BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 118-]23
Vol. 180, No. 1, 1991
October 15, 1991
I N H I B I T I O N OF M I C R O T U B U L E A S S E M B L Y IS A P O S S I B L E M E C H A N I S M OF A C T I O N OF M I T O X A N T R O N E Chi-Kuan
Ho*,Sai-Lung Law, H u n g C h i a n g * * , M a i - L i n g Chien-Chia Wang and Sheng-Yuan Wang
Hsu
Departments of Medical Research and Pathology** Veterans General Hospital, Taipei and Yang-Ming Medical College, Taipei Taiwan, Republic of China
Received August 28, 1991 SUMMARY: We have found that mitoxantrone can inhibit the polymerization of brain tubulinin a dose dependent manner. MXT hadrelatively high affinity for tubulin but had no appreciable effect on tubulin associated guanosinetriphosphatase (GTPase) activity nor couldit compete with vinblastine (VB) and colchicine (Col) for tubulinbinding sites. Furthermore, MXT (0.1-10 ~tM) is antiprolifcrative to cold-treated (0 ° C) epithelial cells afteronly brief exposure (30 rain). These results indicated that MXTis a microtubule inhibitory agent and can exert its anticellular effect through modulation of microtubule
assembly.
@ 1991 Academic Press, Inc.
Mitoxantrone broad
spectrum
(MXT) is a n e w a n t h r a c e n e d i o n e whiuh
antineoplastic
models and clinical
trials against
other malignant diseases than doxorubicin
activity
in
both
breast cancer
but has much
less potential
(5) and is a b l e to induce DNA breakage, (1,6). However, there
is no
between the potent
anticellular
of
effect
(2,3). Therefore, its
remains to be determined.
In the
exact
accumulative
an i n t e r c a l a t o r of
and i n h i b i t
acid b i o s y n t h e s i s
e f f e c t in v i v a
a n d a v a r i e t y of
(1-3) with p o t e n c y equal to or s u p e r i o r
cardiatoxic effect (4). MXT is d e s i g n e d to b e DNA
has p r o v e n
experimental
nucleic
direct c o r r e l a t i o n
M~r and its a n t i t u m o r mechanism
of
action
p r e s e n t s t u d y we h a v e s h o w n t h a t
*To whom all correspondence should bc addressed. A b b r e v i a t i o n s : MXT, m i t o x a n t r o n e ; T L C , t h i n l a y e r c h r o m a t o g r a p y ; D E A E , d i e t h y l a m i n o e t h y l ; P-G buffer, p h o s p h a t e - g l u t a m a t e buffer ; G T P a s e , guanosinetriphosphatase ;DMEM, Dubolco minimal essential medium; VB,vinblastine ; Col, colchicine. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
Vol. 180, No. 1, 1991
MXT can
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibit
reattachment
the p o l y m e r i z a t i o n
of e p i t h e l i a l cells
results
imply
that
mediated
through
of b r a i n t u b u l i n
and subsequent
the anti-cellular
restriction MATERIALS
AND
delay
the
cell g r o w t h . T h e s e
effect
of m i c r o t u b u l e
and
of
MXT
may
be
assembly.
