molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory...

$: Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells$
Molecular Mechanisms for Apigenin-InducedCell-Cycle Arrest and Apoptosis of HormoneRefractory Human Prostate CarcinomaDU145 Cells

Sanjeev Shukla1,2 and Sanjay Gupta1,2,3*1Department of Urology, The James and Eilleen Dicke Research Laboratory, Case Western Reserve University, Cleveland, Ohio2Department of Urology, The James and Eilleen Dicke Research Laboratory, University Hospitals of Cleveland, Cleveland,Ohio3Department of Urology, The James and Eilleen Dicke Research Laboratory, Ireland Cancer Center, Cleveland, Ohio

Development of effective agents for treatment of hormone-refractory prostate cancer has become a nationalmedical priority. We have reported recently that apigenin (40,5,7-trihydroxyflavone), found in many common fruitsand vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human

prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin onandrogen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony

formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the proteinexpression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, withconcomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its

growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenintreatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release ofcytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in asignificant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin

treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-kB)/p65 and NF-kB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IkBa) in the cytosol.Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction

of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function,and (3) NF-kB inhibition. � 2004 Wiley-Liss, Inc.

Key words: prostate cancer; Bcl2; nuclear factor-kappaB; cyclin; cyclin-dependent kinase inhibitor; caspase;cytochrome c

INTRODUCTION

Because of high prevalence, mortality, and unsa-tisfactory treatment options available at this time,hormone-refractory prostate cancer remains thesecond leading cause of cancer-related deaths inmen in the United States [1–3]. In fact, most cases ofprostate cancer are relatively indolent; however, ifnot detected early, more aggressive forms, character-ized by invasion of the seminal vesicle, followed bymetastasis primarily to the bone, usually result inlethality [4]. This transition to metastatic disease isgenerally followed by a shift from androgen depen-dence to androgen independence, which is oftenprovoked by androgen-ablation therapy [5]. Therecurrence of androgen independence often leadsto genetic alterations marked by loss of function ormutation of tumor suppressor genes and oncogenes,transforming the cells so that they evade apoptosisand achieve a chemotherapy-resistant state [6,7].

Therefore, new approaches for the management ofthis type of cancer are urgently needed.

In recent years, apigenin (40,5,7-trihydroxyfla-vone; Figure 1), the most common nonmutagenicflavonoid, which is widely distributed in many fruits

MOLECULAR CARCINOGENESIS 39:114–126 (2004)

� 2004 WILEY-LISS, INC.

*Correspondence to: Department of Urology, The James andEilleen Dicke Research Laboratory, Case Western Reserve University,10900 Euclid Avenue, Cleveland, OH 44106.

Received 8 May 2003; Revised 8 October 2003; Accepted 6November 2003

Abbreviations: NF-kB, nuclear factor-kappaB; Rb, retinoblastoma;cki, cyclin-dependent kinase inhibitor; cdk, cyclin-dependent kinase;Apaf-1, apoptotic protease-activating factor-1; PARP, poly(ADP-ribose) polymerase; IkBa, IkappaB-alpha; DMSO, dimethyl sulfoxide;MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide;PBS, phosphate buffered saline.

DOI 10.1002/mc.10168

and vegetables, has shown remarkable effects ininhibiting cancer cell growth both in cell culturesystems and in in vivo tumor models [8–10].Apigenin has shown to possess anti-inflammatoryand free radical scavenging properties in manyin vitro systems [11,12]. Apigenin possesses anti-mutagenic properties against nitropyrene-inducedgenotoxicity in Chinese hamster ovary cells [13] andhas been shown to inhibit mitogen-activated proteinkinase and the down-stream oncogenes, c-jun andc-fos in v-Ha-ras–transformed NIH3T3 cells [14].Studies have shown that apigenin increases gapjunction in rat epithelial cells and maintains cell-to-cell communication [15]. Apigenin is a stronginhibitor of cyclooxygenase-2 and ornithine decar-boxylase, enzymes responsible for playing a majorrole in tumor promotion [12,16]. Further, it has beenshown to increase the intracellular concentration ofglutathione, enhancing the endogenous defenseagainst oxidative stress [17]. Apigenin under in vivosituations has shown to inhibit tumor necrosisfactor-induced intercellular adhesion molecule-1upregulation [18]. Exposure to apigenin prior tocarcinogenic insult has shown to afford a protectiveeffect in murine skin and colon model systems[19,20]. More recently, apigenin has shown promiseof inhibiting tumor cell invasion and metastasis byregulating protease production [21]. Apigenin treat-ment has been shown to induce apoptosis in a widerange of human carcinoma cells, including thyroid,leukemia, melanoma, breast, colon, and prostate[22–27] and to induce a reversible G2/M arrest inepidermal cells and fibroblast by inhibition ofp34cdc2 kinase activity which is accompanied by anincrease in p53 protein stability [26,28]. Recently,apigenin has shown potential to downregulate theactivation of nuclear transcription factor nuclearfactor-kappaB (NF-kB), responsible for the regulationof critical genes for cell proliferation, metastasis,apoptosis, and chemotherapy resistance [29].

