In Vitro Effects of Soybean Protease Inhibitors - ACS Publications

22 In Vitro Effects of Soybean Protease Inhibitors

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JONATHAN YAVELOW1, KENNETH A. BECK1, MORTIMER LEVITZ2, and WALTER TROLL3 Department of Biology/Biochemistry, Rider College, Lawrenceville, NJ 08648 Department of Obstetrics and Gynecology, New York University Medical Center, New York, NY 10022 3 Institute of Environmental Medicine, New York University Medical Center, New York, NY 10016 1

2

We have recently reported that the action of protease inhibitors as dietary anticarcinogens may work via two mechanisms: 1) An indirect effect on protein absorption; and 2) A direct effect on cell transformation (Cancer Res., Suppl. 43: 2454S-2459S, 1983). The direct effect on cell transformation has been demonstrated in vitro using several protease inhibitors. The following studies have been designed to assess the effect of protease inhibitors on transformed cell growth and to determine which protease inhibitory activity (antitrypsin or antichymotrypsin) is responsible for these effects. A number of protease inhibitors have been tested, among them the Kunitz soybean trypsin inhibitor and the Bowman-Birk soybean trypsin and chymotrypsin inhibitor. The cell lines which have been examined are LoVo (human colon cancer) and MCF-7 (human breast cancer). In cells tested, the Bowman-Birk inhibitor is more potent than the Kunitz inhibitor in decreasing cell growth. Thus, i t appears that the antichymotrypsin activity is modulating growth of these cells. The role of diet in cancer causation and prevention was originally derived from epidemiological data. More recently these studies have been followed up with laboratory experiments where variables can be more carefully controlled. For example, the correlation between meat consumption and elevated colon and breast cancer incidence Q) has been confirmed in animal studies showing high fat and/or protein dietary levels result in an increased number of colon tumors (2). Also mutagens have been isolated from cooked beef (3_). With regard to cancer prevention recent work has defined several classes of dietary anti-carcinogens; for example, retinoids (4^f antioxidants (5^), prostaglandin antagonists (6); and protease in0097-6156/85/0277-0283$06.00/0 © 1985 American Chemical Society

Finley and Schwass; Xenobiotic Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

