[cancer research 59, 653–660, february 1, 1999 ...€¦ · laulimalide and 5 mg of isolaulimalide...

[CANCER RESEARCH 59, 653–660, February 1, 1999]

Laulimalide and Isolaulimalide, New Paclitaxel-Like Microtubule-Stabilizing Agents1

Susan L. Mooberry,2 Georgia Tien, Anne H. Hernandez, Anuchit Plubrukarn, and Bradley S. DavidsonNatural Products Program, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii 96813 [S. L. M., G. T., A. H. H.], and Department of Chemistryand Biochemistry, Utah State University, Logan, Utah 84322-0300 [A. P., B. S. D.]

ABSTRACT

A mechanism-based screening program aimed at the discovery of newantimicrotubule agents from natural products yielded laulimalide andisolaulimalide, two compounds with paclitaxel-like microtubule-stabiliz-ing activity. Treatment of A-10 cells with laulimalide resulted in a dose-dependent reorganization of the cellular microtubule network and theformation of microtubule bundles and abnormal mitotic spindles. Coin-cident with the microtubule changes, these two compounds induced nu-clear convolution and the formation of multiple micronuclei. Laulimalideis a potent inhibitor of cellular proliferation with IC 50 values in the lownanomolar range, whereas isolaulimalide is much less potent with IC50

values in the low micromolar range. In contrast to paclitaxel, both lauli-malide and isolaulimalide inhibited the proliferation of SKVLB-1 cells, aP-glycoprotein overexpressing multidrug-resistant cell line, suggestingthat they are poor substrates for transport by P-glycoprotein. Incubationof MDA-MB-435 cells with laulimalide resulted in mitotic arrest andactivation of the caspase cascade of proteolytic enzymes that accompanyapoptotic cell death. Laulimalide stimulated tubulin polymerization and,although less potent than paclitaxel, it was more effective. Laulimalide-induced tubulin polymers resembled paclitaxel-induced polymers, al-though the laulimalide-induced polymers appeared notably longer. Lauli-malide and isolaulimalide represent a new class of microtubule-stabilizingagents with activities that may provide therapeutic utility.

INTRODUCTION

Microtubules play key roles in many cellular processes includingintracellular transport, motility, architecture, and division. Normal celldivision is dependent on the dynamic instability of microtubulesduring the course of mitosis and cytokinesis. Interruption of micro-tubule dynamics has proven to be an effective target for cancerchemotherapy (1). Agents that target microtubules, such as theVincaalkaloids, have been used in the treatment of cancer for over 30 years.New agents such as paclitaxel (TaxolTM) have demonstrated effec-tiveness against a broad spectrum of tumors including ovarian, breast,and lung carcinomas. Paclitaxel has a mechanism of action unlike anyof the tubulin-targeting agents that preceded it to the clinic (2). Itpromotes tubulin polymerization, stabilizes microtubules, and therebyalters normal microtubule dynamics, leading to the formation ofabnormal mitotic spindles, mitotic arrest, and the initiation of apop-tosis (1, 2). At high concentrations, paclitaxel causes thick microtu-bule bundles to form, whereas agents such as theVincaalkaloids bringabout the complete loss of cellular microtubules (2).

The clinical successes of the taxanes, paclitaxel and the semisyn-thetic derivative docetaxel (Taxotere) prompted the search for newagents with similar mechanisms of action. The search for new pacli-taxel-like agents has been vigorous with the goal of providing new

agents with advantages over paclitaxel, specifically those that cancircumvent transport by drug efflux pumps that confer multidrugresistance (3). Since the discovery of the mechanism of action ofpaclitaxel, only three other nontaxane chemical classes (epothilones Aand B, discodermolide, and eleutherobin) have been identified thatpossess a similar mode of action. The epothilones A and B wereisolated from the myxobacteriumSorangium cellulosumas a result ofa large-scale screening effort (4). The epothilones have generatedsignificant interest, as they retain activity against drug-resistant celllines (5, 6). Discodermolide was purified from the marine spongeDiscodermia dissolutaas an immunosuppressant and was screened forantimitotic activity on the basis of a predictive structure-activityrelationship when compared with other tubulin-interacting drugs (6).Discodermolide promotes tubulin assembly more potently than pacli-taxel (6, 7), and it is an effective inhibitor of cell growth in paclitaxel-resistant cells (7). Eleutherobin, a potent cytotoxin from the soft coralEleutherobiasp., promotes tubulin polymerization but exhibits cross-resistance to paclitaxel-resistant cell lines (8, 9). The potential thera-peutic usefulness of these new microtubule-stabilizing compoundsand whether they will provide advantages over the taxanes have yet tobe determined.

