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Carcinogenesis Advance Access originally published online on October 27, 2006
Carcinogenesis 2007 28(4):858-864; doi:10.1093/carcin/bgl205
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Apigenin inhibits tumor angiogenesis through decreasing HIF-1{alpha} and VEGF expression

Jing Fang1, Qiong Zhou1, Ling-Zhi Liu1, Chang Xia2, Xiaowen Hu1, Xianglin Shi1 and Bing-Hua Jiang1,2,*

1 The Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences Shanghai 200031, China
2 Mary Babb Randolph Cancer Center, Department of Microbiology, Immunology and Cell Biology, West Virginia University Morgantown, WV 26506, USA

*To whom correspondence should be addressed. Email: bhjiang{at}hsc.wvu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apigenin is a non-toxic dietary flavonoid with anti-tumor properties. We recently showed that apigenin-inhibited hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells under normoxic condition. However, the effect of apigenin in angiogenesis remains to be elucidated. Angiogenesis is the formation of new blood vessels and is required for tumor growth and metastasis. In this study, we showed that apigenin-inhibited expression of HIF-1 and VEGF in different cancer cells under both normoxic and hypoxic conditions. We demonstrated that apigenin significantly inhibited tumor angiogenesis in vivo, by using both the chicken chorioallantoic membrane and Matrigel plug assays. The inhibition of tumor angiogenesis was associated with the decrease of HIF-1 and VEGF in tumor tissues. Taken together, our results show that apigenin suppresses tumor angiogenesis through HIF-1 and VEGF expression.

Abbreviations: ß-gal, ß-galactosidase; CAM, chicken chorioallantoic membrane; CHX, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsp90, heat shock protein 90; HIF-1, hypoxia-inducible factor 1; Luc, luciferase; PI3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Angiogenesis is the formation of new blood vessels from preexisting ones, and is required for tumor growth and metastasis (1). Tumor angiogenesis is stimulated by angiogenic inducers such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF) and interleukin 8 (IL-8). VEGF and its receptors are major regulators of angiogenesis (2), and are important in tumor progression (3,4). VEGF expression and its receptor function are required for tumor growth, invasion and metastasis (5–8), and are associated with the development of many human cancers (9). VEGF may regulate both tumor growth and angiogenesis (9–11). Thus, inhibition of VEGF production and/or function is a promising strategy for cancer therapy and prevention.

Hypoxia-inducible factor-1 (HIF-1) activates the transcription of many genes including VEGF (12). HIF-1 activates the expression of VEGF gene by binding to the hypoxia response element (HRE) in the VEGF promoter region (13). HIF-1 expression is elevated in many human cancers (14), and HIF-1 levels in cells correlate with tumorigenicity and angiogenesis (15). HIF-1 is composed of HIF-1{alpha} and HIF-1ß subunits (16,17). In most experimental systems, HIF-1{alpha} protein is constitutively expressed, but rapidly degraded by the ubiquitin–proteasome pathway under normoxia condition (18,19), which is mediated by the specific binding of the von Hippel–Lindau (VHL) tumor suppressor to HIF-1{alpha} (20). The prolyl hydroxylation of HIF-1{alpha}, is required for the HIF-1{alpha}–pVHL physical interaction, which is critical in degrading HIF-1{alpha} in the cells (21,22). Under hypoxic condition, the absence of oxygen inhibits the hydroxylase activities for modifying HIF-1{alpha}, and pVHL can not bind to HIF-1{alpha}.

