The effect of withaferin A on plant cells

Europ. J. Cancer Vol. 7, pp. 561-563. Pergamon Press 1971. Printed in Great Britain

The Effect of Withaferin A on Plant Cells B. SHOHAT and H. J O S H U A The Clinical Laboratory, Beilinson Medical Centre, Petah Tikva, Israel A b s t r a c t - - W i t h a f i ~ n A in a concentration of 0.01-0.5% growth of Allium cepa roots. Furthermore, withaferin A induced lateral root formation on treated roots. Microscopical examination root tip cells showed a clear arrest at metaphase as soon as 2 hr

INTRODUCTION

solutions were changed every two days. Daily measurements of the Allium cepa roots were performed during a period of 14 days. At 2, 24 and 48 hr intervals several root tips from each experiment were carefully removed, fixed in Carnoy solution and stained with acetocarmine. About 1000 cells were counted for each treatment. The mitotic index expresses the number of mitoses per 1000 cells counted.

WITHAFERINA C28H3806, a steroidal lactone obtained from Withania somnifera Dun, has been shown to have carcinostatic properties [1, 2]. More recently it was demonstrated that

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in water inhibited the c-tumor formation and of withaferin A-treated after treatment.

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RESULTS Treatment with withaferin A in a concentration of 0.01 ~o highly inhibited the growth of Allium cepa roots 2 days after treatment (Fig. 1). The inhibition of root tip growth obtained by the highest concentration of

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HO withaferin A produced a mitotic arrest in the metaphase of dividing Ehrlich ascites carcinoma cells as well as in Hela cells [3-5]. In order to clarify further the effects of withaferin A on cell division the actions of this chemical on the structure and behaviour of root tip cells of Allium cepa was investigated.

Control

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MATERIAL AND METHODS

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Small bulbs of Allium cepa were grown in tap water at 20°C i n t h e dark. When roots were 1 cm long they were divided into several series of 2-4 bulbs each, placed into jars containing the following solution (1) tap water, (2) withaferin A in concentration of 0.01 to 0"5~o in water, (3) colchicine 0.01 ~o in water, (4) colchicine and withaferin A in concentration as above in water. These series of experiments were repeated three times. The

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Fig. 2. Graph of the growth in length of roots treated by withaferin A 0.5%, withaferin A and colchicine, and colchidne alone, as compared with control.

Accepted 17 May 1971. 561

562

B. Shohat and H. Joshua

withaferin A tested (0"570) as compared to withaferin and colchicine, colchicine alone and control, is shown in Fig. 2. All points shown on reaction curves are the means of not less than three root tips per point. Microscopical examination of aceto-carmine stained smears preparations of Allium cepa roots (Fig. 3) revealed numerous mitoses at metaphase with scattered chromosomes 2 hr after withaferin A treatment. Structural abnormalities such as aberrant chromosomes at anaphase as well as enlargement of cells and micronuclei were seen at the end of 48 hr of treatment. The mitotic index (Fig. 4) was elevated already 2 hr after withaferin A treatment, and a high percentage of cells in metaphase was still present after 24 hr. The mitotic index started to drop at the end of 4 8 h r , probably due to escape of mitotic inhibition, as suggested by the reappearance of anaphase figures at this time. [ ] Withoferin A [ ] Control

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Fig. 4.

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Mitotic indices of Allium cepa roots exposedfor 48 hr to 0.01% withaferin A as compared to control.

Twelve days after exposure to withaferin A set at the highest concentration used (0.5 7o), numerous lateral roots appeared (Fig. 5). Table 1.

Three days later, enlargements of root tips were seen (Fig. 6), similar to the so called c-tumours obtained after colchicine treatment and attributed to undiscriminated multidirectional growth of the cells. Allium cepa roots treated with colchicine alone showed c-tumor formation at the end of the second day but no side roots were ever formed. Addition of withaferin A 0.5 7o simultaneously with colchicine 0.01 70 greatly inhibited c-tumor formation in number and in time of appearance (Table 1 and Fig. 7). DISCUSSION