METHODS
Inhibition o f Tubulin a s s e m b l y : Highly purified bovine brain t u b u l i n w a s p r e p a r e d as d e s c r i b e d b y Hamel and L i n (7) u s i n g D E 52 i o n i c e x c h a n g e chromatography followed by 3 cycles of polymerization and depolymerization and assembly of t u b u l i n w a s monitored by theviscometric m e t h o d of K u r i y a m a & S a k a i (8) i n the p r e s e n c e a n d a b s e n c e of v a r i o u s c o n c e n t r a t i o n s of M X T ( 2 - 5 0 ~M) or v i n b l a s t i n e (5 o r 10 ~M) a n d t h e e f f e c t o f M X T o n t u b u l i n associatedGTPase a c t i v i t y was d e t e r m i n e d b y T L C a s d e s c r i b e d b y D a v i d - P f e u t y et al (9) Electron Microscopy : Highly purified t u b u l i n (1-2 mg/ml) i n a s s e m b l y b u f f e r was i n c u b a t e d at 37°C for 30 m i n i n t h e p r e s e n c e o r a b s e n c e of M X T (10 ~M) and s a m p l e s w e r e d i l u t e d ( 1 : 1 ) . w i t h a buffer containing 2 Mhexylene g l y c o l (in 0 . 2 M M e s p H 6.9 + 1 . 0 m M M g C I 2) a n d m i c r o t u b u l e was r e v e a l e d u s i n g a s t a n d a r d negative s t a i n i n g t e c h n i q u e (8). Bindinu of M X T t o t u b u l i n :Tubulin was subjected to 7.5% polyacrylamide gel electrophoresis (PAGE) u n d e r n o n - d e n a t u r i n g conditions in T r i s - H C I b u f f e r pH 8 . 6 . F o l l o w i n g electrophor e s i s , i n d i v i d u a l slots w e r e s l i c e d a n d then t r e a t e d as d e s c r i b e d in t h e f i g u r e legend. The b i n d i n g of 3 H _ v i n b l a s t i n e (2 ~Ci/~M) or 3 H - c o l c h i c i n e (2 ~ C i / ~ M ) in the p r e s e n c e or a b s e n c e cf v a r i o u s d o s e s of M X T (1-100 f o l d excess) t o t u b u l i n w a s d e t e r m i n e d b y t h e DE-81 p a p e r discs m e t h o d of W e i s e n b e r g et al (10). A n t i c e l l u l a r effect : T L ( c y t o t r o p h o b l a s t i c ) , KB a n d W I S H c e l l s w e r e r e s u s p e n d e d at 2 x ~ 5/ml in D M E M plus 1 0 % f e t a l b o v i n e serum and gentamycin (20 ~g/ml) and t h e n i n c u b a t e d on ice for 6 h. The cells w e r e then i n c u b a t e d for an a d d i t i o n a l 30 m i n in t h e p r e s e n c e or a b s e n c e of M X T (0.1-10 ~M), w a s h e d 3X a n d p l a t e d o n t o 60 m m t i s s u e c u l t u r e s p l a t e s and t h e n i n c u b a t e d a t 3 7 ° C i n 5% CO 2. At v a r i o u s t i m e s , c e l l s w e r e observed u n d e r an i n v e r t e d m i c r o s c o p e f o r c e l l a t t a c h m e n t a n d c e l l n u m b e r cf a l l c u l t u r e s was d e t e r m i n e d at 48 h f o l l o w i n g i n c u b a t i o n by t r y p a n b l u e stain. RESULTS
Inhibition
of t u b u l i n
_
~
:
~s c o u l d b e
p o l y m e r i z a t i o n of t u b u l i n w a s or 10 ~M) similar effect
in p r o p o r t i o n to
of
that MXT
microscope.
of
A dense
observed when tubulin of M X T t h e Unlike enhance
VB
extent and
to d o s a g e . T h e
VB
could
inhibited
but
is
also
tubular
Col,
under
of MXT
inhibition
The
i, (2 is
inhibitory
the
structure
electron could
be
p o l y m e r i z e d (Fig 2A), however, in t h e p r e s e n c e
of p o l y m e r i z a t i o n
nor suppress
k i n e t i c s of
less efficient.
of
in F i g u r e
in t h e p r e s e n c e
be o b s e r v e d
network
seen
was greatly
over a
wide
GTPase
activity
dose
119
range, (Fig.4).
reduced MXT
could
(Fig.2B) . neither
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0 1 0.75~ ~ i , ~ ~ I ~50 "4 025 00Ir-----'''~ 5, 10 15 20 25 30 ,
,
rm
L
,
,
(m~)
F i q . i. The inhibitory effect of mitoxantrone on t h e p o l y m e r i z a t i o n of tubulin as a s s a y e d by the v i s c o m e t r i c method. P u r i f i e d bovine b r a i n t u b u l i n w a s a s s e m b l e d i n M e s b u f f e r at 37°C in the absence (i) and p r e s e n c e of 2,10 and 50 JiM (2-4) of MXT respectively or 10 ~ M of VB (5) . R e l a t i v e v i s c o s i t y was arbitrarily set at 0-i unit.
Binding
o f MXT
andtubulin
to
the intense blue tubulin
tubulin
was assessed
(in
concentration with albumin
color
15-25 but or
physical
modified
of MXT.
min under
:The by a
interaction
immunoblot
Fig 3 indicates
post-incubation) the same
transferrinwhich
conditions, are
known
.in MXT
between
assay that
MXT
based
MXT binds
proportion does
MXT-binding
not
to t o
react
proteins.
