Induction of caspase-dependent, p53-mediated apoptosis by ...mct.aacrjournals.org/content/molcanther/4/1/1.full.pdf · Induction of caspase-dependent, p53-mediated apoptosis by apigenin

Induction of caspase-dependent, p53-mediated apoptosisby apigenin in human neuroblastoma

Risa Torkin,1,2 Jean-Francois Lavoie,1,3

David R. Kaplan,1,3,4 and Herman Yeger1,2

1Cancer Research Program, The Hospital for Sick Children;2Department of Laboratory Medicine and Pathobiology; 3Instituteof Medical Sciences; and 4Department of Medical Genetics andMicrobiology, University of Toronto, Toronto, Ontario, Canada

AbstractNeuroblastoma is a pediatric tumor accounting for 15% ofchildhood cancer deaths and has a poor prognosis inchildren > 1 year of age. We investigated the ability ofapigenin, a nonmutagenic dietary flavonoid that has beenshown to have antitumor effects in various tumor cell lines,to inhibit growth and induce apoptosis of the humanneuroblastoma cell lines NUB-7, LAN-5, and SK-N-BE(2).Apigenin inhibited colony-forming ability and survival, andinduced apoptosis of NUB-7 and LAN-5 cells. The presenceof the C2-C3 double bond and the 4V-OH group on theflavonoid structure correlated with the growth-inhibitorypotential of apigenin. Furthermore, apigenin inhibitedNUB-7 xenograft tumor growth in a nonobese diabetic/severe combined immunodeficiency mouse model, likelyby inducing apoptosis. Apigenin did not inhibit survival ofprimary sympathetic neurons, suggesting that it is nottoxic to nontransformed cells. The mechanism of actionof apigenin seems to involve p53, as it increased thelevels of p53 and the p53-induced gene productsp21WAF1/CIP1 and Bax. Furthermore, apigenin (15–60Mmol/L) induced cell death and apoptosis of neuroblasto-ma cells expressing wild-type but not mutant p53.Apigenin increased caspase-3 activity and PARP cleav-age, and Z-VAD-FMK, a broad-spectrum caspase-3inhibitor, rescued NUB-7 cells from apigenin-mediatedapoptosis indicating that apigenin induced apoptosis in

a caspase-dependent manner. Overexpression of Bcl-XL

rescued NUB-7 from apigenin-induced cell death, sug-gesting that Bax activity is important for the action ofapigenin. Apigenin is thus a candidate therapeutic forneuroblastoma that likely acts by regulating a p53-Bax-caspase-3 apoptotic pathway. [Mol Cancer Ther2005;4(1):1–11]

IntroductionNeuroblastoma, a neuroectodermal tumor of the peripheralnervous system, is the most common extracranial solidtumor of childhood, accounting for 15% of pediatric cancer-related deaths (1). Neuroblastoma is thought to be derivedfrom embryonic neural crest cells (2, 3) that form theperipheral nervous system (4). Despite aggressive multi-modal therapy, this devastating tumor often acquires drugresistance and metastasizes (5). Therefore, our goal is toidentify agents that will promote apoptosis of neuroblas-toma cells without toxic effects to nontransformed cells.

One candidate agent is apigenin and related dietaryflavonoids, which have been shown to have antitumoreffects in several human adult tumor cell lines, includingthose derived from prostate, colon, and breast cancer(6–12). Apigenin also inhibits UV-induced tumor promo-tion (13), and decreases melanoma lung metastasis (14) inmouse models of melanoma. In addition, apigenin medi-ates the stabilization and transcriptional activation of thetumor suppressor p53 in keratinocytes (15), and inducesmorphologic differentiation and G1 arrest in a rat neuro-blastoma cell line (16). Apigenin is of particular interestas an anticancer agent because it exhibits low intrinsictoxicity and is not mutagenic compared with other struc-turally related flavonoids (17). The exact molecular targetsof apigenin and related flavonoids are currently unknown,but they have been shown to bind ATPase domains (18)and may act as kinase inhibitors (9, 11, 12, 19, 20). Othertargets may include heat shock proteins (21), telomerase(22), ornithine decarboxylase (23), and aryl hydrocarbonreceptor activity (24).

The goal of this study is to determine whether apigenin isapoptotic for neuroblastoma in vitro and in vivo, and todetermine whether it induces apoptosis by activatingcaspases and p53. Although p53 is frequently mutated inmany human cancers, it is wild-type in neuroblastoma butfunctionally inactive due to cytoplasmic sequestration(25, 26). We, along with others, have shown that nucleartranslocation and activation of p53 can be induced inneuroblastoma cells by various stimuli, including chemo-therapeutic agents (27–30). Nontoxic agents that stimulatep53 activity may be ideal candidates as therapeutics forneuroblastoma.

Received 9/14/04; revised 10/28/04; accepted 11/8/04.

Grant support: This study was supported by operating grants to H. Yegerfrom the Canadian Cancer Society, National Cancer Institute of Canada,the Canadian Institutes for Health Research, and the James BirrellNeuroblastoma Research Fund; and to D.R. Kaplan from the NationalCancer Institute of Canada; R. Torkin was supported by RESTRACOMfrom the Hospital for Sick Children Research Institute. D.R. Kaplan isa recipient of a Canada Research Chair.

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: Herman Yeger, Cancer Research Program,Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,Canada, M5G 1X8. Phone: 416-813-5958; Fax: 416-813-5974.E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

Molecular Cancer Therapeutics 1

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In this study, we report that apigenin inhibits tumorgrowth and induces caspase-dependent apoptosis inneuroblastoma cell lines in vitro and in a nonobesediabetic/severe combined immunodeficient (NOD/SCID)mouse xenograft model with NUB-7 s.c. tumors. We showthat the mechanism by which apigenin induces its anti-tumor effects is by activating caspases, concurrent withincreased expression of nuclear p53 and of the p53 targetgene products p21WAF1/CIP1 and Bax.

