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The FASEB Journal Research Communication

The FASEB Journal • Research Communication

Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways

Jing Fang, Chang Xia, Zongxian Cao, Jenny Z. Zheng, Eddie Reed, and Bing-Hua Jiang 1

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

ABSTRACT Apigenin is a nontoxic dietary flavonoid that has been shown to possess anti-tumor properties and therefore poses special interest for the develop- ment of a novel chemopreventive and/or chemothera- peutic agent for cancer. Ovarian cancer is one of the most common causes of cancer death among women. Here we demonstrate that apigenin inhibits expression of vascular endothelial growth factor (VEGF) in human ovarian cancer cells. VEGF plays an important role in tumor angiogenesis and growth. We found that apige- nin inhibited VEGF expression at the transcriptional level through expression of hypoxia-inducible factor 1 (HIF-1 ). Apigenin inhibited expression of HIF-1 and VEGF via the PI3K/AKT/p70S6K1 and HDM2/ p53 pathways. Apigenin inhibited tube formation in vitro by endothelial cells. These findings reveal a novel role of apigenin in inhibiting HIF-1 and VEGF expres- sion that is important for tumor angiogenesis and growth, 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 and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/ p53 pathways. FASEB J. 19, 342–353 (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 from gynecological 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 cancer has spread to the surface of the peritoneal cavity. At this stage, it is impossible to surgically remove all apparent lesions, and this accounts for the high rate of cancer recurrence after surgery; hence, ovarian cancer is a prime target in chemoprevention research. Although the mechanism(s) involved in the progression of ovar- ian cancer are still unclear, increasing evidence in cancer prevention literature point to a role of autocrine and paracrine factors in the development of ovarian tumorigenesis, indicating that the vascular endothelial growth factor (VEGF) plays an important role in ovar- ian cancer development. VEGF plays a critical role in tumor angiogenesis.

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Angiogenesis is the formation of new blood vessels from preexisting ones and is required for tumor growth and metastasis (3). Tumor angiogenesis is stimulated by angiogenic growth factors like VEGF, basic fibroblast growth factor (bFGF), transforming growth factor (TGF), and interleukin 8 (IL-8). VEGF and its receptors have been described as the fundamental regulators of angiogenesis (4) and play an important role in tumor progression (5, 6). VEGF expression and its receptor function are required for tumor growth, invasion, and metastasis in animal models (7–10). VEGF and VEGF receptors were found to be expressed in human ovarian carcinoma (11, 12). Inhibition of VEGF blocked the cell proliferation of ovarian cancer cells in vitro and tumor 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 growth factor withdrawal and provided resistance to apoptosis induced by cisplatin (13). Thus, inhibiting the role of VEGF in promoting angiogenesis and tumor growth is becoming a target for ovarian cancer therapy. Hypoxia inducible factor 1 (HIF-1) activates the transcription of many genes, including VEGF (15). HIF-1 activates the expression of the VEGF gene by binding to the hypoxia response element (HRE) in the VEGF promoter (16). HIF-1 is overexpressed in many human cancers (17), and levels of its activity in cells correlate 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 protein levels are constitutively expressed but rapidly degraded by the ubiquitin-proteasome pathway under normoxia (21, 22), a process mediated by specific binding of pVHL, the product of the von Hippel–Lindau (VHL) tumor suppressor gene (23). By controlling HIF-1– pVHL physical interaction, prolyl hydroxylation of HIF-1 is critical in the regulation of HIF-1 steady- state levels (24, 25). Under hypoxia, the absence of oxygen prevents the hydroxylases from modifying HIF-