In addition to the microtubule-stabilizing agents discodermolideand eleutherobin, there are many other microtubule-targeting marinenatural products (reviewed in Ref. 10). Because marine organismshave proven to be a rich source of compounds that target eukaryoticmicrotubules, we tested extracts from marine invertebrates collectedin the Marshall Islands in a mechanism-based screening programaimed at the discovery of new antimicrotubule agents. Strong pacli-taxel-like microtubule-stabilizing activity was found in the crudelipophilic extract from the marine spongeCacospongia mycofijiensis.Bioassay-directed purification of the extract yielded the compoundslaulimalide and isolaulimalide, 18-membered macrocyclic lactonesthat had been previously isolated from sponges collected in Indonesia(11), Vanuatu (12), and Okinawa (13). Isolaulimalide was shown to bea laulimalide rearrangement product, formed through the acid-cata-lyzed attack of the side chain hydroxyl group on the epoxide ring. Atthe time of isolation, both compounds were known to be cytotoxic,however, the mechanism of action was not known. We report here thatlaulimalide and isolaulimalide are paclitaxel-like microtubule-stabi-lizing agents. These two compounds, in contrast to paclitaxel, seem tobe poor substrates for the drug efflux pump P-glycoprotein.

MATERIALS AND METHODS

Sponge Collection and Identification. The sponge was collected at depthsof 10–30 m on the exposed ocean reef-slopes of Majuro and Arno Atolls,Republic of the Marshall Islands, and kept frozen until use. The sponge ispresently namedC. mycofijiensis(14), but was previously classified asSpongia mycofijiensis.

Chemical Isolation of Laulimalide and Isolaulimalide. Frozen spongesamples were lyophilized and extracted with methanol. After concentration,the residue was dissolved in 20% aqueous methanol and extracted withchloroform. The remaining aqueous methanol solution was concentrated, andthe residue was partitioned between water and extracted with n-butanol. Thecombined chloroform and butanol fractions were chromatographed over Seph-

Received 7/15/98; accepted 12/2/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by faculty development funds from NIH Grants CCSG-P30CA 71789 (toS. L. M.) and ACS JFRA-528 (to B. S. D.).

2 To whom requests for reprints should be addressed, at Natural Products Program,Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813.

3 The abbreviations used are: DAPI, 4,6-diamidino-2-phenylindole; PARP, poly-(ADP-ribose) polymerase; GTP, guanosine 59-triphosphate.

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adex LH-20 (20% methanol in dichloromethane), followed by reversed-phasehigh-performance liquid chromatography (octadecyl saline, gradient 20–80%acetonitrile in H2O) and normal-phase high-performance liquid chromatogra-phy (silica, 5% methanol in dichloromethane) yielding pure samples of lauli-malide and isolaulimalide. From 100 g of dry sponge tissue, 16 mg oflaulimalide and 5 mg of isolaulimalide were obtained. The structures oflaulimalide and isolaulimalide were confirmed through the comparison ofspectral data with published data (15, 16) and by comparison with an authenticsample kindly supplied to us by Drs. J. Tanaka and T. Higa (University of theRyukyus, Nishinara, Japan).

Laulimalide and isolaulimalide stocks were solubilized in 100% ethanol andstored at270°C to inhibit the spontaneous conversion of laulimalide intoisolaulimalide. Working dilutions were stored at220°C. Drug stocks wereused within 4 months of dilution, and no loss of potency, suggesting conver-sion, was seen over this time period.

Cell Culture. The A-10 rat aortic smooth muscle cell line and SK-OV-3ovarian carcinoma cell line were purchased from American Type CultureCollection (Manassas, VA). SKVLB-1 cells were the generous gift of Dr.Victor Ling (British Columbia Cancer Center, Vancouver, British Columbia).The SKVLB-1 line is a P-glycoprotein overexpressing subline of SK-OV-3that was selected for resistance to vinblastine (15). The MDA-MB-435 cellline, a human breast adenocarcinoma line, was kindly provided by Dr. MaiHigazi (Georgetown University, Washington, DC). SK-OV-3, SKVLB-1, andA-10 cells were cultured in BME supplemented with 10% fetal bovine serum(HyClone Laboratories, Logan, UT) and 50mg/ml gentamicin. SKVLB-1 cellswere grown in the presence of 1mg/ml vinblastine to maintain selectionpressure for the overexpression of P-glycoprotein. MDA-MB-435 cells weremaintained in Richters medium (Biofluids, Inc., Rockville, MD) with 10%fetal bovine serum and 50mg/ml gentamicin.

