Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathway in human osteosarcoma U‐2 OS cells

Abstract

Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti-proliferation activities and induces apoptosis in many cancer cell lines. However, there are no reports on human osteosarcoma. In this study, we investigate the anti-cancer effects and molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate that kaempferol significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, especially U-2 OS cells in a dose-dependent manner, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis confirm the effects of DNA damage and apoptosis in U-2 OS cells. Flow cytometry detects the increase of cytoplasmic Ca(2+) levels and the decrease of mitochondria membrane potential. Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences the time-dependent expression of proteins involved in the endoplasmic reticulum stress pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The anti-cancer effects of kaempferol in vivo are evaluated in BALB/c(nu/nu) mice inoculated with U-2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol inhibits human osteosarcoma cells in vivo and in vitro.

Full text

RESEARCH ARTICLE
Kaempferol induced apoptosis via endoplasmic
reticulum stress and mitochondria-dependent pathway
in human osteosarcoma U-2 OS cells
Wen-Wen Huang
1
, Yu-Jen Chiu
2
, Ming-Jen Fan
3
, Hsu-Feng Lu
4,5
, Hsiu-Feng Yeh
6
,
Kun-Hong Li
2
, Po-Yuan Chen
1
, Jing-Gung Chung
1,3
and Jai-Sing Yang
7

1
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
2
School of Medicine, China Medical University, Taichung, Taiwan
3
Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan
4
Department of Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, Taipei, Taiwan
5
Department of Clinical Pathology, Cheng-Hsin General Hospital, Taipei, Taiwan
6
School of Pharmacy, China Medical University, Taichung, Taiwan
7
Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan
Received: January 6, 2010
Revised: February 23, 2010
Accepted: March 25, 2010
Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti-
proliferation activities and induces apoptosis in many cancer cell lines. However, there are no
reports on human osteosarcoma. In this study, we investigate the anti-cancer effects and
molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate
that kaempferol significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, espe-
cially U-2 OS cells in a dose-dependent manner, but exerts low cytotoxicity on human fetal
osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis
confirm the effects of DNA damage and apoptosis in U-2 OS cells. Flow cytometry detects the
increase of cytoplasmic Ca
21
levels and the decrease of mitochondria membrane potential.
Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences
the time-dependent expression of proteins involved in the endoplasmic reticulum stress
pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase
inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The
anti-cancer effects of kaempferol in vivo are evaluated in BALB/c
nu/nu
mice inoculated with
U-2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol
inhibits human osteosarcoma cells in vivo and in vitro.
Keywords:
Apoptosis / Endoplasmic reticulum stress / Kaempferol / Mitochondria-dependent/
U-2 OS
1 Introduction
Flavonoids are a class of plant secondary metabolites,
assorted into flavones, flavonols, flavanones, isoflavones,
and anthocyanidins [1]. It has been reported that intake of
flavonoids is associated with many biological properties,
such as antiviral [2], antitumor [3], anti-oxidative [4],
anti-inflammatory [5], hepatoprotective activities [6] and the
Correspondence: Professor Jing-Gung Chung, Department of
Biological Science and Technology, China Medical University,
No 91, Hsueh-Shih Road, Taichung City 404, Taiwan
E-mail: jgchung@mail.cmu.edu.tw
Fax: 1886-4-2205-3764
Abbreviations: PI, propidium iodide; DMSO, dimethyl sulfoxide;
FBS, fetal bovine serum; Z-LEHD-FMK, z-Leu-Glu-His-Asp-fluoro-
methyl ketone; Z-DEVDFMK, z-Asp-Met-Gln-Asp-fluoromethyl
ketone; hFOB, human fetal osteoblast; MTT, 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide; Dcm, mitochon-
drial membrane potential; ER, endoplasmic reticulum; EDTA,
ethylenediaminetetraacetic acid; DAPI, 4’,6-diamidino-2-phenyl-
indole