Apigenin (4',5,7,-trihydroxyflavone) is a common dietary flavonoid which is widely distributed in many fruits and vegetables (23). Apigenin has recently been shown to possess anti-tumor properties against cancers, especially prostate cancer (24–28). We found recently that apigenin is able to inhibit HIF-1 and VEGF expression in ovarian cancer cells under normoxia condition (29). Hypoxia is a major inducer of HIF-1 and VEGF expression. In the present work, we tested whether apigenin (i) inhibits HIF-1 and VEGF expression in other cancer cells; (ii) affects hypoxia-induced HIF-1 and VEGF expression; and (iii) affects tumor angiogenesis in vivo.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents and antibodies
Apigenin and cycloheximide (CHX), an inhibitor of protein synthesis, were purchased from Sigma (St Louis, MO). Apigenin was dissolved in DMSO and stored at –20°C. The antibodies against ß-actin and FITC-conjugated second antibodies were also from Sigma. Antibodies against VEGF were obtained from Santa Cruz (Santa Cruz, CA). Antibodies against HIF-1{alpha}, HIF-1ß, heat shock protein-90 (Hsp90), and the growth-factor-reduced Matrigel were from BD Biosciences (Bedford, MA).

Cell culture
The human prostate cancer cells, PC-3, DU145 and LNCaP; human ovarian cancer cells, OVCAR-3, and human colon cancer cells, HCT-8, were cultured in RPMI 1640 medium. Human breast cancer cells, MCF-7, were cultured in DMEM medium. Both media were supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco BRL, Grant Island, NY), 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were incubated in a 5% CO2 incubator at 37°C. For hypoxia treatment, the cells were incubated in a chamber containing 1% oxygen, 5% carbon dioxide and 94% nitrogen at 37°C.

Construction of plasmids
VEGF promoter reporter pMAP11wt containing 47 bp of human VEGF 5'-flanking sequence (from –985 to –939) was inserted into the pGL2 basic luciferase vector as described previously (13). The mutant VEGF promoter reporter pMAP11mut was constructed by introducing a 3-bp substitution at the HIF-1 binding site (13). The plasmid encoding human HIF-1{alpha} was inserted into pCEP4 vector as we described previously (13,16).

Immunoblotting, immunoprecipitation, and ELISA analysis
Immunoblotting analysis of HIF-1{alpha} and other proteins was performed as described previously (30). For immunoprecipitation, cellular proteins (500 µg) were incubated with 1 µg of anti-Hsp90 antibodies at 4°C for 3 h, then with 30 µl of protein A/G plus-agarose at 4°C overnight. The beads were washed four times with ice-cold phosphate-buffered saline (PBS), then incubated with 30 µl of SDS–PAGE loading buffer and heated at 100°C for 3 min. The protein samples were resolved on 8% SDS–PAGE for immunoblotting. VEGF protein levels were determined using the Quantikine human VEGF ELISA kit from R&D Systems (Minneapolis, MN) as described previously (30). In brief, the cells were seeded in 12-well plates, and cultured to 90–100% confluence. The cells were switched to fresh medium in the presence or absence of apigenin, and incubated under hypoxic or normoxic conditions for the time period as indicated. After the treatment, the supernatants were collected and the cells in each well were counted. Relative VEGF concentrations in the supernatant (100 µl) were determined and normalized to the cell number in each well with a serial dilution of human recombinant VEGF as a standard curve.

Transient transfection and luciferase assay
The cells were seeded in 6-well plates, and cultured to 60–70% confluence. To determine the effect of apigenin on VEGF transcriptional activation, the cells were transiently transfected with VEGF reporter and pCMV-ß-galactosidase plasmid using Lipofectamine (Invitrogen, Carlsbad, CA). The transfected cells were cultured for 20 h, followed by incubation with apigenin for 15 h. Cells were then washed once with PBS, and lysed with reporter lysis buffer from Promega (Madison, WI). The luciferase (Luc) activities were determined as described by the authors previously (30). The relative Luc activity was calculated as the ratio of Luc/ß-gal activity, and normalized to that of the control.

Tumor angiogenesis assay using chicken chorioallantoic membrane (CAM)
To investigate the effect of apigenin on tumor-induced angiogenesis, a modified CAM assay was carried out. Briefly, the cells were trypsinized, washed and re-suspended in serum-free RPMI 1640 medium at 4°C. The cells were mixed with 50% (v/v) of growth-factor-reduced Matrigel in the absence or presence of apigenin. The mixture (30 µl) was loaded onto the CAM of a 9-day-old chicken embryo. After the incubation for 4 days, the area around the implantation was photographed with a Nikon digital camera, and the blood vessels were determined by two observers in a double-blind manner.

