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Lignans and flavonoids inhibit aromatase enzyme in human preadipocytes
J. SteroidBiochem.Molec.Biol. Vol. 50, No. 3/4, pp. 205-212, 1994
Pergamon
0960-0760(94)E0084-X
Copyright~) 1994ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00
Lignans and Flavonoids Inhibit A r o m a t a s e E n z y m e in H u m a n P r e a d i p o c y t e s Chuanfeng
W a n g , t T a r u M~ikel/i, 2 T a p i o H a s e , 2 H e r m a n a n d M i n d y S. K u r z e r 1.
Adlercreutz 3
1Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St Paul, M N 55108, U.S.A.; 2Department of Chemistry, University of Helsinki and 3Department of Clinical Chemistry, University of Helsinki, Meilahti Hospital, Helsinki, Finland L i g n a n s a n d f l a v o n o i d s a r e n a t u r a l l y - o c c u r r i n g d i p h e n o l i c c o m p o u n d s f o u n d in h i g h c o n c e n t r a t i o n s in w h o l e g r a i n s , l e g u m e s , f r u i t s a n d v e g e t a b l e s . S e v e n l i g n a n s a n d six f l a v o n o i d s w e r e e v a l u a t e d f o r t h e i r a b i l i t i e s to i n h i b i t a r o m a t a s e e n z y m e a c t i v i t y in a h u m a n p r e a d i p o s e cell c u l t u r e s y s t e m . T h e l i g n a n , e n t e r o l a c t o n e ( E n l ) a n d its t h e o r e t i c a l p r e c u r s o r s , 3 ' - d e m e t h o x y - 3 0 - d e m e t h y l m a t a i r e s i n o l (DMDM) and didemethoxymatairesinol ( D D M M ) d e c r e a s e d a r o m a t a s e e n z y m e a c t i v i t y , w i t h Ki v a l u e s o f 14.4, 5.0 a n d 7.3/~M, r e s p e c t i v e l y . T h e f l a v o n o i d s , c o u m e s t r o l , l u t e o l i n a n d k a e m p f e r o l a l s o d e c r e a s e d a r o m a t a s e e n z y m e a c t i v i t y , w i t h Ki v a l u e s o f 1.3, 4.8 a n d 27.2 g M , r e s p e c t i v e l y . A m i n o g l u t e t h i m i d e , a p h a r m a c e u t i c a l a r o m a t a s e i n h i b i t o r , s h o w e d a Ki v a l u e o f 0.5 p M . K i n e t i c s t u d i e s s h o w e d t h e i n h i b i t i o n b y all c o m p o u n d s to b e c o m p e t i t i v e . S m a l l e r d e c r e a s e s in a r o m a t a s e a c t i v i t y w e r e o b s e r v e d w i t h t h e l i g n a n , e n t e r o d i o l ( E n d ) a n d its t h e o r e t i c a l p r e c u r s o r s , O - d e m e t h y l s e c o i s o lariciresinol (ODSI), demethoxysecoisolariciresinol (DMSI) and didemethylsecoisolariciresinol (DDSI). The flavonoids, O-demethylangolensin (O-Dma), fisetin and morin showed no inhibitory effects. T h e i n h i b i t i o n o f h u m a n p r e a d i p o c y t e a r o m a t a s e a c t i v i t y b y l i g n a n s a n d f l a v o n o i d s s u g g e s t s a m e c h a n i s m b y w h i c h c o n s u m p t i o n o f l i g n a n - a n d f l a v o n o i d - r i c h p l a n t f o o d s m a y c o n t r i b u t e to r e d u c t i o n o f e s t r o g e n - d e p e n d e n t d i s e a s e , s u c h as b r e a s t c a n c e r .
J. Steroid Biochem. Molec. Biol., Vol. 50, No. 3/4, pp. 205-212, 1994
INTRODUCTION
[8]. Specific flavonoids have been shown to inhibit oxidase enzyme activity [9, 10], suppress oxygenLignans and flavonoids are naturally-occurring com- induced cytotoxicity [11, 12] and suppress viral reverse pounds present in fruits, vegetables, legumes and transcriptase [13]. T h e y also have been shown to whole grains. Lignans are diphenolic c o m p o u n d s possess anti-viral [14-16] and anti-mutagenic activity formed from plant precursors by the actions of intesti[17-19] and to inhibit t u m o r induction by carcinogens nal bacteria [1]. T h e p r i m a r y m a m m a l i a n lignans are and growth promoters. enterolactone (Enl) and enterodiol (End), formed from It has been suggested that lignans and flavonoids plant precursors matairesinol and secoisolariciresinol, may contribute to the prevention of breast cancer in respectively. Fiber-rich foods such as whole grains are part as a result of their antiestrogenic properties [6]. rich in plant precursors [2-4], such that the urinary Specific lignans and flavonoids have been shown to excretion of m a m m a l i a n lignans is proportional to inhibit estrogen synthesis in h u m a n placental microdietary fiber consumption [5,6]. Epidemiological somes [20-22] and preadipocytes [23] and to compete studies showing low urinary lignan excretion in breast with estradiol for the estrogen receptor and type I I cancer patients [3, 6, 7] suggest that consumption of binding sites [24-27]. Lignans also have been shown to lignans m a y exert cancer-preventive effects. stimulate sex h o r m o n e binding globulin production in Flavonoids are diphenolic c o m p o u n d s widespread in the liver [27, 28] and to inhibit in vivo estrogen-stimuplant foods. It is estimated that a normal h u m a n diet lated R N A synthesis [29], proliferation of ZR-75-1 contains an average of 1 g of these c o m p o u n d s per day breast cancer cells [30] and estradiol-induced proliferation of M C F - 7 cells [31]. T h e major source of estrogen in postmenopausal *Correspondence to M. S. Kurzer. Received 30 Dec. 1993; accepted 8 Apr. 1994. w o m e n is aromatization of androstenedione to estrone 205
Chuanfeng Wang et al.
206
in peripheral tissue such as adipose [32, 33]. Inhibition of aromatase enzyme activity, with subsequent reduction in estrogen synthesis, is thought to contribute to prevention of estrogen-dependent cancers, such as breast cancer, in postmenopausal women. In fact, several pharmaceutical aromatase inhibitors have been used successfully in the treatment of estrogen-dependent carcinoma [34]. It has been suggested that naturally-occurring dietary phytoestrogens may contribute to prevention of estrogen-dependent cancers by some of the same mechanisms as pharmaceutical antiestrogens. It is therefore of great interest to identify dietary phytoestrogens, to evaluate their antiestrogenic potencies and to elucidate the mechanisms by which they exert their effects. T h e purpose of this study was to evaluate the specific effects of lignans and flavonoids on estrogen synthesis in h u m a n preadipocytes by measuring aromatase enzyme activity. Although lignans and flavonoids have been shown to inhibit aromatase enzyme activity in placental microsomes [20-22] and lignans have been shown to inhibit aromatase activity in a h u m a n choriocarcinoma cell line [20], little work has been done in whole cells derived from primary cultures of h u m a n tissue. We previously reported the flavonoids, ct-naphthoflavone (ANF), chrysin, flavone and biochanin A to be inhibitors of aromatase enzyme activity in h u m a n preadipocyes [23]. For the present study, we evaluated the effects of six other commonly consumed flavonoids as well as seven lignans, on aromatase enzyme activity in a preadipose cell culture system. T h e following lignans were studied: Enl and its theoretical precursors, 3'd e m e t h o x y - 3 0 - d e m e t h y l m a t a i r e s i n o l ( D M D M ) and didemethoxymatairesinol ( D D M M ) ; and End and its theoretical precursors, O-demethylsecoisolariciresinol ( O D S I ) , demethoxysecoisolariciresinol ( D M S I ) and didemethylsecoisolariciresinol ( D D S I ) . T h e flavonoids studied were: the flavonols, morin, fisetin and kaempferol; the flavone, luteolin; and the isoflavones, coumestrol and O-demethylangolensin ( O - D m a ) . T h e pharmaceutical aromatase inhibitor, aminoglutethimide (AG), was studied for comparison of inhibitor potency. EXPERIMENTAL
Chemicals Aminoglutethimide [3-(4-aminophenyl)-3-ethyl2,6-piperidinedione], collagenase (clostridiopeptidase A), dexamethasone (9e-fluoro-16ct-methylprednisolone), trypan blue stain (0.4%), kaempferol and morin were purchased from Sigma Chemical C o m p a n y , St Louis, M O , U.S.A. Dulbecco's Modified Eagle M e d i u m ( D M E M ) , Dulbecco's Phosphate Buffered Saline (PBS), antibiotic-antimycotic and calf serum were purchased from Gibco B R L , G r a n d Island, NY. 3 ml disposable extraction columns packed with
reversed phase octadecylsilane (C18) bonded to silica gel were purchased from J. T. Baker Inc., Phillipsburg, NY. Tritiated androstenedione ([1/~-3H(N)]androst-4 ene-3,17-dione) was purchased from N E N / D u Pont, Wilmington, DE. Fisetin was purchased from Aldrich Chemical Company, Inc., Milwaukee, W I . Luteolin was purchased from Carl Roth G m b H and Co., Karlsruhe, G e r m a n y and coumestrol was purchased from Eastman Kodak, Rochester, NY. All lignans and O - D m a were synthesized in the D e p a r t m e n t of Chemistry, University of Helsinki, Finland.
