Cardnogenesis vol.17 no.ll pp.2367-2375, 1996

The chemopreventive flavonoid apigenin induces G2/M arrest inkeratinocytes

Denise M.Lepley1, Boyong Li1, Diane F.Birt1 andJill C.Pelling^3

'University of Nebraska Medical Center, Eppley Institute for CancerResearch, Omaha, NE 68198 and 2University of Kansas Medical Center,Department of Pathology and Laboratory Medicine, Kansas City, KS 66160,USA

•'To whom correspondence should be addressed

Apigenin is a plant flavonoid which has been shown tosignificantly inhibit UV-induced mouse skin tumorigenesiswhen applied topically, and may represent an alternativesunscreen agent in humans. We have investigated themolecular mechanism(s) by which apigenin inhibits skintumorigenesis. Initial studies examined the effects of api-genin on the cell cycle. DNA flow cytometric analysisindicated that culturing cells for 24 h in medium containingapigenin induced a G2/M arrest in two mouse skin derivedcell lines, C50 and 308, as well as in human HL-60 cells.The G2/M arrest was fully reversible after an additional24 h in medium without apigenin. We investigated theeffects of apigenin on cyclin Bl and p34cdc2, since cyclinBl/p34cdc2 complexes regulate G2/M progression. Westernblot and immune complex kinase assays using whole celllysates from 308 and C50 cells treated for 24 h with0—70 (iM doses of apigenin demonstrated that apigenintreatment did not change the steady-state level of pM"102

protein, but did inhibit p34edc2 HI kinase activity in 308cells. Western blot analysis showed that apigenin treatmentof C50 cells and 308 cells inhibited the accumulation ofcyclin Bl protein in a dose-dependent manner. The apigeninlevels detected in cultured keratinocytes were relevant tothose detected in epidermal cells of Sencar mice treatedwith tumor inhibitory doses of apigenin. In conclusion, wepresent evidence that apigenin induces a reversible G2/Marrest in cultured keratinocytes, the mechanism of whichis in part due to inhibition of the mitotic kinase activity ofp34cdc2, and perturbation of cyclin Bl levels.

Introduction

Apigenin is a natural, plant flavonoid present in the leavesand stems of vascular plants, including fruits and vegetables(1). It is relatively nontoxic and nonmutagenic in comparisonwith other flavonoids such as quercetin. Apigenin and otherflavonoids are strong antioxidants which exert a number ofeffects on cell biology (1). A classic example is their abilityto scavenge free radicals, which are involved in cell damageand tumor promotion (2). As a member of the flavonoid family,apigenin is present in foods associated with a decreased risk

•Abbreviations: CHO, Chinese hamster ovary; TLB, triton lysis buffer; ICK,immune complex kinase assay; DMSO, dimethyl sulfoxide; DMBA, 7,12-dimcthylbenz[a]anthracene; TPA, 12-0-tetradecanoylphorbol-13-acetate; Thr,threonine; Tyr, tyrosine; FBS, fetal bovine serum; EMEM, Earle's modifiedeagle's medium; RPMI, Roswell Park Memorial Institute; SDS, sodiumdodccyl sulfate; PVDF, polyvinylidene difluoride; HI, histone HI.

(0 Oxford University Press

of cancer, and flavonoids are believed to play an importantrole in delaying or preventing carcinogenesis, although theirmechanism(s) of action is not fully understood.

The chemopreventive aspects of apigenin have been investi-gated both in vitro and in vivo (3-5). Apigenin has been shownto be antimutagenic against nitropyrene-induced genotoxicityin Chinese hamster ovary (CHO*) cells in vitro (3). In addition,Wei et al. demonstrated that apigenin is a strong inhibitor ofornithine decarboxylase activity, a marker of tumor promotion,induced by TPA in epidermal cells (4). The flavonoid apigeninhas also been shown to increase gap junctions in rat liverepithelial cells, a property which may contribute to itschemopreventive ability by maintaining cell to cell communica-tion (5).

In vivo studies carried out in our laboratory using themouse skin two-stage carcinogenesis model initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted with 12-0-tetradecanoylphorbol-13-acetate (TPA), demonstrated thattopical application of apigenin significantly reduced the numberof benign papillomas and squamous cell carcinomas in SENCARmice (4). We also demonstrated that apigenin was equallyeffective at reducing UV-B radiation-induced squamous cellcarcinomas in hairless SKH mice (Birt et al., submitted).

