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Preclinical Development

Targeting the Microtubular Network as a NewAntimyeloma Strategy

Rentian Feng1, Shirong Li1, Caisheng Lu1, Carrie Andreas1, Donna B. Stolz2, Markus Y. Mapara1,and Suzanne Lentzsch1

AbstractWe identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the

Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a

broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited

growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and

in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase

arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression

of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1L levels were involved in

nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of

cell death, triggered by the activation of c-jun-NH2 kinase (JNK) instead of p38 kinase or extracellular signal–

regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced

nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma

tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole

has potent antimyeloma activity and that targeting the microtubular network might be a promising new

treatment approach for multiple myeloma. Mol Cancer Ther; 10(10); 1886–96. �2011 AACR.

Introduction

Multiple myeloma is the second most prevalent hema-tologic malignancy and is uniformly fatal, very often as aresult of development of drug resistance. To overcomethe chemoresistance to current therapies and improvepatient outcome, novel treatment agents are needed totarget mechanisms whereby multiple myeloma cellsgrow and survive.

The coordinatedprocesses of cell-cycle progression, cellgrowth, and apoptosis are dysfunctional in cancer (1, 2).During cell-cycle progression, microtubule assembly is aproven target for anticancer drug development becauseof its critical role for mitotic spindle formation and theseparation of chromosomes duringmitosis (3). It has beenshown that the c-jun-NH2 kinase (JNK)/stress-activated

protein kinase (SAPK) pathway is involved in cell-cycleregulation and that microtubule-interfering agentsactivate this pathway inducing G2–M arrest that resultsin apoptosis in a variety of human cancer cells (4).

The Bcl-2 family of proteins includes both pro- andantiapoptotic molecules and their ratio determines thefate of cells. Bcl-2 protein is regulated at transcriptionaland posttranslational levels including phosphorylationwithin the flexible loop regulatory domain. Thesemodifications induce conformational changes in theBcl-2 protein and regulate its active forms in responseto cell death signaling (5, 6). It has been further found thatBcl-2 is phosphorylated/inactivated by JNK/SAPK (7),suggesting that Bcl-2 protein is a target of microtubule-damaging agents, resulting in G2–M cell-cycle block (8, 9).

Benzimidazoles, including albendazole, fenbendazole,mebendazole, and nocodazole, have been used as anthel-mintics and fungicides on the basis of their antimicrotu-bule activity (10) and have been reported to elicitpromising antitumor effect (11–13). Although nocodazolehas been recently categorized as an antineoplastic agent,the antimyeloma effects and its underlyingmechanism ofaction have not been examined yet. By using multiplexcytokine array on a chemical library containing 1,000compounds provided by theMultiple Myeloma ResearchFoundation, we identified nocodazole as a potent anti-myeloma agent and showed that benzimidazoles, andespecially nocodazole, significantly reduce the secretionof cytokines essential for multiple myeloma survival (14).

Authors' Affiliations: 1Division of Hematology/Oncology, Department ofMedicine, University of Pittsburgh Cancer Institute; and 2The Center forBiologic Imaging, Department of Cell Biology & Physiology, University ofPittsburgh, Pittsburgh, Pennsylvania

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Suzanne Lentzsch, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute, 5150 Centre Avenue,Suite 568, Pittsburgh, PA 15232. Phone: 412-648-6578; Fax: 412-648-6579; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0234

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(10) October 20111886

on January 18, 2021. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 8, 2011; DOI: 10.1158/1535-7163.MCT-11-0234

http://mct.aacrjournals.org/

In the present study,we show that benzimidazoles inducecell death in multiple myeloma cell lines and in primaryCD138þmyelomacells andovercomedrug resistance. Theinduction of apoptosis by nocodazolewas associatedwithG2–M phase of cell-cycle arrest and was induced by JNK-mediated Bcl-2 phosphorylation in both primary andmultiple myeloma cell lines. Subsequent induction ofapoptosis by nocodazole could be abrogated by a specificJNK inhibitor. Nocodazole, alone or combined withdexamethasone, effectively suppressed myeloma tumorgrowth in a human multiple myeloma xenograft mousemodel, suggesting that nocodazole is a potent andpromis-ing new antimyeloma agent.