The findings of the present study clearly indicate that withaferin A acts as a mitotic inhibitor on plant cells even at the low concentration of 0.01~. The mitotic arrest at metaphase and the appearance of c-tumors closely resemble the effect of colchieine [6]. However, the lagging chromosomes at anaphase suggested that even after a prolonged arrest at metaphase, some of the cells could continue to anaphase and telophase. The striking formation of lateral roots in all experiments with withaferin A could be compared to the effects of naphthalene acetic acid as described by Levan and Lofty [7]. These authors have shown that phytohormones such as naphthalene acetic acid and indole acetic acid initiate the secondary root meristem formation along the pericycle, and induce transversely oriented mitoses. Another point of similarity between these compounds and withaferin A resides in the antagonistic effects to colchicine with respect to c-tumor formation. Although witha-

Effect of withaferin A on growth of Allium cepa roots

Time (days) Control 2 days 6 days 14 days Colchicine 0.01% 2 days 6 days 14 days Withaferin A 0.5% 2 days 6 days 14 days Colchicine 0.01% and withaferin A 0.5% 2 days 6 days 14 days

Length of roots (era)

No. of c-tumors on each bulb

Appearance of lateral roots

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0 0 Lateral roots on c-tumors

Fig. 1. Inhibition of growth of Al&m cepa root after 48 hr of treatment with 0.01% withaferin A (right root) as compared to control (left root).

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Aceto-carmine A. B. C. D.

stained smear preparations

of root tip exposed to 0.01%

withaferin

Withaferin A treated cells arrested at metaphase 2 hr after treatment, Lagging chromosomes at anapha.re 48 hr after mithaferin A treatment, Micronuclei 4% hr after treatment,

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48 hr x 1000.

Fig. 5. Bulb of Allium cepa exfjosed IO @So, withaferin A for 12 days. Laferal root,formntion.

Fi,q. 6. Bulb qf‘ Allium cepa exposed to 0.5O,, zcithqferin A,for I5 dqw. c- Tumorfo~mation.

Fig. 7. Bulbs of Allium cepa exposed to 0.01 yi colchicine and to 0.0 196 colchicine and @40,/, withafeerin A for 48 hr. c- Tumors are already formed on the c&hi&e treated bulbs while no c-tumors appeared in the withaferin A and colchicine treated bulbs.

The Effect of Withaferin A on Plant Cells ferin A per se causes c-tumor formation, the combined treatment results in a delay of c-tumor formation. These findings m a y indicate that the c-tumors of withaferin A and colchicine are induced by different mechanisms. The lagging chromosomes at anaphase and formation of micronuclei induced by withaferin A m a y be due to the arrest in metaphase "per se" (colchicine like effect) and/or to a direct deleterious effect on the chromosomes similar to the

1. 2.

3. 4. 5. 6. 7. 8.

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changes induced by nitrogen mustard, T E M , azaserine, actinomycin C and actinomycin D on meristem of Allium cepa, as described by T r u h a u t and Deysson [8].

Acknowledgements--Our thanks are due to Prof. D. Lavie from the Weizman Institute of Science, Rehovoth, for providing us with withaferin A, and to Mrs. Kay Norton, Head of Department of Photography of Beilinson Hospital, for the photographs.

REFERENCES B. SHOHAT,S. GITTER, A. ABRAHAMand D. LAVXE,Anti-tumor activity of withafcrin A. Cancer Chemother. Rep. 51, 271 (1967). S . M . KUPCHAN, R. W. DOSKOTCH, P. BOLLINGER,A. T. McPHAIL, G. A. SIM and J. A. S. RENAULD, The isolation and structural elucidation of a novel steroidal tumor inhibitor from Acnistus arborescens. J. Amer. chem. Soc. 87, 5825 (1965). B. SHOHAT,S. GITTER and D. LAVIE, Effect ofwithaferin A on Ehrlich ascites tumor cells--cytological observations. Int. J. Cancer 5~ 244 (1970). B. SHOHAT, S. GITTER and D. LAVIE, Proceedings of Second International Symposium on Animal and Plant Toxins, Israel (Feb., 1970). J. PALYI, E. TYIHAKand V. PALYI, Cytological effects of compounds isolated from Withania somnifera Dun. Herba Hungarica 8, 73 (1969). A. LEVAN, The effect of colchicine on the root mitosis in Allium. Hereditas 24, 471 (1938). A. LEVANand T. LOTFY, Naphthalene acetic acid in the Allium test. Hereditas 35, 337 (1949). R.. TRUHAUT and G. DEYSSON, Sur l'int6rdt de l'utilisation de la cellule v6gfitale comme r~actifd'dtude des compos~s dits radiomimdtiques employ6s en chimiothdrapie anticancdreuse. Bull. Cancer 44, 22 (1957).