induction of apoptosis by flavopiridol in human ... · lytic site of caspases 1 to -9 inhibiting...

Induction of Apoptosis by Flavopiridol in Human NeuroblastomaCells Is Enhanced under Hypoxia and Associated With N-mycProto-oncogene Down-Regulation

Maura Puppo,1 Sandra Pastorino,4

Giovanni Melillo,3 Annalisa Pezzolo,2

Luigi Varesio,1 and Maria Carla Bosco1

1Laboratory of Molecular Biology, and 2Laboratory of Oncology,Giannina Gaslini Institute, Genoa, Italy; 3Developmental TherapeuticsProgram-Tumor Hypoxia Laboratory, Science ApplicationsInternational Corp.-Frederick, Inc., National Cancer Institute-Frederick Cancer Research and Development Center, Frederick,Maryland; and 4Neuro-Oncology Branch, National Cancer Institute,NIH, Bethesda, Maryland

ABSTRACTPurpose: Neuroblastoma is the most common extracra-

nial solid tumor of children that arises from the sympatheticnervous system. Survival rates for neuroblastoma patients islow despite intensive therapeutic intervention, and the iden-tification of new effective drugs remains a primary goal. Thecyclin-dependent kinase inhibitor, flavopiridol, has demon-strated growth-inhibitory and cytotoxic activity against var-ious tumor types. Our aim was to investigate flavopiridoleffects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of itsactivity by hypoxia, a critical determinant of tumor progres-sion and a major challenge of therapy.

Experimental Design: Cell viability was monitored by3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazoliumbromide (MTT) and trypan blue dye exclusion assays; DNAsynthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa stain-ing, DNA fragmentation, terminal deoxynucleotidyl-trans-ferase-mediated dUTP nick end labeling reaction, flowcytometric determination of hypodiploid DNA content, andevaluation of caspase activity and cytochrome c (CytC) re-lease; MYCN expression was determined by Northern andWestern blotting.

Results: Flavopiridol caused dose- and time-dependentdecreases in neuroblastoma viability by inducing apoptosis,as confirmed by morphologic and biochemical criteria. Celldeath was preceded by DNA synthesis inhibition and G1-G2

arrest, reversed by the pancaspase inhibitor, zVAD-fmk,and associated with caspase-3 and -2 activation and CytCincrease. Moreover, flavopiridol strongly down-regulatedMYCN mRNA and protein expression. Exposure to hypoxiaenhanced both the extent of apoptosis and flavopiridol ef-fects on CytC, caspase 3, and MYCN.

Conclusions: These results indicate that flavopiridol hasgrowth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further in-vestigation for the treatment of this disease.

INTRODUCTIONNeuroblastoma is the most common pediatric solid tumor;

it derives from the neural crest and arises in the adrenal medullaor paraspinal ganglia (1). Neuroblastomas exhibit clinical andbiological heterogeneity, ranging from rapid progression asso-ciated with metastatic spread and poor clinical outcome tooccasional, spontaneous, or therapy-induced regression or dif-ferentiation into benign ganglioneuroma (2). Several transfor-mation-linked genetic changes have been identified in neuro-blastoma (1, 2). Amplification of the N-myc proto-oncogene(MYCN), which occurs in a subset of tumors and results inenhanced MYCN expression, is the most typical genetic featureof advanced-stage neuroblastoma and an adverse prognosticindicator (1–3). MYCN amplification correlates with a moremalignant course of the disease, induction of angiogenesis,resistance to therapy, and poor clinical outcome (3–6), suggest-ing that it may be a progression-related event and a potentialtherapeutic target (2).

Rapidly expanding neuroblastoma tumors present areas oflow O2 tension (hypoxia) and metastasize to hypoxic sites, suchas bone and bone marrow (7). Hypoxia is a common denomi-nator of many pathologic processes and a critical determinant oftumor cell growth, susceptibility to apoptosis, and resistanceto radio- and chemotherapy (8–10). Hypoxia activates thehypoxia-inducible transcription factor-1 (HIF-1), a hetero-dimeric complex composed by the basic-helix-loop-helix PASproteins, HIF-1� (or the homologues HIF-2�/3�), which is thehypoxia-responsive subunit, and HIF-1�, also known as the arylhydrocarbon receptor nuclear translocator (ARNT), and consti-tutively expressed in unstimulated cells (9, 11). HIF-1 trans-activates the hypoxia-responsive element (HRE) present in thepromoter or enhancer elements of many genes encoding angio-genic, metabolic and metastatic factors (10, 12, 13), inducingtheir expression and contributing to the acquisition of tumorcell aggressive phenotypic changes (8–10). Accumulation ofHIF-1�/2� proteins was observed in vitro in advanced-stage,

Received 10/20/03; revised 7/9/04; accepted 7/14/04.Grant support: Supported by grants from the “Fondazione Italiana perla Lotta al Neuroblastoma” and the Italian Association for CancerResearch (AIRC), The San Paolo Company; FIRB-Ministero dellaSanita Italiana.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 toindicate this fact.Requests for reprints: Maria Carla Bosco, Laboratorio di BiologiaMolecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini 5, 16148Genova Quarto, Italy. Phone: 39-010-5636633; Fax: 39-010-3733346;E-mail: [email protected].

©2004 American Association for Cancer Research.

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metastatic human neuroblastoma cell lines exposed to hypoxiaand in the hypoxic areas of experimental neuroblastoma xe-nografts grown in mice (14, 15). Recent findings have demon-strated that hypoxia has a profound impact on neuroblastomaaggressive behavior. Increased production of the angiogenicmediator, vascular endothelial growth factor (VEGF), and of theId2 protein, an inhibitor of the antiproliferative function of theretinoblastoma (Rb) tumor suppressor gene (16), was observedin hypoxic neuroblastoma cells both in vitro and in vivo (14, 17).Furthermore, hypoxia down-regulates several neuronal/neu-roendocrine marker genes in neuroblastoma cells and,conversely, up-regulates genes expressed in neural crestsympathetic progenitors, leading to neuroblastoma cell de-differentiation and acquisition of an immature and moreaggressive neural-crest-like phenotype (14).

Despite the array of chemotherapeutic agents presentlyavailable (2, 18) and multimodality therapeutic protocols thatuse high-dose chemotherapy associated with autologous bone-marrow or stem-cell transplantation (2, 7, 19), the prognosis ofchildren who develop metastatic, stage IV neuroblastoma overthe age of 1 year has only marginally improved, and the overalllong-term disease-free survival rate of this tumor remains low(2, 7, 19–21). Thus, the search for effective treatments forneuroblastoma, either at advanced stage or at minimal residualdisease, remains a primary goal. The regulation of neuroblas-toma growth, differentiation, and apoptosis has become an areaof active research in the last decade with the aim of identifyingnovel and more specific therapeutic agents. Advanced-stage,MYCN-amplified neuroblastomas usually present defects in theexpression and function of elements of the apoptotic machinery,such as deletion/inactivation of genes involved in cell deathinduction and/or constitutive activation/overexpression of genescounteracting the apoptotic process (for a review, see ref. 22)that have been implicated in neuroblastoma development, ma-lignant progression, and resistance to traditional cytotoxicagents (22–25). Much work has consequently focused on thepossibility of restoring the normal rate of apoptosis in neuro-blastoma, and the identification of novel agents capable oftriggering programmed cell death in this type of tumor hasbecome an important therapeutic objective (2, 22).

