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Kaempferol Inhibits Angiogenesis and VEGF Expression Through
Both HIF Dependent and Independent Pathways in Human Ovarian
Cancer Cells
Haitao Luo,
Alderson-Broaddus College, Philippi, West Virginia, USA
Gary O. Rankin,
Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia, USA
Lingzhi Liu,
Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, USA
Matthew K. Daddysman,
Alderson-Broaddus College, Philippi, West Virginia, USA
Bing-Hua Jiang, and
Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, USA
Yi Charlie Chen
Alderson-Broaddus College, Philippi, West Virginia, USA
Abstract
Ovarian cancer is 1 of the most significant malignancies in the Western world, and the
antiangiogenesis strategy has been postulated for prevention and treatment of ovarian cancers.
Kaempferol is a natural flavonoid present in many fruits and vegetables. The antiangiogenesis
potential of kaempferol and its underlying mechanisms were investigated in two ovarian cancer cell
lines, OVCAR-3 and A2780/CP70. Kaempferol mildly inhibits cell viability but significantly reduces
VEGF gene expression at mRNA and protein levels in both ovarian cancer cell lines. In
chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits OVCAR-3-
induced angiogenesis and tumor growth. HIF-1α, a regulator of VEGF, is downregulated by
kaempferol treatment in both ovarian cancer cell lines. Kaempferol also represses AKT
phosphorylation dose dependently at 5 to 20 μM concentrations. ESRRA is a HIF-independent VEGF
regulator, and it is also downregulated by kaempferol in a dose-dependent manner. Overall, this study
demonstrated that kaempferol is low in cytotoxicity but inhibits angiogenesis and VEGF expression
in human ovarian cancer cells through both HIF-dependent (Akt/HIF) and HIF-independent
(ESRRA) pathways and deserves further studies for possible application in angio prevention and
treatment of ovarian cancers.
INTRODUCTION
Ovarian cancer is one of the most important malignancies for women in the Western world,
ranking as the fifth leading cause of cancer-related deaths (1). Due to a lack of effective
Copyright © 2009, Taylor & Francis Group, LLC
Address correspondence to Yi Charlie Chen, Natural Science Division, Alderson-Broaddus College, 101 College Hill Drive, Philippi,
WV 26416. Phone: 304-457-6277. Fax: 304-457-6239. chen@ab.edu.
NIH Public Access
Author Manuscript
Nutr Cancer. Author manuscript; available in PMC 2009 October 30.
Published in final edited form as:
Nutr Cancer. 2009 ; 61(4): 554–563. doi:10.1080/01635580802666281.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
biomarkers for screening, nearly 60–70% of ovarian cancers are diagnosed at advanced stages
(2), with a poor prognosis of about 30% for a 5-yr survival rate (3). Treatment of ovarian
cancers involves surgery and chemotherapy, but is often not effective because of problems
with drug resistance (4) and later relapse in patients (5).
Angiogenesis is the process of developing new blood vessels and plays an important role in
tumor growth (6). In a normal adult, angiogenesis is virtually quiescent, with only 0.01% of
endothelial cells undergoing cell division (7). In contrast, tumor growth requires active
angiogenesis, and antiangiogenesis becomes a rational anticancer strategy (7). Vascular
endothelial growth factor (VEGF) is the most pivotal positive regulator of angiogenesis (6),
and VEGF gene expression is found in many human tumors including ovarian cancers (8).
VEGF gene expression is regulated by oxygen tension, growth factors, hormones, and
oncogenes (9). Hypoxia induces VEGF expression through hypoxia-inducible factor 1 (HIF-1),
which is composed of HIF-1α and HIF-1β subunits, with the former one being inducible (10).
In normoxia, the PI3 kinase/AKT pathway is implicated in the regulation of HIF-mediated
VEGF responses (11). Growth factors and inflammatory cytokines, including epidermal
growth factor, transforming growth factor (TGF), interleukin-1 (IL-1), IL-6, also stimulate
expression of the VEGF gene (9). Estrogens activate VEGF expression through estrogen
receptors (ERs) and the estrogen response element (ERE) (12). Proto-oncogene c-Myc enforces
cellular proliferation and growth in tumors (13) and cooperates with HIF-1 in inducing VEGF
expression (14). Myc has been recently reported to be regulated by PI3K/AKT pathways
(15). Whereas regulation of VEGF through PI3Kinase/AKT and HIF is considered the classical
pathway, a pathway involving peroxisome proliferator-activated receptor gamma coactivator
1 alpha (PPARGC1A) and estrogen-related receptor alpha (ESRRA) was recently discovered
to be independent of HIF (16). This pathway goes through ESRRA, an orphan nuclear receptor
that has a high degree of sequence similarity and intense cross-talk to ERs (17).
