Apigenin Inhibits VEGF and HIF-1 Expression Via

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The FASEB Journal Research Communication

Apigenin inhibits VEGF and HIF-1 expression viaPI3K/AKT/p70S6K1 and HDM2/p53 pathways

Jing Fang, Chang Xia, Zongxian Cao, Jenny Z. Zheng, Eddie Reed, and Bing-Hua Jiang1

The Mary Babb Randolph Cancer Center, Department of Microbiology, Immunology and CellBiology, West Virginia University, Morgantown, West Virginia, USA

ABSTRACT Apigenin is a nontoxic dietary flavonoidthat has been shown to possess anti-tumor propertiesand therefore poses special interest for the develop-ment of a novel chemopreventive and/or chemothera- peutic agent for cancer. Ovarian cancer is one of themost common causes of cancer death among women.Here we demonstrate that apigenin inhibits expressionof vascular endothelial growth factor (VEGF) in humanovarian cancer cells. VEGF plays an important role in

tumor angiogenesis and growth. We found that apige-nin inhibited VEGF expression at the transcriptionallevel through expression of hypoxia-inducible factor1 (HIF-1). Apigenin inhibited expression of HIF-1and VEGF via the PI3K/AKT/p70S6K1 and HDM2/ p53 pathways. Apigenin inhibited tube formation invitro by endothelial cells. These findings reveal a novelrole of apigenin in inhibiting HIF-1 and VEGF expres-sion that is important for tumor angiogenesis andgrowth, identifying new signaling molecules that medi-ate this regulation.Fang, J., Xia, C., Cao, Z., Zheng,J. Z., Reed, E., Jiang, B.-H. Apigenin inhibits VEGF andHIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/

p53 pathways. FASEB J. 19, 342353 (2005)

Key Words: vascular endothelial growth factor ovarian can-cer hypoxia inducible factor 1 tumor growth HUVEC

Ovarian cancer is the leading cause of death fromgynecological malignancy and the fourth most com-mon cause of cancer death among American women(1). The 5-year survival rate of ovarian cancer is 30%for advanced stage disease (2). The symptoms of ovar-ian cancer are generally observed only after the cancerhas spread to the surface of the peritoneal cavity. At this

stage, it is impossible to surgically remove all apparentlesions, and this accounts for the high rate of cancerrecurrence after surgery; hence, ovarian cancer is aprime target in chemoprevention research. Althoughthe mechanism(s) involved in the progression of ovar-ian cancer are still unclear, increasing evidence incancer prevention literature point to a role of autocrineand paracrine factors in the development of ovariantumorigenesis, indicating that the vascular endothelialgrowth factor (VEGF) plays an important role in ovar-ian cancer development.

VEGF plays a critical role in tumor angiogenesis.

Angiogenesis is the formation of new blood vesselsfrom preexisting ones and is required for tumor growthand metastasis (3). Tumor angiogenesis is stimulated byangiogenic growth factors like VEGF, basic fibroblastgrowth factor (bFGF), transforming growth factor(TGF), and interleukin 8 (IL-8). VEGF and its receptorshave been described as the fundamental regulators ofangiogenesis (4) and play an important role in tumorprogression (5, 6). VEGF expression and its receptor

function are required for tumor growth, invasion, andmetastasis in animal models (710). VEGF and VEGFreceptors were found to be expressed in human ovariancarcinoma (11, 12). Inhibition of VEGF blocked thecell proliferation of ovarian cancer cells in vitro andtumor growth in vivo through an autocrine mechanism(12, 13). VEGF-trap decreases tumor burden and inhib-its ascites formation (14). However, overexpression of VEGF significantly enhanced cell survival after growthfactor withdrawal and provided resistance to apoptosisinduced by cisplatin (13). Thus, inhibiting the role ofVEGF in promoting angiogenesis and tumor growth isbecoming a target for ovarian cancer therapy.

Hypoxia inducible factor 1 (HIF-1) activates thetranscription of many genes, including VEGF (15).HIF-1 activates the expression of the VEGF gene bybinding to the hypoxia response element (HRE) in theVEGF promoter (16). HIF-1 is overexpressed in manyhuman cancers (17), and levels of its activity in cellscorrelate with tumorigenicity and angiogenesis (18).HIF-1 is composed of HIF-1 and HIF-1 subunits (19,20). In most experimental systems, the HIF-1 proteinlevels are constitutively expressed but rapidly degradedby the ubiquitin-proteasome pathway under normoxia(21, 22), a process mediated by specific binding of

pVHL, the product of the von HippelLindau (VHL)tumor suppressor gene (23). By controlling HIF-1pVHL physical interaction, prolyl hydroxylation ofHIF-1 is critical in the regulation of HIF-1 steady-state levels (24, 25). Under hypoxia, the absence ofoxygen prevents the hydroxylases from modifying HIF-

1 Correspondence: MBR Cancer Center, Department ofMicrobiology, Immunology and Cell Biology, West VirginiaUniversity, Morgantown, WV 26506-9300, USA. E-mail:[email protected]

doi: 10.1096/fj.04-2175com

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1; therefore pVHL fails to recognize HIF-1, allowingHIF-1 to accumulate.

