Study of the action of tamoxifen on the mammary gland epithelium of premenopausal patients by lysosome quantification

Study of the action of tamoxifen on the mammary gland

epithelium of premenopausal patients by

lysosome quantification

D e p a r t m . e n t o f G y n e c o l o g y a n d O b s t e t r i c s , F e d e r a l U n i v e r s i t y o f S ã o P a u l o , E s c o l a P a u l i s t a d e M e d i c i n a

-S ã o P a u l o , S p , B r a z i l

T am oxifen is an antiestrogen drug w idely utilized for the adjuvant horm onal treatm ent of breast carcinom a. Its use in the prim ary prophylaxis of this disease is currently being proposed. A lthough the drug has few side effects, its precise action on breast tissue that has not undergone neoplastic transform ation has not been fully elucidated. T his prospective, random ized study assessed the estro-gen activity of tam oxifen on the m am m ary gland epithelium of prem enopausal patients using a quantitative analysis of m am m ary epithelium Iysosom es identified by the cytochem ical technique of G O M O R I for acid phosphatase and by light m icroscopy. T am oxifen significantly increased the num ber of Iysosom es only during the secretory phase of the m enstrual cycle. W e concluded that the early effect of the drug on norm al m am m ary tissue is synergistic w ith the effect of estrogen during the prem enopausal period.

IN T R O D U C T IO N

T

amoxifen

nonsteroid

is considered

drug that inhibits the growth of breast

to be an antiestrogen

carcinoma,

thus representing

the. drug of choice

for hormonal treatment of this disease.

t

_{Recent studies have}

shown a lower incidence of primary cancer and cyclic pain

in the contralateral breast of patients with carcinoma treated

with tamoxifen.

t

-3

Thus,

it is of great interest to know how

the drug acts upon normal breasts, to the end of it being

used as a primary

prophylaxis

of breast carcinoma

in

women at high-risk for developing

this disease.

3-S

Lysosomes

are cytoplasmic

organelIes

containing

lytic

enzymes

and

covered

with

semiperlTIeable

A d d re s s fo r c o rre s p o n d e n c ~ : C a rlo s ltio T a n a k a

A v . S e n a d o r C a s e m iro d a R o c h a , 1257,A p to . 5 4 -B , S ã o P a u lo /S P - B ra s il - C E P 0 4 0 4 7 -0 0 3

membranes,

and were first studied by

DE DUVE

et aI.ó

These investigators

identified

these organelIes

by celI

fractionation techniques foIlowed by centrifugation,

which

led to the observation

of vesicles

rich in hydrolytic

enzymes.

Studies relating the mamlTIary gland to lysosomes

have shown a larger number of these organelIes in breast

tUlTIOr ceIls

compared

to

the

normal

adjacent

parenchyma.

7

•X

Lysosomes

are important

markers

of

estrogen-mediated

ceIl proliferation

in the breast

parenchyma.

7.X

GIRÃO et aI.,9 studying the numerical

variation of

lysosomes

in the mammary

epithelium

during

the

menstrual cycle, observed a significantly

greater amount

of lysosomes

during the first phase than in the second

phase, demonstrating that the number of lysosomes refiects

estrogen activity.

The objecti ve of this study was to assess the estrogen

activity of talTIoxifen on the epitheliulTI of the malTImary

gland

of

premenopausal

WOlTIen by

lysosome

qua~tification.

S ão P aulo M edicai JournallR P M 115(2): 1390-1394, 1997 T A N A K A , C .I.; G E B R IM , L.H .; LIM A , G .R . et aI. - S tudy of the action of tam oxifen on the

m am m ary gland epithelium of prem enopausal patients by

OBJECTIVE

c o m p le te ly tille d w ith e p ith e lia l c e lls . D a ta w e r e a n a ly z e d s ta tis tic a lly b y th e M a n n - W h itn e y a n d F is h e r e x a c t te s ts .1 3

RESULTS

T a m o x if e n is th e m o s t w ie le ly - u s e d e lr u g f o r

c h e m o p r o p h y la x is o f b r e a s t c a r c in o m a . H o w e v e r , its a c tio n

o n th e n o r ln a l m a m m a r y lo b u le b e f o r e a n d a f te r

m e n o p a u s e is s till u n k n o w n .1 4 -ló

I t is d if f ic u lt to c r e a te a n e x p e r im e n ta l m o d e I th a t

w ill. r e p r o d u c e th e c o m p le x e n d o c r in e in te r a c tio n o f th e

h U ln a n b r e a s t, a n d th e r e a r e o b v io u s e th ic a I r e s tr ic tio n s

w ith r e .s p e c t to ta k in g b io p s ie s f r o ln th e n O r I n a l b r e a s t. T a m o x if e n s ig n if ic a n tly in c r e a s e d th e n u m b e r o f

ly s o s o m e s in th e m a m m a r y e p ith e liu m d u r in g th e s e c o n d

p h a s e o f th e m e n s tr u a l c y c le ( F ig s . 1 a n d 2 ) .