1 Correspondence: MBR Cancer Center, Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA. E-mail:

bhjiang@hsc.wvu.edu doi: 10.1096/fj.04-2175com

0892-6638/05/0019-0342 © FASEB

1 ; therefore pVHL fails to recognize HIF-1 , allowing

HIF-1 to accumulate. The regulation of HIF-1 can also be independent of the oxygen environment. Oncogenic mutations such as the loss of function of VHL (23), p53 (26), and PTEN (27) induce HIF-1 expression. Growth factors, cyto- kines, and other signaling molecules can stimulate HIF-1 protein synthesis via activation of the phospha- tidylinositol 3-kinase (PI3K)/AKT or mitogen-activated protein kinase (MAPK) pathways (28 –30). PI3K is a heterodimeric enzyme composed of a 110 kDa catalytic subunit and an 85 kDa regulatory subunit (31). The best-known downstream target of PI3K is the serine- threonine kinase AKT, which transmits survival signals from growth factors (32, 33). We and others recently demonstrated that PI3K/AKT signaling is required for VEGF expression through HIF-1 in response to growth factor stimulation and oncogene activation (27, 28, 34 –36). Apigenin (4 ,5,7,-trihydroxyflavone) is a common dietary 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 is used as a healthy food supplement and has recently been shown to possess anti-tumor properties (38 – 41). Nevertheless, its mechanism is unclear. In ovarian cancer, the catalytic subunit p110 of PI3K is increased in copy numbers (42), and PI3K catalytic subunit expression positively correlated with the expression of

VEGF in ovarian cancers (43). VEGF and HIF-1 are both expressed in epithelial ovarian cancer; VEGF expression correlates with HIF-1 expression, suggest- ing that HIF-1 contributes to the overexpression of VEGF in ovarian cancer (44). In this study, we have used ovarian cancer cells A2780/CP70 and OVCAR-3 as a model system to investigate the mechanism of the anti-tumor properties of apigenin. We found that api- genin significantly inhibited the expression of HIF-1 and of VEGF in the ovarian cancer cells. Therefore, we investigated the possible mechanism by which apigenin inhibited 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 at –20°C. Antibodies against HIF-1 and HIF-1 were from BD Biosciences (Bedford, MA, USA). The antibodies against phospho-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 from Santa 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-

APIGENIN INHIBITS VEGF AND HIF-1 EXPRESSION

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

Cell culture

The human ovarian cancer cells OVCAR-3 and A2780/CP70 were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grant Island, NY, USA), 100 u/mL penicillin, and 100 g/mL streptomycin in 5% CO 2 incubator at 37°C. For cell culture under hypoxia, the cells were grown in a chamber containing 1% oxygen, 5% carbon dioxide, and 94% nitrogen at 37°C. 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 from Cambrex Company (Walkersille, MD, USA).

Construction of plasmids

VEGF promoter reporter pGL-Stu1 containing a 2.65 Kb KpnI-BssHII fragment of the human VEGF gene promoter and VEGF promoter reporter pMAP11wt, which contains only 47 bp of VEGF 5 -flanking sequence (from –985 to –939), were cloned into the pGL2 basic luciferase vector as described previously (16). The mutant VEGF promoter reporter pMAP11mut 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 into pCEP4 vector (16, 19). Plasmids encoding active myr-AKT (45), an active p110 subunit of PI3K, p110E227K (46), an active p70S6K1 (47), and wild-type HDM2 (48) have been described.

Immunoblot analysis

Cells at 80 –90% confluence were exposed to apigenin for specific times. Cells were lysed on ice for 30 min in RIPA buffer (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 mM

dithiothreitol, and 1 mM pepstatin A. The supernatant was collected after centrifugation at 12,000 g for 15 min and protein concentration was determined using protein assay reagent from Bio-Rad (Hercules, CA, USA). Equal amounts of protein were resolved on SDS-PAGE and transferred to a nitro-cellulose membrane. Proteins of interest were detected by immunoblotting using specific antibodies.

RNA isolation and Northern blot analysis

Cells at 80 –90% confluence were treated with apigenin for

6 h. Total cellular RNA was extracted using Trizol reagent

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Aliquots of total RNA were de- natured at 65°C 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 VEGF cDNA fragment was used as a probe. The probe was labeled with [ - 32 P]dATP using RadPrime DNA labeling system (In- vitrogen) and purified with the ProbeQuant G-50 Micro- columns from Amersham Biosciences. Hybridizations were performed at 48°C in Northern Max hybridization buffer from Ambion (Austin, TX, USA). The membrane was stripped off and probed with [ - 32 P]-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 plates and cultured to 60 –70% confluence. To determine the effects of apigenin on transcriptional activation of VEGF, cells were transiently transfected with VEGF reporter and pCMV- -galactosidase plasmid using Lipofectamine from Invitrogen according to the manufacturer’s instructions. The transfected cells were cultured for 20 h, followed by incubation with apigenin 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 was measured in assay buffer (100 mM phosphate, pH 7.5, 2 mM MgCl 2 , 100 mM -mercaptoethanol, 1.33 mg/mL o -nitrophe- nyl - d -galactopyranoside). Relative Luc activity (defined as VEGF reporter activity) was calculated as the ratio of Luc/ - gal activity.

