Aryltetralin Lignans Inhibit Plant Growth by Affecting the Formation of Mitotic Microtubular Organizing Centers

Pesticide Biochemistry and Physiology 72, 45–54 (2002) doi:10.1006/pest.2002.2582, available online at http://www.idealibrary.com on

Aryltetralin Lignans Inhibit Plant Growth by Affecting the Formation of Mitotic Microtubular Organizing Centers A. Oliva,* R. M. Moraes,† S. B. Watson,‡ S. O. Duke,‡ and F. E. Dayan‡,1 *Department of Life Science, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy; †National Center for the Development of Natural Products, School of Pharmacy, University of Mississippi, University, Mississippi 38677; and ‡United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, P.O. Box 8048, University, Mississippi 38677 Received July 5, 2001; accepted October 17, 2001 The aryltetralin lignans podophyllotoxin, ␣ -peltatin, and ␤ -peltatin, their respective O-␤ -D-glucosides, and the semisynthetic derivative etoposide were tested for phytotoxicity. The aglycones were more potent inhibitors than their respective glucosides, and podophyllotoxin was the most active natural lignan tested. These compounds were more active against rye (Lolium multiflorum L.) and onion (Allium cepa L.) than lettuce (Lactuca sativa L.). The semisynthetic lignan etoposide was more active than any of the natural analogues and was phytotoxic to both monocotyledonous and dicotyledonous species. Inhibition of root growth was the main developmental response observed on plants tested with the lignans. At the cellular level, podophyllotoxin and etoposide caused similar symptoms in actively dividing meristematic cells of onion root tips. All phases of mitosis were inhibited by nearly 50%, relative to the controls. Both compounds also induced abnormal star anaphase chromosomal configurations. While the precise molecular mechanism of action of these compounds remains to be identified in plants, a primary effect is the alteration of the formation of the spindle microtubular organization centers, resulting in the formation of multiple spindle poles and an asymmetrical convergence of the chromosomes. 䉷 2002 Elsevier Science (USA) Key Words: podophyllotoxin; ␣ -peltatin; ␤ -peltatin; etoposide; natural products mode of action; mitotic inhibitors.

INTRODUCTION

recently, the antimalarial sesquiterpene lactone artemisinin and certain anticancer quassinoids were found to cause abnormal mitotic configurations in onion root tips (10, 11). Many lignans have antimicrobial, antiviral, herbicidal, or antifeedant activities that are thought to participate in plant defense mechanisms against biotic stresses (12–16). For example, physical damage to the leaves of mayapple (Podophyllum peltatum L.) causes an accumulation of podophyllotoxin glucoside (2) and its more potent aglycone (1) (Fig. 1), suggesting that 1 may play a role as a feeding deterrent. Lignans are derived from the phenylpropanoid pathway and are widely distributed in plants (17). Aryltetralins, a particular class of lignans, are particularly abundant in plants of the genus Podophyllum (Berberidaceae). Lignans commonly found in Podophyllum species are shown

Thousands of potential herbicide target sites exist. However, commercial herbicides have only about 20 mechanisms of action, with approximately a quarter of the marketed products directly disrupting mitosis. While some herbicides affect mitosis indirectly by disrupting cell wall/cell plate formation (1–4), most mitotic inhibitors affect microtubule formation directly (5). The dinitroaniline herbicides prevent the polymerization of the free tubulin subunits into microtubules (6, 7), whereas carbamate herbicides affect the organization of spindle microtubules (8). Natural phytotoxins are also known to affect mitosis of plants. These have been reviewed by Vaughn and Vaughan (9). More 1 To whom correspondence should be addressed. Fax: (662) 915-1035. E-mail: [email protected].

45 0048-3575/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.

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ARYLTETRALIN LIGNANS INHIBIT PLANT GROWTH

FIG. 1. Structures of aryltetralin lignans and their O-␤ -D-glucosides (1–6) present in Podophyllum peltatum and the antineoplastic drug etoposide (7) derived from podophyllotoxin.

in Fig. 1. These lignans can exist as aglycones, but are often stored O-␤ -D-glucosides in plants. Mayapple is a perennial dicotyledonous species that forms colonies or patches of hundreds of plants interconnected with a highly branched rhizome system. Despite self-incompatibility between individuals in a colony, the genetic diversity within mayapple is highly restricted because of relatively high rates of flower and fruit abortion, low rates of germination, and high seedling mortality (18). Podophyllin, the resin extracted from the rhizomes of Podophyllum, has been used in traditional medicine. The aryltetralin 1 is the active ingredient of podophyllin and this compound has been studied extensively because of its ability to inhibit microtubule assembly in mammalian cells and its strong antiviral activity (19). Numerous semisynthetic derivatives of podophyllotoxin, such as etoposide (7) and teniposide, having inhibitory activity against DNAtopoisomerase II, have been developed as effective antineoplastic drugs (20, 21). In our search for natural product-derived herbicides (22–24), we investigated the phytotoxic activity of the aryltetralin lignans of mayapple and their O-␤ -D-glucoside derivatives. The effect of 1 and 7 on microtubule assembly/organization and function of mitotically active plant cells was investigated.

