Enantioselective Effects of (+)- and (−)-Citronellal on Animal andPlant MicrotubulesOsnat Altshuler,† Mohamad Abu-Abied,† David Chaimovitsh,‡ Alona Shechter,‡ Hilla Frucht,‡

Nativ Dudai,‡ and Einat Sadot*,†

†The Institute of Plant Sciences, ARO, Volcani Center, PO Box 6, Bet-Dagan 50250, Israel‡Division of Aromatic Plants, ARO, Newe Ya’ar, PO Box 1021, Ramat Yishai 30095, Israel

*S Supporting Information

ABSTRACT: Citronellal is a major component of Corymbiacitriodora and Cymbopogon nardus essential oils. Herein it isshown that whereas (+)-citronellal (1) is an effectivemicrotubule (MT)-disrupting compound, (−)-citronellal (2)is not. Quantitative image analysis of fibroblast cells treatedwith 1 showed total fluorescence associated with fibersresembling that in cells treated with the MT-disrupting agentscolchicine and vinblastine; in the presence of 2, thefluorescence more closely resembled that in control cells.The distribution of tubulin in soluble and insoluble fractions inthe presence of 1 also resembled that in the presence ofcolchicine, whereas similar tubulin distribution was obtained inthe presence of 2 and in control cells. In vitro polymerizationof MTs was inhibited by 1 but not 2. Measurements of MT dynamics in plant cells showed similar MT elongation and shorteningrates in control and 2-treated cells, whereas in the presence of 1, much fewer and shorter MTs were observed and no elongationor shrinkage was detected. Taken together, the MT system is suggested to be able to discriminate between different enantiomersof the same compound. In addition, the activity of essential oils rich in citronellal is affected by the relative content of the twoenantiomers of this monoterpenoid.

Many of the essential oils produced by aromatic plants arecomposed of volatile aromatic monoterpenes. Some of

these monoterpenes occur as enantiomers.1 Examples of chiralvolatile aromatic monoterpenes are limonene,2 carvone,2

pinene,3 linalool,4,5 citronellol,6 and citronellal.7

Among the essential oils produced by aromatic plants areknown growth inhibitors,8,9 termed allelochemicals.10,11,12 Themechanism governing their activity against bacteria and otherpathogens has been studied widely, but not much is knownabout their mechanism of action against plant or mammaliancells. Essential oils have been found to act as typical lipophilesthat interfere with the cell wall and membranes of prokaryotes,leading to leakage of macromolecules and lysis.13 In eukaryoticcells, aromatic oils have been shown to cause permeabilizationof the plasma and mitochondrial membranes and to lead toapoptosis and necrosis.13,14 In plants, aromatic oils have beenfound to inhibit cell division, leading to membrane disruptionand oxidative stress,15−19 and also to have an effect on hormonebalance.20,21Data have been accumulated regarding themolecular mode of action of some components of aromaticessential oils. For example, citral was shown to act as amicrotubule (MT)-disrupting compound in Arabidopsis seed-lings as well as in rat fibroblasts.22

MTs are dynamic polymers of α- and β-tubulin heterodimersarranged to form hollow tubes of 25 nm in diameter and up to

several micrometers in length. MT dynamics are tightlyregulated23,24 and crucial for MT-specific functions such asmaintenance of cell shape, cell division, cell signaling,intracellular vesicle and organelle transport, and cell polarityand locomotion.25,26 The critical role of MTs in cell divisionmakes them a very attractive target in the screening ofanticancer drugs, and, over the years, numerous compoundsaffecting MTs have been found.27−29

Herein, the two enantiomers of citronellal (1 and 2)naturally present in the essential oils of Corymbia citriodora(Hook.) K.D. Hill & L.A.S. Johnson (Myrtaceae) andCymbopogon nardus L. Rendle (Poaceae) were tested for theirability to disrupt either MTs or the actin cytoskeleton in animaland plant cells. Citronellal was found to exhibit enantioselectiveactivity, with 1 disrupting MTs efficiently, while 2 at the sameconcentration did not.