Materials and Methods

Reagents and cell cultureU0126 and PD98059 [mitogen-activated protein

kinase (MAPK) inhibitor], SB203580 (p38-MAPK inhib-itor), and SP600125 (JNK inhibitor) were purchasedfrom Calbiochem. Fetal calf serum (FCS) and other cellculture reagents were purchased from Sigma-Aldrich.Albendazole, fenbendazole, mebendazole, and nocoda-zole (Fig. 1B) were prepared in dimethyl sulfoxide asstock solutions, stored at �20�C, and subsequentlydiluted in RPMI-1640 medium before use. All drugswere provided by Prestwick Chemicals and obtainedwithin a grant awarded by the Multiple MyelomaResearch Foundation.The following primary antibodies were purchased

from Cell Signaling Technology: phospho-c-Jun NH2-terminal kinase, phospho-p38 and phospho-p44/42MAPKs, phospho-Bcl-2, Bax, Bim, X-linked inhibitor ofapoptosis (XIAP), survivin, and Bid. Mcl-1L and deathreceptor 4 (DR4) antibodies were purchased from SantaCruz Biotechnology, and actin and b-tubulin conjugatedwith Alexa Fluor 555 antibodies were purchased fromSigma-Aldrich.Multiple myeloma cell lines H929 and U266 were

purchased from American Type Culture Collection inMay 2010 and August 2009, respectively. Dr. WilliamDalton (H. Lee Moffitt Cancer Center & Research Insti-tute, Tampa, FL) kindly provided the human multiplemyeloma cell line RPMI-8226/S and its sublines RPMI-8226/Dox40 (resistant to doxorubicin), RPMI-8226/MR20 (resistant to mitoxantrone), and RPMI-8226/LR5(resistant to melphalan) in August 2009. Dexamethasone-sensitive and -resistant cell linesMM.1S andMM.1Rwereprovided by Dr. Klaus Podar (Dana Farber CancerInstitute, Harvard Medical School, Boston, MA) inAugust 2009. These cell lines were tested for their drugresistance, but no cell line authentication was done by theauthors. Multiple myeloma cell lines were cultured inRPMI-1640 mediumwith 10% FCS, 2 mmol/L glutamine,and 100 U/mL penicillin–streptomycin (Sigma-Aldrich)at 37�C with 5% CO2. The chemoresistant multiplemyeloma cell lines were cultured in the presence ofdoxorubicin, mitoxantrone, melphalan, or dexametha-

sone, and resistance was confirmed by cell proliferationassays (Supplementary Fig. S1).

Bone marrow mononuclear cells were obtained fromuntreated multiple myeloma patients and were iso-lated by separation on Ficoll-Hypaque gradients asdescribed previously (15). CD138þ bone marrow cellsfrom multiple myeloma patients were purified byCD138 (syndecan-1) microbeads using a magnetic cellsorting system (Miltenyi Biotec) as described previously(16) and used as primary multiple myeloma cells.Unlabeled cells were used as CD138� cells. All studieswere approved by the Institutional Review Board of theUniversity of Pittsburgh, and all subjects signedapproved consent forms.

Cell proliferation assay (3H-thymidineincorporation)

U266, H929, RPMI-8226 (3 � 104 cells per well), MM.1Scell line (6 � 104 cells per well), and their resistantsublines were cultured in 96-well culture plates (Costar)in RPMI-1640 medium containing 10% FCS with or with-out drugs for 48 hours at 37�C with 5% CO2. For bonemarrow stromal cell coculture experiments, human pri-mary bone marrow stromal cells (3 � 103 cells per well)were cultured for 24 hours in Dulbecco’s ModifiedEagle’s Medium containing 10% FCS in 96-well plates.Then, multiple myeloma cells (3� 104 cells per well) wereseeded on bone marrow stromal cells and cultured for 48hours in the presence or absence of nocodazole at differ-ent concentrations. Cells were pulsed with 1 mCi/well3H-thymidine during the last 8 to 10 hours of culture,harvested onto glass fiber filter mats (Wallac) with anautomatic cell harvester and counted using a WallacTriLux Beta plate scintillation counter.

Cell-cycle analysesAfter incubation with nocodazole (100 nmol/L for 0–24

hours or 0–160 nmol/L for 16 hours), human multiplemyeloma cells were harvested and washed with coldPBS, fixed in 70% ethanol at �20�C, treated withDNase-free RNase A (Sigma-Aldrich), and stained with50 mg/mL propidium iodide (Sigma) at 37�C for 30minutes. Analyses were conducted on a BD FACSCaliburflow cytometer and analyzed using ModFit LT 2.0 andCell Quest software (BD Biosciences).

Assessment of apoptotic cell death and cell viabilityApoptosis was assessedmorphologically by evaluation

of nuclear condensation and fragmentation usingHoechst 33258 staining as described previously (17).Treated multiple myeloma cells were incubated with2.5 mg/mL Hoechst 33258 (Molecular Probes, Life Tech-nologies Corp.) for 20 minutes followed by examinationunder a fluorescence microscope (Olympus CKX41).Cells (�200 per condition) were randomly selected andassessed. Hoechst 33258–positive cells with apoptoticbodies or condensed and fragmented nuclei wereconsidered and counted as apoptotic cells.

