Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase

CANCER LETTERS Cancer Letters 75 (1993) 175-182

Tobacco

Nobuyuki Endocrine

fliochemisrr~

alkaloid derivatives as inhibitors breast cancer aromataset Kadohama*, Department,

Keiji Shintan&

Medical

Foundation

Buffalo,

N. Y. 14203. USA

of Buj‘aio

(Received 21 May 1993; revision received 6 October

Yoshio

Osawa

Research Institute.

1993; accepted

of

8 October

73 High St.,

1993)

Abstract The inhibition of estrogen biosynthesis by the use of aromatase inhibitors is emerging as a valuable approach to breast cancer therapy. B,ecause smoking has a profound effect on estrogen-related processes we examined the ability of tobacco constituents to suppress estrogen production by breast cancer aromatase. N-n-octanoylnornicotine and N-(4-hydroxyundecanoyl) anabasine suppressed aromatase activity in culture of two human breast cancer cell lines, MDA-MB-231 (I(&, of 310 and 20 PM, respectively) and SK-BR-3 (IC,, of 450 and approximately 2 PM, respectively). MDA-MB-231 cells induced by 250 nM dexamethasone or 1 mM (Bt),cAMP were slightly more sensitive to both inhibitors. Kinetic analyses showed that inhibition by N-(4-hydroxyundecanoyl)anabasine is competitive with respect to androstenedione as substrate, with apparent K, values of 0.2 PM against microsomal aromatase activity derived from both (Bt)+AMP-induced MDA-MB-231 cells and human breast tumor tissue. The corresponding apparent Ki against human placental microsomal aromatase activity was 0.4 PM. These results indicate that acyl derivatives of nornicotine and anabasine block estrogen formation in breast tumor cells and tissue and could contribute to the decreased intra-tissue estrogen levels in women who smoke. Key

words:

Aromatase;

Breast cancer; Tobacco

alkaloid;

Inhibitor;

Estrogen

biosynthesis

1. Introduction

action of anti-estrogens [26,37]. The primary ther-

Endocrine treatment of breast cancer is based on the fact that approximately one-third of breast carcinomas respond to the growth-inhibiting

apeutic strategy is to block the mitogenic action of estrogen through the use of antiestrogens, such as tamoxifen, that interfere with the receptor-mediated growth mechanisms [21]. A form of endocrine therapy which has been gaining acceptance is the

* Corresponding author. tThis researchwas supported

by USPHS NIH Research Grant HDO4945. Grant 1643Bfrom the Council for Tobacco Research-USA. Inc., and a grant from the Charlotte Geyer Foundation. $p&doctoral Research Fellow on leave from Okayama University Medical School, Department of Obstetrics and Gynecology. Okayama,

Japan.

0304-3835/93/$06.00 0 1993 Elsevier Scientific SSDI 0304-3835(93)0321(1-V

Publishers

Ireland

Ltd. All rights reserved.

176

use of agents that specifically suppress estrogen biosynthesis by inhibiting the aromatization of androgens to estrogens [8,13,35-371. Such drugs presently in clinical use include aminoglutethimide and 4-hydroxyandrostenedione. More than 2200 compounds are known to be present in tobacco and tobacco smoke [38]. Although nicotine, nornicotine and anabasine comprise the most abundant of tobacco alkaloids, numerous acylated derivatives have been identified [5,10,25]. Our initial discovery of natural inhibitors in tobacco [40] led to the isolation, characterization, and synthesis of tobacco alkaloid derivatives that suppress aromatase enzyme activity in vitro as well as exhibit in vivo effects on endocrine function in the rat model [30]. Acylnornicotine and acylanabasine derivatives caused a delay in the estrus cycle, suppressed estrogendependent mammary tumor growth [30] and affected fetal brain aromatase activity during pregnancy (unpublished data). Our current study focuses on the inhibitory effects of tobacco alkaloids on tumor aromatase. Reported herein are the results of our investigation of aromatase activity expressed in two breast cancer cell lines and human breast tumor tissue. The effects of N-acyl derivatives of nornicotine and anabasine were characterized in terms of potency and inhibition kinetics. 2. Materials and methods 2.1. Chemicals and reagents Androstenedione and progesterone were purchased from Steraloids, Inc. (Wilton, NH) and [ 1p- 3H]androstenedione from DuPont (New England Nuclear, Boston, MA). Culture media, fetal calf serum and antibiotics were purchased from GIBCO (Grand Island, NY). Dexamethasone (DEX), dibutyryl cyclic adenosine monophosphate ((Bt)2cAMP), reduced /3-nicotinamide adenine dinucleotide phosphate (NADPH), dithiothreitol (DTT), nicotine and anabasine were obtained from Sigma (St. Louis, MO). N-n-Octanoylnornicotine was a gift from Central Research Institute of Japan Tobacco, Inc. AJ-(4-Hydroxyundecanoyl)anabasine was synthesized as previously described [28]. Briefly, anabasine was reacted with