Materials andMethodsCell CultureNUB-7, developed in our laboratory, was established from

a primary neuroblastoma tumor (31) and has beenpreviously characterized (32). LAN-5 was kindly providedby Dr. R.C. Seeger (Department of Pediatrics, University ofCalifornia at Los Angeles School of Medicine, Los Angeles,CA). Both NUB-7 and LAN-5 are MYCN-amplified andrepresent high-risk, metastatic tumors (32, 33). SK-N-BE(2)(ATCC #CRL-2271) was derived from disseminated neuro-blastoma located in the bone marrow (34, 35). The celllines were cultured in a-MEM supplemented with fetalbovine serum, and penicillin/streptomycin at 37jC, 5%CO2. Cells were plated in T25 or T75 culture flasks (Falcon,Becton Dickinson, Oakville, ON, Canada), 24- or 6-wellplates (Costar, VWR CanLab, Mississauga, ON, Canada) or96-well black walled plates (Falcon) prior to treatment. Theprotocol for superior cervical ganglia (SCG) culture wasadapted from Ma et al. (36).

FlavonoidsCells were treated with apigenin (Sigma, St. Louis, MO),

quercetin, naringenin, diosmetin, chrysin, baicalein, andflavone (Indofine Chemical, Belle Mead, NJ) at indicatedconcentrations, or the equivalent volume of vehicle (DMSO).

AntibodiesPrimary antibodies were obtained as follows: anti-

phospho mitogen-activated protein kinase (Promega,Madison, WI), anti-Bcl-2, anti-Bcl-XL, anti-p53 (DOI), andanti-p53 (FL393; Santa Cruz Biotechnology, Santa Cruz,CA), anti-p53 (Ab-8) (Neomarkers, Montreal, PQ, Canada),anti-cleaved PARP (Asp214) and anti-cleaved caspase-3(Cell Signaling, Beverly, MA), anti-p21WAF1/CIP1 and anti-Bax (Upstate Biotechnology, Lake Placid, NY), anti-h actin(Sigma). Secondary antibodies: goat anti-mouse horse-radish peroxidase (Bio-Rad, Hercules, CA), goat anti-rabbithorseradish peroxidase (Chemicon International, Temecula,CA) and goat anti-rabbit Cy3 (Jackson ImmunoResearchLaboratories, West Grove, PA).

CellViabilityAssaysCell viability was determined using three methods: cell

death assay, survival assay, and terminal nucleotidyl trans-ferase (TdT)–mediated nick end labeling (TUNEL) assay.Cell Death Assay. Following indicated treatments, cells

were incubated with trypan blue (Sigma; final volumesec20% added to media) for 10 minutes, 37jC. Trypanblue–positive and total cells were counted per microscopefield, for a total of four fields per condition (a minimum of

3,000 cells were counted per condition). The proportion ofcell death was calculated by dividing the number of deadcells by total cell number/field. Alternatively, viable cellnumber was counted. The error bars correspond to SE.Results are representative of two or three experiments.Survival Assay. AlamarBlue (Biosource International,

Camarillo, CA), which measures mitochondrial activity,was used to quantitatively measure cell survival (protocolfrom Biosource). Preliminary experiments determined thatrelative fluorescence of AlamarBlue correlated with cellnumber (data not shown). Fluorescence was measured withexcitation at 540 nm, and emission at 590 nm, with a cutoffat 550 nm. The average percentage of survival is reportedwith its associated SE.TUNEL Assay. The protocol was adapted from Li et al.

(37). Cells were plated in 24-well plates and treated withvarying doses of apigenin for 24 hours. Floating cells werecollected and pelleted and added to the trypsinizedadhered cells. Cells were washed twice in PBS andcentrifuged onto glass cover slips by Cytospin (Shandon,Inc., Pittsburgh, PA). The cells were fixed in 1% parafor-maldehyde for 10 minutes and stored in PBS at 4jC.Nuclear DNA was denatured with a 2:1 mixture of ethanoland acetic acid for 10 minutes. The cells were washed twicein PBS for 5 minutes and incubated for 1 hour at 37jC in50AL of TdT reaction buffer containing: 1� concentratedbuffer solution, 2.5 mmol cobalt chloride, 12.5 units of TdT(Roche, Indianapolis, IN), and 0.25 nmol of ChromaTideBODIPY FL-14-dUTP (Molecular Probes, Eugene, OR) indistilled water. For control, TdT was substituted withdistilled water. The cells were washed twice with 15 mmolEDTA (pH 8.0), once with 0.1% Triton X-100 in PBS, andtwice with PBS. Cells were counterstained with 4V,6-diamidino-2-phenylindole (DAPI) and mounted in ProlongAntifade (Molecular Probes) mounting media. The slideswere examined under epifluorescence microscopy andphotographed on Kodak EL 135-36, ASA 400. A totalof six photographs were taken of each slide—three photo-graphs were taken under the UV filter to obtain a totalcell count with the DAPI counterstain, and a blue filterwas then used to photograph the BODIPY-stained cells.Total cells and TUNEL-positive cells were counted inthree photographed fields, and the experiment was donein duplicate.

Flavonoid Structure/Function AssayCells were incubated with varying concentrations (0–60

Amol/L) of flavonoid analogues for 48 hours, and cellsurvival was determined by AlamarBlue.

Effect of Serum ConcentrationCells were seeded in 96-well black walled plates (Falcon)

at 10,000 cells/well. Apigenin (0–100 Amol/L) in combi-nation with 2.5%, 5%, 10%, or 20% fetal bovine serum wasadded 24 hours after seeding. For the 6-day treatment, freshapigenin was added every other day. Cell survival wasmeasured by AlamarBlue.

