Direct Inhibition of Retinoblastoma Phosphorylation by ...induce cell-cycle arrest by stabilizing p21 and p53 (5, 6), whereas curcumin and resveratrol (both in cancer clinical trials)

Cancer Therapy: Preclinical

Direct Inhibition of Retinoblastoma Phosphorylation byNimbolide Causes Cell-Cycle Arrest and SuppressesGlioblastoma Growth

Swagata Karkare1,3, Rishi Raj Chhipa1, Jane Anderson1, Xiaona Liu1, Heather Henry1, Anjelika Gasilina2,Nicholas Nassar2, Jayeeta Roychoudhury2, Jason P Clark2, Ashish Kumar2, Giovanni M. Pauletti3,Pradip K. Ghosh4, and Biplab Dasgupta1

AbstractPurpose:Classical pharmacology allows the use and development of conventional phytomedicine faster

and more economically than conventional drugs. This approach should be tested for their efficacy in terms

of complementarity and disease control. The purpose of this study was to determine the molecular

mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta

indica, controls glioblastoma growth.

Experimental Design: Using in vitro signaling, anchorage-independent growth, kinase assays, and

xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.

Results: We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains

nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastomamultiforme in vitro and in

vivo. Azt caused cell-cycle arrest, most prominently at the G1–S stage in glioblastoma multiforme cells

expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/

nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retino-

blastoma protein, cell-cycle arrest at G1—S, and cell death. Independent of retinoblastoma hypopho-

sphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastomamultiforme

cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced

phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific

because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation

and inhibited progression of glioblastoma multiforme growth.

Conclusions:Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks

cell cycle and inhibits glioma growth in vitro and in vivo. Clin Cancer Res; 20(1); 199–212. �2013 AACR.

IntroductionGlioblastoma multiforme is one of the most common

primary brain tumors in the adult population. Despitesubstantial progress in patient outcome in other humancancers and significant advancement in our understandingof the genetics and pathology of glioblastoma multiforme,

decades of therapy have failed to drastically change survival.The most frequent (>40%) gain-of-function event in pri-mary glioblastoma multiforme is amplification of the EGFreceptor (EGFR), often associated with a ligand-indepen-dent constitutively active mutant receptor called EGFRvIII(1). Two tumor suppressors frequently lost in glioblastomamultiforme are INK4a/ARF that regulate the retinoblastoma(RB) protein and PTEN that regulates phosphoinositide 3-kinase (PI3K; refs. 2, 3). Because multiple growth factorpathways are often upregulated in glioblastoma multi-formes, including the PI3K-Akt, MEK-Erk1/2, and theJAK-STAT3 pathways (4), it is being argued that for certaintypes of cancers (including glioblastoma multiforme),development of drugs that target multiple pathways couldbe more effective than pathway-specific drugs.

Phytochemicals derived from medicinal plants are timetested for their curative properties against a plethora ofchronic human diseases. Because of their safety, long-termuse, and their ability to target multiple pathways, there is arenewed interest to understand their molecular mechan-isms of action. Phenolic compounds and isothiocyanates

Authors' Affiliations: Departments of 1Oncology, 2Experimental Hema-tology and Cancer Biology, Cincinnati Children's Hospital Medical Center;3James L.Winkle College of Pharmacy, University of Cincinnati, Cincinnati,Ohio; and 4Maryville University, St. Louis, Missouri

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

S. Karkare and R.R. Chhipa contributed equally to this article.

CorrespondingAuthor:BiplabDasgupta, Division ofOncology,CincinnatiChildren's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH45229. Phone: 513-803-1370; Fax: 513-803-1083; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-0762

�2013 American Association for Cancer Research.

ClinicalCancer

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induce cell-cycle arrest by stabilizing p21 and p53 (5, 6),whereas curcumin and resveratrol (both in cancer clinicaltrials) induce apoptosis by downregulating Bcl2 and upre-gulating Bax (6, 7). Organosulphur derivatives from garlicalso exert anticancer effects by downregulating NF-kB (8).Recently, trabectedin, a natural product of marine origin(also in clinical trial), induced apoptosis specifically intumor macrophages (9). Azadirachta Indica, is one suchmedicinal plant with tremendous commercial interest andwith long history of use for over 2,000 years in primaryhealth care in India and neighboring countries (10, 11).Fractionated extracts of its leaves (known as IRAB) withregistered U.S. Pat. No. 5370873 is registered andmarketedin Africa as IRACARP for its broad-spectrum anti-cytoadhe-sion activity and its beneficial effects in HIV/AIDS (12, 13).By comparing the cytotoxic potency of the bioactive com-pounds isolated from the leaf extract, it was found thatnimbolide is the principal cytotoxic component of the leafextract (14). Nimbolide is a tetranortriterpenoid consistingof a classical limonoid skeleton with a a,b-unsaturatedketone system and a d-lactonic ring (13, 14). The etha-nol-soluble fraction ofAzadirachta Indica leaves (henceforthcalled Azt) as well as nimbolide has been shown to exertseveral biologic activities, including anti-satiety response(15), antimalarial (16), anti-HIV (13), and anticancerresponse (17).

Azt/nimbolide exhibits anticancer properties against avariety of tumor cells, including neuroblastoma, osteosar-

coma, leukemia, andmelanoma cells (18–21). These cancercells are variously affected, likely due to the interaction ofAzt with the unique pathways mutated in these cells. Someof the pathways involved in Azt action include cell-cyclearrest at G0–G1 (21), increased reactive oxygen speciesproduction (19), activation of caspases, modulation of thelevels of cell-cycle inhibitors (22), and suppression of NF-kB activity (20). In animal tumor models, nimbolide (10–100 mg/kg) has been shown to exhibit chemopreventiveactivity against 7,12-dimethylbenz(a)anthracene-inducedoral carcinogenesis (17, 23). The ab- unsaturated ketonestructure of nimbolide is linked to its anticancer property,whereas amide derivatives modified on the lactone ringenhanced its cytotoxicity (14). Because the cytotoxic prop-erties of Azt/nimbolide has not been thoroughly tested inglioblastoma multiforme, we examined its effectivenessagainst humanglioma cells, especially cellswith overexpres-sion of the oncogene EGFRvIII, found in up to 25% ofprimary glioblastoma multiforme patients (1).

In this study, we report that by inhibiting retinoblastomaphosphorylation and blockingmultiple growth factor path-ways relevant to glioblastomamultiforme, Azt/nimbolide isan extremely potent cytotoxic agent that kills glioblastomamultiforme cells in vitro and suppresses tumor initiationand progression in vivo.