Antimyeloma Activity of Nocodazole

www.aacrjournals.org Mol Cancer Ther; 10(10) October 2011 1887




Viability of cells was determined by trypan blue stain-ing, which distinguishes the membrane-defective deadcells from the viable cells.

Immunofluorescence staining and electronmicroscopy

Multiple myeloma cells (H929 or RPMI-8226)were fixed in PBS containing 3% formaldehyde for15 minutes and washed in PBS. Cytospin slideswere prepared using Cytospin (Thermo ShandonInc.) and permeabilized in 0.3% Triton X-100/PBS

(30 minutes at 4�C). After blocking with 2.5% bovineserum albumin in PBS, the samples were incubatedwith Alexa Fluor 555–conjugated antibody againstb-tubulin overnight at 4�C. Counterstaining withHoechst 33258 for nuclear location and integritywas conducted before sealing the slides withFluoro-Gel (Electron Microscopy Sciences). Slideswere examined under a filter-combined fluorescencemicroscope (Olympus CKX41) equipped with a �20/0.40 numeric aperture objective lens (OlympusAmerica, Inc.).

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Figure 1. Effects ofbenzimidazoles on multiplemyeloma cell growth and viability.A, U266 multiple myeloma cellswere treatedwith concentrations ofbenzimidazole for 48 hours. H929multiple myeloma cells weretreated with the indicatedconcentrations of benzimidazolesfor 48 hours. DNA synthesis wasmeasured by 3H-thymidineincorporation. A, bottom, RPMI-8226 multiple myeloma cells weretreated with benzimidazoles for 16hours: albendazole, fenbendazole,mebendazole, and nocodazole.Percentages of the apoptotic celldeathwith typical apoptotic nuclearmorphology were determined bystaining the cells with Hoechst33258 fluorochrome. B, chemicalstructure of nocodazole. PrimaryCD138þ andCD138�bonemarrowmononuclear cells were treatedwith indicated concentrations ofnocodazole for 48 and 72 hours.Cell viability was determined usingtrypan blue staining. C, bonemarrow stromal cells were grown in96-well plates in Dulbecco'sModified Eagle's Mediumcontaining 10% FCS. H929multiplemyeloma cells were addedto bone marrow stromal cells(BMSC) and cultured for another48 hours in the presence orabsence of the indicatedconcentrations of nocodazole.DNA synthesis was measured by3H-thymidine incorporation. D,RPMI-8226 cell line (RPMI-8226/S)and its chemoresistant sublines(RPMI-8226/Dox40, RPMI-8226/MR20, or RPMI-8226/LR5) andMM.1S and its dexamethasone-resistant subline (MM.1R) weretreated with indicatedconcentrations of nocodazole for48 hours. Cell proliferation wasmeasured by 3H-thymidineincorporation. All experimentswereconducted in triplicates. Resultsare shownasmean�SD. *,P < .05.

Feng et al.

Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics1888




For electron microscopy, multiple myeloma cells werefixed with 2% paraformaldehyde and 2% glutaraldehydein 0.1 mol/L PBS (pH ¼ 7.4) followed by 1% OsO4. Afterdehydration, sections were stained with uranyl acetateand lead citrate and examined under a JEM 1011 (JEOL)electron microscope. To examine and quantify cellularmorphologic changes, digital phase-contrast images wererecorded.

SDS-PAGE and Western blottingWestern blotting was conducted as previously de-

scribed (18). Briefly, cells were harvested, lysed withradioimmunoprecipitation assay buffer (Pierce) con-taining phosphatase and protease inhibitors (Halt Pro-tease Inhibitor Cocktail Kit; Pierce). Lysates weresubjected to 12% SDS-PAGE and transferred to poly-vinylidene fluoride membrane. Following probing withspecific primary antibodies plus horseradish peroxi-dase–conjugated secondary antibody, the protein bandswere detected using SuperSignal West Pico Chemilu-minescent Substrate (Pierce).

Human xenograft mouse modelBeige/nude/X-linked immunodeficient mice were

purchased from Charles River Laboratories at 6 to8 weeks of age with a weight between 20 and 25 grams.For human tumor xenograft studies, 1 � 107 H929myeloma cells in 50 mL RPMI-1640 together with anequal volume of Matrigel basement membrane matrix(BD Biosciences) were injected subcutaneously asdescribed previously (19). When tumors were palpable,mice were assigned randomly to treatment groupsreceiving 5 or 20 mg/kg nocodazole intraperitoneally3 times weekly on Monday, Wednesday, and Friday.Combination studies with dexamethasone were carriedout by treating mice with 12 mg/kg nocodazole (n ¼ 5)intraperitoneally on Monday, Wednesday, and Friday;2 mg/kg dexamethasone (n ¼ 6) subcutaneously onMonday and Friday; or their combination (n ¼ 5). Thecontrol group (n ¼ 6) received the same volume of vehi-cle alone with the same treatment frequency. Mice wereweighed twice weekly and observed daily for diarrheaor any changes in behavior and condition. Tumor sizeswere measured whenever the drug was given using acaliper and calculated by the formula: 0.5 � width2 �length, representing the 3-dimensional volume of anellipse. Animals fromboth cohortswere euthanizedwhentheir tumors reached 2 cm in one diameter and/or wereulcerated (endpoint). Survival was evaluated from thefirst day of treatment until animals were euthanized.This was defined as endpoint of the study.All animal studies were approved by the Institutional