Previous reports from our laboratory and else-where have demonstrated the ability of apigenin tocause cell growth inhibition and apoptosis of severalhuman prostate carcinoma cells [27,30,31]. Extend-

ing these studies, we have further shown themolecular mechanism(s) responsible for such effectsin androgen-sensitive human prostate carcinomaLNCaP cells [32]. In the present study, we evaluatedthe effect of apigenin on androgen-refractory humanprostate carcinoma DU145 cells, which are highlytumorigenic and chemotherapy-resistant [33,34].Importantly, this cell line has several nonfunctionalgenes responsible for cell-cycle control and carriesmutations in the tumor suppressor gene productsp53 and pRb [35,36]. Here we showed that apigenininhibited growth and colony formation of DU145cells via a G1 arrest during cell-cycle progressionby modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery.The induction of WAF1/p21 and its growth inhibi-tory effects by apigenin appeared to be independentof p53 and pRb status of these cells. Theseevents were associated with alterations in the levelof Bcl2 shifting the Bax/Bcl2 ratio more towardsapoptosis accompanied with release of cytochromec, induction of apoptotic protease-activatingfactor-1 (Apaf-1), and cleavage of caspase-9, -3, andpoly(ADP-ribose) polymerase (PARP) along withNF-kB inhibition.

MATERIALS AND METHODS

Materials

Apigenin (>95% purity) was obtained from SigmaChemical Co. (St. Louis, MO). The mono- and poly-clonal antibodies (human reactive anti-cdk2, 4, and6, WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18,p53, pRb, cytochrome c, Apaf-1, caspase-9) wereobtained from Santa Cruz Biotechnology, Inc. (SantaCruz, CA). The human reactive monoclonal andpolyclonal antibodies for anticyclin D1, D2, and Ewere obtained from Neomarker (Fremont, CA).The other human reactive monoclonal and poly-clonal antibodies for anti-PARP, caspase-3, Bax, Bcl2,NF-kB/p65, NF-kB/p50, and IkappaB-alpha (IkBa)were purchased from Upstate Biotechnology (LakePlacid, NY).

Cell Culture

The human prostate carcinoma DU145 cells wereobtained from American Type Culture Collection(Manassas, VA) and cultured in RPMI 1640 medium(Gibco, Rockville, MD), supplemented with 5% fetalbovine serum, 1% penicillin/streptomycin in a 5%CO2 atmosphere at 378C.

Treatment of Cells

Apigenin dissolved in dimethyl sulfoxide (DMSO,maximum concentration 0.2%) was employed forthe treatment of cells. The cells (70–80% confluent)were treated with 1-, 5-, 10-, and 20-mM concentra-tion of apigenin for 24 h in complete cell culturemedium, whereas cells treated with only DMSO

Figure 1. Structure of apigenin, a common dietary flavonoidabundantly present in fruits and vegetables. Apigenin is composed ofa common phenylchromanone structure (C6-C3-C6) with one ormore hydroxyl substituents.

APIGENIN-INDUCED CELL-CYCLE DEREGULATION AND APOPTOSIS OF DU145 CELLS 115

served as control. For time-dependent studies, thecells were treated with 10 mM dose of apigenin fordesired time intervals.

Cell Viability

The effect of apigenin on the viability of cells wasdetermined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay. The cellswere plated at 1� 104 cells per well in 200 mL ofcomplete culture medium containing 1-, 5-, 10-, 20-,40-, and 80-mM concentrations of apigenin in 96-wellmicrotiter plates for 24, 48, and 72 h, respectively.Apigenin stock solutions were prepared in DMSO at10 mM concentration and mixed with fresh mediumto achieve the desired final concentration. Eachconcentration of apigenin was repeated in 10-well.After incubation for the specified time at 378C in ahumidified incubator, cell viability was determined.MTT (5 mg/mL in phosphate buffered saline (PBS))was added to each well and incubated for 2 h afterwhich the plate was centrifuged at 1800 rpm for5 min at 48C. The MTT solution was removed fromthe wells by aspiration. After careful removal of themedium, 0.1 mL of buffered DMSO was added toeach well, and plates were shaken. The absorbancewas recorded on a microplate reader at the wave-length of 540 nm. The effect of apigenin on growthinhibition was assessed as percent cell viability wherevehicle-treated cells were taken as 100% viable.

Soft Agar Colony Formation Assay

DU145 cells were cultured under the conditionspreviously described [37]. For anchorage-indepen-dent cell growth, soft agar colony formation assaywas performed in a 6-well plate. Each well contained2 mL of 0.5% agar in medium as the bottom layer,1 mL of 0.38% agar in medium, and�1200 cells at thefeeder layer treated with various concentrations ofapigenin (5–20 mM in medium). Cultures weremaintained at 378C in a humidified 5% CO2 atmo-sphere. The number of colonies was determined after2 weeks by counting them under an inverted phase-contrast microscope at �400 magnification and agroup of �20 cells was counted as a colony.