The regulation of HIF-1 can also be independent ofthe oxygen environment. Oncogenic mutations such asthe loss of function of VHL (23), p53 (26), and PTEN(27) induce HIF-1 expression. Growth factors, cyto-kines, and other signaling molecules can stimulateHIF-1 protein synthesis via activation of the phospha-tidylinositol 3-kinase (PI3K)/AKT or mitogen-activatedprotein kinase (MAPK) pathways (2830). PI3K is aheterodimeric enzyme composed of a 110 kDa catalyticsubunit and an 85 kDa regulatory subunit (31). Thebest-known downstream target of PI3K is the serine-threonine kinase AKT, which transmits survival signalsfrom growth factors (32, 33). We and others recentlydemonstrated that PI3K/AKT signaling is required forVEGF expression through HIF-1 in response to growthfactor stimulation and oncogene activation (27, 28,3436).

Apigenin (4,5,7,-trihydroxyflavone) is a commondietary flavonoid. It has low toxicity, is nonmutagenic,and is widely distributed in many fruits and vegetables

including parsley, onions, oranges, tea, chamomile,wheat sprouts, and in some seasonings (37). Apigenin isused as a healthy food supplement and has recentlybeen shown to possess anti-tumor properties (3841).Nevertheless, its mechanism is unclear. In ovariancancer, the catalytic subunit p110 of PI3K is increasedin copy numbers (42), and PI3K catalytic subunitexpression positively correlated with the expression ofVEGF in ovarian cancers (43). VEGF and HIF-1 areboth expressed in epithelial ovarian cancer; VEGFexpression correlates with HIF-1 expression, suggest-ing that HIF-1 contributes to the overexpression ofVEGF in ovarian cancer (44). In this study, we have

used ovarian cancer cells A2780/CP70 and OVCAR-3 asa model system to investigate the mechanism of theanti-tumor properties of apigenin. We found that api-genin significantly inhibited the expression of HIF-1and of VEGF in the ovarian cancer cells. Therefore, weinvestigated the possible mechanism by which apigenininhibited VEGF production.

MATERIALS AND METHODS

Reagents and antibodies

The apigenin, cycloheximide (CHX), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and N,N-di-methylformamide were purchased from Sigma (St. Louis,MO, USA). Apigenin was dissolved in DMSO and stored at20C. Antibodies against HIF-1 and HIF-1 were from BDBiosciences (Bedford, MA, USA). The antibodies againstphospho-AKT (Ser473), AKT, phospho-ERK1/2 (extracellu-lar signal-related protein kinases 1/2), ERK1/2, and phos-pho-HDM2 were from Cell Signaling (Beverly, MA, USA).The antibodies against p53, HDM2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased fromSanta Cruz (Santa Cruz, CA, USA). VEGF neutralizing anti-body, VEGF receptor KDR neutralizing antibody, and non-immune control IgG were purchased from R&D (Minneapo-

lis, MN, USA). The growth factor-reduced Matrigel was fromBD Biosciences.

Cell culture

The human ovarian cancer cells OVCAR-3 and A2780/CP70were cultured in RPMI 1640 medium supplemented with 10%heat-inactivated fetal bovine serum (FBS; Gibco BRL, GrantIsland, NY, USA), 100 u/mL penicillin, and 100 g/mLstreptomycin in 5% CO

2incubator at 37C. For cell culture

under hypoxia, the cells were grown in a chamber containing1% oxygen, 5% carbon dioxide, and 94% nitrogen at 37C.Human umbilical vein endothelial cells (HUVEC) were cul-tured in EBM-2 medium supplemented with EGM-2 Single-Quots. EBM-2 and EGM-2 SingleQuots were purchased fromCambrex Company (Walkersille, MD, USA).

Construction of plasmids

VEGF promoter reporter pGL-Stu1 containing a 2.65 KbKpnI-BssHII fragment of the human VEGF gene promoterand VEGF promoter reporter pMAP11wt, which contains only47 bp of VEGF 5-flanking sequence (from 985 to 939),

were cloned into the pGL2 basic luciferase vector as describedpreviously (16). The mutant VEGF promoter reporterpMAP11mut was constructed by introducing a 3-bp substitu-tion into pMAP11wt that abolishes the HIF-1 binding site(16). Plasmid encoding human HIF-1 was inserted intopCEP4 vector (16, 19). Plasmids encoding active myr-AKT(45), an active p110 subunit of PI3K, p110E227K (46), anactive p70S6K1 (47), and wild-type HDM2 (48) have beendescribed.

Immunoblot analysis

Cells at 8090% confluence were exposed to apigenin forspecific times. Cells were lysed on ice for 30 min in RIPAbuffer (100 mM Tris, 150 mM NaCl, 1% Triton, 1% deoxy-cholic acid, 0.1% SDS, 1 mM EDTA, and 2 mM NaF)

supplemented with 1 mM sodium vanadate, 1 mM leupeptin,1 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mMdithiothreitol, and 1 mM pepstatin A. The supernatant wascollected after centrifugation at 12,000 g for 15 min andprotein concentration was determined using protein assayreagent from Bio-Rad (Hercules, CA, USA). Equal amountsof protein were resolved on SDS-PAGE and transferred to anitro-cellulose membrane. Proteins of interest were detectedby immunoblotting using specific antibodies.

RNA isolation and Northern blot analysis

Cells at 8090% confluence were treated with apigenin for6 h. Total cellular RNA was extracted using Trizol reagent

(Invitrogen, Carlsbad, CA, USA) according to themanufacturer's instructions. Aliquots of total RNA were de-natured at 65C for 10 min and separated in agarose gel(1%). RNA was transferred to Hybond-N membranes from

Amersham Biosciences (Piscataway, NJ, USA) and cross-linked to the membrane by UV radiation. Human VEGFcDNA fragment was used as a probe. The probe was labeled

with [-32P]dATP using RadPrime DNA labeling system (In- vitrogen) and purified with the ProbeQuant G-50 Micro-columns from Amersham Biosciences. Hybridizations wereperformed at 48C in Northern Max hybridization bufferfrom Ambion (Austin, TX, USA). The membrane wasstripped off and probed with [-32P]-labeled HIF-1 cDNA.mRNA levels of GAPDH were used as the control.