The antitumor properties of other flavonoids have beenstudied with respect to apoptosis and the cell cycle. Quercetinand genistein caused apoptosis when cultured with severalmyeloid and lymphoid cell lines (6-8). Other flavonoids havebeen found to perturb the G\ and G2 phases of the cell cycle.A recent report by Sato et al. demonstrated that apigenincaused a G2/M arrest and induced differentiation in rat neuronalcells (9). Genistein, an isoconformer of apigenin, was shownto cause a G2/M arrest in several human and murine cell lines(6-8,10). Quercetin has been demonstrated to inhibit cell cycleprogression at the G r S phase boundary in a human gastriccancer cell line HGC-27 (11). The synthetic flavone L86-8275was also shown to block cell cycle progression at either G| orG2 phase depending on the method used to synchronize thecell culture (12). Questions as to the mechanism of cell cyclearrest by flavonoids and DNA damaging agents remain largelyunanswered, but appear to involve multiple cell cycle regu-latory proteins.

Cyclin-cyclin dependent kinase (cdk) complexes regulatethe passage through the eukaryotic cell cycle checkpoints byinitiating a phosphorylation cascade leading to the transcriptionof genes needed for proliferation, as well as the phosphorylationof nuclear lamins and histones for nuclear membrane break-down and chromosome condensation, respectively (13). TheG2 to M phase progression is governed by the cyclin B-p34cdc2

complex (14). Cyclin B is expressed during late S and G2

phase and immediately binds to p34cdc2. P34cdc2 is activated,in part, by binding to cyclin B and by phosphorylation ofp34cdc2 at Thrl61 by the cdk-activating kinase CAK (15).Cyclin B-p34cdc2 complexes are negatively regulated until Mphase by phosphorylation of Thrl4 and Tyrl5 (the latter

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residue being phosphorylated by Weel kinase) in the ATPbinding domain (16). At the onset of M phase, the complexis translocated from the cytoplasm to the nucleus in concertwith an abrupt increase in HI kinase activity by cyclinB-p34cdc2 due to the rapid dephosphorylation of Thrl4 andTyrl5 by the phosphatase cdc25 (17,18). Inactivation of cyclinB-p34cdc2 is required for exit from mitosis and results fromubiquitin-mediated degradation of cyclin B (19).

Based upon our in vivo studies demonstrating that apigeninwas an effective and potent inhibitor of both chemical andUV-induced skin tumorigenesis, we were interested in elucidat-ing the molecular mechanism(s) of apigenin's chemopreventiveactivity. In view of previous reports by other laboratories thatvarious flavonoids can perturb the cell cycle, we began byinvestigating the affects of apigenin on the keratinocyte cellcycle and some of the regulatory proteins involved in the G2to M phase progression.

Materials and methods

Cell culture

C50 cells are an immortalized murine keratinocyte cell line (20). The 308keratinocyte cell line contains an activated Ha-ras oncogene and formspapillomas upon engraftment in nude mice (21). C50 and 308 cells werecultured in 0.05 mM Ca2+ EMEM medium with 8% chelexed fetal bovineserum (FBS). HL-60 human myelogenous leukemia cells were cultured inRPMI-1640 (Gibco, Grand Island, NY) containing 10% FBS.

Cell cycle analysis

Asynchronous C50 and 308 cells were cultured in either 60 or 100 mm dishesto 50% confiuency at which time the culture medium was replaced and analiquot of apigenin in DMSO was added. Stock solutions of apigenin orgenistein (Sigma, St Louis, MO) were dissolved in dimethyl sulfoxide (DMSO)and an aliquot was added to each culture dish to achieve the desired finalconcentration of apigenin. HL-60 cells were cultured in six-well dishes at adensity of 1-3X 105 cells/ml in fresh media supplemented with various dosesof apigenin (or genistein). Cells were cultured with apigenin or genistein fora minimum of 24 h at 37°C in 5% CO2. Control cultures received an equivalentamount of DMSO and it had no measurable effect on the cell cycle for allcell lines tested. After the allotted time in culture, C50 and 308 cells wereharvested by trypsin release and resuspended in 1 ml of modified Vindelov'sDNA staining solution (10 (ig/ml RNase A, 0.1% Nonidet P-40, 85 u.g/mlpropidium iodide, in PBS) 1-2 h prior to flow cytometric analysis. HL-60cells were collected by centrifugation at 900 rpm for 5 min and resuspendedin 1 ml Vindelov's solution. The fluorescence of 10 000 cells was measuredwith a Becton-Dickinson F A C " Plus flow cytometer with excitation at488 nm. The percentage of cells in Gl, S, and 62/M phase of the cell cyclewas determined using the CellHt Cell-Cycle Analysis computer programversion 2.01.2.