DNA Cell-Cycle Analysis

Asynchronous DU145 cells (70% confluent) weretreated with apigenin (1-, 5-, 10-, and 20-mM doses) inRPMI-1640 complete media for 24 h. The cells weretrypsinized thereafter, washed twice with cold PBS,and centrifuged. The pellet was resuspended in 50 mLcold PBS and 450mL cold methanol for 1 h at 48C. Thecells were centrifuged at 1100 rpm for 5 min, pelletwashed twice with cold PBS, suspended in 500 mLPBS, and incubated with 5 mL RNAse (20 mg/mL finalconcentration) at 378C for 30 min. The cells werechilled over ice for 10 min and stained withpropidium iodide (50 mg/mL final concentration)for 1 h and analyzed by flow cytometry.

Protein Extraction and Western Blotting

The DU145 cells (70% confluent) were treated withapigenin (1-, 5-, 10-, and 20-mM doses) in RPMI-1640complete media for 24 h. For time-dependent assay,the cells (50–60% confluent) were treated for 24, 48,and 72 h with 10 mM of apigenin. After which themedia was aspirated, the cells were washed with coldPBS (10 mM, pH 7.4) and ice-cold RIPA buffer [50 mMTris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA,20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1%Triton X-100, 1 mM PMSF (pH 7.4)] with freshlyadded protease inhibitor cocktail (Protease InhibitorCocktail Set III, Calbiochem, La Jolla, CA) over ice for30 min. The cells were scraped and the lysate wascollected in a microfuge tube and passed through a21½ G needle to break up the cell aggregates. Thelysate was cleared by centrifugation at 14 000� g for15 min at 48C and the supernatant (total cell lysate)was used immediately or stored at �808C. For NF-kBassay the cells were processed for cytosolic andnuclear fractions as described previously [38]. Theprotein concentration was determined by DC Bio-Rad assay according to the manufacturer’s protocol(Bio Rad Laboratories, Hercules, CA).

For Western blotting, 50 mg protein was resolvedover 4–20% polyacrylamide gel and transferred to anitrocellulose membrane. The blot was blocked inblocking buffer (5% nonfat dry milk/1% Tween-20;in 20 mM TBS, pH 7.6) for 1 h at room temperature,incubated with appropriate primary antibody inblocking buffer for 1 h to overnight at 48C followedby incubation with appropriate secondary antibodyhorseradish peroxidase conjugate obtained fromAmersham Life Science, Inc. (Arlington Height, IL)and detected by chemiluminescence and autoradio-graphy with XAR-5 film (Eastman Kodak Co.,Rochester, NY). Densitometric measurements ofthe bands in Western blot analysis were performedwith digitalized scientific software program UN-SCAN-IT (Silk Scientific Corporation, Orem, UT).

DNA Fragmentation Assay

The DU145 cells were grown to about 70%confluence and treated with apigenin (1-, 5-, 10-,and 20-mM concentration) for 48 h. For time-dependent assay, the cells were treated with 10 mMdose of apigenin for desired time intervals. Followingthese treatments, the cells were washed twice withphosphate-buffered saline (10 mM Tris, pH 7.5,150 mM NaCl, 5 mM MgCl2, and 0.5% Triton X-100), left on ice for 15 min, and pelleted bycentrifugation (14 000� g) at 48C. The pellet wasincubated with DNA lysis buffer (10 mM Tris, pH7.5,400 mM NaCl, 1 mM EDTA, and 1% Triton X-100) for30 min on ice and then centrifuged at 14 000� g at48C. The supernatant obtained was incubated over-night with RNAse (0.2 mg/mL) at room temperatureand then with Proteinase K (0.1 mg/mL) for 2 h at

116 SHUKLA AND GUPTA

378C. DNA was extracted with phenol:chloroform(1:1) and precipitated with 95% ethanol for 2 h at�808C. The DNA precipitate was centrifuged at14 000� g at 48C for 15 min and the pellet was air-dried and dissolved in 20 mL of Tris-EDTA buffer(10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Totalamount of DNA was resolved over 1.5% agarose gel,containing 0.3 mg/mL ethidium bromide in 1� TBEbuffer (pH 8.3, 89 mM Tris, 89 mM boric acid, and2 mM EDTA) (Bio Wittaker, Inc., Walkersville, MD).The bands were visualized under UV transillumina-tor (Model # TM-36, UVP, Inc., San Gabriel, CA)followed by polaroid photography (MP-4 Photo-graphic System, Fotodyne, Inc., Hartland, WI).

Apoptosis Detection by Fluorescence Microscopy

The ApopNexin apoptosis detection kit (Oncor,Gaithersburg, MD) was used for the detection ofapoptotic cells. This kit uses a dual-staining protocolin which the apoptotic cells are stained with annexinV (green fluorescence) and the necrotic cells arestained with propidium iodide (red fluorescence).Briefly, the DU145 cells were grown to about 50%confluence in a 6-well plate and then treated withapigenin (5-, 10-, and 20-mM concentration) for 24 h.Apoptosis was detected by the use of the kitaccording to the vendor’s protocol with a ZeissAxiovert 100 microscope. Briefly, the samples wereexcited at 330–380 nm, and the image was observedand photographed under a combination of a 400-nmdichoric mirror and then 420-nm high-pass filter.Cells with a green, condensed chromatin patternwere considered apoptotic, whereas those with rednuclei without nuclear condensation were consid-ered necrotic.