Flavonoids are natural polyphenols present in a wide variety of fruits and vegetables (19) and
have been shown to inhibit cancer development (20,21). It has been reported that dietary
flavonoids reduce the risk to cardiovascular disease (22), prostate cancer (23), colorectal cancer
(24), and renal cancer (19) in humans. Flavonoids have also been found to inhibit cell growth
and proliferation (25) and induce cell toxicity (26,27) in cancer cells. Kaempferol [3,5,7-
trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is among the most common dietary
flavonoids and recently has been shown to possess antioxidant and antitumor properties.
Kaempferol was found to inhibit proliferation of human breast and lung cancer cells (28,29),
inhibit ER-α expression in breast cancer cells (28), and induce apoptosis in glioblastoma cells
(30) and in lung cancer cells by activation of MEK-MAPK (29). Kaempferol also exhibited an
anti-inflammatory effect through inhibition of IL-4 (31), COX-2 and CRP expression and
downregulation of NFκ B pathway in liver cells (32). In human studies, a significant 40%
decrease in ovarian cancer incidence was found for the highest quintile of kaempferol intake
as compared to the lowest quintile (33). However, the effect and mechanism by which
kaempferol inhibits ovarian cancer cell proliferation and tumor formation is not yet clear. In
this study, kaempferol was investigated for its effects on angiogenesis and VEGF expression
in ovarian cancer cells and the underlying mechanisms for this effect, including the
conventional AKT-HIF and novel PPARGC1A-ESRRA pathways.
MATERIALS AND METHODS
Cell Culture
Two human ovarian cancer cell lines, OVCAR-3 (mutant p53) and A2780/CP70 (wild-type
p53) (34,35), were maintained in RPMI 1640 medium supplemented with 100 units/ml
penicillin, 100 μg/ml streptomycin (VWR, West Chester, PA), and 10% US-qualified fetal
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bovine serum (Invitrogen, Grand Island, NY) in a humidified incubator with 5% CO2 at 37°
C.
Cell Viability Assay
Kaempferol’s effects on OVCAR-3 and A2780/CP70 cell viability were colorimetrically
determined with a “CellTiter 96 Aqueous One Solution Cell Proliferation Assay” kit from
Promega (Madison, WI). Cells (8 × 103/well) were seeded into 96-well plates and incubated
for 16 h before being treated with 0 to 80 μM kaempferol (Sigma, St. Louis, MO) in triplicates
for another 24 h with DMSO as solvent control. Cells were then washed twice with phosphate-
buffered saline (PBS), introduced with 100 μl medium containing 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS),
incubated at 37°C for 2 h, and measured for OD values at 490 nm. A linear standard curve was
generated by seeding different amount of cells (0 to 1 × 104) and treating with medium
containing DMSO only. Cell viability was expressed as percentage of control from 3
independent experiments.
qRT-PCR
The effects of kaempferol on several genes’ mRNA level were determined by quantitative
reverse-transcription PCR (qRT-PCR). Cells (5 × 105) were seeded in 60-mm dishes and
incubated for 16 h before treatment with kaempferol. For time course of VEGF mRNA
expression, both cell lines were treated with 40 μM kaempferol for 0 to 24 h, and cells were
harvested in TRIzol reagent (Invitrogen, Grand Island, NY) and stored in −80°C until analysis.