MATERIALS AND METHODS

Chemicals and Standards Dr. D. Nanayakkara, chemist at the National Center for Natural Products Research, School of Pharmacy of the University of Mississippi, provided the standard compounds isolated from mayapple. Identity and purity were confirmed by chromatographic (TLC, HPLC) and spectral (IR, 1D- and 2D-NMR, HRESIMS) methods (25). Etoposide was purchased from SigmaAldrich (St. Louis, MO). Phytotoxicity of Podophyllotoxin and Related Lignans The natural lignans podophyllotoxin (1), ␣ -peltatin (3), and ␤ -peltatin (5), their O-␤ -Dglucosides (2, 4, and 6, respectively), and the synthetic analogue etoposide (7) were tested for herbicidal activity (Table 1). Biological activity of these compounds was tested in 24-well plates as described by Dayan et al. (10, 26). Each treatment consisted of four replicates and two controls. All compounds were dissolved in 10 ␮l of acetone, diluted with water containing 1 ml/L Tween 20, and tested at final concentrations of 2.5, 25, 62.5, 125, and 250 ␮M. Controls consisted of seeds germinated with a similar amount of solvent without test compound. Species tested were lettuce (Lactuca sativa L. cv.

OLIVA ET AL.

Iceberg) and rye (Lolium multiflorum L. cv. Gulf). A 200-␮l volume of each test solution was applied to each well. Plates were incubated at 25⬚C ⫾ 2⬚C under fluorescent lights maintaining a 16-h photoperiod at 400 ␮mol m⫺2s⫺1 PAR. Root lengths of lettuce and rye were measured on 7-day-old seedlings. Effect of Podophyllotoxin and Etoposide on Onion Root Cell Division (Mitotic Index) Onion (Allium cepa L. cv. Evergreen bunching) seeds were germinated for 7 days as previously described (10) in the presence of 100 ␮M 1 or 7 at 25⬚C under a 14-h photoperiod. The test compounds were prepared in 100⫻ stock solutions (10 mM) and acetone. Control samples received the same amount of solvent as those receiving the test compounds. The amount of acetone in the tests accounted for 1% of the total volume. Root tips were prepared according to Armbruster et al. (27) and mitotic analysis was performed on 1000 cells per slide (3000 cells per treatment) as described by Dayan et al. (10, 26). The mitotic phases were determined according to Hess (28), and abnormal mitotic configurations were counted as a separate class. Micrographs representative of the effects caused by the aryltetralin toxins were obtained at 40X magnification on an Olympus BX-60 microscope mounted with a PM-C35X camera connected to PM-30 automatic exposure unit. Data were analyzed with SAS (29). Immunofluorescence Microscopic Analysis Germination, growth, and treatment of onion seedlings were performed according to Vaughn and Vaughan (9). Stock solutions (100 ␮M) of 1 and 7 were prepared as described above. Controls were grown in the same concentration of solvent for the same duration as for the treated seedlings. Roots were prepared for indirect immunofluorescent localization of tubulin using a slight modification of the method of Sherman and Vaughn (30). Roots were fixed for 1 h at room temperature in 4% (w/v) paraformaldehyde in 50 mM piperazine-N,N ⬘-bis(2-ethanesulfonic acid)