Additional corresponding author: Assistant Professor Jai-Sing Yang
E-mail: jaising@mail.cmu.edu.tw
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
Mol. Nutr. Food Res. 2010, 54, 1585–1595 1585DOI 10.1002/mnfr.201000005

prevention of cardiovascular diseases [7]. Kaempferol, 3, 4’,
5, 7-tetra hydroxyflavone, a natural flavonoid, has been
isolated from various plant sources [8]. Kaempferol is
commonly known for antioxidant activity and is used for
cyto-protection agents. Previous studies have reported that
kaempferol has anti-proliferation activity and induces
apoptosis in various human cancer cell lines in vitro, such as
non-small cell lung cancer [9], leukemia [10], esophageal
cancer [11], prostate cancer [12], oral cavity cancer [13] and
colon cancer [14], but no reports on osteosarcoma.
High-grade osteosarcoma is the most common bone
malignancy, accounting for about 60% of malignant bone
tumors diagnosed in the first two decades of life, with an
aggressive local pattern of growth and high metastatic
potential [15]. Current standard treatment is to use chemo-
therapy followed by surgical resection [16]. The survival rate
of patients with localized osteosarcoma is about 11% with
surgery alone, compared to approximately 70% when
combined with chemotherapy [17]. Despite the success of
frontline therapy, about 40% of patients have progression
and further therapy with additional chemotherapy is
palliative and toxic. It is estimated that less than 30% of
patients with recurrent metastasis will be cured [18].
Chemotherapy-resistant cancer is one of the most serious
obstacles. Therefore, in this study, we focus on identifying
new agents to treat osteosarcoma.
Apoptosis is the process of programmed cell death that
occur in multi-cellular organisms, playing an important role
in normal physiology in animals [19]. However, impairment
of apoptotic function has been associated with several
diseases [20], such as neurodegenerative disorders and
cancers [21]. The perturbation of this process is considered a
crucial part of cancer prevention and therapy.
To date, one of the most effective anti-cancer strategies is
through the induction of apoptosis. Although previous
studies have reported that kaempferol has anti-cancer
activity in various human cancer cell lines [9–14], little is
known of the mechanisms exerting cytotoxicity in human
osteosarcoma. Therefore, the purpose of this study is to
investigate the apoptotic effects and the molecular
mechanisms of kaemperol in osteosarcoma cell lines in vitro
and in vivo.
2 Materials and methods
2.1 Chemicals and reagents
Kaempferol, propidium iodide (PI), Tris-HCl, calpeptin and
Triton X-100 were obtained from Sigma Chemical Co.
(St. Louis, MO, USA). BAPTA, dimethyl sulfoxide (DMSO)
and potassium phosphates were purchased from Merck Co.
(Darmstadt, Germany). Eagle’s minimum essential medium
(MEM), penicillin-streptomycin, trypsin-EDTA, fetal bovine
serum (FBS) and glutamine were obtained from Gibco BRL
(Grand Island, NY, USA). Caspase activity assay kit was
bought from OncoImmunin (MD, USA). Caspase-9 inhi-
bitor z-Leu-Glu-His-Asp-fluoromethyl ketone (Z-LEHD-
FMK) and caspase-3 inhibitor z-Asp-Met-Gln-Asp-fluoro-
methyl ketone (Z-DEVDFMK) were bought from R&D.
2.2 Human osteosarcoma cell lines (U-2 OS, HOB,
143B) and human fetal osteoblast progenitor cell
line (hFOB)
Human osteosarcoma U-2 OS, HOB, 143B cells and
conditionally immortalized human fetal osteoblast
progenitor hFOB cells were purchased from American Type
Culture Collection (ATCC). U-2 OS cells were cultured in
McCoy’s 5A medium (GIBCO-BRL) with 10% FBS and
antibiotics (100 U/ml of penicillin G and 100 mg/ml of
streptomycin) at 371C in a humidified atmosphere of 5%
CO
2
/95% air. HOB cells were cultured in minimum
essential medium (GIBCO-BRL) with 10% FBS and
antibiotics (100 U/ml of penicillin G and 100 mg/ml of
streptomycin) and 1.5 g/L sodium bicarbonate, 0.1 mM non-
essential amino acids at 371C in a humidified atmosphere of
5% CO
2
/95% air. 143B cells were cultured in minimum
essential medium (GIBCO-BRL) with 10% FBS and anti-
biotics (100 U/ml of penicillin G and 100 mg/ml of strepto-
mycin) and 0.015 mg/ml 5-bromo-20-deoxyuridine at 371Cin
a humidified atmosphere of 5% CO
2
/95% air. hFOB cells
were cultured in DMEM/Ham’s F12 medium (GIBCO-BRL)
with 10% FBS and antibiotics (100 U/ml of penicillin G,
100 mg/ml of streptomycin and 300 mg/ml geneticin) at
33.51C in a humidified atmosphere of 5% CO
2
/95% air [22].
2.3 Cell viability assay
The cell viability was determined by 3-(4,5-dimethylthiazol-
2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.
Human osteosarcoma U-2 OS, HOB, 143B cells and
conditionally immortalized human fetal osteoblast
progenitor hFOB cells were cultured in 96-well culture
plates and allowed to attach for hours before treated with
various concentrations (0, 25 50, 100, 150 or 200 mM) of
kaempferol. After cultivation for 24 h, 0.5 mg/ml of MTT
was then added to each well and the mixture was incubated
for 4 h at 371C. Culture medium was then replaced with an
equal volume of 0.04N HCl/isopropanol to dissolve forma-