Western blot analysis

C50 and 308 cells at 50% confluency were cultured in EMEM containingvarious doses of apigenin (or DMSO alone) for 24 h with each dish containingthe same total amount of DMSO (<0.6% v/v). After 24 h in culture, cellswere harvested in 0.4 ml Triton Lysis Buffer (TLB) (20 mM Tns pH 7.4,137 mM NaCl, 25 mM (J-glycerophosphate, 2 mM EDTA, 1 mM Na3VO4,1% triton X-100, 10% glycerol, 1 mM PMSF, 5 ug/ml leupeptin, 5 ug/mlaprotinin, 2 mM benzamidine, and 0.5 mM DTT) using a cell scraper. Wholecell lysates were incubated on ice for 15 min before clearing the insolublematerial by centrifugation at maximum speed in a microcentrifuge for 10 min,at 4°C. Protein concentration of supematants was determined using a modifiedLowry assay (Biorad). One hundred micrograms of whole cell extract waselectrophoresed on 12% SDS-PAGE gels and transferred in 25 mM Tris,192 mM glycine, 15% methanol, 0.02% SDS to polyvinylidene difluoride(PVDF) membrane (Immobilon-P, Millipore) at 4°C. Rainbow molecularweight markers (Amersham) were visible on the membrane after transfer.Membranes were cut into two pieces horizontally at the 46 kD mark andsubsequent steps performed separately. Nonspecific binding sites were blockedfor 1 h or overnight in blocking buffer (10 mM Tris pH 7.5, 100 mM NaCl,0.1% Tween 20, and 1% Bovine serum albumin [BSA]). The lower half ofmembranes containing proteins <46 kD were first incubated with a 1:1000dilution of rabbit, polyclonal anti-p34cdc2 (generously provided by Dr R.Lock)in blocking buffer for 3 h at room temperature. The top portion of membranes

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was incubated with a mouse monoclonal anti-Cyclin Bl antibody (Santa Cruz)at a 1:300 dilution in blocking buffer for 1 h at room temperature. Unboundprimary antibody was removed with six washes of 5 rain each with washbuffer (blocking buffer minus BSA). Membranes were incubated for 1 h atroom temperature with secondary antibody [horse radish peroxidase conjugatedgoat anti-rabbit IgG (1:3000) or anti-mouse IgG (1:1000; Sigma)] in blockingbuffer. Following six washes of 5 min each, bound antibody was detectedusing enhanced chenuluminescence (Amersham) according to manufacturer'sinstructions. Membranes were exposed to ECL-Hyperfilm (Amersham).

Immune complex p34alc2 kinase assays

C50 or 308 cells were treated with apigenin and harvested exactly as describedabove for Western blot analysis. P34 was immunoprecipitated from 100 ngwhole cell extract using 0.5 (ig mouse anti-p34cdc2(Santa Cruz) prebound to20 nl Protein A-sepharose. Immunoprecipitation reactions employing anti-p34«Jc2 w e r e carried out at room temperature for 2 h. Immunoprecipitatedcomplexes were resuspended in H1 kinase reaction mix containing 40 Hi of20 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT, 10 mM MgCl2, 5 HiHI solution (1 mg/ml solution in 200 mM NaCl, 0.1 mM EGTA, 1 mM DTT,20 mM Tris-HCl, pH 7.5, 10 (Jg of both leupeptin and aprotinin), and 1 |il(10 u.Ci) [32PJyATP. Kinase reactions proceeded for 15 min at room temperatureand were stopped by the addition of 25 uj 2 X SDS-PAGE loading buffer.Radioactive kinase reaction products were separated by electrophoresis on 10%SDS-PAGE gels, dried under vacuum at 80°C, and exposed to Phosphorimager(Molecular Dynamics). Histone band intensities were quantitated using thePhosphorimager. Relative kinase activities for each treatment were normalizedto solvent control.