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Transient transfection and luciferase assay

A2780/CP70 and OVCAR-3 cells were seeded in 6-well platesand cultured to 6070% confluence. To determine theeffects of apigenin on transcriptional activation of VEGF, cells

were transiently transfected with VEGF reporter and pCMV--galactosidase plasmid using Lipofectamine from Invitrogenaccording to the manufacturers instructions. The transfectedcells were cultured for 20 h, followed by incubation withapigenin for 15 h. Cells were washed once with phosphate-buffered saline (PBS) and lysed with Reporter Lysis Buffer

from Promega (Madison, WI, USA). Luciferase (Luc) activi-ties of the cell extracts were determined using the Luciferase

Assay System (Promega). -Galactosidase (-gal) activity wasmeasured in assay buffer (100 mM phosphate, pH 7.5, 2 mMMgCl

2, 100 mM -mercaptoethanol, 1.33 mg/mL o-nitrophe-

nyl -d-galactopyranoside). Relative Luc activity (defined asVEGF reporter activity) was calculated as the ratio of Luc/-gal activity.

Quantification of VEGF protein

VEGF protein was measured using the Quantikine humanVEGF ELISA kit from R&D Systems (Minneapolis, MN, USA), which had been calibrated against recombinant human

VEGF165. In short, the cells were seeded in 12-well plates andcultured to 90100% confluence. Cells were switched to freshmedium in the presence or absence of apigenin. In 15 h, thesupernatants were collected and cell numbers of each well

were counted. VEGF in the supernatant (100 L) was deter-mined and normalized to the remaining cell numbers. Aserial dilution of human recombinant VEGF was included ineach assay to obtain a standard curve.

Cell proliferation and cell death assay

Cell proliferation was determined using MTT reductionmethod. In brief, 100 L of the cells were cultured in 96-wellplates and treated with different concentrations of apigeninfor specified times. After treatment, 10 L of MTT reagent (5mg/mL) was added to each well. In 2 h, the reaction wasstopped by addition of 100 L of solubilization solution (50%N, N-dimethylformamide, 20% SDS). The absorbance at 590nm of solubilized MTT formazan products was measured in6 h. For determining cell death, cells were collected andstained with 0.4% of Trypan blue for 5 min at room temper-ature before examination under the microscope. The num-bers of viable cells were determined by Trypan blue exclu-sion. Dead cells stained blue were scored positive andcounted against the total number of cells to determine thepercentage of cell death.

Tube formation assay

We determined the tube formation of endothelial cells in thepresence of conditioned medium prepared from A2780/CP70 cells. A2780/CP70 cells were cultured to 90100%confluence. The old medium was discarded and the cells wereprovided with serum-reduced medium (1% FBS) in thepresence or absence of apigenin (10 M). The cells wereincubated for 15 h and the medium was collected and storedat 80C. HUVEC cells at subconfluence were switched toEBM-2 basic medium containing 0.2% FBS. In 24 h, thestarved HUVEC cells were trypsinized, collected, counted,and resuspended in EBM-2 basic medium. The cells weremixed with equal volume of the conditioned medium andseeded to Matrigel-pretreated 96-well plate at 104 cells/well.In 18 h, tube formation was examined under light micro-

scope. The length of the tubes was measured using the SoftImaging System (Soft Imaging System GmbH, Germany). Topretreat the 96-well plate, 50 L of growth factor-reducedMatrigel thawed on ice was added to each well. The plate wasthen placed in an incubator to allow the gel to solidify at 37Cfor 1 h.

Statistical analysis

The data represent mean se from three independent

experiments except where indicated. Statistical analysis wasperformed by Students t test at a significance level of P0.05.

RESULTS

Apigenin inhibited VEGF expression

We determined the effects of apigenin on VEGF mRNAlevels in ovarian cancer cells. As shown in Fig. 1A, B,apigenin inhibited expression of VEGF mRNA in adose-dependent manner in A2780/CP70 and OVCAR-3

cells. The ELISA data indicated that apigenin inhibitedproduction of VEGF protein in both cell lines (Fig. 1C).To exclude the possibility that the decrease in VEGFproduction is due to inhibition of cell proliferation, wedetermined the cell proliferation by an MTT methodunder the same experimental conditions. Apigenintreatment for 15 h had little effect on proliferation ofA2780/CP70 and OVCAR-3 cells (Fig. 1D). We detectedthe effects of apigenin on cell viability and did notobserve any difference of cell viability between thecontrol and apigenin-treated groups (data not shown).These results suggest that the inhibition of VEGFproduction by apigenin is not through inhibition of cell

proliferation or death.To determine whether apigenin inhibits VEGF tran-

scriptional activation, we tested the effect of apigeninon VEGF promoter reporter containing a 2.65 kbfragment of human VEGF gene promoter (16) inA2780/CP70 ovarian cancer cells, and found that api-genin at 10 M inhibited significantly the VEGF re-porter activity (Fig. 2A). Overexpression of HIF-1completely reversed the apigenin-inhibited reporteractivity (Fig. 2A). To determine whether apigenininhibits VEGF expression through HIF-1 DNA bindingsite of VEGF promoter, we analyzed the effects ofapigenin on a VEGF promoter reporter, pMAP11wt,

containing the HIF-1 binding site. Apigenin inhibitedthe pMAP11wt reporter activity in A2780/CP70 andOVCAR-3 cells in a dose-dependent manner (Fig. 2B).Overexpression of HIF-1 reversed the pMAP11wt re-porter activities inhibited by apigenin (Fig. 2C), sug-gesting that apigenin inhibits VEGF transcriptionalactivation via HIF-1 DNA binding site and HIF-1protein expression. Next, we introduced 3 bp substitu-tion at the HIF-1 binding site to generate a mutantpMAP11mut VEGF promoter reporter (16) and testedthe effect of apigenin on this reporter activity. Treat-ment of apigenin did not inhibit the mutant VEGF

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reporter activity (data not shown), further indicatingthat apigenin affects the VEGF reporter activitythrough the HIF-1 binding site.