N. Kadohama et al. / Cancer Lett. 75 ( 1993) 175-182

4-oxoundecanoic acid in the presence of dicyclohexylcarboxamide to give N-(4-oxoundecanoyl)anabasine. Reduction of this compound by NaBH, yielded N-(4-hydroxyundecanoyl)anabasine. 2.2. Tissue, cells and cell culture Breast tumor tissue was supplied by Dr. Dietmar Braun of Ciba-Geigy, Ltd., Basel, Switzerland. Established ccl! lines, SK-BR-3 and MDA-MB-23 1, derived from human breast adenocarcinoma, were obtained from American Type Culture Collection (Rockville, MD). The SK-BR3 line was cultured in Dulbecco’s Modified Eagle Medium and MDA-MB-23 1 in RPMI- 1640 medium. The media were supplemented with 10% fetal calf serum, 100 units penicillin G/ml, 100 pg streptomycin sulfate/ml and 0.25 fig amphotericin B/ml. For ‘in cell’ aromatase assays, 100 mm diameter culture dishes (Falcon, Cockeysville, MD) were inoculated with approximately 2 x lo5 cells and the medium (3 ml/dish) was changed at 48 h intervals until the cells reached confluence. As a preparatory step for the aromatase assay, the substrate ([ lfi-3H]androstenedione, 100 nM, 9 x IO’ disintegrationslmin 3Hlpg), progesterone (5 PM) and inhibitors, when used, were concentrated under nitrogen to a small volume of propylene glyco1 and dissolved in culture medium. In the case of induced MDA-MB-23 1 cells, dexamethasone (250 nM) or (Bt),cAMP (1 mM) was added to the standard medium for a 24-h induction period after the cells reached confluence followed by exchange with fresh medium containing [ l&‘H]androstenedione (100 nM) and progesterone (5 PM). For non-induced cells the medium was replaced with standard medium for 24 h followed by exposure to fresh medium containing [ l/3-3H]androstenedione (100 nM) and progesterone (5 PM). One hour after the addition of [ l&3H]androstenedione, an aliquot of the medium was quantitated for aromatase activity by the 3H-water method described in the aromatase assay section. 2.3. Preparation of microsomes Cell lines were propagated in multiple T-225 culture flasks (Corning, Corning, NY) and after centrifugation, the cells from successive passages were

177

N. Kadohama et al. /Canter Lett. 75 (1993) 175-182

stored at -80°C. Microsomes were prepared from 5-10 x lo8 ceils as previously described [44]. Briefly, frozen cultured cells were thawed and suspended in SDP buffer then disrupted by sonication. In the case of breast tumor tissue, two separate specimens having identical histopathology (invasive ductal carcinoma) were pooled and 10 g of the combined tissue was minced in SDP buffer and homogenized with a Polytron homogenizer. The cultured cell sonicates and tumor tissue homogenate were centrifuged at 12 000 x g for 45 min at 4°C and the supernatants recentrifuged at 105 000 x g for 50 min at 4°C. The microsomal pellets were resuspended by hand homogenization in 67 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.5 mM EDTA, and 0.5 mM DTT. Protein content was determined by the Pierce Micro BCA method (Pierce, Rockford, IL). 2.4. Aromatase assay The aromatase assay is based on ‘H released from the l/3 position of the substrate [l/3-3H]androstenedione. Microsomal preparations ranged from 12 fig protein for human placenta to 500 pg protein for breast tumor tissue. An ethanolic solution of the substrate (150 nM, 9 x IO7 disintegrations/min ‘Hlpg), 10 /mM progesterone, and O-120 PM inhibitor was evaporated under nitrogen in the presence aof propylene glycol (20 ~1). Subsequent addition:; were: 67 mM potassium phosphate buffer (pH 7.4), 0.1% BSA, and microsomal preparation. Following a 10 min preincubation at room temperature, the reaction was started with the addition of NADPH (1.2 mM) in a total incubation volume of 0.5 ml. After a 10 min (placental microsomes) or 30 min (others) incubation at 37°C in a shaking water bath, the reaction was terminated by the addition of 0.5 ml of 5% trichloroacetic acid. Following charcoal absorption and filtration through a cotton-plugged Pasteur pipet, a l-ml aliquot was taken and quantitated. Controls consisted of incubation without NADPH or with an equivalent amount of BSA in place of the microsomal protein. Aromatase activity in cultured cells was measured on aliquots of media after precipitation by 15% trichloroacetic acid. The supernatant was then processed in the :same manner as described in