Apigenin uptake

C50 cells were cultured in 100 mm dishes to 50% confluency. The culturemedium (10 ml) was replaced with fresh medium and an aliquot of apigenin(with 26.8 Ci/mol [G-3H]apigenin) in DMSO or DMSO alone was added toeach dish to achieve the predetermined apigenin concentration in the medium.In all experiments, the volume of solvent DMSO was the same for all doses.The cells were cultured in the apigenin-containing medium for a predeterminedtime at 37°C and 5% CO2- Then the medium was removed and the cells wererinsed with 2X5 ml PBS. After removing the PBS completely, the cells wereharvested into ethanol by scraping and homogenized mechanically. Followingcentrifugation the supernate was saved and the residue was extracted withethanol for another two times. The ethanol extracts were combined andassayed for radioactivity by scintillation counting. The cell number for eachgroup was obtained from a separate replicate dish. For cell counting, the cellswere treated with cold apigenin only and harvested by trypsin release. Theapigenin uptake was calculated from the radioactivity, cell number, andpercentage of radioactivity associated with apigenin determined by HPLC-scintillation analysis. For HPLC-scintillation analysis, the cell extract wasinjected into HPLC system equipped with Alltima Cl8 column (250X2.1 mm).The column was eluted with 0.1% TEA in H2O/acetonitrile (52:48) at a flowrate of 0.3 ml/min. The eluent was collected as 0.5 min fractions and assayedfor radioactivity by scintillation counting. The medium was extracted withether and the extract was converted into ethanol solution for HPLC-scintilla-tion analysis.

Results

Apigenin induced a G-/M arrest in keratinocytesWe investigated the affects of apigenin on the keratinocytecell cycle using two murine skin cell lines, C50 and 308.Figure la presents the results of the DNA flow cytometricanalysis of C50 keratinocytes cultured for 24 h in mediumcontaining various doses of apigenin. As can be seen in Figure1A, we observed a dose-responsive accumulation of C50 cellsin the G2/M phase of the cell cycle after culturing the cellsfor 24 h in media containing apigenin. Maximum G2/M arrestof C50 cells was achieved with 30 |i.M apigenin treatment. Noaffect on the cell cycle was observed with medium containingDMSO alone (0 ^lM). We also tested the affect of apigenin onanother keratinocyte cell line, the 308 papilloma line. 308cells were cultured for 24 h in medium containing 10, 30, 50,or 70 }iM apigenin (or DMSO alone). The results in FigureIB show that 10 and 30 \iM doses of apigenin produced a25% and 44% G2/M accumulation, respectively. No furtherarrest was achieved at higher doses of 50 and 70 (iM. In order

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OuM 5uM 15U.M

Gl: 14S: 16

C2/M: 69

45uM

B.

OftM 10//M 30/tM 50//M 70//M

Table

24 h

Dose

010305070

I. Time course analysis

%G1

5448434950

1 %S

2427131020

%G2/M

2225444030

of the

36 h

%G1

5554423854

cell

%S

221714155

cycle in apigenin-treated

%G2/M

2330444842

48 h

%G1

5954413151

%S

2326181410

1 308 cells

%G2/M

1820415540

Fig. 1. DNA flow cytometnc analysis of keratinocytes cultured with increasing doses of apigenin for 24 h. The percentages of cells in G|, S, and G2/M phasewere calculated using Cellfit computer software and are displayed within each histogram. A, C50 cells. B, 308 cells.

in medium with 18.5 |iM (5 |ig/ml) or 37 |iM (10 (ig/ml)apigenin. Our results as shown in Figure 2, indicate that 37 \iMapigenin treatment caused a profound G2/M arrest in HL-60cells (up to 93%) compared to solvent control cultures.

G/M arrest by apigenin is reversible

We next determined whether G2/M arrest induced by apigeninis reversible. C50 keratinocytes were cultured in mediumcontaining 30 |iM apigenin for 24 h at which time the cultureswere washed three times with PBS, replenished with freshmedia without apigenin, and returned to the incubator at 37°Cfor an additional 6, 12, 24, or 36 h. DNA cell cycle analysisof C50 cells harvested at time points post release are depictedin Figure 3. After 24 h in culture with 30 (iM apigenin (T =0), 68% of the cells were blocked in G2/M phase. After release,the percentage of cells in G(/G| phase increased to a maximumof 51 % at 24 h post release. These data indicate that between12 and 24 h post release, the majority of G2/M arrested cellshad progressed through mitosis and exhibited a normal DNAcell cycle profile. Of the cells still remaining in G2/M at 24 hpost release, nearly all had progressed through mitosis by 36 has indicated by the fact that only 6% of cells were in G2/M at36 h. These results clearly reflect a full reversal of apigenin-induced G2/M arrest in keratinocytes. In fact, the cells becamesynchronized by apigenin treatment as indicated by the factthat nearly 70% were going through S phase at 36 h post release.