For VEGF mRNA expression in response to kaempferol doses, both cell lines were treated
with 0 to 40 μM kaempferol for 24 h before RNA was extracted, reconstituted in DEPC-treated
water, and checked for integrity by agarose-gel electrophoresis. RNA samples were quantitated
at OD 260/280, and 1 μg RNA was introduced to reverse transcription with AMV reverse
transcriptase from Promega (Madison, WI). cDNA equivalent to 80 ng RNA was amplified by
real-time PCR for various genes in triplicate with RT2 SYBR Green qPCR Master Mix
(SuperArray, Frederick, MD) and a Chromo4™ real-time detector coupled to a DNA Engine®
thermal cycler (Bia-Rad, Hercules, CA). Primers for GAPDH, HIF-1α, HIF-1β, and ESRRA
were chosen from the Primer-Bank Web site (http://pga.mgh.harvard.edu/primerbank/), and
primers for VEGF and PPARGC1A were designed from the Primer3 Web site
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer sequences are listed in
Table 1. The PCR program was set as follows: 95°C 10 min (95°C 20s, 58°C 45 s, 72°C 20 s,
77°C 1 s, read plate) × 50; 72°C 5 min; 58°C 1 min; melting curve (65°C to 95°C by 0.5°C
increments). A standard curve for each gene was generated from serial dilutions of PCR
products to monitor amplification efficiency and to relatively quantify mRNA abundance.
RNA samples without reverse transcription served as a non-reverse-transcription (−RT)
control, and water served as a nontemplate control (NTC). Arbitrary units of each gene were
derived from a corresponding standard curve, and mRNA abundance was normalized to
GAPDH levels and expressed as percentages of control for statistical analysis.
ELISA
Secreted VEGF protein levels were analyzed by sandwich ELISA with a Quantikine Human
VEGF Immunoassay Kit from R&D Systems (Minneapolis, MN) targeting VEGF165 in cell
culture supernates. Cells (8 × 103/well) were seeded into 96-well plates and incubated for 16
h before treatment with kaempferol for 24 h. Culture supernates were collected for VEGF
assay, and cell numbers were quantitated with MTS-based assay as mentioned above. VEGF
levels, as determined following the manufacturer’s instructions, were normalized to cell
numbers for each treatment. A total of 4 independent experiments, each in duplicates, was
assayed, and the mean VEGF protein level from each duplicate was used for statistical analysis.
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For phosphorylated ERK1/ERK2 and AKT, DuoSet IC kits (R&D Systems, Minneapolis, MN)
were used to develop sandwich ELISA, measuring phospho-ERK1 (T202/Y204)/phospho-
ERK2 (T185/Y187), and phospho-AKT (Pan) (S473) in cell lysates, respectively. Cells (5 ×
105) were seeded into 60-mm dishes and incubated for 16 h before treatment with kaempferol
for 24 h. Cell lysates were analyzed for p-ERK and p-AKT levels as per instructions, and total
protein levels in lysates were determined with BCA Protein Assay Kit (Pierce, Rockford, IL)
to normalize p-ERK and p-AKT abundance. A total of 4 independent experiments with
triplicates each were performed, and averages from each triplicate were used for statistical
analysis.
Western Blot
Cells (5 × 105) were seeded in 60-mm dishes and incubated for 16 h before treatment with
kaempferol for 24 h. After double wash with cold PBS, cells were harvested with RIPA buffer
freshly supplemented with 3 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mM
sodium vanadate (activated), and 1 mM PMSF (Sigma, St. Louis, MO). Total protein levels
were assayed with a BCA Protein Assay Kit (Pierce, Rockford, IL), and lysates (50 μg total
protein) were separated by 10% SDS-PAGE and blotted into nitrocellulose membrane with a
Mini-Protean 3 System (Bio-Rad Laboratories, Hercules, CA). For immunodetection,
antibodies against HIF-1α, HIF-1β (BD Biosciences, San Jose, CA), and GAPDH (Santa Cruz
Biotechnology, Santa Cruz, CA) were applied and signals visualized with a SuperSignal West
Pico Complete Mouse IgG Detection Kit and x-ray film (Pierce Biotechnology, Rockford, IL).
Protein bands were quantitated with Quantity One software (Bio-Rad Laboratories, Hercules,
CA), normalized to corresponding GAPDH bands, and expressed as percentages of control. A
total of 3 independent experiments were carried out for statistical analysis.
Chicken Embryo Chorioallantoic Membrane (CAM) Assay
OVCAR-3 cells (3 × 106) were suspended in 100 μl medium (serum-free, 4°C), mixed with
50 μl liquid BD Matrigel Basement Membrane Matrix High Concentration (BD Biosciences,
San Jose, CA), supplemented with or without 20-μM kaempferol at 4°C, and implanted in the
CAM of 9-day-old chicken embryos. After 5-day incubation at 37.5°C, tumor implants were
dissected out, photographed, weighed, and counted under a dissecting microscope for
branching blood vessels. A total of 5 chicken embryos were included in each treatment group.