Fiu. 2. E l e c t r o n m i c r o g r a p h s of negatively s t a i n e d s p e c i m e n s of t u b u l i n p o l y m e r s s h o w i n g a dense n e t w o r k of m i o r o t u b u l e s t r u c t u r e f o l l o w i n g i n c u b a t i o n at 37°C for 30 m i n (A) a n d r e l a t i v e l y few t u b u l a r f i l a m e n t s w h e n t u b u l i n was a s s e m b l e d in the p r e s e n c e of i0 ~M of MXT (B). x16,000,
120
on
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
32p--G D P
32p--G T P
®
1
2
1
2
3
4
5
6
7
8
9
10
Fig. 3. The binding of MXT to tubulin as determined by a m o d i f i e d immunoblot assay. Tubulin in semi-preparative 7.5% polyacrylamide gel was electrophoresed under non-denatured conditions in TrisHCl buffer pH 8.6. Individual slots of the same gel were treated separately as follows: Lane i, stained withcoomassie blue stain; Lane 2, immunoblotted with a m o u s e anti-rat tubulin monoclonal a n t i b o d y using EIA and i n c u b a t e d with MXT (i00 ~g/ml) a f t e r transfer onto nitro-cellulose membrane (Lane 3). Three major a n d one minor MXT-reactive p r o t e i n bands as indicated by arrowheads were consistently observed. Fiq. 4. Assay for polymerization-dependent t u b u l i n - a s s o c i a t e d GTPase activity by TLCbased on the conversion of 32p-GTP to m p _ GDP. Bovine tubulin (2-5 mg/ml) was i n c u b a t e d with a s s e m b l y buffer only (Lane i) a n d in the presence of 2, i0 a n d 50 ~ M of VB (Lanes 2-4), MXT (Lanes 5-7) or Col (Lanes 8-10) respectively. Individual spots were revealed by autoradiography.
Furthermore,
protein-bound
indicating
that
preparation
is
the
M X T c o u l d be
binding
homogenous
reactive band), but
the
i s
in
same
elutedwith
reversible.
SDS-PAGE
preparation
(3 m a j o r a n d 1 m i n o r
; arrowheads)
lane
represent
which
concatamers. All
of t h e s e
are
anti-tubulin
reactive to
2).However,
based
not compete
with either
at a 1 0 0 - f o l d Effect
on
on the
excess
cell
bands
_:
structures of indirect
Following all cells
andanti-tubulin
a d h e r e n t in a b o u t
most
MXT-treated
least 8
h at
all
cells
the c y t o t o x i c
appreciable increase after
antibody
and
l a n e 3)
and
(Fig.3,
drugs,
lane
MXT
does
binding sites
even
cold were
immunofluorescence
however
these cultures
(Fig.3
tubulin
4
(Not shown).
revealed
withoutMXTbecame
reveals
in n o n - d e n a t u r e d
radioactive
Col for
tubulin
heterodimers
bindMXT
monoclonal
VB or
growth
cytospin cell smears
can
t r a c i n g of
cytoskeletal by
may
buffer
anti-tubulin
consistently
gel
i)
Our
( a single
protein bands (Fig.3,
P-G
48h
(Not
using
in
disrupted
suspension
tested.
.
as
C e l l s in c u l t u r e s
or cell death
shown)
the
acetone-fixed
2 h after incubation
doses
121
totally
antibody.
remained
in c e l l n u m b e r
treatment,
In
at 37°C for
addition,
was observed
at no in
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
Despite the is not
intense interest
completely
s u g g e s t s that
in MXT, its m e c h a n i s m
understood.
Accumulated
MXT is an a n t i - t o p o i s o m e r a s e
of a c t i o n
evidence II
thus
(11) d r u g
far
and c a n
inhibit nuclei acid synthesis (6,12). However,these
a c t i v i t i e s do
not
effect
correlate
well
with
its
in vivo
antitumor
(2,3)
suggesting that other mechanisms may be involved. We
have
shown in the p r e s e n t study that MXT c a n
p o l y m e r i z a t i o n of p u r i f i e d bovine b r a i n H o w e v e r , u n l i k e V B or C o l , M X T h a s n o associatedGTPase two t u b u l i n
has high a f f i n i t y
sites
(Not
of MXT for t u b u l i n
and
this
may be
it c o m p e t e w i t h
the
shown).In addition,
MXT
c o m p a r i n g to o t h e r k n o w n M X T
b i n d i n g p r o t e i n s such as a l b u m i n and shown)
tubulin (Figs 1&2) . effect on tubulin-
activity (Fig 4), nor does
drugs for binding
s u p p r e s s the
transferrin
theprobable
cause
of
(Fig 3 a n d
not
its a b i l i t y
to
interfere with tubulin assembly. The m i c r o t u b u l e supporting
system plays
network
conductor
for
for
cellular
s p e c u l a t i v e that MXT restriction
cell
surface
receptors
communication
can
e x e r t its
of m i c r o t u b u l e
membrane activity.