One potential candidate in this line of research is flavopiri-dol (Flp), a semisynthetic flavone derivative of the alkaloidrohitukine (26) that has raised considerable interest in the pastfew years because of its potent antineoplastic activity against avariety of tumors both in vitro (27–31) and in vivo in humanxenografts models (27–29), including melanomas and small-celllung carcinomas, that share with neuroblastoma the neuroecto-dermal origin (28, 32, 33). Preclinical evidence also indicatesthat this agent can potentiate the therapeutic effects of conven-tional chemotherapeutics (34, 35) and is endowed with antian-giogenic and antimetastatic properties that may contribute to itscytotoxic activity in vivo (13, 17, 36). Flavopiridol is currentlyundergoing Phase II/III clinical trials for the treatment of vari-ous refractory neoplasms, both as a single agent and in combi-nation with other antineoplastic drugs (for a review, see ref. 36),and promising therapeutic activity associated with mild toxicityhas been observed (36–38). Numerous in vitro studies haveshown that flavopiridol can inhibit tumor cell growth either bycausing cell cycle arrest at the G1 and G2 phases, through the

suppression of cyclin-dependent kinase (CDK) activity (36, 39,40), or by triggering apoptosis (27, 29–31, 33, 35, 41). Remark-ably, flavopiridol apoptotic activity can be exerted on bothcycling and noncycling tumor cells (30, 40) and is refractory tovarious genetic alterations commonly found in human neopla-sias (42–44), a property that makes this compound a promisingtherapeutic agent for the treatment of malignancies resistant toother chemotherapeutic drugs.

The purpose of this study was to assess the effects offlavopiridol on neuroblastoma cells, with particular attention tothe induction of apoptosis and the biochemical mechanisms thatmay regulate this process. Moreover, we were interested ininvestigating the modulation of flavopiridol activity by hypoxia.We demonstrate that pharmacological concentrations of fla-vopiridol (in the 200–300 nmol/L range) arrest cell growth andtrigger programmed cell death in advanced-stage, MYCN-amplified neuroblastoma cells lines. Flavopiridol-induced apo-ptosis involves both caspase activation and cytochrome c (CytC)release and is enhanced on cell exposure to hypoxia. Moreover,we show a correlation between flavopiridol cytotoxic activityand MYCN down-regulation.

MATERIALS AND METHODSCells and Culture Conditions. The human neuroblas-

toma cell lines GI-LI-N, LAN-5, ACN, GI-ME-N, IMR-32(purchased from the Bank of Biological Material Interlab CellLine Collection, Advanced Biotechnology Center, Genoa, It-aly), and HTLA-230 (45) were isolated from patients withmetastatic, stage IV neuroblastoma tumors and were character-ized for the presence of molecular alterations (1). With regard toMYCN status, amplification was observed in GI-LI-N, LAN-5,IMR-32, HTLA-230, but not in ACN and GI-ME-N cell lines (1,45). Cells were maintained in the logarithmic phase of growth inRPMI 16140 (Euroclone Ltd., Celbio, Milan, Italy), supple-mented with 10% heat-inactivated fetal bovine serum (Sigma,Milan Italy), 2 mmol/L L-glutamine, 100 units/mL penicillin,and 100 �g/mL streptomycin (Euroclone Ltd), at 37°C in ahumidified incubator containing 20% O2, 5% CO2, and 75% N2.Hypoxic conditions (i.e., 1% O2) were achieved by culturing thecells in an anaerobic work station incubator (BUG BOX, Jouan,ALC International S.r.l., Cologno Monzese, Milano, Italy)flushed with a gas mixture containing 1% O2, 5% CO2, andbalanced N2 at 37°C in a humidified atmosphere. Oxygen ten-sion in the medium was measured with a portable, trace oxygenanalyzer (Oxi 315i/set, WTW; VWR International, Milano, It-aly). For cell growth and apoptosis determination, adherent cellswere detached from culture plates with a solution of 0.05%trypsin and 0.02% EDTA in PBS (Euroclone Ltd) and wereplated at subconfluent densities 2 days before the addition of thereagents.

Reagents. Flavopiridol (obtained from the Developmen-tal Therapeutics Program, Drug Synthesis and ChemistryBranch, Division of Cancer Treatment and Diagnosis, NationalCancer Institute, Bethesda, MD), was dissolved in DMSO as a10-mmol/L stock solution and was stored in aliquots at �80°C.Flavopiridol was diluted in culture medium and was used atfinal concentrations ranging from 50 to 300 nmol/L. zVAD-fmk, a pancaspase inhibitor that irreversibly binds to the cata-

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lytic site of caspases 1 to -9 inhibiting apoptosis, was purchasedfrom Becton Dickinson (Milan, Italy), dissolved in DMSO at aconcentration of 10 mmol/L, and stored in aliquots at �20°C.zVAD-fmk was used at a final concentration of 50 �mol/L.

MTT Assay. Exponentially growing LAN-5 and GI-LI-N cells were plated in triplicate wells of 96 flat-bottomed-well Corning plates (Celbio) at a density of 7 � 103 and 2 � 104

per well, respectively, and were exposed to increasing flavopiri-dol concentrations or to DMSO for different time points. Afterthe incubation period, 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphen-yl-2H tetrazolium bromide solution (MTT; Sigma) was added toeach well to a final concentration of 0.5 mg/mL and wasincubated for 4 hours to allow MTT reduction. The formazancrystals were dissolved by adding a solubilization solution con-taining 0.01 M HCl/10%SDS, and absorbance was measured atthe dual-wavelengths of 570 and 630 nm with a SPECTRAFluorPlus spectrophotometer (TECAN Italia, S.r.l., Cologno Monz-ese, Milan, Italy). The data, subtracted of background MTTabsorbance, are expressed as percentages of viable cells relativeto control cells at each time point. In some experiments, cellviability was assessed by trypan blue dye exclusion test. Cellswere harvested at the indicated time points after drug exposureand were stained with 0.2% trypan blue. Live (trypan blue-excluded) and dead cells (trypan blue-stained) were thencounted on a hemocytometer.

Morphologic Evaluation of Apoptosis. One � 105 cellswere applied to Polysine glass slides by cytocentrifuging at 900rpm for 10 minutes, were fixed in methanol, and were stainedwith May-Gruenvald-Giemsa dye (Sigma). Stained cytospinpreparations were examined with a phase contrast microscope(Olympus Italia second s.r.l., Segrate, Milan, Italy). Photomi-crographs were taken with a Zeiss camera (Zeiss, Jena, Ger-many).

DNA Fragmentation Analysis. Cells (2 � 106) werewashed with ice-cold PBS, were lysed with TE buffer [10mmol/L Tris-HCl (pH 8), 1 mmol/L EDTA] containing 0.2%Triton X-100, and were centrifuged for 30 minutes at 13,000rpm to isolate the cytoplasmic DNA fragments (soluble) fromintact chromatin (nuclear pellets). Supernatants were treatedwith proteinase K (0.1 mg/mL) at 37°C overnight, and the DNAwas extracted sequentially with phenol:chloroform (1:1) andchloroform:isoamyl alcohol (24:1). DNA was then precipitatedwith 3 mol/L sodium acetate (pH 5.2), 1 mol/L MgCl2, and100% isopropanol, and was resuspended in TE buffer. DNase-free RNase A (50 �g/mL; Roche Molecular Chemicals, Milan,Italy) was then added for 2 hours at 37°C for RNA digestion.DNA samples were electrophoresed on 1.2% agarose slab gelcontaining 0.1 �g/mL ethidium bromide. A 1-kb DNA ladder(New England Biolabs, Celbio, Milan) was run in parallel toprovide molecular-sized markers. DNA fragments in a ladderpattern were visualized by UV light trans-illumination.

Flow Cytometric Analysis of Hypodiploid DNA Con-tent. Cells (5 � 105) were washed with PBS, fixed in ice-cold70% EtOH, and stained for 1 hour in the dark with a solutioncontaining 0.1% sodium citrate, 0.1% Triton X-100, 50 �g/mLpropidium iodide (PI; Sigma), and 100 �g/mL DNase-freeRNase in PBS. Monovariate distributions of cell number versusDNA content (PI) were analyzed with a FACScan flow cytom-eter (Becton Dickinson, Milano, Italy) equipped with a xenon

lamp and a filter set for excitation at 488 nm. PI fluorescenceintensity was recorded through a 575-nm high pass filter. Atleast 20,000 events were collected in each histogram, and datawere analyzed with CellQuest software (Becton Dickinson).Apoptotic cells could be observed as a “sub-G1” peak on theDNA histogram.