Processing of the adipose tissue T h e protocol for this experiment was approved by the University of Minnesota Institutional Review Board: H u m a n Subjects Committee. Subcutaneous adipose tissue from a b d o m e n and thigh regions was obtained by liposuction from female patients aged 23-57 years. T h e procedures for the tissue processing have been described in detail previously [23]. T h e fresh tissue was digested with collagenase for 3 0 - 6 0 m i n . After standing for 10 min at 37°C, the aqueous layer was removed and centrifuged. T h e resulting pellet of vascular/stromal cells was seeded in polystyrene tissue culture flasks containing D M E M and 15% calf serum and grown to confluence in a 5% CO2 humidified chamber. After the primary culture, cells were passaged approximately once per week.
Aromatase assay For the aromatase assay, cells were grown to confluence in 25 ml flasks containing D M E M supplemented with 10% calf serum. Prior to the assay, the cells were treated with 1 0 0 n M dexamethasone for 1 8 - 2 4 h to induce aromatase activity. At the end of the pre-incubation, the cells were washed twice with PBS and incubated at 37°C for 4 h in the experimental medium, consisting of D M E M containing 1% antibiotic-antimycotic, the desired concentration of 3H-androstenedione and the desired concentration of lignan or flavonoid. In the dose-response experiments, the concentration of 3H-androstenedione was 100 n M and the concentration of lignan or flavonoid ranged from 0 to 1 0 0 p M . W h e n inhibition was demonstrated, kinetic studies were carried out to determine the mechanism of inhibition and inhibitor effectiveness. For the kinetic studies, 3H-androstenedione concentrations were 25, 40, 75 and 150 n M and inhibitor concentrations were three levels between 0 and 100 p M. A modification of the tritiated water method of T h o m p s o n and Siiteri [35] was used to measure aromatase activity. T h i s modification has been described in detail previously [23]. Following the 4 h incubation with ~H-androstenedione and lignan or flavonoid, the experimental m e d i u m was removed and extracted with chloroform. After centrifugation at 1000g for 10 min, the aqueous u p p e r layer was transferred to individual C18 columns for final extraction. Aliquots were
Phytoestrogen Inhibition of Fat Cell Aromatase a.
207
Table 1. Kinetic data for aromatase inhibitors
b.
Percent control Vm~ R4
0
R4
OH
Re 1:13
Enl OH DMDM OH DDMM H
Inhibitor
R4, R3'
OH H H
R4
R,I.
H H OH OH OH OH
R3 End DDSI DMSI ODSI
Fla'
OH OH OH OH H OCHa OH OCH3
P.4
R4.
H OH OH OH
H OH OH OH
I # M 10#M 100#M Is0(#M )
Enterolactone DMDM DDMM Enterodiol ODSI DMSI DDSI Coumestrol Luteolin Kaempferol AG
Fig. 1. T h e s t r u c t u r e s o f E n l (a), End (b) a n d t h e i r t h e o r e t i c a l precursors.
c o u n t e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r ( B e c k m a n Instruments, Inc. F u l l e r t o n , C A ) . Cell p r o t e i n was s o l u b i l i z e d in s o d i u m h y d r o x i d e a n d q u a n t i f i e d b y t h e m e t h o d o f B r a d f o r d [36]. U n d e r t h e s e c o n d i t i o n s , t h e r e s u l t s fall in t h e linear r a n g e o f a r o m a t a s e e n z y m e a c t i v i t y for t i m e a n d cell number. T h e effects o f t h e i n h i b i t o r s o n cell v i a b i l i t y w e r e e v a l u a t e d via t r y p a n b l u e e x c l u s i o n . T e s t s w e r e p e r f o r m e d after i n c u b a t i o n o f t h e cells for 24 h r w i t h D M E M c o n t a i n i n g 10% calf s e r u m a n d the h i g h e s t c o n c e n t r a t i o n ( 1 0 0 p M ) o f i n h i b i t o r u s e d in t h e dose-response experiments.
97 104 71 100 95 81 100 119 90 127 76
89 96 68 90 96 80 84 61 72 107 39
43 45 44 75 87 77 78 14 17 35 7
K~(/~M)
74 84 60 > 100 > 100 > 100 > 100 17 25 61 5
RESULTS In the dose-response studies, a number of lignans a n d f l a v o n o i d s w e r e f o u n d to be a r o m a t a s e i n h i b i t o r s . T h e lignan, E n l a n d its t h e o r e t i c a l p r e c u r s o r s , D M D M a n d D D M M ( F i g . 1), w e r e s h o w n to be m o d e r a t e a r o m a t a s e i n h i b i t o r s ( F i g . 2, T a b l e 1), w i t h I50 values ( i n h i b i t o r c o n c e n t r a t i o n b l o c k i n g 5 0 % o f Vmax) of 74,84 and 60#M, r e s p e c t i v e l y . T h e flavonoids, c o u m e s t r o l , l u t e o l i n a n d k a e m p f e r o l (Fig. 3) w e r e also f o u n d to be a r o m a t a s e i n h i b i t o r s ( F i g . 2, T a b l e 1), w i t h I50 values o f 17, 25 a n d 61 p M , r e s p e c t i v e l y . F o r c o m p a r i s o n , the I50 value o f A G was 5 p M . K i n e t i c s t u d i e s w e r e p e r f o r m e d to i n v e s t i g a t e the mechanism by which the lignans and flavonoids inhibit a r o m a t a s e e n z y m e activity. I n the a b s e n c e o f i n h i b i t o r , t h e Km o f a n d r o s t e n e d i o n e a v e r a g e d 30 n M . I n t h e p r e s e n c e o f 50 a n d 100 p M Enl, the a p p a r e n t K m ( K app m ) value i n c r e a s e d to 144 a n d 2 2 1 n M , r e s p e c t i v e l y (Fig. 4). I n the p r e s e n c e o f 50 a n d 100 # M D M D M , t h e K ~P i n c r e a s e d to 94 a n d 265 n M , r e s p e c t i v e l y . I n
D a t a analysis D o u b l e - r e c i p r o c a l p l o t s w e r e u s e d to c h a r a c t e r i z e t h e t y p e o f i n h i b i t i o n . T h e K i values, reflecting i n h i b i t o r effectiveness, w e r e d e t e r m i n e d b y g r a p h i n g t h e slopes o f t h e d o u b l e - r e c i p r o c a l p l o t s vs i n h i b i t o r c o n c e n tration.
