Pharmacology & Pharmacy, 2013, 4, 689-695
Published Online December 2013 (
Open Access PP
Effect of Farnesyl Caffeate-Induced Apoptosis of Lung
Carcinoma Cell Line from Damage to DNA
Kyu Sik Kim1, Akemi Umeyama2, Toshihiro Hashimoto2, Hyun-Ju Cho1, Je-Jung Lee3,4, Masao Takei3*
1Department of Pulmonary Medicine, Chonnam National University Medical School, Gwangiu, South Korea; 2Faculty of Pharma-
ceutical Sciences, Tokushima University, Tokushima, Japan; 3Research Center for Cancer Immunotherapy, Chonnam National Uni-
versity Hwasun Hospital, Hwasun-eup, Hwasun-gun, South Korea; 4Department of Hematology-Oncology, Chonnam National Uni-
versity Medical School, Gwangiu, South Korea.
Email: *
Received October 26th, 2013; revised November 27th, 2013; accepted December 12th, 2013
Copyright © 2013 Kyu Sik Kim et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Farnesyl caffeate, synthesis of propolis and polar bud excretion, has been reported to exhibit anti-allergic effects in mice.
However, little is known about anti-tumor effects. In this study, we investigated the effect of Farnesyl caffeate in cell
proliferation of lung carcinoma cell line (H157). Antiproliferative effect and apoptosis on H157 cell were evaluated
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) and DNA fragmentation assay, re-
spectively. Farnesyl caffeate inhibited the growth of H157 cell in dose-dependent manner. Morphological changes of
nuclei by staining Hoechst 33258 and DNA fragmentation suggested that Farnesyl caffeate induced death involved in a
mechanism of apoptosis. Moreover, caspase-3, caspase-7 and caspase-9 were activated by Farnesyl caffeate on H157
cell. The protein expressions of Bax and Bcl-2 were down-regulated by Farnesyl caffeate, resulting in cytochrome c
release from the mitochondria. Farnesyl caffeate significantly increased the expression of p53 proteins which indicates
that p53 plays a pivotal role in the initiation phase of Farnesyl caffeate-induced HepG2 cell apoptosis. These results
demonstrated Farnesyl caffeate-induced apoptosis in human lung carcinoma cell line. More detailed mechanism of Far-
nesyl caffeate-induced H157 apoptosis remains to be elucidated.
Keywords: Apoptosis; Propolis; Farnesyl Caffeate; DNA Fragmentation; Lung Cancer
1. Introduction
Various natural products have been reported to exert
their anti-tumor effects by induction of cancer cell apop-
tosis. Propolis is a resinous substance that is collected by
honeybees from plant sources and is thought to play a
protective role against potential predators [1,2]. Propolis
has been used in folk medicine and has been reported to
possess therapeutic and prophylactic effects against in-
flammation, diabetes mellitus and cancer. Propolis con-
tains up to 30 or more different flavonoid aglycones as
well as a variety of phenolic compounds [3-6]. Recently,
esters of cinnamic acids were identified from propolis [7].
One of these, Farnesyl caffeate has been shown by sensi-
tizing experiments to be the main contact allergen in
propolis, causing a contact dermatitis that is observed in
bee-keepers and increasingly in persons using propolis
preparations in “natural cures” and “biocosmetics” [8].
Apoptosis is a genetically engineered self-destructive
mechanism of cell death. It is accompanied by various
morphological changes like nuclear condensation and
fragmentation, isolation of cytoplasmic organelles into
discrete regions, formation of surface membrane blebs
and fragmentation of the dead or dying cell into mem-
brane-bound bodies [9-11]. Apoptosis is executed by
caspases, a family of intracellular aspartate-specific cys-
teine proteases, which amplify the apoptotic signal and
proteolytically process numerous cellular target mole-
cules with different functions [12]. A hallmark of DNA
damage triggered apoptosis is reduced Bcl-2 expression,
which was followed by caspase-9/caspase-3 activation
and DNA degradation. Many Bcl-2 family proteins re-
side the mitochondrial outer membrane. The balance
between Bax and Bcl-2 determines the fate of cells in
many apoptotic systems. Bcl-2 and Bcl-xL can be
cleaved by caspase-3 and cleavage of these proteins ap-
*Corresponding author.
Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA
pears to inactivate their survival function. In response to
the death stimuli, the mitochondorial membranes are
permeabilized, resulting in the release of cytochrome c.
In the cytosol, cytochrome c activates apoptosis by bind-
ing and activating apoptotic protease activating factor-1
(Apaf-1)-caspase-9-complex, which forms an apoptosome
acting as a processing/activation center for the down-
stream caspase-3 [13-16]. However, the detailed antipro-
liferative and molecular mechanism behind the apoptosis
of carcinoma cell line is not clear yet. Therefore, the
present study was designed to investigate if p53 contrib-
uted to Farnesyl caffeate-induced cell apoptosis, the in-
volvement of Bax, Bcl-xL and ERK pathway in the re-
lease of cytochrome c amplified activation of caspase
cascade, in promoting apoptosis in lung carcinoma cell
line (H157).
2. Materials and Methods
2.1. Chemicals and Reagents
Farnesyl caffeate was dissolved in dimethyl sulfoxide
(DMSO). Hoechst 33258, Ribonuclease A, MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide)
and propidium iodide (PI) were purchased from Sigma
Aldrich Company (St. Louis, MO). The MTT assay kit
(Cell Titer 96 Aqueous One Solution Cell Proliferation
Assay) was purchased from Promega Corp. (Madison,
WI, USA). Polyclonal antibody against cleaved caspase-
3, cleaved caspase-7 and cleaved caspase-9, and cleaved
PARP, and caspase-3, caspase-7 and caspase-9, and PARP,
Bcl-xL (54H6), p21, phosphor-p44/42 MAPK, and p44/42
MAP kinase were purchased from Cell Signaling Tech-
nology (Danvers, MA, USA). GAPDH (FL-335), p53
(DO-1), Bcl-2 (C-2) and Bax (2D2) antibodies were pur-
chased from Santa Cruz (Santa Cruz, CA, USA).
2.2. Synthesis of Farnesyl Caffeate
Synthesis of Farnesyl caffeate was prepared as described
previously [17].
2.3. Cell Line and Cell Culture Conditions
H157 human lung carcinoma cell were supplied by the
American Type Culture Collection (Rockville, MD).
Cells were maintained in RPMI 1640 (Gibco BRL, Grand
Island, NY) supplemented with 10% heat-inactivated
fetal bovine serum, 20 mM HEPES (pH 7.4), penicillin
(100 IU/ml), streptomycin (100 μg/ml), and 4 mM glu-
tamine (Invitrogen Corp., Carlsbad, CA, USA) in a hu-
midified atmosphere of 95% air and 5% CO2 at 37˚C.
2.4. Cell Growth Inhibition Test
Growth inhibition was evaluated by MTT (Thiazolyl blue,
Sigma) methods. Briefly, H157 cell (3 - 5 × 103/cell)
seeded in 96 well plates were cultured for 24 h, then
various concentrations of HA5 were added and cultured
for 24 h. MTT test were performed to detect cell growth
using an enzyme-liked imunosorbent assay (ELISA)
plate reader. Each experiment was performed in six rep-
licate wells for each drug concentration and was inde-
pendently performed four times. The IC50 value was de-
fined as the concentration needed for a 50% reduction in
the absorbance, as calculated based on the survival curves.
2.5. Cell Morphological Characteristics
H157 cells in PRMI-1640 containing 10% FBS were
seeded into 25 ml culture bottles and incubated overnight.
2.5 - 10 μM of HA5 was added to the cell culture, and
the cellular morphology was observed using Olympus
microscope (Olympus, Japan) at 24 h.
2.6. Nuclear Damage Observed by Hoechst
33258 Staining
Apoptotic nuclear morphology was assessed using Hoechst
33258 as described previously [18]. Cells were fixed
with 3.7% paraformaldehyde at room temperature for 2 h,
then washed and stained with 167 mM of Hoechst 33258
at 37˚C for 10 min. At the end of incubation, cells were
washed and re-suspended in PBS for observation of nu-
clear morphology using fluorescence microscope (Olym-
pus, Japan).