Materials and MethodsCell culture

T98G, A172, andU87 cells were obtained fromAmericanType Culture Collection and G2 cells were obtained fromPeter Houghton, Nationwide Children’s Hospital (Colum-bus,OH).U87EGFR andU87EGFRvIII cells (obtained fromPaul Mischel, University of California, Los Angeles, LosAngeles, CA) were maintained in puromycin (1 mg/mL)and hygromycin (150 mg/mL), respectively. P9 is a primaryadult glioblastoma multiforme neurosphere line obtainedfrom John Ohlfest, University of Minnesota (Minneapolis,MN). All glioma cells except P9 were cultured in Dulbecco’sModified EagleMedium (DMEM)with 10% fetal calf serum(FCS). P9 cells were cultured in ultra-low attachment dishesin DMEM/F12 supplemented with B27, N2, EGF, andfibroblast growth factor. Cell viability was determined byTrypan blue exclusion method.

PhotomicrographyImages of soft agar colonies and whole brain coronal

sectionswere takenwith aNikonAZ-100multizoommicro-scope attached with DS-RI1 12 MP color camera, andhistology images were taken with a Leica DM2500 bright-field upright microscope attached with a DFC 500 12 MPcolor camera.

Preparation of AztDried powder of A. indica leaves was prepared (by P.K.

Ghosh) by drying fresh leaves at 37�C for 24 hours andgrinding them into a powder using a mortar and pestle.Azt extract was prepared as before (23) with minor mod-ifications. To prevent batch to batch variation, a single batch

Translational RelevanceDespite our understanding of the core genetic path-

ways of glioblastoma multiforme and despite consider-able advancement in therapy and success in other can-cers, the median survival in glioblastoma multiformeranges from9 to 12months. Decades of surgical therapy,chemotherapy, and radiotherapy have essentially failedto significantly change the outcome of the disease.Therefore, there is a desperate need to find newapproaches that might work together with standard-of-care therapy to improve survival of patients withglioblastoma multiforme. In our study, we demonstratethat Azt/nimbolide is a very potent agent in glioblasto-ma. In fact, we found that at least in vitro, this agent ismore cytotoxic than nearly all small molecules currentlybeing tested in glioblastoma multiforme preclinicalmodels. It also inhibited tumor progression in vivo.Glioblastoma multiforme is characterized by hyperacti-vation of multiple growth factor pathways and Azt/nimbolide inhibited three key growth factor pathwaysrelevant in glioblastomamultiforme. A vitally importantissue will be to conduct more rigorous and extensiveanalysis to determine the bioavailability, pharmacoki-netics, and pharmacodynamics of nimbolide andimprove blood-brain-barrier diffusion of this potentcytotoxic agent.

Karkare et al.

Clin Cancer Res; 20(1) January 1, 2014 Clinical Cancer Research200




of Azt extract was prepared by soaking 40 g of dry powder in200mL95%ethanol (200mg/mL), andAztwas extracted at4�C on a shaker for 5 days. The extract was centrifuged andclear supernatant was filtered through a 0.2 micron filterand stored in aliquots at�20�C. Appropriate volumeof thisstock (200 mg/mL) was added to the culture medium toachieve 1, 2, and 4 mg/mL final concentration (e.g., 5, 10,or20mL of stockwas added to 1mL culturemedium to achieve1, 2, or 4 mg/mL final concentration).

Flow cytometryCell-cycle distribution was performed by flow cytometry.

Cells were treated with EtOH (control) or Azt (2 mg/mL for12 hours and 1 mg/mL for 24 hour), harvested, fixed with70% ice cold EtOHat�20�C for 1 hour, and resuspended in0.5 mL of PI/RNAse staining buffer. Cell death analysis wasdone by Annexin V staining. Following labeling, cells werefiltered through a 70 mm Sefar Nylon Lab Pak Mesh. DNAcontent was analyzed on a Beckman Coulter Quanta SCMPL Flow Cytometer.

Anchorage-independent growthFor anchorage-independent growth, 2 � 104 glioblasto-

ma multiforme cells were mixed with 0.7% top agar andlayered on top of 1% bottom agar made in 2�DMEMwith20% FCS and antibiotics. Cells were fed with mediumcontaining EtOH, dimethyl sulfoxide (DMSO; control) orAzt, nimbolide (purchased fromBio Vision) every third dayandallowed to grow for 2weeks. Colonieswere stainedwithcrystal violet and imaged. Colony quantitation was doneusing ImageJ software.

Western blot analysisGlioma cells were lysed with radioimmunoprecipitation

assay (RIPA) lysis buffer (20 mmol/L Tris, 10 mmol/LEGTA, 40 mmol/L b-glycerophosphate, 1% NP40, 2.5mmol/L MgCl2, 2 mmol/L orthovanadate, 1 mmol/L phe-nylmethylsulfonylfluoride, 1 mmol/L DTT and proteaseinhibitor cocktail). The following antibodies (all from CellSignaling Technology) were used: PARP, Bcl2, pRB, pAkt,Akt, pGSK-3b, GSK3-ab, pCyclinD1, cyclin D1, pErk1/2,Erk1/2, pSTAT3, STAT3, pS6, S6, p4EBP1, 4EBP1, cyclin B1,P27, P21, and actin.

Nonradioactive in vitro kinase assayNonradioactive in vitro kinase assay was performed by

examining phosphorylation on a GST-RB fusion peptide(residues 736–840).GST-RB1 construct containing residues736–840 of RB (Addgene) was purified according to stan-dard protocol. GST-RB1 was expressed in Rosetta 2 (DE3)pLys cells. Cells were lysed by sonication in buffer A (20mmol/L Tris, pH8.0, 300mmol/L NaCl). Lysate was loadedon glutathione Superflow beads (Qiagen), equilibratedwith buffer A. Following extensive wash in buffer A, GST-RB1 was eluted with buffer A supplemented with 50mmol/L glutathione. Fractions containing GST-RB1, as judged bySDS-PAGE, were pooled, dialyzed against 20 mmol/L Tris,pH 8.0, 200 mmol/L NaCl, 0.1% b-mercaptoethanol, and

concentrated to 2 mg/mL (Amicon, 10 kDa cutoff). Kinaseassay was conducted as previously described (24). Briefly,U87EGFRvIII cells were lysed with RIPA buffer, and CDK4/6 was immunoprecipitated with cyclin D1 antibody,washed twice with RIPA buffer, once with 10 mmol/LTris-Hcl (pH 7.5) and 0.5 mol/L LiCl, once with kinaseassay buffer and suspended in kinase buffer (25 mmol/LTris-HCl, pH 7.5, 5mmol/L b-glycerophosphate, 2mmol/LDTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2). Reactionswere carried out at 30�C for 45 minutes by adding GST-RBfusion protein and 250 mmol/L ATP to the reaction mix.Reaction was terminated by adding Laemmli buffer andboiling for 5 minutes. Samples were resolved in 14% SDS-PAGE, andWestern blot analysis was performed with phos-pho-RBSer807/811 antibody (Cell Signaling Technology).

