Aromatase inhibition in JAr choriocarcinoma cells by 7α-arylaliphatic androgens

J. Steroid Biochem. Molec. Biol. Vol. 61, No. 1/2, pp. 73-77, 1997

Pergamon PII:

S0960-0760(97)00006-X

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A r o m a t a s e Inhibition in JAr C h o r i o c a r c i n o m a Cells by im[ j -Arylahphauc A n d r o g e n s •

•

Robert W. Brueggemeier, Nancy E. Gilbert, Xinyu Gu, e Jill M. O'Reilly and Carl J. Lovely ~College of Pharmacy, The Ohio State University, Columbus, OH 43210, U.S.A. and ~Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, U.S.A.

T h e J Ar c h o r l o c a r c i n o m a cell c u l t u r e s ha v e d e m o n s t r a t e d high levels o f a r o m a t a s e activity a n d h a v e b e e n useful for assaying a wide v a r i e t y o f a r o m a t a s e i n h i b i t o r s for a r o m a t a s e i n h i b i t i o n in i n t a c t cells. Recently, sever.-d 7~-arylaliphatic a n d r o g e n s have show n effective i n h i b i t i o n o f h u m a n p l a c e n tal m i c r o s o m a l a r o m a t a s e in vitro, with a p p a r e n t Ki values r a n g i n g f r o m 10 to 20 nM. A series o f 7~-arylaliphatic a n d r o s t - 4 - e n e - 3 , 1 7 - d i o n e c o m p o u n d s d e m o n s t r a t e d p o t e n t c o m p e t i t i v e inhibition, a n d 7~-arylaliphatic a n d r o s t a - l , 4 - d i e n e - 3 , 1 7 - d i o n e s w e r e e n z y m e - a c t i v a t e d i r r e v e r s i b l e inhibitors. B o t h series o f th es e p o t e n t i n h i b i t o r s w ere i nvest i gat ed for t he ability to i nhi bi t a r o m a t a s e activity in J Ar cells b y m e a s u r i n g the c o n v e r s i o n o f [ l f l - 3 H ] - a n d r o s t e n e d i o n e to 3H20 a n d u n l a b e l l e d est r o n e . J Ar cell c u l t u r e s w e r e i n c u b a t e d for 2 h at 37°C with t he a r o m a t a s e i n h i b i t o r s at c o n c e n t r a t i o n s o f 10 p M to l 0 / t M , the p e r c e n t a g e o f e n z y m e i nhi bi t i on was d e t e r m i n e d , a n d ICs0 values for i n h i b i t o r s w e r e c a l c u l a t e d . B o t h s e r i e s o f s y n t h e t i c c o m p o u n d s d e m o n s t r a t e d g o o d to e x c e l l e n t a r o m a t a s e i n h i b i t i o n , a n d t h e m o s t e f f e c t i v e i n h i b i t o r s in b o t h s e r i e s w e r e t h o s e c o m p o u n d s w i t h a p h e n y l p r o p y l substi~Lent at t he 7~-position o f the s t e r o i d nucleus. T h e 7~-arylallphatic a n d r o s t - 4 e n e- 3 ,1 7 - d io n es e x h i b i t e d i nhi bi t i on o f J A r a r o m a t a s e activity with ICs0 values f r o m 300 to 434 riM. M o r e p o t e n t aromat¢~se i n h i b i t i o n was o b s e r v e d with the 7~-arylaliphatic a n d r o s t a - l , 4 - d i e n e - 3 , 1 7 diones, wh ich exhi bi t ed ICs0 values f r o m 64 to 232 nM. E n h a n c e d efficacy o f st eroi dal e n z y m e - a c t i v a t e d i r r e v e r s i b l e i n h i b i t o r s c o m p a r e d to c o m p e t i t i v e i n h i b i t o r s was o b s e r v e d in these studies a n d is c o n s i s t e n t with p r e v i o u s r e p o r t s . T h e s e results suggest t h a t JA r c h o r i o c a r c i n o m a cells with high levels o f a r o m a t a s e activity m a y be useful in d i f f e r e n t i a t i n g s t e r o i d a l a r o m a t a s e i n h i b i t o r s exhibiting d i f f e r e n t mechanism.,; o f e n z y m e inhibition. In s u m m a r y , the 7 a - p h e n y l p r o p y l a n d r o s t a - l , 4 - d i e n e 3,17-dione analogs, w h i c h a r e e n z y m e - a c t i v a t e d i r r e v e r s i b l e i nhi bi t ors, d e m o n s t r a t e d t he m o s t effective in h ib iti on of a r o m a t a s e activity p r e s e n t in the JAr cell c u l t u r e s a m o n g the v a r i o u s 7a-arylaHphatic a n d r o g e n s . ~) 1997 E l s e vi e r Sc i e nc e Ltd.