Matrigel plug assay
Male and female BALB/cA-nu nude mice (4-weeks-old) were purchased from Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai, China), and maintained in pathogen-free conditions. The cells at subconfluence were harvested, washed with PBS and re-suspended in serum-free medium. Aliquots of the cells (0.2 ml) were mixed with 0.4 ml of Matrigel in the presence or absence of apigenin. The mixture was immediately injected into both flanks of nude mice. Male and female nude mice were injected with PC-3 and OVCAR3 cells, respectively. The mice were sacrificed when tumors were visible, and the Matrigel plugs were carefully taken out of the mice without other adjacent tissues. The excised plugs were placed in cold PBS at 4°C overnight to be liquefied. Specimens were centrifuged at 14 000 r.p.m. to collect the supernatant. Hemoglobin contents were quantified using Drabkin's reagent kit (Sigma-Aldrich, St Louis, MO) as described previously (31).

Immunohistochemical staining for VEGF
Tumor tissues were fixed in 4% buffered formalin for 24 h, and processed by conventional paraffin-embedded method. The paraffin-embedded tumor sections (5-µm thick) were heat-immobilized, deparaffinized using xylene, then rehydrated in a series of increasing ethanol concentrations. Antigen retrieval was done by incubating the sections in 10 mM citrate buffer (pH 6.0) in microwave for 10 min, followed by the incubation with 1.5% block serum for 1 h. Sections were then incubated with antibodies against VEGF at 4°C overnight, followed by the incubation with the FITC-conjugated second antibodies for 1 h at room temperature in the dark. The tissue sections were stained with propidium iodide for a short time, and subsequently detected under a confocal microscope.

Statistical analysis
The data represent mean ± SD from three independent experiments except those that are specifically indicated. Statistical analysis was performed using Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Apigenin inhibited HIF-1{alpha} and VEGF expression in PC-3 and DU145 cells under normoxia and hypoxia conditions
It has been reported that overexpression of HIF-1{alpha} is commonly observed in many human cancers including prostate, breast, colon, and ovarian cancer cells (32,33). To test whether apigenin could suppress HIF-1{alpha} expression in human prostate cancer cells, PC-3 and DU145 cells were treated with apigenin. As shown in Figure 1A, PC-3 and DU145 cells produced high levels of HIF-1{alpha} protein under normoxic condition (21% O2), and apigenin significantly inhibited HIF-1{alpha} expression. Apigenin had little effect on HIF-1ß expression. Similarly, PC-3 and DU145 cells produced high levels of VEGF protein and apigenin suppressed VEGF expression (Figure 1B). To further assess the effect of apigenin under hypoxic condition (1% oxygen), HIF-1{alpha} and VEGF protein levels were determined in PC-3 cells. Hypoxia increased the production of VEGF protein and apigenin treatment inhibited the hypoxia-induced VEGF production (Figure 1C). Immunoblotting analysis showed that hypoxia-induced HIF-1{alpha} expression, which was inhibited by apigenin treatment (Figure 1C). These data indicated that apigenin exerts its inhibitory effect on HIF-1{alpha} and VEGF expression under both normoxic and hypoxic conditions.


