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The effect of withaferin a, a natural steroidal lactone, on the fine structure of S-180 tumor cells
Cancer Letters, 2 (1976) 71--78 © Elsevier/North-Holland,BiomedicalPress
T H E EFFECT O F W I T H A F E R I N A, A N A T U R A L S T E R O I D A L LACTONE, O N T H E FINE S T R U C T U R E O F S-180 T U M O R CELLS
B. SHOHAT, A. SHALTIEL,* M. BEN-BASSATand H. JOSHUA Clinical Laboratory and J. Casper Department o f Pathology, Tel-Aviv University Snottier School of Medicine, Beilinson Medical Center, Petah Tiqva (Is;,acl)
(Received 22 March 1976) (Revised version received 14 May 1976)
SUMMARY Mouse sarcoma 180 (S-180) solid and ascites tumor cells were treated in vivo and in vitro with withaferin A and observed with the electron microscope. The compound wa~ found to effect the spindle microtubules of ceils in metaphase. An interesting finding was the double membranes surrounding the chromosomes in the treated ceils; probably the nuclei were reconstructed directly from the metaphase s~age in ~he in vivo withaferin A-treated cells. In addition the membranes of the cells in i~terphase were affected by in vivo or in vitro treatment with the c o m p o u n d
INTRODUCTION Withaferin A, a steroidal lactone isolated from the leaves of Withania s o m n i f e r ~ Dun. has been found to retaxd the growth of various experimental mouse tumors and to arrest the rapid proliferation of tumor ceils in metaphase [6,7]. In the present study the effect of this compound on the uItrastructure of resting and dividing cells was investigated by the aid of electron microscopy, The study was performed with S-180 solid and ascites tumor cells. MATERIALS ANDMETHODS
The solid S-180 tumor was transplanted by the standard ~rocar procedure, with tumor fragments implanted subcutaneously in the axillary region of Swiss or DBA/2 mice weighing t 8 - - 2 2 g. The a~ci~ic form of S-180 was inoculated intraperigoneally (i.p.) using 0.1 ml of tumor suspension (2 X 106/mouse). .(n vivo t r e a t m e n t
The S-180 solid and ascites-tumor-bearing mice were treated i.p. w,.th one single or several daily doses of withaferin A, 30 mg/kg or 20 mg/kg body *' Thiswork is part of tha~ done towards the M.Sc. degree by A. ShaltieL
72 weight respectively. Treatment was started 24 h after tumor implantation. Groups of 113--30 mice were used for each dose level. Samples of solid or ascites t,~,mor cells were taken 2, 4, 6, 8, 24, 48, 72 and 120 h after withaferin A administration and the mice were observed until death.
L~ vitro treatment Freshly-harvested, 7 to 10-day-old S-180 ascites cells were sedimented and resuspen;ded in Tc-199 medium supplemented with 10% fetal calf serum. A]icluo~s of 2 × I 0 ~ cells/ml were incubated for 2--8 h with different concentmti[ons of withaferin A from 5 ug to 50 #g/cc in a shaking water bath at 37°C. Samples of the t u m o r cells were taken after 2, 4, 6 and 8 h.