Reversibility experiments were also performed to comparethe ability of HL-60 cells to recover from G2/M arrest inducedby apigenin and an isoconformer, genistein. HL-60 cells werecultured in medium with the optimal dose of apigenin (37 \iM)or genistein (55 (iM). After 24 h the cells were washed withPBS, replenished with fresh media without any flavonoids,

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308 cells were treated with increasing doses of apigenin for 24, 36, or 48 hat which times they were harvested and analyzed for cell cycle distributionon the flow cytometer.

to investigate whether the accumulation of 308 cells in G2/Mwas time-dependent rather than dose-dependent, 308 cells werecultured for 24, 36, and 48 h in medium containing apigenin.Table I presents the results of this study. (Data for the 24 htime point is the same as that displayed in the DNA histogramin Figure IB.) Our results show that after a 48 h culture with50 (iM apigenin, only 55% of the cells had accumulated inG2/M phase, compared with C50 cells in which 74% of thecells were blocked at G2/M at 24 h with 30 (iM apigenin.Thus, apigenin caused cell cycle arrest in both keratinocytecell lines, although the 308 cell line was less responsive toapigenin than C50 cells.

G/M arrest by apigenin is not cell-type specificExperiments were carried out to determine if apigenin couldinduce G2/M arrest in cells of non-keratinocyte origin. Weinvestigated the affect of apigenin on the pro-myelocytichuman cell line, HL-60. HL-60 cells were cultured for 24 h

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G 2 / M - 1 0 % G2/M= 2 3 %

Vie ' : e*e ' ' aie

G2/M- 93%

0//M 18.5//M 37/*M

Fig. 2. DNA flow cytometric analysis of HL-60 cells treated with apigenin. The percent of cells in G^fM phase of the cell cycle were calculated using Cellfitcomputer software.

immortalization and transformation of human keratinocytes(23).

Effects of apigenin on p34cdc2 kinase activityWe next tested the hypothesis that apigenin might induce aG2/M arrest by inhibiting p34cdc2 HI kinase activity. C50 and308 cells were cultured with increasing doses of apigenin for24 h as described previously, and whole cell extracts wereprepared and assayed for HI kinase activity using an immunecomplex kinase assay. Results of HI kinase assay of apigenin-treated C50 cells in Figure 6A demonstrated a 55% kinaseinhibition when the dose of apigenin reached 45 \iM. lowerdoses of apigenin had no effect on kinase activity in C50 cells.Figure 6B demonstrates the dose responsive inhibition ofp34«ic2 kjuase activity by apigenin in 308 cells, with doses of10, 30, and 50 (iM resulting in cdc2 kinase inhibition by 28%,88%, and 95%, respectively. These results taken in conjunctionwith Western blot experiments presented in Figure 5 lead usto conclude that apigenin inhibited p34cdc2 kinase activity bychanging the activation state, rather than altering the amount

present.

' 12 24

Hours Post Release

Fig. 3. G2/M arrest reversal in 30 |iM apigenin-treated C50 keratinocytes.

and returned to culture at 37°C for an additional 6, 12, or24 h. Distinct G2/M arrest by both apigenin (85%) and genistein(90%) was evident at 0 h post release (Figure 4). As early as12 h post release the majority of HL-60 cells treated withapigenin had progressed through mitosis and entered Go/G|phase, and by 24 h post release apigenin treated HL-60 cellshad resumed a normal cell cycle profile. In contrast, only asmall percentage of genistein-arrested HL-60 cells entered GQ/G| after 24 h. Therefore our results indicate that unlikegenistein, apigenin induces a G2/M arrest which is fullyreversible after 24 h.

Effects of apigenin on p34cdc2 steady-state levelsIn view of the fact that the major regulatory cyclin dependent

kinase(cdk), p34cdc2, is responsible for G2 to M phase progres-sion in all eukaryotic cells, we next investigated whetherapigenin induces G2/M arrest by altering the protein level orinhibiting kinase activity of pSi"102 in keratinocytes. P34cdc2

is normally present at approximately constant levels throughoutthe cell cycle, but its kinase activity is cyclically modulated.We performed western blot analysis of cell extracts preparedfrom apigenin-treated C50 and 308 cells (Figure 5). Our resultsshowed that apigenin treatment did not change the steady-statelevel of p34c°c2 compared to DMSO-treated cultures (Figure5). Western blots indicated a triplet band migrating at 34 kDthat is believed to represent different phosphorylated forms ofp34cdc2 (22). We observed no change in the relative intensityof the triplet bands, which suggests apigenin had no effect onphosphorylation status. Although the steady-state level ofp34«ic2 ^ n o t change wjthjn the same cell line, we observedthat 308 cells expressed higher steady-state levels of p34cdc2

than C50 cells. Our data concurs with that of Rice et al.who described an increase in p34cdc2 during the spontaneous