Animal Care and Use Committee of the University ofPittsburgh.

Statistical analysesAll quantitative data are presented as mean � SD of at

least triplicates. Statistical differences were determined

by Student’s t test. Kaplan–Meier survival analysis andlog-rank test were conducted to estimate the survivalrates and survival differences. Results were consideredsignificantly different if P < 0.05.

Results

Nocodazole inhibits myeloma cell growth andovercomes chemoresistance

We tested the direct effects of benzimidazoles onhuman multiple myeloma cell proliferation and celldeath induction. Albendazole, fenbendazole, and noco-dazole significantly inhibited multiple myeloma cellproliferation with an IC50 value of 25 to 94 nmol/L forU266 multiple myeloma cells and an IC50 value of 63to 380 nmol/L for H929 multiple myeloma cells. Further-more, benzimidazoles induced nuclear fragmentation,a typical sign of cell apoptosis, with an IC50 value of30 to 250 nmol/L in RPMI-8226 myeloma cells (Fig. 1A).On the basis of our finding that nocodazole exhibitedthe strongest antimyeloma effect, we focused our furtherstudies on nocodazole (Fig. 1B, left). Following 48- and72-hour exposure to nocodazole, the viability of primaryCD138þ multiple myeloma cells decreased in a dose-dependent manner as monitored by trypan blue staining(IC50 ¼ 125–185 nmol/L). The viability of non–multiplemyeloma (CD138�) cells obtained from the same pati-ents was affected only at much higher concentration(IC50 � 500 nmol/L), indicating the relative selectivityof nocodazole for malignant plasma cells (Fig. 1B, right).In addition, the strong inhibition of proliferation ofmultiple myeloma cells treated with nocodazole wasnot reversed if multiple myeloma cells were coculturedwith bone marrow stromal cells (Fig. 1C).

Because drug resistance is a prevalent problemin multiple myeloma treatment (20), we determinedwhether nocodazole overcomes drug resistance in mul-tiple myeloma. First, we confirmed the chemoresistantphenotypes of RPMI-8226 sublines (8226/Dox40, 8226/MR20, or 8226/LR5) by culturing the RPMI-8226 cellswith doxorubicin, mitoxantrone, or melphalan andanalyzing cell proliferation (Supplementary Fig. S1).The drug-resistant sublines exhibited similar responsesto nocodazole treatment (IC50 ¼ 25–65 nmol/L) as theirparental cell lines. Similar results were observed withdexamethasone-sensitive and -resistant cell lines MM.1Sand MM.1R (Fig. 1D), suggesting that nocodazole over-comes chemoresistance.

Nocodazole-induced prometaphase arrest isassociated with disruption of microtubular networkarray in multiple myeloma cells

Treatment of H929 multiple myeloma cells withnocodazole resulted in G2–M phase cell-cycle arrest.After treatment with nocodazole for 7 and 16 hours,the percentage of G2 population increased to 50% and35%, respectively, in comparison with control (21%).Prolonged treatment was associated with increased






apoptosis of multiple myeloma cells, as evidenced bythe increased population of pro-G0 phase cells as well asthe appearance of polyploid cells (Fig. 2A, top). In accor-dance with the time-dependent induction of G2 arrest,we also observed a dose-dependent G2 arrest with anincrease of G2 population to 40% at 80 nmol/L and 56% at160 nmol/L in comparison with control (19%; Fig. 2A,bottom).

To identify mitotic phase arrest in nocodazole-treatedmultiple myeloma cells, we examined microtubuledynamic change and chromosome status as well asnuclear membrane integrity. Immunofluorescence stain-ing of the vehicle-treated cells for tubulin showed theregular structure of microtubules with cells in anaphase/telophase. However, nocodazole-treated multiple mye-loma cells displayed a disordered microtubule structure.

Spindle formation was disrupted and cells exhibited anirregular staining pattern for tubulin along with somesmall punctate areas (Fig. 2B). The mitosis-like chromo-somes with disordered orientation were condensed andscattered inside the cells. The peak of destruction of themicrotubular network was observed after 16 hours ofnocodazole treatment (Fig. 2B and SupplementaryFig. S2A). Electron microscopy studies indicated thatthe nuclear membrane had collapsed and disappeared(Fig. 2C), which is a typical phenomenon of prophaseand prometaphase, suggesting that the cells arrested inprophase/prometaphase.