D u r in g ta m o x if e n a d m in is tr a tio n , th e n u m b e r o f

ly s o s o m e s r e m a in e d s ta b le in b o th p h a s e s o f th e m e n s tr u a l

c y c le ( T a b le 1 ) .

P r o g e s te r o n e le v e I s w e r e s ig n if ic a n tly h ig h e r in

g r o u p B p a tie n ts d u r in g th e I u te a l p h a s e o f th e m e n s tr u a l

c y c le ( T a b le 2 ) .

MATERIALS

ANO METHOOS

T o e v a lu a te q u a n tita tiv e ly th e ly s o s o m e s o f th e

h u m a n m a ln m a r y g la n d e p ith e liu m in th e p r o lif e r a tiv e a n d

s e c r e to r y p h a s e o f th e m e n s tr u a l c y c le d u r in g ta m o x if e n

a d m in is tr a tio n , a n d to c o m p a r e th e v a lu e s o b ta in e d w ith

th o s e d e te c te d in u n tr e a te d w o m e n d u r in g th e s e s a m e

p h a s e s .

T h e s tu d y w a s c a r r ie d o u t o n 3 3 w o m e n a g e d 1 5 - 3 7

w ith f ib r o a d e n o m a s ] - 3 c m in d ia m e te r w h ic h w e r e

r e m o v e d a n d s u b m itte d to b io p s y . N o r m a l p a r e n c h y m a

s a m p le s w e r e o b ta in e d f r o m b r e a s t tis s u e s itu a te d a t le a s t

2 c m f r o m th e f ib r o a d e n o m a a t th e tim e o f s u r g ic a l e x e r e s is

u n d e r lo c a l a n e s th e s ia . A lI p a tie n ts g a v e in f o r m e d w r itte n

OISCUSSION

c o n s e n t a n d th e s tu d y w a s a p p r o v e d b y th e E th ic s

C o m m itte e o f H o s p ita l S ã o P a u lo .

T h e p a tie n ts w e r e d iv id e d r a n d o m ly in to tw o g r o u p s :

G r o u p A ( c o n tr o I ) c o n s is te d o f 1 6 p a tie n ts , a n d G r o u p B

c o n s is te d o f 1 7 p a tie n ts w h o to o k ta m o x if e n ( 2 0 m g /d a y )

f o r 3 0 d a y s b e f o r e s u r g e r y .

A ll p a tie n ts h a d n o r m a l m e n s tr u a l c y c le s . T h e c y c le

p h a s e w a s c h a r a c te r iz e d o n th e b a s is o f th e d a te o f la s t

m e n s tr u a tio n a n d m e a s u r e m e n t o f p la s m a p r o g e s te r o n e .

P a tie n ts w ith e n d o c r in e d is e a s e , p r e g n a n t p a tie n ts o r th o s e

w h o h a d ta k e n h o r m o n e s d u r in g th e

p r io r 1 2 m o n th s w e r e e x c lu d e d f r o m

th e s tu d y , a s w e r e a ls o p a tie n ts w h o s e

b io p s y d id n o t c o n f ir m th e p r e s e n c e

o f a f ib r o a d e n o m a in th e n o d u le o r

n o r m a lity o f th e a d ja c e n t p a r e n c h y m a .

T h e c h ü ic e o f m e n s tr u a l c y c le

p h a s e f o r e x e r e s is o f th e n o d u le w a s

a le a to r y f o r b o th g r o u p s , b u t th e d a te s

w e r e f ix e d f o r a p e r io d b e tw e e n th e

7 th a n d 1 0 th d a y d u r in g th e f ir ~ t

p h a s e , a n d b e tw e e n th e 2 1 s t a n d 2 3 r d

d a y d u r in g th e s e c o n d .

L y s o s o ln e s w e r e id e n tif ie d b y th e

c y to c h e m ic a l m e th o d o f a c ie l

p h o s p h a ta s e d e te c tio n , lO a n e l. c o u n ts

w e r e c a r r ie e l o u t u s in g th e P R I C O L I "

a n e l M A R T I N S 1 2m e th o e ls . C o u n ts w e r e

Figure 1 -P h o to m ic ro g ra p h o f a h is to lo g ic a l s e c tio n o f h u m a n m a m m a ry g la n d e p ith e liu m

c a r r ie e l o u t in a d o u b le - b lin d m a n n e r o n d u rin g th e s e c re to ry p h a s e fro m a p a tie n t N O T tre a te d w ith ta m o x ife n (G o m o ri m e th o d

2 5 r e tic u la te d a r e a s p e r 4 0 0 x f ie ld fo r a c id p h o s p h a ta s e . M a g n ific a tio n .:t 1 .4 4 0 X ).