Clonogenic AssayThe soft agarose method was used to measure colony

formation by neuroblastoma cell lines (31). Briefly, a bottom

Apigenin Induces Apoptosis of Neuroblastoma2




layer of agarose solution containing 1.5% agarose (FMCColloids, Rockland, MA) in a final concentration of 1�McCoy’s medium and various concentrations of apigeninwas poured into gridded 35 mm dishes (Nunclon, FisherScientific, Napean, ON, Canada) (1 mL/dish) and allowedto gel. The top layer contained trypsinized, counted cells,1.2% agarose, 1� McCoy’s medium, and apigenin. After 1to 2 weeks, the cultures were fixed with formalin andcolonies were scored under phase microscopy.

Fluorescence-Activated Cell Sorting AnalysisPropidium iodide, staining of DNA, and fluorescence-

activated cell sorting (FACS) were used to determine theeffect of apigenin on DNA content. The protocol wasadapted from Matteucci et al. (38). Propidium iodide fluo-rescence was used to determine DNA content. Cells wereanalyzed by flow cytometry with FACScan (Becton Dick-inson), acquisition software Cell Quest (Becton Dickinson),and the DNA curve fitting program Modfit LT (VeritySoftware, Becton Dickinson).

Xenograft MouseModelAll animal studies were approved by our institutional

animal care committee in accordance with accepted guide-lines for animal care and experimentation. NUB-7 (5 � 106

cells) were suspended in Matrigel (BD Biosciences, Bed-ford, MA) and injected s.c. in the scapular region of femaleNOD/SCID mice (five mice per condition). Tumors weregrown for 1 week. To prepare the test concentration of25 mg/kg, 25 mg apigenin was dissolved in 5% DMSO,10% PEG400, and 1% Tween 80, mixing well between eachaddition (adapted from ref. 14). Sterile PBS was added toa final volume of 10 mL with a working concentration of2.5 mg/mL. Apigenin (200 AL) or vehicle control was in-jected i.p. daily for 5 days. Mice were sacrificed and tumorsexcised and weighed. Tumor slices were dissociated to asingle cell suspension by trypsinization, gentle vortexingand trituration, and stained with propidium iodide andanalyzed by FACS.

Caspase-3 ActivityAssayCells were treated with apigenin for 24 hours in 96-well

plates. Caspase-3 activity assay was adapted from Carrascoet al. (39). Briefly, caspase assay buffer containing a caspase-3-specific cleavage substrate linked to a fluorophore (Ac-DEVD-AMC; Biomol, Plymouth Meeting, PA) was addedto each well. Staurosporine (Biomol; 60 Amol/L, 4 hours)was used for positive control and the broad-spectrumcaspase inhibitor Z-VAD-FMK (Biomol; 100 nmol/L,24 hours) was used for negative control. Caspase-3 activitywas quantified by AMC fluorescence read every 2 minutesfor 90 minutes. Activity was normalized to the untreatedcontrol.

Bcl-XL OverexpressionNUB-7 cells were trypsinized, counted, and plated at

200,000 cells/well in six-well plates coated with poly-D-lysine (Sigma). Sixteen hours later, cells were infectedwith Bcl-XL Ad5 (multiplicity of infection, 50) or GFP Ad5(multiplicity of infection, 5) adenoviruses that expresscytomegalovirus promoter–driven Bcl-XL or GFP (Aegera,

Iles-Des-Soeurs, PQ, Canada). Twenty-four hours post-infection, cells were treated with 60 Amol/L apigenin fora subsequent 24 hours. Bcl-XL and GFP expression wasconfirmed by Western blotting and cell death wasmeasured by trypan blue exclusion assay.

Western Blot AnalysisCells were treated or infected as above, and lysed in

lysis buffer (Cell Signaling) with added protease inhibitorcocktail (Roche, Mannheim, Germany). Cell lysates werequantified by bicinchoninic acid protein assay kit (Pierce,Brockville, ON, Canada). Equalized lysates were denaturedin sample loading buffer [1.67% SDS, 8.3% glycerol, 1.3%DTT (Sigma), trace bromophenol blue (Fisher)], boiled andrun on 7% to 15% gradient SDS-PAGE. Protein was trans-ferred to nitrocellulose membrane, and the membranewas blocked in 5% bovine serum albumin (Calbiochem,VWR Can Lab)/TBS-T [TBS with 0.2% Tween-20 (Fisher)]for antibodies against phosphorylated proteins, or 5%skim milk powder (Carnation)/TBS-T for other antibodies,for 1 hour at room temperature. Primary antibodies werediluted in TBS-T and incubated with the membrane over-night at 4jC. Following washes, membranes were incubatedin secondary antibodies diluted in TBS-T for 1 hour at roomtemperature. Antibodies were visualized with enhancedchemiluminescence kit (Amersham Biosciences, Piscat-away, NJ). For reprobes, membranes were stripped with2% SDS, 62 mmol Tris (pH 6.8), 0.7% h-mercaptoethanol for15 minutes at 55jC, rinsed in TBS, blocked and reprobed.Densitometry analysis was done with Total Lab software(Amersham Biosciences).

Immunofluorescence StainingCells were plated in two-well chamber slides coated with

poly-D-lysine (Sigma), treated with 60 Amol/L apigenin forindicated times and fixed for 15 minutes in ice-cold 4%paraformaldehyde. Cells were washed thrice in HBSS,permeabilized for 5 minutes with 0.5% NP40, washed fourtimes in HBSS and blocked in 3% normal goat serum for1 hour. Cells were incubated with rabbit anti-p53 (FL393)overnight at 4jC and washed four times. Results wereconfirmed with an additional p53 antibody, Ab-8 (datanot shown). Primary antibodies were detected using goatanti-rabbit Cy3-coupled secondary antibody and nucleiwere stained for 2 minutes with Hoechst 33258 (Sigma).Immunostained cells were photographed using constantexposure time with a 100� lens and NortonEclipse soft-ware version 6.1.