TUNEL Assay. DNA cleavage was assessed by enzy-matic end-labeling of DNA strand breaks with a commercial kit(In Situ Cell Death Detection kit, Fluorescent, Roche MolecularChemicals). Labeling was carried out according to the manu-facturer’s instructions. Briefly, 5 � 104 neuroblastoma cellswere cytocentrifuged onto Polysine glass slides, fixed in 4%paraformaldehyde in PBS for 1 hour at room temperature, andwashed with PBS. Cells were, then, permeabilized with 0.1%Triton X-100 and 0.1% sodium citrate for 2 minutes at 4°C.After rinsing, slides were incubated with 50 �L of terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabeling (TUNEL) reaction mixture, containing TdT- and FITC-labeled dUTP, in a humidified atmosphere for 1 hour at 37°C inthe dark. Rinsed slides were then coverslipped with Vectashieldmounting medium containing 4�,6�-diamidino-2-phenylindole(DAPI; Vector Laboratories, Burlingame, CA) for nuclear coun-terstaining. TUNEL� apoptotic cells, which fluoresce brightgreen, were viewed with a Nikon Eclipse E1000 fluorescentmicroscope (Nikon Corp., Tokyo, Japan) equipped with a FITCfilter. The percentage of apoptotic cells on total cell number wasdetermined for each sample in a blind fashion by counting thenumber of green fluorescent nuclei among a total of 300 or moreDAPI-stained blue nuclei in randomly chosen fields, at a mag-nification of �20/0.5 (objective). Photomicrographs were takenwith a Zeiss camera. In some experiments, TUNEL labeling wascarried out on cells in suspension (2 � 106 per sample) fixed inice-cold 70% EtOH. Total DNA was then counterstained byresuspending the cells in 0.5 mL PBS containing 5 �g/mL PIand 100 �g/mL DNase-free RNase for 30 minutes at roomtemperature in the dark. Cells were analyzed for fluoresceinateddUTP incorporation (FITC) versus DNA content (PI) by two-color flow cytometry with a FACScan. FITC and PI fluores-

Fig. 1 Southern blot analysis of MYCN gene in GI-LI-N, LAN-5, andACN cell lines. Ten �g of genomic DNA isolated from the indicatedcell lines were digested with EcoRI, electrophoresed on a 0.6% agarosegel, and subjected to Southern blot analysis with a probe specific for thehuman MYCN oncogene. The ACN cell line, which carries a singleMYCN copy, was used as a control. A 2-kb EcoRI band correspondingto the amplified MYCN oncogene is shown in the GI-LI-N and LAN-5cells. A 1-kb marker was run as a molecular-sized standard (M).

8706 Flavopiridol Induces Apoptosis in Neuroblastoma



cence intensities were recorded through a 520/530 nm and575-nm filters, respectively. At least 20,000 events were col-lected in each plot. Data were analyzed with the CellQuestprogram.

Analysis of DNA Synthesis by BromodeoxyuridinePulse-Labeling Technique. Esponentially growing cells (2 �106) were pulsed-labeled with 10 �mol/L bromodeoxyuridine(BrdUrd; Sigma), 4 or 18 hours before harvesting, were washedwith 1% bovine serum albumin (BSA)/PBS, and fixed with 70%ice-cold EtOH for 30 minutes. Cells were then incubated for anadditional 30 minutes with 2M HCl/0.5% Triton X-100. topartially denature the DNA, were washed, and were resus-pended in PBS/1%BSA/0.5% Tween 20 solution. IncorporatedBrdUrd was stained with 20 �L of FITC-conjugated anti-BrdUrd mouse monoclonal antibody (mAb; Becton Dickinson)for 30 minutes at room temperature. Samples were then washedand resuspended in PBS containing 5 �g/mL PI. Bivariatedistributions of BrdUrd amounts (FITC) versus DNA content(PI) were analyzed by a FACScan flow cytometer with CellQuest software. A total of 20,000 to 50,000 events were col-lected in each final plot.

Evaluation of Cytochrome c Release. CytC levels incytoplasmic extracts were assessed with a specific ELISA(Bender MedSystems, Vienna, Austria), according to the man-

ufacturer’s instructions. Briefly, 1.5 � 105 cells were washed incold PBS and were lysed with lysis buffer. Cell lysates werecleared by centrifugation and were assessed for protein concen-tration with the Bradford assay (Bio-Rad, Richmond, CA).Equal amounts of proteins diluted in assay buffer were loadedonto a 96-well plate coated with an anti-CytC mAb and weresubjected to ELISA. CytC concentrations were determined bymeasuring the absorbance values at 450 nm with a SPEC-TRAFluor Plus spectrophotometer and plotting them onto astandard CytC curve.

Determination of Caspase Activity. The protease activ-ity of caspases 3, -8, -9, and -2 was determined simultaneouslywith the BD ApoAlert Caspase Assay plates (Becton Dickin-son), according to the manufacturer’s instructions. Briefly, neu-roblastoma cells (2 � 105 per sample) were washed with coldPBS and lysed with ice-cold cell lysis buffer. Cell lysates werecleared by centrifugation, and the supernatants were transferredto 96-well caspase assay plates, containing immobilized selec-tive caspase substrates covalently linked to the fluorogenic dyeAMC (7-amino-4-methyl coumarin), and incubated for 2 hoursat 37°C. Caspase activity in each tested lysate was determinedby measuring the intensity of the fluorescent signal generated bysubstrate cleavage and consequent AMC release with a SPEC-

Fig. 2 Flavopiridol (Flp) re-duces neuroblastoma cell via-bility. In A, exponentiallygrowing GI-LI-N and LAN-5cells were treated in triplicatewith increasing concentrationsof Flp or the vehicle (DMSO)alone for the indicated timepoints [h, hour(s)] and were as-sessed for viability by MTT as-say. The results of one repre-sentative experiment of four arepresented as percentages ofcontrol versus Flp concentra-tion. In B, cells were incubatedfor 24 hours with 300 nmol/LFlp (f) or DMSO ( ��, Control),and cell death was determinedby counting live (trypan blueexcluded) and dead cells(trypan blue stained) on a he-macytometer. Results are plot-ted on a bar graph as percent-ages of cell death on the totalcell number. Bars, the mean �SEM of four independent ex-periments.




TRAFluor Plus spectrofluorometer with excitation at 388 nmand emission at 460 nm.

Southern and Northern Blot Analysis. Genomic DNAwas isolated from GI-LI-N, LAN-5, and ACN cells, cultured in15-cm Costar plates at a density of 1 � 106 (LAN-5 and ACN)or 1.5 � 106 cells/mL (GI-LI-N) with the DNAzol reagent (LifeTechnologies, Celbio), according to the manufacturer’s instruc-tions. Ten �g of DNA from each sample were digested withEcoRI restriction enzyme (New England Biolabs [NEB]), andthe DNA fragments were electrophoresed on a 0.6% agarosegel, denatured by treatment with a solution of NaOH 0.4M/NaCl 0.6 mol/L, and transferred to Nytran membranes(Schleichen & Schuell Inc., Keen, NH). A 1-kb marker was runin parallel as a molecular-sized standard. Total cellular RNAwas purified from LAN-5 and GI-LI-N cells with the TriZOLRNA reagent (Life Technologies), according to the manufactur-er’s instructions. Twenty �g of RNA from each sample waselectrophoresed under denaturing conditions on a 1.2% agarosegel containing 2.2 mol/L formaldehyde and transferred to Nyt-ran membranes. A RNA marker was run in parallel as a molec-ular-sized standard. Filter hybridization was done with 2 � 106