Plasmids. Retinoblastoma RBS811E mutant was madefrom wild-type mouse Rb cDNA purchased from OpenBiosystems and using Quickchange II-XL site-directedmutagenesis kit (Agilent Technologies).

In vivo experimentsAll animal procedures were carried out in accordance

with the Institutional Animal Care and Use Committee(IACUC)-approved protocol of Cincinnati Children’s Hos-pital Medical Center (CCHMC; Cincinnati, OH). Animalsweremonitored daily by animal care personnel. For flank invivo xenografts experiments, 1.5 � 106 U87EGFRvIII cellswere injected into the flanks of nu/nu mice. For gliomainitiation experiment, cell were pretreated with EtOH (con-trol) or Azt for 1 hour and injected into the flanks. Mice didnot receive any further injections of EtOHorAzt. For gliomaprogression experiment, similar numbers of untreated cellswere injected and tumors were allowed to grow for 7 days.Following this, 62.5 mL of 95% ethanol or Azt (0.34 mg/gbody weight) was injected directly into the tumor bed ofthe left and right flank, respectively, every other day for8 days. To examine the systemic effect of nimbolide,we injected 1.5 � 106 U87EGFRvIII cells into the flanks ofnu/nu mice, allowed tumor seeding for 3 days, and treatedanimals with nimbolide (@10 mg/kg body wt, intravenous-ly) or vehicle (DMSO) every day for 7 days. Tumor growthwas monitored by measuring tumor volume every otherday by using the formula p/6� A� B2 (A¼ larger diameter;B ¼ smaller diameter). Tumors were dissected out andimaged using an iPAD 3. For intracranial xenograft experi-ments, U87EGFRvIII cells were infected with LeGo-Cer2lentivirus expressing firefly luciferase or lentivirus expressingDsRed fluorescent reporter. A total of 1 � 105 cells, unla-beled or labeled cells (depending on experimental group–see Results) were injected into the cerebral cortex of malenu/numice (2mmcaudal to bregma; 2mmright ofmidline;3 mm deep) using a stereotactic device. Tumor seeding wasallowed for 3 days following which mice received nimbo-lide (@ 200 mg/kg body wt, intraperitoneally) or vehicle(DMSO). All animals were imaged twice—once, three daysafter tumor seeding before start of treatment to ensure theyhad similar starting signals and next, on day 14. Fiveminutes before imaging, mice were anesthetized and

Glioblastoma Growth Suppression by Nimbolide

www.aacrjournals.org Clin Cancer Res; 20(1) January 1, 2014 201




injected (intraperitoneally) with luciferin (150 mg/kg) andimaged using IVIS (Xenogen). All mice were euthanizedfollowing observation of lethargy and/or neurologic symp-toms. For DsRed-labeled tumors, 2 mm thick coronal brainsections were dissected and tumor area was measured usingthe ImageJ software.

ImmunohistochemistryImmunohistochemistry was done as previously

described (24). Mice were anesthetized, perfused intracar-dially with 4% PBS, tumors were dissected out and pro-cessed for paraffin embedding and sectioning. Tissue sec-tions were stained with hematoxylin and eosin (H&E).Immunohistochemistry was performed on adjacent depar-affinized 6 micron sections by using standard antigenretrieval methods. The following primary antibodies wereused: Ki-67 (Vector Laboratories), cleaved caspase-3, pAkt,pErk1/2, and pSTAT3 (Cell Signaling Technology). Vectas-tain ABC kit (Vector Laboratories) was used formicroscopicvisualization.

Statistical analysisStudent’s t test was used to calculate statistical significance

with P < 0.05 representing a statistically significant differ-ence. Kaplan–Meier analysis with log-rank posthoc test wasused for survival studies.

ResultsAzt/nimbolide kills glioblastoma multiforme cells in adose-dependent manner

We first compared the cytotoxic efficacy of an aqueousand an organic solvent extract of A. indica leaves. Althoughthe aqueous extract had no effect, a 95%EtOH extract killedT98G glioblastoma multiforme cells in a dose-dependentmanner (Fig. 1A). At 4 mg/mL, Azt killed 100% of the cells in20 hours under normal culture conditions. As a compari-son, treatment of glioblastoma multiforme cells for 20hours with temozolomide, the adjuvant used together withradiation as part of standard of care in glioblastoma multi-forme therapy had no effect at 50 and 100 mmol/L (Fig. 1B).Temozolomide had a slight effect at 72 hour (Supplemen-tary Fig. S1A). This is not surprising because as shown byothers (25), T98G and U87 cells are not significantly sus-ceptible to even long exposure of 100 mmol/L temozolo-mide alone, but are susceptible only when used togetherwith radiation. We also compared the inhibitory efficiencyof Azt/nimbolide with other standard chemotherapeuticagents, including DNA topoisomerase inhibitors likeetoposide, camptothecin, another alkylating agentmitomy-cin C, mTOR complex (mTORC)-1 inhibitor rapamycin,mTORC1/C2 inhibitor PP242, and the PI3K/mTOR dualkinase inhibitor BEZ235. We used these compounds atdoses higher than the reported range of IC50 values forvarious cancer cell lines at 72 hours of growth (e.g., etopo-side 5–10 mmol/L, camptothecin 0.5–1 mmol/L, mitomycinC 3–30 nmol/L, rapamycin 2.5–10 nmol/L, PP242 0.5–2mmol/L, BEZ235 4–75 nmol/L). Because nimbolide killednearly 100% of cells at 72 hours, we compared viability at