We found that nocodazole also caused significant(P < 0.05) morphologic elongation of multiple myelomacells (Fig. 2B and C and Supplementary Fig. S2A–C).Mostof the elongated cells only had one irregular nucleus,

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Figure 2. Nocodazole inducesprometaphase arrest associatedwith disruption of the microtubularnetwork. A, H929 multiplemyeloma cells were treated withnocodazole as indicated. Cellcycle was analyzed by propidiumiodide staining and flowcytometry, measuring the cellularDNA content. Results arerepresentative of 2 independentexperiments. B, H929 multiplemyeloma cells were treated withnocodazole (100 nmol/L) for0 to 16 hours andimmunofluorescence stainingagainst tubulin was conducted.Nuclear location and integritywere detected by counterstainingwith Hoechst 33258. Disarray ofmicrotubular network is shown bywhite arrows and prometaphasenuclei are shown by yellowarrows. C, H929 multiple myelomacells treated with nocodazole(100 nmol/L) were subjected toelectron microscopy study.Prometaphase nuclei morphologyand apoptotic bodies are detectedafter 16 and 24 hours ofnocodazole treatment.

Feng et al.





consisting of clusters of condensed chromosomes, sug-gesting that the cells failed to progress into mitosis. Im-portantly, immunofluorescence staining for tubulinshowed that the disordered structure of microtubuleswas only observed in the elongated cells (Fig. 2B andSupplementary Fig. S2A). These data suggest that noco-dazole induces damage of the microtubular network anddisrupts the interaction of microtubules and kinetochore,resulting in the prevention of the formation of the mitoticspindle and the arrest inmitosisprophase/prometaphase.

JNK-mediated Bcl-2 phosphorylation contributes tonocodazole-induced cell deathTo study the molecular mechanism underlying noco-

dazole-induced myeloma cell death, we focused on theapoptosis-regulatory protein Bcl-2. Bcl-2 has beensuggested to regulate microtubule integrity (21) and israpidly phosphorylated and inactivated in response toexposure of cells to microtubular disrupting agents. Bcl-2phosphorylation was strongly induced in a dose- andtime-dependent manner at both Ser70 and Thr56 siteswhereas total Bcl-2 protein was unaffected by nocoda-zole treatment in myeloma cells (Fig. 3A–C). Important-ly, upregulation of Bcl-2 phosphorylation andproapoptotic BH3-only protein Bim isoforms (BimEL,BimL, and BimS) was also observed when primaryCD138þ myeloma cells were treated with nocodazole(Fig. 3B and C, left). On the contrary, nocodazole

exposure had no significant effect on the Bcl-2 phos-phorylation and Bim isoforms in nonmyeloma CD138�

cells (Fig. 3B and C, right). Nocodazole decreased theprotein levels of antiapoptotic XIAP and Mcl-1L but hadno effect on proapoptotic molecules such as Bid and Baxas well as death receptor 4 and the apoptosis inhibitor,survivin (Fig. 3A and D).

Several protein kinases have been identified to becritical for Bcl-2 phosphorylation (5). We found thatnocodazole phosphorylates JNK and p38-MAPK anddecreases the phosphorylation of the extracellularsignal–regulated kinases (ERK) in a dose- and time-de-pendent manner (Fig. 4A). JNKs, in contrast to p38-MAPK or ERKs, contribute primarily to the inductionof Bcl-2 phosphorylation. To confirm whether only JNKinduces phosphorylation of Bcl-2 in nocodazole-treatedmultiple myeloma cells, we treated RPMI-8226 multiplemyeloma cells with nocodazole in the presence of specificMAPK inhibitors. Inhibition of MEK-ERK (UO126 andPD98059) and p38-MAPK (SB202190) pathways hadno effect on the phosphorylation of Bcl-2 protein. Onlythe JNK inhibitor (SP600125) was able to abrogate Bcl-2phosphorylation at both Ser70 and Thr56 sites (Fig. 4B). Inaccordance with this is the finding that only JNK inhibitorSP600125 prevented nocodazole-induced cell nuclearfragmentation. Compared with vehicle control, nocoda-zole-induced cell death was significantly decreasedby 50% and 64% when JNK pathway was blocked by

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Figure 3. Nocodazole induces phosphorylation of Bcl accompanied by a decreased expression of antiapoptotic proteins such as Mcl-1L and XIAP.RPMI-8226 (A and D) and CD138þ primary multiple myeloma cells and CD138� cells (B and C) were treated with vehicle control or nocodazole atindicated concentrations for indicated times. Whole-cell lysates were subjected to Western blot assay and detected with indicated antibodies. b-Actin servedas loading control.