hibitors (7). I n d e e d , i t may be t h a t o u r r i s k f o r d e v e l o p i n g cancer r e l a t e s to the b a l a n c e o f i n g e s t e d c a r c i n o g e n s / p r o m o t e r s and a n t i c a r c i n o g e n s ( € 0 . As w e l l a s b e i n g o f p o t e n t i a l p u b l i c h e a l t h i n t e r e s t , a n t i c a r c i n o g e n s c a n s e r v e as u s e f u l p r o b e s i n t o t h e m e c h a n i s m o f c a r c i n o g e n e s i s . For example, a n t i o x i d a n t s , p r o s t a g l a n d i n a n t a g o n i s t s and p r o t e a s e i n h i b i t o r s ( c l a s s e s o f c h e m o p r e v e n t i v e agents) s t r o n g l y suggest f r e e oxygen r a d i c a l s , p r o s t a g l a n d i n s and p r o t e a s e s , r e s p e c t i v e l y , as i m p o r t a n t c h e m i c a l m e d i a t o r s o f t r a n s f o r m a t i o n . With r e s p e c t to p r o t e a s e s , tumor c e l l s e l a b o r a t e t h e means f o r i n v a s i o n t h r o u g h t h e c o n n e c t i v e t i s s u e and i n t o t h e v a s c u l a t u r e . I n v i t r o s t u d i e s h a v e a l s o r e v e a l e d t h a t tumor c e l l s s e c r e t e p r o t e a s e s ( e . g . l y s o s o m a l h y d r o l a s e s , c o l l a g e n a s e , plasminogen activator). These e n z y m e s may m e d i a t e i n v a s i o n and m e t a s t a s i s . ( S e e 9_ f o r r e view). In a d d i t i o n t o s e c r e t e d p r o t e a s e s , i n t r a c e l l u l a r a n d / o r m e m b r a n e - a s s o c i a t e d p r o t e a s e s may be i m p o r t a n t i n t h e e s t a b l i s h m e n t and m a i n t e n a n c e o f t h e t r a n s f o r m e d c e l l . This c o u l d o c c u r v i a a c t i v a t i o n o f p r e c u r s o r enzymes o r h o r m o n e s w h i c h may n o t be p r e s e n t o r p r e s e n t a t d i f f e r e n t l e v e l s i n normal c e l l s . P r o t e a s e i n h i b i t o r s may t h e r e f o r e be e f f e c t i v e a n t i c a r c i n o g e n s or a n t i t u m o r d r u g s v i a e f f e c t i n g p r o t e a s e s a t different sites. The Bowman-Birk t y p e p r o t e a s e i n h i b i t o r s r e p r e s e n t a c l a s s o f low m o l e c u l a r w e i g h t , c y s t e i n e - r i c h p r o t e i n s found i n legume seeds ( 1 0 ) . The m a j o r Bowman-Birk i n h i b i t o r i n s o y b e a n seeds i s a double-headed p r o t e i n c a p a b l e o f b l o c k i n g the a c t i v i t y o f b o t h t r y p s i n and c h y m o t r y p s i n . This protein re p r e s e n t s a p p r o x i m a t e l y 4% o f t h e t o t a l p r o t e i n i n s o y b e a n seeds (JJ_). In c o n t r a s t t o t h e s o y b e a n t r y p s i n inhibitor ( K u n i t z ) , t h e " d o u b l e - h e a d e d " i n h i b i t o r ( r e f e r r e d t o as BB) i s t y p i c a l o f p r o t e a s e i n h i b i t o r s p r e s e n t i n a l a r g e number o f legume s e e d s : f o r example, peanuts (12); c h i c k peas ( 1 3 ) ; k i d n e y b e a n s (_14_); a d z u k i b e a n s (15_); l i m a b e a n s ( 1 6 ) . The BB p r o t e a s e i n h i b i t o r s a r e h e a t and a c i d s t a b l e , t h u s t h e y s u r v i v e b o t h c o o k i n g ( ] J ) and d i g e s t i o n (,18). BB com p l e x e s w i t h p r o t e a s e s i n t h e s m a l l i n t e s t i n e o f r o d e n t s and i s e x c r e t e d p r i m a r i l y as p r o t e a s e - p r o t e a s e i n h i b i t o r complexes i n the f e c e s . The d e c r e a s e d e f f i c i e n c y o f p r o t e i n u t i l i z a t i o n f r o m f o o d s r i c h i n p r o t e a s e i n h i b i t o r s may i n d i r e c t l y l o w e r an i n d i v i d u a l ' s e f f e c t i v e p r o t e i n intake (12). T h i s may, in p a r t , p r o t e c t a n i m a l s on p r o t e a s e i n h i b i t o r - r i c h d i e t s a s shown i n a l o w e r i n c i d e n c e o f s k i n , b r e a s t and l i v e r t u m o r s (20,21 ) . The Bowman-Birk i n h i b i t o r a l s o b l o c k s t h e t r a n s f o r m a t i o n o f C H/10T1/2 c e l l s ( 1 8 ) . T h i s r a i s e s t h e s p e c u l a t i o n t h a t BB may r e p r e s e n t a d i r e c t a c t i n g n u t r i t i o n a l l y r e l e v a n t a n t i c a r cinogen p a r t i c u l a r l y i n the case o f colon cancer. In t h i s r e g a r d i t was r e c e n t l y r e p o r t e d t h a t ε - a m i n o c a p r o i c a c i d ( a trypsin inhibitor) i n h i b i t s dimethylhydrazine-induced colon tumors i n mice ( 2 2 ) . Does t h e a c t i v e f o r m o f t h e Bowman-Birk i n h i b i t o r r e a c h the colon? I f s o , what i s t h e e f f e c t on c o l o n t u m o r c e l l s ? I s t h e e f f e c t o f BB s p e c i f i c o r m i g h t i t e f f e c t many c e l l types? S t u d i e s d e s c r i b e d b e l o w l o c a l i z e a c t i v e BB i n t h e