Quantification of VEGF protein

VEGF protein was measured using the Quantikine human VEGF 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 and cultured to 90 –100% confluence. Cells were switched to fresh medium in the presence or absence of apigenin. In 15 h, the supernatants 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. A serial dilution of human recombinant VEGF was included in each assay to obtain a standard curve.

Cell proliferation and cell death assay

Cell proliferation was determined using MTT reduction method. In brief, 100 L of the cells were cultured in 96-well plates and treated with different concentrations of apigenin for specified times. After treatment, 10 L of MTT reagent (5 mg/mL) was added to each well. In 2 h, the reaction was stopped by addition of 100 L of solubilization solution (50% N, N -dimethylformamide, 20% SDS). The absorbance at 590 nm of solubilized MTT formazan products was measured in 6 h. For determining cell death, cells were collected and stained 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 and counted against the total number of cells to determine the percentage of cell death.

Tube formation assay

We determined the tube formation of endothelial cells in the presence of conditioned medium prepared from A2780/ CP70 cells. A2780/CP70 cells were cultured to 90 –100% confluence. The old medium was discarded and the cells were provided with serum-reduced medium (1% FBS) in the presence or absence of apigenin (10 M). The cells were incubated for 15 h and the medium was collected and stored at – 80°C. HUVEC cells at subconfluence were switched to EBM-2 basic medium containing 0.2% FBS. In 24 h, the starved HUVEC cells were trypsinized, collected, counted, and resuspended in EBM-2 basic medium. The cells were mixed with equal volume of the conditioned medium and seeded to Matrigel-pretreated 96-well plate at 10 4 cells/well. In 18 h, tube formation was examined under light micro-

scope. The length of the tubes was measured using the Soft Imaging System (Soft Imaging System GmbH, Germany). To pretreat the 96-well plate, 50 L of growth factor-reduced Matrigel thawed on ice was added to each well. The plate was then placed in an incubator to allow the gel to solidify at 37°C for 1 h.

Statistical analysis

The data represent mean se from three independent experiments except where indicated. Statistical analysis was performed by Student’s t test at a significance level of P 0.05.

RESULTS

Apigenin inhibited VEGF expression

We determined the effects of apigenin on VEGF mRNA levels in ovarian cancer cells. As shown in Fig. 1A, B, apigenin inhibited expression of VEGF mRNA in a dose-dependent manner in A2780/CP70 and OVCAR-3 cells. The ELISA data indicated that apigenin inhibited production of VEGF protein in both cell lines (Fig. 1C ). To exclude the possibility that the decrease in VEGF production is due to inhibition of cell proliferation, we determined the cell proliferation by an MTT method under the same experimental conditions. Apigenin treatment for 15 h had little effect on proliferation of A2780/CP70 and OVCAR-3 cells (Fig. 1D ). We detected the effects of apigenin on cell viability and did not observe any difference of cell viability between the control and apigenin-treated groups (data not shown). These results suggest that the inhibition of VEGF production 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 apigenin on VEGF promoter reporter containing a 2.65 kb fragment of human VEGF gene promoter (16) in A2780/CP70 ovarian cancer cells, and found that api- genin at 10 M inhibited significantly the VEGF re- porter activity (Fig. 2A ). Overexpression of HIF-1 completely reversed the apigenin-inhibited reporter activity (Fig. 2A ). To determine whether apigenin inhibits VEGF expression through HIF-1 DNA binding site of VEGF promoter, we analyzed the effects of apigenin on a VEGF promoter reporter, pMAP11wt, containing the HIF-1 binding site. Apigenin inhibited the pMAP11wt reporter activity in A2780/CP70 and OVCAR-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 transcriptional activation via HIF-1 DNA binding site and HIF-1 protein expression. Next, we introduced 3 bp substitu- tion at the HIF-1 binding site to generate a mutant pMAP11mut VEGF promoter reporter (16) and tested the effect of apigenin on this reporter activity. Treat- ment of apigenin did not inhibit the mutant VEGF