Statistical AnalysisGraphing, and statistical analysis by ANOVA and t test

were done with GraphPad Prism software.

ResultsApigenin Inhibits the Survival of Neuroblastoma Cell

LinesPrevious reports have shown that structurally, the C2-

C3 double bond and the 4V-hydroxy group on the flavonediphenyl ring of flavonoids correlate with increasedgrowth-inhibitory effects in tumor cell line models





(6, 14; Fig. 1A). We tested whether these structuralcharacteristics of flavonoids are implicated in inhibitionof growth and/or survival of human neuroblastoma. NUB-7 and LAN-5, two MYCNamplified,wild-type p53,humanneuroblastomacelllines,thatrepresenttheI-type(intermediate)and N-type (neuronal) morphology, respectively, and themetastatic, high stage phenotypes (40), were treated for24 hours with a series of structural analogues of apigeninincluding: naringenin, which differs from apigenin by thelack of the C2-C3 double bond, diosmetin which has a 4V-methoxy group, baicalein and chrysin, which lack the 4V-substitution, quercetin that has an additional 3VOH group,and flavone (unsubstituted; Fig. 1A). The analogues wereranked as follows, from the strongest to weakest inhibitorof cell survival in NUB-7: apigenin, quercetin, diosmetin,chrysin > baicalein > flavone > naringenin (Fig. 1B). Atthe concentrations tested, naringenin had no effect oncell survival (Fig. 1B). Similar results were observed inLAN-5 (data not shown). Our data was in agreementwith previous reports indicating that the presence of theC2-C3 double bond and (to a lesser extent in our model)the 4V-OH substitution group are important structuralrequirements for inhibition of melanoma cell survival(6, 14). Although quercetin, diosmetin, and chrysin hadsimilar inhibitory potencies as apigenin, apigenin waschosen for further study as it is prevalent in the diet andhas low intrinsic cytotoxicity (17).

We next tested apigenin for dose-dependent effects oncell survival. Apigenin (24 hours) inhibited cell viability in adose-dependent manner in human neuroblastoma cell lineswith an EC50 = 35 Amol/L in NUB-7, and EC50 = 22 Amol/Lin LAN-5 as determined by trypan blue exclusion assay(Fig. 2A). The dose-dependent effect was also observed byAlamarBlue survival assay (Fig. 2C).

An important factor that might influence the survivalresponse of cells to a particular drug is the method ofculture growth. Cells grown in monolayer culture are oftenmore sensitive to death-inducing agents compared withcells grown as colonies in three dimensions (41). Therefore,we asked whether apigenin could inhibit neuroblastomacolony formation in an agarose culture model. Cells wereseeded in agarose that was premixed with 5 to 60 Amol/Lapigenin or vehicle control, and colonies were counted after2 weeks. Apigenin induced a dose-dependent decrease incolony number from f25% at 5 Amol/L to 100% at60 Amol/L relative to control in NUB-7 and by f30% at5 Amol/L and 100% at 60 Amol/L in LAN-5 (Fig. 2B). Takentogether, the monolayer and colony assay results showthat apigenin effectively induced dose-dependent reduc-tion in the survival of these neuroblastoma cell lines. Inaddition, in monolayer culture, increasing serum con-centration in the culture medium partially rescued NUB-7cells from cell death over long-term treatment (6 days;Fig. 2C). However, the presence of high serum concentra-tion (20%) did not completely protect these cells fromapigenin-induced death (Fig. 2C). This suggests that arelatively high concentration of serum is not sufficientto block apigenin from inducing cell death, however, it

does mediate moderate resistance to cell killing. We there-fore chose 10% fetal bovine serum for all following ex-periments to facilitate the study of apoptotic mechanismsin this model.

Apigenin ReducesTumor Mass in a Mouse XenograftModel of Neuroblastoma

Our data indicated that apigenin inhibited in vitro sur-vival of neuroblastoma cell lines. We therefore asked ifapigenin inhibited neuroblastoma survival in a NOD/SCIDxenograft model. Initial trials indicated that mice bearingscapular NUB-7 tumors treated with 25 mg/kg apigeninfor 5 days did not display overt toxicity relative to un-treated mice, as determined by monitoring mice for signsof morbidity, weight gain, and by histopathologic exami-nation of major organs. Mean tumor mass in the treatedgroup decreased by 50% (Table 1). Portions of the excisedtumors were dissociated, stained with propidium iodide,and analyzed by FACS and showed a significant increase

Figure 1. A, the structure of apigenin. Analogues of flavone have sub-stitutions (OH, OCH3) at various positions of the carbon ring structure. B,presence of the C2-C3 double bond and the 4V-OH group correlate withgrowth inhibitory effects in NUB-7. Cells were cultured in 24-well platesand treated with flavonoids for 2 d. Cell survival was quantified byAlamarBlue. Each condition was tested in quadruplicate and the results arerepresentative of two independent experiments. Bars, SE.





in the subdiploid fraction of tumors from apigenin-treatedmice (Table 1). This preliminary evaluation of apigeninin an in vivo model shows that tumors from treated micewere substantially smaller than control, and that thesetumors had significantly increased apoptosis, suggestingthat apigenin affected tumor cell survival at a well-tolerated dose.