zan crystals. Absorbance of each well was determined at
570 nm wavelength using ELISA reader [23].
2.4 Phase-contrast microscopy of morphological
changes
U-2 OS cells were plated in 24-well plates at a density of
2.5 10
5
cells/well. The 50, 100 or 150 mM of kaempferol
were added, and the cells were incubated for 24 h. A phase-
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contrast microscope was used for photography to determine
morphological changes as described elsewhere [24].
2.5 Comet assay and DAPI staining
After treated with 50, 100 or 150 mM of kaempferol, U-2 OS
cells were harvested and mixed with low melting point
(LMP) agarose at 371C. This mixture was placed on the top
of previous layer of 5% agarose (normal melting point) on
the slide, and then covered with a covership at 41C until
solid. Subsequently, the covership was removed gently and
some agarose was added onto this slide, and then covered
with the covership again. The slide was placed at 41C until
the mixture was solid, and put in chilled alkaline lysis buffer
for electrophoresis. Afterwards, the slide was gently washed
with neutralized buffer, and stained with DAPI [25].
2.6 Agarose gel electrophoresis
After treated with 150 mMofkaempferol,U-2OScellswere
harvested then lysed in lysis buffer (20 mM Tris, 10 mM
EDTA, 0.2 % Triton X-100, pH 8.0) at 41C for 15 min, and then
the lysate was centrifuged for 13,000 rpm, 10 min at 41C. The
supernatant containing fragmented DNA was collected and
incubated at 501C overnight with proteinase K (0.1 mg/ml) to
digest protein, followed by RNase A (50 mg/ml) digestion at
371C for 30 min. After extracted with phenol/chloroform/
isoamyl alcohol (25:24:1), the DNA was precipitated in 50%
isopropanol with 1 mlofglycogen(20mg/ml) at 201Cover-
night. The precipitated DNA was centrifuged at 14,000 rpm for
30 min, dried, and dissolved in 10 mlH
2
O. After electrophor-
esis in a 1.5% agarose gel containing ethidium bromide
(0.5 mg/ml) in TAE buffer (40 mM Tris-acetate, 1 mM EDTA,
pH 8.0), the DNA in gel was resolved with UV light [26].
2.7 Terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) assay
After treated with 150 and 200 mM of kaempferol, U-2 OS
and hFOB cells were harvested. Evaluation of apoptosis in
the U-2 OS and hFOB cells were accomplished by flow
cytometry to detect cells labeled by TUNEL, using fluor-
escein-labeled dUTP (treatment). Controls consisted of cells
incubated with fluorescein dUTP without Tdt (In Situ Death
Kit, Boehringer-Mannheim Biochemicals) [27].
2.8 Intracellular Ca21levels assay
U-2 OS cells (2.5 10
5
/well) in 12-well plate were treated
with 150 mM of kaempferol and incubated for 0, 6, 12 or
24 h. Cells were harvested, washed twice, re-suspended in
3mg/ml of Indo 1/AM (Calbiochem; La Jolla, CA) at 371C for
30 min and analyzed by flow cytometry (Becton Dickinson
FACS Calibur) [26].
2.9 Calpain activity assays
U-2 OS cells were prepared on 24-well plates and pretreated
with BAPTA, a Ca
21
chelator or calpeptin and an inhibitor
of calpain for 1 h. Then, cells were loaded with 40 M Suc-
Leu-Leu-Val-Tyr-AMC calpain protease substrate (Biomol)
and treated with 150 mM of kaempferol to the indicated time
at 371C in a humidified 5% CO
2
incubator. Proteolysis of the
fluorescent probe was monitored by a fluorescent plate
reading system (HTS-7000 Plus Series BioAssay, Perkin
Elmer) with filter settings of 360720 nm for excitation and
460720 nm for emission [28].
2.10 Determination of mitochondrial membrane
potential (Dcm)
The mitochondrial membrane potential (Dc
m
) of the U-2
OS cells was determined by flow cytometry using DiOC6
(Molecular Probes). U-2 OS cells were treated with 150 mM
of kaempferol for 0, 6, 12 or 24 h to detect the changes of
Dc
m
. The cells were harvested and washed twice, re-
suspended in 500 ml of DiOC6 (4 mmol/L) and incubated at
371C for 30 min before analyzed by flow cytometry (Becton
Dickinson FACSCalibur) [29].
2.11 Caspase-3, -8 and -9 activities assay
Caspase-3, -8 and -9 activities were assessed according to
manufacturer’s instruction of caspase colorimetric kit (R&D
system Inc., MN, USA). U-2 OS cells were seeded in 12-well
cell culture plates at an initial density of 5.0 10
6
cells and
pretreated with caspase-3 inhibitor (Z-DEVD-FMK), caspase-
8 inhibitor (Z-IETD-FMK) or caspase-9 inhibitor (Z-LEHD-
FMK) for 1 h prior to treatment with 150 mM of kaempferol
for 0, 6, 12 or 24 h. Cells were harvested and lysed for 10 min
in 50 ml lysis buffer which contained 2 mM DTT.
After centrifugation, the supernatant containing 100 mg
protein were incubated with caspase-3 substrate (Ac-DEVD-
pNA), caspase-8 substrate (Ac-IETD-pNA) and caspase-9
substrate (Ac-LEHD-pNA) respectively in reaction buffer.
Then all samples were incubated in 96-well flat bottom
microplate at 371C for 1 h. Levels of released pNA
were measured at O.D.405 nm with ELISA reader (Anthos
2001) [23].
2.12 Western blot analysis
Briefly, the cytosolic and total proteins were collected from
U-2 OS cells which were treated with 150 mM of kaempferol
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Mol. Nutr. Food Res. 2010, 54, 1585–1595 1587