20 hours. Nimbolide was much more effective in reducingviability of U87EGFRvIII cells than any of these agents at 20hours of growth (Supplementary Fig. S1B). This cytotoxiceffect of Azt was universal because it killed six other celllines, including two adult glioblastoma multiforme cellsthat overexpresses EGFR or EGFRvIII, one adult primaryglioblastoma multiforme neurosphere line (P9), and onepediatric glioblastoma multiforme line (G2; Fig. 1C).Photomicrographs of A172 and U87 EGFRvIII cells treatedwith Azt for 20 hours are shown in Fig. 1D and E. TheU87EGFRvIII cells rounded up (as happens to dying cells)quicker than other cells even at the lowest dose of Azt (1 mg/mL). Accordingly, although the parental U87 cells did notlose viability at lower doses, 50% to 70%U87EGFRvIII cellswere killed at 0.75 and 0.875 mg/mL Azt, respectively (Fig.1F). This suggests that constitutively active signalingthrough the EGFRvIII oncogene likely induces oncogenicstress and enhances vulnerability of glioblastoma multi-forme cells toward Azt. Next, we conducted similar experi-ments with nimbolide (Fig. 1G), the principal cytotoxiccomponent of Azt. Similar to Azt, nimbolide considerablyreduced the viability of glioblastoma multiforme cells in adose-dependent manner, and again glioblastoma multi-forme cells expressing the EGFRvIII oncogene were moresensitive than other cells at 10 mmol/L concentration (Fig.1H). Taken together, these results show that nimbolideeither in its purified form or as an EtOH-soluble fractionin Azt is a potent cytotoxic agent that abolishes growth ofglioblastoma multiforme cells in vitro.

Glioblastomamultiforme cells expressing the EGFRvIIIoncogene are highly susceptible to G1–S arrest byAzt/nimbolide

To examine how Azt reduces viability of glioblastomamultiforme cells, we first conducted cell-cycle analysis byflow cytometry. The most dramatic effect of Azt was on theU87EGFRvIII cells. At 12 hours, it caused a massive accu-mulation of cells at S and depletion at G1 and G2M(compare Fig. 2A with B). At 24 hours, compared withEtOH-treated cells, more than 90% of Azt-treated cellsaccumulated at G1, concomitant with a depletion of cellsfrom S and G2M (compare Fig. 2C with D). Importantly, asshown in (Fig. 2E–H), Azt did not affect the cell cycle ofnormal astrocytes at 12 or 24 hours (compare cells in S–G2M in B versus F andD versus H). Cell death in U87EGFR-vIII cells was verified by flow cytometry analysis inwhichweobserved that about 70% of the Azt-treated U87EGFRvIIIcells stained positive for Annexin V (Fig. 2I). Comparedwith EtOH, Azt also caused substantial increase in the Sub-G0 population of T98G cells at both 12 and 24 hours(compare cells in Sub-G0 in J vs. K and L vs. M in Fig. 2)and to some extent in the U87EGFRvIII cells (compare cellsin Sub-G0 in A vs. B with C vs. D in Fig. 2), but not inastrocytes (Fig. 2F and H). We could not detect Sub-G0

population in the Azt-treated parental U87 cells (Fig. 2N–Q). Compared with EtOH-treated cells, twice as many Azt-treated T98G cells showed accumulation in the S phase(compare S phase cells in J vs. K with L vs. M in Fig. 2).

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Figure 1. Azt/nimbolide is a potent cytotoxic agent in glioblastoma. A, cytotoxic effect of aqueous or EtOH extracts (Azt) of A. indica on T98G humanglioblastoma multiforme (GBM) cells (A) and that of temozolomide (TMZ) on T98G, U87, and U87EGFRvIII cells (B). C, dose-dependent cytotoxicity ofAzt in apanel of humanglioblastomamultiforme (GBM)cells treatedwithAzt or ethanol (control) for 20hours. DandE, photomicrographs showingmorphologyof glioblastoma multiforme cells (control- EtOH treated, attached) but lifting from tissue culture plates following Azt treatment for 20 hours. F, glioblastomamultiforme cells expressing the EGFRvIII oncogene are sensitive to lower doses of Azt than parental cells. Cytotoxicity of nimbolide (G), the principalcytotoxic component of Azt on a panel of glioblastoma multiforme cells (H). Note: U87 EGFRvIII cells are more sensitive to 10 mmol/L nimbolide(circled asterisk). Data are representative of two to four experiments. �, P � 0.001.






Azt-treated parental U87 cells showed a slight increase inG1

and a reduction in the S phase (Fig. 2N–Q). Together, theseresults suggest that perhaps genetic variations (mutations)in glioblastoma multiforme cells influence the nature ofcell-cycle arrest induced by Azt, and cells with the EGFRvIIImutation are considerably more vulnerable to Azt-mediat-ed G1 arrest and the effect of Azt on cell cycle is specific toglioblastoma multiforme cells.

Azt/nimbolide inhibits anchorage-independentgrowth of glioblastoma multiforme cells

We showed that Azt reduces viability of glioblastomamultiforme cells grown inmonolayers. To examinewhetherAzt is capable of inhibiting anchorage-independent growth(a key test for tumor aggressiveness andmetastatic potentialin vivo), we conducted soft agar colony formation assay.Following 2 weeks of growth in the presence of EtOH

(control) or Azt, control cells rapidly formed large colonies,whereas Azt treatment significantly (P < 0.001) preventedcolony formation in all glioblastoma multiforme cells in adose-dependent manner (Fig. 3A–D). When added after 7days of growth, Azt still inhibited colony formation (notshown) suggesting its inhibitory effect on the progression ofcolony growth. Similar toAzt, nimbolide potently inhibitedcolony formation in a dose-dependentmanner (Fig. 3E–H).Thus, Azt and its principal cytotoxic component nimbolideare highly potent agents that inhibit anchorage-indepen-dent growth of glioma colonies in vitro.

Azt/nimbolide downregulates Bcl2, induces apoptosis,and blocks serum-stimulated Akt and retinoblastomaphosphorylation

Because Azt shifted cells to the sub-G0 stage, a featurecharacteristic of cell death, we examined whether Azt

Figure 2. Azt/nimbolide arrests glioblastoma multiforme (GBM) cells at G1–S. Flow cytometry using propidium iodide showing the effect of Azt/nimbolideon cell cycle of U87EGFRvIII cells (A–D), astrocytes (E–H), and Annexin V staining showing apoptosis of Azt-treated U87EGFRvIII cells (I) is shown. Effectsof Azt on cell cycle of T98G cells (J–M) and U87 cells (N–Q) are also shown. Note that Azt treatment caused complete depletion of U87EGFRvIII cells from G2

M at 12 hours and nearly 100% G1 arrest at 24 hours. Data are representative of three independent experiments.