a key r o l e in m i t o s i s
as well as
(13). T h e r e f o r e
anticellular
effect
formation and m o d u l a t i o n
This h y p o t h e s i s
is
supported b y
observation that MXT is p r e f e r e n t i a l l y e n c a p s u l a t e d charged liposomes
which then become p o s i t i v e l y
a d d i t i o n , it
been
carcinoma system
has
cell
reported
line has
a n d is t h e
that
structural
it
is
through
of
plasma
our r e c e n t
by n e g a t i v e l y -
c h a r g e d (14).
a MXT-resistent
changes
the
In
gastric
in its m i c r o t u b u l e
(15).
Taken
together,
results
others s t r o n g l y suggest that to p e r t u r b
nucleic
s o l e l y by
that
doxorubicin
with our suggestion
with
can
be useful
for the
its
122
on
Aprevious
cytotoxic
action
(16)
accord
is in
related. Our
to a c c o u n t
of M X T a n d p r o v i d e cf more e f f e c t i v e
protocals against various neoplastic diseases.
of
ability
also t a r g e t
are c l o s e l y
explanation
design
and those
cytoskeleton.
surface
since the two drugs
d i v e r s i t y of the b i o l o g i c a l effects which may
MXT can
exert
the c e l l
a more d e t a i l e d
study
from its e s t a b l i s h e d
and the a s s o c i a t e d
interaction
findings offer
apart
acid b i o s y n t h e s i s ,
membranous Structures observation
of the p r e s e n t
for t h e
some c l u e s treatment
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT The authors fromthe
wish
National
to express g r a t i t u d e Science Council,
for t h e
financial support
Taiwan,R.O.C.
(#NSC 8 0 - 0 4 1 2 -
B075-12). REFERENCES
i. Durr,
F.E., Wallace, R.E.and C i t a r e l l a , R.V. (1983) C a n c e r (suppl B) i0, 3-11. 2. Koeller, J.and Eble, M. (1988) ~ 7, 574-581. 3. Shenkenberg, T . D . a n d Von Hoff, D.D. (1986) Ann. Intern.Med. 105, 67-81. 4 . L e n k , H . , Muller, U.and Janneber, S.(1987). A n t l c a n c e r Res. 7, 1257-1264. 5. Cheng, C.C.and Zee-Cheng, R.K.(1983) P _ r / l g ~ 2 0 , 83-118. 6. T e w e y , K . M . , R o w e , T.C., Yang, L.,Halligan, B.D.and Liu,L.F.(1984) Science 226, 466-468. 7. Hamel, E.,Lin, C.M. (1981) Arch. Biochem.BioDhvs.209, 29-40. 8. Kuriyama, R.and Sakai, H. (1974) J _ ~ 75, 463-471. 9.David-pfeuty, T., Simon, C.and Pantolon, D.(1979) 254, 11696-11702. 10. W e i s e n b e r g , R.C., Borisy, G.G.and Taylor, E.W. (1968) ~ J _ ~ 7, 4466-4478. 11. Evenson, D.P.Darznkiewicz,Z.,Stariano-Coico, L. T r a g a n o s , F . a n d Melamed, M.R.(1979) Cancer ~es. 39, 2574-2581. 12. K a p u s c i n s k i , J . , D a r z y n k i e w i c z , Z . , T r a g a n o s , F.and M e l a m e d M.R.(1984) Proc. Natl. Acad. Sci.. U.S.A.BI, 1361-1365. 13. Otto, A.M. (1986) Int. Rev. Cvtol. 109, 113-158. 14. Law, S.L., Chang, P . a n d Lin, C.H. (1991) Int. J. Pharm. 70, 1-7. 15. Dietel, M., Arps, H., Niendorf, A. (1989) [ A b s t r a c t ] Proc. Am. Assoc. Cancer Res. 30, 178. 16. Trittion, T.R.and Yee, G. (1982) ~ 217, 248-250.