140
~ Luteolin
120
¢
IO0
Coumestrol
Kaempferol AG Enl DMDM
40 t3~
DDMVl
2O
I 0
0.01
I
I
I
I
0.1
1
10
100
Inhibitor
14.4 5.0 7.3 ----1.3 4.8 27.2 0.5
(ixM)
Fig. 2. D o s e - r e s p o n s e c u r v e s s h o w i n g t h e effects o f i n h i b i t o r c o n c e n t r a t i o n s o n a r o m a t a s e e n z y m e a c t i v i t y in h u m a n p r e a d i p o c y t e s . A r o m a t a s e a c t i v i t y w a s m e a s u r e d a f t e r i n c u b a t i o n o f t h e c e l l s f o r 18-24 h w i t h 100 n M d e x a m e t h a s o n e . D u r i n g t h e 4 h a s s a y , c e l l s w e r e i n c u b a t e d w i t h 100 n M 3 H - a n d r o s t e n e d i o n e a n d 0 - 1 0 0 / ~ M inhibitor. Each point represents the mean of three samples.
208
Chuanfeng Wang et al. a Rs. Rz.,~.4.
' Rs
P~'
O Ravonoid
R7 Quemetin OH Idol'in OH Fisetin OH Luteolin OH Kaempferol OH
R5 OH OH H OH OH
R3 OH OH OH H OH
R2' H OH H H H
R3' OH H OH OH H
b
R4' OH OH OH OH OH
c
"OH Coumestrol
" ~ O H
O-Domethylangolensin
Fig. 3. Structures of quercetin and the flavonoids studied. (a) flavonols and luteolin; (b) coumestrol; (c) O-Dma. the presence of 50 and 100/~M D D M M , the K ~ p increased to 62 and 94 n M , respectively. I n the presence o f 15 and 3 0 p M coumestrol, the a p p a r e n t Km ( K ~ p) increased to 199 and 4 2 2 n M , respectively (Fig. 5). I n the presence of 25 and 5 0 / ~ M luteolin, the K ~P increased to 206 and 435 n M , respectively. I n the presence o f 50 and 1 0 0 p M kaempferol, the K ~ p of androstenedione increased to 162 and 300 n M , respectively. I n the presence of 5 and 1 0 # M A G , the K ~ p increased to 277 and 916 n M , respectively. All inhibitors exerted competitive inhibition, as indi-
cated by the increasing K ~P values and constant Vmax in the presence of increasing inhibitor concentrations. D M D M was the strongest lignan inhibitor, with a Ki value o f 5.0 # M (Table 1). F o r Enl (Fig. 4, inset) and D D M M , Ki values were 14.4 and 7.3 p M , respectively (Table 1). C o u m e s t r o l was f o u n d to be the strongest flavonoid inhibitor, with a Ki value o f 1.3 p M (Fig. 5, T a b l e 1). L u t e o l i n was next, with a K~ o f 4 . 8 p M , followed by kaempferol, with a K~ of 27.2 # M . U n d e r the same experimental conditions, A G showed a K~ value of 0.5 p M . T h e lignan, E n d and its theoretical precursors, O D S I , D M S I and D D S I (Fig. 1) were weak inhibitors o f aromatase e n z y m e activity. I50 values for these c o m p o u n d s were all greater than 1 0 0 p M , and Ki values were not determined. T h e flavonoids, morin, fisetin, quercetin and O - D m a showed no inhibitory effects on aromatase e n z y m e activity. N o obvious m o r p h o l o g i c a l changes in lignan- or flavonoid-treated cells were f o u n d u n d e r microscopic examination. After incubation with 100 p M inhibitor for 24 h, the t r y p a n blue exclusion test showed less than 6 % cytotoxicity, with no differences between inhibitortreated cells and controls. It thus appears that cell viability was not affected by exposure to lignans or flavonoids.
DISCUSSION W e have shown that n a t u r a l l y - o c c u r r i n g lignans and flavonoids are able to pass t h r o u g h the cell m e m b r a n e
O.S-
n
0
0.02-
0.015 -
0.01
0,005
/
0
c2.
0.2Enl 0tM)
-
0
I 0'
2 '0
30
! 40
l/Androstenedione (F.M)" 1 F i g . 4. Double-reciprocal plot showing the effect of substrate (3H-androstenedione) c o n c e n t r a t i o n o n aromatase activity i n t h e presence of 0 (C)), 50 (A) and 100 ([])/~M Enl. The K m is the negative, inverted x intercept and the Vm. ~ is the inversion of the y intercept. Vma~ values were 13, 14 and 12 pM, and Km values were 30, 144 and 221 riM, for 0, 50 and 100 gM Ent, respectively. Each point represents the mean of three samples. I n s e t : Graph showing the effect of Enl concentration on the slope of the double-reciprocal plot lines. The K i value (negative x intercept), reflecting the inhibitor effectiveness, was 14.4 pM.
Phytoestrogen Inhibition of Fat Cell Aromatase
209
6-
L
5-
0.15 40.1 u)
0.05 O.
o
1'o
2'0
Coumestrol
(p.M)
2-
3'0 T--
T 10
T 20
I 30
"1 40
Androstenedione (gM)- 1 Fig. 5. D o u b l e - r e c i p r o c a l p l o t s h o w i n g t h e effect o f s u b s t r a t e ( 3 H - a n d r o s t e n e d i o n e ) c o n c e n t r a t i o n o n a r o m a t a s e a c t i v i t y in t h e p r e s e n c e o f 0 ( O ) , 15 ( A ) a n d 30 ([~) p M c o n m e s t r o l . T h e K m is t h e n e g a t i v e , i n v e r t e d x i n t e r c e p t a n d t h e Vma~ is t h e i n v e r s i o n o f t h e y i n t e r c e p t . T h e Vm,x w a s 3.2 p M , a n d t h e Km w a s 25, 199 a n d 422 n M , f o r 0, 15 a n d 30 p M c o u m e s t r o l , r e s p e c t i v e l y . I n s e t . T h e effect o f c o u m e s t r o l c o n c e n t r a t i o n o n t h e s l o p e of the double-reciprocal plot lines. The K I value (negative x intercept), reflecting inhibitor effectiveness, was 1.3 # M .
to competitively inhibit aromatase enzyme in h u m a n predipocytes. Although D M D M and D D M M are theoretical intermediates between matairesinol and Enl and D D S I , D M S ! and O D S I are theoretical intermediates between secoisolariciresinol and End, similar intermediates have been identified in primate urine, suggesting that they are true physiological intermediates [37]. Enl and its theoretical precursors were moderate inhibitors, while End and its theoretical precursors were weak inhibitors. T h e Ki values of the moderate inhibitors were between 10 and 30 times the K i of AG, suggesting that the affinities of these lignans to aromatase were 10-30-fold weaker than that of AG. At the same time, the K i values were between 150 and 500 times the Km of androstenedione, indicating that these c o m p o u n d s bind preadipocyte aromatase less than 1% as tightly as androstenedione binds aromatase. Using h u m a n placental microsomes, Adlercreutz et al. [20] first reported that Enl and its theoretical precursors were competitive inhibitors of aromatase. T h e I50 values of Enl and D D M M (4,4'-dihydroxyenterolactone) were 14 and 6/~M, respectively, 5-10 times lower than our results. T h e K i value of Enl in placental microsomes was 6 p M , less than half of our finding of 14.4 # M in preadipocytes. T h e affinity of Enl for aromatase in their study was 1.3% of that of androstenedione, 3-10-fold greater than in our study. T h u s , the inhibitor affinity and potency we observed in preadipocytes was 2-10-fold lower than that observed by Adlercreutz et al. in placental microsomes. T h e s e differences between whole cell and placental microsome systems are similar to those we reported pre-
viously in studies of flavonoid aromatase inhibitors [23]. As we suggested earlier, the lower potency of lignans in preadipocytes is possibly due to the incomplete passage of lignans through the cell m e m b r a n e barrier in whole cells. T h e intracellular concentration of inhibitor would likely be lower than that in the culture medium. T h e preadipocyte microsomes would therefore be exposed to lower concentrations of lignan than the placental microsomes, leading to lower inhibitory affinity and efficiency. T h e I50 value of D D M M (6 # M ) was lower than that of Enl (14 p M ) in the experiments of Adlercreutz et al., suggesting greater potency with D D M M . Our studies also showed this relationship in preadipocytes, with Is0 values of 60 and 7 4 # M , and Ki values of 7.3 and 14.4 # M for D D M M and Enl, respectively. Adlercreutz et al. also tested the effect of Enl on aromatase activity in the h u m a n choriocarcinoma cell line J E G - 3 [20]. After 1-2 h incubation with Enl, estrone production was 73-78, 61-63 and 2 4 - 2 7 % of the control level when Enl concentrations were 1, 10 and 100/~M, respectively. T h e inhibition in J E G cells was greater than in preadipocytes (aromatase activities of 97%, 89% and 37% of control level at 1, 10 and 100 (/~M Enl), most likely reflecting differences in cell type. Our study confirmed the observations of Adlercreutz et al. that End is a somewhat weaker aromatase inhibitor than Enl in placental microsomes [20]. Structural differences may contribute to this observed difference in inhibitor potency between Enl and End. While both are diphenolic compounds, Enl and its theoretical precursors contain a keto-tetrahydrofuran ring, and
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Chuanfeng Wang et al.