for 0, 6, 12 or 24 h. Protein concentrations were determined
by the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA,
USA). Protein samples (30 mg each) were boiled with gel
loading buffer for 5 min. Protein extracts were separated on
10% SDS-polyacrylamide electrophoresis gels (SDS-PAGE)
and transferred onto a polyvinylidene difluoride (PVDF)
membrane. After blocking with TBST (0.05% Triton X-100
in PBS ) buffer of 5% non-fat milk for 1 h, the membrane
was exposed to the primary antibody: GADD153, GRP78,
GRP94, ATF-6a, ATF-6b, calpain 1, calpain 2, Fas, FasL,
Bax, Bcl-2, cytochrome c, Apaf-1, AIF, caspase-4, caspase-9,
caspase-3, caspase-7, caspase-8, caspase-12 and b-actin,
primary antibodies were diluted in PBST (0.05% Triton
X-100 in PBS ) buffer of 5% non-fat milk, and incubated at
41C overnight. The secondary antibodies were coupled
to horseradish peroxidase. Finally, they were detected by
ECL [27].
2.13 In vivo tumor xenograft model
Fifteen BALB/c
nu/nu
mice eight-week-old (approximately
22–28 g) were purchased from the Laboratory Animal Center,
National Taiwan University, College of Medicine (Taipei,
Taiwan). U-2 OS cells (1 10
7
) in culture medium were
subcutaneously injected into the flank of each mouse. Mice
with tumors were randomly assigned to three groups and
each group contained five animals. The treatment was initi-
ated when xenografts reached a volume of about 100 mm
3
and
these mice were treated orally every day with olive oil (control
vehicle), 25 mg/kg or 50 mg/kg of k aempferol in olive oil.
After xenograft tumor transplantation, mice were closely
monitored, counted and weighted. The tumor sizes were
measured every four days using calipers and tumor volume
was estimated according to the following formula: tumor
volume (mm
3
)5LxW
2
/2 (L: length and W: width). At the end
of the study, animals were sacrificed. Tumors were removed,
measured and weighted individually [29, 30].
2.14 Densitometry and statistical analysis
All data were expressed as mean7SEM from at least three
separate experiments. Statistical calculations of the data
were performed using an unpaired Student’s t-test. Statis-
tical significance was set at

Po0.05;

Po0.01;