J. Steroid Biochem. Molec. Biol., Vol. 61, No. 1/2, pp. 73-77, 1997

INTRODUCTION

irreversible inhibitors. One of the most potent competitive inhibitors, 7~-(4'-amino)phenylthio-4-androstene-3,17-dione (7~-APTA), exhibits an apparent Ki of 1 8 n M [1]. The introduction of an additional double bond in the A ring resulted in inhibitors that inactivated aromatase by an enzyme-catalyzed process [6-8]. The introduction of a 7~-substituent on 1,4androstadiene-3,17-dione yielded a potent mechanism-based irreversible inhibitor of aromatase, 7~-(4'amino)phenylthio- 1,4-androstadiene-3,17-dione (7~APTADD) [5]. Both inhibitors have demonstrated

Several 7~-thiosubstituted derivatives of androstenedione have demonstrated enhanced affinity for aromatase, and have produced very effective inhibition of aromatase activity present in human placental microsomes [1-5]. This group of inhibitors includes competitive, affinity, photoaffmity, and enzyme-activated *Correspondence to D r R. W. Brueggemeier. Tel: +1 614 292 5231; Fax: +1 614 292 2435; e-mail: [email protected]. Received 19 Aug. 1996; accepted 18 Dec. 1996. 73

74

R.W. Brueggemeier et al.

effectiveness in inhibiting aromatase in cell cultures [9, 10] and in treating hormone-dependent rat mammary tumors [11]. Current research activities in our laboratory have focused on the synthesis, biochemistry, and pharmacology of 7~-arylaliphatic androst-4-ene-3,17-diones and 7~-arylaliphatic androsta-l,4-diene-3,17-diones (Fig. 1). Bioisosteric replacement of the thioether linkage of the 7~-thiosubstituted androgens with a carbon-carbon bond in the 7~-arylaliphatic androgens provided inhibitors with enhanced chemical and metabolic stability [12,13]. Several 7~-arylaliphatic androgens have shown effective inhibition of human placental microsomal aromatase in vitro, with apparent Ki values ranging from 10 to 20 nM. The 7~-arylaliphatic androst-4-ene-3,17-dione compounds demonstrated potent competitive inhibition, and 7~arylaliphatic androsta- 1,4-diene-3,17-diones were enzyme-activated irreversible inhibitors. This study reports the evaluation of aromatase inhibition by these agents (compounds 3 - 1 1 ) in the JAr human choriocarcinoma line. The JAr human choriocarcinoma cell line exhibits high levels of aromatase activity in culture [14-16], and has thus been utilized to evaluate aromatase inhibition [10,17,18]. Using this cell line, small numbers of cells, less media and short incubation times are employed in measuring aromatase activity [10, 18]. In our laboratory, the level of aromatase activity in the JAr cells is approximately

7~-Thiosubstituted Androgens:

eight to 10 times higher than levels in MCF-7 cells. Investigations with aromatase inhibitors in the JAr cell have also suggested that mechanism-based inhibitors, e.g. 7~-APTADD and 4-hydroxy-androstenedione, are more effective (lower ICso values and steeper dose-response curves) than competitive inhibitors [10, 18]. MATERIALS AND METHODS Commercial steroids were obtained from Steraloids (Wilton, NH, U.S.A.) and checked for purity by melting point and thin layer chromatography (TLC). 7~Arylaliphatic androst-4-ene-3,17-diones and 7~-arylaliphatic androsta-1,4-diene-3,17-diones were prepared as previously described [ 12, 13]. [ 1fl-3H]-4Androstene-3,17-dione was purchased from New England Nuclear (Boston, MA, U.S.A.) and purity checked by TLC. JAr human choriocarcinoma cells were obtained from American Tissue Cell Culture, Bethesda, MD, U.S.A. RPMI media was obtained in powdered form from Gibco (Long Island, NY, U.S.A.). The sterilized liquid media was prepared by the Ohio State University Cell Culture Service, OSU Comprehensive Cancer Center, by dissolving the powder in water containing sodium chloride (0.487 g/ 1), pyruvic acid (0.11 g/l), sodium bicarbonate (1.5 g/ 1) and phenol red (0.01%) and the pH adjusted to 6.8. Fetal calf serum (FCS) was obtained from