Preparation of samples for electron and light microscopy 7.'u,n0r samples obtained from ~he in vitro and in rive experiments were divided into two portions: one was used for light microscopy and one for electron microscopy. For the light microscopy the ascitic tumor samples were prepared with a Shandon cytocentrffuge; touch preparations were made from the solid tumors. They were stained with May-Gr~nwald-Giemsa stain. For the electron microscopy, both ascitic cells and solid tumor tissues were fixed in 2% bu~fered glutaraldehyde, post fixed in 1% osmium tetroxide, dehydrated am[ er.~bedded in Epon 812. About 1000 treated mitotic ceils were studied. Five [ locks containing mitotic figures were chosen at random for serial sectioJ.ail~,g. RF-&~ULT~
Light microscopy Th.~ untreated (control) $-180 solid t u m o r cells exhibited an irregular shape, I,~ge nuclei and mitotic activi'~y. The untreated S-180 ascitic cells had a rounded shape with small cytoplasmic vacuoles at the periphery. The nuclei were rotmd or oval and co~Ltained one to several nucleoli. After in rive treatment of S~180 ascites-beaxing mice w:ith one single dose of withafer;[n A (30 mg/kg body weight), a marked decrease of tumor cell population wlith an increase of polymorphonuclears and macrophages was always found. This treatment resulted in a significant prc,longation (50%) in the surviwl time of treated mice as compared with the un~reated controls (Fig. t). Furthermore, 7 oul: of 54 mice remained without tumors. After in rive or in vitro treatment oi' S-1S0 ascites cells with withaferin A, there was vacuolization of cytoplasm~ karyolysis of resting ceils and ~ r e s t at m,etaphase of cell divi:;ion. Vacuolizati.on of cytoplasm was seen as early as 2 h after in rive treatment and 30 rain after in vitro treatment. Karyolysis of nuclei was found 24 h after in rive tm~tment and 3 h after in vitro ~Lreatment. A:rres~;at metaphase was found 4 h after in rive treatment. A longer exposure and repeated administration in rive were nece~sary to obtain similar degenerative changes in ~Lhesolid form of the tumor.
73
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I
80
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b
...,
,,, ~o
>=
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i
I
2o-
i i
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30
40
DAYS
Fig.~1. Effect of withaferin A, 30 mg/kg body weight in a single dose, on the survivaltime of ,¢l-180a~e~tes-bearingmice.
Electron microscopy Control solid S-1S0 cells. The ultrastructure of these cells was similar to those recently described by Zuckerberg [9] except that desmosomes were n o t found. Mitotic figures of S-180 cells were common. The ultrastructure of the ascitic form of S-180 was similar to that described by Molnar et aL [ 51 except that v~'us-like particles were not observed. Effect of withaferin A on S-180 cells. The electron micrographs of in vivo trevted S-180 solid and ascites cel~s showed marked changes of the memhranat system of cells !in in~erphase, consilsting of: ( l ) separation of the two membranes of the nucleus with formation of membranai sacs {Fig. 2); (2) distension or dissolution of mi:~ochondrial cristae; (3) labilisation of inner cytoplasm[Lc membranes with the consequent appearaace of autophagic vacuoles followed by complete autolysis of the cytoplasm and rupture of the outer plasma membrane. The nuclei showed condensa*;ion of the chromatin and/or a marked loss of chromatin and vacuolisation (Fig, 8).
74
Fig. 2. Withaferin A-treated S-180 ascites cell (10 mg/kg body weight) 24 h after treatment. Separation of me~mbr~.nessurrounding nucleus with formation of mcmbranal sae~,;(X 10,260}.
Cell:s in m e t a p b a s e s h o w e d n o p o l e - t o - p o l e or p o l e - t o - c h r o m o s o m e spindle mic~.~otubules; h o w e v e r , f r a g m e n t s o f t h e m i c r o t u b u l e s , s o m e o f t h e m connec~:ed t o t h e k i n e t o c h o r e , were o b s e r v e d . F o u r o f t h e five cells s t u d i e d b y serial sectioning did r~ot s h o w m i c r o t u b u l e s b u t t h e f i f t h cell s h o w e d fragm e n t s of miclro:~ubules c o n n e c t e d t o t h e centriole, Also seen were d o u b l e m e r a b r a n e s s u n ' o u n d i n g t h e c h r o m o s o m e s , or p~-t o f t h e m , in metap:hase (Fig, 4). tn prerJarations o b t a i n e d f r o m w i t h a f e r i n A t r e a t e d S-1S0 ascitesbearing m i c e , wlhich s h o w e d n o t u m o r g r o w t h , t h e r e was a clustering o f m a c r o p h a g e s a r o u n d t h e t u m o r cells (Fig. 5) a n d d e s t r u c t i o n of t h e t u m o r cells t h u s s u n o u n d e d was evident.
Fig. 3. WithaferJ~n-~k-treated S-180 ascitcs cell 4 days after treatment. Autophaglc wmuole, dissolution of mitoehondrial crlstae, autolysis of cytoplasm and rupture of outer pl:asma membrane (× 6800). Fig. ,t. Withai~erln A-treated S-180 ~eitea tumor 48 h after treatment. Doable sheeted membranes around chromosomes (× 10,200).