cpm/mL of 5�-[�32P]dCTP-labeled human MYCN cDNA,kindly provided by Dr. Tonini (Advanced Biotechnology Cen-ter, Genoa, Italy), in Hybrisol I hybridization solution (Oncor,Gaithersburg, MD) as described previously (17). Blots wereautoradiographed with Kodak XAR-5 film (Eastman Kodak,Rochester, NY), and quantitative assessment of the band inten-sities was carried out with the VersaDoc Image Analyzer (Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis. Neuroblastoma cells (1 � 107/mL) were solubilized in TBS-E lysis buffer [20 mmol/L Tris(pH 8), 50 mmol/L NaCl, 5 mmol/L EDTA) containing 1%NP40, 10% glycerol, 10 �g/mL leupeptin, 10 �g/mL aprotinin,10 �g/mL pepstatin, 10 mmol/L DTT, and 1 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF;Roche). One hundred micrograms from each cleared cell lysatewere resolved on 8% SDS-PAGE and electroblotted to Immo-bilon-P nitrocellulose membranes (Millipore Corp., Bedford,MA). A prestained protein marker (175–6.5 kDa; NEB) was runas a molecular-size standard. Membranes were immunoblottedovernight with biotin-conjugated antihuman MYCN mAb(Oncogene Research Products, Boston, MA), which recognizestwo nuclear phosphoproteins of molecular mass 64,000 Daltonsand 67,000 Daltons coded by the human MYCN gene, in block-ing buffer (TBS supplemented with 0.5% Tween 20 and 5%

BSA), washed in TBS supplemented with 0.05% Tween 20,and incubated for 30 minutes with horseradish peroxidase-conjugated-conjugated streptavidin (Kirkegaard and Perry Lab-oratories, Inc., Gaithersburg, MD) in washing buffer containing2.5% BSA. An anti-�-actin mAb (Sigma) was used as aninternal control for loading. Immunoreactivity was detected withenhanced chemoluminescence (ECL, Pierce, Celbio, Milan, It-aly) and was autoradiographed.

Statistical Analysis. Data are the means � SE of at leastthree independent experiments. The Student’s t test was used todetermine the significance of the results (significance at P 0.05).

RESULTSFlavopiridol Is Cytotoxic to Neuroblastoma Cells. To

study flavopiridol activity on advanced-stage neuroblastomacells, the human neuroblastoma cell lines, GI-LI-N and LAN-5,which exhibit MYCN gene amplification (Fig. 1) and expressMYCN protein (1), were exposed to increasing concentrationsof flavopiridol (ranging from 0 to 300 nmol/L), and relative cellnumbers were then assessed at different time points by MTTassay (Fig. 2A). Both cell lines displayed dose- and time-dependent decreases in viability, detectable as early as 24 hoursafter culture with 50 nmol/L of the drug. Seventy-two hours ofcontinuous exposure to 100 nmol/L flavopiridol resulted in 82and 67% reduction in GI-LI-N and LAN-5 cell number, respec-tively, whereas 300 nmol/L were required to achieve total inhi-bition in both cell lines (Fig. 2A). Increased cell death wasobserved by trypan blue dye exclusion in flavopiridol-treatedcells relative to control cultures (Fig. 2B), suggesting a cytotoxicactivity of the drug.

Flavopiridol Cytotoxicity Occurs by Programmed CellDeath. Experiments were then carried out to establish whetherneuroblastoma response to flavopiridol was apoptotic. As evi-denced by phase-contrast microscopy of Giemsa-stained cyto-spin preparations (Fig. 3A), 15% of GI-LI-N and LAN-5 cells,treated for 24 hours with 300 nmol/L flavopiridol, displayedtypical morphologic hallmarks of apoptosis, including intenseshrinkage, progressive cytoplasm and chromatin condensation,and nuclear fragmentation (22). Conversely, less than 4% of thecontrol cells exhibited apoptotic features (Fig. 3A). Flavopiri-dol-treated neuroblastoma cells eventually rounded up and de-tached from the dish (data not shown).

Internucleosomal cleavage of genomic DNA is typical of

Fig. 3 Flavopiridol (Flp) induces apoptosis in neuroblastoma cells. A, morphologic assessment of apoptosis. GI-LI-N and LAN-5 cells wereincubated for 24 hours with 300 nmol/L Flp (�) or DMSO (�), were cytocentrifuged, and were stained with May-Gruenvald-Giemsa. Stainedcytocentrifuge slide preparations were examined under a phase-contrast microscope and photomicrographed (�40). Arrows, representative cells withapoptotic morphology. B, dose-dependent DNA laddering. GI-LI-N and LAN-5 cells were exposed to the indicated concentrations of Flp for 24 hours,and genomic DNA was then extracted, resolved by electrophoresis on a 1.2% agarose gel, and visualized by ethidium bromide staining and UV lighttrans-illumination. A 1-kb marker was run as a molecular-sized standard (M). C and D, analysis of DNA cleavage by TUNEL assay. C, GI-LI-N(panels a, c, e, g) and LAN-5 (panels b, d, f, h) cells treated for 24 hours (panels c, d), 48 hours (panels e f), and 72 hours (panels g, h) with 300nmol/L Flp or for 72 hours with DMSO alone (panels a ,b), were centrifuged onto microscope slides and stained with the TUNEL reaction mixture.Apoptotic nuclei displaying green fluorescence and viable DAPI-counter-stained blue nuclei were visualized by fluorescence microscopy. Photomi-crographs are shown (�20). Inset, apoptotic bodies are detectable (�100). D, the indicated neuroblastoma cell lines were treated for 48 hours with300 nmol/L Flp or with DMSO, and TUNEL reaction was performed as detailed in the Materials and Methods. Results are presented as a bar graphand expressed as percentages of TUNEL� cells. Data shown are the mean � SEM of the results obtained in three independent experiments, with SEMless than 10% of the mean.




Fig. 4 Dose- and time-dependent induction of hypodiploid DNA in GI-LI-N and LAN-5 cells by flavopiridol (Flp). GI-LI-N cells were stimulated:(A) for 24 hours with increasing concentrations of Flp (50–300 nmol/L) or the vehicle alone (�); (B) with 300 nmol/L Flp for 48 hours (panel b)and 72 hours (panel c) or with DMSO for 72 hours (panel a). In C, LAN-5 cells were exposed for the indicated time points to different doses of Flp.Hypodiploid changes were evaluated by flow cytometric analysis after PI staining. A and B, histograms of total DNA content, representative of oneof three different determinations. Horizontal axis, linear fluorescence intensity. Vertical axis, relative cell number. In each panel (A and B), thepercentage of cells with sub-G1 DNA content is indicated. In C, the percentages of cells containing hypodiploid DNA are plotted on a bar graph (h,hours). Bars are the mean � SEM of three independent experiments. 2N, diploid DNA content; 4N, tetraploid DNA content.




cells undergoing apoptosis (22). As shown in Fig. 3B, a char-acteristic pattern of nucleosome-sized ladder of DNA fragmentswas detectable by agarose gel electrophoresis in both cell linesafter a 24-hour flavopiridol treatment, with as little as 100nmol/L able to induce DNA fragmentation in GI-LI-N and 200nmol/L effective in LAN-5. Almost no DNA fragmentation wasobserved in control cells (Fig. 3B).