Statistical Analysis
Average values of replicates were collected from independent experiments and analyzed by
analysis of variance (ANOVA) and post hoc test (2-sided Dunnett’s t) with SPSS 15.0 (SPSS
Inc., Chicago, IL) to test both overall differences and specific differences between each
treatment and control. A P value of less than 0.05 was considered significant.
RESULTS
Kaempferol Mildly Inhibits Cell Viability in Ovarian Cancer Cell Lines
OVCAR-3 and A2780/CP70 cells were treated with kaempferol for 24 h and assayed for cell
viability. As shown in Fig. 1A, OVCAR-3 cell viability was inhibited to 91% by 20-μM
kaempferol treatment (P < 0.01), and to 74% at 80-μM kaempferol concentration (P < 0.001).
For A2780/CP70 cells, the viability was slightly promoted to 102% at 20-μM kaempferol
concentration (P > 0.38), and then inhibited down to 94% and 79% by 40-μM and 80-μM
kaempferol treatments, respectively (P < 0.001). An overall inhibitory effect on cell viability
was observed for both cell lines, and A2780/CP70 cells appeared more resistant than OVCAR-3
cells to the inhibiting effect of kaempferol (P < 0.05).
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Kaempferol Inhibits VEGF Expression in Ovarian Cancer Cell Lines
Both cell lines were treated with kaempferol and assayed for VEGF mRNA and protein levels
by qRT-PCR and ELISA, respectively. As shown in Fig. 1B, 40-μM kaempferol treatment did
not influence VEGF mRNA expression within 8 h. However, VEGF mRNA levels were
downregulated to 57% in OVCAR-3 cells (P < 0.05) and 72% in A2780/CP70 cells (P < 0.05)
at Hour 16 and remained at close levels at Hour 24. For 24-h kaempferol treatment (Fig. 1C),
VEGF mRNA expression was downregulated by 20-μM kaempferol treatment to 73% (P <
0.05) and 81% (P < 0.001) in OVCAR-3 and A2780/CP70 cells, respectively. Significant
further downregulation was also observed for 40-μM kaempferol treatment in both cell lines.
Both cell lines showed concentration dependent inhibition on VEGF mRNA levels by
kaempferol treatment, and no significant difference between OVCAR-3 and A2780/CP70 cells
was observed.
VEGF protein levels in cell culture supernates were also downregulated by kaempferol
treatments (Fig. 1D). The levels of VEGF protein were 80% (P < 0.05) and 82% (P < 0.05) of
controls in OVCAR-3 and A2780/CP70 cells, respectively, at 10-μM kaempferol
concentration. Further inhibition was observed for higher kaempferol treatments in a
concentration-dependent and significant manner, and both cell lines appeared similar in their
response to kaempferol’s inhibition on VEGF protein levels.
Kaempferol Inhibits Tumorigenesis and Angiogenesis in Chicken Embryo CAMs
Kaempferol was tested for its effects on in vivo angiogenesis. Chicken embryo CAMs were
implanted with OVCAR-3 cells suspended in cold liquid matrigel, which gels quickly at raised
temperature. This cancer cell-containing gel becomes a tumor in chicken embryo and continues
to grow and induce angiogenesis within the vasculature-rich CAM due to the lack of an immune
system in chicken embryos. Growth and angiogenesis of tumor implants are inhibited by
inclusion of 20-μM kaempferol. As shown in Fig. 1E, the implanted cancer cells grow to a
tumor of 70 mg, with 26 blood vessels counted. Inclusion of 20-μM kaempferol in this implant,
however, reduced tumor growth down to 38 mg (P < 0.01) and inhibited blood vessel
development to 16 (P < 0.05). A typical photograph (Fig. 1E) was shown to contrast the 2
tumors with or without kaempferol in terms of both tumor size and angiogenesis.