7~-Arylaliphatic Androgens:

0

Oo n__O 7(~-APTA 1

n = 1,2, or 3 3-5

0

O ~ . _ _ O _ _ N H 7o~-APTADD 2

0

0

2 n = 1,2, or 3 6-11

Fig. 1.7~-Thiosubstituted and androgens and 7~-arylaliphatic androgens.

75

Aromatase Inhibition by 7~-Arylaliphatic Androgens

Table 1. IC5o values for the inhibition of JAr aromatase activ#y by 7~-arylaliphatic androst-4-ene-3,17-diones

cpd.

n

R

3 4 5

1 2 3

H H H

Substitution at C-7 ct-benzyl a-phenethyl ~-phenylpropyl

IC5o

Log IC5o

S.E.

328 nM 138 nM 208 nM

-6.484 -6.859 -6.681

0.0202 0.0612 0.0678

Gibco. Steroids were removed from the F C S by t w o treatments with dextran-coated charcoal [18]. Tissue culture flasks and supplies were obtained from C o m i n g Glass Works (Coming, NY, U.S.A.). Biochemicals were purchased from Sigma Chemical Co. (St Louis, M O , U.S.A.). Radioactive samples were detected with Beckman LS6800 or L S 6 0 0 0 I C scintillation counters using Formula 963 (New England Nuclear) as the counting solution. IC5o values represent the concentration of inhibitor required to produce a half-maximal inhibition of aromatase activity in the JAr cell cultures and were calculated by a non-linear regression analysis using the M a r q u a r d t m e t h o d (SAS Institute, Cary, N C , U.S.A.).

the tubes were incubated at 4°C for 15 min, then centrifuged for 10 min at 2600g. T h e s u p e m a n t was mixed with F o r m u l a 963 cocktail and counted by liquid scintillation. T h e D N A content of the cultures was determined by the diphenylamine assay [18]. T h e percentage inhibition was determined by dividing the total a m o u n t of 3 H 2 0 formed (pmol/#g D N A ) in the inhibited sample by the a m o u n t of 3 H 2 0 formed in the uninhibited (control) samples. ICso values for inhibitors are derived from log ICso values calculated by a non-linear regression analysis using the M a r q u a r d t m e t h o d (SAS Institute, Cary, N C , U.S.A.).

Inhibition of JAr aromatase activity

T h e efficacies of 77-arylaliphatic androstenediones to inhibit aromatase activity were evaluated in JAr trophoblastic choriocarcinoma cells. Aromatase activity was determined by measuring the conversion of [lfl-3H]-androstenedione to 3 H 2 0 as previously reported [17, 18]. T h e level of aromatase activity present in the JAr cell cultures was 6.31 (_+2.17)pmol product formed per 10 6 cells per hour, or approximately 44.15 p m o l per 9.2 cm 2 well in 2 h. Aromatase inhibition was determined by dividing the total a m o u n t of estradiol formed in the particular inhibitor

JAr cells were grown in 9.4 cm 2 wells at 37°C in R P M I media (2 ml) containing 10% F C S and gentamycin (20 mg/1). W h e n cultures reached 90% confluency (approximately 1 x 106 cells), media was changed and varying concentrations of aromatase inhibitors (10 p M to 10 # M in 5 #1 95% ethanol) were added. Aromatase activity was determined by measuring the conversion of [lfl-3H]-androstenedione to 3 H 2 0 [10, 17, 18]. F o r all cell culture studies, experiments were carried out in triplicate, and inhibitors were evaluated in experiments performed at least three different times. For aromatase inhibition, [lfl-3H]-androstenediorte ( 5 0 n M , 0 . 2 5 # C i , in 5#1 95% ethanol) was added to the cultures at the same time as the inhibitor. Control cultures received 3Handrostenedione, 95% ethanol, and no inhibitor. Blank samples contained 3H-androstenedione and 95% ethanol in media only (no cells). After 2 h the flasks were removed from the incubator and the media transferred into 35 ml centrifuge tubes. Chloroform (10 ml) was added to the media, the sample vortexed, incubated at r o o m temperature for 30 min, then centrifuged at 500g for 10 min. T h r e e aliquots (500 vl) of the s u p e m a t a n t were transferred into 12 x 75 m m culture tubes and 1% dextran coated charcoal solution was added (500/4). After vortexing,