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Figure 1. Apigenin decreased VEGF expression in ovarian cancer cells. A2780/CP70 ( A ) and

Figure 1. Apigenin decreased VEGF expression in ovarian cancer cells. A2780/CP70 (A ) and OVCAR-3 (B ) ovarian cancer cells were treated with apigenin for 6 h. Cells treated with solvent DMSO alone were used as the control. Total RNA was isolated and VEGF mRNA was detected as described in Materials and Methods. GAPDH mRNA was detected and used as control. Left panels show 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 90 –100% confluence. The cells were switched to fresh medium with or without apigenin and incubated for 15 h. The concentrations of VEGF in the supernatants were determined by ELISA. Data are mean se from 3 independent experiments; each experiment was performed with triplicate cultures. *Significantly decreased VEGF compared with that of control, P 0.05. D) A2780/CP70 and OVCAR-3 cells were cultured to 90 –100% confluence. The cells were provided with fresh medium (with apigenin) and incubated for 15 h. Cell proliferation was determined as described in Materials and Methods.

reporter activity (data not shown), further indicating that apigenin affects the VEGF reporter activity through the HIF-1 binding site.

Apigenin inhibited HIF-1 expression

To determine whether apigenin regulates VEGF ex- pression through HIF-1 expression, we examined the effects of apigenin on HIF-1 and HIF-1 protein levels. Expression of HIF-1 in both A2780/CP70 and OVCAR-3 cells was inhibited by apigenin in a dose- and time-dependent manner (Fig. 3). However, apigenin had no effect on HIF-1 protein levels (Fig. 3). These results suggest that apigenin inhibits VEGF transcrip- tional activation by specifically inhibiting HIF-1 but not HIF-1 expression. We determined the possible mechanism by which apigenin inhibits HIF-1 expression. Apigenin had some effect on the HIF-1 mRNA levels in ovarian cancer cells (Fig. 4A ). We next determined the effects of apigenin on the stability of HIF-1 protein using CHX to inhibit new protein synthesis in the cells. A2780/CP70 cells were treated with CHX or CHX plus apigenin as indicated in Fig. 4B . The half-life of HIF-1 protein in the cells was 8 min when the cells were treated with CHX alone (Fig. 4B ) and 5 min when cells were pretreated with apigenin (Fig. 4B ). These data suggest that apigenin inhibits HIF-1 expression par- tially through decreasing HIF-1 protein stability.

APIGENIN INHIBITS VEGF AND HIF-1 EXPRESSION

Apigenin inhibited VEGF transcriptional activation through the PI3K/AKT/p70S6K1 pathway

Recent studies indicate that expression of HIF-1 and VEGF can be regulated through the PI3K/AKT path- way (28, 29, 34 –36). To know whether apigenin inhibits expression of HIF-1 and VEGF through this signaling pathway, we determined the effects of apigenin on activation 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 signaling pathway, 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 of phospho-ERK1/2 levels (Fig. 5). These results suggest that apigenin affects HIF-1 and VEGF expression through PI3K/AKT, but not ERK1/2, in ovarian cancer cells. To confirm that apigenin inhibits VEGF expression through the PI3K/AKT pathway, A2780/CP70 cells were cotransfected with VEGF promoter reporter and active forms of p110 , the catalytic subunit of PI3K, or myr-AKT construct. Forced expression of p110 or myr-AKT reversed the apigenin-inhibited reporter ac- tivity (Fig. 6A ), indicating that the inhibition of VEGF transcriptional activation by apigenin is via the PI3K/ AKT pathway. p70S6K1 is a downstream target of PI3K/AKT. We found that apigenin inhibited phos-

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Figure 2. Apigenin inhibited VEGF transcriptional activation via HIF-1. Cells were transfected with the vectors

Figure 2. Apigenin inhibited VEGF transcriptional activation via HIF-1. Cells were transfected with the vectors indicated and cultured for 20 h, followed by treatment with apigenin for 15 h. Cells were lysed and the supernatants were subjected to Luc

and -gal activity assay. Relative Luc activities in the cell extracts were assayed by the ratio of Luc/ -gal activity and normalized to 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 of pGL-StuI reporter plus empty vector, P 0.05. *Significant increase of activity compared with that of reporter empty vector in the presence of apigenin, P 0.05. B) A2780/CP70 and OVCAR-3 cells were transfected with 1 g of pMAP11wt reporter