Apigenin Does Not Induce the Death of Nontrans-formed Primary Cells

Having established that apigenin has a potent effect onneuroblastoma tumor cell growth, we sought to determinewhether apigenin-induced death is specific to neuroblasto-ma cells. We therefore asked whether apigenin inhibitssurvival of nontransformed cells. Because neuroblastomacells are derived from the sympathoadrenal precursorlineage of the embryonic neural crest, we tested the effect ofapigenin on differentiated peripheral sympathetic neuronsisolated from primary cultures of rat SCG cells. Treatmentfor 24 hours with 15 to 120 Amol/L apigenin, which in-hibited the survival of neuroblastoma cells, did not affectthe survival of SCG cells, as determined by AlamarBluesurvival assay (Table 2). These results show that apigeninspecifically induced dose-dependent death of neuroblas-toma cells without toxicity to nontransformed neuronalcells. Furthermore, induction of morphologic differentia-tion of NUB-7 with retinoic acid (32) inhibited apigenin-induced cell death, suggesting that apigenin preferentiallyinduces death of undifferentiated neuroblastoma cells (datanot shown).

Apigenin Mediates Cell Death by Induction of Apo-ptosis

To determine whether the observed apigenin-inducedcell death in neuroblastoma cell lines occurred viainduction of apoptosis, TUNEL and FACS analysis weredone. NUB-7 and LAN-5 cells were treated with 0 to 100Amol/L apigenin for 24 hours and double-stained withTUNEL and DAPI to calculate the percentage of TUNEL-positive cells per microscopic field. Cells treated with50 Amol/L apigenin had an 8% apoptotic fraction ascompared with 14% in 100 Amol/L and f1% in untreatedcells (Fig. 3A). This result is also supported by FACSanalysis of NUB-7 cells which showed that compared withcontrol, 60 Amol/L apigenin induced a 50% increase in the

Figure 2. A, apigenin induces dose-dependent cell death in neuroblas-toma cell lines. Cells were treated for 24 h and NUB-7 and LAN-5 celldeath was quantified by trypan blue exclusion. Each concentration wastested in triplicate and repeated thrice. Results are expressed as thepercent of viable cells relative to untreated control. *, significant differencefrom control, P < 0.05. Bars, SE. B, apigenin inhibits colony-formingability of NUB-7 and LAN-5. Cells were seeded in soft agarose F apigeninand cultured for 2 wks. Colonies were counted under phase contrastmicroscopy. The results represent three independent experiments. *,significant difference from control, P < 0.05. Bars, SE. C, serum concen-tration inversely correlates with apigenin-induced cell death. NUB-7 cellswere cultured in the 2.5% to 20% FBS, F10 to 100 Amol/L apigenin for6 d. Cell survival was quantified by AlamarBlue assay. D, apigenin doesnot induce cell death in SK-N-BE(2), a p53 mutant cell line. Cells weretreated with apigenin for 24 h and cell death was assessed by trypan blueexclusion assay. Each concentration was tested in quadruplicate, and theresults are representative of two independent experiments.

Table 1. Apigenin inhibited tumor growth and increased thenumber of apoptotic cells in NUB-7 xenograft tumors in aNOD/SCID mouse model of neuroblastoma

NUB-7 Xenograftmouse treatment group

Tumorweight (g),mean F SE

% Cells insubdiploid peak,

mean F SE

Vehicle control 1.986 F 0.172 7.519 F 1.014Apigenin* 1.092 F 0.22c 11.96 F 0.98c

*NOD/SCID mice bearing subcutaneous NUB-7 xenograft tumors weretreated with 25 mg/kg apigenin for 5 days.cSignificant difference from control, P < 0.05.





percentage of gated cells in the subdiploid peak (i.e.,apoptotic cells; data not shown).

Caspase-3 activation and PARP cleavage are character-istic indicators of apoptosis. Caspase-3 is a downstreameffector in the caspase cascade, activation of whichrepresents the apoptotic ‘‘point of no return’’ (42). First,to determine whether apigenin activated caspase-3, cellstreated with 15 to 60 Amol/L apigenin for 24 hours werelysed in the presence of Ac-DEVD-AMC that fluorescesupon specific cleavage by caspase-3. In NUB-7, caspase-3activity increased by 5-fold at 60 Amol/L apigenin after24 hours of treatment (Table 3). Staurosporine and Z-VAD-FMK, a broad-spectrum caspase inhibitor (43), wereused as positive and negative controls, respectively, forcaspase activation (Table 3). Second, we assessed PARPcleavage, a nuclear protein that is specifically cleaved byactivated caspases (44), by Western blot. In NUB-7 cells,cleaved PARP increased in a dose-dependent manner withapigenin treatment (15–60 Amol/L) after 24 hours (Fig. 3B).Naringenin, the structurally related, inactive analogue(Fig. 1A and B) did not induce PARP cleavage (Fig. 3B).Furthermore, cells that were pre-treated with Z-VAD-FMKfollowed by treatment with apigenin had no cleaved PARP.The trypan blue cell death assay also showed that Z-VAD-FMK significantly inhibited apigenin (15–120 Amol/L)–induced cell death (Fig. 3C). Taken together, these resultsshow that apigenin induced apoptosis in a dose-dependentmanner, and that apoptosis required caspase activation.

Apigenin Induces Apoptosis in a p53-MediatedManner

p53 is most commonly wild-type in neuroblastomatumors and cell lines, albeit inactive due to sequestrationin the cytoplasm (28). It has also been shown inneuroblastoma that cytoplasmic p53 can be induced totranslocate to the nucleus, where it activates gene tran-scription and apoptosis upstream from mitochondrialevents related to caspase activation (27, 28). We thereforeasked whether apigenin required wild-type p53 forinduction of caspase-mediated apoptosis. NUB-7 (wild-type p53,5 additional cytogenetic information available inref. 45) and SK-N-BE(2), a rare neuroblastoma cell lineexpressing a nonfunctional mutant p53 (6), were treated

with 60 Amol/L apigenin and lysed after 4, 8, 16, and24 hours, as this concentration of apigenin induced PARPcleavage at 24 hours post-treatment (Fig. 3B). Western blotanalysis of NUB-7 lysates showed that starting at 8 hourspost-treatment, the cleaved PARP product level rose andpeaked at 16 hours (Fig. 4A), suggesting caspase activationat or before 8 hours post-treatment. Consistent with p53being upstream of caspase activation, p53 levels increasedat 4 hours post-treatment, followed by the increase of twop53 target genes, p21WAF1/CIP1 and Bax (Fig. 4A). Immu-nofluorescence staining showed that in untreated NUB-7,p53 expression is low and sparsely distributed in the