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of p34cdc2

Apigenin treatment reduces the steady-state of cyclin Bl proteinWe were interested in determining whether or not the G2/M

arrest and p34cdc2 kinase inhibition were due to the fact thatapigenin may be limiting the supply of cyclin B in apigenin-treated keratinocytes. When Western blot analysis was per-formed with 100 |ig aliquots, we observed no major changein the level of cyclin Bl protein until the highest dose of45 |iM (Figure 7A). However, we noted that the percentageof cells in G2/M was increasing with apigenin dose (thepercentage of cells in G2/M is indicated under each lane inFigure 7). When the band intensity of cyclin B1 was normalizedto the percentage of cells in G2/M, the level of cyclin B1 didnot increase as one would expect in a population of cellscontaining a higher proportion in G2/M (Figure 7B). In fact,after normalizing the band intensity to the percent of cells inG2/M at each drug concentration, a dose-dependent reductionof cyclin B1 was clearly evident (Figure 7B). (This patternwas observed in duplicate experiments.) In 308 cells, westernblot analysis was performed and we observed that the level ofcyclin Bl protein was nearly undetectable at 50 (iM and 70(iM doses of apigenin (Figure 7C). Again, note the percentageof cells in G2/M is indicated below each lane. After normalizingthe band intensity of cyclin Bl with the percentage of cells inG2/M for each treatment, a reduction in the supply of cyclinBl was clearly evident (Figure 7D). (These findings were

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APK3ENIN

62/M = 85X

hours afterremoval'

GENISTEING2/H = 90X

hours aftarremoval.

62/N • 65X

Fig. 4. Comparison of G2/M arrest reversal in apigenin vs. genistein-treated HL-60 cells. Top panels represent HL-60 cells that were G^M arrested with37 (iM apigenin. Bottom panels represent HL-60 cells G2/M arrested with 55 ^M genistein. See Results section for expenmental design.

A.

0Apigenin15 30 45

B.

0 10Apigenin30 50 70

Apigenin uptake is comparable in vivo and in vitroWe determined the uptake of apigenin by C50 cells in relationto dose and time. The uptake of apigenin by C50 cells wasdose-responsive although the intracellular accumulation wasnot linear (Figure 8A). The uptake increased slowly at lowdoses of apigenin and much more quickly after the doseexceeded 30 (iM. Regression analysis showed that the dose-response curve fit a trinomial model (y = O.OOtSj^-O.DSSx2 /2.149A:-0.9966, R2 = 0.9998). The time course experimentshown in Figure 8B indicated that the uptake of 20 (iMapigenin increased quickly in the first few hours, reached amaximum at 8 h, and remained steady thereafter. HPLC-scintillation analysis indicated that 100% of the radioactiveisotope recovered from cell extract and medium was apigenin.The effect of apigenin on cell viability was minimal, as testedby trypan blue dye exclusion. Cells treated with 10 |XMapigenin showed no change in viability with respect to control.Cells treated with 30 ^iM, 50 \iM, and 70 \LM were 96%, 96%,and 89% of control viability, respectively. We have alsodemonstrated that intracellular levels of apigenin are compar-able to those achieved in mouse epidermal cells after topicalapplication (manuscript in preparation).

Fig. 5. Western blot analysis of the steady-state level of p34cdc2 in apigenin-treated keratinocytes. A, C50 cells. B, 308 cells.

repeated three times.) Since the level of cyclin Bl did notincrease in either cell line with a corresponding increase inthe percentage of G2/M cells, we conclude that apigenintreatment reduces the steady-state level of cyclin Bl duringthe keratinocyte cell cycle.

Discussion

Several epidemiologic studies have demonstrated that theconsumption of vegetables correlates negatively with cancerincidence for colon, breast, and lung cancers, as well as othertypes of cancer (24-26). Indoles, thiocyanates, isothiocyanates,and flavonoids such as apigenin, are among the nonnutritivevegetable components identified as having antitumorigenicproperties (27).

We have studied the flavonoid apigenin in vivo using UV-Bradiation and DMBA/TPA-induced mouse skin carcinogenesismodels, and demonstrated that topical application of 5 |imolapigenin significantly reduced the number of benign papillomasand squamous cell carcinomas (4, Birt et al., submitted).