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caused apoptosis. Indeed, Western blot analysis usingPARP antibody showed active caspase-dependent cleav-age of PARP in Azt-treated glioblastoma multiforme cells(Fig. 4A). Because Azt reduced cell viability, we examinedwhether it affected expression of antiapoptotic cell sur-

vival proteins. Indeed, Azt robustly downregulated theantiapoptotic protein Bcl2 in a time-dependent manner(Fig. 4A). Together, these results indicate that Azt/nim-bolide reduces glioma viability both by inducing cell-cycle arrest and cell death.

Figure 3. Azt/nimbolide inhibitsanchorage-independent growth ofglioblastoma multiforme cells.Photomicrographs showingcolony formation of T98G cells (A),U87 cells (B) andU87EGFRvIII cells(C) grown in the presence of EtOH(control) or Azt for 2 weeks. Theinset shows magnified view of thecolonies. D, quantitation of colonycounts in A–C. Similar experimentswere done using various doses ofnimbolide or DMSO (control).Colony formation of the same cellsare shown in E–G. Quantitation ofcolony counts in E–G is shown inH.The inset shows magnified view ofthe colonies. �, P � 0.001.






To investigate the mechanism of G1—S arrest by Azt, weexamined the key regulators of the G1–S phase. Growthfactor withdrawal inactivates cyclin dependent kinases(CDK) that upon growth factor stimulation phosphorylatesand inhibits the retinoblastoma tumor suppressor proteinto allow G1–S transition. As expected, serum-stimulationcaused robust retinoblastoma phosphorylation, which wasconsiderably inhibited in the presence of Azt, and this effectwas maximal in the U87EGFRvIII cells (Fig. 4B). Azt alsosuppressed serum-stimulated Akt phosphorylation (Fig.4B). We initially hypothesized that probably upstreamevents like Akt inhibition leads to hypophosphorylationand activation of the Akt substrate GSK3-b leading tophosphorylation of the GSK3-b site on cyclin D1. Phos-phorylation of cyclin D1 by GSK3-b at threonine 286enhances its ubiquitination and proteasomal degradationpreventing its interactionwith CDK4 andCDK4-dependentretinoblastoma phosphorylation. However, despite inhibi-

tion of Akt phosphorylation by Azt, we did not observe anychange inGSK3-b and cyclinD1 phosphorylation (Fig. 4B).No change in protein levels of cyclin D1, (a crucial G1

cyclin), cyclin B1 (a crucial G2 cyclin), and P27 and P21(two key negative regulators of the G1–S phase) wereobserved.

We therefore questioned whether Azt/nimbolide directlyinhibits the kinase activity of CDK4/6. To test this, CDK4/6was immunoprecipitated from U87EGFRvIII cells withcyclin D1 antibody and was used in a nonradioactivein vitro kinase reaction with a GST-RB (736–840) peptidein the presence of EtOH or DMSO (control) and Azt orpurified nimbolide. Indeed, Azt/nimbolide robustly inhib-itedCDK4/6 activity in a dose-dependentmanner (Fig. 4C).The highly orchestrated, cyclical phosphorylation ofretinoblastoma throughout the cell-cycle makes retinoblas-toma overexpression studies difficult. Nevertheless, wegenerated lentiviruses expressing retinoblastoma (S811E),

Figure 4. Azt/nimbolide inducesapoptosis, downregulates Bcl2,inhibits Akt and CDK4/6 kinaseactivity. A, immunoblot analysesshowing the dose-dependenteffects of Azt in induction ofapoptosis (note: smaller bands ofPARP cleaved by active caspasesat 6 and 12 hours of Azt treatment),and downregulation of Bcl2. Actinwas used as a loading control. B,immunoblot analyses showingretinoblastoma and Aktphosphorylation by serum inserum-starved glioblastomamultiforme cells and inhibition ofphosphorylation by Azt but not byEtOH (control). Phosphorylationstate of Akt substrate GSK3-b,GSK3-b substrate cyclin D1, andtotal levels of GSK3-ab, Akt, cyclinD1, cyclin B1, p21 and p27 are alsoshown (note; Azt did not changetotal levels of proteins or that ofG1–S cell-cycle inhibitors p21 andp27). C, in vitro kinase assayshowing dose-dependentinhibition of CDK4/6 activity by Azt(1, 2, and 4mg/mL) and nimbolide (5,10, and 20 mmol/L) but not by EtOHor DMSO (vehicle controls). D,rescue experiment showing theeffect of Azt and nimbolide on theproliferation of U87EGFRvIII cellstransduced with control lentivirusor lentivirus expressingphosphomimetic retinoblastoma.�, P < 0.005. Data arerepresentative of two to fourindependent experiments.

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potentially creating a phosphomimetic. We used controlGFP virus or RBS811E to examinewhether a retinoblastomaphosphomimeticmutantwould rescueAzt andnimbolide’sgrowth inhibitory effects. DMSO-treated RBS811E-expres-sing U87EGFRvIII cells grew similar to control virus-infected cells and both Azt and nimbolide reduced prolif-eration of control virus-infected cells. However, RBS811E-expressing cells showed nearly 70% rescue when treatedwith Azt/nimbolide (Fig. 4D) suggesting that CDK4/6 inhi-bition is likely an importantmechanismof Azt/nimbolide’sgrowth inhibitory actions. Our results demonstrate thatby directly inhibiting CDK4/6 activity, Azt/nimbolidecauses a sustained (chronic) inhibition of retinoblastomaphosphorylation and cell-cycle arrest at G1–S and gliomacells carrying the EGFRvIII oncogene are particularly sus-ceptible to this arrest by Azt/nimbolide.