Figure 3. A, apigenin induces apoptosis in NUB-7 and LAN-5. Cellswere treated with increasing concentrations of apigenin for 24 h. TUNELlabeling was performed in conjunction with a DAPI counterstain. Totalcells were counted in three fields photographed under a UV filter.Apoptotic cells were enumerated and expressed as a percentage of thetotal DAPI-positive cells per field. Bars, SE. B, apigenin induces PARPcleavage in a dose- and caspase-dependent manner. NUB-7 cells weretreated with 15 to 60 Amol/L apigenin, 60 Amol/L naringenin or 100 nmol/LZ-VAD-FMK F60 Amol/L apigenin. Staurosporine (60 Amol/L, 4 h) servedas a positive control for PARP cleavage. The cleaved form of PARP wasanalyzed by Western blot with an antibody against cleaved PARP. C,apigenin induces cell death in a caspase-dependent manner. NUB-7 cellswere pretreated with Z-VAD-FMK (100 nmol/L) for 24 h prior to addition ofapigenin for 24 h. Cell survival was quantified by trypan blue assay andexpressed as dead/total cells counted per field �100. Each condition wasquantified in quadruplicate. P < 0.05. Bars, SE.

Table 2. Apigenin does not inhibit survival of nontransformedrat SCG

Apigenin (Amol/L) AlamarBlue relative fluorescence*

15 102.57 F 0.87630 102.241 F 0.99860 97.62 F 3.3209120 97.884 F 1.857

NOTE: Rat SCG cells were treated with apigenin for 24 hours and cellsurvival was quantified by AlamarBlue.*Normalized to untreated control F SE.

5 Dr. J. Squire, personal communication.





cytoplasm and nucleus, but seems to be excluded from thenucleolus (Fig. 4B). In contrast, NUB-7 cells treated for 4hours with 60 Amol/L apigenin expressed high levels ofp53 relative to control, with most of the protein locatedthroughout the nucleus. This suggests that apigeninmediated nuclear accumulation of p53, where it activatedtranscription of p21WAF1/CIP1 and Bax. Results were con-firmed with an additional p53 antibody, Ab-8 (data notshown). We then asked if wild-type p53 was required forapigenin-induced apoptosis. In apigenin-treated SK-N-BE(2), caspase-3 was not activated as in NUB-7 (Table 3),and there was no PARP cleavage (Fig. 4C). In addition,apigenin did not induce increased p21WAF1/CIP1 or Baxlevels in SK-N-BE(2) (Fig. 4C). Apigenin (15–60 Amol/L) didnot induce overt cell death of SK-N-BE(2), the mutantp53 cell line (Fig. 2D); there were small increases in death at60 Amol/L by f20% and at 120 Amol/L by f25% whichmight indicate that at higher concentrations, apigenininduces death by a p53-independent mechanism. How-ever, the lack of caspase activity and PARP cleavage inSK-N-BE(2) at 60 Amol/L suggests that apigenin requiredwild-type p53 for induction of apoptosis.

We also observed that after 4 hours, apigenin induced anincrease in extracellular signal-regulated kinase (ERK)activity, as assessed by using an antibody to phosphory-lated and activated ERK (Fig. 4A), suggesting that apigeninrequires activated ERK for apoptosis. To test this, NUB-7cells were pre-treated with the selective ERK inhibitor,U0126, at 20 Amol/L, a concentration that inhibited ERKphosphorylation, for 4 hours and cell viability wasassessed. The presence of the ERK inhibitor did not rescuethe cells from apigenin-induced cell death (data notshown), indicating that apigenin does not require ERKactivity to induce apoptosis.

Overexpression of Bcl-XL Rescues NeuroblastomaCells from Apigenin-Induced Apoptosis

The mitochondrial apoptotic pathway is activated bychanges in the ratio of proapoptotic versus antiapoptoticproteins of the Bcl-2 family such as Bax and Bcl-2/Bcl-XL,

respectively. Therefore, we examined the Bax/Bcl-XL ratioby densitometry as an indicator of the apoptotic/antiapoptotic balance (Fig. 4A). Bax, but not Bcl-XL, beganto increase at 8 hours and reached a maximum level of3.5-fold from baseline at 16 hours. Comparatively, Bcl-XL

protein transiently increased to a maximum level only by 2-fold at 16 hours post-treatment, possibly as a compensatorysurvival response. Therefore, the late increase in Bcl-XL

might have been insufficient to inhibit the effects of Bax oncytochrome c release by and the initiation of the caspasecascade (46).

Expression of Bcl-2 and Bcl-XL in neuroblastoma cells hasbeen shown to suppress chemotherapy-induced apoptosis(47–49). Therefore, we hypothesized that overexpression ofBcl-XL would rescue NUB-7 cells from apigenin-inducedapoptosis. To test this, we infected NUB-7 cells with anadenovirus construct encoding Bcl-XL for 24 hours,followed by treatment with 60 Amol/L apigenin. Bcl-XL

overexpression inhibited the cleavage of both caspase-3and PARP as determined by Western blot (Fig. 5A),indicating that Bcl-XL overexpression was sufficient torescue cells from induction of apoptosis. Bcl-XL over-expression had no effect on p53 or p21WAF1/CIP1 proteinlevels, suggesting that the effects of Bcl-XL suppressedapoptosis downstream of p53. Furthermore, apigenininduced a shift in hyperphosphorylated to hypophosphory-lated pRb, which was not affected by Bcl-XL overexpression.Finally, overexpression of Bcl-XL inhibited apigenin-induced cell death as determined by trypan blue survivalassay (Fig. 5B), indicating that the activity of BH3-containing Bcl-2 family members such as BAX are requiredfor apigenin-induced cell death.