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c o l o n w a l l and a s s e s s t h e i n h i b i t o r y e f f e c t o f BB on human c o l o n and b r e a s t c a n c e r c e l l g r o w t h . Studies concerning i t s mechanism o f a c t i o n suggest t h e a n t i c h y m o t r y p s i n activity a s s o c i a t e d w i t h BB i s a f f e c t i n g g r o w t h i n h i b i t i o n . M e t h o d s and M a t e r i a l s I s o l a t i o n , i o d i n a t i o n and m e t a b o l i c s t u d i e s w i t h 1 2 5 I - B B : The Bowman-Birk i n h i b i t o r was i s o l a t e d a s p r e v i o u s l y d e s c r i b e d (jj.). F o r m e t a b o l i c s t u d i e s BB was i o d i n a t e d and p u r i f i e d u s i n g c h y m o t r y p s i n - s e p h a r o s e Q 8 ) . C-H/HEN m i c e were a d m i n istered I-BB b y g a v a g e and s a c r i f i c e d a f t e r one h o u r . C o l o n was e x c i s e d , i r r i g a t e d f r e e o f f e c e s , h o m o g e n i z e d and c h r o m a t o g r a p h e d on c h y m o t r y p s i n s e p h a r o s e ( 1 8 ) . Cell Culture: Human c o l o n c a n c e r c e l l s ( L o V o ) were o b t a i n e d from D r . M. L i p k i n ( C o r n e l l U n i v e r s i t y M e d i c a l C e n t e r ) and human b r e a s t c a n c e r c e l l s (MCF-7) were o b t a i n e d f r o m D r . H. Leon B r a d l o w ( R o c k e f e l l e r U n i v e r s i t y ) . C e l l s were grown i n RPMI 1640 s u p p l e m e n t e d w i t h 10% F e t a l C a l f Serum ( G i b c o ) and pen/strep/ fungizone. E x p e r i m e n t s were p e r f o r m e d i n m i c r o t i t e r w e l l s o r 35mm t i s s u e c u l t u r e d i s h e s . C e l l s were enumerated u s i n g a hemocytometer. P r o t e a s e i n h i b i t o r s and a s s a y s : BB m o d i f i e d e n z y m a t i c a l l y by s e q u e n t i a l t r y p s i n and c a r b o x y p e p t i d a s e Β d i g e s t i o n s was a k i n d g i f t from D r . Y. B i r k (23_). T h i s i n h i b i t o r w h i c h c o n t a i n s only chymotrypsin i n h i b i t o r y a c t i v i t y i s r e f e r r e d t o as BBchy. Soybean t r y p s i n i n h i b i t o r ( K u n i t z ) (Worthington, F r e e h o l d , N J ) , BB and BBchy were a s s a y e d f o r t r y p s i n and chymotrypsin i n h i b i t o r y a c t i v i t i e s using benzoyl a r g i n i n e para n i t r o a n i l i d e (ΒΑΡΝΑ) and b e n z o y l t y r o s i n e p a r a n i t r o a n i l i d e (BTPNA) r e s p e c t i v e l y ( S i g m a ) ( 2 4 ) . Protease a c t i v i t y secreted i n t o s e r u m - f r e e medium was a s s a y e d u s i n g Η casein (17). Results The Bowman-Birk p r o t e a s e i n h i b i t o r was p u r i f i e d from d e f a t t e d soybean f l o u r . A c t i v i t y and p u r i t y were a s s e s s e d b y i n h i b i t i o n o f t r y p s i n and c h y m o t r y p s i n , SDS and n a t i v e p o l y a c r y lamide g e l e l e c t r o p h o r e s i s , high performance l i q u i d chro matography and amino a c i d a n a l y s i s ( d a t a n o t s h o w n ) . The I-BB d e r i v a t i v e was a d m i n i s t e r e d t o m i c e b y g a v a g e and a f t e r 1 hour t h e l o w e r i n t e s t i n e was d i s s e c t e d and i r r i g a t e d free o f feces. A p p r o x i m a t e l y 1% o f t h e i n g e s t e d BB i s a s s o ciated with colon w a l l . Figure 1 i l l u s t r a t e s t h e chymotryp s i n - s e p h a r o s e e l u t i o n p r o f i l e o f a c l a r i f i e d homogenate o f intestinal wall. The I-BB peak t h a t i s n o t s t i c k i n g t o t h e a f f i n i t y c o l u m n most l i k e l y r e p r e s e n t s a p r o t e a s e - p r o t e a s e i n h i b i t o r c o m p l e x b e c a u s e t h i s élûtes a s a 33 OOO d a l t o n peak on HPLC ( 1 8 ) . C o u n t s e l u t i n g a t pH 2.6 r e p r e s e n t f r e e BB c a p able o f binding protease. The m o l e c u l a r w e i g h t o f t h e peak t u b e e l u t i n g w i t h a c i d was s i m i l a r t o t h e m a t e r i a l a d m i n i s t e r e d t o t h e animals by gavage. (Data n o t shown). Human c o l o n c a n c e r c e l l g r o w t h ( L o V o ) i s e f f e c t e d by p r o t e a s e i n h i b i t o r s . I n t h e p r e s e n c e o f 62.5 μΜ BB b o t h t h e d o u b l i n g t i m e and s a t u r a t i o n d e n s i t y a r e c o n s i d e r a b l y t