0.3 g of -gal plasmid. *P 0.05 vs. control. C) Cells were transfected with 1 g of the pMAP11wt reporter, 0.3 g of -gal

plasmid, and 0.5 g (or 1 g) of plasmid encoding wild-type HIF-1 . The empty vector was added, if necessary, to make total transfected DNA 2.3 g. The concentration of apigenin used was 10 M. #P 0.05 vs. pMAP11wt empty vector. *P 0.05 vs. pMAP11wt empty vector in the presence of apigenin. Data are mean se from 3 independent experiments. Each experiment was performed with triplicate cultures.

phorylation of p70S6K1 (data not shown). Thus, we hypothesized 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 of p70S6K1 (CA-p70S6K1) plasmid. As shown in Fig. 6A , forced expression of p70S6K1 reversed the apigenin- inhibited VEGF transcriptional activation. To know whether the reverse of VEGF transcriptional activation is via expression of HIF-1 , we determined the effects of overexpression of these constructs on HIF-1 pro- tein levels. A2780/CP70 cells were transfected with the empty vector, p110 , Myr-AKT, or CA-p70S6K1 plas- mid, and treated with apigenin. Forced expression of p110 , 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 expression through 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 in A2780/CP70 cells. Apigenin induced p53 protein ex- pression in a dose- and time-dependent manner (Fig.

7 A, B). As is well known, the amount of p53 protein in

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Figure 3. Apigenin inhibited HIF-1 protein expression. A2780/CP70 and OVCAR-3 ovarian cancer cells were cul-

Figure 3. Apigenin inhibited HIF-1 protein expression. A2780/CP70 and OVCAR-3 ovarian cancer cells were cul- tured to 80 –90% confluence, followed by treatment with apigenin. The cells treated with solvent alone were used as a control. 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 and transfer efficiency. A) A2780/CP70 and OVCAR-3 cells were treated with apigenin (2.5, 5, 10, and 25 M) for 6 h. B) A2780/CP70 and OVCAR-3 cells were exposed to 20 M of apigenin for different times as indicated.

cells is determined mainly by the rate at which it is degraded rather than the rate at which it is made. HDM2 is the protein that mediates p53 degradation by binding p53 and stimulating the addition of ubiquitin

to 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.

7 A, B, apigenin impaired the expression of HDM2

protein. Similar results were obtained with OVCAR-3 cells treated with apigenin (data not shown). These results suggest that apigenin induces p53 through down-regulation of HDM2. It is reported that inhibi- tion of AKT activity will impair the phosphorylation of HDM2, which may result in the destabilization of HDM2 (50). In A2780/CP70 cells, addition of LY294002 inhibited phosphorylation of AKT and HDM2 (Fig. 7C ). The total HDM2 protein was de- creased whereas p53 expression was induced (Fig. 7C). These results suggest that the PI3K/AKT signaling plays an important role in regulating HDM2 and p53 expres-

sion in ovarian cancer cells. Apigenin could inhibit the phosphorylation of HDM2 by AKT in ovarian cancer cells (Fig. 7D). Considering the inhibitory effects of apigenin on phospho-AKT (Fig. 5) and HDM2 (Fig. 7A,

B ), our results suggest that apigenin regulates HDM2

probably via PI3K/AKT signaling. Blockade of HDM2 phosphorylation is a possible mechanism through which apigenin decreased HDM2 protein levels. Based on previous results (26) and our data, we hypothesized that HDM2/p53 signaling might be in- volved in the regulation of VEGF expression in ovarian

APIGENIN INHIBITS VEGF AND HIF-1 EXPRESSION

cancer cells. To confirm this, we determined the effects of overexpression of HDM2 on the VEGF promoter reporter activities in A2780/CP70 cells. Forced expres- sion of HDM2 reversed the reporter activity inhibited by apigenin (Fig. 7E ), suggesting that HDM2 is in- volved in the regulation of VEGF expression by apige- nin. To ascertain whether this regulation is through expression 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-1 protein levels inhibited by apigenin. These results

HIF-1 protein levels inhibited by apigenin. These results Figure 4. Apigenin regulated HIF-1 expression partially

