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Effect of natural human tumor necrosis factor (nHuTNF) and natural human interferon (nHuIFN) on arachidonic acid metabolism
WS11-15
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C Y T O T O X I C E F F E C T S O F N A T U R A L H U ~ _ N T U M O R I~ECROSIS F A C T O R (niluTNF) - ~ , AND/OR NATURAL HUMAN INTERFERON (nHuIFN) - e , - 7 S.Shiiki,S.Fuchimoto,H.Iwagaki,M.Miyake,Y.Yasui,T.Maeda and K . O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a U n i v e r s i t y M e d i c a l S c h o o l , O k a y a m a , Japan T h i s s t u d y w a s c o n d u c t e d to a s s e s s the c y t o t o x i c e f f e c t s of n H u T N F - e , - 6 and n H u I F N - e, - 7 , e i t h e r a l o n e or in c o m b i n a t i o n , on ten h u m a n c a n c e r c ~ l lines. The c e l l l i n e s t e s t e d w e r e R P M I 4 7 8 8 , M K N - I , M K N - 2 8 , K - 5 6 2 , H-7, D a u d i , KB, P C - 1 0 , Z R - 1 0 , Z R - 7 5 - 1 and Z R - 7 5 - 3 0 . A f t e r 3 - d a y i n c u b a t i o n in 9 6 - w e l l m i c r o t i t e r p l a t e , the c y t o t o x i c e f f e c t s w e r e a s s e s s e d b y M T T assay. F o u r c e l l l i n e s ( R P M I 4 7 8 8 , P C - 1 0 , ~ { N - I and ~ N - 2 8 ) w e r e s e n s i t i v e (>30% c y t o t o x i c i t y , at t h e h i g h e s t c o n c e n t r a t i o n s t e s t e d ) to n H u T N F - e or -£ alone. n H u I F N - e s h o w e d c y t o t o x i c e f f e c t s on R P M I 4 7 8 8 , P C - 1 0 , ~ N - I and ~ N - 2 8 , and n H u I F N - 7 h a d s e n s i t i v i t y to R P M I 4 7 8 8 , P C ' I 0 a n d KB . A synergistic a n t i t u m o r e f f e c t o c c u r e d in f o u r c e l l l i n e s I R P M I 4 7 8 8 , P C - 1 0 , D a u d i and ZR75-1) t r e a t e d w i t h a c o m b i n a t i o n of n H u I F N - ~ a n d n H u T N F - ~ or - ~ . In s e v e n cell l i n e s ( R P M I 4 7 8 8 , P C - 1 0 , KB, M K N - I , M K N - 2 8 , D a u d i a n d ZR-75-1) , the c o m b i n a t i o n o f n H u I F N - 7 a n d n H u T N F - e o r - 6 s h o w e d the s y n e r g i s t i c e n h a n c e ment. A l t h o u g h f u r t h e r i n v e s t i g a t i o n s n e e d to b e c o n d u c t e d , o u r r e s u l t s may have therapeutic implications.
WSl1-16 THE E A R L Y E V E N T O F I N T E R F E R O N ( I F N - ~ , ¥ ) INDUCED CYTOPLASMIC FREE CALCIUM ION M O B I L I Z A T I O N A N D M E M B R A N E P O T E N T I A L R E S P O N S E I N C A N C E R C E L L S H. I w a g a k i , S . F u c h i m o t o , M . M i y a k e , S . S h i i k i , A . S a s a k i , Y . Y a s u i , A . G o u c h i , K . O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a University Medical School,Okayam? Japan Interferon(IFN-B,7) is r e p o r t e d to h a v e an a n t i t u m o r e f f e c t in c a n c e r cells. H o w e v e r , l i t t l e is k n o w n of the m e c h a n i s m b y w h i c h I F N t r a n s m i t s its e f f e c t f r o m the c e l l s u r f a c e i n t o the cell w h e r e i t s a c t i o n s are m a n i fested. The Ca 2+, w i d e l y r e c o g n i z e d as an i m p o r t a n t i n t r a c e l l u l a r m e s s e n ger, c o n t r i b u t e s t o c o n t r o l a v a r i e t y of c e l l u l a r p r o c e s s e s . W e u n d e r t o o k to s t u d y the e a r l y e v e n t s o f the c y t o p l a s m i c free C a 2 + m o b i l i z a t i o n and m e m b r a n e p o t e n t i a l r e s p o n s e t h a t o c c u r in c a n c e r c e l l s s e c o n d s a f t e r e x p o s u r e to i n d e n t i f y the c h a n g e s a s s o c i a t e d w i t h the t r a n s m e m b r a n e p r o c e s s i n g of the I F N s i g n a l , u s i n g Ca 2+ i n d i c a t o r ( F u r a 2-~) and membrane potential sensitive dye(dis-C3-(5)) . IFN-~,7 stimulated a transient about 1.6 - 1.8 fold i n c r e a s e in the c o n c e n t r a t i o n of Ca 2~ ([Ca 2+] i ) w i t h i n 15 to 30 s e c o n d s of c e l l e x p o s u r e to IFN. T h e [Ca2+]i r e t u r n e d to b a s a l levels by a b o u t 60 s e c o n d s . The m e m b r a n e w a s d e p o l a r i z e d s e c o n d s a f t e r exp o s u r e to IFN-7 , b u t w a s n o t a f t e r e x p o s u r e to IFN-S . T h e s e r e s u l t s s u g g e s t e d t h a t I F N - 7 i n d u c e d Ca 2+ s i g n a l w a s c a u s e d b y the m e m b r a n e potential-dependent C a 2+ c h a n n e l .