T A N A K A , C .I.; G E B R IM , L .H .; L IM A , G .R . e t a I. - S tu d y o f th e a c tio n o f ta m o x ife n o n th e m a m m a ry g la n d e p ith e liu m o f p re m e n o p a u s a l p a tie n ts b y

S e c re to ry

U c a lc = 1 2 *

U c rit= 1 2

tis s u e s . T h e ln e th o d u s e d fo r b re a s t tis s u e is re p ro d u c ib le a s d e m o n s tra te d b y Z A M IT H e t a 1 .2 0a n d G IR Ã O e t a lY T h e s e in v e s tig a to rs o b s e rv e d a la rg e r n u m b e r o f ly s o s o m e s d u rin g th e firs t p h a s e o f th e c y c le in b re a s t tis s u e fro m p re m e n o p a u s a l w o m e n , in a g re e lT Ie n t w ith th e s e ru m le v e I o f e s tra d io l.

T a m o x ife n a d ln in is te re d b e fo re m e n o p a u s e firs t c a u s e s a n in c re a s e in s e ru m e s tro g e n a n d p ro g e s te ro n e le v e Is , p o s s ib ly in itia lin g th e p ro life ra tio n o f . th e m a IT Im a ry e p ith e liu m .2 1 T h e

in c re a s e d n u m b e r o f ly s o s o ln e s o b s e rv e d d u rin g th e s e c o n d p h a s e o f th e c y c le in w o m e n ta k in g ta m o x ife n c o n firm s th e e s tro g e n a g o n is t e ffe c t o f th e d ru g o n p re m e n o p a u s a l W O IT Ie n .

O n th e o th e r h a n d , th e in c re a s e d c o n c e n tra tio n o f a c tiv e s e ru m p ro g e s te ro n e a c ti v a te s th e e n z y m e 1 7 -b e ta -h y d ro x y s te ro id o x id o re d u c ta s e , w h ic h fa v o rs th e c o n v e rs io n o f e s tro n e to e s tra d io l in b re a s t tis s u e , w h ic h in tu rn s in c re a s e s e s tro g e n a c tiv ity .2 2

R e c e n t s tu d ie s u s in g IT Io le c u la r te c h n iq u e s h a v e d e m o n s tra te d a lte ra tio n s . in th e g e n e e x p re s s io n o f th e e s tra d io l re c e p to rs w h ic h IT Ia y e x p la in th e a n ti p ro life ra ti v e a c tio n o f ta In o x ife n d e s p ite th e a g o n is t e s tro g e n a c ti v i ty . 2 3 .2 4

T h e e x p la n a tio n o f th e a n tip ro life ra tiv e e ffe c t o n th e b re a s t d llrin g lo n g -te n n tre a tn le n t ln a y b e c lo w n -re g llla tio n

1 2 3

G ro u p A x B

P ro life ra tiv e

U c a lc = 2 8

U c rit= 1 5 G ro u p B

P ro life ra tiv e S e c re to ry

86

75

1 0 4 1 2 6

1 3 1 1 1 3

1 2 1 1 3 6

1 3 7 1 1 3

1 1 7 1 3 9

1 5 0 1 7 5

1 4 1 1 3 4

1 8 8

1 3 3

2 n d )

99

G ro u p A

P ro life ra tiv e S e c re to ry

1 4 7 9 4

1 3 1 1 3 1

1 3 8 8 1

1 1 9 8 7

1 2 8 8 5

1 4 0 1 1 5

1 6 9 1 0 6

1 0 1

1 3 1

1 3 3

T h e tis s u e fra g m e n t u tiliz e d in th e p re s e n t s tu d y w a s o b ta in e d fro In p a tie n ts w ith fib ro a d e n o m a w h o d id n o t re s p o n d to ta m o x ife n tre a tm e n t, a fte r a u th o riz a tio n b y th e M e d ic a I E th ic s C o m m itte e o f H o s p ita l S ã o P a u lo .