Induction of apoptosis was confirmed by the TUNEL assay(Fig. 3C), which measures DNA strand breaks in individualcells (22). We detected a time-dependent increase in the numberof cells displaying small, bright green fluorescent and con-densed nuclei after exposure to 300 nmol/L flavopiridol (Fig.3C). The percentages of TUNEL� cells were 17 and 15% inGI-LI-N and LAN-5 cells, respectively, after 24 hours (Fig. 3C,c and d), correlating with cell death evaluated by trypan blueexclusion, and increased progressively with time in culture,reaching 35–40% and 50–60% after 48 (Fig. 3C, e and f) and72 hours (Fig. 3C, g and h). Apoptotic bodies were also ob-served (Fig. 3C, d, inset). Conversely, only a few control cellsexhibited a faint fluorescent signal after 72 hours of culture (Fig.3C, a and b). No TUNEL� cells were detectable in culturestreated with flavopiridol for 12 hours or at earlier time points(data not shown). Induction of apoptosis was also triggered byflavopiridol in another four human neuroblastoma cell linesderived from metastatic, stage IV neuroblastoma tumors, twoexhibiting high levels of MYCN gene amplification and MYCNprotein expression (IMR-32 and HTLA-230; ref. 1, 45) and twowithout amplified MYCN gene (ACN and GI-ME-N; ref. 1),ranging from 30 to 72% after 48 hours of treatment (Fig. 3D),thus providing clear evidence that programmed cell death is ageneral response of neuroblastoma cells to flavopiridol.

To better quantify the percentage of cells dying of apopto-sis, GI-LI-N and LAN-5 cells were exposed to increasing drugconcentrations for various time points and were analyzed byflow cytometry after PI staining (Fig. 4), which allowed us toidentify cells with a sub-G1 DNA content (22). A dose-depen-dent increase in the percentage of cells containing hypodiploidDNA was detected in GI-LI-N cells after 24 hours of exposureto the drug, with 17% of cells undergoing apoptosis in re-sponse to 300 nmol/L (Fig. 4A). Longer exposure to flavopiridolfurther increased the apoptotic fraction of cells, causing up to56% cell death after 72 hours (Fig. 4B), consistent with theTUNEL results. Conversely, less than 6% apoptotic cells weredetected in control cultures (Fig. 4A and B). Comparable resultswere obtained in flavopiridol-treated LAN-5 cells (Fig. 4C),with only slight differences in the extent of cell death.

We conclude that flavopiridol triggers apoptotic cell deathin neuroblastoma cells.

Flavopiridol-Induced Apoptosis Is Preceded by CellCycle Arrest. Given the ability of flavopiridol to impose ablock to cell cycle progression in several tumor types (36, 39,40), we were interested in determining whether induction ofneuroblastoma apoptosis was coupled with growth arrest. Totest this possibility, we exposed exponentially growing LAN-5(Fig. 5A) and GI-LI-N (Fig. 5B and C) cells to flavopiridol andpulse-labeled them with BrdUrd for 4 (Fig. 5A and B) or 18hours (Fig. 5C). The amount of BrdUrd incorporated into DNAwas then evaluated by fluorescence-activated cell sorting as ameasure of DNA synthesis. As depicted in Fig. 5A and B, which

shows BrdUrd uptake (FITC) versus total cellular DNA content(PI), 40% of control cells actively incorporated BrdUrd duringthe last 4 hours of a 24-hour incubation period. BrdUrd incor-poration rate showed a dose-dependent decline on treatmentwith flavopiridol, with the concentrations required for a 50%reduction (IC50) ranging between 100 and 200 nmol/L (Fig. 5A)and almost complete blockage occurring at 300 nmol/L (Fig. 5Aand B). DNA synthesis inhibition was an early response toflavopiridol, because a 12-hour treatment with 300 nmol/L wassufficient to inhibit BrdUrd uptake by 80% (Fig. 5B). Thedecrease in BrdUrd-incorporation rate was paralleled by a 3- to5-fold increase in the percentage of cells with a G2 DNAcontent, suggesting flavopiridol-mediated arrest of cell cycleprogression at the G2 checkpoint. Although no significantchange in the G1 cell fraction was apparent within the 4-hourpulse-labeling period (Fig. 5A and B), we detected cell accumu-lation in G1 during a 18-hour BrdUrd pulse (Fig. 5C), suggestiveof G1 block from entry into S phase. Two-color flow cytometricmeasurement of dUTP incorporation (FITC) versus cellularDNA content (PI) evidenced apoptotic cell death in both G1 andG2 populations in response to longer drug exposure (Fig. 5D). Aprogressive increase in the fraction of TUNEL� G1 and G2 cellswas, in fact, detectable during a 24- to 72-hour treatment with300 nmol/L flavopiridol, which, accordingly, decreased thenumber of viable cells in each population and increased thepercentage of sub-G1 cells relative to control cultures.

These results suggest that flavopiridol-treated neuroblas-toma cells arrest in G1 and G2 before undergoing apoptotic celldeath.

Hypoxia Enhances Flavopiridol-Induced Apoptosis.Next, we investigated the effects of O2 deprivation on neuro-blastoma response to flavopiridol. GI-LI-N and LAN-5 cellswere cultured for various time points under normoxic (20% O2)or hypoxic conditions (1% O2), in the presence or absence offlavopiridol, and relative cell numbers were assessed by MTT.Hypoxia led to a time-dependent increase in neuroblastoma cellnumber, which was 1.5- and 2-fold higher than that of normoxiccontrols by 48 and 72 hours of culture, respectively (data notshown). In accordance to what was shown under normoxia,flavopiridol caused dose- and time-dependent decreases in hy-poxic cell viability, with almost complete inhibition achievedafter 72 hours’ exposure to 300 nmol/L. The drug IC50s underhypoxia and normoxia are compared in Table 1 for each of thetime points studied. Under normal oxygen tension, the IC50

values were within the 50- to 120-nmol/L and the 75- to 175-nmol/L range in GI-LI-N and LAN-5 cells, respectively, de-pending on the length of exposure, which suggests a greatersensitivity of GI-LI-N cells to flavopiridol. Exposure to 1% O2

caused some decrease of the IC50 values, although this effect didnot appear to be statistically significant (P � 0.05).

The effects of hypoxia on apoptosis induction by flavopiri-dol were then evaluated. As shown in Fig. 6A, which depictselectrophoretic analysis of genomic DNA in flavopiridol-treatedGI-LI-N and LAN-5 cells cultured under 1% O2 tension, DNAfragmentation was effectively elicited in both cell lines by aslow a concentration as 100 nmol/L flavopiridol, suggesting ahigher sensitivity of hypoxic than normoxic LAN-5 cells toflavopiridol, in agreement with the MTT assay. Moreover, asdetermined by TUNEL reaction, combined exposure to fla-




vopiridol and hypoxia resulted in a 1.6-fold increase in thepercentage of cells undergoing apoptosis relative to flavopiridolalone (Fig. 6B). As expected, no DNA laddering was detectablein cells exposed to hypoxia alone, which, accordingly, did notsubstantially modulate the number of TUNEL� cells (Fig. 6Aand B). Essentially equivalent results were obtained when apo-ptosis was evaluated by PI staining. The time course of appear-ance of apoptotic LAN-5 cells is depicted in Fig. 6C, whichshows a representative experiment of three experiments per-formed. Combined exposure of LAN-5 cell to hypoxia andflavopiridol increased the number of cells with a sub-G1 DNAcontent relative to cells treated with flavopiridol alone. Apo-ptosis enhancement by hypoxia was detectable after 24 hoursculture and was still evident at later time points, with thepercentage of apoptotic cells augmented from 50% to almost80% after 72 hours. Similar results were observed in GI-LI-Ncells (data not shown).

These results demonstrate that hypoxia enhances flavopiri-dol cytotoxic effects on neuroblastoma cells.