RESULTS AND DISCUSSION

t 8o

=

cpd. 3 cpd. 4

60

~

2

-11

-'to

-b

-~ -~' log [I]

-~

25

Fig. 2. Inhibition of JAr aromatase activity by 7~=benzylandrost-4-ene-3,17-dione (3) and 7~-phenethylandrost-4-ene3,17-dlone (4).

76

R.W. Bmeggemeier et al.

Table 2. IC5o values for the inhibition of JAr aromatase activity by 7~-arylaliphatic androsta-l,4-diene-3,17-diones

cpd.

n

R

Substitution at C-7

6 7 8 9 10 11

1 2 2 3 3 3

H H NO 2 NH2 H NO2

~-benzyl ~-phenethyl ~-(4'-nitro)phenethyl ~- (4'-amino)phenethyl ~-phenylpropyl ~-(4'-nitro)phenylpropyl

sample by the amount of estradiol formed in the uninhibited (control) samples, and ICso values for inhibitors are derived from log ICso values calculated by a non-linear regression analysis [10, 18]. T h e 7~-arylaliphatic androst-4-ene-3,17-diones (35) exhibited ICso values ranging from 138 to 328 n M for the inhibition of JAr aromatase activity (Table 1). T h e most effective agent of this group of compounds was 7~-phenethylandrost-4-ene-3,17-dione (4), with an ICso of 138 n M (Fig. 2). As expected, the weakest inhibitor of this series was the 7~-benzyl derivative, which also exhibited the weakest apparent Ki value in enzymatic assays [12]. All three of these 7~-arylaliphatic androgens were weaker than the 7a-thiosubstituted compound, 7~-APTA. T h e 7~-arylaliphatic androsta-l,4-diene-3,17-diones (6-11) exhibited potent inhibition of JAr aromatase activity, with ICso values from 64 to 232 n M (Table 2, Fig. 3). In this series, the most potent analogs were the 7~-phenylpropyl analogs, compounds 10 and 11. T h e ICso value for unsubstituted 7~-phenylpropylandrosta-l,4-diene-3,17-dione (10) was found to be 63.7 nM. T h e introduction of a nitro functionality on

~, 100•-> 80-

cpd. 6 = cpd. 7 cpd. 10

~,

6040200 -I I

-1'0

-9

-8

-7

-6

75

log [I] Fig, 3. Inhibition of JAr aromatase activity by 7 = - b e n z y l a n -

drosta-l,4-diene-3,17-dione (6), 7a-phenethylandrosta-l,4diene-3,17-dlone (7) and 7a-phenylpropylandrosta-l,4-diene3,17-dione (10).

ICso 232.0 154.8 132.7 157.6 63.7 75.6

nM nM nM nM nM nM

Log ICso

S.E.