Indirect Immunofluorescence. A-10 cells were plated onto glass cover-slips and grown until 70–85% confluent, then treated with drugs, as describedin the figure legends. The cells were fixed with ice-cold methanol for 5 min,blocked for 20 min with 10% calf serum in PBS, and incubated for 90 min withmonoclonalb-tubulin antibody (T-4026; Sigma Chemical Co., St. Louis, MO).After a series of washes, the cells were incubated with FITC-conjugated sheepantimouse IgG (F-3008; Sigma Chemical Co.) for 1 h. The coverslips werewashed, stained with 0.1mg/ml DAPI for 10 min, and mounted. Cellularmicrotubules and chromatin were visualized and photographed using a ZeissAxioplan fluorescence microscope with optics for fluorescein and DAPI.

Inhibition of Cell Proliferation. The IC50 for inhibition of cellular pro-liferation was determined by measuring cell-associated protein after drugtreatment using the sulforhodamine B assay (16, 17).

Cell Cycle Analysis. MDA-MB-435 cells were treated with 20 nM lauli-malide for the times indicated. The cells were stained with propidium iodide,as described previously (18), and DNA content was measured using a CoulterEPICS XL-MCL flow cytometer and plotted as the number of eventsversuspropidium iodide fluorescence intensity.

Tubulin Assembly. The assembly of purified bovine brain tubulin wasmonitored using the CytoDYNAMIX Screen (Cytoskeleton, Denver, CO).This assay uses a 96-well assay plate format with 200mg of lyophilizedpurified tubulin in each well. The tubulin was reconstituted with ice-cold 180ml G-PEM buffer [80 mM NaPIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA, and1 mM GTP] containing laulimalide (1–20mM), paclitaxel (0.5–20mM), orvehicle control (1% ethanol). The assay was conducted at 37°C in a temper-ature-controlled microtiter plate reader. Tubulin polymerization was monitoredspectrophotometrically by the change in absorbance at 340 nm. The absorb-ance was measured at 1-min intervals for 60 min.

Immunoblot Analysis. After laulimalide treatment, the cells were har-vested and protein extracted in radioimmunoprecipitation buffer in the pres-ence of protease inhibitors, as described previously (18). The protein concen-tration of each sample was determined (Pierce Reagent, Rockford, IL), andsamples containing equal amounts of protein were separated by PAGE withSDS using standard Laemmli techniques. The proteins were transferred ontoImmobilon membranes in Tris/glycine buffer. The proteins of interest wereprobed with specific antibodies and detected by chemiluminescence (ECL;Amersham Corp., Arlington Heights, IL). The PARP antibody was obtainedfrom Boehringer Mannheim (Indianapolis, IN), and the caspase 3 antibody waspurchased from PharMingen (San Diego, CA).

Electron Microscopy. Bovine brain tubulin (glycerol-free) was purchasedfrom Cytoskeleton and was used at a concentration of 1 mg/ml in 0.1 M2-[N-morpholino]ethanesulfonic acid buffer (pH 6.9) with 0.1 mM MgCl2 and0.1 mM residual GTP from the tubulin supply buffer. Either paclitaxel orlaulimalide was added to achieve a final concentration of 10mM, the temper-ature was increased from 18°C to 37°C, and absorbance at 350 nm wasmonitored over time. When maximal polymerization had been achieved,aliquots of tubulin were removed and applied to 300 mesh carbon-coatedFormvar-treated copper grids. The tubulin polymers were stained with 1%uranyl acetate and examined and photographed using a Zeiss model 10Belectron microscope.

Statistical Analysis. The Kruskal-Wallis test (19) was used to statisticallycompare the average percentage of cells with micronuclei between the pacli-taxel- and laulimalide-treated groups. The means between the laulimalide- andpaclitaxel-treated groups were compared at each drug concentration.