Quantification of Apoptosis

For quantification of apoptosis, the DU145 cellswere grown at a density of 1�106 cells in 100-mmculture dishes and were treated with apigenin (1-, 5-,10-, and 20-mM concentration) for 24 h. The cellswere trypsinized, washed with PBS, and processed forlabeling with fluorescein-tagged deoxyuridine tri-phosphate nucleotide and propidium iodide by useof an APO-DIRECT apoptosis kit obtained fromPhoenix Flow Systems (San Diego, CA) as permanufacturer’s protocol. The labeled cells were thenanalyzed by flow cytometry.

Statistical Analysis

As required, Student’s two-tailed t-test wasemployed to assess the statistical significancebetween the control- and apigenin-treated groups.

RESULTS

Apigenin Induces Cell Growth Inhibition in DU145 Cells

Our aim was to investigate whether apigenintreatment imparts antiproliferative effects against

androgen-refractory human prostate carcinomacells. Therefore, we employed DU145 cells andevaluated the effect of apigenin on cell growth byMTT assay. As shown in Figure 2, apigenin treatment(5- to 80-mM) resulted in a dose-dependent inhibi-tion of cell growth, as compared to vehicle-treatedcontrols. Apigenin treatment also resulted in time-dependent inhibition of cell growth. This effect wasmore pronounced at 48 and 72 h post-treatment(Figure 2).

Apigenin Inhibits Anchorage-Independent Growthand Colony Formation of DU145 Cells

In the next series of experiments, we investigatedthe effect of apigenin on anchorage-independentgrowth by soft agar colony formation. As shown inFigure 3, compared with vehicle treated control(42 colonies/field), treatment of DU145 cells withapigenin resulted in a significant decrease in ancho-rage-independent growth and colony formation to26-, 14-, and 8-colonies/field at 5-, 10-, and 20-mMconcentration.

Apigenin Induces G1 Cell-Cycle Arrest and Alterations

in G1 Cell-Cycle Regulatory Proteins in DU145 Cells

To assess whether apigenin-induced growth inhi-bition of cells is mediated via alterations in cell cycle,we evaluated the effect of apigenin on cell-cycledistribution. We performed DNA cell-cycle analysiswith growing DU145 cells followed by treatmentwith varying concentrations of apigenin for 24 h.Compared with vehicle-treated controls (34% cellsin G0-G1 phase), apigenin treatment resulted in an

Figure 2. Effect of apigenin on the viability of DU145 cells. Thecells were treated with vehicle (dimethyl sulfoxide, DMSO) only orspecified concentrations of apigenin for 24, 48, and 72 h, and cellviability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. The values are represented as thepercentage cell inhibition where vehicle-treated cells were regardedas 100%. The details are described under Materials and Methods.Each data represent the mean� SE of two different experimentseach conducted in triplicate. *P< 0.05, **P<0.01, and {P< 0.001versus control.


appreciable arrest of cells in G0-G1 phase of cell cycleafter 24 h of treatment. The treatment caused anarrest of 39% cells in G0-G1 phase of cell cycle at 5 mMconcentration that further increased to 52% at10 mM, and 56% at highest dose of 20 mM in thesecells (Figure 4). This increase in G0-G1 cell populationwas accompanied with a concomitant decrease ofcell number in S-phase and G2-M phase of the cellcycle (Figure 4).

Next, we assessed the protein expression of cyclinsand cdks, which are known to be operative in G1-phase of the cell cycle. Treatment of DU145 cells withapigenin resulted in a dose-dependent decreasein protein expression of cyclin D1, cyclin D2, andcyclin E (Figure 5A). The decrease in cyclin E proteinexpression was more pronounced than that of cyclinD1 and cyclin D2. Similar time-dependent inhibi-tion was observed in cyclin D1, cyclin D2, and cyclinE after treatment of cells with 10mM concentration ofapigenin for 24, 48, and 72 h (Figure 5A).

The effect of apigenin was also observed in theprotein levels of cdks. Treatment of DU145 cells with

apigenin resulted in a dose-dependent decrease incdk2, cdk4, and cdk6 (Figure 5B). Similar time-dependent inhibition was observed in cdk2, cdk4,and cdk6 after treatment of cells with 10 mMconcentration of apigenin for 24, 48, and 72 h(Figure 5B). The decrease in protein expression ofcdk2 was much more pronounced at 48 and 72 hthan at 24 h postapigenin treatment (Figure 5B).