Azt/nimbolide-induced cytotoxicity is irreversibleWe have shown that chronic exposure of glioblastoma

multiforme cells to Azt/nimbolide suppresses growth. Tointerrogate whether Azt-induced growth suppression isreversible, we exposed glioblastomamultiforme cells to Azt(2 mg/mL) or 95% EtOH (1% final) or PBS (control) for 1 or2 hours. Cells were washed in drug-free medium, and equalnumbers of live cells were reseeded in drug-free growthmedium for 72 hours. Although EtOH- treated cells grewsimilar to PBS-treated cells (although there was some effectof EtOH itself), nearly 100%of the Azt-treated cells failed tosurvive and proliferate (Fig. 5A), suggesting that acuteexposure to Azt/nimbolide causes irreversible changes thatare detrimental to the growth and survival of glioblastomamultiforme cells.To investigate the signaling pathways that might be

acutely affected by Azt, we examined several growth factorpathways. Glioblastoma multiforme cells were serumstarved and stimulated with serum for 1 or 2 hours in thepresence of EtOH (control) or Azt (2 mg/mL). Azt stronglyinhibited phosphorylation of Akt, extracellular signal—regulated kinase (Erk)-1/2, STAT3, and retinoblastoma(Fig. 5B)-signaling pathways vital for proliferation andsurvival of glioblastoma multiforme cells. This inhibitionshowed some specificity because the mTOR pathwayremained uninhibited as revealed by unchanged phosphor-ylation of S6 and 4EBP1, two bonafide downstream effectorsof mTOR (Fig. 5B).We next tested whether the acute in vitro irreversible

growth suppressive effect of Azt is also true in vivo. Wepredicted that probably tissue microenvironment wouldallow Azt-treated cells to survive and form tumor masses.A total of 1.5 � 106 live U87EGFRvIII cells (treated asabove) were injected into the flank of nude mice.Although EtOH-treated cells formed large tumor mass,contrary to our prediction, glioblastoma multiforme cellsacutely treated with Azt completely failed to survive andgrow as xenografts (Fig. 5C and D). Together, our resultsdemonstrate that Azt exerts an irreversible cytotoxic effecton glioblastoma multiforme cells in vitro that cannot berescued in vivo.

Azt/nimbolide inhibits glioblastoma multiformetumor growth in vivo

So far we have shown that Azt/nimbolide markedlyreduces glioblastoma multiforme cell viability in vitro. Toverify, whether Azt/nimbolide can repress tumor growth invivo, we took three different approaches. All in vivoapproaches were in compliance with institutional andIACUC guidelines. In the first approach, we created flankxenografts usingU87EGFRvIII cells and injectedAzt or EtoH(control) directly into the tumor bed. In the secondapproach, we injected nimbolide or DMSO (control)through the tail vein (intravenously) after creating flankxenografts of U87EGFRvIII cells. In the third approach,U87EGFRvIII were seeded orthotopically in the cortex ofNu/Nu mice and nimbolide or DMSO (control) wasinjected (intravenously). In the first model, tumors wereallowed to grow for 7 days following which 62.5 mL of 95%EtOH or Azt (0.34 mg/g body weight) was injected into thetumor bed of the left and right flank, respectively, everyother day for 8 days. Other than the tumor burden, miceinjected with EtOH or Azt were healthy, and did not showlethargy, locomotion, or behavioral abnormalities. EtOHexposure caused skin reddening (the reddish area next to thetumor in Fig. 6A). Azt, which is extracted with ethanol fromleaves is dark green in color and when injected into tumors,gave a dark green hue to tumors (Fig. 6A), but did not causetumor necrosis as judged by histologic analysis of tumorsections (Fig. 6E and G). However, Azt significantly (P ¼0.00013) reduced growth of glioblastoma multiformetumors (Fig. 6A–C). Following three injections, the volumeof Azt-injected tumors were 50% less than control tumors,whereas at the end of the study, Azt-treated tumorswere lessthan 10% of the volume of control tumors (Fig. 6C).

Histologic examination using H&E staining revealed thatAzt-treated tumors had markedly reduced cellularity (com-pare density of nuclei in Fig. 6D and E). Furthermore, Aztmarkedly reduced the number of tumor cells in differentstages of cell division (Fig. 6D and E; arrow heads), whereasit increased tumor cells with condensed/fragmented chro-matin and pycnotic nuclei (indicative of dead cells; Fig. 6Fand G and inset; arrow heads and circle). Immunohis-tochemistry using Ki-67 antibody showed significant (P¼ 0.00002) reduction of proliferative index of Azt-treatedtumors (Fig. 6H–J). Azt also caused apoptosis as shown byincreased immunoreactivity with cleaved caspase-3 anti-body (Fig. 6K–M). Control tumors overexpressed activatedAkt, Erk1/2, and STAT3 as indicated by their phosphoryla-tion status (Fig. 6N, P, and R). Importantly, similar to our invitro findings, Azt markedly reduced activation of all threepathways in vivo (Fig. 6O, Q, and S).

We next examined whether systemic administration ofnimbolide will be sufficiently bioavailable to have an effecton the growth of flank glioblastomamultiforme xenografts.Indeed, compared with DMSO treatment (control), nim-bolide (10 mg/kg body wt) had a significant (P < 0.0001)inhibitory effect on growth of glioblastoma multiformetumors (Fig. 7A and B). When the study ended (day 11),we observed a 3- to 4-fold reduction of tumor volume in






nimbolide-treated animals. Many preclinical drugs in trialfail because of their inability to cross the blood–brainbarrier at quantities sufficient to have an effect on intracra-nial tumors. Therefore, we examined whether nimbolidehad any effect on orthotopic glioblastoma multiformexenografts. We used three groups of animals—in the firstgroup, we injectedU87EGFRvIII cells labeledwith lentiviralluciferase, treated mice with DMSO or nimbolide (intrave-nously) every other day (n ¼ 4/subgroup), and measuredtumor growth on day 14 (Fig. 7C). In the second group, weinjected U87EGFRvIII cells labeled DsRed, treated micewith DMSO or nimbolide (intravenously) every other day

(n ¼ 3/subgroup), euthanized mice on day 14, and mon-itored tumor growth by measuring tumor area in 2 mmthick coronal brain sections (Fig. 7D and E). In the thirdgroup, we injected unlabeled U87EGFRvIII cells, treatedmice with DMSO or nimbolide (intravenously) every otherday (n ¼ 5/subgroup), and followed survival (Fig. 7F). Allanimals ultimately died; however, as revealed by biolumi-nescence assay (Fig. 7C) and fluorescence microscopy ofbrain sections (Fig. 7D), nimbolide reduced tumor growthand prolonged survival of mice (Fig. 7E; log-rank P ¼0.017). By measuring tumor area (total area within yellowcontour line inD),we found that nimbolide-treated tumors

Figure 5. Cytotoxicity of Azt/nimbolide is irreversible. A, growthassay showing viability ofU87EGFRvIII cells followingpretreatment with medium(control), EtOH (control), or Azt(2 mg/mL). B, immunoblot analysisshowing activation of growth factorpathways by serum and acuteinhibition of the pathways byAzt/nimbolide. Note:phosphorylation of Akt, Erk1/2,and STAT3 and retinoblastoma byserum in the presence of EtOH(control) and robust inhibition ofphosphorylation in the presence ofAzt. Also note that mTORC1activation, indicated byphosphorylation of S6 and 4EBP1is unaffected by Azt. Actin wasused as a loading control. Data arerepresentative of at least threeindependent experiments. C,representative digital photographshowing tumor growth in miceinjected with EtOH-pretreated (redarrowhead) or Azt-pretreated (bluearrowhead) U87EGFRvIII cells. D,growth of tumor xenografts inNu/Nu mice (n ¼ 8) initiated byU87EGFRvIII cells pretreated withEtOH or Azt. Tumor volume wasmeasured at indicated time pointsand the mean tumor volume wascalculated. �, P � 0.0002.