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(Pipes) buffer (pH 6.9) containing 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N ⬘N ⬘tetraacetic acid (EGTA), 5 mM MgSO4, and 10% (v/v) dimethyl sulfoxide. The fixative was removed by three 10-min rinses with 50 mM Pipes buffer containing 5 mM EGTA and 5 mM MgSO4. Samples were subsequently digested for 15 min with 1% (w/v) cellulysin (Calbiochem, La Jolla, CA) in 50 mM Pipes buffer containing 5 mM MgSO4, 100 ␮m CaCl2, and 1% (w/v) bovine serum albumin (BSA), pH 6.9, at room temperature. The roots were rinsed as above, incubated for 1 h in rinse buffer containing 0.1– 0.25% (v/v) Triton X-100, and rinsed again. The terminal 1–2 mm of the roots was macerated in phosphate-buffered saline solution (PBS) containing 1% (w/v) BSA on a microscope slide with a razor blade. The root cells were covered with a coverslip and squashed gently between layers of filter paper. Cells adhering to the slides and coverslips were incubated in a 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated mouse IgG monoclonal anti-␤ -tubulin (Sigma) in PBS/BSA for 1 h at room temperature in a moist chamber. The root cells were rinsed four times with PBS (5 min per rinse) and mounted in PBS/50% (v/v) glycerol, containing 1.5% (w/v) propyl gallate (to retard fading of the fluorescence) and 0.01 mg/ml 4⬘-6-diamidino-2phenylindole (DAPI) (Sigma) to stain the chromosomes. Samples were viewed under oil immersion at 100X magnification using an Olympus BX60 epifluorescence microscope described above, with U-MNU and U-MNB filters for FITC and DAPI fluorescence, respectively. The U-MNU filter is a narrow-band cube for UV excitation with a 360- to 370-nm transmission range excitation filter, a dichroic mirror with DM400 splitting wavelength, and a barrier filter with a ⬎420nm transmission range. The U-MNB filter is a narrow-band cube for blue excitation with a 470to 490-nm transmission range excitation filter, a dichroic mirror with DM500 splitting wavelength, and a barrier filter of ⬎515-nm transmission range. Micrographs were recorded on Super HG Fujicolor film at 1600 ASA using the photographic setup described above.

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Each of the experiments described herein has been repeated at least three times and four to six samples in each treatment were examined by light microscopy. At least four to six root tips per slide and several slides were examined in each treatment. RESULTS

TABLE 1 Preemergence Herbicidal Activity of Podophyllotoxin and Various Aryltetralin Lignan Analogues

Compound None 1

Phytotoxicity of Aryltetralin Lignans from Mayapple 2

Podophyllotoxin (1) was the most phytotoxic natural product among lignans extracted from mayapple, inhibiting 75 to 94% of Lolium root growth at 125 to 250 ␮M concentrations (Table 1). The glycosidic moiety on 2 reduced the phytotoxicity against monocotyledonous species. Compounds 5 and 6 had the same selectivity profile but were less toxic than podophyllotoxin, and the activities of 3 and its O-␤ -D-glucoside (4) were marginal (Table 1). These results were similar to those of Bedows and Hatfield (12) with regard to mayapple lignan antiviral activity, in which 1 was the most potent lignan and 5 produced just a marginal antiviral effect. The antineoplastic drug 7, the semisynthetic derivative of 1, was highly toxic to both monocotyledonous and dicotyledonous species tested, even though rye was more sensitive than lettuce. Compound 7 was about 10-fold more active against Lolium than 1.

3

4

5

6

7

Effect of Podophyllotoxin and Etoposide on Mitosis The meristematic region of untreated onion roots had a normal mitotic distribution, with all of the mitotic stages being represented (Fig. 2A). Most of the actively dividing cells were in prophase, followed by metaphase, anaphase, and telophase (Fig. 3). No abnormal chromosomal conformations were observed. During cell division, chromosomes lined up at the equator and were pulled evenly toward spindle poles. The normal arrangement of microtubules of the spindle apparatus during metaphase and anaphase is visualized in the immunofluorescence micrographs of Figs. 4A and 5A, respectively. As with

Root inhibition (%)

Concentration (␮M)

Lettuce

Rye

0 250 125 62.5 25 2.5 250 125 62.5 25 2.5 250 125 62.5 25 2.5 250 125 62.5 25 2.5 250 125 62.5 25 2.5 250 125 62.5 25 2.5 250 125 62.5 25 2.5

0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 67 50 30 17

0 94 75 11 0 0 59 33 27 0 0 38 30 30 0 0 24 12 0 0 0 84 51 9 0 0 84 51 9 0 0 99 99 96 86 27

Note. Data were calculated using four replications, containing four seeds each, for each treatment.