Po0.001
was taken as significant.
3 Results
3.1 Effects of kaempferol on cell viability in human
osteosarcoma U-2 OS, HOS and 143B cells
We treated human osteosarcoma U-2 OS, HOB, 143B
and human fetal osteoblast progenitor hFOB cells
with kaempferol at different concentrations from 0 to
200 mM for 24 h. The number of viable cells was counted by
MTT method. As shown in Fig. 1A, the viability
was significantly decreased in the kaempferol-treated
human osteosarcoma cells groups, but not in hFOB
cells (IC
50
4200 mM). The IC
50
for U-2 OS cells was
148.36 mM. This therefore indicated that kaempferol
reduced the proportion of viable osteogenic cancer cells in
dose-dependent manner, but with low toxicity to hFOB
cells.
3.2 Effects of kaempferol on cell morphological
changes, DNA damage and apoptosis in human
osteosarcoma U-2 OS cells
To investigate the occurrence of morphological changes
and DNA damage in human osteosarcoma cells, we
predominantly focused on U-2 OS cells and treated
them with kaempferol at different concentrations from 0 to
150 mM for 24 h. In Fig. 1B, morphological examinations
of U-2 OS cells showed the difference between the kaemp-
ferol-treated groups and the control. In the kaempferol-
treated groups, cancer cells were detached from the
surface and contained some debris, whereas the control
group was well spread with a flattened morphology. In
Fig. 1C–D, the data showed that U-2 OS cells induced DNA
fragmentation and DNA damage was determined by
DAPI staining and comet assay. In Fig. 1E, in order to
reconfirm the induction of DNA damage, we isolated
DNA from the cells after treatment with 150 mM kaempferol
for 24 h, and then they were harvested for DNA fragmen-
tation determination in DNA gel electrophoresis. The
results showed that kaempferol induced apoptosis because
of the occurrence of DNA ladder. We investigated whether
or not kaempferol induces U-2 OS cell death through an
apoptotic mechanism. TUNEL assay was used for the
detection of DNA fragmentation in apoptosis. In Fig. 1F,
compared with control cells, U-2 OS cell were treated with
kaempferol showed significant cell apoptosis. However,
hFOB cell were showed non-significant cell apoptosis. We
suggested that kaempferol represented a promising candi-
date as an anti-osteosarcoma drug with low toxicity to
normal cells.
3.3 Effects of kaempferol on the cytoplasmic Ca21
and mitochondria membrane potential (Dcm)
levels in human osteosarcoma U-2 OS cells
In order to elucidate the possible signaling pathways of
kaempferol-induced apoptosis in U-2 OS cells,we examined
intracellular Ca
21
levels and mitochondria membrane
potential by flow cytometry analysis. As shown in Fig. 2A
and Fig. 3A, U-2 OS cells were treated with 150 mM
of kaempferol for 24 h and this significantly increased
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cytoplasmic Ca
21
levels and decreased Dcmin time-depen-
dent manner. These results suggested that kaempferol-
induced apoptotic response might be mediated by endo-
plasmic reticulum stress and mitochondrial-dependent
apoptotic pathways.
3.4 Effects of kaempferol on the levels of
endoplasmic reticulum stress related proteins in
human osteosarcoma U-2 OS cells
To be more detail in the molecular mechanisms of
endoplasmic reticulum stress pathway, we investigated
these related protein levels: GADD153, GRP78, GRP94,
ATF-6a, ATF-6b, caspase-4, caspase-12, calpain 1 and
calpain 2 by western blotting. As shown in Fig. 2B,
kaempferol increased these protein levels in time-depen-
dent manner, but caspase-12 has no statistical influence.
These results suggested that kaempferol-induced apop-
tosis was mediated via endoplasmic reticulum stress
pathway.
3.5 Effects of kaempferol with BAPTA or Calpeptin
on the levels of cytoplasmic Ca21and Calpain
activity and cell viability in human
osteosarcoma U-2 OS cells
In order to confirm that kaempferol-induced apoptosis
was mediated by ER stress pathway, we pretreated U-2 OS
cells with BAPTA, a Ca
21
chelator or calpeptin, an inhibitor
of calpain, after exposure to kaempferol. As shown in
Figure 1. Effects of kaempferol on cell
viability and apoptosis in osteo-
sarcoma cell lines. After treatment with
various concentrations of kaempferol
for 24 h, the cell viabilities of U-2 OS,
HOB, 143B osteosarcoma cell lines and
the conditionally immortalized human
fetal osteoblast progenitor hFOB
cells are shown (A). Data represent
mean7SD of three experiments.