123
Vol. 180, No. 1, 1991
October 15, 1991
I N H I B I T I O N OF M I C R O T U B U L E A S S E M B L Y IS A P O S S I B L E M E C H A N I S M OF A C T I O N OF M I T O X A N T R O N E Chi-Kuan
Ho*,Sai-Lung Law, H u n g C h i a n g * * , M a i - L i n g Chien-Chia Wang and Sheng-Yuan Wang
Hsu
Departments of Medical Research and Pathology** Veterans General Hospital, Taipei and Yang-Ming Medical College, Taipei Taiwan, Republic of China
Received August 28, 1991 SUMMARY: We have found that mitoxantrone can inhibit the polymerization of brain tubulinin a dose dependent manner. MXT hadrelatively high affinity for tubulin but had no appreciable effect on tubulin associated guanosinetriphosphatase (GTPase) activity nor couldit compete with vinblastine (VB) and colchicine (Col) for tubulinbinding sites. Furthermore, MXT (0.1-10 ~tM) is antiprolifcrative to cold-treated (0 ° C) epithelial cells afteronly brief exposure (30 rain). These results indicated that MXTis a microtubule inhibitory agent and can exert its anticellular effect through modulation of microtubule
assembly.
@ 1991 Academic Press, Inc.
Mitoxantrone broad
spectrum
(MXT) is a n e w a n t h r a c e n e d i o n e whiuh
antineoplastic
models and clinical
trials against
other malignant diseases than doxorubicin
activity
in
both
breast cancer
but has much
less potential
(5) and is a b l e to induce DNA breakage, (1,6). However, there
is no
between the potent
anticellular
of
effect
(2,3). Therefore, its
remains to be determined.
In the
exact
accumulative
an i n t e r c a l a t o r of
and i n h i b i t
acid b i o s y n t h e s i s
e f f e c t in v i v a
a n d a v a r i e t y of
(1-3) with p o t e n c y equal to or s u p e r i o r
cardiatoxic effect (4). MXT is d e s i g n e d to b e DNA
has p r o v e n
experimental
nucleic
direct c o r r e l a t i o n
M~r and its a n t i t u m o r mechanism
of
action
p r e s e n t s t u d y we h a v e s h o w n t h a t
*To whom all correspondence should bc addressed. A b b r e v i a t i o n s : MXT, m i t o x a n t r o n e ; T L C , t h i n l a y e r c h r o m a t o g r a p y ; D E A E , d i e t h y l a m i n o e t h y l ; P-G buffer, p h o s p h a t e - g l u t a m a t e buffer ; G T P a s e , guanosinetriphosphatase ;DMEM, Dubolco minimal essential medium; VB,vinblastine ; Col, colchicine. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
Vol. 180, No. 1, 1991
MXT can
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibit
reattachment
the p o l y m e r i z a t i o n
of e p i t h e l i a l cells
results
imply
that
mediated
through
of b r a i n t u b u l i n
and subsequent
the anti-cellular
restriction MATERIALS
AND
delay
the
cell g r o w t h . T h e s e
effect
of m i c r o t u b u l e
and
of
MXT
may
be
assembly.