Figure 1
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  		Fig. 1 Apigenin inhibited HIF-1{alpha} and VEGF expression in PC-3 and DU145 cells. (A) PC-3 and DU145 cells were cultured to 80–90% confluence, followed by a 6-h treatment with apigenin as indicated. The cells treated with solvent alone were used as a control. HIF-1{alpha} and HIF-1ß protein levels were detected by immunoblotting as described in Materials and methods. (B) Apigenin suppresses VEGF production in PC-3 and DU145 cells. The cells were seeded in 12-well plates, and cultured to 90–100% confluence. The old medium was discarded and fresh medium with or without apigenin was added. The cells were incubated for 15 h. The VEGF concentrations in the medium were measure by ELISA. *, Indicates significant decrease of VEGF when compared to that of control (P < 0.05). (C) Apigenin (Api)-inhibited HIF-1{alpha} and VEGF expression under hypoxic condition. For determination of VEGF production, the cells were seeded in 12-well plates, and cultured to 90–100% confluence. The old medium was discarded and fresh medium with or without apigenin was added. The cells were incubated under hypoxia for 18 h. VEGF was measured as described previously. For immunoblotting analysis, PC-3 cells were pretreated with apigenin for 1 h, followed by the incubation for 6 h under hypoxic condition. The cells were harvested and lysed. Total proteins were analyzed by immunoblotting. *, Indicates significant decrease of VEGF levels when compared to that of the control (P < 0.05). (D) Apigenin inhibited VEGF transcriptional activation. The cells were transfected with 1 µg of human VEGF promoter reporter, pMAP11wt and 0.3 µg of ß-gal plasmid, and cultured for 20 h, then treated with apigenin for 15 h. Cells were lysed and the supernatants were used for Luc and ß-gal activity assay. The relative Luc activities in the cell extracts were assayed by the ratio of Luc/ß-gal activity, and normalized to the value of the solvent DMSO control. *, Indicates significant difference when the value is compared to the control (P < 0.05). (E) Overexpression of HIF-1{alpha} reversed inhibitory effects of apigenin on VEGF transcriptional activation. PC-3 and DU145 cells were transfected with 1 µg of pMAP11wt reporter, 0.3 µg of ß-gal plasmid and 0.5 µg (or 1 µg) of wild-type HIF-1{alpha} plasmid. The empty vector was added to make total transfected DNA to 2.3 µg. The concentration of apigenin used was 20 µM. *, Indicates significant difference when the value is compared to the control, and, hash symbol, indicates significant difference when the value is compared to the apigenin treatment alone (P < 0.05).

 
To know whether apigenin inhibited VEGF transcriptional activation, PC-3 and DU145 cells were transfected with VEGF promoter reporter and treated with apigenin. Apigenin inhibited VEGF reporter activity in both PC-3 and DU145 cells (Figure 1D). Apigenin did not affect cell viability under the same experimental conditions (data not shown) as described previously (29). To test whether apigenin inhibits VEGF through HIF-1{alpha} expression, the cells were co-transfected with VEGF reporter and HIF-1{alpha} plasmids. Overexpression of HIF-1{alpha} reversed apigenin inhibited VEGF reporter activities in both PC-3 and DU145 cells (Figure 1E). In order to determine whether apigenin suppresses VEGF transcriptional activation through HIF-1{alpha} binding site at the VEGF promoter, a mutant VEGF promoter reporter, pMAP11mut was used to test the effect of apigenin (13). Treatment of the cells with apigenin did not inhibit the mutant reporter activity (data not shown), indicating that apigenin affects VEGF transcriptional activation through the HIF-1 binding site. These results suggest that apigenin inhibits VEGF transcriptional activation through HIF-1{alpha} expression.

Apigenin inhibited hypoxia-induced HIF-1{alpha} and VEGF expression in different cancer cells
To test whether apigenin affects hypoxia-induced HIF-1{alpha} and VEGF expression, we use cancer cell lines with low level of HIF-1{alpha} expression under normoxia condition. Human prostate cancer, LNCaP cells, human colon cancer, HCT-8 cells, and human breast cancer, MCF-7 cells, produced low levels of HIF-1{alpha} and VEGF proteins in normal cultured condition (Figure 2). However, when cells were incubated under hypoxic condition, levels of HIF-1{alpha} protein were significantly induced in the cells (Figure 2A). Treatment of the cells with apigenin abrogated the hypoxia induction of HIF-1{alpha} protein (Figure 2A). Similarly, hypoxia dramatically induced VEGF production and apigenin inhibited hypoxia-induced expression of VEGF (Figure 2B). Since hypoxia is associated with tumor growth, the inhibitory effect of apigenin on HIF-1{alpha} and VEGF expression is an important factor for inhibiting tumor growth.