Kaempferol Inhibits HIF-1α Protein Expression in Ovarian Cancer Cell Lines
The effect of kaempferol on HIF-1α and HIF-1β gene expression was investigated at both the
mRNA and protein levels by qRT-PCR and Western blot, respectively. As shown in Fig. 2A,
kaempferol treatment did not have an obvious effect on HIF-1α mRNA levels except for an
80-μM treatment on OVCAR-3 cells, which downregulated HIF-1α mRNA level to 80% (P =
0.010). For HIF-1β mRNA, no appreciable effects were found in both cell lines (P > 0.40),
leaving a random distribution and an inconsistent pattern (Fig. 2B).
HIF-1α protein levels showed intense and consistent down-regulation by kaempferol treatment
(Fig. 2C). A 5-μM kaempferol treatment led to inhibition of HIF-1α protein to 75% in
OVCAR-3 cells (P < 0.01), and a 10-μM treatment reduced HIF-1α protein to 70% in A2780/
CP70 cells (P < 0.05). Higher concentrations of kaempferol resulted in greater inhibition, with
the levels of HIF-1α protein at 80-μM kaempferol down to 9% and 4% in OVCAR-3 and
A2780/CP70 cancer cells, respectively (P < 0.001). For the concentration-dependent inhibition
of HIF-1α protein levels, no significant difference between OVCAR-3 and A2780/CP70 cells
was observed. HIF-1β proteins were not affected by kaempferol treatment, with levels
distributed from 91% to 118% (P > 0.50) randomly for both cell lines (Fig. 2D).
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Kaempferol Inhibits p-AKT But Not p-ERK Levels in Ovarian Cancer Cell Lines
The levels of the signal transduction molecules, phosphorylated AKT and ERK, were
determined with ELISA. As shown in Fig. 3A, p-AKT levels were downregulated from 21.8
ng/mg total protein (TP) at control to 11.2 ng/mg TP at 20-μM kaempferol treatment in
OVCAR-3 cells, and from 8.8 ng/mg TP at control to 7.1 ng/mg TP at 20-μM kaempferol
treatment in A2780/CP70 cells, showing a concentration dependent and significant inhibition
in both cell lines (P < 0.05). Phosphorylation of ERK is promoted by kaempferol treatment
(Fig. 3B). The p-ERK levels were increased from 4.7 ng/mg TP at control to 6.7 ng/mg TP at
20 μM in OVCAR-3 cells (P < 0.05), showing a concentration-dependent promotion effect by
kaempferol treatment; however, kaempferol did not affect p-ERK level in A2780/CP70 cells
(P = 0.18). Both signaling molecules were found at much higher levels in OVCAR-3 cells than
in A2780/CP70 cells, and kaempferol’s effect on these signaling molecules was also more
pronounced in OVCAR-3 cells (Fig. 3).
Kaempferol Inhibits ESRRA mRNA Expression in Ovarian Cancer Cell Lines
Kaempferol’s effect on PPARGC1A and ESRRA was examined by qRT-PCR in both cell lines.
As shown in Fig. 4A, no significant change was found on PPARGC1A mRNA levels, resulting
in a random fluctuation of mRNA levels with increasing doses of kaempferol. For ESRRA,
the mRNA levels were inhibited to 62% in OVCAR-3 cells by a 20-μM treatment (P < 0.01)
and to 68% in A2780/CP70 cells at 40-μM kaempferol (P < 0.01; Fig. 4B). Further inhibition
effects were observed for higher kaempferol treatments in a concentration-dependent manner.
A2780/CP70 cells also show higher resistance than OVCAR-3 cells to kaempferol treatment
(P < 0.01), with a significant inhibition of ESRRA mRNA levels observed only at 40- and 80-
μM treatments.
DISCUSSION
The risk of ovarian cancer increases with age and with the use of oral contraceptive pills
(18). Few studies have been done to relate lifestyles to ovarian cancer risks, although ovarian
cancer risk has been consistently associated with a high intake of saturated fat and a low intake
of vegetables (36). Flavonoids, as an abundant ingredient in fruits and vegetables, are believed
to play an important role in anticarcinogenesis through their antioxidant, antiestrogenic,
antiproliferative, antiangiogenic, and anti-inflammatory properties (33). Kaempferol is a
flavonoid widely and abundantly distributed in diet, and an impressive 40% decrease in ovarian
cancer incidence with kaempferol intake reported by a recent study suggests kaempferol as a
potential chemoprevention agent but calls for more intense studies on its effect and mechanism
of action in ovarian cancers (33).