We next examined the effect of apigenin on ckioperative in G0/G1-phase of the cell cycle. Manystudies have shown that these ckis regulate progres-sion of cells in G0/G1-phase and an induction ofthese molecules causes a blockade of G1! S transi-tion thereby resulting in cell-cycle arrest [39,40]. Asshown by Western blot analysis, apigenin treatment(1-, 5-, 10-, 20-mM for 24 h) of DU145 cells resulted insignificant dose-dependent upregulation of cyclinkinase inhibitors WAF1/p21 and KIP1/p27. A modestincrease in the protein expression of INK4a/p16, andINK4c/p18 was observed after apigenin treatment(Figure 5C). Almost similar results were obtainedwith time-dependent assay where treatment ofapigenin resulted in increase in protein expressionof WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18(Figure 5C). We also performed the assay for proteinexpression of p53 and pRb and their phosphoryla-tion. No significant alteration in protein expressionor their phosphorylated forms of p53 and pRb wasobserved with apigenin treatment in these cells (datanot shown). These results indicated that upregula-tion of WAF1/p21 after treatment with apigenin wasindependent of p53 and pRb status.

Apigenin Induced Apoptosis in DU145 Cells

In the next series of experiments, we assessed theeffect of apigenin treatment on apoptosis in DU145cells. The cells were treated with varying concentra-tion of apigenin (1–20 mM). As shown in Figure 6A,compared with vehicle-treated control, apigenintreatment (10- and 20-mM for 48 h) resulted in theformation of DNA fragments in DU145 cells. Simi-larly, treatment with 10-mM concentration of api-genin resulted in the formation of DNA ladder at48 and 72 h post-treatment. The induction ofapoptosis by apigenin was also evident from themorphology of cells as assessed by fluorescencemicroscopy after labeling the cells with annexin V(Figure 6B). We used this method because it identifiesapoptotic (green fluorescence) as well as necrotic (redfluorescence) cells. As shown by data in Figure 6B,apigenin treatment resulted in a dose-dependentapoptosis in DU145 cells. These data indicated thatapigenin treatment also resulted in necrosis of thesecells. This is possibly due to apoptosis induced byapigenin, which is preceded by secondary necrosis inthese cells.

We next quantified the extent of apoptosis by flowcytometric analysis of DU145 cells labeled withdeoxyuridine triphosphate and propidium iodide.

Figure 3. Effect of apigenin on anchorage-independent growthassay estimated by soft agar colony formation. The cells were grownover 0.38% agar in medium along with vehicle (DMSO) only orspecified concentration of apigenin. The number of colonies wasrecorded after 14 days after treatment. (A) Number of colonies onsoft agar. Each data represent the mean� SE of three differentassays. *P< 0.01 and **P< 0.001 versus control. (B) Photograph ofthe colonies (�400 magnification). The details are described underMaterials and Methods.


The cells were treated with 5–20 mM of apigenin for24 h. As shown by data in Figure 7, apigenintreatment resulted in 6.5, 12.2, and 14.8 of apoptoticcells at 5- 10-, and 20-mM. While induction ofapoptosis was almost negligible (3.4% compared to1.5% of control) at the lowest dose of 1 mM (data notshown), the highest dose (20 mM) resulted in asignificant increase in apoptosis as determined byflow cytometry.

Apigenin Alters Bcl2 Protein Expression in DU145 Cells

Bax and Bcl2 are known to play a crucial role inapoptosis [41]; therefore, we next studied the dose-

and time-dependent effects of apigenin on constitu-tive protein levels of Bax and Bcl2 in DU145 cells. Theprotein expression of Bcl2 was significantly decreasedby apigenin treatment at 10- and 20-mM concentra-tion (Figure 8A). Similar decrease in Bcl2 proteinexpression was observed during time-dependentstudy with 10 mM concentration of apigenin for 24,48, and 72 h (Figure 8A). However, we did not observeany significant alterations in level of Bax afterapigenin treatment (Figure 8A). Overall, treatmentof cells with apigenin resulted in a significant dose-and time-dependent shift in Bax/Bcl2 ratio, indicat-ing the induction of apoptotic process (Figure 8B).

Figure 4. Effect of apigenin on the DNA cell-cycle analysis ingrowing DU145 cells. The cells were treated with vehicle (DMSO)only or specified concentrations of apigenin for 24 h, stained with PI(50 mg/mL) and analyzed by flow cytometry. Percentage of cells inG0-G1, S, and G2-M phase was calculated with cell-fit computer

software and are represented in the right side of the histograms. Thedetails are described under Materials and Methods. Data shown hereare from a representative experiment repeated three times with lessthan 10% variation.


Apigenin Induced Cytochrome c Release, Induction of

Apaf-1, and Cleavage of Caspase-9, -3, and PARP inDU145 Cells

The process of cell death may involve disruption ofmitochondrial function through the release ofcytochrome c which interacts with Apaf-1 leadingto caspase activation and subsequent apoptosis [41].Treatment of DU145 cells with apigenin (1–20 mM)resulted in a dose-dependent increase in the proteinexpression of cytochrome c, Apaf-1, and the cleavedproduct of caspase-9, -3, and PARP (Figure 9). This

effect was more pronounced at �10 and �20 mM ofapigenin. Similar time-dependent inhibition wasobserved in cytochrome c, Apaf-1, and the cleavedproduct of caspase-9, -3, and PARP after treatment ofcells with 10mM concentration of apigenin for 24, 48,and 72 h (Figure 9).