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FRACTION NUMBER

Figure

1 - Chymotrypsin-sepharose e l u t i o n p r o f i l e of c o l o n w a l l h o m o g e n a t e 1 hour a f t e r i . g . administration of I-BB. Arrow (Ψ) d e n o t e s c h a n g i n g e l u t i o n b u f f e r t o pH 2.6. 5


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d e p r e s s e d ( F i g u r e 2 ) . An i n d e p e n d e n t s e r i e s o f e x p e r i m e n t s p e r f o r m e d i n t h e p r e s e n c e o f 25,50 and 62.5 μΜ BB d e m o n s t r a t e d 25%, 3 5 % and 4 0 % i n h i b i t i o n , r e s p e c t i v e l y i n c e l l number a f t e r 4 days o f growth. ( 6 2 . 5 μΜ b o v i n e serum a l b u m i n i n h i b i t e d d a y 4 c e l l number b y 5 % ) . A d d i t i o n a l s t u d i e s a r e needed t o p r e c i s e l y d e f i n e d e c r e a s e s i n d o u b l i n g t i m e a s a f u n c t i o n o f BB concentration. H i g h e r c o n c e n t r a t i o n s o f BB f u r t h e r d e p r e s s g r o w t h , h o w e v e r , t h e p r o t e a s e i n h i b i t o r y e f f e c t s c a n n o t be d i f f e r e n t i a t e d from a g e n e r a l p r o t e i n i n h i b i t o r y e f f e c t . I n o r d e r t o a s s e s s t h e r e l a t i v e i n h i b i t i o n o f BB and t h e K u n i t z s o y b e a n t r y p s i n i n h i b i t o r ( S B T I ) p l a t i n g e f f i c i e n c y and c e l l g r o w t h e x p e r i m e n t s were p e r f o r m e d . BB b l o c k s g r o w t h and p l a t i n g e f f i c i e n c y 50 and 5 3 % r e s p e c t i v e l y a t 6 2 . 5 PM w h e r e a s 47.5 UM SBTI b l o c k s g r o w t h and p l a t i n g e f f i c i e n c y 14 and 2 0 % , r e s p e c t i v e l y . ( T a b l e I ) A t 190 μΜ t h e SBTI b l o c k e d c e l l g r o w t h 30% a f t e r 3 d a y s . ( D a t a n o t shown) f

Table I .