75
Fig.
Fig. 4
76
Fig. 5. Clustering of macrophages around S-180 tumor cell in withaferin A-treated sample. Advanced degenerative changes in the tumor cell (× 3300). In vitro treatment of the t u m o r cells with withaferin A resulted in identical signs of damage. A striking effect on the membranal system was clearly evident, There was separation of the two nuclear membranes, autophagic vacuole formation, and rupture of the outer cell membrane with cytoplasmic material visible outside 15hecells.. DISCUSSION
The stathmokinetic effect of withaferin A was established in previous investigations [6,7 ]; however, nothing was known about the organelle changes which occur when cell division is arrested at metaphase. The present study has shown that withaferin A ,disrupts the microtubules of the mitotic spindle. This compound may therefore be added to the list of well..known inhibitors of the mi¢otic spindle, e.g., colchicine, vinblastine and griseofutvin [2--4]. In some of the cells arrested at metaphase a double membrane was found around the dispersed chromosomes. Fvesumably, reconstruction of the nuclei from the metaphase stage had taken place. The effect of withafelcin A was not limited to cells in metaphttse but was also seen in the membrane systems of
77 t h e resting cells. Similar changes have b e e n o b s e r v e d in lysolecithin-tzeated t u m o r cells [8], T h e interaction b e t w e e n t h e m a c r o p h a g e s and t u m o r cells is similar t o t h a t described b y Brandes e t al. [1] in l e u k e m i c cells t r e a t e d w i t h c y c l o p h o s p h a m i d e and vitamin A. All o f t h e above-described findings p r o v i d e e v i d e n c e clarifying t h e antit u m o r action previously r e p o r t e d t o b e e x e r t e d b y w i t h a f e r i n A. ACKNOWLEDGEMENTS Our t h a n k s are due t o t h e N a t i o n a l C a n c e r I n s t i t u t e , National I n s t i t u t e o f Health, U.S. ( A g r e e m e n t No. 06-124-1) a n d t h e U n i t e d States--Israel Binational Science F o u n d a t i o n , J e r u s a l e m , Israel ( G r a n t No. 560) f o r t h e i r financial s u p p c r t . REFERENCES 1 Brandes, D., Anton, E. and Lain, K.W. (1967) Studies of L1210 leukemia. II. Ultrastructural and cytochemical changes after treatment with eyclophosphamide and vitamin A. J. Natl. Cancer Inst., 39, 385--421. 2 Brinkley, B.R., Stubblefield, E. and Hsu, T.C. (1967) The effects of eolcemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro. J. Ultrastrueture Res., 19, 1--18. 3 Brinkley, B.R. and Cartwright, J. (1971) UItra~tructural analysis of mitotic spindle elongation in mammalian cells in vitro. J. Cell Biol., 50, 416--431: 4 Malawista, S.E., Sato, H. and Bensch, K.G. (1968) Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science, 160, 770--772. 5 Molnar, Z. and I~ekesi, J.G. (1972) Effects of D-glucosamine, D-mannosarnine, and 2-deoxy-D-glucose on the ultrastructure of ascltes tumor cells in vitro. Cancer Res., 32, 380--389. 6 Shohat, R., Gitter, S. and Lavie, D. (1970) Effect cf withaferin A on Ehrtich ascites tumor cells -- cytological observations. Intl. J. Cancer,, 5, 244--252. 7 Shohat, B. and Joshua, H. (1971) Effect of withaferin A on Ehrlich ascites tumor ceils. II. Target tumor cell destruction in vlvo by immune activation. Intl. J~ Cancer, 8, 487--496. 8 Wilkinson, P.J. and Cater, D.B. (1969) An electron-microscopestudy of the effects of lysolceithln on B.P. 8 aseites tumor ceils and phagocytes of mice compared with the effects of a specific anti-tumor serum plus complement. J. Pathol., 97, 219--230. 9 Zuekerberg, C. (1973) Ultra.structure of Sarcoma 180. Cancer Res., 33, 2278--2282.