Apigenin Inhibits Constitutive-NF-kB Expression inDU145 Cells

In the next series of experiment, we investigatedthe effect of apigenin on constitutive expression ofNF-kB/p65, NF-kB/p50, and IkBa in DU145 cells. Anumber of studies have suggested the role of NF-kBin cell survival by inhibiting apoptosis and by

Figure 5. Effect of apigenin on protein expression of (A) cyclin D1,cyclin D2, and cyclin E. (B) Cdk2, Cdk4, and Cdk6. (C) WAF1/p21,KIP1/p27, INK4a/p16, and INK4c/p18 in DU145 cells. The cells weretreated with vehicle (DMSO) only or specified concentrations ofapigenin for 24 h and then harvested. For time-dependent studies,the cells were treated with vehicle only or apigenin (10 mM) forspecified times and then harvested. The details are described underMaterials and Methods. The values below the figures representchange in protein expression of the bands normalized to a-tubulin.

Figure 6. Effect of apigenin on the induction of apoptosis inDU145 cells. (A) DNA fragmentation induced by apigenin in DU145cells. For dose-dependent studies, the cells were treated with vehicle(DMSO) only or with the specified concentrations of apigenin for48 h. For time-dependent studies, the cells were treated with vehicleonly or apigenin (10 mM) for specified times and then harvested. DNAwas isolated and subjected to agarose gel electrophoresis, followedby visualization of bands and polaroid photography. (B) Fluorescencemicroscopy of DU145 cells treated with apigenin. The cells weretreated with vehicle alone or specified concentration of apigenin for24 h. The ApopNexin apoptosis detection kit (Oncor, Gaithersburg,MD) was used for the detection of apoptotic and necrotic cells.Apoptosis and necrosis were detected by the kit according to thevendor’s protocol with a Zeiss Axiovert 100 microscope. The sampleswere excited at 330–380 nm, and the image was observed andphotographed under a combination of a 400-nm dichoric mirror andthen 420-nm high-pass filter. The details are described underMaterials and Methods.


regulating multitude of critical genes responsible forcell proliferation, metastasis, and chemotherapyresistance [42, 43 and references therein]. EmployingWestern blot analysis, we investigated the effect ofapigenin (1–20 mM concentration for 24 h) onconstitutively expressed NF-kB/p65, and NF-kB/p50in nuclear fractions obtained from these cells. Asshown in Figure 10, apigenin treatment resulted in adose- and time-dependent inhibition of constitutiveNF-kB/p65, NF-kB/p50 protein expression in thesecells. The magnitude of change in protein expressionof NF-kB/p65 was more than NF-kB/p50. Almostsimilar results were observed with NF-kB expressionin the cytosolic fractions of cells treated withapigenin (data not shown). The inhibition of NF-kBin the nuclear fraction correlated with increase inprotein expression of IkBa in cytosol both in dose-and time-dependent fashion (Figure 10).

DISCUSSION

The search for promising agents that could reducethe incidence and burden of cancer has becomeincreasingly important in recent years [44,45].Immense interest has been generated for biologicallyactive dietary supplements in view of their putativerole in attenuating the risk of developing cancer [8–10,44,45]. In this regard, apigenin is a promisingagent that has shown to afford protection againstseveral major human epithelial and nonepithelialcancers [8,10]. Apigenin was first evaluated in vitroand reported to inhibit carcinogen-induced bacterialmutagenesis, and in vivo tumor promotion on 12-O-tetradecanoylphorbol-13-acetate-induced ornithinedecarboxylase activity [46]. Subsequently, divergentbeneficial modulating effects of apigenin on varioustypes of diseases have been reported in the literature

Figure 7. Effect of apigenin on quantitation of apoptosis inDU145 cells. The cells were treated with vehicle (DMSO) only orwith the specified concentrations of apigenin for 24 h, labeled withdeoxyuridine triphosphate with terminal deoxynucleotide transferaseand PI by with apoptosis kit obtained from Phoenix Flow Systems(San Diego, CA) as per vendor’s protocol followed by flow cytometry.

Cells showing deoxyuridine triphosphate fluorescence above that ofcontrol population, as indicated by the line in each histogram, wereconsidered as apoptotic cells and their percentage population isshown in each box. The data shown here are from a representativeexperiment repeated three times with less than 10% variation. Thedetails are described under Materials and Methods.