the 3H-water assay. The cells were detached from the dish by trypsinization and cell count was determined using a hemocytometer. 3. Results Human breast cancer cells MDA-MB-231 and SK-BR-3 at confluence were exposed to [l/33H]androstenedione added to fresh culture media. Based on the ‘in cell’ 3H-water assay, the aromatase specific activity of SK-BR-3 cells was 350 fmol/h/106 cells. Although the aromatase in MDA-MB-231 cells was relatively low (approximately 15 fmol/h/106 cells), it could be induced by DEX and (Bt)2cAMP. Initial dose response profiles showed that the optimal induction concentrations were 250 nM for DEX (Fig. 1A) and 1 mM for (Bt),cAMP (Fig. 1B). The time course of aromatase induction shows maximum effect at 24-36 h after exposure (Fig. 2). In contrast, aromatase activity in SK-BR-3 cells was not induced by DEX or (Bt),cAMP. (data not shown).

0

I

.I

.Ol

Dexamethasone

(PM)

_YV

0

0

.Ol

.I

Dibutyryl

I

IO

1

CAMP (mM)

Fig. I. Dose-response relationship in the induction of aromatase activity in cultured MDA-MB-231 cells by (A) DEX and (B) (Bt),cAMP. Data represent the mean of 2 experiments.

N. Kadohuma et al. /Cancer

178

Fig, 2. Time course of induction of aromatase activity in MDAMB-231 cells. DEX (250 PM, A). (Bt),cAMP (I mM. 0). Data represent the mean of 2 experiments.

The IC,, values reported previously showed that the in vitro inhibitory effect against human placental aromatase activity was a function of increasing acyl carbon chain length with a maximum at eleven carbons [30]. In the present study. N-n-octanoylnornicotine (relative potency 72 times greater than nicotine) and N-(Chydroxyundecanoyl)anabasine (relative potency of 220) were evaluated against two human breast cancer cell lines. A comparison of the dose response patterns of uninduced MDA-MB-231 cells (Fig. 3) shows that N-(4-hydroxyundecanoyl)anabasine, ( ICso of 20 PM) was 15 times more effective than N-noctanoylnornicotine (IC50 of 310 PM). In SK-BR3 cells the difference in potency of these compounds was even greater, with the ICsO values for

IO

100 Inhibitor

IlXlC

Letr. 75 (19931 175-182

N-n-octanoylnornicotine and N-(4-hydroxyundecanoyl)anabasine of 450 PM and 2 PM, respectively (Fig. 4). At 50 PM concentration N-(4hydroxyundecanoyl)anabasine showed 93% inhibition of aromatase activity in MDA-MB-231 cells and 96% inhibition in SK-BR-3 cells. A summary of the inhibitory effects of the two synthetic tobacco alkaloids is presented in Table 1. As a point of reference. the IC,, values obtained against aromatase activity in human placental 12 000 x gpellet solubilized in assay buffer [30] are included. Inhibition of cultured breast cancer aromatase by N-noctanoylnornicotine was similar to the placental enzyme. Aromatase activity in MDA-MB-231 cells cultivated in the presence of DEX (250 nM) or (Bt& (1 mM) was inhibited to a greater degree than activity in noninduced cells, with ICso values of 64 and 48’%,, respectively, of that for non-induced MDA cultured cells. N-(4-Hydroxyundecanoyl)anabasine was approximately ten times more potent than N-n-octanoylnornicotine. Aromatase activity expressed by SK-BR-3 cells appeared to be the most sensitive to inhibition by N-(Chydroxyundecanoyl)anabasine (IC50 of approximately 2 PM) even though it was the least sensitive to N-n-octanoylnornicotine (IC,,, of 450 PM). We selected N-(4-hydroxyundecanoyl)anabasine for kinetic evaluation of aromatase inhibition in microsomal preparations of (Bt)?cAMP-induced MDA-MB-231 cultured cells as well as in microsomes prepared from a specimen of human breast carcinoma. Dose response curves were re-evaluated

10000

(FM)

Fig. 3. Inhibition of aromatase activity in cultured MDA-MB23 I cells by N-n-octanoylnornicotine (0) and N-(4-hydroxyundecanoyl)anabasine (0).

Inhibitor

(PM)

Fig. 4. Inhibition of aromatase activity in cultured SK-BR-3 cells by N-n-octanoylnornicotine (0) and N-(4-hydroxyundecanoyl)anabasine (0).