po0.001. U-2 OS cells in response
to various concentrations of kaemp-
ferol for 24 h showed morphological
changes (B) which indicated kaemp-
ferol-induced cell death, and DAPI
staining (C), comet assay (D), gel elec-
trophoresis (E) and TUNEL assay (F)
revealing kaempferol-induced DNA
damage, fragmentation and apoptosis,
which was another hallmark of cells
undergoing apoptosis.
Mol. Nutr. Food Res. 2010, 54, 1585–1595 1589
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Fig. 2C–D, the levels of calpain activity and cell viability
were significantly influenced. Overall, in Fig. 2, these data
demonstrated that activation of ER stress pathway played an
important role in kaempferol-induced apoptosis in U-2 OS
cells.
3.6 Effects of kaempferol on the levels of Bcl-2
family in human osteosarcoma U-2 OS cells
Previous studies have demonstrated that Bcl-2 and Bax
locate in the mitochondrial outer-membrane and the
Bcl-2/Bax ratio regulate the release of mitochondrial
cytochrome cto cytosol [31, 32]. We investigated expression
levels of Bcl-2 and Bax in kaempferol-treated U-2 OS cells by
western blotting. As shown in Fig. 3B, the pro-apoptotic
protein level of Bax was up-regulated, whereas the anti-
apoptotic protein level of Bcl-2 was down-regulated in time-
dependent manner.
3.7 Effects of kaempferol on the levels of
mitochondrial caspase-dependent and caspase-
independent pathway related proteins in human
osteosarcoma U-2 OS cells
To be more detail in the molecular mechanisms of mito-
chondrial-dependent apoptotic pathway, we examined the
expression levels of cytochrome c, Apaf-1, caspase-9, caspase-3,
caspase-7 and AIF by western blotting. As shown in Fig. 3B,
these protein levels were increased in time-dependent manner.
Our results suggested that kaempferol-induced apoptotic
response was mediated by mitochondrial-dependent cascade.
3.8 Effects of kaempferol on the caspase-9 and caspase
-3 activities in human osteosarcoma U-2 OS cells
In order to confirm that kaempferol-induced apoptosis was
mediated by caspase-dependent pathway, we investigated the
Figure 2. Effects of kaempferol
on U-2 OS cells in endoplasmic
reticulum stress apoptotic
pathway. The intracellular Ca
21
levels in kaempferol-treated
U-2 OS cells from each time
point were measured by flow
cytometric analysis (A). Cells
were treated with 150 mMof
kaempferol for the indicated
time, cytosolic proteins or
whole cell lysate were
prepared, and subjected to
Western blotting. The resulting
blots were probed for
GADD153, GRP78, GRP94, ATF-
6a,ATF-6b, caspase-4, caspase-
12, calpain1 and calpain2
(whole cell lysate). b-actin
served as the loading control.
Levels of the associated
proteins in endoplasmic reticu-
lum stress apoptotic pathway
were affected (B). Cells were
pretreated with BAPTA, a Ca
21
chelator or calpeptin, an inhi-
bitor of calpain for 1 h after
exposure to kaempferol, then
incubated for 24 h. The whole-
cell lysates were subjected to
calpain activity assay (C) and
cells were collected to deter-
mine the percentage of viable
cells (D). Data from three inde-
pendent experiments were
presented (