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A.

1.2

£> 0.8~ 0.6

••= O.A

< 0.2• Him0 15 30

Apigenin

45

B.

0 10 30 50 70

Apigenin

Fig. 6. Immune complex kinase assay of p34cdc2/cyclin Bl in apigenin-treated keratinocytes. Phosphorylated histone band intensity was quantitatedand normalized to 0 |xM apigenin (DMSO control) for each treatment.A, p34cdc2 ICK in C50 cells. B, pM1^2 ICK in 308 cells.

Apigenin levels used in C50 cells were similar to those wehave detected in the epidermal cells in vivo from mice treatedtopically with 5 (amol apigenin (Li et al., unpublished).Thus, the fact that similar cellular apigenin concentrations areobserved in vitro and in vivo provides a connection betweenour in vitro mechanistic results and the in vivo chemopreventiveactivity of apigenin. There was also no evidence for apigeninmetabolism in cultured keratinocytes, which is consistent withour in vivo and in vitro metabolism studies with the epidermisof SENCAR mice (Li et al., unpublished).

In the data presented herein, we report that apigenin exertsits chemopreventive activity, in part, by causing a G2/M cellcycle arrest in keratinocytes that is fully reversible. Apigeninwas effective at causing a reversible G2/M arrest in cells atdifferent stages of tumorigenesis and in cells of differentlineages. The variation between cell lines in the percent ofcells in G2/M after a 24 h culture with apigenin may beexplained by the fact that 308 cells may also be undergoing aGr/Gi arrest as well as a G2/M arrest. We are currentlyinvestigating this possibility with respect to stage of tumori-genesis and sensitivity to apigenin treatment. Worthwhile

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noting is the fact that DNA flow cytometric and agarose gelanalysis show no signs of apigenin-induced apoptosis inkeratinocytes.

To further elucidate the molecular mechanism of apigeninwith respect to its ability to cause a G2/M cell cycle arrest,we began investigating the major regulatory proteins governingG2 to M phase progression. We found that apigenin does notalter the steady-state level of the mitotic cdk, p34cdc2, inkeratinocytes (Figure 5). Since we established that apigenintreatment did not alter the steady-state level of p34cdc2, anychange in HI kinase activity would accurately reflect changesin activation state, rather than an absence of p34cdc2 proteinpresent in immune complex kinase assays. We demonstratedthat apigenin treatment inhibited p34cdc2 kinase activity in C50and 308 keratinocyte cell lines (Figure 6A and B, respectively).A dose of 45 U.M apigenin inhibited cdc2 kinase activity by48%, while 15 (iM and 30 U.M apigenin treatment of C50 cellsresulted in a substantial G2/M arrest (75%) with little kinaseinhibition. Possible explanations for this discrepancy includealterations in cyclin Bl subcellular localization and effects oncyclin Bl/p34cdc2 complex formation. Alternatively, apigeninmay produce a G2/M arrest in C50 cells by a mechanismindependent of cyclin-cdk involvement, for example by inhibit-ing microtubule formation.

In contrast to C50 cells, apigenin inhibited cdc2 kinaseactivity in 308 cells in a dose-dependent manner. Inhibition ofkinase activity was apparently not due to perturbation of thecdc2 phosphorylation status, since no evidence for alteredband migration was observed in Western blot analysis (Figure5). Other laboratories using various agents to perturb the cellcycle or damage DNA have noted changes in the phosphoryl-ation state of p34cdc2. In CHO cells, etoposide inhibits p34cdc2

kinase activity and maintains the enzyme in a tyrosine phos-phorylated state (28). Tyrosine phosphorylation of p34cdc2 isalso evident in HL-60 cells after they are subjected to ionizingradiation (29). In contrast, a decrease in tyrosine phosphoryl-ated p34cdc2 is observed with G2 arrest by the synthetic flavoneL86-8275 (30). Thus, the mechanistic differences in inducingG2/M arrest may be due to the activity of other cell cycleregulatory proteins. To date there have been no describedcyclin dependent kinase inhibitors of p34cdc2 in vivo (14).