DiscussionIn this study, we show that apigenin, a nonmutagenicantitumor flavonoid, exhibits tumor cell–specific effects onsurvival of neuroblastoma both in vitro and in vivo. Weprovide the first evidence that apigenin given to mice withneuroblastoma xenograft tumors reduced tumor masspossibly by induction of apoptosis. Subcutaneous xenografttumor drug testing models are limited to measurement ofgrowth-inhibitory effects of the drug in question, whereasorthotopic models provide information on effects of a drugon growth in a more clinically relevant environment andprovide a model for assessment of metastatic effects (50).Because this apoptotic effect was limited to neoplastic cellswith no evident toxicity to normal tissue, apigeninrepresents a potential compound for further developmentas a neuroblastoma therapeutic agent. In this regard,another flavonoid, flavopiridol, a semisynthetic polyhy-droxylated flavone, exhibits in vitro activity and is currentlyin clinical trials as an antitumor agent for various cancers(51, 52). The mechanism whereby apigenin acts seemsto involve p53; apigenin modulated p53 protein levelsand accumulation in the nucleus, and induced theexpression of the p53 target proteins p21WAF1/CIP1 andBax. Thus, neuroblastoma tumors, which in virtually all

Table 3. The effects of apigenin (15–60 Amol/L) on caspase-3activity in NUB-7 and SK-N-BE(2)

Treatment Caspase-3 activity*

NUB-7 SK-N-BE(2)

15 Amol/L AP 1.02 F 0.02 2.03 F 0.1230 Amol/L AP 1.48 F 0.27 1.46 F 0.3360 Amol/L AP 4.91 F 1.23 2.32 F 0.1760 Amol/L NA 0.83 F 0.11 1.30 F 0.64Staurosporine 9.59 F 4.26 5.75 F 0.75Z-VAD-FMK 0.17 F 1.22 0.10 F 0.35

NOTE: Cells were treated with apigenin (AP), naringenin (NA), staur-osporine (positive control), and Z-VAD-FMK (broad-spectrum caspaseinhibitor).*Numbers represent relative fluorescence resulting from caspase-mediatedspecific cleavage of the fluorogenic substrate Ac-DEVD-AMC, normalizedto control F SE.





cases examined express wild-type p53 that is inactivepossibly due to sequestration in the cytoplasm (26, 28, 53,54), can be chemically targeted by apigenin for activationresulting in apoptosis.

It has been shown that the in vitro growth inhibitoryeffects of flavonoids correlate with variations in chemicalstructure (6, 19). Flavonoids are characterized by a benzenering (A) fused with a pyrone ring (C), that carries a phenylring (B) in position 2 or 3 (see Fig. 1A). Previous studieshave shown that the C ring with oxo function at position 4,and C2-C3 double bond correlate with antiproliferativeactivity (6, 55). The presence of the C2-C3 double bondstrongly correlated with the anti-survival effect in NUB-7and LAN-5. Naringenin, which is identical to apigenin butlacks the C2-C3 double bond, had no effects on survivalor PARP cleavage, further suggesting that the presence

of the C2-C3 double bond is necessary for activation ofapoptosis. In addition, the presence of hydroxy or methoxysubstitutions on the flavone ring structure have been shownto correlate with increased inhibitory activity (6, 7, 18, 19,56 – 58), as also shown here by the lack of growth-inhibitory activity of nonhydroxylated flavone and thestrong anti-survival effects of hydroxylated apigenin,quercetin, diosmetin, and chrysin. Structural characteristicsof flavonoids may also contribute to their modulatoryeffects on P-glycoprotein (18), protein kinase C (56),phosphatidylinositol 3-kinase (19), cyclin-dependentkinases 1 and 2 (6), mitogen-activated protein kinase(20), and tyrosine kinases (e.g., EGF-R and Her2/neu;refs. 11, 19). A recent study reported that aminoflavone,a compound based on the flavone parent structure ofapigenin (Fig. 1A), had potent antiproliferative effects in

Figure 4. A, apigenin induces temporal changes in apoptotic andsurvival protein expression in NUB-7 (wild-type p53), but not in SK-N-BE(2) (mutant p53). NUB-7 cells were treated with 60 Amol/L apigeninand protein was extracted at 4, 6, 8, 16, and 24 h. Protein wasseparated by SDS-PAGE and analyzed by Western blot for cleavedPARP, phospho-ERK, p53, p21WAF1/CIP1, Bax, Bcl-XL, and h-actin(loading control). B, apigenin mediates nuclear accumulation of p53in NUB-7. NUB-7 were treated with 60 Amol/L apigenin followed byimmunostaining with anti-p53 and Hoechst nuclear stain. Cells werephotographed under 100� magnification, constant exposure time of1 s. C, SK-N-BE(2) cells were treated with 60 Amol/L apigenin for 4, 8,16, and 24 h and protein extracts were analyzed by Western blot forcleaved PARP, p53, p21, Bax, and h-actin (loading control).





a breast cancer cell line by activation of aryl hydrocarbonreceptor signaling (59). Thus, modifications to the chemi-cal structure might modulate the antitumor activity andpotency of apigenin.