Flavopiridol Induces Caspase Activation and Cyto-chrome c Release. Experiments were carried out to elucidatethe role of caspases (46) in apoptosis induction by flavopiridol.Figure 7A depicts the kinetics of caspase 2, -3, -8, and -9 activityin GI-LI-N cells. Exposure to flavopiridol resulted in a markedand rapid activation of caspase 3, which was already evidentafter 3 hours and was maximal after 6 hours, decreasing butremaining elevated at later time points. Induction of caspase 2activity was also triggered by flavopiridol with a similar kinet-ics, although the extent of activation was lower than that ofcaspase 3. In contrast, under the same experimental conditionsflavopiridol failed to induce caspase 9 and -8 enzymatic activity.The effects of hypoxia on caspase activation by flavopiridolwere evaluated in parallel experiments. Although hypoxia alonedid not affect protease activity at any of the time points ana-lyzed, it enhanced flavopiridol effects on caspase 3, leading toapproximately a 1.5 increase in its activation over that inducedby flavopiridol alone after 6 hours of stimulation (data notshown). A similar pattern of results was observed in LAN-5cells (data not shown), suggesting the involvement of caspases3 and -2 in apoptosis induction by flavopiridol.

We next analyzed the effects of the cell-permeable pan-caspase inhibitor, zVAD-fmk. We first determined the concen-tration of zVAD-fmk necessary to inhibit caspase activation byflavopiridol. As shown in Fig. 7A, caspases 3 and -2 activity wassuppressed by preincubation for 2 hours with 50 �mol/L zVAD-fmk. Accordingly, both flavopiridol-mediated cell killing (Fig.7B) and internucleosomal DNA fragmentation (Fig. 7C) were

Fig. 5 Flavopiridol (Flp) induces growth arrest in neuroblastoma cells.In A, exponentially growing LAN-5 cells were stimulated for 24 hourswith 50 to 300 nmol/L Flp or with the vehicle alone (�); in B and C,GI-LI-N cells were treated with 300 nmol/L Flp or with DMSO for 12(12h) and 24 hours (24h). Cells were pulse-labeled with BrdUrd for thelast 4 hours (A, B) or 18 hours (C), were fixed, and were stained withFITC-conjugated anti-BrdUrd mouse mAb and PI. BrdUrd uptake(FITC) versus DNA content (PI) was evaluated by two-color flowcytometry. Representative density plots of one of three independent

determinations are shown. PI and FITC fluorescence intensities areplotted on the X and Y axes, respectively. A–C, % numbers in boxes, thepercentages of BrdUrd-incorporating cells. In D, LAN-5 cells weretreated with DMSO for 72 hours or 300 nmol/L Flp for the indicatedtime points (h, hours; 24h, 48h, 72h) and were analyzed for dUTPstaining (FITC) versus DNA content (PI) by two-color flow cytometry.Representative density plots of one of three different experiments areshown. PI and FITC fluorescence intensities are plotted on the X and Yaxes, respectively. % numbers, the percentages of TUNEL� or sub-G1

cells.




blocked by zVAD-fmk, suggesting that caspases 3 and -2 acti-vation was instrumental in these processes.

The possibility that mitochondrial events were also in-volved in apoptosis induction by flavopiridol was next ad-dressed by determining flavopiridol effects on CytC. CytCtranslocation from mitochondria to the cytosol occurs at an earlystep of programmed cell death and is causally related to thisprocess (47). CytC cytoplasmic content was determined at dif-ferent time points in both LAN-5 (Fig. 7D) and GI-LI-N cells(data not shown) by a specific ELISA. Flavopiridol did notsignificantly affect CytC cytosolic concentration during the first3 hours of treatment but led to a 4.5- and 6-fold increase after 6and 12 hours, respectively, relative to control cells. Althoughhypoxia alone was ineffective in augmenting CytC content, itenhanced the effects of flavopiridol (Fig. 7D) up-regulating

CytC basal levels after 3 hours’ exposure. Similar results wereobtained in the GI-LI-N cell line (data not shown), suggestingthat CytC is released in response to flavopiridol and that hy-poxia can cooperatively interact with flavopiridol in elicitingthis effect.

Taken together, these results indicate that caspase 3 and -2activation associated with CytC release is involved in apoptosisinduction by flavopiridol in neuroblastoma.

Flavopiridol Down-Regulates MYCN Expression inNeuroblastoma Cells. MYCN down-regulation was found tocorrelate with drug-induced apoptosis and growth inhibition inneuroblastoma (48–51). We were, thus, interested in determin-ing whether flavopiridol modulated MYCN expression. To ad-dress this question, we examined the changes in MYCN mRNAand protein in GI-LI-N (Fig. 8) and LAN-5 (data not shown)

Fig. 5 Continued




after exposure to increasing concentrations of flavopiridol fordifferent time points. GI-LI-N cells constitutively expressedhigh levels of MYCN transcript, which remained essentiallyconstant for up to 24 hours (Fig. 8A and B). Flavopiridol

induced a rapid and dose-dependent mRNA down-regulation,detectable with as low a concentration as 100 nmol/L flavopiri-dol (Fig. 8A) and within the first hour of culture (Fig. 8B).mRNA levels continued to decrease with time in culture, untilthey became barely detectable. Conversely, culture under hy-poxia alone only marginally affected MYCN expression (Fig.8A and B). Interestingly, however, hypoxia further enhancedflavopiridol inhibitory effects, inducing complete inhibition ofmRNA expression after 24 hours’ exposure to 300 nmol/Lflavopiridol (Fig. 8A and B). Comparable down-regulation wasdetected in LAN-5 cells (data not shown). mRNA reduction wasparalleled by decreased protein expression, which was morepronounced in hypoxic versus normoxic cells (Fig. 8C).

These data indicate that flavopiridol down-regulatesMYCN expression in MYCN-amplified neuroblastoma cells.

DISCUSSIONImpaired apoptosis is involved in the pathogenesis of can-

cer, and triggering this process in tumor cells is an importanttarget of therapy (52, 53). Advanced-stage, MYCN-amplifiedneuroblastoma are often resistant to conventional chemothera-peutic drugs because of aberrations/dysfunctions in their apop-

Fig. 6 Enhancement by hypoxia of flavopiridol (Flp) cytotoxic activity on neuroblastoma cells. A, dose-dependent DNA fragmentation. GI-LI-N andLAN-5 cells were treated for 24 hours under hypoxia alone (�) or in the presence of the indicated concentrations of Flp, and DNA fragmentationassay was carried out. The 1-kb ladder is shown as molecular-sized marker (M). B, detection of apoptosis by TUNEL assay. TUNEL assay was carriedout on cells treated for 24 hours with 300 nmol/L Flp (�), or with the vehicle alone (�), under hypoxic (Hy, �) or normoxic conditions (Hy, �),as described in the legend to Fig. 3C and D. Results (mean � SEM of three independent experiments) are presented as a bar graph and expressedas percentages of TUNEL� cells. C, determination of hypodiploid DNA content. LAN-5 cells were cultured for 24 hours (panels b, f), 48 hours(panels c, g), and 72 hours (panels d, h) with 300 nmol/L Flp, or for 72 hours with DMSO alone (panels a, e), under normoxic (Normo, a, b, c, d)and hypoxic (Hy, e, f, g, h) conditions. Representative DNA histograms generated from flow cytometric analysis of PI-stained cells are shown. % ineach panel, the percentages of cells containing hypodiploid DNA.

Table 1 Comparison of Flp effects on neuroblastoma viability undernormoxia and hypoxia

Cell linesO2 (%)

IC50 (nmol/L)

24 hours 48 hours 72 hours

GI-LI-N20 120 � 25 80 � 10 50 � 71 110 � 30 72 � 11 49 � 5

LAN-520 175 � 45 110 � 20 75 � 111 160 � 25 80 � 16 50 � 6

NOTE. Exponentially growing GI-LI-N and LAN-5 cells were seededinto 96-well plates and treated for the indicated time points with increasingconcentrations of Flp (0 to 300 nmol/L) under normoxic (20% O2) orhypoxic (1% O2) conditions. MTT assay was then used to assess relativecell numbers. Concentrations of Flp required to reduce by 50% viable cellnumbers (IC50) at the different time points are presented as the mean � SEof four independent experiments, each performed in triplicate.