Figure 2
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  		Fig. 2 Apigenin inhibited HIF-1{alpha} and VEGF expression in HCT-8, MCF-7, and LNCaP cells under hypoxia. (A) LNCaP, HCT-8 and MCF-7 cells at 80–90% confluence were pretreated with apigenin for 1 h, then incubated under hypoxic condition for 6 h. HIF-1expression was determined by immnoblotting. (B) The cells were seeded in 12-well plates and cultured to 90–100% confluence. The old medium was discarded and fresh medium with or without apigenin was added. The cells were incubated for 18 h under hypoxia. VEGF in the medium was determined by ELISA. Asterisks, hash symbols and open squares indicate significant decrease of VEGF level when compared to the solvent control of each cell line (P < 0.05).

 
To study the potential mechanism that apigenin inhibits HIF-1{alpha} expression, the cells were treated by apigenin in the presence of cycloheximide for inhibiting de novo protein synthesis. Apigenin treatment reduced the stability of HIF-1{alpha} in PC-3 cells (Figure 3A). The binding of Hsp90 and HIF-1{alpha} is known to stabilize HIF-1{alpha} protein (34–36). We therefore determined the effects of apigenin on the interaction between Hsp90 and HIF-1{alpha}. As shown in Figure 3B, apigenin inhibited the binding of HIF-1{alpha} and Hsp90. These results suggest that apigenin decreases HIF-1{alpha} level by interfering the binding of HIF-1{alpha} and Hsp90 in the cancer cells.


Figure 3
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  		Fig. 3 Apigenin reduced stability of HIF-1{alpha} and interfered the binding of HIF-1{alpha} and Hsp90. (A) PC-3 cells were cultured to 80–90% confluence in normal medium. The cells were pretreated without or with apigenin (20 µM) for 1 h, then incubated with CHX (100 µM) in the presence or absence of apigenin for different times as indicated. HIF-1{alpha} and ß-actin levels were detected by immunoblotting. Relative levels of HIF-1{alpha} protein were determined by measuring the density of the HIF-1{alpha} protein band and normalized to that of ß-actin. The relative HIF-1{alpha} protein level in the control is defined as 100%. (B) PC-3 cells cultured in normal medium were harvested when the cells were at 80–90% confluence. The cell lysates (500 µg) were incubated with 1 µg Hsp90 antibodies in the presence of solvent alone or apigenin (40 µM) for 3 h at 4°C. Aliquots of the Protein A/G plus-agarose beads were added to the lysates, and incubated overnight. The beads were washed four times with ice-cold PBS, then incubated with SDS–PAGE loading buffer, and heated at 100°C for 3 min. The proteins were resolved on 8% SDS–PAGE, and analyzed by immunoblotting.

 
Apigenin inhibited tumor angiogenesis in vivo
To assess whether apigenin inhibits angiogenesis in vivo, we used CAM model to study the effect of apigenin on tumor-induced angiogenesis. As indicated in Figure 4A, PC-3 and OVCAR-3 cells induced angiogenesis on the CAM. Treatment of the cells with apigenin inhibited the tumor-induced angiogenesis (Figure 4A). To quantify the relative angiogenesis on the CAM, the blood vessels were counted in replicate experiments. As shown in Figure 4B, PC-3 and OVCAR-3 cells increased the relative angiogenesis 3- and 4-fold, respectively. The tumor-induced angiogenesis was significantly suppressed by apigenin treatment in a dose-dependent manner (Figure 4B). We analyzed VEGF expression in the tumor tissues by immunohistochemistry staining. Apigenin treatment greatly decreased the VEGF-positive signals in the tumor tissues (Figure 4C). We also analyzed HIF-1{alpha} protein levels in tumor tissues using the extracts of the tumor tissues by immunoblotting. Apigenin treatment inhibited HIF-1{alpha} expression in the tumors (Figure 4D). These results suggest that apigeinin suppress tumor angiogenesis by inhibiting expression of HIF-1{alpha} and VEGF in the tumors. To determine the signaling molecules that might be involved, we determined the phosphorylation of AKT and p70S6K1 in the extracts prepared from tumor tissues, and found that the phosphorylation levels of AKT and p70S6K1 were suppressed by apigenin treatment (Figure 4D).