Feulgen-stained micrographs, the DAPI-labeled chromosomes lined up at the equator and were pulled evenly toward the poles (Figs. 4B and 5B, respectively). After 24-h exposure to 100 ␮M 1 or 7, the number of actively dividing cells decreased, and many cells had abnormal mitotic organization,

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4–5% of the actively dividing cells of treated tissues had abnormal configurations (Fig. 3). Some cells exhibited c-metaphase with condensed chromosomes arrested in their prometaphase stage, but this abnormal mitotic configuration was not prevalent (data not shown). While the appearance of the most chromosomal arrangement at metaphase appeared normal (Figs. 4D and 4F), the formation of multiple spindle poles can be visualized (Figs. 4C and 4E). The effect of the toxins on the formation of the spindle apparatus is more clearly revealed during anaphase. Multiple spindle poles (Figs. 5C and 5D) caused uneven separation of the chromosomes (Figs. 5D and 5F). The few cells in telophase in controls exhibited normal configuration. However, treatment with 1 or 7 led to even fewer cells in telophase (Fig. 3) and none of them reacted properly (despite several attempts) with the fluorescent ␤ -tubulin antibody, precluding the visualization of the microtubule arrangement in later stages of mitosis. DISCUSSION

The phytotoxic effects of the natural aryltetralin lignans, their O-␤ -D-glucosides, and the

FIG. 2. Light micrographs of (A) control, (B) podophyllotoxin-treated (100 ␮M), and (C) etoposide-treated (100 ␮M) onion root tips. Cells at various stages of mitosis are indicated in A as follows: p, prophase; m, metaphase; a, anaphase; t, telophase. Abnormal mitotic stages (ab) are indicated in podophyllotoxin-treated (B) and etoposidetreated (C) root tips. Onion root tip squashes were fixed in acetic acid/ethanol (1:3) and stained with Feulgen reagent after 5 days growth. Bars represent 100 ␮m.

with chromosomes being pulled unevenly, suggesting the presence of multiple spindle poles (Figs. 2B and 2C). Those results were confirmed in the mitotic index data. The number of cells in prophase is approximately half that in the control for all the mitotic stages. As much as

FIG. 3. Distribution of phases of mitosis in control (䡲) and in podophyllotoxin- (⬅) and etoposide- (▫) treated onion root tips. Data were obtained by counting at least 3000 cells per treatment. Cells with chromosomal arrangements deviating from those associated with normal mitotic phases were classified as abnormal. LSD at P ⫽ 0.05.

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FIG. 4. Double-fluorescence labeling with antitubulin (A, C, and E) and DAPI (B, D, and F) of onion root cells in metaphase. A and B illustrate normal metaphase microtubular and chromosomal configurations in control cells. In cells treated with 100 ␮M podophyllotoxin (C and D) or 100 ␮M etoposide (E and F), microtubule arrangements appeared normal, although the spindle poles (brackets) (C and E) were broader than those in the control (A). No abnormal chromosomal conformation could be observed at the metaphase stage (D and F). Bars represent 30 ␮m.

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FIG. 5. Double-fluorescence labeling with antitubulin (A, C, and E) and DAPI (B, D, and F) of onion root cells in anaphase. (A) In untreated cells, microtubules radiated toward the cell equator from single spindle poles (asterisks); (B) chromosomes are pulled toward the poles in a symmetrical manner and their kinetochores are converging at the spindle pole (arrows). In cells treated with 100 ␮M podophyllotoxin (C and D) or 100 ␮M etoposide (E and F), microtubules radiated from multiple spindle poles (asterisks) in tripolar (C) and bipolar (E) star anaphase conformations. Abnormal chromosomal configurations (D and F) are the results of the contraction of the spindle apparatus toward multiple poles. Bars represent 30 ␮m.

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semisynthetic derivative etoposide were tested on lettuce and onion. For all the compounds, the aglycones were more potent than their respective glucosides, suggesting that the presence of the glucoside was detrimental to their phytotoxicity, although the presence of this sugar moiety enhanced the water solubility of these compounds. Early investigations in the use of aryltetralin lignans for pharmaceutical purposes also indicated that the aglycones were more cytotoxic than the glucosides on animal cells. The phytotoxicity of 1 was similar to that reported earlier (31) and the semisynthetic etoposide (7) was the most phytotoxic molecule tested in this study. All of the aryltetralin tested possessed a 3,4,5trimethoxyphenyl ring, except for ␣ -peltatin and etoposide, which have a 4-hydroxy-3,5-dimethoxyphenyl ring. It has been shown that the presence of the 4-hydroxy group is essential, but not sufficient for antineoplastic activity, for compound 7. Inhibition of root growth observed in the bioassays suggested that these compounds affected either cell division or cell elongation. The inhibitory effects of 1 and 7 on the different mitotic phases and especially on prophase indicated that these compounds affect mitosis. Inhibition of an early stage of mitosis, such as prophase, is normally associated with mechanisms of action that alter the condensation of chromosomes. DNA-topoisomerase II inhibitors are known to affect chromosome condensation by arresting the cell cycle at the G2 stage (19); so the effect observed with 7 is consistent with its reported mode of action in animal cells. However, 1 also caused a decrease in the number of cells entering mitosis, although its primary effect is reported to be inhibition of microtubule assembly, resulting in mitotic arrest at prometaphase in a manner similar to that of colchicine and vinblastine (9). The cell cycle of meristematic plant cells can be affected by altering a number of physiological processes (5, 28). In fact, certain herbicides indirectly prevent normal cell division either by affecting protein synthesis, as was observed with