Apigenin inhibited HIF-1 expression

To determine whether apigenin regulates VEGF ex-pression through HIF-1 expression, we examined theeffects of apigenin on HIF-1 and HIF-1 proteinlevels. Expression of HIF-1 in both A2780/CP70 andOVCAR-3 cells was inhibited by apigenin in a dose- andtime-dependent manner (Fig. 3). However, apigeninhad no effect on HIF-1 protein levels (Fig. 3). Theseresults suggest that apigenin inhibits VEGF transcrip-tional activation by specifically inhibiting HIF-1 butnot HIF-1 expression.

We determined the possible mechanism by which

apigenin inhibits HIF-1 expression. Apigenin hadsome effect on the HIF-1 mRNA levels in ovariancancer cells (Fig. 4A). We next determined the effectsof apigenin on the stability of HIF-1 protein usingCHX to inhibit new protein synthesis in the cells.A2780/CP70 cells were treated with CHX or CHX plusapigenin as indicated in Fig. 4B. The half-life of HIF-1protein in the cells was 8 min when the cells weretreated with CHX alone (Fig. 4B) and 5 min when cells were pretreated with apigenin (Fig. 4B). These datasuggest that apigenin inhibits HIF-1 expression par-tially through decreasing HIF-1 protein stability.

Apigenin inhibited VEGF transcriptional activationthrough the PI3K/AKT/p70S6K1 pathway

Recent studies indicate that expression of HIF-1 andVEGF can be regulated through the PI3K/AKT path-

way (28, 29, 3436). To know whether apigenin inhibitsexpression of HIF-1 and VEGF through this signalingpathway, we determined the effects of apigenin onactivation of AKT by immunoblotting of phospho-AKT. We found that apigenin inhibited AKT phosphoryla-tion in both A2780/CP70 and OVCAR-3 cells (Fig. 5).We determined the effects of apigenin on phosphory-lation of ERK1/2, two kinases of the MAPK signalingpathway, as ERK1/2 has been implicated in the regula-tion of HIF-1 expression (36, 49). Incubation of cells with apigenin resulted in no significant change ofphospho-ERK1/2 levels (Fig. 5). These results suggestthat apigenin affects HIF-1 and VEGF expression

through PI3K/AKT, but not ERK1/2, in ovarian cancercells.

To confirm that apigenin inhibits VEGF expressionthrough the PI3K/AKT pathway, A2780/CP70 cellswere cotransfected with VEGF promoter reporter andactive forms of p110, the catalytic subunit of PI3K, ormyr-AKT construct. Forced expression of p110 ormyr-AKT reversed the apigenin-inhibited reporter ac-tivity (Fig. 6A), indicating that the inhibition of VEGFtranscriptional activation by apigenin is via the PI3K/ AKT pathway. p70S6K1 is a downstream target ofPI3K/AKT. We found that apigenin inhibited phos-

Figure 1. Apigenin decreased VEGF expression in ovarian cancer cells. A2780/CP70 (A) and OVCAR-3 (B) ovarian cancer cellswere treated with apigenin for 6 h. Cells treated with solvent DMSO alone were used as the control. Total RNA was isolated andVEGF mRNA was detected as described in Materials and Methods. GAPDH mRNA was detected and used as control. Left panelsshow Northern blot of VEGF mRNA. Right panels show relative VEGF mRNA levels. Values are mean from 2 experiments. C)

A2780/CP70 and OVCAR-3 cells were plated in 12-well plates and cultured to 90100% confluence. The cells were switched tofresh medium with or without apigenin and incubated for 15 h. The concentrations of VEGF in the supernatants weredetermined by ELISA. Data are mean se from 3 independent experiments; each experiment was performed with triplicatecultures. *Significantly decreased VEGF compared with that of control, P0.05. D) A2780/CP70 and OVCAR-3 cells werecultured to 90100% confluence. The cells were provided with fresh medium (with apigenin) and incubated for 15 h. Cellproliferation was determined as described in Materials and Methods.



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phorylation of p70S6K1 (data not shown). Thus, wehypothesized that p70S6K1 was involved in the inhibi-tion of VEGF expression by apigenin. To test this, A2780/CP70 cells were transfected with the VEGF

promoter reporter and a constitutively active form ofp70S6K1 (CA-p70S6K1) plasmid. As shown in Fig. 6A,forced expression of p70S6K1 reversed the apigenin-inhibited VEGF transcriptional activation. To knowwhether the reverse of VEGF transcriptional activationis via expression of HIF-1, we determined the effectsof overexpression of these constructs on HIF-1 pro-tein levels. A2780/CP70 cells were transfected with theempty vector, p110, Myr-AKT, or CA-p70S6K1 plas-mid, and treated with apigenin. Forced expression ofp110, AKT, or p70S6K1 in the cells increased the

levels of HIF-1 protein inhibited by apigenin (Fig. 6B).These results suggest that apigenin blocks VEGF tran-scriptional activation by inhibiting HIF-1 expressionthrough the PI3K/AKT/p70S6K1 pathway.