Figure 4
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  		Fig. 4 Apigenin inhibited in vivo tumor angiogenesis. (A) Apigenin inhibited tumor angiogenesis in the CAM model. PC-3 and OVCAR-3 cells were harvested, and re-suspended in serum-free medium at 1.5 x 107 cells/ml. Aliquots of the cells (15 µl) were mixed with 15 µl of growth-factor-reduced Matrigel in the absence or presence of apigenin as indicated. The cell mixture was implanted onto the CAM of a 9-day-old chick embryo to grow tumor. The total blood vessels around the Matrigel were counted double-blindly under a microscope 4 days after the implantation. The pictures are representatives of each treatment. (B) Quantification of blood vessels on the CAM. The number of blood vessels was normalized to that of Matrigel alone, and the data was the mean ± SD from two independent experiments (n = 8). *, Indicated significant difference when the blood vessel number was compared to that of the cells without apigenin (P < 0.05). (C) Immunohistochemical staining of VEGF expression in the CAM tumor tissues induced by OVCAR-3 cells. (D) Phosphorylation of AKT and p70S6K1 in the tissue extracts. The OVCAR-3-induced tumors on the CAM were snap-frozen in liquid nitrogen, and total tissue extracts were prepared in RIPA lysis buffer and analyzed by immunoblotting.

 
To further analyze the effect of apigenin in angiogenesis in a different animal model, we studied angiogenesis by Matrigel plug assay in nude mice. Similarly, we used PC-3 and OVCAR-3 cells for the study, and found that PC-3 and OVCAR-3 cells greatly induced the formation of blood vessels in the Matrigel plug (Figure 5A). Apigenin treatment inhibited the tumor cell-induced angiogenesis (Figure 5A). Hemoglobin contents in the plug were determined as a relative angiogenesis index. There was little hemoglobin in the Matrigel plug alone. The hemoglobin contents in the plugs with PC-3 and OVCAR-3 cells were 7 and 12 mg/g, respectively (Figure 5B). Apigenin treatment inhibited the hemoglobin levels by >50% in both PC-3- and OVCAR-3-induced tumors (Figure 5B). Taken together, these results indicated that apigenin is a potent inhibitor of tumor angiogenesis.