chlorsulfuron and imazethapyr (32) and cinmethylin (33, 34), or by inhibiting cell wall formation, as was demonstrated with dichlobenil and isoxaben (2). There are, however, inhibitors that alter the process of meristematic cell division more specifically. Compounds affecting DNA replication prevent mitotic entry, such as hydantocidin, but such compounds are often difficult to develop as herbicides because of the possible toxicology problems toward mammals (35). Aryltetralin lignans have been studied extensively because of their ability to inhibit mitosis in mammalian cells. Interestingly, 1 primarily inhibits microtubule assembly, whereas its semisynthetic derivative 7 primarily inhibits DNAtopoisomerase II. The orientation of the oxygen atom on the 4-carbon, and the presence of a 4hydroxy instead of a 4-methoxy on the pending phenyl moiety, has been associated with the difference in the molecular target site of these aryltetralin lignans (36). An early study reported that podophyllin, the resin extracted from the rhizomes of Podophyllum, affected the formation of the spindle apparatus in a manner similar to that observed in tissues exposed to colchicine (37). More recently, Vaughn and Vaughan (9) reported that podophyllotoxin arrested mitosis at the prometaphase. While colchicine and podophyllotoxin compete for the same binding site on tubulin in animal cells, these two compounds do not share the same binding site in plant tubulin (9). However, the symptoms observed with podophyllotoxin and etoposide were more similar to those caused by vinblastine and colchicine reported by Hillmann and Ruthmann (38) on bean root tips and Segawa and Kondo (39) on Allium root tips, where abnormal anaphase configurations were observed along with cells in c-metaphase. Colchicine normally prevents the assembly of tubulin into microtubules by blocking the formation of the microtubule cap. As a result, the chromosomes condense but do not proceed past the prometaphase for the lack of spindle microtubules, leading to a polymorphic nucleus (40). While podophyllotoxin has been classified as a colchicine-type mitotic inhibitor, the work of

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Hillmann and Ruthmann (38) on vinblastine and our present work suggests that this natural toxin may also affect microtubule organization, as was evident in the immunofluorescence micrographs (Figs. 4 and 5). Other phytotoxic natural products such as the sesquiterpene endoperoxide lactones and quassinoids have similar inhibitory activities on early stages of mitosis (9, 10). In addition, abnormal mitotic stages observed in root cells exposed to 100 ␮M 1 and 7 suggest that those compounds act as natural mitotic disrupters (9). Effects were noted only on the cell division stages past the prometaphase level. At this time, the normal spindle microtubular organizing center seems to be affected by the tested compounds, so that the microtubules are orientated toward multiple poles where the chromosomes appear in “star anaphase” figures. This display is similar to that found when the herbicides terbutol, sindone B, pronamide, and dithiopyr and some carbamate herbicides are tested at low concentrations (0.1–30 ␮M) (8, 41, 42). Visual observations of the chromosomes stained with Feulgen reagent or DAPI, or the microtubules labeled by immunofluorescence, did not enable the differentiation between the mechanisms of action of 1 and 7, although the literature indicates that the two compounds have different mechanisms of action in animal cells. In particular, etoposide was not expected to cause abnormal assembly of the spindle microtubules if its activity was limited to the inhibition of DNA-topoisomerase II. It is, therefore, likely that this highly potent inhibitor may also affect microtubule assembly as a secondary mechanism of action, but at higher concentration than that of topoisomerase II inhibition. Also, it is possible that the compounds tested may have additional secondary target sites, such as ␥ tubulin, that would lead to abnormal or multiple spindle pole formation. However, the specific role of these putative sites remains to be tested. The antimitotic effect of aryltetralin lignans on mammalian systems will most likely preclude their use in agricultural settings. However, this study reveals that these natural compounds can inhibit plant growth by affecting the formation of

mitotic microtubular organizing centers in roots. Therefore, plants producing these lignans may have allelopathic effects on other species growing in their immediate surroundings. ACKNOWLEDGMENTS This work was partially funded by USDA seed Grant 9735501-4886, strengthening award Grant 99-01739. We thank Dr. N. P. D. Nanayakkara for providing the lignan standards.

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