The first requirement for G2 to M phase progression is thatcyclin B must accumulate above a threshold level during G2

to enter mitosis (31). Without adequate levels of cyclin Bpresent, cells arrest in G2. For example, Maity et al. demon-strated this checkpoint feature by y-irradiating HeLa cells,which resulted in a reduction of cyclin Bl protein andsubsequent G2 block (32). They further reported that decreasedRNA stability contributes to the reduction in cyclin B1 mRNAfollowing y-irradiation and subsequent G2 delay (32). Ourresults presented herein indicate that the level of cyclin Blprotein is reduced in apigenin-treated C50 and 308 cells whennormalized to the percent of cells in G2/M (Figure 7). We donot yet know whether this reduction in cyclin B protein is dueto an effect on cyclin B mRNA. Studies are in progress toinvestigate this possibility since during a normal cell cycle thelevel of cyclin Bl mRNA parallels that of the protein, whichstarts to rise in S phase, peaks in G2/M phase and then rapidlydisappears in early Gl (33).

Equally as important as the transcription and stability ofcyclin B1 mRNA is the programmed degradation of cyclin B1protein at the metaphase-anaphase transition, which is arequired event for cells to exit mitosis (19). A predominant

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Apigenin induces Gj/M arrest In keratinocytes

A. B. 1.25-1

0Apigenin15 30 45 O

0.75-

%G2/M: 31 37 49 74 69.S 0.25 -

0 5 15 30 45

//M Apigenin

c. D.L2S-I

Apigenin10 30 50 70

%G2/M: 22 25 44 40 30

0 10 30 SO 70

ftM Apigenin

Fig. 7. Western blot analysis of cyclin Bl levels in apigenin-treated keratinocytes. A, cyclin Bl Western blot of apigenin-treated C50 cells. Percent of G2/Mcells for each treatment shown. B, level of cyclin Bl in C50 cells normalized to the percent of C50 cells in G2/M. C, cyclin Bl Western blot of apigenin-treated 308 cells. Percent of G2/M cells for each treatment shown. D, level of cyclin Bl in 308 cells normalized to the percent of 308 cells in G2/M.

theory is that cyclin degradation occurs by ubiquitin-mediatedproteolysis (34). Although the signal transduction events lead-ing to ubiquitin-mediated cyclin destruction are unresolved, itis plausible that apigenin causes aberrant cyclin B1 degradationby perturbing a ubiquitin signal transduction cascade. Experi-ments are underway to determine if the lack of cyclin Blprotein in apigenin-treated cells is due to transcriptional, post-transcriptional, or post-translational regulation.

Gi/S and G2/M checkpoint controls prevent the acquisitionof multiple genetic changes by ensuring that DNA is repairedbefore replication in S phase and properly segregated during

M phase of the cell cycle, respectively. The majority of cancerscharacterized to date have defects in at least one cell cyclecheckpoint For example, p53 is a tumor suppressor gene thatis frequently mutated, especially in human skin cancer (35).P53 has been extensively studied for its role in inducing thep21WAFl/CIPl cdk inhibitor, which results in G, arrestfollowing DNA damage (36,37). Incompletely replicated anddamaged DNA signals a G2/M arrest in order to preventmutations, translocations, and/or chromosome loss. Attenuationof this G2 delay has recently been demonstrated to precedeimmortilization of human fibroblasts (38). Furthermore, the

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10 20 30 40Apigenin in medium (uM)

50

B.

H 1 1 1 1 1 1 1—I 1 1

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hr)

Fig. 8. Apigenin uptake by C50 cells. The data are expressed as the means(dots) ± SE (bars). Points without bars had SE within the dots. A, Doseresponse. C50 cells were cultured for 24 h with increasing doses of apigeninin the medium, n = 3. B, Time course. C50 cells were cultured with 20 uMapigenin in the medium for 0-24 h, n = 2.

loss of important cell cycle checkpoints selects cells that havenot only a growth advantage but also a predisposition foracquiring more chromosome aberrations. Cancer cells that losecell cycle checkpoints evolve into drug resistant, invasive, andmetastatic variants (39). In light of our results that apigenincauses a G2/M arrest, the essence of apigenin's chemopreven-tive activity may be to slow cancer development by ensuringthat sufficient intrinsic and artificially imposed cell cyclecheckpoints exist in the presence of DNA damaging and tumorpromoting agents. By deciphering the molecular mechanismsof apigenin, it may be possible to interfere with cancerdevelopment through good nutrition or local delivery of purifiedfiavonoids, especially for those who are genetically predisposedto certain types of cancer (40). Also in view of the fact thatapigenin may represent an alternative sunscreen agent inhumans, studies to further define apigenin's molecular mechan-ism of chemoprevention are warranted.

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Received on March 28, 1996; revised on August 16, 1996; accepted on August26, 1996

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