N. Kadohama et al. /Cancer

Lett. 75 (1993)

Table 1 Inhibition of aromatase activity by tobacco alkaloid deriv.atives

Human placenta” SK-BR-3 Non-induced MDAb DEX-induced MDAC CAMP-induced MDA‘)

in cultured

175-182

179

breast cancer cells

N-n-cctanoylnornicotine

N-(Chydroxyundecanoyl)anabasine

(l&i> irM)

(I&.

360 450

30 2

310

20

200

15

150

I1

300 z

PM)

200

0 -20

-40

0

20

JO

80

60

IN

“Solubilized fraction of 12 000 x g pellet of placental tissue homogenate. bMDA-MB-231 cells in standard medium. CMDA-MB-231 cultured in medium containing DEX (250 nM). dMDA-MB-231 cultured in medium containing (Bt& (I

Fig. 6. Inhibition kinetics of N-(4-hydroxyundecanoyl)anabasine against (Bt)*cAMP-induced MDA-MB-231 aromatase activity. Inhibitor concentrations were: 0 pM (0). 1.O PM (A), 2.0 gM &I), 3.5 (0). Y = pmol/min/mg. [S] = PM androstenedione The Ki was derived from the plot of K,,,N,,, vs. inhibitor concentration (inset).

mM).

under these in vitro incubation conditions (Fig. 5). Compared to human placental microsomal aromatase (ICsO of 5.0 l&M), the corresponding ICsO values for microsomes from (Bt)*cAMP-induced MDA cells and breast cancer tissue were 3.0 and 4.4 PM, respectively. In preliminary kinetic analysis, N-(Chydroxyundecanoyl)anabasine was a competitive inhibitor of human placental microsomal aromatase (K, of 9.4 nM) with an apparent Ki of 0.4 PM. The results of subsequent inhibition kinetic studies on

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20 0 : 0

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loo

6 u

microsomes prepared from (Bt),cAMP-MDA cultured cells and breast tumor tissue are presented in Figs. 6 and 7, respectively. Competitive inhibition was observed. The apparent Ki value was 0.2 PM for both (Bt&-MDA cells (K,,, = 27 nM) and tumor tissue (K,,, = 11.3 nM). A summary of the inhibition kinetic parameters is presented in Table 2. The results show that aromatase activity in both breast carcinoma tissue and in cultured cells exhibits a similar order of K,,, for aromatization as the placental enzyme. Based on apparent

IO N-14.llydroxyundecanoyl)anabasine

100

1000

(IIM)

Fig. 5. Inhibition of microsomal aromatase activity by N-(4hydroxyundecanoyl)anabasine. Microsomes were derived from human placental tissue(n), human breast carcinoma tissue (0). and (Bt)2cAMP-induced cultured MDA-MB-231 cells (0).

-80

-60

-40

-20

0

20

40

60

80

WI

Fig. 7. Inhibition kinetics of N-(4-hydroxyundecanoyl)anabasine against human breast carcinoma tissue aromatase activity. Inhibitor concentrations were: 0 PM (0). 1.0 PM (A). 2.0 PM (17). 3.5 pM (0). 7.0 1M (A). Y = fmol/min/mg, [S] = PM androstenedione. The K, was derived from the plot of KJV,,, vs. inhibitor concentration (inset).

N. Kadohama et al. /Cancer

180

Table 2 Effect of N-(4-hydroxyundecanoyl)anabasine activity in microsomal preparations

Human placenta” (Bt),cAMP-induced TumorC

MDAb

on aromatase

Kl?l

4

‘C,U

(nM)

(FM)

(PM)

9.4 27.0 11.3

0.4 0.2 0.2

5.0 3.0 4.4

“Microsomes prepared from normal full-term human placenta. bMicrosomes from MDA-MB-231 cells cultured in the presence of (Bt),cAMP (I mM). ‘Microsomes from human breast tumor tissue.