RESULTS

Effects of Laulimalide, Isolaulimalide, and Paclitaxel on Cellu-lar Microtubules. Strong paclitaxel-like microtubule-stabilizing ac-tivity was found in the crude lipophylic extract from the marinespongeC. mycofijiensis. The extract was cytotoxic, and after treat-ment the only cell remnants that remained were thick, short microtu-bule bundles. Bioassay-directed purification of the extract yielded themicrotubule active-compounds laulimalide and isolaulimalide (Fig.1), as well as the microfilament disrupter latrunculin A. Studies withthe purified compounds showed that both laulimalide and isolauli-malide caused dramatic reorganization of cellular microtubules.

A-10 cells were treated with laulimalide, isolaulimalide, or pacli-taxel for 18 h and the morphological effects on microtubules exam-ined by indirect immunofluorescence techniques. The control cellsexhibited normal microtubule arrays with filamentous microtubulesradiating from the microtubule organizing center to the cell periphery(Fig. 2A). Treatment of the cells with laulimalide disrupted the normalmicrotubule array; the microtubules were more numerous and ap-peared to occupy more of the cytoplasm. A 2-mM concentration oflaulimalide caused microtubule bundles to form throughout the cyto-plasm (Fig. 2B). Cells treated with 20mM laulimalide exhibitedbundles of short tufts of microtubules (Fig. 2C) that were prevalent inthe cell periphery and seemed to be independent of nucleation from

Fig. 1. Chemical structures of laulimalide and isolaulimalide.

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LAULIMALIDE AND ISOLAULIMALIDE



microtubule organizing centers. Isolaulimalide at concentrations be-tween 2–20mM caused an increase in the density of cellular micro-tubules, but no microtubule bundles were present (data not shown).Paclitaxel at concentrations between 1–20mM initiated the formationof a highly organized array of microtubules, some of which formedthick microtubule bundles. Long thick microtubule bundles oftensurrounded the nucleus (Fig. 2D). The extensive long microtubulehoops and bundles that formed after treatment with 2mM paclitaxel(Fig. 2D) were not seen with laulimalide. Cellular microtubules sta-bilized with paclitaxel, laulimalide or isolaulimalide were resistant tovinblastine-induced depolymerization (data not shown).

Effects on Nuclear Structure. A hallmark of the both A-10 andSK-OV-3 cells treated with a wide range of concentrations of lauli-malide and isolaulimalide was the formation of multiple micronuclei.The effects of laulimalide on nuclear structure are visible in Fig. 2.The normal rounded shape of the nucleus, which is devoid of micro-tubules, can be detected (Fig. 2,A and D), whereas in laulimalide-treated cells this distinct microtubule-free area containing the discretecentral nucleus was lost and only vesicle-like areas devoid of micro-tubules remained (Fig. 2,B andC). Nuclear staining of control cellsrevealed a central compact nucleus (Fig. 3A), whereas laulimalide-treated cells exhibited a dramatic breakdown of the nucleus intomicronuclei (Fig. 3B). Similar nuclear changes occurred with isolauli-malide (data not shown). Paclitaxel also initiated the formation ofmicronuclei, as has been reported by others (20). Analysis of theincidence of micronuclei formation showed that a higher percentageof laulimalide-treated cells contained micronuclei than cells treatedwith the same concentrations of paclitaxel. The data in Fig. 4 show

that in A-10 cells 0.02–2mM laulimalide caused micronuclei forma-tion in approximately 60% of the cells. Paclitaxel at the same con-centrations caused approximately 40% of the cells to exhibit thisabnormal restructuring of the nucleus. At all concentrations tested,laulimalide caused a higher percentage of cells to exhibit micronucleiwhen compared with paclitaxel-treated cells. The difference betweenthe two drugs was statistically significant at all five concentrationstested (P,0.0001).

Effects of Laulimalide on Cell Cycle Progression and MitoticSpindles. A common characteristic of antimicrotubule agents is theirability to initiate mitotic arrest. Flow cytometric analysis revealed thatlaulimalide-induced cell cycle arrest in G2-M in MDA-MB-435 breastcarcinoma cells within 9 h of treatment (Fig. 5). This is consistentwith the effects of other antimicrotubule agents, where disruption ofmicrotubule dynamics prevents normal mitotic progression and leadsto mitotic arrest. Abnormal mitotic spindles were seen in both A-10cells and SK-OV-3 cells after treatment with laulimalide and isolauli-malide. The mitotic cells were rounded, and the spindles formed acircular pattern with spindle microtubules radiating outward from amicrotubule-free core in the center of the cell (Fig. 6A). In the mitoticcells, the nuclear membranes were not apparent, and the chromatinwas condensed and aligned in a circular pattern. Abnormal mitoticspindles were visible in paclitaxel-treated cells and were typicallytri-or tetra-polar (Fig. 6B) and did not exhibit the morphology seenwith the laulimalides.