WS11-17 E F F E C T OF N A T U R A L H U M A ~ T U M O R N E C R O S I S F A C T O R (nHuTNF) A N D N A T U R A L H U ~ N I N T E R F E R O N (nHuIFN) ON A R A C H I D O N I C A C I D ~ T A B O L I S M Y. Y a s u i , S. F u c h i m o t o , F. H a m a d a , S. S h i i k i , H. I w a g a k i , M. M i y a k e , T. M a e d a a n d K. O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a U n i v e r s i t y M e d i c a l S c h o o l , O k a y a m a , Japan T h i s s t u d y w a s c o n d u c t e d to e v a l u a t e the e f f e c t of n H u T N F s and n H u I F N s on a r a c h i d o n i c a c i d (AA) m e t o b o l i s m of i m m u n o s u p p r e s s i v e state. We measured t h e p r o d u c t i o n o f p r o s t a g l a n d i n E2 (PGE2) of p e r i p h e r a l b l o o d m o n o c u c l e o c y t e (PBMC) of g a s t r o i n t e s t i n a l cancer patient, which were incubated with various cytokines, n H u T N F - ~ and n H u T N F - B p r o m o t e d the p r o d u c t i o n , b u t n H u I F N - e, n H u I F N - ~ , or c o m b i n a t i o n o f n H u T N F and ~ H u I F N (I:I) s u p p r e s s e d t h e p r o d u c t i o n of PGE2. T h e l e v e l of P G E 2 w a s a b o u t 25% of the c o n t r o l w h e n n H u I F N w a s a d d e d a l o n e , a n d t h a t w a s a b o u t 30% in c o m b i n a t i o n . It w a s r e p o r t e d t h a t P G E 2 i n h i b i t s the N K a c t i v i t y , so t h e s e r e s u l t s s u g g e s t t h a t n H u I F N s m a k e u p t h e n e g a t i v e e f f e c t of n H u T N F s in the i m m u n o s u p p r e s s i v e s t a t e b y m e a n s o f the i n h i b i t i o n of A A m e t a b o l i s m .
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C Y T O T O X I C E F F E C T S O F N A T U R A L H U ~ _ N T U M O R I~ECROSIS F A C T O R (niluTNF) - ~ , AND/OR NATURAL HUMAN INTERFERON (nHuIFN) - e , - 7 S.Shiiki,S.Fuchimoto,H.Iwagaki,M.Miyake,Y.Yasui,T.Maeda and K . O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a U n i v e r s i t y M e d i c a l S c h o o l , O k a y a m a , Japan T h i s s t u d y w a s c o n d u c t e d to a s s e s s the c y t o t o x i c e f f e c t s of n H u T N F - e , - 6 and n H u I F N - e, - 7 , e i t h e r a l o n e or in c o m b i n a t i o n , on ten h u m a n c a n c e r c ~ l lines. The c e l l l i n e s t e s t e d w e r e R P M I 4 7 8 8 , M K N - I , M K N - 2 8 , K - 5 6 2 , H-7, D a u d i , KB, P C - 1 0 , Z R - 1 0 , Z R - 7 5 - 1 and Z R - 7 5 - 3 0 . A f t e r 3 - d a y i n c u b a t i o n in 9 6 - w e l l m i c r o t i t e r p l a t e , the c y t o t o x i c e f f e c t s w e r e a s s e s s e d b y M T T assay. F o u r c e l l l i n e s ( R P M I 4 7 8 8 , P C - 1 0 , ~ { N - I and ~ N - 2 8 ) w e r e s e n s i t i v e (>30% c y t o t o x i c i t y , at t h e h i g h e s t c o n c e n t r a t i o n s t e s t e d ) to n H u T N F - e or -£ alone. n H u I F N - e s h o w e d c y t o t o x i c e f f e c t s on R P M I 4 7 8 8 , P C - 1 0 , ~ N - I and ~ N - 2 8 , and n H u I F N - 7 h a d s e n s i t i v i t y to R P M I 4 7 8 8 , P C ' I 0 a n d KB . A synergistic a n t i t u m o r e f f e c t o c c u r e d in f o u r c e l l l i n e s I R P M I 4 7 8 8 , P C - 1 0 , D a u d i and ZR75-1) t r e a t e d w i t h a c o m b i n a t i o n of n H u I F N - ~ a n d n H u T N F - ~ or - ~ . In s e v e n cell l i n e s ( R P M I 4 7 8 8 , P C - 1 0 , KB, M K N - I , M K N - 2 8 , D a u d i a n d ZR-75-1) , the c o m b i n a t i o n o f n H u I F N - 7 a n d n H u T N F - e o r - 6 s h o w e d the s y n e r g i s t i c e n h a n c e ment. A l t h o u g h f u r t h e r i n v e s t i g a t i o n s n e e d to b e c o n d u c t e d , o u r r e s u l t s may have therapeutic implications.