2.7. Determination of DNA Fragmentation by
Agarose Gel Electrophoresis
DNA extraction and electrophoresis were performed as
described previously [18]. Briefly, H157 cells were
treated with 10 μM HA-5 for 24, 48 and 72 h. The cell
pellet was suspended in cell lysis buffer and kept at 4˚C
for 10 min. The lysate was centrifuged at 12,000 × g for
20 min. The supernatant was incubated with RNase a 40
mg/ml (Sigma) at 37˚C for 60 min. The supernatant was
mixed with 0.5 M NaCl and 50% 2-isopropanol at 20˚C
overnight, then centrifuged at 12,000 × g for 20 min. Af-
ter drying, DNA was dissolved in TE buffer and sepa-
rated by 2% agarose gel electrophoresis at 80 V for 2 h
and stained with 0.1 mg/ml ethidium bromide. DNA
fragments were visualized by ultraviolet transillumina-
2.8. The Cell Cycle Analysis by Flow Cytometry
After indicated treatments, cells were rinsed with ice-
cold PBS (pH 7.4) and fixed with 70% cold ethanol at
4˚C for overnight. Before flow cytometric assays, cells
were washed twice with ice-cold PBS and fixed cells
were resuspended in PBS containing 0.1 mg/ml RNAase
at room temperature for 30 min, and then cells were
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Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA 691
added 50 μg/ml propidium iodide (Sigma-Aldrich). The
stained nuclei were analyzed using a FACSCalibur (BD
Biosciences, NJ, USA). At least 10,000 events were ana-
lyzed, and the percentage of cells in the sub-G1 popula-
tion calculated. Aggregates of cell debris at the origin of
the histogram were excluded from the sub-G1 cells. DNA
histograms were created using Cell Quest TM software
for Apple Macintosh (Becton Dickinson).
2.9. Western Blotting Analysis
H157 cells were treated with 2.5 μM Farnesyl caffeate
for 0, 24, 48 and 72 h. Cells were collected and frozen at
80˚C. Western blot analysis was performed as described
previously [18]. Cells were washed with PBS and lyses
at 0˚C for 30 min in lysis buffer (20 mM Hepes (pH 7.4)
2 mM EGTA, 50 mM glycerol phosphate, 1% Triton
X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM Phenyl-
methylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml
aprotinin, 1 mM Na3VO4, and 5 mM NaF). Equal
amounts of protein were separated by 6% - 15% SDS-
PAGE followed by electrotransfer onto a polyvinylidene
difluoride membrane (Millipore, Bedford, MA, USA).
The membranes were blocked for 4 h with TBS-T (10
mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) con-
taining 5% nonfat milk and then incubated at room tem-
perature with primary antibodies. The blots were washed
four times for 15 min with 0.1% Tween 20-containing
TBS-T and then incubated for 1 h with peroxidase-con-
jugated secondary antibodies (1:5000, Jackson Immuno
Research Inc., West Grove, PA, USA). After three more
washes, proteins were detected by chemiluminescence
detection system (iNtRON Biotech, Seoul, Korea) and
the images were obtained with LAS-4000 mini (Fuji,
Tokyo, Japan).
2.10. Statistical Analysis
Statistical analysis of the results was performed by
ANOVA. Differences were considered statistically sig-
nificant then p values were less than 0.05.
Farnesyl caffate
3. Results
3.1. Farnesyl Caffeate Induces Apoptotic Cell
Death in H157 Cells
To determine whether Farnesyl caffeate-induced H157
cell death was caused by apoptosis, we examined the
morphological changes and DNA fragmentation. We
observed a decrease in the total number of cells and an
accumulation of cells floating in the culture medium after
24 h treatment with 0.5 - 20 μM of Farnesyl caffeate,
indicating Farnesyl caffeate-induced cell death in dose-
dependent manner (Figure 1). Nuclear morphological
changes were also observed by Hoechst 33,258 staining.
In contrast, H157 cell in control were round in shape and
stained homogeneously. After 24 h treatment with 2.5 -
10 μM of Farnesyl caffeate, blabbing nuclei and granular
apoptotic bodies appeared (Figure 2). DNA fragmenta-
tion appeared obviously after 2.5 - 10 μM of Farnesyl
caffeate treatment for 24 h on agarose gel electrophoresis
(Figure 3(a)). To determine whether the decrease in cell
viability was attributable to apoptosis, cells were stained
with FITC conjugated Annexin V plus PI and evaluated
by FACS (Figure 3(b)). Cell underwent apoptotic cell
death in response to Farnesyl caffeate.