Po0.001, as
compared with control treat-
ments).
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caspase-9, -3 and -8 activities by fluorogenic enzymatic assay.
As shown in Fig. 3C–D, both caspase-9 and caspase-3 activ-
ities were significantly increased. Moreover, pre-incubation
with specific inhibitors of caspases-9 (Z-LEHD-FMK), caspase-
3 (Z-DEVE-FMK) strongly reduced the caspase-9 or caspase-3
activities and increased U-2 OS cell viability. However,
caspase-8 activity has no significant influence. Overall, in
Fig. 3, these data demonstrated that caspase-dependent
mitochondrial pathway played an important role in kaemp-
ferol-induced apoptosis in U-2 OS cells.
3.9 Effects of kaempferol on anti-proliferative
activity in BALB/c
nu/nu
mice after injection with
human osteosarcoma U-2 OS cells
Three groups of mice were respectively treated with DMSO
control vehicle, 25 mg/kg or 50 mg/kg of kaempferol. These
representative animals with tumors were shown in Fig. 4A.
In Fig. 4B–C, kaempferol significantly decreased the
tumor weight and tumor volume compared to the control
group.
Figure 3. Effects of kaempferol
on U-2 OS cells in mitochon-
drial-dependent apoptotic pa-
thway. The mitochondrial
membrane potential (Dc
m
)of
kaempferol-treated U-2 OS
cells from each time point was
measured by staining with
DiOC6 (A). Cells were treated
with 150 mM of kaempferol for
the indicated time, cytosolic
proteins or whole cell lysate
were prepared, and subjected
to Western blotting. The
resulting blots were probed for
cytochrome c, Apaf-1, AIF,
caspase-9, caspase-3, caspase-
7 (cytosolic proteins), and Bcl-
2,Bax (whole cell lysate).
b-actin served as the loading
control. Levels of the asso-
ciated proteins in mitochon-
drial-dependent apoptotic
pathway were affected (B).
Cells were pretreated with
specific inhibitors of caspases-
9 (Z-LEHD-FMK), caspase-3
(Z-DEVE-FMK) or caspase-8
inhibitor (Z-IETD-FMK) for 1 h
after exposure to kaempferol,
then incubated for 24 h.
The whole-cell lysates were
subjected to caspase activity
assay (C) and cells were
collected to determine the
percentage of viable cells (D).
Data from three independent
experiments were presented
(

Po0.001, as compared
with control treatments).
Mol. Nutr. Food Res. 2010, 54, 1585–1595 1591
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4 Discussion
Kaempferol, a natural flavonoid, has been reported to induce
apoptosis and inhibit proliferation in various human cancer
cell lines, including non-small cell lung cancer [9], leukemia
[10], esophageal cancer [11], prostate cancer [12], oral cavity
cancer [33] and colon cancer [14]. Furthermore, Zhang et al.
demonstrated that kaempferol not only effectively inhibited
pancreatic cancer cell proliferation and induced apoptosis,
but also may sensitized pancreatic tumor cells to chemo-
therapy [34]. However, little is known in human osteo-
sarcoma cell lines. In contrast to beneficial effects, there are
still some question marks about the toxic side-effects to
normal tissue. Li et al. showed cytotoxicities of kaempferol at
higher doses in human normal liver L-02 cells (IC
50
5
57.05 mM, cultivation for 48 h) and human hepatoma
HepG2 cells (IC
50
584.72 mM, cultivation for 48 h) in vitro
[35]. Soares et al. showed that the viability of kaempferol-
treated mouse fibroblast McCoy cells was fell, without the
hepatic S9 microsomal fraction; but low toxicity occurred
(IC
50
4500 mM) when the S9 mixture metabolized these
compounds [36]. In this study, we first reported that
kaempferol was active against human osteosarcoma U-2 OS,
HOB and 143B cell lines in vitro and U-2 OS in vivo.In
Fig. 1, it was shown that kaempferol reduced the percentage
of viable cancer cells in a dose-dependent manner and
induced apoptotic cell death in U-2 OS cells; however, it
exhibited low toxicity to human fetal osteoblast progenitor
hFOB cells (IC
50
4200 mM).
Inducing apoptosis in cancer cells is one of the major
strategies of cancer therapeutics. Three major pathways lead to
apoptosis [37]. First, the death receptor pathway is triggered by
the binding of extrinsic signals to surface receptors, resulting
in activation of caspase-8 followed by the activation of caspase-3
and -7 [19]. Second, the mitochondrial pathway is triggered by
various stimuli damage inside the cell. When an excess of pro-
apoptotic over anti-apoptotic signals, it initiates mitochondrial
outer membrane permeabilization and results in caspase
dependent and independent apoptotic pathway [31, 32]. Kang
et al. demonstrated that kaempferol and quercetin, compo-
nents of ginkgo biloba extract, induced caspase-3-dependent
apoptosis in oral cavity cancer cell lines, SCC-1483, SCC-25
and SCC-QLL1 [33]. Leung et al. showed that kaempferol-
induced apoptosis in human lung non-small carcinoma H460
cells was through caspase-3 (caspase-dependent) and AIF
(caspase-independent) pathways [9]. Zhang et al. reported that
kaempferol exerted cytotoxic effects on OE33, a human
esophageal adenocarcinoma cell line, causing G2/M arrest and
inducing caspase-dependent apoptosis [11]. Furthermore,
Marfe et al. demonstrated that kaempferol induced apoptosis
in K562 and U937 leukemia cell lines via Akt inactivation and
mitochondrial dysfunction [10]. Our results are in agreement
with previous studies. In Fig. 3, these data indicated that
kaempferol up-regulated the level of pro-apoptotic protein Bax
and down-regulated anti-apoptotic protein Bcl-2, accompanied
with the loss of Dc
m
, and then promoting activities of caspase-
9,-3,and-7,butnotcaspase-8.Specificinhibitorsofcaspase-9
and -3 which decreased caspase activities and increased the
kaempferol-treated cell viability suggested that kaempferol
induced apoptosis through the mitochondrial-dependent
pathway in U-2 OS cells. Also, up-regulating the protein level
of AIF indicated that apoptosis was also undergone via case-
pase-independent mitochondrial pathway. Third, the novel
endoplasmic reticulum (ER)-specific apoptotic pathway, it is
induced by accumulation of unfolded/misfolded protein
aggregating in ER or by excessive protein traffic. Increasing the
proteins level of GADD153, GRP78, GRP94 and ATF which
are the hallmarks of ER stress induces a rise in intracellular
Ca
21
level, mitochondrial membrane depolarization and
Figure 4. In vivo anti-tumor activity of kaempferol. BALB/c
nu/nu
mice were administered 25 and 50 mg/kg of kaempferol orally.
Representative animals with tumors (A), tumor weight (B) and
total tumor volume of BALB/c
nu/nu
mice (C). Data were presented
(