Comparison

o f BB and SBTI o n LoVo G r o w t h and Plating Efficiency

P.E. (%) Control BB 62. 50UM SBTI 47.50μΜ

44.25 21.0 35.5

(%I)

+ Γ75 + 4 . 5 (53%) + 4.5 (20%)

Growth

(Day 3)

4.43 x 10^ + .475 2 . 2 8 χ ίο!* + .98 3.80 χ 10^ + .10

(%I)

(50%) (14%)

P l a t i n g e f f i c i e n c y e x p e r i m e n t s were p e r f o r m e d by p l a t i n g 200 v i a b l e c e l l s i n t o 60 mm t i s s u e c u l t u r e d i s h e s and c o u n t i n g c o l o n i e s a f t e r 7-10 d a y s . Growth e x p e r i m e n t s were p e r f o r m e d u s i n g m i c r o t i t e r w e l l s w i t h 1 χ 10 c e l l s / w e l l o n day 0 (N=3).

In an e f f o r t t o a s s e s s t h e s i t e o f a c t i o n o f BB on LoVo c e l l g r o w t h , p r o t e a s e s s e c r e t e d from t h e s e c e l l s i n t o s e r u m - f r e e medium were a n a l y z e d i n t h e p r e s e n c e and a b s e n c e o f BB and t h e a l protease i n h i b i t o r . BB i n h i b i t e d s e c r e t e d p r o t e a s e a c t i v i t y 80% and t h e a l p r o t e a s e i n h i b i t o r b l o c k e d s e c r e t e d p r o t e a s e a c t i v i t y 99% ( T a b l e I I ) . Table

II.

E f f e c t o f BB and a l P I on P r o t e a s e s S e c r e t e d f r o m LoVo C e l l s

CPM Control BB αϊ PI

5685 + 545 1158 + 189 70 + 7

%I

80% 99%

H 10 c e l l s were p l a t e d i n m i c r o t i t e r w e l l s ( o . 2 m l ) . Serum was r e m o v e d , c e l l s washed w i t h PBS and i n c u b a t e d i n s e r u m - f r e e medium. 100 μΐ o f c o n d i t i o n e d medium was i n c u b a t e d w i t h Hc a s e i n + 10 μΐ o f 1 μg/ml s o l u t i o n s o f p r o t e a s e i n h i b i t o r .


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2 - L o V o c e l l s were grown i n m i c r o t i t e r w e l l s i n t h e p r e s e n c e and a b s e n c e o f 62.5 BB. T r i p l i c a t e c u l t u r e s were c o u n t e d on d a y s indicated. 10 c e l l s were p l a t e d o n day 0. D a t a i s e x p r e s s e d as mean + S.D. ( N = 3 ) .