Effects of Laulimalide, Isolaulimalide, and Paclitaxel on CellProliferation of Drug-Sensitive and Multidrug-Resistant Cell

Fig. 2. Effects of laulimalide on microtubules.Microtubules were visualized by indirect immuno-fluorescence techniques in A-10 cells after 18-htreatments with vehicle control (A), 2 mM lauli-malide (B), 20mM laulimalide (C), or 2mM pacli-taxel (D).

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Lines. The early literature on laulimalide reported that it was acytotoxin (11, 13). In this study, experiments were conducted todetermine the IC50 values for laulimalide and isolaulimalide in twodrug-sensitive cell lines, MDA-MB-435 and SK-OV-3, and in amultidrug-resistant cell line, SKVLB-1. Laulimalide is a potent in-hibitor of cell proliferation with IC50 values between 5–12 nM (Table1). Isolaulimalide is less potent with IC50 values in the lowmM range.Both laulimalide and isolaulimalide inhibited the proliferation of theSKVLB-1 cell line that overexpresses the drug efflux pump P-glyco-

protein. The resistance factors between the parental, drug-sensitiveline (SK-OV-3) and the drug-resistant cell line (SKVLB-1) were 105and 1.03 for laulimalide and isolaulimalide, respectively (Table 1).We did not achieve.70% inhibition of the SKVLB-1 cell line withconcentrations of paclitaxel up to 100mM. Laulimalide and isolauli-malide are significantly more effective against the SKVLB-1 cell linethan paclitaxel. These data confirm that these new agents are poorsubstrates for transport by P-glycoprotein.

Fig. 4. Effects of laulimalide and paclitaxel on micronuclei formation. A-10 cells weretreated for 18 h with paclitaxel or laulimalide. The cells were fixed, and nuclei werevisualized by DAPI staining. The percentages of cells containing micronuclei werecounted in 20 microscope fields/treatment in two experiments.

Fig. 5. Effects of laulimalide on cell cycle distribution. Log phase growth cultures ofMDA-MB-435 cells were treated with vehicle control (A) or 20 nM laulimalide for 9 h (B)or 18 h (C). After treatment, the cells were fixed, stained, and analyzed on a CoulterEPICS XL-MCL flow cytometer and plotted as eventsversuspropidium iodide fluores-cence intensity.

Table 1 Inhibition of proliferation in drug sensitive and resistant cells

Cells were treated with varying concentrations of the drugs for 48 h, and cell-associated protein was determined using the SRB assay. The IC50 for each compound wascalculated for each cell line. The values represent the means of three (laulimalide,isolaulimalide) or four (paclitaxel) experiments6 SD.

IC50 (nM)

Cell line Laulimalide Isolaulimalide Paclitaxel

MDA-MB-435 5.746 0.58 1,9706 97 1.026 0.25SK-OV-3 11.536 0.53 2,5706 290 1.716 1.07SKVLB 1,2106 490 2,6506 1,384 .100,000Resistance factor 105 1.03 .58,480

Fig. 3. Effects of laulimalide on nuclear structure. DNA was visualized by stainingwith DAPI in A-10 cells after 18-h treatments with a vehicle control (A) or 2 mM

laulimalide (B).

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The IC50 for inhibition of proliferation was also determined in theA-10 cell line, a nontransformed line that was used to show the effectsof microtubule-stabilizing agents on cellular structures (Figs. 2 and 6).The IC50 for laulimalide was 51mM and for paclitaxel was 40mm.The nontransformed A-10 cell line was much less sensitive to theinhibitory effects of both paclitaxel and laulimalide, illustrating theneed to use micromolar concentrations in these cells to induce abnor-mal microtubules.

Initiation of Apoptosis by Laulimalide. The ultimate mechanismof action of many cytotoxic cancer chemotherapeutic agents is theinitiation of pathways of gene and protein expression leading toapoptosis (reviewed in Refs. 21 and 22). Antimicrotubule drugsincluding paclitaxel, vinblastine, and cryptophycin 1 initiate apoptosisboth in vitro and in vivo (18, 23–25). The flow cytometry data (Fig.5) show a doubling of the subdiploid peak at 18 h (Fig. 5C), suggest-ing the initiation of apoptosis. The loss of cellular DNA is detected bythe appearance of the subdiploid peak when apoptotic cells are ana-lyzed by flow cytometry (26). Studies were undertaken to determinewhether laulimalide initiates a gene-driven program of cellular sui-cide.