Obviously, kaempferol’s effects in reducing ovarian cancer risks can not be explained solely
for its direct cytotoxicity on ovarian cancer cells, as our experiments on ovarian cancer cell
lines only revealed a very mild inhibitory effect on cell viability. Besides, very few chemicals
can distinguish well between tumor cells and normal tissues, and a strong cytotoxicity is
normally associated with severe side effects because of the killing of healthy cells. As a widely
distributed dietary flavonoid, kaempferol does not cause any severe side effects in either
healthy people or ovarian cancer patients, nor does it kill ovarian cancer cells directly and
strongly. However, kaempferol could possibly inhibit ovarian cancer cells through an indirect
mechanism, antiangiogenesis.
Ovarian cancer cells are known to secrete VEGF to recruit vascular endothelial cells for
angiogenesis (37,38). Angiogenesis is critical in tumor growth, invasion, and metastasis (39,
40). Therefore, VEGF is one of the most significant and direct targets in an antiangiogenesis
strategy. Our experiments discovered time- and dose-dependent inhibition on VEGF
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expression in ovarian cancer cells by kaempferol, with a greater effect at protein levels than
mRNA levels. A 10-μM kaempferol treatment significantly inhibited VEGF protein secretion
down to 80%; but for VEGF mRNA, a concentration of 20 μM was needed to reach similar
VEGF inhibition. This difference possibly reflects an amplification effect in translating mRNA
to proteins in ovarian cancer cells where the signal for VEGF expression was amplified along
the central dogma in genetics. By comparing kaempferol’s inhibitory effects on cell viability
and VEGF expression, it further proves that kaempferol mainly performs its function through
antiangiogenesis rather than killing cells directly: a 10-μM kaempferol treatment inhibits
VEGF protein down to 80%, a slightly higher 20-μM kaempferol treatment inhibits VEGF
mRNA down to 80%, but a eight-fold higher concentration (80 μM) of kaempferol is needed
to inhibit cell viability down to 80% in A2780/CP70 ovarian cancer cells. In our chicken
embryo CAM assay, kaempferol also reduced tumor size significantly. This should be
explained more as a result of antiangiogenesis rather than direct inhibition of tumor growth
because a 20 μM kaempferol only inhibits OVCAR-3 cell viability down to 91% in our cell
viability assay, whereas the tumor weight was reduced down to 54% (from 70 to 38 mg).
Although our results indicate that kaempferol works on ovarian cancer cells through inhibiting
VEGF expression rather than killing cancer cells directly, inhibition of VEGF is more than just
antiangiogenesis. It, in turn, has an indirect effect in suppressing growth of cancer cells.
Originally thought to recruit vascular endothelial cells through a paracrine mode, VEGF was
later found to also act in an autocrine manner to promote growth of tumor cells themselves
(41). Ovarian tumors are known to express VEGF receptors (42), and VEGF secreted by
ovarian cancer cells can act in both autocrine and paracrine fashions to promote growth of
tumor cells. Moreover, VEGF can act as a survival factor through enhanced expression of B-
cl-2 and survivin, protecting cancer cells against apoptosis (6). By targeting VEGF in ovarian
cancer cells, kaempferol inhibits angiogenesis in tumors and represses tumor growth and
survival indirectly.
Our experiments further explored mechanisms through which kaempferol inhibits VEGF
expression and angiogenesis in ovarian cancer cells. It is known that hypoxia stimulates VEGF
expression through inducing HIF-1 transcriptional factors; and under normoxia, the PI3 kinase
and AKT signaling pathway is implicated in HIF-1-mediated responses (43). Like apigenin,
the other dietary flavonoid studied for this pathway (10), kaempferol inhibits HIF-1α protein
levels in a concentration dependent manner; but HIF-1α mRNA levels are only inhibited at
high kaempferol concentration (80 μM) in ovarian cancer cells. This again indicates an
amplification effect in the translation process. HIF-β is a constitutively expressed subunit and
as expected is not affected by kaempferol treatment at mRNA or protein levels. Phosphorylated
AKT in this pathway was downregulated by kaempferol treatment up to 20 μM in a
concentration-dependent manner, and it proves AKT-HIF-VEGF as a working mechanism at
physiologically relevant kaempferol concentrations, which is typically below 20 μM in vivo.