METHODS
Inhibition o f Tubulin a s s e m b l y : Highly purified bovine brain t u b u l i n w a s p r e p a r e d as d e s c r i b e d b y Hamel and L i n (7) u s i n g D E 52 i o n i c e x c h a n g e chromatography followed by 3 cycles of polymerization and depolymerization and assembly of t u b u l i n w a s monitored by theviscometric m e t h o d of K u r i y a m a & S a k a i (8) i n the p r e s e n c e a n d a b s e n c e of v a r i o u s c o n c e n t r a t i o n s of M X T ( 2 - 5 0 ~M) or v i n b l a s t i n e (5 o r 10 ~M) a n d t h e e f f e c t o f M X T o n t u b u l i n associatedGTPase a c t i v i t y was d e t e r m i n e d b y T L C a s d e s c r i b e d b y D a v i d - P f e u t y et al (9) Electron Microscopy : Highly purified t u b u l i n (1-2 mg/ml) i n a s s e m b l y b u f f e r was i n c u b a t e d at 37°C for 30 m i n i n t h e p r e s e n c e o r a b s e n c e of M X T (10 ~M) and s a m p l e s w e r e d i l u t e d ( 1 : 1 ) . w i t h a buffer containing 2 Mhexylene g l y c o l (in 0 . 2 M M e s p H 6.9 + 1 . 0 m M M g C I 2) a n d m i c r o t u b u l e was r e v e a l e d u s i n g a s t a n d a r d negative s t a i n i n g t e c h n i q u e (8). Bindinu of M X T t o t u b u l i n :Tubulin was subjected to 7.5% polyacrylamide gel electrophoresis (PAGE) u n d e r n o n - d e n a t u r i n g conditions in T r i s - H C I b u f f e r pH 8 . 6 . F o l l o w i n g electrophor e s i s , i n d i v i d u a l slots w e r e s l i c e d a n d then t r e a t e d as d e s c r i b e d in t h e f i g u r e legend. The b i n d i n g of 3 H _ v i n b l a s t i n e (2 ~Ci/~M) or 3 H - c o l c h i c i n e (2 ~ C i / ~ M ) in the p r e s e n c e or a b s e n c e cf v a r i o u s d o s e s of M X T (1-100 f o l d excess) t o t u b u l i n w a s d e t e r m i n e d b y t h e DE-81 p a p e r discs m e t h o d of W e i s e n b e r g et al (10). A n t i c e l l u l a r effect : T L ( c y t o t r o p h o b l a s t i c ) , KB a n d W I S H c e l l s w e r e r e s u s p e n d e d at 2 x ~ 5/ml in D M E M plus 1 0 % f e t a l b o v i n e serum and gentamycin (20 ~g/ml) and t h e n i n c u b a t e d on ice for 6 h. The cells w e r e then i n c u b a t e d for an a d d i t i o n a l 30 m i n in t h e p r e s e n c e or a b s e n c e of M X T (0.1-10 ~M), w a s h e d 3X a n d p l a t e d o n t o 60 m m t i s s u e c u l t u r e s p l a t e s and t h e n i n c u b a t e d a t 3 7 ° C i n 5% CO 2. At v a r i o u s t i m e s , c e l l s w e r e observed u n d e r an i n v e r t e d m i c r o s c o p e f o r c e l l a t t a c h m e n t a n d c e l l n u m b e r cf a l l c u l t u r e s was d e t e r m i n e d at 48 h f o l l o w i n g i n c u b a t i o n by t r y p a n b l u e stain. RESULTS
Inhibition
of t u b u l i n
_
~
:
~s c o u l d b e
p o l y m e r i z a t i o n of t u b u l i n w a s or 10 ~M) similar effect
in p r o p o r t i o n to
of
that MXT
microscope.
of
A dense
observed when tubulin of M X T t h e Unlike enhance
VB
extent and
to d o s a g e . T h e
VB
could
inhibited
but
is
also
tubular
Col,
under
of MXT
inhibition
The
i, (2 is
inhibitory
the
structure
electron could
be
p o l y m e r i z e d (Fig 2A), however, in t h e p r e s e n c e
of p o l y m e r i z a t i o n
nor suppress
k i n e t i c s of
less efficient.
of
in F i g u r e
in t h e p r e s e n c e
be o b s e r v e d
network
seen
was greatly
over a
wide
GTPase
activity
dose
119
range, (Fig.4).
reduced MXT
could
(Fig.2B) . neither
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0 1 0.75~ ~ i , ~ ~ I ~50 "4 025 00Ir-----'''~ 5, 10 15 20 25 30 ,
,
rm
L
,
,
(m~)
F i q . i. The inhibitory effect of mitoxantrone on t h e p o l y m e r i z a t i o n of tubulin as a s s a y e d by the v i s c o m e t r i c method. P u r i f i e d bovine b r a i n t u b u l i n w a s a s s e m b l e d i n M e s b u f f e r at 37°C in the absence (i) and p r e s e n c e of 2,10 and 50 JiM (2-4) of MXT respectively or 10 ~ M of VB (5) . R e l a t i v e v i s c o s i t y was arbitrarily set at 0-i unit.
Binding
o f MXT
andtubulin
to
the intense blue tubulin
tubulin
was assessed
(in
concentration with albumin
color
15-25 but or
physical
modified
of MXT.
min under
:The by a
interaction
immunoblot
Fig 3 indicates
post-incubation) the same
transferrinwhich
conditions, are
known
.in MXT
between
assay that
MXT
based
MXT binds
proportion does
MXT-binding
not
to t o
react
proteins.