T H E EFFECT O F W I T H A F E R I N A, A N A T U R A L S T E R O I D A L LACTONE, O N T H E FINE S T R U C T U R E O F S-180 T U M O R CELLS
B. SHOHAT, A. SHALTIEL,* M. BEN-BASSATand H. JOSHUA Clinical Laboratory and J. Casper Department o f Pathology, Tel-Aviv University Snottier School of Medicine, Beilinson Medical Center, Petah Tiqva (Is;,acl)
(Received 22 March 1976) (Revised version received 14 May 1976)
SUMMARY Mouse sarcoma 180 (S-180) solid and ascites tumor cells were treated in vivo and in vitro with withaferin A and observed with the electron microscope. The compound wa~ found to effect the spindle microtubules of ceils in metaphase. An interesting finding was the double membranes surrounding the chromosomes in the treated ceils; probably the nuclei were reconstructed directly from the metaphase s~age in ~he in vivo withaferin A-treated cells. In addition the membranes of the cells in i~terphase were affected by in vivo or in vitro treatment with the c o m p o u n d
INTRODUCTION Withaferin A, a steroidal lactone isolated from the leaves of Withania s o m n i f e r ~ Dun. has been found to retaxd the growth of various experimental mouse tumors and to arrest the rapid proliferation of tumor ceils in metaphase [6,7]. In the present study the effect of this compound on the uItrastructure of resting and dividing cells was investigated by the aid of electron microscopy, The study was performed with S-180 solid and ascites tumor cells. MATERIALS ANDMETHODS
The solid S-180 tumor was transplanted by the standard ~rocar procedure, with tumor fragments implanted subcutaneously in the axillary region of Swiss or DBA/2 mice weighing t 8 - - 2 2 g. The a~ci~ic form of S-180 was inoculated intraperigoneally (i.p.) using 0.1 ml of tumor suspension (2 X 106/mouse). .(n vivo t r e a t m e n t
The S-180 solid and ascites-tumor-bearing mice were treated i.p. w,.th one single or several daily doses of withaferin A, 30 mg/kg or 20 mg/kg body *' Thiswork is part of tha~ done towards the M.Sc. degree by A. ShaltieL
72 weight respectively. Treatment was started 24 h after tumor implantation. Groups of 113--30 mice were used for each dose level. Samples of solid or ascites t,~,mor cells were taken 2, 4, 6, 8, 24, 48, 72 and 120 h after withaferin A administration and the mice were observed until death.
L~ vitro treatment Freshly-harvested, 7 to 10-day-old S-180 ascites cells were sedimented and resuspen;ded in Tc-199 medium supplemented with 10% fetal calf serum. A]icluo~s of 2 × I 0 ~ cells/ml were incubated for 2--8 h with different concentmti[ons of withaferin A from 5 ug to 50 #g/cc in a shaking water bath at 37°C. Samples of the t u m o r cells were taken after 2, 4, 6 and 8 h.
Preparation of samples for electron and light microscopy 7.'u,n0r samples obtained from ~he in vitro and in rive experiments were divided into two portions: one was used for light microscopy and one for electron microscopy. For the light microscopy the ascitic tumor samples were prepared with a Shandon cytocentrffuge; touch preparations were made from the solid tumors. They were stained with May-Gr~nwald-Giemsa stain. For the electron microscopy, both ascitic cells and solid tumor tissues were fixed in 2% bu~fered glutaraldehyde, post fixed in 1% osmium tetroxide, dehydrated am[ er.~bedded in Epon 812. About 1000 treated mitotic ceils were studied. Five [ locks containing mitotic figures were chosen at random for serial sectioJ.ail~,g. RF-&~ULT~
Light microscopy Th.~ untreated (control) $-180 solid t u m o r cells exhibited an irregular shape, I,~ge nuclei and mitotic activi'~y. The untreated S-180 ascitic cells had a rounded shape with small cytoplasmic vacuoles at the periphery. The nuclei were rotmd or oval and co~Ltained one to several nucleoli. After in rive treatment of S~180 ascites-beaxing mice w:ith one single dose of withafer;[n A (30 mg/kg body weight), a marked decrease of tumor cell population wlith an increase of polymorphonuclears and macrophages was always found. This treatment resulted in a significant prc,longation (50%) in the surviwl time of treated mice as compared with the un~reated controls (Fig. t). Furthermore, 7 oul: of 54 mice remained without tumors. After in rive or in vitro treatment oi' S-1S0 ascites cells with withaferin A, there was vacuolization of cytoplasm~ karyolysis of resting ceils and ~ r e s t at m,etaphase of cell divi:;ion. Vacuolizati.on of cytoplasm was seen as early as 2 h after in rive treatment and 30 rain after in vitro treatment. Karyolysis of nuclei was found 24 h after in rive tm~tment and 3 h after in vitro ~Lreatment. A:rres~;at metaphase was found 4 h after in rive treatment. A longer exposure and repeated administration in rive were nece~sary to obtain similar degenerative changes in ~Lhesolid form of the tumor.