Normal population kaempferol intake is assumed to be the 0.8 to 11.0 mg/day in the Nurses’
Health Study (33). We also examined another signaling molecule, p-ERK, and our results
showed that ERK pathway is not involved in kaempferol’s effect on VEGF inhibition.
Independent of HIF transcription factors, the PPARGC1A/ESRRA pathway is newly
discovered that affects VEGF expression and angiogenesis (16). Kaempferol treatment has no
effect on PPARGC1A mRNA levels, but it concentration dependently repressed ESRRA
mRNA levels in ovarian cancer cells. This might be another mechanism through which
kaempferol inhibits VEGF expression Estrogens bind to nuclear receptors ERα and ERβ, which
are ligand-inducible transcription factors and stimulate transcription of many genes. Although
no classical EREs are found in the 5′ regulatory regions, VEGF is an estrogen responsive gene
with several AP-1 and Sp1 sites in this region to mediate estrogens’ effect (44) and a variant
ERE 1.5 kb away from the transcription start (6). In fact, estrogens have been shown to induce
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VEGF mRNA levels in ER+ breast tumor cells (12), and kaempferol has been reported to
inhibit ERα expression at both mRNA and protein levels in breast cancer cells (28). Although
ESRRA does not respond to an estrogen stimulus, it is a downstream effector of PPARGC1A.
ESRRA is also evolutionarily related to ERs and can efficiently bind to EREs that are
commonly shared by many target genes (45). In fact, ESRRA expression has already been
suggested as a negative prognostic factor for disease-free survival of ovarian cancer patients
(17). Inhibition of ESRRA mRNA levels in ovarian cancer cells has suggested another pathway
as kaempferol’s working mechanism in antiangiogenesis.
Overall, the dietary flavonoid, kaempferol, was found to be effective in inhibiting VEGF
expression, angiogenesis, and cell viability through both the HIF-1α dependent and the
HIF-1α independent pathways in ovarian cancer cells (Fig. 5). As a widely distributed natural
product, kaempferol is low in toxicity, potent in antiangiogenesis, and deserves further study
on possible applications in angio-prevention and therapy of ovarian cancers.
Acknowledgments
This research was supported by Grant P20 RR16477 from the National Center for Research Resources awarded to the
West Virginia IDeA Network for Biomedical Research Excellence.
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FIG. 1.
Kaempferol’s effect on cell viability, VEGF expression, tumorigenesis, and angiogenesis in
ovarian cancer cell lines. A: Cells (8 × 103/well) were seeded in 96-well plates, incubated for
16 h, and treated with kaempferol for 24 h. Cell viability was colorimetrically determined by
a MTS-based method and expressed as percentages of control. Data represents mean ± SE from
3 independent experiments. B: Cells (5 × 105) were seeded in 60-mm dishes, incubated 16 h,
and treated with 40-μM kaempferol for 0 to 24 h. Cells were harvested in TRIzol Reagent and
stored in −80 °C until analysis. RNA was extracted; reverse transcribed with AMV reverse
transcriptase; and quantitated by SYBR Green-based, real-time PCR. VEGF mRNA levels
were normalized by GAPDH abundance and expressed as percentages of control. Data
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represents mean ± SE from 2 independent experiments. C: Cells (5 × 105) were seeded in 60-
mm dishes, incubated 16 h, and treated with 0 to 40 μM kaempferol for 24 h. RNA was extracted
with TRIzol Reagent, reverse-transcribed with AMV reverse transcriptase, and quantitated by
SYBR Green-based, real-time PCR. VEGF mRNA levels were normalized by GAPDH
abundance and expressed as percentages of control. Data represents mean ± SE from 3
independent experiments. D: Cells (8 × 103/well) were seeded in 96-well plates, incubated for
16 h, and treated with kaempferol for 24 h. Culture supernates were collected and analyzed for
VEGF165 by ELISA, and cell numbers were determined by MTS-based assay. VEGF levels
were normalized by cell numbers and expressed as percentages of control. Data represents
mean ± SE from 4 independent experiments. E: OVCAR-3 cells (3 × 106) were suspended in
100 μ l serum-free medium at 4°C, mixed with 50 μl cold BD Matrigel, supplemented with or
without 20-μM kaempferol at 4°C, implanted in the chorioallantoic membrane of 9-day-old
chicken embryos, and incubated for 5 days at 37.5°C before tumors were weighed and counted
for blood vessels. Data represents mean ± SE from 5 chicken embryos. *P < 0.05 as compared
to control. **P < 0.01 as compared to control.