Fiu. 2. E l e c t r o n m i c r o g r a p h s of negatively s t a i n e d s p e c i m e n s of t u b u l i n p o l y m e r s s h o w i n g a dense n e t w o r k of m i o r o t u b u l e s t r u c t u r e f o l l o w i n g i n c u b a t i o n at 37°C for 30 m i n (A) a n d r e l a t i v e l y few t u b u l a r f i l a m e n t s w h e n t u b u l i n was a s s e m b l e d in the p r e s e n c e of i0 ~M of MXT (B). x16,000,
120
on
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
32p--G D P
32p--G T P
®
1
2
1
2
3
4
5
6
7
8
9
10
Fig. 3. The binding of MXT to tubulin as determined by a m o d i f i e d immunoblot assay. Tubulin in semi-preparative 7.5% polyacrylamide gel was electrophoresed under non-denatured conditions in TrisHCl buffer pH 8.6. Individual slots of the same gel were treated separately as follows: Lane i, stained withcoomassie blue stain; Lane 2, immunoblotted with a m o u s e anti-rat tubulin monoclonal a n t i b o d y using EIA and i n c u b a t e d with MXT (i00 ~g/ml) a f t e r transfer onto nitro-cellulose membrane (Lane 3). Three major a n d one minor MXT-reactive p r o t e i n bands as indicated by arrowheads were consistently observed. Fiq. 4. Assay for polymerization-dependent t u b u l i n - a s s o c i a t e d GTPase activity by TLCbased on the conversion of 32p-GTP to m p _ GDP. Bovine tubulin (2-5 mg/ml) was i n c u b a t e d with a s s e m b l y buffer only (Lane i) a n d in the presence of 2, i0 a n d 50 ~ M of VB (Lanes 2-4), MXT (Lanes 5-7) or Col (Lanes 8-10) respectively. Individual spots were revealed by autoradiography.
Furthermore,
protein-bound
indicating
that
preparation
is
the
M X T c o u l d be
binding
homogenous
reactive band), but
the
i s
in
same
elutedwith
reversible.
SDS-PAGE
preparation
(3 m a j o r a n d 1 m i n o r
; arrowheads)
lane
represent
which
concatamers. All
of t h e s e
are
anti-tubulin
reactive to
2).However,
based
not compete
with either
at a 1 0 0 - f o l d Effect
on
on the
excess
cell
bands
_:
structures of indirect
Following all cells
andanti-tubulin
a d h e r e n t in a b o u t
most
MXT-treated
least 8
h at
all
cells
the c y t o t o x i c
appreciable increase after
antibody
and
l a n e 3)
and
(Fig.3,
drugs,
lane
MXT
does
binding sites
even
cold were
immunofluorescence
however
these cultures
(Fig.3
tubulin
4
(Not shown).
revealed
withoutMXTbecame
reveals
in n o n - d e n a t u r e d
radioactive
Col for
tubulin
heterodimers
bindMXT
monoclonal
VB or
growth
cytospin cell smears
can
t r a c i n g of
cytoskeletal by
may
buffer
anti-tubulin
consistently
gel
i)
Our
( a single
protein bands (Fig.3,
P-G
48h
(Not
using
in
disrupted
suspension
tested.
.
as
C e l l s in c u l t u r e s
or cell death
shown)
the
acetone-fixed
2 h after incubation
doses
121
totally
antibody.
remained
in c e l l n u m b e r
treatment,
In
at 37°C for
addition,
was observed
at no in
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
Despite the is not
intense interest
completely
s u g g e s t s that
in MXT, its m e c h a n i s m
understood.
Accumulated
MXT is an a n t i - t o p o i s o m e r a s e
of a c t i o n
evidence II
thus
(11) d r u g
far
and c a n
inhibit nuclei acid synthesis (6,12). However,these
a c t i v i t i e s do
not
effect
correlate
well
with
its
in vivo
antitumor
(2,3)
suggesting that other mechanisms may be involved. We
have
shown in the p r e s e n t study that MXT c a n
p o l y m e r i z a t i o n of p u r i f i e d bovine b r a i n H o w e v e r , u n l i k e V B or C o l , M X T h a s n o associatedGTPase two t u b u l i n
has high a f f i n i t y
sites
(Not
of MXT for t u b u l i n
and
this
may be
it c o m p e t e w i t h
the
shown).In addition,
MXT
c o m p a r i n g to o t h e r k n o w n M X T
b i n d i n g p r o t e i n s such as a l b u m i n and shown)
tubulin (Figs 1&2) . effect on tubulin-
activity (Fig 4), nor does
drugs for binding
s u p p r e s s the
transferrin
theprobable
cause
of
(Fig 3 a n d
not
its a b i l i t y
to
interfere with tubulin assembly. The m i c r o t u b u l e supporting
system plays
network
conductor
for
for
cellular
s p e c u l a t i v e that MXT restriction
cell
surface
receptors
communication
can
e x e r t its
of m i c r o t u b u l e
membrane activity.