-6.633 -6.809 -6.876 -6.801 -7.194 -7.120

0.081 0.105 0.111 0.056 0.147 0.162

the aromatic ring, providing 7a-(4'-nitro)phenylpropylandrosta-l,4-diene-3,17-dione (11), did not significantly alter inhibition. Introduction of the nitro or the amino functionality at the 4'-position of the aromatic ring in the phenethyl analogs, compounds 8 and 9, also did not affect the ICso values. These results are consistent with those of the enzyme inhibition studies and apparent Ki values [13], and the results of JAr aromatase inhibition by 7-substituted androsta-l,4,6triene-3,17-diones [ 18]. For comparison, 7~-APTA, 7~-APTADD, and 4hydroxyandrostenedione (4-OH-A) inhibited aromatase activity in JAr cells in a dose-dependent fashion, with ICs0 values of 105 nM, 7.3 n M and 3.5 nM, respectively [10]. In microsomal assays, the two 7~-thiosubstituted aromatase inhibitors exhibited similar apparent Kis and thus similar affinity for the enzyme complex, whereas the apparent Ki for 4-OH-A is slightly higher. Interestingly, inhibition in the JAr cultures by 7~-APTA, a competitive inhibitor, was approximately 10 times less active than 7 a - A P T A D D and 4-OH-A, both mechanism-based inhibitors. T h e introduction of a 1,2-double bond in the A ring of the 7a-arylaliphatic androst-4-ene-3,17-diones (3-5) provided the 7a-arylaliphatic androsta-l,4diene-3,17-diones (6-11), which demonstrated mechanism-based irreversible inhibition in placental microsomal assays [13]. T h e best inactivator of the series was the 7a-phenylpropylandrosta- 1,4-diene-3,17dione, which exhibited a T 1 / 2 of 6.08 min and an apparent kinact of 1.90 X 10-3/S [13]. These mechanism-based compounds were very effective in inhibiting aromatase activity in the JAr cell cultures, and the addition of the 1,2-double bond in these steroidal inhibitors resulted in increased inhibitory activities and enhanced IC5o values by as much as 3.5-fold. In summary, the introduction of bulky, aryl substitutions at the C-7 position of the B-ring of androstenedione have provided several effective aromatase inhibitors. In JAr cell cultures, the 7a-arylaliphatic

Aromatase Inhibition by 7~-Arylaliphatic Androgens androst-4-ene-3,17-diones exhibited inhibition of a r o m a t a s e a c t i v i t y w i t h I C s 0 v a l u e s f r o m 3 0 0 to 434 nM. More potent aromatase i n h i b i t i o n was observed with the 7~-arylaliphatic androsta-l,4-diene3 , 1 7 - d i o n e s , w h i c h e x h i b i t e d I C s o v a l u e s f r o m 64 to 155 n M . I n c o n t r a s t , t h e d i f f e r e n c e s a m o n g t h e 11 inh i b i t o r s w e r e s i g n i f i c a n t l y s m a l l e r in t h e h u m a n p l a c e n t a l m i c r o s o m a l assays, w i t h a p p a r e n t Ki v a l u e s r a n g i n g f r o m 13 to 2 0 n M [12, 13]. A g r e a t e r r a n g e o f a r o m a t a s e i n h i b i t i o n b y t h e s e c o m p o u n d s t h u s exists in t h e J A r cells, a n d d i f f e r e n c e s in p o t e n c y a m o n g s p e c i f i c i n h i b i t o r s are o b s e r v e d in t h e cell c u l t u r e s w h i c h w e r e n o t s e e n in p l a c e n t a l m i c r o s o m a l assays. T h e s e r e s u l t s s u g g e s t t h a t o t h e r p a r a m e t e r s , s u c h as lipophilicity, membrane permeability, and non-specific p r o t e i n b i n d i n g , will irLfluence i n h i b i t o r y a c t i v i t y in i n t a c t cells. T h e enhanced efficacy of steroidal e n z y m e - a c t i v a t e d i r r e v e r s i b l e i n h i b i t o r s , also r e f e r r e d to as m e c h a n i s m - b a s e d i n h i b i t o r s , c o m p a r e d to c o m p e t i t i v e i n h i b i t o r s w a s o b s e r v e d in t h e s e s t u d i e s a n d is consistent with previous reports. These results of the inhibition of aromatase activity in J A r cells b y v a r i o u s 7c~-substituted s t e r o i d a l i n h i b i tots confirm and extend structure-activity relationships on these agents. The 7~-arylaliphatic androstenediones and 7~-arylaliphatic androsta-l,4d i e n e - 3 , 1 7 - d i o n e s are e q u a l l y e f f e c t i v e as t h e p r e v i o u s l y r e p o r t e d 7 c ~ - t h i o s u b s t i t u t e d a n a l o g s [12, 13], and may offer an advantage of greater metabolic stability in v i v o . T h e s e r e s u l t s s u g g e s t t h a t a d d i t i o n a l int e r a c t i o n s o c c u r b e t w e e n t h e p h e n y l r i n g at t h e 7~p o s i t i o n a n d a m i n o a c i d s at o r n e a r t h e e n z y m a t i c site o f a r o m a t a s e to r e s u l t i:n e n h a n c e d affinity o f t h e inhibitors. Acknowledgements--This reseazch work was supported in part by a

gift from the Ohio Chapter of the Ladies Auxiliary of Veterans of Foreign War, and by NIH Grants R01 CA58462, T32 CA09498, and R21 CA66193.

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