End and its theoretical precursors are without such a ring. T h e presence of this ring structure may either directly increase the affinity of Enl to aromatase enzyme, or it may increase lipid solubility, allowing Enl and its theoretical precursors to enter the cell more easily and gain access to the binding site of aromatase. An effect of the ring on lipid solubility may also explain our finding that the difference in potency between End and Enl is greater in whole cells than in microsomes, with End being a far weaker inhibitor in whole cells. O u r results with flavonoids are the first to d e m o n strate that the isoflavone, coumestrol, the flavone, luteolin, and the flavonol, kaempferol, are competitive inhibitors of aromatase enzyme activity. Ki values for the flavonoids were 2.6, 9.6 and 54 times the K i of AG, for coumestrol, luteolin and kaempferol, respectively, refecting inhibitor potencies that were about 40%, 10°,/o and 2% of the potency of AG. T h e i r affinities to aromatase enzyme were less than 3% that of androstenedione. Coumestrol is a phytoestrogen known to bind the estrogen receptor [38] and exert estrogenic effects. T h e s e include induction of vaginal opening, cornification and cycling; stimulations of uterine metaplasia and promotion of hemorrhagic follicles [39, 40]; stimulation of cytosolic progestin receptors [40]; and proliferation of M C F - 7 [41] and other m a m m a r y t u m o r cells [38]. Our findings suggest that the estrogenic effects of coumestrol may be opposed in some cases by its inhibition of estrogen synthesis. T h e isoflavone, O - D m a , did not inhibit aromatase enzyme in preadipocytes. T h i s was not surprising, as the structure of O - D m a is similar to that of other isoflavones, such as daidzein and genistein, that we previously reported to be noninhibitors [23]. On the other hand, Adlercreutz et al. [20] found O - D m a to be a weak aromatase inhibitor in h u m a n placental microsomes, with an I50 of 160 p M . U n d e r our conditions in whole cells, we have generally found I50 and Ki values to be 10-fold greater than those reported in placental microsomes. With an I50 of 160 # M in placental microsomes [20], we would be unable to detect inhibition by O - D m a in our system. T h e flavonols, morin and fisetin, were found to be noninhibitors of aromatase enzyme in our system. Our results with morin differ from those of Moochhala et al., who found morin to bind aromatase enzyme in placental microsomes [42]. These investigators found morin to bind aromatase to a similar degree as does quercetin, a flavonol that we previously reported to be a noninhibitor in preadipocytes [23]. Although morin binds aromatase enzyme weakly in placental microsomes, as do quercetin and O - D m a , limited passage through the cell m e m b r a n e causes these weak inhibitors to fall below the limits of sensitivity of our whole cell system. Several reports have explored the relationship between flavonoid structure and ability to inhibit aroma-
tase enzyme activity. I b r a h i m and Abul-Hajj suggested the importance of C-7 hydroxylation for binding to aromatase [21], while Moochhala et al. suggested the importance of 5,7-dihydroxylation [42]. In our study, all of the compounds have a C-7 hydroxyl, while two of the three inhibitors and one of the three noninhibitors have a C-5 hydroxyl. Quercetin, a flavonol previously reported to be a non-inhibitor [23], also has a C-5 hydroxyl. T h u s , for this group of compounds, hydroxylation at the 5 or 7 position does not appear to be a major determinant of inhibitory effect. Our findings do, however, support previous reports of decreased inhibition by C-3 hydroxylated flavonoids [21,23,42]. In our study, all of the noninhibitor flavonols, as well as the weak inhibitor, kaempferol, have a C-3 hydroxyl, while the stronger inhibitor, luteolin, does not. T h e increased inhibitory capacity of luteolin, relative to quercetin, strongly supports this hypothesis. In addition, the increased inhibitory capacity of kaempferol relative to quercetin suggests that the presence of a C - Y hydroxyl reduces inhibitory capacity and the increased inhibitory capacity ot kaempferol relative to morin suggests that the presence of a C-2' hydroxyl reduces inhibitory capacity. T h e r e are no reports of the effects of lignans or flavonoids on aromatase enzyme activity in living organisms. Considering the m u c h lower affinities of these compounds to aromatase than that of the substrate, it is reasonable to assume that very high levels of inhibitor would be required to successfully compete with androstenedione and testosterone. Few data have been reported, however, on the normal circulating levels of these compounds in humans. Adlercreutz et al. reported mean plasma levels of End and Enl of 2.5 and 33 nM, respectively, in omnivorous w o m e n and 17 and 253 n M in vegetarian w o m e n [43]. At 253 nM, the circulating level of Enl in vegetarians is about 50-fold lower than the Ki of Enl, suggesting that the physiological significance of inhibition by Enl alone is unlikely. Although the concentration of any one c o m p o u n d may be low relative to the individual Ki value, the total effect of m a n y aromatase inhibitors circulating in v i v o over a lifetime, may be of physiological significance. Additional dietary aromatase inhibitors have been reported, including other lignans [20] and flavonoids such as chrysin and biochanin A [23], hydroxyflavones [21] and apigenin and flavanone [22]. T h e estrogenreducing effects of these compounds may be enhanced by their other reported antiestrogenic properties, such as suppression of estrogen action and bioavailability. T h e s e effects would be more important in vegetarians, as a result of their higher consumption of plant foods containing lignans and phytoestrogens. T h e s e results provide further evidence that naturally-occurring dietary compounds inhibit aromatase enzyme activity in intact preadipose cells. Our findings suggest that lignans and flavonoids may contribute to
Phytoestrogen Inhibition of Fat Cell Aromatase
the cancer-preventive properties of plant-based diets by inhibiting estrogen synthesis in adipose tissue. Although it is unlikely that any one compound alone inhibits aromatase inhibitor significantly in vivo, the physiological significance of long-term consumption of many such compounds has yet to be explored. Acknowledgements--This work was supported by Minnesota Agricultural Experiment Station project 18-34 and an International Life Science Institute-Nutrition Foundation Future Leader Award (MSK). Synthesis of the lignan intermediates and O-Dma was supported by NIH grant CA56289-01 to H.A. The authors are grateful to Dr Bruce Cunningham (Department of Surgery, University of Minnesota) and the staff of the St Paul Surgical Center, St Paul Ramsey Medical Center (St Paul, MN) for assistance in obtaining adipose tissue and to Dr K. W~ih~il~i (Department of Chemistry, University of Helsinki, Finland) for the synthesis of O-Dma.
14.
15. 16. 17. 18. 19. 20.