WSl1-16 THE E A R L Y E V E N T O F I N T E R F E R O N ( I F N - ~ , ¥ ) INDUCED CYTOPLASMIC FREE CALCIUM ION M O B I L I Z A T I O N A N D M E M B R A N E P O T E N T I A L R E S P O N S E I N C A N C E R C E L L S H. I w a g a k i , S . F u c h i m o t o , M . M i y a k e , S . S h i i k i , A . S a s a k i , Y . Y a s u i , A . G o u c h i , K . O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a University Medical School,Okayam? Japan Interferon(IFN-B,7) is r e p o r t e d to h a v e an a n t i t u m o r e f f e c t in c a n c e r cells. H o w e v e r , l i t t l e is k n o w n of the m e c h a n i s m b y w h i c h I F N t r a n s m i t s its e f f e c t f r o m the c e l l s u r f a c e i n t o the cell w h e r e i t s a c t i o n s are m a n i fested. The Ca 2+, w i d e l y r e c o g n i z e d as an i m p o r t a n t i n t r a c e l l u l a r m e s s e n ger, c o n t r i b u t e s t o c o n t r o l a v a r i e t y of c e l l u l a r p r o c e s s e s . W e u n d e r t o o k to s t u d y the e a r l y e v e n t s o f the c y t o p l a s m i c free C a 2 + m o b i l i z a t i o n and m e m b r a n e p o t e n t i a l r e s p o n s e t h a t o c c u r in c a n c e r c e l l s s e c o n d s a f t e r e x p o s u r e to i n d e n t i f y the c h a n g e s a s s o c i a t e d w i t h the t r a n s m e m b r a n e p r o c e s s i n g of the I F N s i g n a l , u s i n g Ca 2+ i n d i c a t o r ( F u r a 2-~) and membrane potential sensitive dye(dis-C3-(5)) . IFN-~,7 stimulated a transient about 1.6 - 1.8 fold i n c r e a s e in the c o n c e n t r a t i o n of Ca 2~ ([Ca 2+] i ) w i t h i n 15 to 30 s e c o n d s of c e l l e x p o s u r e to IFN. T h e [Ca2+]i r e t u r n e d to b a s a l levels by a b o u t 60 s e c o n d s . The m e m b r a n e w a s d e p o l a r i z e d s e c o n d s a f t e r exp o s u r e to IFN-7 , b u t w a s n o t a f t e r e x p o s u r e to IFN-S . T h e s e r e s u l t s s u g g e s t e d t h a t I F N - 7 i n d u c e d Ca 2+ s i g n a l w a s c a u s e d b y the m e m b r a n e potential-dependent C a 2+ c h a n n e l .
WS11-17 E F F E C T OF N A T U R A L H U M A ~ T U M O R N E C R O S I S F A C T O R (nHuTNF) A N D N A T U R A L H U ~ N I N T E R F E R O N (nHuIFN) ON A R A C H I D O N I C A C I D ~ T A B O L I S M Y. Y a s u i , S. F u c h i m o t o , F. H a m a d a , S. S h i i k i , H. I w a g a k i , M. M i y a k e , T. M a e d a a n d K. O r i t a F i r s t D e p a r t m e n t of S u r g e r y , O k a y a m a U n i v e r s i t y M e d i c a l S c h o o l , O k a y a m a , Japan T h i s s t u d y w a s c o n d u c t e d to e v a l u a t e the e f f e c t of n H u T N F s and n H u I F N s on a r a c h i d o n i c a c i d (AA) m e t o b o l i s m of i m m u n o s u p p r e s s i v e state. We measured t h e p r o d u c t i o n o f p r o s t a g l a n d i n E2 (PGE2) of p e r i p h e r a l b l o o d m o n o c u c l e o c y t e (PBMC) of g a s t r o i n t e s t i n a l cancer patient, which were incubated with various cytokines, n H u T N F - ~ and n H u T N F - B p r o m o t e d the p r o d u c t i o n , b u t n H u I F N - e, n H u I F N - ~ , or c o m b i n a t i o n o f n H u T N F and ~ H u I F N (I:I) s u p p r e s s e d t h e p r o d u c t i o n of PGE2. T h e l e v e l of P G E 2 w a s a b o u t 25% of the c o n t r o l w h e n n H u I F N w a s a d d e d a l o n e , a n d t h a t w a s a b o u t 30% in c o m b i n a t i o n . It w a s r e p o r t e d t h a t P G E 2 i n h i b i t s the N K a c t i v i t y , so t h e s e r e s u l t s s u g g e s t t h a t n H u I F N s m a k e u p t h e n e g a t i v e e f f e c t of n H u T N F s in the i m m u n o s u p p r e s s i v e s t a t e b y m e a n s o f the i n h i b i t i o n of A A m e t a b o l i s m .
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