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The e f f e c t s o f BB on c e l l s i n v i t r o a r e n o t c o n f i n e d t o L o V o . S e v e r a l o t h e r t r a n s f o r m e d c e l l l i n e s a r e a l s o i n h i b i t e d by BB ( h a m s t e r a m e l a n o t i c melanoma, AM, and i t s r e v e r t a n t , F F , m e t h y l c h o l a n t h a e n e t r a n s f o r m e d C H/10T1/2 c e l l s , MCA6, ( d a t a not shown). I n a l l c a s e s BB i s a more p o t e n t i n h i b i t o r t h a n equimolar c o n c e n t r a t i o n s o f the K u n i t z SBTI. The e f f e c t s o f BB and K u n i t z SBTI on MCF-7 c e l l g r o w t h a r e s u m m a r i z e d i n T a b l e I I I . The c e l l number a f t e r 10 d a y s o f g r o w t h i n t h e p r e s e n c e o f e s t r a d i o l was d e c r e a s e d from 45 t o 79% by 200 y g / m l BB, w h e r e a s K u n i t z S B T I had no e f f e c t . In t h e a b s e n c e o f e s t r a d i o l t h e e f f e c t o f BB was more v a r i a b l e r e s u l t i n g i n no e f f e c t i n 3 e x p e r i m e n t s and a p p r o x i m a t e l y 5 0 % i n h i b i t i o n i n the other 2 experiments. Parameters e f f e c t i n g t h i s v a r i a b i l i t y are c u r r e n t l y being investigated. The Bowman-Birk i n h i b i t o r a l s o e f f e c t s MCF-7 c e l l g r o w t h i n m e d i a c o n t a i n i n g u n s t r i p p e d serum. In a s i n g l e experiment ( w i t h t r i p l i c a t e p l a t e s ) we o b s e r v e d a d e c r e a s e i n MCF-7 c e l l number a f t e r 6 d a y s o f g r o w t h ( a p p r o x i m a t e l y 70% o f c o n t r o l ) . BB d e r i v a t i z e d s u c h t h a t i t was a c h y m o t r y p s i n i n h i b i t o r o n l y and n a t i v e BB e q u a l l y e f f e c t e d g r o w t h m o d u l a t i o n . Equimolar c o n c e n t r a t i o n s o f t h e K u n i t z SBTI was i n e f f e c t i v e i n modulating c e l l growth. Discussion The Bowman-Birk s o y b e a n t r y p s i n and c h y m o t r y p s i n i n h i b i t o r m o d u l a t e s t h e g r o w t h o f human c o l o n and b r e a s t c a n c e r c e l l s . In p a r a l l e l e x p e r i m e n t s t h e K u n i t z s o y b e a n t r y p s i n i n h i b i t o r proved i n e f f e c t i v e i n modulating c e l l growth. These r e s u l t s s u g g e s t t h e c h y m o t r y p s i n i n h i b i t o r y a c t i v i t y i s more i m p o r t a n t i n e f f e c t i n g growth i n h i b i t i o n . One must b e c a u t i o u s i n i n t e r p r e t i n g t h i s d a t a b e c a u s e i t must be c o n f i r m e d w i t h t h e i s o l a t i o n o f t h e c e l l u l a r t a r g e t o f BB. U n f o r t u n a t e l y one i s u n a b l e t o a s s e s s t h e e f f e c t s o f BB o n g r o w t h o f " n o r m a l " c o l o n and b r e a s t e p i t h e l i a i_n v i t r o b e c a u s e t h e y do n o t grow i n culture. S c h n e b l i and B e r g e r ( 2 5 ) o b s e r v e d s e l e c t i v e g r o w t h i n h i b i t i o n o f o n l y SV40 t r a n s f o r m e d 3T3 c e l l s w i t h TLCK ( t o s y l l y s i n e c h l o r o m e t h y l k e t o n e ) . T h i s s e l e c t i v e i n h i b i t i o n has b e e n d i s c u s s e d by Q u i g l e y (9_) a s r e s u l t i n g f r o m c y t o t o x i c events independent o f a protease s p e c i f i c to transformed cells. T h e r e a r e t h r e e p o s s i b l e s i t e s where BB may a c t t o m o d u l a t e c e l l g r o w t h : 1) p r o t e a s e s s e c r e t e d f r o m c e l l s ; 2) p r o t e a s e s on t h e p l a s m a membrane; 3) i n t r a c e l l u l a r p r o t e a s e s . The p o s s i b i l i t y o f s e c r e t e d p r o t e a s e s a s m e d i a t i n g c e l l g r o w t h seems n o t t o be t h e c a s e b e c a u s e t h e e n d o g e n o u s serum a l p r o tease i n h i b i t o r i s c a p a b l e o f b l o c k i n g s e c r e t e d p r o t e a s e s from LoVo c e l l s more e f f i c i e n t l y t h a n BB. ( C e l l s a l s o grow n o r m a l l y i n serum c o n t a i n i n g a c t i v e p r o t e a s e i n h i b i t o r ) . P r e l i m i n a r y studies using I-BB s u g g e s t t h e p r o t e a s e i n h i b i t o r i s n o t b e i n g t a k e n up b y c e l l s ( Y a v e l o w - u n p u b l i s h e d d a t a ) . The p o s s i b i l i t y r e m a i n s t h a t BB i s i n t e r a c t i n g w i t h t h e p l a s m a membrane. Indeed i t has been r e p o r t e d t h a t ovomucoid ( a t s i m i l a r c o n c e n t r a t i o n s t o t h o s e used i n t h e a b o v e s t u d i e s ) b l o c k s g r o w t h o f p o l y o m a t r a n s f o r m e d 3T3 c e l l s and o v o m u c o i d



