E.M. (1990). Mol. Endocrinol. 4:22-28. Pietras, R.J., Szego, C.M. (1977) Nature 265:69-72. Pietras, R.J., Szego, C.M. (1980) Biochem. J. 191:743-760. Steinsapir, J., Evans, A.C. Jr., McDonald, T., Muldoon, T.G. (1990) Biol. Reprod. 421337-349. Milgrom, E., Atger, M., Baulieu, E.-E. (1973) Biochem Biophys. Acta 320:267-283. Wilding, G., Gelmann, E.P., Freter, C.E. (1990) The Prostate 16:15-27.

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