Apigenin inhibited VEGF expression through HDM2/p53 expression

p53 functions to promote degradation of HIF-1 (26).This prompted us to determine whether apigenin in-hibits HIF-1 and VEGF through p53. We first deter-mined the effects of apigenin on p53 expression inA2780/CP70 cells. Apigenin induced p53 protein ex-pression in a dose- and time-dependent manner (Fig.7A, B). As is well known, the amount of p53 protein in

Figure 2. Apigenin inhibited VEGF transcriptional activation via HIF-1. Cells were transfected with the vectors indicated andcultured for 20 h, followed by treatment with apigenin for 15 h. Cells were lysed and the supernatants were subjected to Lucand -gal activity assay. Relative Luc activities in the cell extracts were assayed by the ratio of Luc/ -gal activity and normalizedto the value in the solvent DMSO control. A)A2780/CP70 cells were transfected with 1 g of pGL-Stu1 VEGF promoter reporter,0.3 g of-gal plasmid, and 0, 0.5 or 1 g of plasmid encoding wild-type HIF-1. The empty vector was added to make total

transfected DNA 2.3 g. 10 M of apigenin was used to treat the cells. #Significantly decreased activity compared with that ofpGL-StuI reporter plus empty vector, P0.05. *Significant increase of activity compared with that of reporter empty vectorin the presence of apigenin, P0.05. B)A2780/CP70 and OVCAR-3 cells were transfected with 1 g of pMAP11wt reporter 0.3 g of-gal plasmid. *P0.05 vs. control. C) Cells were transfected with 1 g of the pMAP11wt reporter, 0.3 g of-galplasmid, and 0.5 g (or 1 g) of plasmid encoding wild-type HIF-1. The empty vector was added, if necessary, to make totaltransfected DNA 2.3 g. The concentration of apigenin used was 10 M. #P0.05 vs. pMAP11wt empty vector. *P0.05 vs.pMAP11wt empty vector in the presence of apigenin. Data are mean se from 3 independent experiments. Each experiment

was performed with triplicate cultures.



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cells is determined mainly by the rate at which it isdegraded rather than the rate at which it is made.HDM2 is the protein that mediates p53 degradation bybinding p53 and stimulating the addition of ubiquitinto the carboxyl terminus of p53 for degradation. There-fore, we determined the effects of apigenin on expres-sion of HDM2 in A2780/CP70 cells. As shown in Fig.

7A, B, apigenin impaired the expression of HDM2protein. Similar results were obtained with OVCAR-3cells treated with apigenin (data not shown). Theseresults suggest that apigenin induces p53 throughdown-regulation of HDM2. It is reported that inhibi-tion of AKT activity will impair the phosphorylation ofHDM2, which may result in the destabilization ofHDM2 (50). In A2780/CP70 cells, addition ofLY294002 inhibited phosphorylation of AKT andHDM2 (Fig. 7C). The total HDM2 protein was de-creased whereas p53 expression was induced (Fig. 7C).These results suggest that the PI3K/AKT signaling playsan important role in regulating HDM2 and p53 expres-

sion in ovarian cancer cells. Apigenin could inhibit thephosphorylation of HDM2 by AKT in ovarian cancercells (Fig. 7D). Considering the inhibitory effects ofapigenin on phospho-AKT (Fig. 5) and HDM2 (Fig. 7A,B), our results suggest that apigenin regulates HDM2probably via PI3K/AKT signaling. Blockade of HDM2phosphorylation is a possible mechanism throughwhich apigenin decreased HDM2 protein levels.

Based on previous results (26) and our data, wehypothesized that HDM2/p53 signaling might be in-volved in the regulation of VEGF expression in ovarian

cancer cells. To confirm this, we determined the effectsof overexpression of HDM2 on the VEGF promoterreporter activities in A2780/CP70 cells. Forced expres-sion of HDM2 reversed the reporter activity inhibitedby apigenin (Fig. 7E), suggesting that HDM2 is in-volved in the regulation of VEGF expression by apige-nin. To ascertain whether this regulation is throughexpression of HIF-1, A2780/CP70 cells were trans-fected with HDM2 and treated with apigenin. As shown

in Fig. 7F, transfection of HDM2 increased HIF-1protein levels inhibited by apigenin. These results

Figure 4. Apigenin regulated HIF-1 expression partiallythrough decreasing protein stability. A)Effects of apigenin onmRNA levels of HIF-1. A2780/CP70 and OVCAR-3 cells

were exposed to apigenin for 6 h. Total RNA was isolated andmRNA levels of HIF-1 were detected by Northern blot. B)Effects of apigenin on the stability of HIF-1. To determinethe half-life of HIF-1, A2780/CP70 cells were incubated with

CHX (100 M) to inhibit new protein synthesis and cells wereharvested at different times. To determine the effects ofapigenin on half-life of HIF-1, cells were pretreated with 20M of apigenin for 30 min, followed by addition of CHX (100M). The cells were then harvested at different times, asindicated. HIF-1 was detected by immunoblotting withGAPDH as an internal control. Levels of HIF-1 protein weredetermined by measuring the density of the HIF-1 proteinband and normalized to that of GAPDH. The relative HIF-1protein level at time zero was defined as 1.0. The experiments

were performed 3 times and the data were mean se.*Significant difference (P0.05) when compared with thetreatment of CHX alone.