Fig. 7 Flavopiridol (Flp) trig-gers caspase activation andCytC release in neuroblastoma.A, time course of caspase acti-vation by Flp in GI-LI-N cells.Cells were treated with 300nmol/L Flp in the presence orabsence of 50 �mol/L zVAD-fmk, and the proteolytic activityof caspases-3, -8, -9, and -2 wasassessed at the indicated timepoints [1h (1 hour), 3h, 6h, 12h,18h] against specific fluoro-genic substrates, as detailed inMaterials and Methods. Valuesare the mean of duplicate reac-tions run in parallel and are ex-pressed as fold induction rela-tive to protease activity incontrol samples (equal to 1) ateach time point. One represent-ative experiments of two per-formed is shown. B and C,inhibition of Flp-induced apo-ptosis by the pancaspase in-hibitor, zVAD-fmk. GI-LI-Nand LAN-5 cells were left un-treated (�) or treated (�) with50 �mol/L zVAD-fmk for 2hours before the addition of 300nmol/L Flp (�) or DMSOalone (�) for 24 hours, and (B)cell killing was determined bycounting live (trypan blue ex-cluded) and dead cells (trypanblue stained) on a hemacytom-eter. Results are presented as abar graph and expressed as per-centage of cell death. The val-ues are the mean � SEM ofthree independent experimentswith SEM less than 10% of themean. In C, DNA fragmenta-tion assay was carried out asdescribed in the legend to Fig.3B. The 1-kb ladder is shown(M). D, assessment of CytC cy-tosolic content. LAN-5 cellswere treated for the indicatedtime points (3h, 6h, 12h) with300 nmol/L Flp (�), or the ve-hicle alone (�) under hypoxic(Hy, �) or normoxic (Hy, �)conditions, and cytosolic ex-tracts were tested for CytC(Cytochrome) concentration byELISA, as described in Materi-als and Methods. Results arepresented as a bar graph and arethe mean � SEM of two differ-ent experiments, each done intriplicate.




totic machinery (22–25), making the search for novel agentscapable of inducing programmed cell death in this type of tumormandatory. The data presented in this paper provide the firstevidence that the synthetic flavone, flavopiridol, causes apopto-sis in neuroblastoma cell lines derived from metastatic, MYCN-amplified, stage IV tumors.

Flavopiridol activity against a variety of tumor types, bothin vitro and in vivo in experimental xenografts, is well docu-mented (26–33, 39, 41), and recent findings have reportedpromising therapeutic results of this agent in patients withrefractory neoplasms (36–38). To our knowledge, studies offlavopiridol antitumor effects have been limited to adult malig-nancies, and this is the first report showing that this compoundis also active on a childhood tumor. We demonstrate that fla-vopiridol activity on neuroblastoma cells is strictly dependenton the concentration of the drug and the duration of exposure.GI-LI-N cells appeared to be slightly more susceptible thanLAN-5 cells to flavopiridol, in agreement with previous obser-vations showing that the LAN-5 cell line is endowed with amore aggressive phenotype (1, 17). It is noteworthy that therange of effective doses of flavopiridol (50–300 nmol/L) iscomparable with that shown to be active in other tumor systems(27, 30, 41), indicating a similar sensitivity of neuroblastomaand other cancer cell types to flavopiridol. The observation thatthese doses correspond to clinically achievable pharmacologicalconcentrations of the drug (26, 36, 38) suggest potential thera-peutic implications of these findings. However, clinical trials offlavopiridol are limited to adults, and studies should be con-

ducted in children to determine its safe doses in pediatric pa-tients.

Flavopiridol was initially characterized as a cytostaticagent, capable of blocking cell cycle progression through theinhibition of multiple CDK activity (36, 39, 40, 43). It was laterfound to be an efficient inducer of apoptosis in a variety ofcancer cell lines (27, 29–31, 33, 41, 43), suggesting the engage-ment of different growth-inhibitory pathways depending on thehistotype of malignant cells. We demonstrate that flavopiridol iscytotoxic to neuroblastoma cells and that cytotoxicity is theresult of programmed cell death. A time- and dose-dependentdecrease in viable cell counts occurred in response to the drug,with complete inhibition achieved after a 72-hour exposure to a300 nmol/L concentration, and was associated with the appear-ance of morphologic hallmarks of apoptosis. Activation of anapoptotic pathway was confirmed by accumulation of cells witha sub-G1 DNA content and by the demonstration of DNAfragmentation, caspase activation, and CytC release. Impor-tantly, flavopiridol cytotoxicity was not unique to GI-LI-N andLAN-5 cells but was also exerted on another four cell linesderived from advanced-stage neuroblastoma tumors, two exhib-iting high levels of MYCN amplification and two without am-plified MYCN gene, indicating that apoptosis is a general re-sponse of neuroblastoma cells to flavopiridol. Interestingly,short exposure (i.e., 12–24 hours) to the drug resulted in growtharrest, manifested by DNA synthesis inhibition and G1-G2 blockof cell cycle progression, which preceded the time at whichextensive cell death occurred (i.e., 48–72 hours). These find-

Fig. 8 Dose- and time-depend-ent down-regulation of MYCNexpression. GI-LI-N cells werestimulated under normoxic(Normo) or hypoxic (Hy) condi-tions for 24 hours with increasingconcentrations of flavopiridol(Flp; A) or for different timepoints with 300 nmol/L Flp (B),and total RNA was assayed forMYCN mRNA expression byNorthern blotting. Ethidium bro-mide staining of the 28S and 18SrRNA is shown as a control ofRNA loading. C, GI-LI-N cellswere cultured for 24 hours undernormoxic (Normo) or hypoxic(Hy) conditions in the presenceof 300 nmol/L Flp. Protein ly-sates were analyzed by Westernblotting with a mAb specific forhuman MYCN. A protein markerwas run as a molecular-sizedstandard. Arrows, the molecularmass 64,000 DA and 67,000 Daproteins coded by the humanMYCN gene. The blot was rehy-bridized with anti-�actin mAb tocontrol for protein loading. Eachblot is representative of three in-dependent experiments.




ings, along with the observation that both G1 and G2 cellpopulations underwent apoptosis during flavopiridol treatment,indicate that cell death was not restricted to one particular phaseof the cell cycle and confirm the ability of flavopiridol to induceapoptosis in noncycling cells (29–31, 33, 40), thus raising thepossibility that this agent can be combined with other chemo-therapeutics causing growth arrest in neuroblastoma to potenti-ate their therapeutic activity, as shown in other tumor systems(34, 35). The mechanisms underlying flavopiridol-induced cellcycle arrest in neuroblastoma cells still remains to be elucidated,and we are currently investigating the possibility that the inhi-bition of CDKs, which are the primary targets of the drug at thedose range studied (36, 39), may account for this effect.