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39. Burroughs C. D., Mills K. T. and Bern H. A.: Long-term genital tract changes in female mice treated neonatally with coumestrol. Reprod. Toxic. 4 (1990) 127-135. 40. Whitten P. L. and Naftolin E.: Effects of a phytoestrogen diet on estrogen-dependent reproductive processes in immature female rats. Steroids 57 (1992) 56--61. 41. Welshons W. V., Rottinghaus G. E., Nonneman D. J., DolanTimpe M. and Ross P. F.: A sensitive bioassay for detection of dietary estrogens in animal feeds. J. Vet. Diagn. Invest. 2 (1990) 268-273. 42. Moochhala S. M., Loke K. H. and Das N. 17.: Spectral perturbation of human microsomal cytochrome P-450 by flavonoid binding. Bioehem. Int. 17 (1988) 755-762. 43. Adlercreutz H., Fotsis T., Lampe J., Wfih~il~i K., M/ikel~ T., Brunow G. and Hase T.: Quantitative determination of lignans and isoflavonoids in plasma of omnivorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry. Scand. J. Clin. Lab. Invest. 53, suppl. 215 (1993) 5-18.
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Lignans and Flavonoids Inhibit A r o m a t a s e E n z y m e in H u m a n P r e a d i p o c y t e s Chuanfeng
W a n g , t T a r u M~ikel/i, 2 T a p i o H a s e , 2 H e r m a n a n d M i n d y S. K u r z e r 1.
Adlercreutz 3
1Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St Paul, M N 55108, U.S.A.; 2Department of Chemistry, University of Helsinki and 3Department of Clinical Chemistry, University of Helsinki, Meilahti Hospital, Helsinki, Finland L i g n a n s a n d f l a v o n o i d s a r e n a t u r a l l y - o c c u r r i n g d i p h e n o l i c c o m p o u n d s f o u n d in h i g h c o n c e n t r a t i o n s in w h o l e g r a i n s , l e g u m e s , f r u i t s a n d v e g e t a b l e s . S e v e n l i g n a n s a n d six f l a v o n o i d s w e r e e v a l u a t e d f o r t h e i r a b i l i t i e s to i n h i b i t a r o m a t a s e e n z y m e a c t i v i t y in a h u m a n p r e a d i p o s e cell c u l t u r e s y s t e m . T h e l i g n a n , e n t e r o l a c t o n e ( E n l ) a n d its t h e o r e t i c a l p r e c u r s o r s , 3 ' - d e m e t h o x y - 3 0 - d e m e t h y l m a t a i r e s i n o l (DMDM) and didemethoxymatairesinol ( D D M M ) d e c r e a s e d a r o m a t a s e e n z y m e a c t i v i t y , w i t h Ki v a l u e s o f 14.4, 5.0 a n d 7.3/~M, r e s p e c t i v e l y . T h e f l a v o n o i d s , c o u m e s t r o l , l u t e o l i n a n d k a e m p f e r o l a l s o d e c r e a s e d a r o m a t a s e e n z y m e a c t i v i t y , w i t h Ki v a l u e s o f 1.3, 4.8 a n d 27.2 g M , r e s p e c t i v e l y . A m i n o g l u t e t h i m i d e , a p h a r m a c e u t i c a l a r o m a t a s e i n h i b i t o r , s h o w e d a Ki v a l u e o f 0.5 p M . K i n e t i c s t u d i e s s h o w e d t h e i n h i b i t i o n b y all c o m p o u n d s to b e c o m p e t i t i v e . S m a l l e r d e c r e a s e s in a r o m a t a s e a c t i v i t y w e r e o b s e r v e d w i t h t h e l i g n a n , e n t e r o d i o l ( E n d ) a n d its t h e o r e t i c a l p r e c u r s o r s , O - d e m e t h y l s e c o i s o lariciresinol (ODSI), demethoxysecoisolariciresinol (DMSI) and didemethylsecoisolariciresinol (DDSI). The flavonoids, O-demethylangolensin (O-Dma), fisetin and morin showed no inhibitory effects. T h e i n h i b i t i o n o f h u m a n p r e a d i p o c y t e a r o m a t a s e a c t i v i t y b y l i g n a n s a n d f l a v o n o i d s s u g g e s t s a m e c h a n i s m b y w h i c h c o n s u m p t i o n o f l i g n a n - a n d f l a v o n o i d - r i c h p l a n t f o o d s m a y c o n t r i b u t e to r e d u c t i o n o f e s t r o g e n - d e p e n d e n t d i s e a s e , s u c h as b r e a s t c a n c e r .
J. Steroid Biochem. Molec. Biol., Vol. 50, No. 3/4, pp. 205-212, 1994
INTRODUCTION
[8]. Specific flavonoids have been shown to inhibit oxidase enzyme activity [9, 10], suppress oxygenLignans and flavonoids are naturally-occurring com- induced cytotoxicity [11, 12] and suppress viral reverse pounds present in fruits, vegetables, legumes and transcriptase [13]. T h e y also have been shown to whole grains. Lignans are diphenolic c o m p o u n d s possess anti-viral [14-16] and anti-mutagenic activity formed from plant precursors by the actions of intesti[17-19] and to inhibit t u m o r induction by carcinogens nal bacteria [1]. T h e p r i m a r y m a m m a l i a n lignans are and growth promoters. enterolactone (Enl) and enterodiol (End), formed from It has been suggested that lignans and flavonoids plant precursors matairesinol and secoisolariciresinol, may contribute to the prevention of breast cancer in respectively. Fiber-rich foods such as whole grains are part as a result of their antiestrogenic properties [6]. rich in plant precursors [2-4], such that the urinary Specific lignans and flavonoids have been shown to excretion of m a m m a l i a n lignans is proportional to inhibit estrogen synthesis in h u m a n placental microdietary fiber consumption [5,6]. Epidemiological somes [20-22] and preadipocytes [23] and to compete studies showing low urinary lignan excretion in breast with estradiol for the estrogen receptor and type I I cancer patients [3, 6, 7] suggest that consumption of binding sites [24-27]. Lignans also have been shown to lignans m a y exert cancer-preventive effects. stimulate sex h o r m o n e binding globulin production in Flavonoids are diphenolic c o m p o u n d s widespread in the liver [27, 28] and to inhibit in vivo estrogen-stimuplant foods. It is estimated that a normal h u m a n diet lated R N A synthesis [29], proliferation of ZR-75-1 contains an average of 1 g of these c o m p o u n d s per day breast cancer cells [30] and estradiol-induced proliferation of M C F - 7 cells [31]. T h e major source of estrogen in postmenopausal *Correspondence to M. S. Kurzer. Received 30 Dec. 1993; accepted 8 Apr. 1994. w o m e n is aromatization of androstenedione to estrone 205
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206
in peripheral tissue such as adipose [32, 33]. Inhibition of aromatase enzyme activity, with subsequent reduction in estrogen synthesis, is thought to contribute to prevention of estrogen-dependent cancers, such as breast cancer, in postmenopausal women. In fact, several pharmaceutical aromatase inhibitors have been used successfully in the treatment of estrogen-dependent carcinoma [34]. It has been suggested that naturally-occurring dietary phytoestrogens may contribute to prevention of estrogen-dependent cancers by some of the same mechanisms as pharmaceutical antiestrogens. It is therefore of great interest to identify dietary phytoestrogens, to evaluate their antiestrogenic potencies and to elucidate the mechanisms by which they exert their effects. T h e purpose of this study was to evaluate the specific effects of lignans and flavonoids on estrogen synthesis in h u m a n preadipocytes by measuring aromatase enzyme activity. Although lignans and flavonoids have been shown to inhibit aromatase enzyme activity in placental microsomes [20-22] and lignans have been shown to inhibit aromatase activity in a h u m a n choriocarcinoma cell line [20], little work has been done in whole cells derived from primary cultures of h u m a n tissue. We previously reported the flavonoids, ct-naphthoflavone (ANF), chrysin, flavone and biochanin A to be inhibitors of aromatase enzyme activity in h u m a n preadipocyes [23]. For the present study, we evaluated the effects of six other commonly consumed flavonoids as well as seven lignans, on aromatase enzyme activity in a preadipose cell culture system. T h e following lignans were studied: Enl and its theoretical precursors, 3'd e m e t h o x y - 3 0 - d e m e t h y l m a t a i r e s i n o l ( D M D M ) and didemethoxymatairesinol ( D D M M ) ; and End and its theoretical precursors, O-demethylsecoisolariciresinol ( O D S I ) , demethoxysecoisolariciresinol ( D M S I ) and didemethylsecoisolariciresinol ( D D S I ) . T h e flavonoids studied were: the flavonols, morin, fisetin and kaempferol; the flavone, luteolin; and the isoflavones, coumestrol and O-demethylangolensin ( O - D m a ) . T h e pharmaceutical aromatase inhibitor, aminoglutethimide (AG), was studied for comparison of inhibitor potency. EXPERIMENTAL
Chemicals Aminoglutethimide [3-(4-aminophenyl)-3-ethyl2,6-piperidinedione], collagenase (clostridiopeptidase A), dexamethasone (9e-fluoro-16ct-methylprednisolone), trypan blue stain (0.4%), kaempferol and morin were purchased from Sigma Chemical C o m p a n y , St Louis, M O , U.S.A. Dulbecco's Modified Eagle M e d i u m ( D M E M ) , Dulbecco's Phosphate Buffered Saline (PBS), antibiotic-antimycotic and calf serum were purchased from Gibco B R L , G r a n d Island, NY. 3 ml disposable extraction columns packed with
reversed phase octadecylsilane (C18) bonded to silica gel were purchased from J. T. Baker Inc., Phillipsburg, NY. Tritiated androstenedione ([1/~-3H(N)]androst-4 ene-3,17-dione) was purchased from N E N / D u Pont, Wilmington, DE. Fisetin was purchased from Aldrich Chemical Company, Inc., Milwaukee, W I . Luteolin was purchased from Carl Roth G m b H and Co., Karlsruhe, G e r m a n y and coumestrol was purchased from Eastman Kodak, Rochester, NY. All lignans and O - D m a were synthesized in the D e p a r t m e n t of Chemistry, University of Helsinki, Finland.