Figure 3. Apigenin inhibited HIF-1 protein expression. A2780/CP70 and OVCAR-3 ovarian cancer cells were cul-tured to 8090% confluence, followed by treatment withapigenin. The cells treated with solvent alone were used as acontrol. HIF-1 and HIF-1 protein levels were detected by

immunoblotting as described in Materials and Methods.GAPDH was used as an internal control to test loading andtransfer efficiency. A) A2780/CP70 and OVCAR-3 cells weretreated with apigenin (2.5, 5, 10, and 25 M) for 6 h. B)

A2780/CP70 and OVCAR-3 cells were exposed to 20 M ofapigenin for different times as indicated.



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suggest that HDM2 regulates VEGF transcriptionalactivation through expression of HIF-1 protein.

Apigenin inhibited HIF-1 and VEGF expressionunder hypoxia

The effect of apigenin on HIF-1 expression underhypoxia was determined in A2780/CP70 cells. As shownin Fig. 8A, apigenin inhibited HIF-1 expression in A2780/CP70 cells in response to 1% O2. We deter-mined the effects of apigenin on VEGF productionunder hypoxia. A2780/CP70 cells at subconfluencewere incubated with apigenin under 1% O2 for 20 h. Asshown in Fig. 8B, apigenin inhibited VEGF proteinlevels in A2780/CP70 in a dose-dependent mannerunder hypoxic condition.

Apigenin inhibited tube formation by HUVEC cells

induced by conditioned medium from ovarian cancercells

To determine whether apigenin has anti-angiogenicactivity, we performed tube formation assay, in vitro.The HUVEC cells in the basic medium could not formtubes (Fig. 9A). Tube formation was induced by condi-tioned medium prepared from A2780/CP70 cells (Fig.9B) and inhibited significantly by medium preparedfrom apigenin-treated cells (Fig. 9C). To test whetherthe presence of apigenin in the medium may result inthe inhibition of tube formation, conditioned medium

prepared from untreated cells was supplemented withapigenin, and used for tube formation assay. Additionof apigenin to the conditioned medium did not signif-icantly decrease the tube formation (Fig. 9D, H). Thisresult suggests that apigenin may inhibit the tubeformation by decreasing VEGF levels in the condi-tioned medium. To further determine whether theinhibition is due to VEGF expression, cells were incu-bated with conditioned medium in the presence ofVEGF or its receptor KDR neutralizing antibodies fortube formation assay. Addition of VEGF (Fig. 9E) orKDR (Fig. 9F) neutralizing antibodies impaired thetube formation whereas addition of the non-immunecontrol IgG (Fig. 9G) had no effect. Tube length wasanalyzed. Similar results were obtained from replicateexperiments (Fig. 9H). These results suggest that the

Figure 5. Apigenin inhibited activation of AKT. A) A2780/CP70 and OVCAR-3 cells were treated with apigenin (2.5, 5,10, and 20 M) for 6 h as described above. Aliquots ofproteins were resolved on SDS-PAGE gel and analyzed byimmunoblotting with antibodies against phospho-AKT(Ser473) or phospho-ERK1/2. Total AKT and ERK1/2 weredetected using antibodies against AKT and ERK1/2. B) Cells

were treated with apigenin (20 M). Phosphorylation of AKTand ERK1/2 were detected as described above.

Figure 6. Apigenin inhibited VEGF expression via PI3K/AKT/p70S6K1. A) A2780/CP70 cells were transfected withthe p11MAPwt VEGF promoter reporter (1 g), -gal plasmid(0.3 g), an active form of p110 (1 g), or myr-Akt (1 g),or a constitutively active form of p70S6K1 (CA-p70S6K1) (1

g). The empty vector was added to equal the amount of totalDNA transfected. The cells were then cultured for 20 h,followed by treatment with 10 M of apigenin for 15 h. Datarepresent mean se from 3 experiments; each experimenthad triplicate cultures. #Significant decreased activity com-pared with that of pMAP11wt empty vector, P 0.05.*Significantly increased activity vs. that of pMAP11wt empty

vector in the presence of apigenin, P0.05. B) A2780/CP70cells were seeded in 60 mm dishes. At 7080% confluence,the cells were transfected with 4 g of p110, myr-Akt, orCA-p70S6K1. Cells transfected with 4 g of empty vector wereused as the control. The cells were cultured for 20 h aftertransfection, followed by treatment with apigenin (25 M) for6 h.



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VEGF levels in the medium and VEGF functionthrough its receptor are significant and confirm thatapigenin inhibits tube formation by decreasing VEGFexpression.

DISCUSSION

Because there is a long latency period for the develop-ment of clinical ovarian cancer, studies of ovariancancer chemoprevention have intensified in recentyears, making ovarian cancer a prime target for chemo-prevention. Chemoprevention involves using nontoxic,naturally occurring or synthetic agents to prevent orinhibit human cancer development. Dietary and epide-miological studies have suggested that the dietary in-take of fruits and vegetables can reduce incidences ofmany types of cancer (5153). Flavonoids are abundantin our diet. Apigenin, the most common flavonoid, has

been shown to possess anti-tumor properties (3840).Most human tumors overexpress VEGF, which stimu-lates angiogenesis and tumor growth (6). Inhibition ofangiogenesis by targeting VEGF is becoming an impor-tant approach for cancer treatment. In this paper wehave demonstrated that apigenin impaired VEGF ex-pression in ovarian cancer cells.