Karkare et al.





were about 40% smaller relative to DMSO-treated tumors(P ¼ 0.018). Extensive proliferation in glioblastoma multi-formes causes metabolic stress and necrosis. Nimbolide-injected tumors were not only smaller, but also did notshow necrosis at the same time point when control tumorsshowed widespread necrosis (asterisks in Fig. 7D). Futurestudies will be required to determine whether nimbolidecould be used at higher doses to increase brain bioavail-ability and whether structural modifications of nimbolidecould make it more stable, bioavailable, and permissivethrough the brain tissue. The collective results from ourthree different in vivo approaches raise the possibility thateither application of nimbolide to the tumor bed post-surgery through wafers and/or systemic administration of

a safe, optimized dose of nimbolide could be therapeuti-cally effective in glioblastoma multiforme.

DiscussionGlioblastomamultiforme remains one of the most lethal

formsofhuman cancers. Less than5%ofpatients diagnosedwith glioblastomamultiforme survive formore than 5 years(26). Despite comprehensive sequencing analysis and ourunderstanding of the core genetic pathways altered inglioblastomamultiforme (27), we do not have a clear graspon the biology of the disease. Targeted therapy has failed sofar and conventional standard-of-care therapy only pro-longs progression-free survival by about 20% (28). There-fore, there is anurgent need todevelopnovel therapeutics to

Figure 6. Administration ofAzt/nimbolide into tumor bedsuppresses tumor growth. A,representative digital photographshowing U87EGFRvIII tumorgrowth in mice injected with EtOH(red arrow) or Azt (blue arrow). B,digital photographs of tumorsinjectedwith EtOHor Azt dissectedout at the end of the experiment.C, growth of tumor xenografts inNu/Nu mice (n ¼ 8) initiated byU87EGFRvIII cells and treated withEtOH or Azt. Treatment startedfollowing tumor growth for 7 days.Injection schedule and tumorvolume measurement at indicateddays are shown. D–G, H&E oftumors injected with EtOH or Azt.Note: cells in different stages of celldivision (yellow arrowheads) in D,which is significantly reduced in E,and marked increase in cells withcondensed/fragmented chromatinand pycnotic nuclei (indicative ofdead cells) in G compared with F.Immunohistochemistry usingKi-67antibody showing proliferatingcells (H and I), quantitation ofKi-67þ cells (J), using cleavedcaspase-3 (CC3) antibody showingapoptotic cells (K and L) andquantitation of CC3þ cells (M) inEtOH and Azt-treated tumors.Immunohistochemistry showingphosphorylated levels of Akt(N and O), Erk1/2 (P and Q), andSTAT3 (R and S) in EtOH and Azttreated tumors. �, P < 0.0001.






treat patients with glioblastoma multiforme. In this study,our primary objective was to examine the cytotoxic efficacyof Azt and its principal cytotoxic component nimbolide onglioblastoma multiforme survival and growth. We demon-strate that Azt/nimbolide, at least in vitro, is an extremelypotent cytotoxic agent. In fact, after comparing the cytotoxiceffects of many other anticancer agents including temozo-lomide, PI3K, and mTOR inhibitors, we found that Azt/nimbolide is themost potent of all the agentswe have testedso far.

Although all glioblastoma multiforme cells were suscep-tible to the cytotoxicity of Azt/nimbolide, those expressingthe EGFRvIII oncogene were particularly vulnerable to thisagent. The IC50 of nimbolide on this cell was found to be3.0 mmol/L. Azt/nimbolide halted glioblastoma multi-forme cells mostly in G1–S. The magnitude of arrest indifferent cell lines was variable perhaps due to the effectof mutations present in individual glioma cell lines thatimpact on cell-cycle length and progression. The cell-cycleeffect was robust in the U87EGFRvIII cells as was the extentof retinoblastoma hypophosphorylation (Figs. 4B and 5B),

which might partly explain the dramatic G1–S arrest ofthese cells. Compared with other cells, the U87EGFRvIIIcells were also susceptible to lower doses of Azt and nim-bolide. Hyperactivation of signaling pathways by onco-genes causes oncogenic stress (29) and cancer cells haveevolvedmechanisms to counter such stress to remain viable.It is possible that Azt/nimbolide perturbs one or more ofthese antistress pathways in the U87EGFRvIII cells. Giventhe fact that Azt/nimbolide had similar cytotoxicity onglioblastomamultiforme cellswe tested, especially at higherdoses, but differential effects on cell cycle and to someextent on retinoblastoma phosphorylation, it seems thatothermechanisms, including a direct impact on survival (asshown by Bcl2 downregulation and PARP cleavage), couldalso be involved.

Anchorage-independent growth is a classical assay tointerrogate invasiveness and metastatic potential of cancercells (30). U87 andU87EGFRvIII cells are highly invasive invivo (data from many other labs and our own data notshown) and forms colonies in vitro. The ability of Azt/nimbolide to prevent colony formation was remarkable

Figure 7. Azt/nimbolide suppressestumor growth in vivo. A, digitalphotographs of tumors from micetreatedwithDMSOor nimbolide. B,growth analysis of tumorxenografts in Nu/Nu mice initiatedby U87EGFRvIII cells and treatedwith EtOH or Azt (�, P < 0.0001). C,bioluminescence signal ofintracranial tumors of threerepresentative mice treated withDMSO or nimbolide and measuredon day 14. D, DsRed fluorescencesignal from coronal sections ofintracranial gliomas prepared frommice treated with DMSO ornimbolide. Tumors are circled inyellow; asterisks, necrosis. E,quantification of tumor area fromD.F, Kaplan–Meier survival analysisof intracranial tumor-bearing micetreated with DMSO or nimbolide(log-rank P ¼ 0.017).