■ RESULTS AND DISCUSSIONDifferential Effects of (+)-Citronellal (1) and (−)-Cit-

ronellal (2) on MTs. To determine the effects of 1 and 2separately on MTs, standard compounds were purchased fromSigma-Aldrich Israel. The sample of 2 was 96% pure, whereas

Received: April 9, 2013Published: August 15, 2013

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that of 1 was about 90% pure. Therefore, 1 was further purifiedas described in the Experimental Section (see also Figure S1,Supporting Information) to make it 96% pure. The twoenantiomers were dissolved in DMSO and diluted in the cellgrowth medium such that the concentration of DMSO was less

than 0.01%. MTs of two types of cells were examined. Incontrast to control cells (Figure S2 A and C, SupportingInformation), MTs of both HeLa and Ref52 cells wereeffectively depolymerized in the presence of 1 (Figure S2 Eand G, Supporting Information). In the presence of 2, somereorganization could be observed (Figure S2 I and K,Supporting Information), but no extensive depolymerization.In the latter cells, more dense or bundled MTs were observedaround the nucleus. Importantly, no changes in actin wereobserved in HeLa or Ref52 cells following treatment with either1 or 2 (compare Figure S2 B, D to F, H and J, M, SupportingInformation). The software FiberScore, which was developed to

Figure 1. Quantitative analysis of the effects of 1 and 2 on MTs. Ref52 cells were treated for 30 min with 0.1 μM colchicine or vinblastine, 2 μMpaclitaxel, or 27.5 μM 1 or 2. The cells were fixed and stained for MTs. Images were analyzed by FiberScore for total fluorescence associated withfibers. A, C, E, G, I, and K are the images of MTs. B, D, F, H, J, and L show the fibers scored by the software. Scale bar = 20 μm. (M) Quantitativeanalysis. Bars (average ± standard error) with different letters differ significantly by Scheffe’s test at p < 0.05.

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quantify cytoskeleton elements,30 was used to quantify thesedifferences. Ref52 cells were treated with 0.01% DMSO aloneor with 1 or 2. As additional controls, cells were treated withdrugs that are known to affect MTs, vinblastine and colchicine,which disrupt them, or paclitaxel, which stabilizes them. MTswere stained, imaged, and analyzed by FiberScore. Figure 1shows the original MT image (Figure 1A,C,E,G,I,K), theFiberScore image (Figure 1B,D,F,H,J,L), and quantitativeanalysis of the total fluorescence associated with fibers in thecells after the various treatments (Figure 1M). The maximumtotal fluorescence associated with fibers was found in thecontrol cells. After treatment with colchicine or vinblastine, thisvalue dropped dramatically. When cells were treated withpaclitaxel, bundled and brighter MTs were observed around thenucleus and less at the cell periphery, and measurementsindicated less total fluorescence associated with fibers.Following treatment with 1, the values of total fluorescenceassociated with fibers resembled those after colchicine orvinblastine treatment. After treatment with 2, on the otherhand, total fluorescence was higher than after paclitaxel andcloser to that of control cells.It was then examined whether the effect of citronellal is dose-

or time-dependent. Cells were treated for 30 min with 6.9, 13.8,27.5, or 55 μM of each of the enantiomers or with 27.5 μM for5, 10, 20, or 30 min (Figure 2). MTs were stained and analyzed

by FiberScore. When cells were treated with 6.9 μM 2 for 30min, total fluorescence associated with fibers increasedsignificantly compared to control cells. This suggestsstabilization and bundling under these conditions. At 13.8μM, 2 did not cause a significant change, but at the higherconcentrations used (27.5 or 55 μM) fluorescence associatedwith fibers decreased to about 50% of that in control cells. Thehigher the concentration of 1 applied to the cells, the less thefluorescence associated with fibers was observed, dropping to20% of control cells at 27.5 and 55 μM. Similarly, when thecells were treated with 27.5 μM 1 for increasing periods of time,the fluorescence associated with fibers decreased gradually andsignificantly, reaching 20% of that in control cells after 30 min.However, when 2 was applied, no significant change in thefluorescence associated with fibers occurred after 5 and 10 min,and after 20 and 30 min, a reduction to only 60% of that of thecontrol cells was observed. Taken together, it could beconcluded that the effects of 1 and 2 on MTs are both dose-and time-dependent and that the effects of these twoenantiomers are markedly different.