We show here that apigenin inhibited VEGF expres-sion in ovarian cancer cells at the transcriptional levelthrough HIF-1 expression. HIF-1 plays an importantrole in VEGF transcriptional activation in ovarian can-cer cells (Figs. 2 and 3). Expression of HIF-1 isregulated via degradation and protein synthesis. Inmost experimental systems, HIF-1 protein subunitsare constitutively expressed but rapidly degraded by theubiquitin-proteasome pathway under normoxia (21,22). We found that OVCAR-3 and A2780/CP70 cellsexpressed constitutively elevated levels of HIF-1 pro-tein under normal conditions (54). Constitutively ele-

Figure 7. Apigenin inhibited VEGF transcriptional activation through HDM2 expression. A2780/CP70 cells were treated withapigenin as indicated. Cellular proteins were prepared and aliquots of proteins were separated on SDS-PAGE and analyzed byimmunoblotting with the antibodies indicated. A) Apigenin increased p53 expression but inhibited HDM2 expression in adose-dependent manner. The cells were incubated with apigenin (0, 5, 10, and 20 M) for 6 h. B) Apigenin induced p53expression but decreased HDM2 protein levels in a time-dependent manner. A2780/CP70 cells were treated with 20 M ofapigenin for different times, as indicated. C) A2780/CP70 cells were treated with 10 M LY294002 for 6 h. Levels of p-AKT,p-HDM2, HDM2, and p53 were determined. D) Apigenin decreased levels of phospho-HDM2. A2780/CP70 cells were treated

with different concentrations of apigenin for 1 h. E) A2780/CP70 cells were transfected with 1 g of pMAP11wt reporter, 0.3g of pCMV--gal, and 1 g of pCMV-HDM2. The empty vector was added to bring the total DNA to 2.3 g. After transfection,

the cells were incubated for 20 h, then exposed to 10 M of apigenin for 15 h. Data represent mean se from 3 experiments;each experiment had triplicate cultures. #P0.05 vs. pMAP11wt empty vector. *P0.05 vs. pMAP11wt empty vector in thepresence of apigenin. F) A2780/CP70 cells were seeded in 6-well plates and transfected with 1 g of pCMV-HDM2 plasmid or1 g of empty vector. The cells were cultured for 20 h and exposed to 25 M of apigenin for 6 h.



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vated levels of HIF-1 protein in ovarian cancer cells were observed in other labs (43, 55). The reason forappreciable levels of HIF-1 in these cells is unknown.The catalytic subunit p110 of PI3K is overexpressed in

more than 40% of ovarian cancers (42). We found thatOVCAR-3 and A2780/CP70 cells both had appreciablelevels of phospho-AKT (Fig. 5) (54). Therefore, onepossibility that would account for the elevated levels ofHIF-1 protein is the elevated PI3K/AKT signaling inthese cells under normal conditions. Studies of the roleof PI3K signaling in hypoxia-induced HIF-1 expres-sion were contradictory based on the cell lines used. Inprostate cancer cells, PI3K and AKT activities wereobserved to be required for HIF-1 expression (34, 56).However, in 1c1c7 mouse hepatocyte cells, inhibition ofPI3K activity did not affect hypoxia-induced HIF-1expression (57). In ovarian carcinoma, the catalytic

unit of PI3K is directly implicated in the control ofHIF-1 protein and VEGF expression (43). We foundin our lab that blockade of PI3K/AKT signaling by itsspecific inhibitor LY294002 significantly inhibitedHIF-1 protein levels, VEGF transcriptional activation,and VEGF protein levels in both OVCAR-3 and A2780/CP70 cells (data not shown). All this suggests that AKTactivity accounts for the expression of HIF-1 andVEGF in ovarian cancer cells. Apigenin inhibited phos-phorylation of AKT in A2780/CP70 and OVCAR-3 cells(Fig. 5). We therefore hypothesized that apigenin reg-ulated HIF-1 and VEGF expression through the PI3K/

AKT signaling. As expected, forced expression of PI3Kor AKT restored apigenin-inhibited VEGF transcrip-tional activation and HIF-1 expression (Fig. 6). There-fore, we wanted to identify the downstream targets of AKT that mediated apigenin-inhibited HIF-1 andVEGF expression. p70S6K1 is a known down-target of AKT. We found that overexpression of p70S6K1 in A2780/CP70 cells restored the VEGF transcriptionalactivity and increased HIF-1 protein levels (Fig. 6).These results suggest that apigenin inhibits HIF-1 andVEGF expression through the PI3K/AKT/p70S6K1 sig-naling pathway. Under hypoxia, the degradation ofHIF-1 protein was blocked due to the inactivation ofhydroxylase under low oxygen concentration, whichpermits the accumulation of HIF-1. We found thatapigenin inhibited expression of HIF-1 protein in

Figure 9. Apigenin inhibited tube formation by HUVEC cellsinduced by conditioned medium prepared from A2780/CP70cells. HUVEC cells were cultured in starve medium for 24 hand suspended in basal EBM-2 medium. To perform the tubeformation assay, cells were mixed with an equal volume ofconditioned medium prepared from A2780/CP70 cells (seeMaterials and Methods) and seeded on the Matrigel. Tubeformation was determined under light microscope (OLYM-PUS, 1X71) in 18 h. Pictures were taken at200 magnifica-tion. HUVEC cells were incubated in A) basal medium; B)

conditioned medium prepared from untreated A2780/CP70cells; C) conditioned medium prepared from apigenin-treated cells; D) conditioned medium prepared from un-treated cells supplemented with 5 M apigenin; E) condi-tioned medium prepared from untreated cells with VEGFneutralizing antibody (1.5 g/mL); F) conditioned mediumprepared from untreated cells with KDR neutralizing anti-body (1.5 g/mL); G) conditioned medium prepared fromuntreated cells with non-immune control IgG (1.5 g/mL).H) Total tube length of each treatment was analyzed asdescribed in the text. Data are mean se from 2 experiments

with 3 replications in each experiment. *Significantly de-creased tube length compared with that of experiment B(P0.05).