Figure 5
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  		Fig. 5 Apigenin inhibited tumor angiogenesis in nude mice model. PC-3 and OVCAR-3 cells were re-suspended in serum-free medium at 1.5 x 107 cells/ml, and aliquots of the cells (0.2 ml) were mixed with 0.4 ml of growth-factor-reduced Matrigel in the absence or presence of apigenin (Api) at 20 µM (PC-3 cells) or at 15 µM (OVCAR-3 cells). The PC-3 and OVCAR-3 cells with Matrigel were injected subcutaneously into the both sites of male and female mice, respectively. The male and female nude mice were euthanized in 11 days and 7 days, respectively, after the implantation. (A) The Matrigel plugs were removed from the mice. The photos are representative Matrigel plugs. (B) Hemoglobin levels of Matrigel plugs. Hemoglobin levels in the supernatant were quantified with Drabkin's reagent kit (Sigma-Aldrich, St Louis, MO). The data are mean ± SD from replicate experiments (n = 10). *, Indicated significant difference when the hemoglobin content was compared to that of the control (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Angiogenesis is required for tumor growth and metastasis. Without new blood vessel formation, the tumor cannot grow larger than 1–2 mm in diameter. Therefore, the development of anti-angiogenesis reagents has become a new attractive strategy to combat cancer. Angiogenesis is stimulated by a few growth factors among which VEGF plays a fundamental role. In human cancers, VEGF expression has been related to a more aggressive (37,38), and metastatic phenotype (39). Similar results were observed using animal models (40). Inhibition of VEGF production is a promising therapeutic approach for cancer. We recently found that apigenin inhibited expression of HIF-1{alpha} and VEGF in ovarian cancer cells in vitro (29). However, it is not known that apigenin also inhibits HIF-1{alpha} and VEGF production in other cancer cells and it is not clear whether apigenin inhibits in vivo tumor angiogenesis. In this study, we tested several human cancer cells including prostate cancer cells PC-3, DU145 and LNCaP, breast cancer cell MCF-7, and colon cancer cell HCT-8. PC-3 and DU145 cells produced high levels of HIF-1{alpha} and VEGF under normoxic conditions (Figure 1A and B). Apigenin inhibited expression of HIF-1{alpha} and VEGF in these cells (Figure 1A and B). We found that apigenin inhibited VEGF transcriptional activation through HIF-1{alpha} expression (Figure 1D and E). During tumor growth, there is low oxygen environment inside the tumor. Therefore, it is interesting to know whether apigenin could inhibit HIF-1{alpha} and VEGF expression induced by hypoxia. Under hypoxia condition, accumulation of HIF-1{alpha} and VEGF proteins was observed in PC-3 cells and addition of apigenin suppressed hypoxia-induced HIF-1{alpha} and VEGF expression (Figure 1C). Similarly, the induction of HIF-1{alpha} and VEGF by hypoxia was abrogated in several different cancer cells by apigenin treatment (Figure 2). These results suggest that apigenin inhibits HIF-1{alpha} and VEGF expression under both normoxic and hypoxic conditions in different cancer cells.

To assess the effects of apigenin on tumor angiogenesis, we performed angiogenesis assay in vivo using two different assay systems. The results showed that apigenin is a potent inhibitor of tumor-induced angiogenesis (Figures 4 and 5). Apigenin also inhibited expression of HIF-1{alpha} and VEGF in tumor tissues (Figure 4C and D), suggesting that apigenin inhibits angiogenesis through HIF-1{alpha} and VEGF expression. Expression of HIF-1{alpha} may be regulated through protein synthesis and degradation pathways (33). Under normoxia condition, HIF-1{alpha} expression is induced via activation of PI3K/AKT and MAPK signaling, which is induced by growth factors, cytokines and other signaling molecules (41–44), and/or oncogenic mutations of some tumor suppressor genes, such as VHL (20,45), PTEN (45) and p53 (46). Under hypoxia condition, the accumulation of HIF-{alpha} is due to the inhibition of HIF-1{alpha} degradation (33). We found that apigenin reduced HIF-1{alpha} stability by blocking the binding of HIF-1{alpha} and Hsp90 (Figure 3). It is known that Hsp90 binding stabilizes HIF-1{alpha} protein (34,36). Out results suggest that apigenin inhibits HIF-1{alpha} expression through interfering the interaction between HIF-1{alpha} and Hsp90. The analysis of tumor tissues indicated that apigenin inhibited phosphorylation of AKT (Figure 4D), suggesting that apigenin may also inhibit HIF-1{alpha} expression via AKT signaling. These in vivo data are consistent with our previous in vivo results (29).

Within the solid tumor, hypoxia commonly develops because the rate of tumor cell proliferation outpaces the rate of blood vessel formation. This study shows that apigenin can inhibit expression of HIF-1{alpha} and VEGF in cancer cells under both hypoxia and normoxic conditions, suggesting that apigenin is a potent inhibitor of tumor angiogenesis. The plant-based diets on prevention of cancer and other chronic diseases have been well documented (24). In the present work, we demonstrate that apigenin inhibited VEGF expression through HIF-1 expression in various cancer cells, and suppressed angiogenesis in vivo. These results suggest that apigenin may be used as a chemopreventive and/or chemotherapeutic agent against human cancer in the future.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 12, 2006; revised October 13, 2006; accepted October 16, 2006.


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