Differential Modification of the Ratio of Soluble toInsoluble Tubulin in the Presence of 1 or 2. The aboveresults were obtained by examining separate cells under themicroscope. To determine the ratio between total soluble andinsoluble tubulin in the presence of the two enantiomers ofcitronellal, proteins from treated cells were separated intosoluble and insoluble fractions, and the presence of tubulin inthe two fractions was determined by Western blot analysis.31 Inthis fractionation, the insoluble fraction contains thecytoskeleton and membrane proteins, while the soluble fractioncontains cytoplasmic proteins. As a control, actin wasdetermined in the same fractions. Figure 3 summarizes fourindependent experiments. In control cells, the ratio of solubleto insoluble tubulin was 1:0.6 (Figure 3A). After treatment withcolchicine, which inhibits polymerization, this ratio became1:0.4. After treatment with paclitaxel, which stabilizes MTs,almost all of the tubulin was found in the insoluble fraction(0.1:1.1). When the cells were treated with 1, the ratio wassimilar to that after colchicine treatment, 1:0.4. However, whenthe cells were treated with 2, the ratio was 1:0.6, similar to thatin control cells. Importantly, the ratio between the amounts ofactin in the soluble and insoluble fractions remained constantthroughout all treatments at 1:1.3 (Figure 3B).

(+)-Citronellal (1), but Not (−)-Citronellal (2), Inter-feres with Polymerization of Tubulin in Vitro. It was thentested whether the effect of citronellal on MTs would occur in acell-free system. Purified fluorescent tubulin was polymerized invitro, in the presence of each enantiomer. The amount andlength of the MTs were determined under the microscope.Figure 4 shows that while 1 interfered with tubulin polymer-ization, leading to both fewer (40% of control) and shorter(30% of control) MTs, in the presence of 2, MTs polymerizedas efficiently as under control conditions.

Differential Effects of 1 and 2 on Plant MTs. Todetermine whether the differential activities of 1 and 2 arespecific to animal MTs, cells from plants expressing GFP-tubulin β632 or GFP-talin33 were exposed to each enantiomer.Whereas 1 led to disruption of MTs in plant cells, 2 did notcause a detectable change in their organization (Movies 1−3,Supporting Information). Under these experimental conditionsno major changes in the organization and dynamics of actinfibers were detected in the presence of either enantiomer(Movies 4−6, Supporting Information). To further investigatethe mechanism by which citronellal affects plant MTs, MTdynamics were monitored in the presence of each enantiomer.MT dynamics were expressed by the rates of growth,shortening, and pausing of their ends.34 More than a hundredindividual MTs were followed in six cells for each treatmentand in control cells by acquiring time-lapse movies. Themeasurements revealed no significant difference in growth or inshortening rates in the control and in cells treated with 2(Figure 5). In addition, no significant differences were found inthe percentage of growing, shortening, or pausing MTsbetween the control cells and cells treated with 2. However,in the cells treated with 1, much fewer and shorter MTs wereobserved, lacking dynamic changes at both ends.Taken together, this suggests that there is an enantioselective

effect of 1 and 2 on MTs. It is worth noting that while 1 wasactive at 27.5 μM, citral disrupted MTs efficiently at aconcentration of 1 μM,22,35 and colchicine or vinblastine at0.1 μM. Therefore, 1 can be considered a weak MT-disruptingagent. Curiously, when cells were treated with 1, remnants ofMT seemed to be stabilized. In animal cells, bright bundles of

Figure 2. Effects of 1 and 2 are dose- and time-dependent. Ref52 cellswere treated with the concentrations indicated in A for 30 min or with27.5 μM for the periods of time indicated in B. The cells were fixedand stained for MTs. Images were analyzed by FiberScore for totalfluorescence associated with fibers. Bars (averages ± standard error)with different letters differ significantly by Scheffe’s test at p < 0.05.