Figure 8. Apigenin inhibited expression of HIF-1 and VEGFin response to hypoxia. A)A2780/CP70 cells were cultured tosubconfluence and pretreated with apigenin for 0.5 h, fol-

lowed by incubation under hypoxia for 6 h. B) A2780/CP70cells were cultured to 90% confluence. Old medium wasdiscarded and new medium was provided in the presence orabsence of apigenin. The cells were then incubated under 1%O2 , as described, for 20 h. The VEGF in the supernatant wasdetermined using ELISA and normalized to the remainingcells. Data represent mean se from 2 experiments; eachexperiment had triplicate cultures. *Significantly decreased

VEGF compared with that of control, P0.05.



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ovarian cancer cells under hypoxic conditions but thatthe concentration of apigenin needed to completelyinhibit HIF-1 expression was higher (Fig. 8A).

We have shown that another important effect ofapigenin is its ability to induce p53 expression andinhibit HDM2 expression (Fig. 7). p53 and HDM2 forman auto-regulatory feedback loop. HDM2 protein has ap53 binding domain and possesses activity of ubiquitinligase capable of targeted ubiquitination of p53. On theother hand, p53 binds to the HDM2 gene and stimu-lates the transcriptional activation of HDM2. Repres-sion of p53 activation is largely due to the action ofHDM2. PI3K/AKT signaling is involved in the regula-tion of HDM2, because AKT is found to phosphorylateHDM2 and increase its stability (50, 58). In ovariancancer cells we found that the PI3K/AKT signalingplays a role in regulating HDM2 expression (Fig. 7C).In our work, two possible explanations for p53 induc-tion by apigenin are 1) apigenin inhibited PI3K/AKTsignaling, which accounts for the decrease of HDM2protein expression; and 2) apigenin prevented thenuclear localization of HDM2, which is required for

HDM2 to degrade the p53 protein because AKT caninduce the HDM2 phosphorylation for nuclear local-ization (58).

It was found that p53 promoted degradation ofHIF-1 (26) and inhibited HIF-1-mediated transcrip-tional activation (59). Inhibition of AKT activationimpaired the HDM2 phosphorylation leading to itsdestabilization and degradation, which accounts for theinduction of the p53 protein. It was reported recentlythat induction of HDM2 positively regulates HIF-1expression (60). In our work, overexpression of HDM2reversed apigenin-inhibited VEGF transcriptional acti-vation (Fig. 7E) and increased the HIF-1 protein level

in ovarian cancer cells (Fig. 7F). This may be explainedby 1) overexpression of HDM2 down-regulated p53,which in turn increased HIF-1 stability and transcrip-tional activity, and 2) overexpression of HDM2 pro-moted expression of HIF-1 . These data suggest thatapigenin regulates HDM2/p53 through PI3K/AKT sig-naling, which mediates HIF-1 and VEGF expression.

Tube formation assay in vitro is frequently used todetermine drug anti-angiogenic effects. In our experi-ments, we found that tube formation was significantlyinhibited when HUVEC cells were cultured in condi-tioned medium prepared from apigenin-treatedA2780/CP70 cells (Fig. 9C). This inhibitory effect was

not due to the presence of apigenin (Fig. 9D). The tubeformation was inhibited when cells were cultured in theconditioned medium with VEGF or its receptor KDRneutralizing antibodies (Fig. 9 E, F), suggesting that A2780/CP70 cells stimulate angiogenesis mainlythrough VEGF expression. These results suggest thatapigenin possesses potent anti-angiogenic activity.

The VEGF promoter region reveals several potentialbinding sites for other transcription factors like SP-1,AP-1, and AP-2 (61). Therefore, apigenin might regu-late VEGF expression through other factors. However,overexpression of HIF-1 is sufficient to reverse the

apigenin-inhibited activity of VEGF promoter reporter(Fig. 2), suggesting that apigenin inhibits VEGF expres-sion primarily through HIF-1 in ovarian cancer cells.Other growth factors, like bFGF and TGF-, may in-duce angiogenesis. We cannot exclude the possibilitythat apigenin inhibited production of these factors andcontributed partly to its anti-angiogenic activity.

We demonstrate here for the first time that apigenininhibits expression of VEGF at the transcriptional levelby HIF-1 expression. We further demonstrated thatapigenin inhibits HIF-1 and VEGF expression throughtwo distinctive signaling pathways: PI3K/AKT/p70S6K1and HDM2/p53. This novel finding provides new in-sight into the potential mechanism of the anti-cancerproperties of apigenin. Based on the daily dietaryconsumption of flavonoids, the concentration of apige-nin used in this work is nontoxic and physiologicallyrelevant in humans (62). Molecular targeting of theVEGF by apigenin may be a useful and novel strategyfor chemoprevention and/or treatment of ovariancancer.

We thank Dr. Faton Agani and Dr. John Blenis for provid-ing cDNA constructs. This work was supported by NationalInstitutes of Health Grant RR16440 to B.H.J. and E.R., and by

American Cancer Society Research Scholar Grant 04-076-01-TBE to B.H.J.

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Apigenin Inhibits VEGF and HIF-1 Expression Via

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