Figure 4. Apigenin regulated HIF-1 expression partially through decreasing protein stability. A) Effects of apigenin on mRNA levels of HIF-1 . A2780/CP70 and OVCAR-3 cells were exposed to apigenin for 6 h. Total RNA was isolated and mRNA levels of HIF-1 were detected by Northern blot. B) Effects of apigenin on the stability of HIF-1 . To determine the half-life of HIF-1 , A2780/CP70 cells were incubated with CHX (100 M) to inhibit new protein synthesis and cells were harvested at different times. To determine the effects of apigenin on half-life of HIF-1 , cells were pretreated with 20 M of apigenin for 30 min, followed by addition of CHX (100 M). The cells were then harvested at different times, as indicated. HIF-1 was detected by immunoblotting with GAPDH as an internal control. Levels of HIF-1 protein were determined by measuring the density of the HIF-1 protein band and normalized to that of GAPDH. The relative HIF-1 protein level at time zero was defined as 1.0. The experiments were performed 3 times and the data were mean se . *Significant difference (P 0.05) when compared with the treatment of CHX alone.

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Figure 5. Apigenin inhibited activation of AKT. A) A2780/ CP70 and OVCAR-3 cells were treated

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 of proteins were resolved on SDS-PAGE gel and analyzed by immunoblotting with antibodies against phospho-AKT (Ser473) or phospho-ERK1/2. Total AKT and ERK1/2 were detected using antibodies against AKT and ERK1/2. B) Cells were treated with apigenin (20 M). Phosphorylation of AKT and ERK1/2 were detected as described above.

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

Apigenin inhibited HIF-1 and VEGF expression under hypoxia

The effect of apigenin on HIF-1 expression under hypoxia was determined in A2780/CP70 cells. As shown in Fig. 8A , apigenin inhibited HIF-1 expression in A2780/CP70 cells in response to 1% O 2 . We deter- mined the effects of apigenin on VEGF production under hypoxia. A2780/CP70 cells at subconfluence were incubated with apigenin under 1% O 2 for 20 h. As shown in Fig. 8B , apigenin inhibited VEGF protein levels in A2780/CP70 in a dose-dependent manner under hypoxic condition.

Apigenin inhibited tube formation by HUVEC cells induced by conditioned medium from ovarian cancer cells

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

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

experiments (Fig. 9 H ). These results suggest that the Figure 6. Apigenin inhibited VEGF expression

Figure 6. Apigenin inhibited VEGF expression via PI3K/ AKT/p70S6K1. A) A2780/CP70 cells were transfected with the 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 total DNA transfected. The cells were then cultured for 20 h, followed by treatment with 10 M of apigenin for 15 h. Data represent mean se from 3 experiments; each experiment had 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, P 0.05. B) A2780/CP70 cells were seeded in 60 mm dishes. At 70 – 80% confluence, the cells were transfected with 4 g of p110 , myr-Akt, or CA-p70S6K1. Cells transfected with 4 g of empty vector were used as the control. The cells were cultured for 20 h after transfection, followed by treatment with apigenin (25 M) for 6 h.

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Figure 7. Apigenin inhibited VEGF transcriptional activation through HDM2 expression. A2780/CP70 cells were treated with

Figure 7. Apigenin inhibited VEGF transcriptional activation through HDM2 expression. A2780/CP70 cells were treated with apigenin as indicated. Cellular proteins were prepared and aliquots of proteins were separated on SDS-PAGE and analyzed by immunoblotting with the antibodies indicated. A) Apigenin increased p53 expression but inhibited HDM2 expression in a dose-dependent manner. The cells were incubated with apigenin (0, 5, 10, and 20 M) for 6 h. B) Apigenin induced p53 expression but decreased HDM2 protein levels in a time-dependent manner. A2780/CP70 cells were treated with 20 M of apigenin 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, 0.3 g 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. #P 0.05 vs. pMAP11wt empty vector. *P 0.05 vs. pMAP11wt empty vector in the presence of apigenin. F) A2780/CP70 cells were seeded in 6-well plates and transfected with 1 g of pCMV-HDM2 plasmid or 1 g of empty vector. The cells were cultured for 20 h and exposed to 25 M of apigenin for 6 h.