73
f 00'~-,.. ,>-.-.--,,.~ wr'I'HAFERIN A
I
80
[
b
...,
,,, ~o
>=
,t
i
I
2o-
i i
OL.
I0
EO
30
40
DAYS
Fig.~1. Effect of withaferin A, 30 mg/kg body weight in a single dose, on the survivaltime of ,¢l-180a~e~tes-bearingmice.
Electron microscopy Control solid S-1S0 cells. The ultrastructure of these cells was similar to those recently described by Zuckerberg [9] except that desmosomes were n o t found. Mitotic figures of S-180 cells were common. The ultrastructure of the ascitic form of S-180 was similar to that described by Molnar et aL [ 51 except that v~'us-like particles were not observed. Effect of withaferin A on S-180 cells. The electron micrographs of in vivo trevted S-180 solid and ascites cel~s showed marked changes of the memhranat system of cells !in in~erphase, consilsting of: ( l ) separation of the two membranes of the nucleus with formation of membranai sacs {Fig. 2); (2) distension or dissolution of mi:~ochondrial cristae; (3) labilisation of inner cytoplasm[Lc membranes with the consequent appearaace of autophagic vacuoles followed by complete autolysis of the cytoplasm and rupture of the outer plasma membrane. The nuclei showed condensa*;ion of the chromatin and/or a marked loss of chromatin and vacuolisation (Fig, 8).
74
Fig. 2. Withaferin A-treated S-180 ascites cell (10 mg/kg body weight) 24 h after treatment. Separation of me~mbr~.nessurrounding nucleus with formation of mcmbranal sae~,;(X 10,260}.
Cell:s in m e t a p b a s e s h o w e d n o p o l e - t o - p o l e or p o l e - t o - c h r o m o s o m e spindle mic~.~otubules; h o w e v e r , f r a g m e n t s o f t h e m i c r o t u b u l e s , s o m e o f t h e m connec~:ed t o t h e k i n e t o c h o r e , were o b s e r v e d . F o u r o f t h e five cells s t u d i e d b y serial sectioning did r~ot s h o w m i c r o t u b u l e s b u t t h e f i f t h cell s h o w e d fragm e n t s of miclro:~ubules c o n n e c t e d t o t h e centriole, Also seen were d o u b l e m e r a b r a n e s s u n ' o u n d i n g t h e c h r o m o s o m e s , or p~-t o f t h e m , in metap:hase (Fig, 4). tn prerJarations o b t a i n e d f r o m w i t h a f e r i n A t r e a t e d S-1S0 ascitesbearing m i c e , wlhich s h o w e d n o t u m o r g r o w t h , t h e r e was a clustering o f m a c r o p h a g e s a r o u n d t h e t u m o r cells (Fig. 5) a n d d e s t r u c t i o n of t h e t u m o r cells t h u s s u n o u n d e d was evident.
Fig. 3. WithaferJ~n-~k-treated S-180 ascitcs cell 4 days after treatment. Autophaglc wmuole, dissolution of mitoehondrial crlstae, autolysis of cytoplasm and rupture of outer pl:asma membrane (× 6800). Fig. ,t. Withai~erln A-treated S-180 ~eitea tumor 48 h after treatment. Doable sheeted membranes around chromosomes (× 10,200).