Po0.001, as compared with control treatments).
1592 W.-W. Huang et al.Mol. Nutr. Food Res. 2010, 54, 1585–1595
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

activation of calpain and caspase-12 in murine systems and/or
caspase-4 in human cells [38–40]. However, there are no
reports about kaempferol-induced ER stress in cancer cells. In
Fig. 2, increased levels of GADD153, GRP78, GRP94, ATF-6a
and ATF-6bwere followed by releasing Ca
21
from ER,
increasing calpain proteins expression and activating caspase-
4, and finally leading to apoptosis. Our result was shown in
fig. 2B and caspase-12 protein expression level has no signif-
icance influence. Caspase-12 has been shown to be involved
ER stress-induced apoptosis pathways, but in humans,
although the caspase-12 gene is transcribed into mRNA,
mature caspase-12 protein would not be produced because the
gene is interrupted by a frame shift and a premature stop
codon. [38–40]. ER stress signaling pathway was reconfirmed
by pre-treating with BAPTA, a Ca
21
chelator, and calpeptin, an
inhibitor of calpain, in kaempferol-treated U-2 OS cells, and it
showed decrease of calpain activity and increase of cell viability.
These accumulating data demonstrated that the activation of
ER stress pathway played an important role in kaempferol-
induced apoptosis in U-2 OS cells.
Concentrated and selected accumulation of anti-cancer
drugs at the tumor site is essential for the success of drug
treatment in vivo. Previous studies have reported that flavonoid
exhibits ability to inhibit human colorectal tumor formation
and block rat glioma tumoral invasion and migration in vivo
[41, 42]. Besides, two cohort studies have showed that high
level of kaempferol intake significantly decreases ovarian
cancer incidence, and intake of flavonol and catechin may be
associated with a decreased colorectal cancer risk in normal
weight women [43, 44]. In Fig. 4, our results showed that both
25 mg/kg and 50 mg/kg of kaempferol significantly reduced
the tumor volume and weight in BALB/c
nu/nu
osteosarcoma
mice. Additional prospective studies are needed to further
evaluate these associations.
In conclusion, with this report, we now show that
kaempferol exhibits direct anti-tumor activity, inducing tumor
cell apoptosis and suppressing tumor cell proliferation.
Moreover, kaempferol induced apoptosis through the mito-
chondria- dependent and ER stress pathways in human
osteosarcoma U-2 OS cells. Finally, we show that kaempferol
profoundly suppresses the in U-2 OS tumor xenograft-bearing
mice in vivo. The proposed signal pathways of kaempferol-
induced apoptosis in human osteosarcoma U-2 OS cells are
shown in Fig. 5. Although there are still some controversy
about the safety and biological effects of flavonoids, these
findings provide important possible molecular mechanisms of
the anti-human osteosarcoma and confirm that kaempferol
may be an anti-osteosarcoma cancer drug candidate.
The investigation was supported by a research grant from
the National Science Council of the Republic of China (NSC
97-2320-B-039 -004 -MY3) and a grant from the China Medical
University (CMU94-056), Taiwan.
The authors have declared no conflict of interest.
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