VEGF levels in the medium and VEGF function through its receptor are significant and confirm that apigenin inhibits tube formation by decreasing VEGF expression.

DISCUSSION

Because there is a long latency period for the develop- ment of clinical ovarian cancer, studies of ovarian cancer chemoprevention have intensified in recent years, making ovarian cancer a prime target for chemo- prevention. Chemoprevention involves using nontoxic, naturally occurring or synthetic agents to prevent or inhibit human cancer development. Dietary and epide- miological studies have suggested that the dietary in- take of fruits and vegetables can reduce incidences of many types of cancer (51–53). Flavonoids are abundant in our diet. Apigenin, the most common flavonoid, has

APIGENIN INHIBITS VEGF AND HIF-1 EXPRESSION

been shown to possess anti-tumor properties (38 – 40). Most human tumors overexpress VEGF, which stimu- lates angiogenesis and tumor growth (6). Inhibition of angiogenesis by targeting VEGF is becoming an impor- tant approach for cancer treatment. In this paper we have 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 level through HIF-1 expression. HIF-1 plays an important role in VEGF transcriptional activation in ovarian can- cer cells (Figs. 2 and 3). Expression of HIF-1 is regulated via degradation and protein synthesis. In most experimental systems, HIF-1 protein subunits are constitutively expressed but rapidly degraded by the ubiquitin-proteasome pathway under normoxia (21, 22). We found that OVCAR-3 and A2780/CP70 cells expressed constitutively elevated levels of HIF-1 pro- tein under normal conditions (54). Constitutively ele-

349

Figure 8. Apigenin inhibited expression of HIF-1 and VEGF in response to hypoxia. A) A2780/CP70

Figure 8. Apigenin inhibited expression of HIF-1 and VEGF in response to hypoxia. A) A2780/CP70 cells were cultured to subconfluence and pretreated with apigenin for 0.5 h, fol- lowed by incubation under hypoxia for 6 h. B) A2780/CP70 cells were cultured to 90% confluence. Old medium was discarded and new medium was provided in the presence or absence of apigenin. The cells were then incubated under 1% O 2 , as described, for 20 h. The VEGF in the supernatant was determined using ELISA and normalized to the remaining cells. Data represent mean se from 2 experiments; each experiment had triplicate cultures. *Significantly decreased VEGF compared with that of control, P 0.05.

vated levels of HIF-1 protein in ovarian cancer cells were observed in other labs (43, 55). The reason for appreciable 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 that OVCAR-3 and A2780/CP70 cells both had appreciable levels of phospho-AKT (Fig. 5) (54). Therefore, one possibility that would account for the elevated levels of HIF-1 protein is the elevated PI3K/AKT signaling in these cells under normal conditions. Studies of the role of PI3K signaling in hypoxia-induced HIF-1 expres- sion were contradictory based on the cell lines used. In prostate cancer cells, PI3K and AKT activities were observed to be required for HIF-1 expression (34, 56). However, in 1c1c7 mouse hepatocyte cells, inhibition of PI3K activity did not affect hypoxia-induced HIF-1 expression (57). In ovarian carcinoma, the catalytic unit of PI3K is directly implicated in the control of HIF-1 protein and VEGF expression (43). We found in our lab that blockade of PI3K/AKT signaling by its specific inhibitor LY294002 significantly inhibited HIF-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 AKT activity accounts for the expression of HIF-1 and VEGF 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 PI3K or 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 and VEGF expression. p70S6K1 is a known down-target of AKT. We found that overexpression of p70S6K1 in A2780/CP70 cells restored the VEGF transcriptional activity and increased HIF-1 protein levels (Fig. 6). These results suggest that apigenin inhibits HIF-1 and VEGF expression through the PI3K/AKT/p70S6K1 sig- naling pathway. Under hypoxia, the degradation of HIF-1 protein was blocked due to the inactivation of hydroxylase under low oxygen concentration, which permits the accumulation of HIF-1 . We found that apigenin inhibited expression of HIF-1 protein in

that apigenin inhibited expression of HIF-1 protein in Figure 9. Apigenin inhibited tube formation by HUVEC