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FIG. 2.
Kaempferol’s effect on HIF gene expression. A and B: Cells (5 × 105) were seeded in 60-mm
dishes, incubated 16 h, and treated with kaempferol for 24 h. RNA was extracted with TRIzol
Reagent, reverse-transcribed with AMV reverse transcriptase, and quantitated by SYBR
Green-based, real-time PCR. HIF-1α and HIF-1β mRNA levels were normalized by GAPDH
abundance and expressed as percentages of control. Data represents mean ± SE from 3
independent experiments. C and D: Cells (5 × 105) were seeded in 60-mm dishes, incubated
for 16 h, and treated with kaempferol for 24 h. Cells were harvested with RIPA buffer, and cell
lysates were separated by SDS-PAGE and blotted into nitrocellulose membrane for
immunodetection. Chemiluminescent signals were captured by x-ray film and quantitated by
imaging software. HIF-1α and HIF-1β protein levels were normalized by GAPDH and
expressed as percentages of control. Typical blots were shown for both cell lines, and data
represents mean ± SE from 3 independent experiments. *P < 0.05 as compared to control.
**P < 0.01 as compared to control.
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FIG. 3.
Kaempferol’s effect on p-AKT and p-ERK levels. A and B: Cells (5 × 105) were seeded in 60-
mm dishes, incubated for 16 h, and treated with kaempferol for 24 h. Cell lysates were analyzed
for p-AKT and p-ERK with ELISA, and for total protein with BCA assay. Levels of p-AKT
and p-ERK were normalized by total protein. Data represents mean ± SE from 4 independent
experiments. *P < 0.05 as compared to control.
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FIG. 4.
Kaempferol’s effect on PPARGC1A and ESRRA mRNA level. A and B: Cells (5 × 105) were
seeded in 60-mm dishes, incubated 16 h, and treated with kaempferol for 24 h. RNA was
extracted with TRIzol Reagent, reverse-transcribed with AMV reverse transcriptase, and
quantitated by SYBR Green-based, real-time PCR. PPARGC1A and ESRRA mRNA levels
were normalized by GAPDH abundance and expressed as percentages of control. Data
represents mean ± SE from 3 independent experiments. **P < 0.01 as compared to control.
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FIG. 5.
Proposed mechanism for kaempferol’s inhibition of angiogenesis and proliferation in ovarian
cancer cells. Connector lines represent established pathways. Arrows indicate regulations by
kaempferol treatment in experimental results. PPARGC1A, peroxisome proliferator-activated
receptor gamma coactivator 1 alpha; ESRRA, estrogen-related receptor alpha; HIF-1α,
hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor.
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TABLE 1
Primer sequences of genes analyzed by qRT-PCRa
Gene GenBank Accession Primers Amplicon Size
VEGF NM 001025368 AAG GAG GAG GGC AGA ATC AT 226
ATC TGC ATG GTG ATG TTG GA
GAPDH NM 002046 CAT GAG AAG TAT GAC AAC AGC CT 113
AGT CCT TCC ACG ATA CCA AAG T
HIF-1αNM 001530 ATC CAT GTG ACC ATG AGG AAA TG 126
CTC GGC TAG TTA GGG TAC ACT T
HIF-1βNM 001668 CTG CCA ACC CCG AAA TGA CAT 117
GCC GCT TAA TAG CCC TCT GG
ESRRA NM 004451 GTC CAA AGG GTT CCT CGG AG 221
GGA TGC CAC ACC ATA GTG GTA
PPARGC1A NM 013261 TGG GTA GCC CAT CAA AAT GT 176
TGG TAC TTA CCA CGG CAT GA
aAbbreviations are as follows: qRT-PCR, quantitative reverse-transcription polymerase chain reaction; VEGF, vascular endothelial growth factor; GAPDH, glyderaldehyde-3-phosphate dehydrogenase;
HIF, hypoxia-inducible factor; ESRRA, estrogen-related receptor alpha; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1 alpha.
Nutr Cancer. Author manuscript; available in PMC 2009 October 30.