The exact molecular targets of apigenin and relatedflavonoids are currently unknown. Many studies (withsome exceptions refs. 11, 18, 56) showed that cells treated inculture respond by modulation of kinase activity, but didnot show direct binding to suspected kinase targets viakinase assays done with lysates from untreated cells.Therefore, the observed effects on kinase activity mighthave been due to an alternate mechanism governinginhibition of cell growth and survival, and not to directkinase inhibition because the protein lysates tested werefrom treated cells that were undergoing death. Wehave determined that apigenin did not directly inhibitAKT by in vitro kinase assays and by Western blot forphosphorylated, active AKT, or tyrosine kinase activity inNUB-7.6 We did observe that the presence of serum in the

culture medium decreased the sensitivity of NUB-7 andLAN-5 to the anti-survival effects of apigenin, suggestingthat factors in serum mediate resistance to apigenin inneuroblastoma cells, possibly by activation of survivalsignaling pathways. For example, growth factor signaltransduction through the IGF1/IGF1R/PI3K/AKT axis hasbeen shown to be essential for neuroblastoma survival(60–62), but does not seem to be targeted by apigenin. Analternative mechanism besides kinase inhibition that maybe targeted by apigenin is inhibition of survivin, an‘‘inhibitor of apoptosis’’ protein whose expression inneuroblastoma tumor cells correlates with the clinicallyaggressive phenotype (63). Silibinin, a flavolignan compo-nent of milk-thistle, inhibited survivin mRNA and proteinexpression in a bladder cancer cell line (64). Further studieswill be required to elucidate the precise targets of apigeninin neuroblastoma cells, in particular with regard to theactivation of p53-mediated apoptosis.

We show that apigenin induced apoptosis via up-regulation of p53 possibly by increasing p53 protein stabilityand/or transcription. Apigenin had similar effects on p53and Bax in human prostate cancer cells (65), which supportsour findings. However, our study presents novel functionaldata (that was not presented in the study by Gupta et al. 65)that the presence of mutant p53 or overexpression of Bcl-XL

rescues from apigenin-induced apoptosis. In keratinocytes,apigenin has been shown to stabilize p53 protein bydisrupting its interaction with mdm2, which targets p53for ubiquitination, without modulating p53 mRNA levels(15). In contrast, preliminary real-time PCR studiesconducted on NUB-7 cells treated with apigenin showedthat p53 mRNA increased at 1 to 4 hours post-treatment.7

However, the mechanism of apigenin-mediated up-regulation and activation of and/or dependence on p53is not yet confirmed and requires further investigation.The effects of apigenin in isogenic cell line pairs, such aswild-type and mutant p53 colorectal carcinoma cell lines(66), may suggest an additional application for apigeninin other tumor types with functional p53.

In addition to the effects on p53, apigenin mediated adecrease in hyperphosphorylated pRb (Fig. 5), therebyinhibiting E2F-mediated cell cycle progression (67). Thehyperphosphorylation of pRb, together with the apigenin-induced increase in the level of the cyclin-dependentkinase inhibitor p21WAF1/CIP1 likely leads to cell cyclearrest. The unscheduled cell cycle arrest in tumor cells thatare constitutively cycling could account for the apoptoticactions of apigenin.

Plant-derived anticancer agents have provided novelprototypes for drug design (68, 69). However, theconcentration of apigenin (60 Amol/L) used to induceovert apoptosis in our study may not be physiologicallyachievable in humans. Because concentrations that induceantitumor effects in vitro and in vivo often differ, futureexperiments need to delineate the specific plasma and

Figure 5. A, overexpression of Bcl-XL in NUB-7 rescues from apigenin-induced death. NUB-7 cells were infected with an adenovirus containingBcl-XL, GFP, or were not infected and treated with apigenin for 24 h.Western blot for cleaved PARP, cleaved caspase-3, p53, p21waf1/cip1, pRb,and h-actin (loading control). B, Bcl-XL overexpressing NUB-7 cells weretreated with apigenin for 24 h. Cell death was quantified by trypanblue exclusion.

7 R. Torkin, S. Der, and H. Yeger, unpublished data.6 R. Torkin and H. Yeger, unpublished data.





intratumoral levels in order to directly correlate reductionin tumor mass with in vivo concentrations of apigenin.

Lower concentrations (<20 Amol/L) also induced apo-ptosis albeit with slower kinetics. Nevertheless, diets highin fruits and vegetables can lead to consumption of > 1 g offlavonoids daily, possibly leading to tissue accumulation(70). Our in vivo mouse study showed that apigenindecreased tumor mass and increased the apoptotic fractionin xenografted tumors, suggesting that apigenin is biolog-ically active after absorption and distribution, possibly viablood circulation. Several studies suggest that flavonoidshave limited bioavailability and are metabolized extensivelyupon ingestion (71). However, epidemiologic data showsthat diets rich in flavonoids correlate with decreasedincidence of certain cancers, suggesting that ingestedflavonoids retain biological activity against tumors andare chemopreventive (69, 71, 72). A recent study showedthat epigallocatechin gallate, a polyphenolic compoundrelated to apigenin, underwent structural rearrangements(autooxidation, oxidative addition, and/or condensation)after brief incubation in human or mouse plasma (pH 6.8).The resulting new structures had dramatically increasedbiological activity, including inhibition of telomeraseactivity (22). This finding suggests that although the parentflavonoid structures might not be detectable in plasma,biologically active breakdown products might achieve highconcentrations and accumulate in tissues. It also suggeststhat combinations of active agents, due to variable intake ofplant-based flavonoids, might yield effective antitumoractivity. Identification of the active products would presentavenues for development of flavonoid-derived antitumordrugs having increased potency and activity at lower andphysiologically achievable doses.

Acknowledgments

We thank Dr. Sandy Der for technical advice, Dr. Olivia Furstoss for SCGcell cultures, Christine Laliberte for technical assistance, Dr. Bikul Das forassistance with the animal study, and Albert Chan for the colony assay.

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2005;4:1-11. Mol Cancer Ther Risa Torkin, Jean-François Lavoie, David R. Kaplan, et al. apigenin in human neuroblastomaInduction of caspase-dependent, p53-mediated apoptosis by

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