During apoptosis, specific cysteine proteases called the caspasesare activated (34). Activation of the caspase cascade leads to theproteolytic degradation of specific cellular proteins. The activationof caspase 3 and the proteolysis of the DNA repair enzyme PARP,a downstream substrate of caspase 3, were examined in cell lysatesfrom laulimalide-treated cells. Activation of caspase 3 leads to theloss of the 32 kDa proenzyme and the formation of the activationproducts p17 and p12 (28). Analysis of immunoblot data from the

cell lysates (Fig. 7) shows the formation of the p17 activationproduct at 24, 42, and 48 h after laulimalide treatment. The loss ofthe p32 proenzyme is seen at 48 h. The specific proteolysis ofPARP by caspase 3 leads to the formation of two products, an 89kDa COOH-terminal fragment and a 24 kDa N-terminal fragment(28). The proteolysis of PARP and the appearance of the 89 kDadegradation product coincided with the activation of caspase 3(Fig. 7). Caspase 3 was activated and PARP proteolytically cleavedin cell lysates from cells treated with laulimalide for 42 and 48 h.These data are consistent with laulimalide-induced apoptotic celldeath.

Effects of Laulimalide on Tubulin Polymerization in Vitro. Onecharacteristic of the microtubule-stabilizing agents paclitaxel, dis-codermolide, epothilones A and B, and eleutherobin is the abilityof these agents to initiate the polymerization of tubulin in theabsence of polymerization promoters, such as glycerol. Studieswere undertaken to determine the effects of laulimalide on tubulinpolymerization. Both isolaulimalide and laulimalide stimulatedtubulin polymerization. Fig. 8A shows the effects of of laulimalideon tubulin polymerization, and the accompanying Fig. 8B showsthe effects of paclitaxel. Comparisons between the effects oflaulimalide and paclitaxel on tubulin polymerization (Fig. 8,A andB) show that, at low micromolar concentrations, paclitaxel is morepotent than laulimalide (Table 2). At low micromolar concentra-tions more tubulin polymer was formed in the presence of pacli-taxel, and the rate of polymerization was faster than was seen withequivalent concentrations of laulimalide.

A very different relationship was seen when comparing the 20-mmconcentrations of laulimalide and paclitaxel. The 20-mM concentrationof laulimalide was more effective at stimulating the formation oftubulin polymer than was the 20-mM concentration of paclitaxel (Fig.8, A and B). Laulimalide promoted the polymerization of approxi-mately 30% more tubulin polymer than was formed in the presence ofpaclitaxel, and the kinetics of tubulin formation were twice as fast asthe rate measured in the presence of 20mM paclitaxel.

The tubulin polymers formed in the presence of laulimalide wereinsensitive to cold and CaCl2-induced depolymerization (data notshown). Neither paclitaxel nor laulimalide promoted the polymeriza-

Fig. 6. Laulimalide-induced mitotic spindles and chromatin alignment. Confluentcultures of A-10 cells were treated with 2mM laulimalide for 18 h, and the microtubuleswere visualized by indirect immunofluorescence. The chromatin was stained with DAPI.

Fig. 7. Laulimalide-induced caspase activation. MDA-MB-435 cells were treated with100 nM laulimalide for 18, 24, 42, or 48 h, followed by protein extraction in lysis buffercontaining protease inhibitors. Samples of equal amounts of protein were applied tobis-acrylamide gels and separated using standard Laemmli techniques. The proteins weretransferred onto polyvinylidene difluoride (Immobilon) membrane, and the blots wereprobed with specific antibodies forcaspase 3(identifying the proenzyme p32, as well asthe p17 activation product) orPARP.

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tion of tubulin at 0°C, and laulimalide stabilized tubulin polymerformed in the presence of GTP.

Samples of the tubulin polymer formed were examined by electronmicroscopy to determine whether the increase in turbidity measuredduring the polymerization experiments was due to the formation ofmicrotubule-like polymers or the formation of other structures. Underhigh magnification (363,000) the tubulin polymers formed by pacli-taxel and laulimalide were indistinguishable (Fig. 9,A andB). Bothagents formed structures resembling tubules with evidence of longi-tudinal symmetry. Examination of tubulin polymers formed in thepresence of the microtubule-stabilizing agents at lower magnification(34,000) showed that the polymers formed with laulimalide werevery long structures with rounded curves (Fig. 9D). The polymersformed with paclitaxel were notably shorter and exhibited no roundedcurves, but instead formed linear structures with angular branches(Fig. 9C).