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short MTs were observed (Figure S2 E and G, SupportingInformation), and in plant cells, short and static MTs weremonitored (Movie 2, Supporting Information). It waspreviously shown that citronellal is rapidly metabolized inplant cells to citronellic acid and citronellol,36 for which theireffect on MTs is still to be determined. Therefore, it may bespeculated that once 1 is being metabolized, depolymerizationof MT slows down and the remnants of truncated MT might bestabilized by its derivatives. Therefore, the combined effect isdifferent from that of other MT-stabilizing agents that alsopromote polymerization.

The search for anti-MT drugs has been ongoing foryears.27−29 MT-targeting agents have been found to interactwith tubulin through mainly four characterized binding sites:colchicine, vinca alkaloid, paclitaxel, and epothilone.28

Colchicine binds to β-tubulin at its interface with α-tubulin.Upon binding, colchicine prevents straightening of tubulin’stypical curved conformation, a conformational change that isnecessary for dimer assembly into the protofilament.37

Vinblastine binds at the interface between two dimers of α-and β-tubulin and inhibits longitudinal head to tail contact inthe protofilament.38 The exact paclitaxel-binding site is on theβ-tubulin subunit, at the inner face of the MT, adjacent to the

Figure 3. Ratio of soluble (S) to insoluble (I) tubulin in the presence of 1 or 2. Ref52 cells were treated with 20 μM 1 or 2 or with 0.1 μM colchicineor 2 μM paclitaxel for 30 min. Control cells were treated with 0.01% DMSO. The cells were washed, and soluble and insoluble protein fractions wereseparated and analyzed for (A) tubulin and (B) actin by SDS-PAGE. The graphs represent four independent experiments. Bars show averages ±standard error.

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binding site between two protofilaments.39−41 The binding siteof epothilone, which stabilizes MTs, partially overlaps with thatof paclitaxel.42 The chemical structures of colchicine,37

vinblastine,43,44 paclitaxel,45 and epothilone46 are very differentfrom each other and more complex than that of citronellal. Themolecular weight (MW) of colchicine is 399.44 g/mol, that ofvinblastine is 811 g/mol, that of paclitaxel is 853.91 g/mol, andthat of epothilone A is 493.66 g/mol. Citronellal is a muchsmaller molecule with a MW of 154.25 g/mol. Although thedirect binding of citronellal to tubulin has yet to be verified, inlight of its small and simple chemical structure, theenantioselective effect on MTs described herein is intriguing.Enantioselective effects of other monoterpenes have been

previously reported. For example, the toxicities of (+)-α-pinene

and (+)-β-pinene toward bacteria and fungi are much higherthan those of (−)-α-pinene and (−)-β-pinene, respectively.3 Inaddition, human odor perception is known to be very sensitiveto chirality,1 and humans have been shown to be much moresensitive to L-carvone than to D-carvone and more sensitive toD-limonene than L-limonene.1,47 Chirality is also very importantin chemical communication via pheromones.48 Thus, (S)-(+)-linalool is more effective than (R)-(−)-linalool as a beemate-attracting pheromone.4 Human odor receptors (ORs) aremade up of a multigene family that includes hundreds ofmembers of the heterotrimeric G-protein-coupled receptors,which bind different odor molecules.49 The general consensusfor ligand specificity of ORs is that each OR has a distinctligand spectrum, and each odorant can be detected by acombination of ORs. How the smell of a specific enantiomer isdistinguished from its chiral counterpart at the molecular levelhas yet to be determined.Monastrol binds to the head domain of the mitotic kinesin