SP600125 at 10 mmol/L and 20 mmol/L, respectively.Other MAPK inhibitors failed to rescue multiple myelo-ma cells from nocodazole-induced cell death (Fig. 4C).

A high amount of phosphorylated Bcl-2 protein wasdetected specifically in G2–M phase within 16 hours ofnocodazole exposure (Figs. 2A and 3A). These results incombination with the known role of Bcl-2 phosphoryla-tion in all cell cycles indicate that nocodazole-inducedBcl-2 phosphorylation is associated with accumulationand arrest of cells in G2–M phase that can be abrogated byJNK inhibitor treatment.

Nocodazole alone and in combination withdexamethasone inhibits multiple myeloma tumorgrowth in vitro and in vivo

To further investigate the effects of nocodazole, wetested nocodazole in combination with other compounds.As shown in Fig. 5A, combination of nocodazole withlenalidomide, dexamethasone, or a novel histone deace-tylase inhibitor inhibitor KD5170 (17) resulted in asignificant (P < 0.05) inhibition of proliferation comparedwith either drug alone. Analysis of nuclear fragmentationas a marker for cell death indicated that dexamethasone(20 nmol/L) alone induced less than 20% cell death. Lowconcentrations (15 and 30 nmol/L) of nocodazole aloneinduced 10% and 16% nuclear fragmentation. However,

combination of 20 nmol/L dexamethasone with 15 or30 nmol/L nocodazole significantly increased the per-centage of apoptotic cells to 67% and 92%, respectively(P < 0.01; Fig. 5B).

To assess the antimyeloma effect of nocodazole in vivo,we used the human xenograft mouse model usinghuman multiple myeloma cells (H929). H929 multiplemyeloma cells grow vigorously and behave as an aggres-sive tumor in vivo. To titrate a treatment dosage, weused high (20 mg/kg) and low (5 mg/kg) concentrationsof nocodazole without dexamethasone. Nocodazole(20 mg/kg) injected intraperitoneally 3 times weeklyshowed significant inhibition of tumor growth as earlyas the tenth day of treatment (P < 0.02) comparedwith thatof the control group (Supplementary Fig. S3).At 20mg/kgnocodazole, no severe side effects, such as weight loss ordiarrhea, were observed but the mice showed mild dryskin with scales. At 5 mg/kg, mice showed no physicalchanges. For further in vivo testing, an intermediate doseof 12 mg/kg nocodazole was chosen. Because our in vitrodata showed that combination of nocodazole and dexa-methasone significantly enhances apoptosis of multiplemyeloma cells (Fig. 5B), combination of nocodazole anddexamethasone was tested in the abovementionedmousemodel. Mice were treated with an intermediate dose ofnocodazole (12 mg/kg intraperitoneally 3 times weekly),

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Figure 4. Nocodazole inducesmultiple myeloma cell apoptosisvia JNK-induced Bcl-2phosphorylation. A, RPMI-8226multiple myeloma cells weretreated with nocodazole atindicated concentrations forindicated times. Whole-celllysates were subjected to Westernblot assay and analyzed withindicated antibodies. B, RPMI-8226 multiple myeloma cells wereincubated with MAPK (U0126, 25mmol/L; PD98059, 25 mmol/L;SB203580, 10 mmol/L); and JNKinhibitor (SP600125, 20 mmol/L)for 30 minutes and withnocodazole for another 16 hours.Cell lysates were subjected toWestern blot assay with indicatedantibodies. C, RPMI-8226multiplemyeloma cells were treated withnocodazole 100 nmol/L incombination with differentphosphorylation inhibitors asindicated. Apoptotic cell deathcharacterized by typical apoptoticnuclear morphology wasdetermined by Hoechst 33258fluorochrome staining. *, P < .02;and **, P <. 01. NC, negativecontrol.

Feng et al.





2 mg/kg dexamethasone (subcutaneously twice weekly),their combination, or vehicle only. Either drug alone hadthe tendency to inhibit tumor growth. Only in the com-bination group, a significant reduction of tumor volumewas observed as early as day 8 that persisted for the entireexperiment (Fig. 6A). Kaplan–Meier survival analysisindicated that the combination treatment significantly(P < 0.01) prolonged survival time in comparison withvehicle treatment and either drug alone. In the vehiclecontrol group, 100% of the animals had to be sacrificed(endpoint) by day 15 whereas in the combination group,only 20% of mice had reached the endpoint (P < 0.01;Fig. 6B). No adverse toxicity was observed throughoutthe treatment period for all the treatment groups asmonitored by bodyweight, behavior, and activity ofmice.