75
Fig.
Fig. 4
76
Fig. 5. Clustering of macrophages around S-180 tumor cell in withaferin A-treated sample. Advanced degenerative changes in the tumor cell (× 3300). In vitro treatment of the t u m o r cells with withaferin A resulted in identical signs of damage. A striking effect on the membranal system was clearly evident, There was separation of the two nuclear membranes, autophagic vacuole formation, and rupture of the outer cell membrane with cytoplasmic material visible outside 15hecells.. DISCUSSION
The stathmokinetic effect of withaferin A was established in previous investigations [6,7 ]; however, nothing was known about the organelle changes which occur when cell division is arrested at metaphase. The present study has shown that withaferin A ,disrupts the microtubules of the mitotic spindle. This compound may therefore be added to the list of well..known inhibitors of the mi¢otic spindle, e.g., colchicine, vinblastine and griseofutvin [2--4]. In some of the cells arrested at metaphase a double membrane was found around the dispersed chromosomes. Fvesumably, reconstruction of the nuclei from the metaphase stage had taken place. The effect of withafelcin A was not limited to cells in metaphttse but was also seen in the membrane systems of
77 t h e resting cells. Similar changes have b e e n o b s e r v e d in lysolecithin-tzeated t u m o r cells [8], T h e interaction b e t w e e n t h e m a c r o p h a g e s and t u m o r cells is similar t o t h a t described b y Brandes e t al. [1] in l e u k e m i c cells t r e a t e d w i t h c y c l o p h o s p h a m i d e and vitamin A. All o f t h e above-described findings p r o v i d e e v i d e n c e clarifying t h e antit u m o r action previously r e p o r t e d t o b e e x e r t e d b y w i t h a f e r i n A. ACKNOWLEDGEMENTS Our t h a n k s are due t o t h e N a t i o n a l C a n c e r I n s t i t u t e , National I n s t i t u t e o f Health, U.S. ( A g r e e m e n t No. 06-124-1) a n d t h e U n i t e d States--Israel Binational Science F o u n d a t i o n , J e r u s a l e m , Israel ( G r a n t No. 560) f o r t h e i r financial s u p p c r t . REFERENCES 1 Brandes, D., Anton, E. and Lain, K.W. (1967) Studies of L1210 leukemia. II. Ultrastructural and cytochemical changes after treatment with eyclophosphamide and vitamin A. J. Natl. Cancer Inst., 39, 385--421. 2 Brinkley, B.R., Stubblefield, E. and Hsu, T.C. (1967) The effects of eolcemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro. J. Ultrastrueture Res., 19, 1--18. 3 Brinkley, B.R. and Cartwright, J. (1971) UItra~tructural analysis of mitotic spindle elongation in mammalian cells in vitro. J. Cell Biol., 50, 416--431: 4 Malawista, S.E., Sato, H. and Bensch, K.G. (1968) Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science, 160, 770--772. 5 Molnar, Z. and I~ekesi, J.G. (1972) Effects of D-glucosamine, D-mannosarnine, and 2-deoxy-D-glucose on the ultrastructure of ascltes tumor cells in vitro. Cancer Res., 32, 380--389. 6 Shohat, R., Gitter, S. and Lavie, D. (1970) Effect cf withaferin A on Ehrtich ascites tumor cells -- cytological observations. Intl. J. Cancer,, 5, 244--252. 7 Shohat, B. and Joshua, H. (1971) Effect of withaferin A on Ehrlich ascites tumor ceils. II. Target tumor cell destruction in vlvo by immune activation. Intl. J~ Cancer, 8, 487--496. 8 Wilkinson, P.J. and Cater, D.B. (1969) An electron-microscopestudy of the effects of lysolceithln on B.P. 8 aseites tumor ceils and phagocytes of mice compared with the effects of a specific anti-tumor serum plus complement. J. Pathol., 97, 219--230. 9 Zuekerberg, C. (1973) Ultra.structure of Sarcoma 180. Cancer Res., 33, 2278--2282.