DISCUSSION

Laulimalide and isolaulimalide were first isolated on the basis oftheir cytotoxicity, however, the mechanism of action was not eluci-

dated. We now report that these agents are paclitaxel-like stabilizersof microtubules that cause alterations of both interphase and mitoticmicrotubules. Laulimalide is a potent inhibitor of cell proliferationand initiates mitotic arrest, micronuclei formation, and ultimatelyapoptosis. These compounds are superior to paclitaxel in their abilityto circumvent P-glycoprotein-mediated drug resistance. The lauli-malides represent a new class of paclitaxel-like microtubule-stabiliz-ing agents with properties that may provide advantages over thetaxanes.

Laulimalide and isolaulimalide are chemically related compounds,with isolaulimalide being a decomposition product of laulimalide. Thedifference between these two compounds is in the size and attachmentpoints of the oxygen-containing ring within the top portion of themolecules. Laulimalide contains a three-membered epoxide ring in-volving carbons C-16 and C-17, whereas isolaulimalide contains afive-membered tetrahydrofuran ring linking carbon C-17 with sidechain carbon C-20. This slight chemical difference between lauli-malide and isolaulimalide results in a difference in potency of greaterthan two orders of magnitude in their ability to inhibit cell prolifer-ation. Furthermore, this site also seems to be crucial for recognition bythe multidrug efflux pump P-glycoprotein. Laulimalide had a resist-ance factor of 105 when comparing the IC50 values in SK-OV-3 cellsand SKVLB-1 cells, whereas isolaulimalide was equally sensitive inboth cell lines. These data suggest that the epoxide moiety of lauli-malide is critical for its interaction with both tubulin and P-glycoprotein.

Among the five groups of known antimicrotubule agents havingpaclitaxel-like microtubule-stabilizing properties, laulimalide mostclosely resembles the epothilones. Although the ring size of lauli-malide is two carbons bigger (18-memberedversus16-membered),both contain a similar structural motif that incorporates the epoxidering, an unsaturated side chain bearing a methylated heterocyclic ring,and the ester of the macrocyclic lactone ring. This similarity seems totranslate to similar activities. Both stabilize microtubules, and likelaulimalide, the epothilones are poor substrates for P-glycoprotein-mediated transport (9).

The cellular effects of laulimalide are similar to, but distinct from,the cellular effects of paclitaxel. The increase in the density of cellularmicrotubules observed at low concentrations of laulimalide closelyresembled the changes induced by paclitaxel at the same concentra-tions. However, at concentrations above 2mM, the effects divergedand laulimalide initiated short thick bundles of microtubules that weremore prevalent in the cell periphery and appeared to form from manynucleation centers. These effects are consistent with the greater effi-cacy of 20mM laulimalide in promoting the polymerization of purifiedtubulin. In contrast, paclitaxel-induced microtubule bundles were longand thick and aligned in the central areas of the cells surrounding thenucleus, consistent with nucleation from one or two centers. Longmicrotubule bundles were not seen in cells treated with a wide rangeof laulimalide concentrations. These data suggest that in cells, lauli-malide was not as effective as paclitaxel at elongation of microtu-bules, but was more effective at stimulating the formation of micro-tubules from multiple nucleation centers, resulting in shortermicrotubule bundles in the periphery rather than the long microtubulebundles surrounding the nucleus.

The microtubules that form the mitotic spindle are highly dy-namic structures and are more sensitive to disruption by antimi-crotubule agents than are the less dynamic interphase microtu-bules. Agents that target microtubules disrupt mitotic spindledynamics, thereby preventing normal mitosis, leading to mitoticarrest (1). Mitotic spindles formed in the presence of laulimalidewere abnormal and formed unique starburst arrays in contrast tothe short thickened tri- and tetra-polar spindles formed in thepresence of paclitaxel. Laulimalide-treated mitotic cells exhibited

Fig. 8. Effect of laulimalide and paclitaxel on tubulin polymerization. The polymeri-zation of bovine brain tubulin was monitored in the presence of laulimalide (A) orpaclitaxel (B). All reagents were equilibrated to 24°C, the drugs were added to a finalconcentration shown, and the temperature was increased to 37°C. The values plotted arethe means of two experiments.