Discussion

Despite the development of novel therapeuticapproaches and combination therapies, multiple myelo-

ma remains an incurable malignancy with a mediansurvival of 3 to 5 years (22). Multiple myeloma is char-acterized by aberrant proliferation of terminally differ-entiated plasma cells and the inability to undergoapoptosis. Some benzimidazole compounds were origi-nally approved by Food and Drug Administration asbroad-spectrum anthelmintic drugs with known toxicityprofiles. Recently Spagnuolo and colleagues describedthe antimyeloma effect of flubendazole, that also is abenzimidazole (13). Monaghan and colleagues reportedthat tubulin polymerization inhibitor CYT997 synergizeswith bortezomib to produce potent antimyeloma activity(23). Although nocodazole has been categorized as abenzimidazole with antineoplastic activity, its effectson multiple myeloma cells are unknown. In this study,we focused on the therapeutic potential of nocodazoleagainst multiple myeloma in vitro and in vivo. The con-centrations we used were in the range of therapeuticachievable levels (24, 25). A study by Woodtli andcolleagues revealed that plasma levels of 0.30 mmol/Lmebendazole are the optimal therapeutic levels for treat-ment of inoperable echinococcosis (26). In our studies,the similar concentration of 0.25 mmol/L mebendazoleinduced apoptosis shown by nuclear fragmentation in65% of the multiple myeloma cells (Fig. 1B). Recently, wehave shown that nocodazole inhibits the production ofcytokines essential for multiple myeloma growth andsurvival (14). Here, we show that nocodazole also inhibitsgrowth and survival of primary multiple myeloma cellsand of multiple myeloma cell lines alone and in coculturewith bone marrow stromal cells by inducing cell-cyclearrest and subsequent apoptosis. Inhibition of cell pro-liferation by nocodazole occurred independent fromMDR of the multiple myeloma cell lines, suggesting thatnocodazole overcomes cell adhesion–mediated resistanceand MDR to conventional therapies. In addition, itsrelative selective cytotoxicity to multiple myeloma celllines and primary myeloma cells, but not to nonmalig-nant bone marrow mononuclear cells, suggests a favor-able therapeutic index. Our in vitro data on combinationstudies show that nocodazole with compounds, such asdexamethasone, significantly increases the antimyelomaeffect (Fig. 5B), but further studies are needed to exploreadditive or even synergistic effect. In vivo nocodazolecombined with dexamethasone significantly inhibitedtumor growth and prolonged survival in a humanxenograft myeloma mouse model. Furthermore, our datashow that in cycling cells, nocodazole affects themicrotubule assembly and causes mitotic arrest, whichin turn leads to apoptosis. The higher selectivity ofnocodazole toward tumor cells might result from target-ing cycling cells compared with the non- or slow-cyclingcells. At prometaphase, the phase of mitosis followingprophase and precedingmetaphase, the nuclear envelopefragments and disappears. The role of prometaphase iscompleted when all of the microtubules have attachedto their kinetochores, at which point metaphase begins(27). In nocodazole-treated multiple myeloma cells, the

**B

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Figure 5. Nocodazole in combination with other agents exhibitssignificantly stronger antimyeloma effects. A, H929multiple myeloma cellswere treated for 48 hours with lenalidomide, dexamethasone, histonedeacetylase inhibitor (HDACi) KD5170 alone or in combination withnocodazole. DNA synthesis wasmeasured by 3H-thymidine incorporation.B, H929 multiple myeloma cells were treated with nocodazole alone or incombination with dexamethasone for 24 hours at the indicatedconcentrations. Cell death was determined using Hoechst 33258fluorochrome staining. Results are shown asmean� SD (n¼ 3). *, P < 0.05compared with the respective drug alone use.






microtubule fails to attach to the kinetochore and activatethe spindle assembly checkpoint. This causes a predom-inant arrest in prometaphase and cells die after prolongedarrest in mitosis, suggesting that mitotic cell death occursin nocodazole-treated myeloma cells (28–30). Based onthe fact that cells treated with nocodazole arrest with G2

or M phase DNA content, nocodazole is frequently usedto synchronize the cell division cycle (24, 31–33), and ourfindings of myeloma cell death after prolonged arrest ofcells in mitotic prometaphase is not unexpected (34).