a key r o l e in m i t o s i s
as well as
(13). T h e r e f o r e
anticellular
effect
formation and m o d u l a t i o n
This h y p o t h e s i s
is
supported b y
observation that MXT is p r e f e r e n t i a l l y e n c a p s u l a t e d charged liposomes
which then become p o s i t i v e l y
a d d i t i o n , it
been
carcinoma system
has
cell
reported
line has
a n d is t h e
that
structural
it
is
through
of
plasma
our r e c e n t
by n e g a t i v e l y -
c h a r g e d (14).
a MXT-resistent
changes
the
In
gastric
in its m i c r o t u b u l e
(15).
Taken
together,
results
others s t r o n g l y suggest that to p e r t u r b
nucleic
s o l e l y by
that
doxorubicin
with our suggestion
with
can
be useful
for the
its
122
on
Aprevious
cytotoxic
action
(16)
accord
is in
related. Our
to a c c o u n t
of M X T a n d p r o v i d e cf more e f f e c t i v e
protocals against various neoplastic diseases.
of
ability
also t a r g e t
are c l o s e l y
explanation
design
and those
cytoskeleton.
surface
since the two drugs
d i v e r s i t y of the b i o l o g i c a l effects which may
MXT can
exert
the c e l l
a more d e t a i l e d
study
from its e s t a b l i s h e d
and the a s s o c i a t e d
interaction
findings offer
apart
acid b i o s y n t h e s i s ,
membranous Structures observation
of the p r e s e n t
for t h e
some c l u e s treatment
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT The authors fromthe
wish
National
to express g r a t i t u d e Science Council,
for t h e
financial support
Taiwan,R.O.C.
(#NSC 8 0 - 0 4 1 2 -
B075-12). REFERENCES
i. Durr,
F.E., Wallace, R.E.and C i t a r e l l a , R.V. (1983) C a n c e r (suppl B) i0, 3-11. 2. Koeller, J.and Eble, M. (1988) ~ 7, 574-581. 3. Shenkenberg, T . D . a n d Von Hoff, D.D. (1986) Ann. Intern.Med. 105, 67-81. 4 . L e n k , H . , Muller, U.and Janneber, S.(1987). A n t l c a n c e r Res. 7, 1257-1264. 5. Cheng, C.C.and Zee-Cheng, R.K.(1983) P _ r / l g ~ 2 0 , 83-118. 6. T e w e y , K . M . , R o w e , T.C., Yang, L.,Halligan, B.D.and Liu,L.F.(1984) Science 226, 466-468. 7. Hamel, E.,Lin, C.M. (1981) Arch. Biochem.BioDhvs.209, 29-40. 8. Kuriyama, R.and Sakai, H. (1974) J _ ~ 75, 463-471. 9.David-pfeuty, T., Simon, C.and Pantolon, D.(1979) 254, 11696-11702. 10. W e i s e n b e r g , R.C., Borisy, G.G.and Taylor, E.W. (1968) ~ J _ ~ 7, 4466-4478. 11. Evenson, D.P.Darznkiewicz,Z.,Stariano-Coico, L. T r a g a n o s , F . a n d Melamed, M.R.(1979) Cancer ~es. 39, 2574-2581. 12. K a p u s c i n s k i , J . , D a r z y n k i e w i c z , Z . , T r a g a n o s , F.and M e l a m e d M.R.(1984) Proc. Natl. Acad. Sci.. U.S.A.BI, 1361-1365. 13. Otto, A.M. (1986) Int. Rev. Cvtol. 109, 113-158. 14. Law, S.L., Chang, P . a n d Lin, C.H. (1991) Int. J. Pharm. 70, 1-7. 15. Dietel, M., Arps, H., Niendorf, A. (1989) [ A b s t r a c t ] Proc. Am. Assoc. Cancer Res. 30, 178. 16. Trittion, T.R.and Yee, G. (1982) ~ 217, 248-250.
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