Eg5 and inhibits its ATPase activity.50 Interestingly, the Senantiomer of monastrol has been found to be more efficient atinhibiting Eg5’s ATPase activity than the R enantiomer orracemic monastrol.50 This demonstrates preferential binding ofone enantiomer over the other.The present finding of enantioselective effects of (+)-cit-

ronellal (1) and (−)-citronellal (2) on MTs is both novel andimportant because it suggests that the MT system, in bothplants and animals, can discriminate between chiral com-pounds. In addition, since citronellal is an allelochemical, therelative concentration of its two enantiomers in essential oilshas consequences on their mechanism of activity.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Enantiomers of citronellal

were purchased from Sigma-Aldrich, Israel, namely, (+)-citronellal (1)

Figure 4. Effects of 1 and 2 on in vitro polymerization of tubulin. Fluorescent purified tubulin was polymerized in the presence of 0.01% DMSO as acontrol (A) or 1 or 2 (B and C). MTs were examined under the microscope and measured by Olympus Cell software. Scale bar = 20 μm. Number(D) and total length (E) of the MTs were quantitated. Bars (averages ± standard error) with different letters differ significantly by Scheffe’s test at p< 0.05.

Figure 5. Effects of 1 and 2 on MT dynamics. Arabidopsis plantsexpressing GFP-tubulin β6 were treated with 25 μM 1 or 2, and thedynamics of individual MTs was followed by time-lapse movies.Quantitative analysis shows growth and shortening rates and relativeamounts of different MT ends in each treatment.

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(cat. number 343641, lot number 07105BSV), technical grade 90%,and (−)-citronellal (2) (cat. number 373753, lot number0001446280), 96% purified.Further Purification of (+)-Citronellal (1). Compound 1 (0.5 g

of 90% purity) was loaded onto a silica column (Merck, 10 g, 7 × 2.5cm) prewashed with hexane. The column was eluted with 50 mL ofhexane, 50 mL of hexane−petroleum ether (50:50), 50 mL ofpetroleum ether, 70 mL of methyl tert-butyl ether, and 20 mL ofmethanol. Fractions of 10 mL were collected and checked under a UVlamp. Fractions 15 and 16, which contained purified citronellal, werecombined to give 350 mg and loaded onto a second silica column(Merck, 10 g, 7 × 2.5 cm) prewashed with hexane. The column waseluted with 50 mL of hexane, 50 mL of hexane−petroleum ether(50:50), 50 mL of hexane−petroleum ether (40:60), 50 mL ofhexane−petroleum ether (30:70), 50 mL of hexane−petroleum ether(20:80), 50 mL of hexane−petroleum ether (10:90), 20 mL ofpetroleum ether, 20 mL of methyl tert-butyl ether, and 20 mL ofmethanol. Fractions of 5 mL were collected and checked under a UVlamp. Fractions containing citronellal were dissolved in petroleumether and injected into a GC-MS. Fractions with similar purity werecombined to give 0.8 mg of 96% purified 1.Cell-Line Tissue Culture and Immunostaining. HeLa cells and

rat embryonic fibroblasts (Ref52) were cultured in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% fetalcalf serum in a 37 °C/5% CO2 incubator. For cell treatments,citronellal and paclitaxel were dissolved in DMSO and colchicine andvinblastine sulfate salt in H2O. The reagents were diluted with serum-free DMEM and added to the cells.For immunostaining, cells were permeabilized with 3% paraformal-

dehyde and 0.5% Triton X-100 for 2 min and then fixed with 3%paraformaldehyde for an additional 30 min. For MT staining, 1%glutaraldehyde was added and its autofluorescence was quenched with0.5 mg/mL sodium borohydride for 15 min in phosphate-bufferedsaline (PBS). Cells were washed with PBS prior to immunostaining.Actin was stained with Alexa 555-phalloidin (Molecular Probes), andMTs with antitubulin monoclonal antibody (DM1A Sigma) and asecondary antibody conjugated to Alexa 488 (Molecular Probes).Cell Fractionation. Cells were treated with 0.1% DMSO as a