Further analysis of prodeath BH3-only moleculesshowed that Bim isoform proteins were increased inboth myeloma cell lines and primary CD138þ cells. Bimis usually sequestered in the cytosol by binding with thedynein light chain-1 and is released from the micro-tubules in response to apoptotic stimuli via microtubu-lar damage (35). This is in accordance with our dataindicating that nocodazole-induced microtubular net-work damage leads to the release of Bim and apoptosisinduction. The functional significance of XIAP andMcl-1L downregulation by nocodazole indicates a linkbetween cell-cycle arrest and cell apoptosis (36, 37).Furthermore, downregulating XIAP has been shownto promote persistent JNK1 activation (38), whichmay induce Bcl-2 phosphorylation. Our data show thatnocodazole-induced death of myeloma cells is associ-ated with JNK activation and Bcl-2 phosphorylation.This is in accordance with data showing that activationof JNK by drug treatment induces Bcl-2 phosphoryla-tion on specific residues including Ser70, which leadsto Bcl-2 inactivation and apoptosis (6, 9, 21). Further-more, it was also suggested that Bcl-2 phosphorylationmight represent a preapoptotic phase after microtubuledamage and subsequently dephosphorylation initiatesapoptosis (39). We found that phosphorylation of Bcl-2

at Ser70, and to a less degree at Thr56, was markedlyinduced after treatment with nocodazole. Specific inhi-bition of JNK1/2 prevented nocodazole-induced Bcl-2phosphorylation (Fig. 4B). In accordance with this,nocodazole-induced myeloma cell apoptosis was alsoreduced by concomitant treatment with JNK1/2 inhib-itor (Fig. 4C). The connection of JNK activation andBcl-2 phosphorylation was further shown by comparingthe overlap between kinetics and dose dependence ofthese kinase-driven events (Figs. 3 and 4A). Further-more, it has been shown that JNK-induced Bcl-2 phos-phorylation diminished the binding activity of Bcl-2to both multidomain and BH3-only proapoptoticfamily members (40, 41), thereby facilitating the proa-poptotic activity of these members. Therefore, ourresults are consistent with findings from other investi-gators showing that the JNK pathway is responsible forphosphorylation of Bcl-2 in the same loop region inresponse to microtubule-damaging agents (42, 43).

In summary, our studies show that nocodazole tar-gets the multiple myeloma cell and its microenviron-ment (14). Nocodazole mediates its antimyelomaactivity through sequential microtubular network dam-age and cell-cycle arrest. JNK-mediated Bcl-2 phosphor-ylation results in multiple myeloma cell apoptosis.Nocodazole overcomes drug resistance, decreases tu-mor growth, and extends survival in vivo in humanxenograft mice model. The known toxicity profile andselective activity against myeloma cells provide therationale for considering nocodazole as future treat-ment for multiple myeloma.

Disclosure of Potential Conflicts of Interest

There are no conflicts of interest to disclose.

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Figure 6.Nocodazole significantly inhibits tumor growth and prolongs survival in the human xenograft mouse model. H929 multiple myeloma cells mixed withMatrigel were injected subcutaneously into the right flank of beige/nude/X-linked immunodeficient mice. A, once tumors became palpable, mice weretreated (intraperitoneally) with vehicle solution, dexamethasone alone (Dex, 2 mg/kg), nocodazole (Noco, 12 mg/kg) alone, or the combination ofdexamethasone and nocodazole (n¼ 5 each group). Results are shown as mean� SD of tumor volume. *, P < 0.02; **, P < 0.01 compared with vehicle-treatedmice. B, survival rate was evaluated using Kaplan–Meier method and log-rank analysis. Combination group (Noco þ Dex) showed significantlyincreased survival (P < 0.01) compared with control.

Feng et al.





Authors' Contributions

R. Feng designed and carried out experiments, analyzed data, and wrotethemanuscript; S. Li andC. Lu carried out experiments; C. Andreas conductedbone marrow collection; D.B. Soltz assisted in electron microscopy studies;M.Y. Mapara analyzed data and reviewed the manuscript; and S. Lentzschdesigned the experiments, analyzed data, and wrote the manuscript.

Acknowledgments

We would like to thank Dr. Jie Han and E. Michael Meyer for assistance withflow cytometric studies. The authors thank Dr. William Dalton (H. Lee MoffittCancer Center & Research Institute, University of South Florida) for providingthe humanmultiple myeloma cell line RPMI-8226/S and its sublines RPMI-8226/Dox40, RPMI-8226/MR20, and RPMI-8226/LR5. Dexamethasone-sensitive and

-resistant cell lines MM.1S and MM.1R were obtained from Dr. Klaus Podar(Dana Farber Cancer Institute, Harvard Medical School). We thank Ms. RitaBhutta for excellent preparation of the manuscript.

Grant Support

The current study was supported in part by research funding from MultipleMyeloma Research Foundation to S. Lentzsch.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received March 29, 2011; revised July 12, 2011; accepted August 1, 2011;published OnlineFirst August 8, 2011.

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2011;10:1886-1896. Published OnlineFirst August 8, 2011.Mol Cancer Ther Rentian Feng, Shirong Li, Caisheng Lu, et al. StrategyTargeting the Microtubular Network as a New Antimyeloma

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