Apoptosis involves activation of members of the caspasefamily of cysteine proteases in a hierarchical cascade, with“initiator caspases” functioning upstream as triggers of theapoptotic process and “executioner caspases” acting down-stream as effector elements (46, 54), and may be regulated byvarious mitochondrial apoptogenic mediators including CytC(47). Flavopiridol can trigger multiple apoptotic pathwaysdepending on the type and stage of the target cell, thuscircumventing drug-resistance mechanisms (42– 44). Initialstudies demonstrated that flavopiridol induced apoptosis viacaspase 8 activation in some tumor cell types (42), whereasmore recent evidence indicated that flavopiridol cytotoxiceffects involved CytC release in others (33, 55, 56). More-over, reports by Alonso et al. (44) and Achenbach et al. (42)demonstrated that flavopiridol apoptotic activity was exertedindependently of both caspase 8 activation and CytC releasein a panel of glioma and small-cell lung carcinoma celllines, suggesting the implication of alternative, unidentifiedapoptotic mechanisms. In this study, we demonstrate thatflavopiridol-mediated apoptosis in neuroblastoma occurredthrough the engagement of the mitochondrial-dependentpathway, as indicated by the marked increase in CytC cyto-solic levels after short (i.e., 6 –12 hours) drug treatment.Moreover, flavopiridol-treated neuroblastoma cells showedextensive activation of caspases-3 and -2, the inhibition ofwhich by the pancaspase inhibitor zVAD-fmk completelyreversed induction of cell death and DNA fragmentation,pointing toward a critical role for these proteases in regulat-ing this process. The implication of caspase 3 in the deathmechanism used by flavopiridol has been described previ-ously in other systems (31, 33, 35, 42), whereas this is thefirst evidence that flavopiridol can trigger caspase 2 activa-tion. Caspases 3 and -2 are known downstream effectors ofapoptosis (46, 54). However, their potential involvement inthe initiation of the caspase cascade has been proposed inrecent studies that demonstrate their colocalization with var-ious apoptogenic proteins within the mitochondrial inter-membrane space in nonapoptotic cells and their rapid releaseand activation on apoptotic challenge (57, 58). Thus, theobservation that flavopiridol induced early caspase 3 and -2activation, before the occurrence of extensive cell death andconcomitant to CytC release, raises the possibility that thesemolecules play an upstream regulatory role in flavopiridol-induced neuroblastoma apoptosis. Moreover, given the abil-ity of caspase 2 to induce CytC release (57) and consequentactivation of caspase 3 (47, 58), which in turn can activate

caspase 2 (57), the temporal correlation between increasedcaspase-2 and -3 activity and CytC release suggests theexistence of a circular positive feedback amplification loopbetween these apoptotic proteins in flavopiridol-treated neu-roblastoma cells. In contrast, the lack of caspase-8 andcaspase-9 activation in response to flavopiridol arguesagainst the requirement for these molecules in the regulatoryphase of flavopiridol apoptotic cascade in neuroblastoma,despite their well-recognized importance as initiator caspasesin apoptosis induced by other stimuli (46, 54, 59, 60).

Recent studies have documented an important role forhypoxia in neuroblastoma cell acquisition of an immature, an-giogenic, and more aggressive neural-crest-like phenotype (14,15, 17). Accordingly, xenografted hypoxia-pretreated neuro-blastoma cells were reported to form palpable tumors early andto grow faster than grafted normoxia-treated cells (14). Becausehypoxia is present at common sites of neuroblastoma relapse (7)and represents a major challenge to therapy (8–10), a criticalquestion was whether flavopiridol retained its activity in areduced O2 environment. Our data demonstrate that not onlywas flavopiridol active on hypoxic neuroblastoma cells but thathypoxia potentiated its cytotoxic activity by 1.6-fold, with upto 80% hypoxic cells undergoing apoptosis after a 72-hourtreatment. The increased sensitivity of hypoxic cells to flavopiri-dol was also indicated by the finding that lower IC50s of thedrug were required under hypoxia than under normoxia toachieve comparable decreases in viable cell counts and that a100 nmol/L concentration of flavopiridol was sufficient to in-duce DNA fragmentation in LAN-5 cells cultured in 1% O2,whereas a 200 nmol/L concentration was necessary in normalO2 tension. The observation that 10% flavopiridol-treatedcells were TUNEL� after 12 hours of culture under hypoxia,whereas they were undistinguishable from control sampleswhen treated under normoxic conditions (data not shown), fur-ther supports this conclusion. These results were unexpectedbecause no cell death was detectable in GI-LI-N and LAN-5cells exposed to 1% O2. Conversely and in accordance with theresults by Jogi et al. (14), the hypoxic stress resulted in in-creased proliferation over that seen at normal O2 tension, furthersupporting the hypothesis of a hypoxia-induced shift toward amore aggressive behavior.

The mechanisms accounting for the increased apoptoticresponse of hypoxic neuroblastoma cells to flavopiridol areunclear, although our results point toward the involvement ofcaspase-3 and CytC in this process. Indeed, we found thathypoxia enhanced flavopiridol effects on caspase-3 activation,and a higher and earlier increase in cytosolic CytC levels overthat induced by flavopiridol alone was observed in response tothe combined treatment. CytC release during apoptosis maydepend on different mechanisms, including mitochondria swell-ing and physical disruption of the outer membrane, loss ofmitochondrial transmembrane potential, or formation of perme-ability transition pores at the site of contact between mitochon-drial inner and outer membrane (47). Whether any of thesemechanisms could explain the synergistic effect of flavopiridoland hypoxia on CytC still remains to be elucidated. Becauseboth flavopiridol and hypoxia can modulate the expression ofsome Bcl-2 family members, which regulate mitochondrialmembrane integrity and the formation of permeability transition




pores (10, 36, 43, 61), it is possible that their interaction maymodify the ratio between pro- and antiapoptotic proteins, thusincreasing mitochondrial permeability and CytC release. Loss ofmitochondrial membrane potential is also known to result fromchanges in the expression of Bcl-2 family members and waspreviously shown to occur in response to flavopiridol (33, 42),indicating another potential mechanism for CytC increase. De-tailed studies will be required to elucidate this issue.

Flavopiridol-treated neuroblastoma cells rapidly and sub-stantially down-regulated MYCN expression. This finding isrelevant, given the correlation between MYCN overexpressionand neuroblastoma aggressive phenotype. Accumulation ofMYCN protein is known to alter neuroblastoma cell cycleprogression by increasing the levels of Id2 protein, with conse-quent inactivation of the Rb-dependent antiproliferative path-way (16), and by enhancing DNA-synthesis through the de-crease of CDK inhibitor p27 expression and consequentinduction of cyclin E-dependent kinase activity (50). Moreover,MYCN overexpression was found associated with neuroblas-toma angiogenesis, metastatic dissemination, and poor progno-sis (4–6). Recent studies demonstrated that MYCN-amplifiedcell lines are tumorigenic when xenografted into immunodefi-cient mice and that MYCN-targeted expression in transgenicmice leads to neuroblastoma tumor development (62). Further-more, growing evidence supports a link between MYCN ampli-fication, impairment of neuroblastoma apoptotic response (23–25), and consequent resistance to therapy both in vitro (22, 49)and in vivo in neuroblastoma patients (63). Inhibition of MYCNexpression has, thus, been proposed as a strategy for neuroblas-toma treatment. In this regard, down-regulation of MYCN ex-pression by chemotherapeutics or antisense cDNA was found tocorrelate with the suppression of neuroblastoma cells growth(50, 51) and the induction of differentiation/apoptosis (48, 49,51). Whether MYCN inhibition plays a role in neuroblastomagrowth arrest and/or apoptosis induction by flavopiridol is con-troversial. The observation that hypoxia enhanced both MYCNdown-regulation and the extent of flavopiridol-triggered celldeath raises the possibility of a causal correlation between thesetwo events. However, the data showing that flavopiridol cyto-toxic activity was not restricted to MYCN-amplified cell linesargue against this hypothesis. This issue is intriguing and iscurrently under investigation.

In conclusion, our data demonstrate that MYCN-amplifiedneuroblastoma cells can be induced to undergo apoptosis bypharmacologically relevant concentrations of flavopiridol andthat this response is preceded by cell cycle arrest, enhanced byhypoxia, and associated with caspase activation, CytC release,and MYCN down-regulation. These findings, together with therecently reported ability of flavopiridol to inhibit constitutiveand hypoxia-induced VEGF production by aggressive neuro-blastoma cell lines (17), make this compound a promisingcandidate for the treatment of advanced-stage neuroblastoma.

ACKNOWLEDGMENTSThe authors thank Anna Negrioli and Paolo Broda for technical

assistance, and Chantal Dabizzi for secretarial work.

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2004;10:8704-8719. Clin Cancer Res Maura Puppo, Sandra Pastorino, Giovanni Melillo, et al.

Proto-oncogene Down-RegulationN-mycAssociated With Neuroblastoma Cells Is Enhanced under Hypoxia and Induction of Apoptosis by Flavopiridol in Human

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