T h e re a re n u m e ro u s . th e o rie s a tte ln p tin g to e x p la in th e a n tip ro life ra tiv e a c tio n o fth is d ru g o n n e o p la s tic tis s lle . T h e m a jo r ln e c h a n is ln o f a c tio n ,

h o w e v e r, is th o u g h t to b e th e c o m b in a tio n o f ta lT Io x ife n w ith n u c le a r e s tro g e n re c e p to r p ro te in (E R P ), w h ic h p ro d u c e s a G ] b lo c k a n d d e c re a s e s p ro d u c tio n o f g ro w th -s tim u la tin g p ro te in s s u c h a s tra n s fo rm in g g ro w th fa c to r a lp h a (T G F -a ), a n d s tim u la tio n o f g ro w th -in h ib it-in g p ro te in s lik e tra n s fo n n in g g ro w th fa c to r b e ta (T G F -B ).1 7 -1 9

T a IT IO x ife n is p re d o m in a n tly a tu m o ris ta tic a g e n t; it d o u b le s th e le v e Is o f s e x h o rln o n e -b in d in g g lo b u lin (S H B G ) a n d m a y th e re fo re e x e rt i ts a n tie s tro g e n e ffe c t b y In a k i n g it u n a v a ila b le to b re a s t c e lls .\

T h e q u a n tita tiv e v a ria tio n in F ig u re 2 - P h o to m ic ro g ra p h o f a h is to lo g ic a l s e c tio n o f h u m a n m a m m a ry g la n d e p ith e liu m

in tra c e llu la r ly s o S O IT Ie S in d ire c tly d u rin g th e s e c re to ry p h a s e fro m a p a tie n t tre a te d w ith ta m o x ife n (G o m o ri m e th o d fo r

m e a s u re s e s tro g e n a c tiv ity o n ta rg e t a c id p h o s p h a ta s e . M a g n ific a tio n .:t 1 .4 4 4 0 X ). M e a n

M a n n -W h itn e y T e s t

1s t) P ro life ra tiv e x s e c re to ry

G ro u p A G ro u p B

U c a lc = 6 * U c a lc = 3 2

U c rit= 1 2 U c rit= 1 5

T a b le 1

N u m b e r o f Iy s o s o m e s in th e b re a s t e p ith e liu m o f p a tie n ts fro m G ro u p A (c o n tro l) a n d G ro u p B (ta m o x ife n ) d u rin g th e p ro life ra tiv e a n d

s e c re to ry p h a s e s o f th e c e ll c y c le .

S ã o P a u lo M e d ic a i J o u rn a l/R P M 1 1 5 (2 ): 1 3 9 0 -1 3 9 4 , 1 9 9 7 T A N A K A , C .I.; G E B R IM , L .H .; L IM A , G .R . e t a I. - S tu d y o f th e a c tio n o f ta m o x ife n o n th e

m a m m a ry g la n d e p ith e liu m o f p re m e n o p a u s a l p a tie n ts b y

Table2

Serum progesterone

leveis (nglml) in patients from Group A (control) and

Group 8 (tamoxifen) during the proliferative and secretory phases of the

menstrual cycle.

G ro u p A

0 .4

0 .9

0 .4

0 .7

0 .6

0.4

0 .5

0 .7

0 .3

0 .5 4

M a n n -W h itn e y T e s t

1s t) P ro life ra tiv e

G ro u p A x 8

U c a le = 3 1

U c rit= 1 5

G ro u p 8

0 .5

0 .8

0 .5

0.6

0 .8

0 .3

0.1

0.1

0 .4 6

2 ) S e c re to ry

G ro u p A x 8

U c a lc = 6 *

U c rit= 1 2

G ro u p A

6.1

7.9

6 .5

6.5

1 2 .7

4 .0

1 6 .3

8 .5

G ro u p 8

8 .9

1 3 .7

2 4 .6

6 .8

1 8 .9

3 6 .9

3 4 .2

4 7 .0

3 3 .0

2 4 .8 9

of estrogen receptors, reduction ofTGF-a

and increase of TGF-B.

Future

studies

on patients

using

talnoxifen

for long periods

of tilne are

needed to better evaluate

the endocrine

effect of the drug on normal breast tissue,

thus pennitting

a Inore rational indication

of the drug

for treatlnent

of benign

diseases and for cancer chelnoprophylaxis.

CONCLUSION

We

conclude

that

talnoxifen

significantly

increased

the nUlnber of

lysosomes in the Inammary epithelium in

the second phase of the lnenstrual

cycle,

with leveIs similar to those detected in the

proliferative

phase for both groups. Thus,

the drug appears

to have a synergistic

effect

with

estrogen

during

the

prelnenopausal

period.

T A N A K A , C .I.; G E B R IM , L .H .; L IM A , G .R . e t a I. - S tu d y o f th e a c tio n o f ta m o x ife n o n th e m a m m a ry g la n d e p ith e liu m o f p re m e n o p a u s a l p a tie n ts b y

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