Processing of the adipose tissue T h e protocol for this experiment was approved by the University of Minnesota Institutional Review Board: H u m a n Subjects Committee. Subcutaneous adipose tissue from a b d o m e n and thigh regions was obtained by liposuction from female patients aged 23-57 years. T h e procedures for the tissue processing have been described in detail previously [23]. T h e fresh tissue was digested with collagenase for 3 0 - 6 0 m i n . After standing for 10 min at 37°C, the aqueous layer was removed and centrifuged. T h e resulting pellet of vascular/stromal cells was seeded in polystyrene tissue culture flasks containing D M E M and 15% calf serum and grown to confluence in a 5% CO2 humidified chamber. After the primary culture, cells were passaged approximately once per week.
Aromatase assay For the aromatase assay, cells were grown to confluence in 25 ml flasks containing D M E M supplemented with 10% calf serum. Prior to the assay, the cells were treated with 1 0 0 n M dexamethasone for 1 8 - 2 4 h to induce aromatase activity. At the end of the pre-incubation, the cells were washed twice with PBS and incubated at 37°C for 4 h in the experimental medium, consisting of D M E M containing 1% antibiotic-antimycotic, the desired concentration of 3H-androstenedione and the desired concentration of lignan or flavonoid. In the dose-response experiments, the concentration of 3H-androstenedione was 100 n M and the concentration of lignan or flavonoid ranged from 0 to 1 0 0 p M . W h e n inhibition was demonstrated, kinetic studies were carried out to determine the mechanism of inhibition and inhibitor effectiveness. For the kinetic studies, 3H-androstenedione concentrations were 25, 40, 75 and 150 n M and inhibitor concentrations were three levels between 0 and 100 p M. A modification of the tritiated water method of T h o m p s o n and Siiteri [35] was used to measure aromatase activity. T h i s modification has been described in detail previously [23]. Following the 4 h incubation with ~H-androstenedione and lignan or flavonoid, the experimental m e d i u m was removed and extracted with chloroform. After centrifugation at 1000g for 10 min, the aqueous u p p e r layer was transferred to individual C18 columns for final extraction. Aliquots were
Phytoestrogen Inhibition of Fat Cell Aromatase a.
207
Table 1. Kinetic data for aromatase inhibitors
b.
Percent control Vm~ R4
0
R4
OH
Re 1:13
Enl OH DMDM OH DDMM H
Inhibitor
R4, R3'
OH H H
R4
R,I.
H H OH OH OH OH
R3 End DDSI DMSI ODSI
Fla'
OH OH OH OH H OCHa OH OCH3
P.4
R4.
H OH OH OH
H OH OH OH
I # M 10#M 100#M Is0(#M )
Enterolactone DMDM DDMM Enterodiol ODSI DMSI DDSI Coumestrol Luteolin Kaempferol AG
Fig. 1. T h e s t r u c t u r e s o f E n l (a), End (b) a n d t h e i r t h e o r e t i c a l precursors.
c o u n t e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r ( B e c k m a n Instruments, Inc. F u l l e r t o n , C A ) . Cell p r o t e i n was s o l u b i l i z e d in s o d i u m h y d r o x i d e a n d q u a n t i f i e d b y t h e m e t h o d o f B r a d f o r d [36]. U n d e r t h e s e c o n d i t i o n s , t h e r e s u l t s fall in t h e linear r a n g e o f a r o m a t a s e e n z y m e a c t i v i t y for t i m e a n d cell number. T h e effects o f t h e i n h i b i t o r s o n cell v i a b i l i t y w e r e e v a l u a t e d via t r y p a n b l u e e x c l u s i o n . T e s t s w e r e p e r f o r m e d after i n c u b a t i o n o f t h e cells for 24 h r w i t h D M E M c o n t a i n i n g 10% calf s e r u m a n d the h i g h e s t c o n c e n t r a t i o n ( 1 0 0 p M ) o f i n h i b i t o r u s e d in t h e dose-response experiments.
97 104 71 100 95 81 100 119 90 127 76
89 96 68 90 96 80 84 61 72 107 39
43 45 44 75 87 77 78 14 17 35 7
K~(/~M)
74 84 60 > 100 > 100 > 100 > 100 17 25 61 5
RESULTS In the dose-response studies, a number of lignans a n d f l a v o n o i d s w e r e f o u n d to be a r o m a t a s e i n h i b i t o r s . T h e lignan, E n l a n d its t h e o r e t i c a l p r e c u r s o r s , D M D M a n d D D M M ( F i g . 1), w e r e s h o w n to be m o d e r a t e a r o m a t a s e i n h i b i t o r s ( F i g . 2, T a b l e 1), w i t h I50 values ( i n h i b i t o r c o n c e n t r a t i o n b l o c k i n g 5 0 % o f Vmax) of 74,84 and 60#M, r e s p e c t i v e l y . T h e flavonoids, c o u m e s t r o l , l u t e o l i n a n d k a e m p f e r o l (Fig. 3) w e r e also f o u n d to be a r o m a t a s e i n h i b i t o r s ( F i g . 2, T a b l e 1), w i t h I50 values o f 17, 25 a n d 61 p M , r e s p e c t i v e l y . F o r c o m p a r i s o n , the I50 value o f A G was 5 p M . K i n e t i c s t u d i e s w e r e p e r f o r m e d to i n v e s t i g a t e the mechanism by which the lignans and flavonoids inhibit a r o m a t a s e e n z y m e activity. I n the a b s e n c e o f i n h i b i t o r , t h e Km o f a n d r o s t e n e d i o n e a v e r a g e d 30 n M . I n t h e p r e s e n c e o f 50 a n d 100 p M Enl, the a p p a r e n t K m ( K app m ) value i n c r e a s e d to 144 a n d 2 2 1 n M , r e s p e c t i v e l y (Fig. 4). I n the p r e s e n c e o f 50 a n d 100 # M D M D M , t h e K ~P i n c r e a s e d to 94 a n d 265 n M , r e s p e c t i v e l y . I n
D a t a analysis D o u b l e - r e c i p r o c a l p l o t s w e r e u s e d to c h a r a c t e r i z e t h e t y p e o f i n h i b i t i o n . T h e K i values, reflecting i n h i b i t o r effectiveness, w e r e d e t e r m i n e d b y g r a p h i n g t h e slopes o f t h e d o u b l e - r e c i p r o c a l p l o t s vs i n h i b i t o r c o n c e n tration.