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chromatin condensation, loss of the nuclear envelope and abnormalchromatin alignment. The aberrant mitotic spindles were associ-ated with circular chromatin arrays, suggesting that the microtu-bules were coordinating a specific, but abnormal structuring of thechromatin. Disruption of the mitotic apparatus by laulimalidetreatment lead to mitotic arrest, followed by the initiation ofapoptosis, as determined by the increase in cells in G2-M and theactivation of the caspase cascade.

Cells treated with laulimalide exhibited vesicle-like structures inthe central region of the cell. DAPI staining revealed that thesestructures were composed of DNA and that laulimalide initiated theformation of multiple micronuclei. In A-10 cells, micronuclei werefound in the majority of cells with treatments of 20 nM-2 mM lauli-malide. Micronuclei formations occur as a consequence of treatment

with either paclitaxel or epothilones and are thought to be the resultof abnormal mitosis leading to abnormal chromosome segregation(4, 20).

One characteristic of the paclitaxel-like microtubule-stabilizingagents is their ability to promote the polymerization of tubulin.Normally tubulin will not polymerize without tubulin promoters;however, laulimalide, like paclitaxel, stimulated the polymeriza-tion of tubulin in the absence of microtubule-associated proteinsand glycerol. The rate and extent of polymerization in the presenceof laulimalide was dependent on concentration and differed sig-nificantly from paclitaxel, suggesting that there are differences intheir mechanisms of action. Further experimentation in the pres-ence of different tubulin and laulimalide concentrations and in thepresence and absence of microtubule-associated proteins will allowmore comprehensive comparisons of the tubulin polymerizing ef-fects of laulimalide as compared with paclitaxel, discodermolide,and the epothilones.

The tubulin polymers formed in the presence of laulimalide wereindistinguishable from paclitaxel-induced polymers under highmagnification, but were noticeably longer than the paclitaxel-induced polymers when visualized under lower magnification.Under these conditions, laulimalide seems to promote polymer-elongating activity more readily than paclitaxel. Thesein vitro

Fig. 9. Paclitaxel- and laulimalide-induced tubulin polymers. Electron micrographs of tubulin polymer formed in the presence of 10mM paclitaxel (AandC) or 10mM laulimalide(B andD). A andB, the original magnification was 63,5003; bar, 200 nm.C andD, the original magnification was 4,0003; bar, 3 mm.

Table 2 Potency of laulimalide and paclitaxel for tubulin polymerization

The EC50s for tubulin polymerization for laulimalide and paclitaxel were calculatedfrom dose-response curves generated using the CytoDYNAMIX Screen 3 assay. Tubulinwas polymerized with paclitaxel or laulimalide for 40 min at 37°C in the presence of drugor vehicle control. Tubulin polymer formed after 40 min in the presence of 20mM drugwas defined as maximum enhancement of polymerization for each compound (n 5 2).

EC50 (mm)

Laulimalide 4.326 0.4Paclitaxel 1.446 0.06

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effects with purified tubulin differ from the effects in cells wherepaclitaxel promotes longer microtubules, whereas laulimalide pro-motes short microtubules. The initial studies suggest that there areintriguing differences in the mechanisms of action of laulimalideand paclitaxel.

The clinical success of the taxanes in treating a wide range oftumors has lead to the search for new agents with a similar mechanismof action. The recent discovery of four additional classes of com-pounds, discodermolide, the epothilones, eleutherobin, and now thelaulimalides, provides hope that new drugs may be found to treatcancer.

ACKNOWLEDGMENTS

We extend special thanks to Bessie Kam of the Pathology Department(Queens Medical Center, Honolulu, HI) for expertise in obtaining the flowcytometry data, Tina Carvalho (University of Hawaii’s Biological ElectronMicroscope Facility, Honolulu, HI) for expert technical assistance with theelectron micrographs, Denise Tambasco [Chaminade University and the Mi-nority Biomedical Research Support (GM 47537) program, Honolulu, HI] forassistance with the caspase activation data, and Serafin Colmenaris III andJennifer A. Hewett for help with the figures. We also thank Dr. MichelleKelly-Borges (Museum of Natural History, London, United Kingdom; pres-ently at the UNITEC Institute of Technology, Auckland, New Zealand) forsponge taxonomy.

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