2.83 1.14 1.94 1.23 3.08

+ + + + +

.13 .04 .15 .06 .64

Control

1.00 0.90 1.38

0.90

2.17

+ + + + +

+BB

.32 .15 .02 .13 .20

(77%) (79%) (52%) (73%) (45%)

E s t r a d i o l (+) -6, 10 c e l l number x l O )

Ν.D. 1.07 + .10 (94%) 1.88 + .15 (97%) Ν. D. Ν.D.

1.73 .51 1.10 1.42 3.37

+ + + + +

.22 .12 .10 .27 .41

1.65 0.59 1.31 0.69 1.71

+ + + + +

+BB .25 (95%) .10 (115%) .25 (119%) .40 (49%) .05 (51%)

Ν. D. 0.61 + .11 1.31 + .25 Ν. D. Ν. D.

+Kunitz

Growth i n t h e Presence and

E s t r a d i o l (-) -6, 10 c e l l number x l O )

Control

(Day

SBTI on m o d u l a t i n g MCF-7 C e l l Absence o f E s t r a d i o l

•Kunitz

The e f f e c t o f BB and K u n i t z

(120%) (120%)

Legend: 5 χ 10 c e l l s were p l a t e d i n 35 mm t i s s u e c u l t u r e d i s h e s c o n t a i n i n g RPMI 1640 + 5%g E s t r o g e n - s t r i p p e d serum. For t h e e s t r a d i o l (+) c o n d i t i o n p l a t e s were supplemented w i t h 10 M estradiol. The f i n a l c o n c e n t r a t i o n o f p r o t e a s e i n h i b i t o r s was 200 yg/ml. Data i s e x p r e s s e d as mean + SD, N=3. Percent o f c o n t r o l c e l l number i s i n p a r e n t h e s i s .

5

1 2 3 4

Expt. #

(Day

Table I I I .

Ο r

DO

si

m

η

Ο es

χ m

22. YAVELOW ET AL.


291

immobilized on polyacrylamide beads affects growth inhibition at lower protease inhibitor concentrations (26). Membranes isolated from transformed chick embryo fibroblasts also possess chymotrypsin- like protease activity (27). These studies establish the possibility that the chymotrypsin inhibitory activity associated with BB may be interacting with a membrane associated protease. Clearly, more studies need to be done to directly assess the nature of the cellular target of BB. From the variety of cells affected by BB i t suggests that the target protease may be common to many cell types. The Bowman-Birk protease inhibitor is exceedingly stable, easy to immobilize to affinity supports and seems not to penentrate cells. For these reasons it is an ideal probe for the putative membrane-associated targets that may mediate cell growth. Until the cellular target is identified it is premature to assume the cellular target of BB to be a protease. Several interesting in vivo studies are suggested from this work, for example: 1) does BB bind to colon cancer cells preferentially over normal colon epithelia in vivo?; 2) does pure BB decrease colon cancer incidence in carcinogen treated animals? Completion of these studies will further assess the role of BB as a dietary anticarcinogen. Acknowledgments This work was supported in part by NIH Grants CA-02071 (M.L.) and CA-16060 (W.T.) The technical assistance of Monica Gidund is greatly appreciated. We thank Ms. Géraldine Holley and Mrs. Helen Hudzina for preparation of the manuscript. Literature Cited 1.