Ki values the inhibitory effect of N-(4hydroxyundecanoyl)anabasine (0.2 PM) on breast cancer aromatase was greater than that reported for aminoglutethimide (0.68 PM), an aromatase inhibitor currently in clinical use [15,14]. 4. Discussion Extraglandular conversion of androgens to estrogens occurs in fat, skin, muscle and brain tissue. Such sites can be important sources of circulating estrogen, particularly in postmenopausal women. In breast cancer, a large amount of circumstantial evidence points to the importance of tumor tissue aromatase in contributing to intratumor estrogen levels which are significantly higher than in normal tissue [6,41,43]. Studies also revealed the existence of a tissue-to-plasma estrogen concentration gradient that is more pronounced in postmenopausal women [41]. In vitro studies have clearly shown that breast tumors have the ability to synthesize estrogens [22,29,32,39,43]. A relationship between tumors possessing in vitro aromatase activity and the likelihood of patients responding to antiaromatase therapy has been shown [4,17,27,4,33]. The association of long-term smoking with early menopause, [3,20,18], increased risk of postmenopausal osteoporosis [16] and decreased levels of urinary estrogens during the luteal phase of the menstrual cycle [24] is evidence that smoking has a profound effect on estrogen-related physiological processes in women. These ‘anti-estrogenic’ effects of smoking, most pronounced in postmenopausal women, have led to studies which attempt to link smoking to a decreased risk of

Lert. 75 (1993)

175-182

breast cancer [ 191. However, an accumulation of epidemiological data appears to indicate that cigarette smoking has no protective effect on breast cancer [7,12,23,3 1,341. The overall association between smoking and breast cancer could possibly be masked by offsetting effects of the carcinogenic effect of cigarette constituents on one hand and a protective effect on the other [18]. Cigarette smoke constituents and tobacco extracts have been shown to inhibit aromatization in vitro [1,2,30,40]. A recent study of postmenopausal smokers showed increased adrenal function, characterized by elevated serum androgen (androstenedione) levels and a smaller decrease in estrogen levels [l l] leading the authors to hypothesize that hypoestrogism is secondary to increased adrenal activity. However, we note that the K,,, for androstenedione (usually in the range of lo-50 nM) far exceeds the reported serum concentration of androstenedione (2.2 nM) in smoking women. A predictable consequence would be increased estrogen via aromatization. The results obtained, indicating a small decrease in estrogen levels [ 111, are in fact consistent with an inhibitory effect of smoking on aromatase activity. Further studies are necessary to determine to what extent decreased estrogens and increased androgens each contribute to changes in endocrine function and disease manifestation. Kinetic analyses of acyl derivatives of nor‘nicotine demonstrated that two derivatives showed competitive inhibition of androstenedione aromatization by human placental microsomal aromatase [9]. The apparent Ki values for N-n-decanoylnornicotine and N-(4-hydroxyundecanoyl)nornicotine of 0.86 PM and 0.24 PM, respectively, were comparable to aminoglutethimide (0.68 PM). In vivo studies showed that the relative toxicity of synthetic acyl derivatives was lo-50-fold less than that for the parent alkaloids. Moreover. they altered the estrus cycle and also suppressed NMUinduced rat mammary carcinoma [30]. The results of the present study show that both SK-BR-3 aromatase and MDA-MB-231 aromatase are inhibited by tobacco alkaloids, but with considerably different sensitivities (Table 1). It remains to be determined whether the molecular basis for isozyme forms is in the primary sequence or in the three-dimensional conformation. Never-

N. Kadohama et al. / Cam-er Letr. 75 (1993)

175-182

theless, variations iri the structure and topology of the active site of aromatase may be useful in the design and synthesis of more effective inhibitors for the treatment of breast cancer. Since cytochrome P-450 enzymes are known to react with, and catalyze the metabolism of, a wide variety of structurally unrelated compounds including steroids, xenobiotics and drugs, it is possible that the tobacco alkaloids may bind to different classes of P-450s. Aromatization of androgens to estrogens can be blocked by specific inhibitors of aromatase activity. Aromatase can also be inhibited by non-specific inhibitors of steroidogenesis [42]. Even though the tobacco alkaloid derivatives we studied exhibit no molecular structural resemblance to the natural substrates, our results show that they compete for the enzyme active site, reflecting some degree of specificity. The ability of acyl derivatives of nicotine and anabasine to block estrogen formation in tumor cells and tissues indicates that they could contribute to the decreased estrogen levels in women who smoke. It is worthwhile to investigate the potential use of aromatase inhibitors naturally present in tobacco an,d other plant leaves as well as synthetic analogs [30] as agents that affect endocrine function in normal and diseased states. Response of breast carcinoma aromatase to novel agents such as described herein can be exploited in the development of new compounds that exhibit important chemotherapeutic properties. 5. Acknowledgments We thank Dr. Dietmar G. Braun of Ciba-Geigy Limited, Basel, Switzerland for the generous gift of breast tumor tissue. We also thank Dr. T. Chuman of Japan Tobacco, Inc. for the gift of acylnornicotine, Mrs. Carol Yarborough and Mrs. Wendy Franke for excellent technical assistance, and Mrs. Margaret Cegielski for help in preparation of this manuscript. 6. References I

2

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