140
~ Luteolin
120
¢
IO0
Coumestrol
Kaempferol AG Enl DMDM
40 t3~
DDMVl
2O
I 0
0.01
I
I
I
I
0.1
1
10
100
Inhibitor
14.4 5.0 7.3 ----1.3 4.8 27.2 0.5
(ixM)
Fig. 2. D o s e - r e s p o n s e c u r v e s s h o w i n g t h e effects o f i n h i b i t o r c o n c e n t r a t i o n s o n a r o m a t a s e e n z y m e a c t i v i t y in h u m a n p r e a d i p o c y t e s . A r o m a t a s e a c t i v i t y w a s m e a s u r e d a f t e r i n c u b a t i o n o f t h e c e l l s f o r 18-24 h w i t h 100 n M d e x a m e t h a s o n e . D u r i n g t h e 4 h a s s a y , c e l l s w e r e i n c u b a t e d w i t h 100 n M 3 H - a n d r o s t e n e d i o n e a n d 0 - 1 0 0 / ~ M inhibitor. Each point represents the mean of three samples.
208
Chuanfeng Wang et al. a Rs. Rz.,~.4.
' Rs
P~'
O Ravonoid
R7 Quemetin OH Idol'in OH Fisetin OH Luteolin OH Kaempferol OH
R5 OH OH H OH OH
R3 OH OH OH H OH
R2' H OH H H H
R3' OH H OH OH H
b
R4' OH OH OH OH OH
c
"OH Coumestrol
" ~ O H
O-Domethylangolensin
Fig. 3. Structures of quercetin and the flavonoids studied. (a) flavonols and luteolin; (b) coumestrol; (c) O-Dma. the presence of 50 and 100/~M D D M M , the K ~ p increased to 62 and 94 n M , respectively. I n the presence o f 15 and 3 0 p M coumestrol, the a p p a r e n t Km ( K ~ p) increased to 199 and 4 2 2 n M , respectively (Fig. 5). I n the presence of 25 and 5 0 / ~ M luteolin, the K ~P increased to 206 and 435 n M , respectively. I n the presence o f 50 and 1 0 0 p M kaempferol, the K ~ p of androstenedione increased to 162 and 300 n M , respectively. I n the presence of 5 and 1 0 # M A G , the K ~ p increased to 277 and 916 n M , respectively. All inhibitors exerted competitive inhibition, as indi-
cated by the increasing K ~P values and constant Vmax in the presence of increasing inhibitor concentrations. D M D M was the strongest lignan inhibitor, with a Ki value o f 5.0 # M (Table 1). F o r Enl (Fig. 4, inset) and D D M M , Ki values were 14.4 and 7.3 p M , respectively (Table 1). C o u m e s t r o l was f o u n d to be the strongest flavonoid inhibitor, with a Ki value o f 1.3 p M (Fig. 5, T a b l e 1). L u t e o l i n was next, with a K~ o f 4 . 8 p M , followed by kaempferol, with a K~ of 27.2 # M . U n d e r the same experimental conditions, A G showed a K~ value of 0.5 p M . T h e lignan, E n d and its theoretical precursors, O D S I , D M S I and D D S I (Fig. 1) were weak inhibitors o f aromatase e n z y m e activity. I50 values for these c o m p o u n d s were all greater than 1 0 0 p M , and Ki values were not determined. T h e flavonoids, morin, fisetin, quercetin and O - D m a showed no inhibitory effects on aromatase e n z y m e activity. N o obvious m o r p h o l o g i c a l changes in lignan- or flavonoid-treated cells were f o u n d u n d e r microscopic examination. After incubation with 100 p M inhibitor for 24 h, the t r y p a n blue exclusion test showed less than 6 % cytotoxicity, with no differences between inhibitortreated cells and controls. It thus appears that cell viability was not affected by exposure to lignans or flavonoids.
DISCUSSION W e have shown that n a t u r a l l y - o c c u r r i n g lignans and flavonoids are able to pass t h r o u g h the cell m e m b r a n e
O.S-
n
0
0.02-
0.015 -
0.01
0,005
/
0
c2.
0.2Enl 0tM)
-
0
I 0'
2 '0
30
! 40
l/Androstenedione (F.M)" 1 F i g . 4. Double-reciprocal plot showing the effect of substrate (3H-androstenedione) c o n c e n t r a t i o n o n aromatase activity i n t h e presence of 0 (C)), 50 (A) and 100 ([])/~M Enl. The K m is the negative, inverted x intercept and the Vm. ~ is the inversion of the y intercept. Vma~ values were 13, 14 and 12 pM, and Km values were 30, 144 and 221 riM, for 0, 50 and 100 gM Ent, respectively. Each point represents the mean of three samples. I n s e t : Graph showing the effect of Enl concentration on the slope of the double-reciprocal plot lines. The K i value (negative x intercept), reflecting the inhibitor effectiveness, was 14.4 pM.
Phytoestrogen Inhibition of Fat Cell Aromatase
209
6-
L
5-
0.15 40.1 u)
0.05 O.
o
1'o
2'0
Coumestrol
(p.M)
2-
3'0 T--
T 10
T 20
I 30
"1 40
Androstenedione (gM)- 1 Fig. 5. D o u b l e - r e c i p r o c a l p l o t s h o w i n g t h e effect o f s u b s t r a t e ( 3 H - a n d r o s t e n e d i o n e ) c o n c e n t r a t i o n o n a r o m a t a s e a c t i v i t y in t h e p r e s e n c e o f 0 ( O ) , 15 ( A ) a n d 30 ([~) p M c o n m e s t r o l . T h e K m is t h e n e g a t i v e , i n v e r t e d x i n t e r c e p t a n d t h e Vma~ is t h e i n v e r s i o n o f t h e y i n t e r c e p t . T h e Vm,x w a s 3.2 p M , a n d t h e Km w a s 25, 199 a n d 422 n M , f o r 0, 15 a n d 30 p M c o u m e s t r o l , r e s p e c t i v e l y . I n s e t . T h e effect o f c o u m e s t r o l c o n c e n t r a t i o n o n t h e s l o p e of the double-reciprocal plot lines. The K I value (negative x intercept), reflecting inhibitor effectiveness, was 1.3 # M .
to competitively inhibit aromatase enzyme in h u m a n predipocytes. Although D M D M and D D M M are theoretical intermediates between matairesinol and Enl and D D S I , D M S ! and O D S I are theoretical intermediates between secoisolariciresinol and End, similar intermediates have been identified in primate urine, suggesting that they are true physiological intermediates [37]. Enl and its theoretical precursors were moderate inhibitors, while End and its theoretical precursors were weak inhibitors. T h e Ki values of the moderate inhibitors were between 10 and 30 times the K i of AG, suggesting that the affinities of these lignans to aromatase were 10-30-fold weaker than that of AG. At the same time, the K i values were between 150 and 500 times the Km of androstenedione, indicating that these c o m p o u n d s bind preadipocyte aromatase less than 1% as tightly as androstenedione binds aromatase. Using h u m a n placental microsomes, Adlercreutz et al. [20] first reported that Enl and its theoretical precursors were competitive inhibitors of aromatase. T h e I50 values of Enl and D D M M (4,4'-dihydroxyenterolactone) were 14 and 6/~M, respectively, 5-10 times lower than our results. T h e K i value of Enl in placental microsomes was 6 p M , less than half of our finding of 14.4 # M in preadipocytes. T h e affinity of Enl for aromatase in their study was 1.3% of that of androstenedione, 3-10-fold greater than in our study. T h u s , the inhibitor affinity and potency we observed in preadipocytes was 2-10-fold lower than that observed by Adlercreutz et al. in placental microsomes. T h e s e differences between whole cell and placental microsome systems are similar to those we reported pre-
viously in studies of flavonoid aromatase inhibitors [23]. As we suggested earlier, the lower potency of lignans in preadipocytes is possibly due to the incomplete passage of lignans through the cell m e m b r a n e barrier in whole cells. T h e intracellular concentration of inhibitor would likely be lower than that in the culture medium. T h e preadipocyte microsomes would therefore be exposed to lower concentrations of lignan than the placental microsomes, leading to lower inhibitory affinity and efficiency. T h e I50 value of D D M M (6 # M ) was lower than that of Enl (14 p M ) in the experiments of Adlercreutz et al., suggesting greater potency with D D M M . Our studies also showed this relationship in preadipocytes, with Is0 values of 60 and 7 4 # M , and Ki values of 7.3 and 14.4 # M for D D M M and Enl, respectively. Adlercreutz et al. also tested the effect of Enl on aromatase activity in the h u m a n choriocarcinoma cell line J E G - 3 [20]. After 1-2 h incubation with Enl, estrone production was 73-78, 61-63 and 2 4 - 2 7 % of the control level when Enl concentrations were 1, 10 and 100/~M, respectively. T h e inhibition in J E G cells was greater than in preadipocytes (aromatase activities of 97%, 89% and 37% of control level at 1, 10 and 100 (/~M Enl), most likely reflecting differences in cell type. Our study confirmed the observations of Adlercreutz et al. that End is a somewhat weaker aromatase inhibitor than Enl in placental microsomes [20]. Structural differences may contribute to this observed difference in inhibitor potency between Enl and End. While both are diphenolic compounds, Enl and its theoretical precursors contain a keto-tetrahydrofuran ring, and
210
Chuanfeng Wang et al.