Armstrong, B. and Doll, R. Int. J. Cancer, 1975, 15, 617. 2. Reddy, B.U., Narisawa, T. and Weisberger, J.H. J. Nat'l Cancer Institute. 1976, 57, 567. 3. Sugimura, T. and Sato, S. Cancer Research (Suppl.) 1983, 45, 2415S. 4. Moon, R.G., McCormick, D.L. and Mehta, R.G. Cancer Research (Suppl.) 1983, 43, 2469S. 5. Wattenberg, L.W. Cancer Research (Suppl.) 1984, 43, 2448S. 6. Narisawa, T., Sato, Μ., Tani, Μ., Kudo, T., Takahashi, T. and Gota, A. Cancer Research, 1981, 41, 1954. 7. Troll, W. and Yavelow, J. In "Current Topics in Nutrition and Disease," Vol. 9. Roe, D.A., Ed., Alan R. Liss, New York, 1983, p. 167-176. 8. Ames, B.N. Science 1983, 221, 1256. 9. Quigley, J.P. In "Surfaces of Normal and Malignant Cells", John Wiley and Sons, New York, 1979, pp. 247-285. 10. Laskowski, M.J. and Kato, I. Ann. Review of Biochem. 1980, 49, 593. 11. Hwang, D.L.R., Davis Lin, K.T., Yang, W-K. and Foard, D.E. Biochem. Biophys. Acta. 1977, 495, 369. 12. Norioka, S. and Ikenaka, T. J. Biochem. 1983, 93, 479.


292

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

Smirnoff, P., Khalef, S., Birk, Y., Applebaum, S.W. I n t ' l . J. Peptide Protein Res. 1979, 14, 186. Pusztai, A. Eur. J. Biochem, 1968, 5, 252. Toshibawa, M., Kiyohava, T., Iasaki, T., Kawata, N., Ohtaki, Y. and Nakkao, C. J. Biochem. 1980, 87, 619. Stevens, F.C., Wuerz, S. and Krahn, J. In Bayer Symposium V "Proteinase Inhibitors", Springer-Verlag, Berlin 1974. pp. 344-354. Yavelow, J., Gidlund, M. and Troll, W. Carcinogensis, 1982, 3, 135. Yavelow, J., Finlay, T.H., Kennedy, A.R. and Troll, W. Cancer Research (Suppl.) 1983, 43, 2454S. Navarette, D.A. and Bressani, R. Am. J. Clinical Nutr. 1981, 34, 1893. Becker, F.F. Carcinogensis, 1981, 2, 1213. Troll, W., Weisner, R., Shellabarger, C.J., Holtzman, S. and Stone, J.P. Carcinogensis 1980, 1, 469. Corasanti, J.G., Hobika, G.H. and Markus, G. Science 1982, 216, 1020. Birk, Y. In Bayer Symposium V "Proteinase Inhibitors", Springer Verlag, Berlin, 1974, pp. 355-361. Abramovitz, A.S., Yavelow, J. Randolph, V. and Troll, W. J. Biol. Chem. 1983, 258, 15153. Schnebli, H.P. and Burger, M.M. Proc. Nat'l Acad. Sci. (USA) 1972, 69, 3825. Talmadge, K.W., Noonan, K.D, and Burger, M.M. In "Control of Proliferation in Animal Cells", Cold Spring Harbor, NY, 1974, pp. 313-325. O'Donnell-Tormey, J. and Quigley, J.P. Proc. Nat'l Acad. Sci. (USA) 1983, 50, 344.

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