control or with 20 μM 1 or 2. Fractionation into Triton X-100-solubleand -insoluble fractions was carried out as follows: cells cultured on100 mm tissue-culture plates were extracted, at room temperature,with 0.5 mL of 50 mM MES buffer pH 6.8 containing 2.5 mM EGTA,5 mM MgCl2, and 0.5% Triton X-100 for 3 min. The Triton X-100-soluble fraction was removed, and the insoluble fraction was scrapedinto 0.5 mL of the same buffer using a rubber policeman. Proteinsample buffer (125 μL) was added, and the samples were boiled for 10min and centrifuged for 10 min at 13 000 rpm. Equal volumes of thetwo fractions were analyzed by SDS-PAGE. For Western blot analysis,the antitubulin DM1A was used to detect tubulin; to visualize actin,the clone C4 mouse anti-chicken actin (ICN Biochemicals, Inc.)antibody was used. The secondary antibodies were goat anti-mouseantibody conjugated to horseradish peroxidase (Jackson ImmunoR-esearch Laboratories). The blots were developed using West PicoChemiluminescent Substrate (Pierce). The film was scanned, and theintensity of the bands was measured by the NIH image software.In Vitro Polymerization of MTs. Purified bovine brain tubulin

(TL238, cytoskeleton) and rhodamine-labeled tubulin (TL331,cytoskeleton) were polymerized as follows: 5 μL of 20 mg/mLtubulin and 0.5 μg of 15 mg/mL fluorescent tubulin were mixed with 2μL of 10 mM GTP, and 0.5 μL of the mixture was transferred to 3.5μL of MRB80 buffer (80 mM PIPES/KOH pH 6.8, 1 mM MgCl2, 1mM EGTA) and 6% glycerol containing either 0.01% DMSO or 20μM 1 or 2. The mixture was incubated at 37 °C for 20 min, and thereaction was stopped by adding 0.5% gluteraldehyde. MTs werediluted to 10 μL with MRB80 buffer, and 1 μL was put under acoverslip and microscopically examined. MT length was measuredusing Olympus Cell software.Plants. Arabidopsis thaliana (L.) Heynh, ecotype Columbia,

(Brassicaceae) plants expressing either GFP-talin as a marker foractin33 or GFP-tubulin β632 were germinated on Murashige and Skoog

medium, incubated for 4 days at 4 °C in the dark, and then transferredto a growth room at 24 °C under 16 h light/8 h darkness. Cotyledonsof 7-day-old seedlings were exposed to 25 μM of either 1 or 2 for 10min. Imaging was carried out under a confocal microscope.

Microscopy. An Olympus IX81/FV500 point scanning confocalmicroscope was used to observe fluorescently labeled cells with thefollowing filter sets: for GFP- or Alexa 488-conjugated antibodies, 488-nm excitation and BA505-525. MT dynamics were followed manuallyby measuring growth or shortening from frame to frame. Lagging endswere defined as exposed ends, opposite fast-growing or fast-shorteningones. Time lapse movies were acquired using a spinning disk confocalmicroscope including a Yokagawa CSU-X1 head, an Andor iXon-EMback-illuminated 512 × 512 CCD camera, and a UPlanSAPO 100×/1.4 na objective. Time-lapse image acquisition was performed every 10s for 3 min; the movies show 10 frames/s. The images of the in vitroassay were acquired on a wide-field inverted Olympus IX81microscope equipped with a Hamamatsu Orca C4742-80-12AGCCD camera and filters from Chroma.

■ ASSOCIATED CONTENT*S Supporting InformationGC-MS analysis of purified compound 1 (Figure S1) and acomparison of the effect of 1 and 2 on MTs and the actincytoskeleton in animal cells (Figure S2). Live cell imaging ofMTs or the actin cytoskeleton in Arabidopsis seedlings eitheruntreated or treated with 1 or 2 (Movies 1−6). Thisinformation is available free-of-charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: 972-3-9683510. Fax: 972-3-9601892. E-mail: vhesadot@agri.gov.il.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSE.S. acknowledges financial support from the Chief Scientist ofthe Ministry of Agriculture, Israel.

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Journal of Natural Products Article

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