End and its theoretical precursors are without such a ring. T h e presence of this ring structure may either directly increase the affinity of Enl to aromatase enzyme, or it may increase lipid solubility, allowing Enl and its theoretical precursors to enter the cell more easily and gain access to the binding site of aromatase. An effect of the ring on lipid solubility may also explain our finding that the difference in potency between End and Enl is greater in whole cells than in microsomes, with End being a far weaker inhibitor in whole cells. O u r results with flavonoids are the first to d e m o n strate that the isoflavone, coumestrol, the flavone, luteolin, and the flavonol, kaempferol, are competitive inhibitors of aromatase enzyme activity. Ki values for the flavonoids were 2.6, 9.6 and 54 times the K i of AG, for coumestrol, luteolin and kaempferol, respectively, refecting inhibitor potencies that were about 40%, 10°,/o and 2% of the potency of AG. T h e i r affinities to aromatase enzyme were less than 3% that of androstenedione. Coumestrol is a phytoestrogen known to bind the estrogen receptor [38] and exert estrogenic effects. T h e s e include induction of vaginal opening, cornification and cycling; stimulations of uterine metaplasia and promotion of hemorrhagic follicles [39, 40]; stimulation of cytosolic progestin receptors [40]; and proliferation of M C F - 7 [41] and other m a m m a r y t u m o r cells [38]. Our findings suggest that the estrogenic effects of coumestrol may be opposed in some cases by its inhibition of estrogen synthesis. T h e isoflavone, O - D m a , did not inhibit aromatase enzyme in preadipocytes. T h i s was not surprising, as the structure of O - D m a is similar to that of other isoflavones, such as daidzein and genistein, that we previously reported to be noninhibitors [23]. On the other hand, Adlercreutz et al. [20] found O - D m a to be a weak aromatase inhibitor in h u m a n placental microsomes, with an I50 of 160 p M . U n d e r our conditions in whole cells, we have generally found I50 and Ki values to be 10-fold greater than those reported in placental microsomes. With an I50 of 160 # M in placental microsomes [20], we would be unable to detect inhibition by O - D m a in our system. T h e flavonols, morin and fisetin, were found to be noninhibitors of aromatase enzyme in our system. Our results with morin differ from those of Moochhala et al., who found morin to bind aromatase enzyme in placental microsomes [42]. These investigators found morin to bind aromatase to a similar degree as does quercetin, a flavonol that we previously reported to be a noninhibitor in preadipocytes [23]. Although morin binds aromatase enzyme weakly in placental microsomes, as do quercetin and O - D m a , limited passage through the cell m e m b r a n e causes these weak inhibitors to fall below the limits of sensitivity of our whole cell system. Several reports have explored the relationship between flavonoid structure and ability to inhibit aroma-
tase enzyme activity. I b r a h i m and Abul-Hajj suggested the importance of C-7 hydroxylation for binding to aromatase [21], while Moochhala et al. suggested the importance of 5,7-dihydroxylation [42]. In our study, all of the compounds have a C-7 hydroxyl, while two of the three inhibitors and one of the three noninhibitors have a C-5 hydroxyl. Quercetin, a flavonol previously reported to be a non-inhibitor [23], also has a C-5 hydroxyl. T h u s , for this group of compounds, hydroxylation at the 5 or 7 position does not appear to be a major determinant of inhibitory effect. Our findings do, however, support previous reports of decreased inhibition by C-3 hydroxylated flavonoids [21,23,42]. In our study, all of the noninhibitor flavonols, as well as the weak inhibitor, kaempferol, have a C-3 hydroxyl, while the stronger inhibitor, luteolin, does not. T h e increased inhibitory capacity of luteolin, relative to quercetin, strongly supports this hypothesis. In addition, the increased inhibitory capacity of kaempferol relative to quercetin suggests that the presence of a C - Y hydroxyl reduces inhibitory capacity and the increased inhibitory capacity ot kaempferol relative to morin suggests that the presence of a C-2' hydroxyl reduces inhibitory capacity. T h e r e are no reports of the effects of lignans or flavonoids on aromatase enzyme activity in living organisms. Considering the m u c h lower affinities of these compounds to aromatase than that of the substrate, it is reasonable to assume that very high levels of inhibitor would be required to successfully compete with androstenedione and testosterone. Few data have been reported, however, on the normal circulating levels of these compounds in humans. Adlercreutz et al. reported mean plasma levels of End and Enl of 2.5 and 33 nM, respectively, in omnivorous w o m e n and 17 and 253 n M in vegetarian w o m e n [43]. At 253 nM, the circulating level of Enl in vegetarians is about 50-fold lower than the Ki of Enl, suggesting that the physiological significance of inhibition by Enl alone is unlikely. Although the concentration of any one c o m p o u n d may be low relative to the individual Ki value, the total effect of m a n y aromatase inhibitors circulating in v i v o over a lifetime, may be of physiological significance. Additional dietary aromatase inhibitors have been reported, including other lignans [20] and flavonoids such as chrysin and biochanin A [23], hydroxyflavones [21] and apigenin and flavanone [22]. T h e estrogenreducing effects of these compounds may be enhanced by their other reported antiestrogenic properties, such as suppression of estrogen action and bioavailability. T h e s e effects would be more important in vegetarians, as a result of their higher consumption of plant foods containing lignans and phytoestrogens. T h e s e results provide further evidence that naturally-occurring dietary compounds inhibit aromatase enzyme activity in intact preadipose cells. Our findings suggest that lignans and flavonoids may contribute to
Phytoestrogen Inhibition of Fat Cell Aromatase
the cancer-preventive properties of plant-based diets by inhibiting estrogen synthesis in adipose tissue. Although it is unlikely that any one compound alone inhibits aromatase inhibitor significantly in vivo, the physiological significance of long-term consumption of many such compounds has yet to be explored. Acknowledgements--This work was supported by Minnesota Agricultural Experiment Station project 18-34 and an International Life Science Institute-Nutrition Foundation Future Leader Award (MSK). Synthesis of the lignan intermediates and O-Dma was supported by NIH grant CA56289-01 to H.A. The authors are grateful to Dr Bruce Cunningham (Department of Surgery, University of Minnesota) and the staff of the St Paul Surgical Center, St Paul Ramsey Medical Center (St Paul, MN) for assistance in obtaining adipose tissue and to Dr K. W~ih~il~i (Department of Chemistry, University of Helsinki, Finland) for the synthesis of O-Dma.
14.
15. 16. 17. 18. 19. 20.
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