[8–20]. Recently, we have shown selective growthinhibitory and apoptotic response of apigenin inhuman prostate carcinoma cells but not in normalcells [27]. More recently, we have provided themolecular understanding of these effects on andro-gen-sensitive human prostate carcinoma LNCaPcells [32]. Further, the role of apigenin on hor-mone-refractory prostate cancer needs to beaddressed as most prostate cancers respond initiallyto androgen ablation and the residual androgen-refractory cells recolonize, expand and ultimatelyestablish hormone-resistant state [4,5]. The recur-rence of androgen independence often leads togenetic alterations in cells marked by loss of functionor mutation of tumor suppressor genes and onco-genes transforming these cells to evade apoptosisand chemo-resistance [6,7]. Like other cancers, lossof various oncogenes and tumor suppressor geneshas been identified in prostatic tumors includingmutations in p53 and pRb [47,48]. In this regard,DU145 cells are a unique in vitro model of prostatecancer that is highly tumorigenic and resistant tochemotherapeutic agents [33,34]. Importantly, thiscell line has several nonfunctional genes responsiblefor cell-cycle control and carries mutation in p53 andpRb [35,36]. By using DU145 cells, we have shownthat apigenin has potential to inhibit growth andcolony formation even with these defects andcharacteristics.

Figure 8. Effect of apigenin on protein expression of (A) Bax andBcl2 in DU145 cells. The cells were treated with vehicle (DMSO) onlyor specified concentrations of apigenin for 24 h and then harvested.For time-dependent studies, the cells were treated with vehicle onlyor apigenin (10 mM) for specified times and then harvested. Thedetails are described under Materials and Methods. The values belowthe figures represent change in protein expression of the bandsnormalized to a-tubulin. (B) Dose- and time-dependent effect ofapigenin on Bax/Bcl2 ratio in DU145 cells.

Figure 9. Effect of apigenin on the protein expression ofcytochrome c, apoptotic protease-activating factor-1 (Apaf-1),caspase-9,-3, and poly(ADP-ribose) polymerase (PARP) cleavage inDU145 cells. The cells were treated with vehicle (DMSO) only orspecified concentrations of apigenin for 24 h and then harvested. Fortime-dependent studies, the cells were treated with vehicle only or

apigenin (10 mM) for specified times and then harvested. Antibodyfor caspase-3 specifically recognizes the pro-(32 kDa) and cleaved-(17 kDa) form, whereas for caspase-9 the antibody recognizes thecleaved (active) product at 35 kDa. The details are described underMaterials and Methods. The values below the figures representchange in protein expression of the bands normalized to a-tubulin.


Studies from our laboratory [27,32] and elsewhere[30,31,49,50] have shown involvement of cell-cycleregulation as a mechanism of cell growth andproliferation; therefore, we investigated the involve-ment of cki-cyclin-cdk machinery during inductionof cell-cycle arrest by apigenin in DU145 cells. Inrecent years, studies have shown an associationbetween cell-cycle regulation and cancer, and inhi-bition of cell cycle has become an appreciated targetfor management of cancer [39,40]. In eukaryotes,passage through the cell cycle is governed byfunction of a family of protein kinase complexesthat is controlled in part by a family of protein kinasecomplex minimally of a catalytic subunit, the cdk,and its essential activating partner, the cyclin [40].Under normal conditions, these complexes areactivated at specific intervals, and through a seriesof events, results in progression of cells throughdifferent phases of the cell cycle, thereby ensuringnormal cell growth. Any defect in this machinerycauses an altered cell-cycle regulation that may resultin unwanted cellular proliferation ultimately culmi-nating in development of cancer [39,51]. Cyclins Dand E along with cdk2, cdk4, or cdk6 are driving forcefor the G1! S phase of cell cycle [52]. Overexpressionof cyclin D is associated with various cancers andtumor-derived cell lines associated with dysregu-lated growth [52,53]. A significant decrease inprotein expression of cyclins D1, D2, and E andcdk2, cdk4, and cdk6 by apigenin suggest itspotential in inhibition of hormone-refractory pros-tate carcinoma cells. This observation is of particular

significance in suggesting apigenin as a promisingagent for prevention and therapy of prostate cancer.

During the progression of the cell cycle, the cdk-cyclin complexes are inhibited via binding to ckissuch as CIP/KIP and INK4 families of proteins[39,40,51]. Numerous studies have shown that theseckis regulate progression of cells in G0/G1-phase andan induction of these molecules causes a blockade ofG1! S transition thereby resulting in a G0/G1-phasearrest of cell cycle [39,40,51–53]. Because our studieshave demonstrated that apigenin treatment ofDU145 cells resulted in a G1-phase arrest, weexamined the effect of apigenin on ckis operativein the G1 phase of cell cycle. Our data demonstrated asignificant upregulation of WAF1/p21, KIP1/p27,and a modest increase in INK4a/p16, and INK4c/p18during G1-phase arrest of these cells by apigenin. Themost important member of cki family is WAF1/p21,which is regarded as an almost universal inhibitor ofcdks and a downstream target of p53 [54]. Manystudies have shown that exogenous stimuli mayresult in p53-dependent as well as p53-independentinduction of WAF1/p21, which may cause a blockadeof G1! S-phase transition resulting in a G1-phasecell-cycle arrest and apoptosis [54,55]. In the absenceof active p53 and pRb in DU145 cells, the observedinduction of WAF1/p21 by apigenin appears to beindependent of wild-type p53 and pRb status.However, the role of mutant p53 and pRb duringthis process remains to be examined.