Figure 9. Apigenin inhibited tube formation by HUVEC cells induced by conditioned medium prepared from A2780/CP70 cells. HUVEC cells were cultured in starve medium for 24 h and suspended in basal EBM-2 medium. To perform the tube formation assay, cells were mixed with an equal volume of conditioned medium prepared from A2780/CP70 cells (see Materials and Methods) and seeded on the Matrigel. Tube formation was determined under light microscope (OLYM- PUS, 1X71) in 18 h. Pictures were taken at 200 magnifica- tion. HUVEC cells were incubated in A) basal medium; B) conditioned medium prepared from untreated A2780/CP70 cells; 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 VEGF neutralizing antibody (1.5 g/mL); F) conditioned medium prepared from untreated cells with KDR neutralizing anti- body (1.5 g/mL); G) conditioned medium prepared from untreated cells with non-immune control IgG (1.5 g/mL). H) Total tube length of each treatment was analyzed as

described 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

( P 0.05).

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FANG ET AL.

ovarian cancer cells under hypoxic conditions but that the concentration of apigenin needed to completely inhibit HIF-1 expression was higher (Fig. 8A ). We have shown that another important effect of apigenin is its ability to induce p53 expression and inhibit HDM2 expression (Fig. 7). p53 and HDM2 form an auto-regulatory feedback loop. HDM2 protein has a p53 binding domain and possesses activity of ubiquitin ligase capable of targeted ubiquitination of p53. On the other 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 of HDM2. PI3K/AKT signaling is involved in the regula- tion of HDM2, because AKT is found to phosphorylate HDM2 and increase its stability (50, 58). In ovarian cancer cells we found that the PI3K/AKT signaling plays 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/AKT signaling, which accounts for the decrease of HDM2 protein expression; and 2) apigenin prevented the nuclear localization of HDM2, which is required for HDM2 to degrade the p53 protein because AKT can induce the HDM2 phosphorylation for nuclear local- ization (58). It was found that p53 promoted degradation of HIF-1 (26) and inhibited HIF-1-mediated transcrip- tional activation (59). Inhibition of AKT activation impaired the HDM2 phosphorylation leading to its destabilization and degradation, which accounts for the induction of the p53 protein. It was reported recently that induction of HDM2 positively regulates HIF-1 expression (60). In our work, overexpression of HDM2 reversed apigenin-inhibited VEGF transcriptional acti- vation (Fig. 7E ) and increased the HIF-1 protein level in ovarian cancer cells (Fig. 7F ). This may be explained by 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 that apigenin regulates HDM2/p53 through PI3K/AKT sig- naling, which mediates HIF-1 and VEGF expression. Tube formation assay in vitro is frequently used to determine drug anti-angiogenic effects. In our experi- ments, we found that tube formation was significantly inhibited when HUVEC cells were cultured in condi- tioned medium prepared from apigenin-treated A2780/CP70 cells (Fig. 9C ). This inhibitory effect was not due to the presence of apigenin (Fig. 9D). The tube formation was inhibited when cells were cultured in the conditioned medium with VEGF or its receptor KDR neutralizing antibodies (Fig. 9E, F), suggesting that A2780/CP70 cells stimulate angiogenesis mainly through VEGF expression. These results suggest that apigenin possesses potent anti-angiogenic activity. The VEGF promoter region reveals several potential binding 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 INHIBITS VEGF AND HIF-1 EXPRESSION

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 possibility that apigenin inhibited production of these factors and contributed partly to its anti-angiogenic activity. We demonstrate here for the first time that apigenin inhibits expression of VEGF at the transcriptional level by HIF-1 expression. We further demonstrated that apigenin inhibits HIF-1 and VEGF expression through two distinctive signaling pathways: PI3K/AKT/p70S6K1 and HDM2/p53. This novel finding provides new in- sight into the potential mechanism of the anti-cancer properties of apigenin. Based on the daily dietary consumption of flavonoids, the concentration of apige- nin used in this work is nontoxic and physiologically relevant in humans (62). Molecular targeting of the VEGF by apigenin may be a useful and novel strategy for chemoprevention and/or treatment of ovarian cancer.

for chemoprevention and/or treatment of ovarian cancer. We thank Dr. Faton Agani and Dr. John Blenis

We thank Dr. Faton Agani and Dr. John Blenis for provid- ing cDNA constructs. This work was supported by National Institutes 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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Received for publication April 27, 2004. Accepted for publication November 12, 2004.

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