Karkare et al.





and dose dependent. At the highest dose (20 mmol/L nim-bolide or 4 mg/mL Azt), large colonies were virtually unde-tectable. This suggests that this agent if retains sufficientsystemic activity, might inhibit tissue invasion andmetastasis.The mechanisms of cell death induced by Azt/nimbolide

remain unclear. Although the A. indica ethanolic extractcontains other bioactive components, it is well establishedthat nimbolide is by far the most cytotoxic component(14, 31). Therefore, although it is possible that some ofthe effects of the extract (Azt) could be potentiated bycomponents other than nimbolide, high-performance liq-uid chromatography analysis and fractionation showedthat the most cytotoxic fraction of the extract containedabout 2.3% nimbolide (31). Furthermore, we did notobserve any additive antiproliferative effect of the extractcompared with pure nimbolide. Importantly, the extent ofretinoblastoma hypophosphorylation and rescue of gliomacell proliferation by expression of phosphomimetic retino-blastoma was very similar in Azt- and nimbolide-treatedcells. Azt/nimbolide caused high to modest apoptosis ofcancer cells, including HeLa (cervical carcinoma), THP1,U937,HL60, B16 (leukemia; refs. 32, 21), andHT29 (coloncancer) cells (22). In one study, Azt/nimbolide causedapoptosis in leukemia, lymphoma, human embryonic kid-ney, multiple myeloma, breast cancer, and human squa-mous cell carcinoma by suppressing cytokine-induced NF-kB activation (20). In our study, Azt/nimbolide causedextensive apoptosis in vitro as well as in vivo. Downstreamof PI3K, Akt plays amajor role in promoting cell survival bystabilizing the Bcl2 protein (33) and increasing its tran-scription (34). It is possible that akin to other studies (17),downregulation of Bcl2 by Azt/nimbolide in glioblastomamultiforme cells is due to its inhibitory action on Aktphosphorylation.Signaling pathways frequently deregulated in glioblas-

toma multiforme include the EGFR/EGFRvIII-drivenPI3K-Akt pathway, RAS-MAP kinase pathway, JAK-STATpathway, and the retinoblastoma pathway (4, 27). It istherefore remarkable that Azt/nimbolide suppressed allthese pathways with the greatest effect observed in glio-blastoma multiforme cells expressing the EGFRvIII onco-gene. Azt/nimbolide did show some specificity becauseit suppressed the above pathways without inhibitingmTOR and without changing the total protein levels ofseveral cell-cycle regulators, including cyclin D, cyclin B,P27, and P21. The three adult glioblastoma multiformecells that we primarily used in this study are all homo-zygous null for the G1–S cell-cycle inhibitor INK4a/ARF(CDKN2A in humans) that inhibits CDK4/6, the primarykinase that phosphorylates and inactivates retinoblasto-ma. Absence of INK4a/ARF therefore leads to uncheckedCDK4/6 activity in these glioblastoma multiforme cells.Thus, it is of high significance that Azt/nimbolide direct-ly inhibits CDK4/6 activity leading to retinoblastomahypophosphorylation and G1–S arrest, particularly inglioblastoma multiforme cells expressing the oncogeneEGFRvIII.

Studies showing the in vivo cytotoxicity of Azt/nimbo-lide are limited. The LD50 of Azt was found to be 4.57mg/g body weight in acute toxicity studies in rodents(35). In this study, we found that acute exposure ofglioblastoma multiforme cells in vitro to Azt preventstumor formation in vivo. We attempted to bring Azt to awater-soluble form, but the resulting extract followingEtOH evaporation was ineffective in killing glioblastomamultiforme cells in vitro. Therefore, we had to rely on theEtOH-soluble fraction for in vivo studies. We however,demonstrated that intratumor injection of a very lowdose of Azt (0.34 mg/g body weight) considerably sup-pressed tumor growth. Importantly, we also show thatsystemic administration of nimbolide, the principal cyto-toxic component of Azt, can significantly inhibit glio-blastoma multiforme growth both in flank and intracra-nial orthotopic glioma models. Grade IV gliomas orglioblastoma multiformes are histologically character-ized by hypercellularity and presence of extensive mitoticactivity. Azt reduced hypercellularity, inhibited prolifer-ation, and induced apoptosis in glioblastoma multi-forme tumor xenografts. It also inhibited growth factorsignaling in vivo as revealed by the reduced staining ofphosphorylated Akt, Erk1/2, and STAT3 in Azt-injectedtumors.

In summary, our results demonstrate that Azt/nimbolideis a potent cytotoxic agent that exerts antiproliferative andapoptotic effect in glioblastoma multiforme in vitro and invivo. It suppresses glioblastoma multiforme viability andsuppresses tumor growth by inhibiting CDK4/6 activityleading to retinoblastoma hypophosphorylation and cell-cycle arrest and by inhibiting growth factor pathways hyper-activated in glioblastoma multiforme, including the PI3K-Akt, MAP kinase, and JAK-STAT pathways. Further studiesare needed to explore solubility of Azt in aqueous solutions,examine the bioavailability of Azt and nimbolide, anddetermine their therapeutic efficacy as a single agent or incombination with chemotherapy and radiation in ortho-topic mouse models of human glioblastoma multiformeand other cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P.K. Ghosh, B. DasguptaDevelopment of methodology: N. Nassar, B. DasguptaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Karkare, R.R. Chhipa, J. Anderson, X. Liu,H. Henry, A. Gasilina, N. Nassar, J. Roychoudhury, J.P. Clark, A. KumarAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S. Karkare, A. KumarWriting, review, and/or revision of the manuscript: S. Karkare, R.R.Chhipa, G.M. Pauletti, B. DasguptaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Karkare, X. LiuStudy supervision: R.R. Chhipa, B. Dasgupta

AcknowledgmentsThe authors thank Paul Mischel, UCLA for U87EGFR and U87EGFRvIII

cells, Peter Houghton, Nationwide Children’s Hospital (Columbus, OH) forthe G2 cells, and John Ohlfest, University of Minnesota for the P9 neuro-sphere line.






Grant SupportThis work was supported by the Division of Oncology, CCHMC, Cancer-

FreeKids, CCHMC Trustee Scholar grant, NIH/NHLBI R01HL111192 (to A.Kumar), and NIH/NINDS R01NS075291 (to B. Dasgupta).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 18, 2013; revised October 8, 2013; accepted October 10,2013; published OnlineFirst October 29, 2013.

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2014;20:199-212. Published OnlineFirst October 29, 2013.Clin Cancer Res Swagata Karkare, Rishi Raj Chhipa, Jane Anderson, et al. Causes Cell-Cycle Arrest and Suppresses Glioblastoma GrowthDirect Inhibition of Retinoblastoma Phosphorylation by Nimbolide

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