Because cell-cycle arrest may lead to induction ofapoptosis, in the next series of the experiment wedetermined the extent of apoptosis caused byapigenin to human prostate carcinoma DU145 cells.Apoptosis is a physiological process by which cellsare removed when an agent damages their DNA [5].Apoptosis represents a discrete manner of cell deaththat differs from necrotic cell death and is regarded asan efficient way to eliminate damaged cells [56,57].Agents that can modulate apoptosis may be able toaffect steady-state cell population, which may beuseful in management and therapy of cancer [57].Our data demonstrated that apigenin resulted inapoptosis of androgen-refractory DU145 cells. Thisobservation was verified by DNA fragmentation,PARP-cleavage, fluorescence microscopy, and flowcytometry. This could be an important observationas modulation of apoptotic response represents anovel mechanism-based approach for preventionand treatment of prostate cancer.

In recent years, Bcl2 family members have beendesignated as critical regulators of apoptotic path-way [41]. Bcl2 has been found in inappropriatelyhigh levels in more than half of all human tumorsand carcinoma cells [58]. Overexpression of Bcl2results in resistance to apoptosis that may lead tochemo-resistance [41,58]. Bcl2 has been shown toform a heterodimer with the proapoptotic memberBax, thereby neutralizing its effects. Importantly, the

Figure 10. Effect of apigenin on the constitutive expression ofnuclear factor-kappaB (NF-kB) in DU145 cells. Protein expression ofNF-kB/p65 and NF-kB/p50 in the nuclear fraction and IkBa in thecytosolic fractions of various treated samples are shown. The cellswere treated with vehicle only or specified concentrations ofapigenin for 24 h and then processed for nuclear and cytosolicfractions. For time-dependent studies, the cells were treated withvehicle only or apigenin (10 mM) for specified times and processed fornuclear and cytosolic fractions. The details are described underMaterials and Methods. The values below the figures representchange in protein expression of bands compared to control. Datashown here are from a representative experiment repeated twicewith similar results.


ratio of Bax/Bcl2 dictates a cell’s susceptibility toundergo apoptosis under experimental conditions[5,41,58]. Alterations in the Bax/Bcl2 ratio results inthe release of cytochrome c, which activates Apaf-1,allowing it to bind to and activate caspase-9.Caspase-9 then cleaves and activates downstreamcaspases, resulting in apoptosis [58]. Importantly,the release of cytochrome c from the mitochondriahas been shown to be an almost universal phenom-enon during apoptosis [58]. In the present study, adecrease in Bcl2 protein observed in DU145 cells afterapigenin treatment, accompanied by the release ofcytochrome c, induction of Apaf-1 and activation ofcaspase-9 and -3 dictated the passage of cells towardsapoptosis. These results suggested that apigenin wascapable of disrupting mitochondrial functions inthese cells.

Previous studies from our laboratory and else-where have demonstrated the ability of apigenin todownregulate NF-kB expression in vitro, leading tothe speculation that it would in turn inhibit criticalgenes regulated by NF-kB and those involved inproliferation and apoptosis [32]. NF-kB is a widelydistributed pleiotropic nuclear factor that is knownto regulate expression of genes responsible for cellproliferation, apoptosis, metastases, and chemother-apy resistance [42,43]. Aberrant NF-kB activation hasbeen associated with pathogenesis of several dis-eases, including cancer [59]. Elevated NF-kB activityhas been shown to activate expression of prostate-specific antigen and facilitates prostate carcinomacells achieving androgen-independent status [60]. Inprostate carcinoma cells, NF-kB is constitutivelyactivated and has shown to promote cell survivalby inhibiting apoptosis and promoting cell invasionand angiogenesis [61]. More recently, NF-kB has beenincreasingly appreciated as a target for anticancerdrug treatment [62]. Our results demonstrated adose- and time-dependent decrease in constitutiveexpression of NF-kB/p65 and NF-kB/p50 in cytosolicand nuclear fraction with subsequent increase inprotein expression of IkBa in cytosol with apigenin.These results corroborate the findings of publishedstudies, as apigenin mediated downmodulation ofNF-kB expression could lead to dysregulation of cellcycle and induction of apoptosis.

Epidemiological studies suggest that regular con-sumption of fruits and vegetables, particularlycruciferous vegetables rich in flavonoids, may reducethe risk of prostate cancer [8–10]. Based on averageintake of flavonoids which ranges from 6 to 64 mgper day [10,63], the concentrations used in ourpresent study are physiologically achievable inhumans. However, limit of flavonoid absorptionand lack of in vivo data raises an important issueconcerning the use of apigenin for prevention andtherapy of cancer [63,64]. Understanding apigenin-mediated molecular events could play a potentiallyimportant role in setting up strategies for prevention

and treatment of prostate cancer. Further mechan-ism-based studies on anticancer activities withapigenin in animal models are warranted.

ACKNOWLEDGMENTS

This work was supported by grants from UnitedStates Public Health Service CA 94248 and CA 99049and funds from Cancer Research Foundation ofAmerica.

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