3.2. Caspases Activation Relevant to Farnesyl
Caffeate-Induced H157 Cell Death
Western blot analysis was performed to confirm the par-
ticipation of caspase-3, caspase-7 and caspase-9. The 17
kDa and 19 kDa band of caspase-3 were degraded after
48 - 72 h treatment with Farnesyl caffeate 25 μM (Figure
4). Similar results were also obtained with caspase-7 and
caspase-9 (Figure 4). The characteristic associated with
the execution phase of the apoptosis pathway is the spe-
cific PARP cleavage by caspases. This cleavage leads to
inactivation of the enzyme, thus preventing futile DNA
repair cycles. It has been reported that capase-3 is the
most efficient processing enzyme for PARP. Cleavage of
PARP was examined after treatment with Farnesyl caf-
feate. As expected, the amount of the 89 kDa degraded
product increased after 48 - 72 h treatment with Farnesyl
caffeate (Figure 4). These results indicate that caspase-3
is activated in Farnesyl caffeate induced apoptosis in
H157 cell.
0.515 101520
Cell Death (%)
Concentration of Farnesylcaffeate(M)
Figure 1. Effect of apoptosis induced by Farnesyl caffeate
on lung carcinoma cell line (H157). H157 cell were treated
Farnesyl caffeate at various concentrations for 24 h. Cell
viability was measured by MTT assay. Data are the mean
.E.M. of four independent experiments. S
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Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA
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HA5 (2.5 M) HA5 (5 M) HA5 (10 M)
Hoechst 33258 stainig
Figure 2. Morphological changes of H157 cell by phase contrast and fluorescence microscope. H157 cell were treated with 2.5
- 10 μM Farnesyl caffeate for 24 h.
3.3. Cytochrome c Release Following Altered
Bax/Bcl-xL and Bax/Bcl-2 Expression in
Farnesyl Caffeate-Treated H157 Cells
The Bcl-2 family members are critical regulators of the
mitochondrial pathway that induces intrinsic activation
of caspases. To confirm whether such a mechanisms is
involved in Farnesyl caffeate induced apoptosis, H157
cells were incubated with Farnesyl caffeate 25 μM for 24,
48 and 72 h. At 72 h, after treatment with Farnesyl caf-
feate the expression of Bcl-2 and Bcl-xL began to de-
crease, whereas that of Bax increased slightly (Figure 5).
These observations seem that protein expression of ration
of Bcl-xL/Bax and Bcl-2/Bax was down-regulated and
Farnesyl caffeate-induced apoptosis involves the initial
phase mediated by the balance among Bcl-xL, Bcl-2 and
Bax proteins, resulting in cytochrome c release from the
3.4. Accumulation of p53 and Down Regulation
of ERK in Farnesyl Caffeate-Induced
To ascertain whether p53 and ERK is required in Farne-
syl caffeate-induced cell apoptosis, H157 cell were
treated with Farnesyl caffeate at 25 μM for 24, 48 and 72
h. The expression of p53 increased at 48 h and 72 h. The
expression of p53 increased at 24 h and then these ex-
pressions declined at 48 - 72 h gradually. In contrast, the
expression of ERK1/2 increased at 24 h and then mark-
edly decreased at 72 h (Figure 6). The results suggest
that ERK and p53 activation were required for Farnesyl
caffeate-induced apoptotic pathway.
4. Discussion
In this study, we showed that Farnesyl caffeate inhibited
the growth of lung carcinoma cell line (H157) in dose-
dependent manner. Morphological changes of nuclei by
staining Hoechst 33258 and DNA fragmentation sug-
gested that Farnesyl caffeate-induced H157 cell death in-
volved in a mechanism of apoptosis. MTT assay sug-
gested significant antiproliferative effect of Farnesyl
caffeate varied depending on the cell line. Further under-
standing the mechanism behind the Farnesyl caffeate-
induced apoptosis in lung carcinoma cells promotes this
compound as a valid anti-cancer agent. Farnesyl caffeate
treated cells displayed characteristic ladder-like patterns
compared to untreated cells. Further flow cytometric
analysis indicated decreased relative size and increased
internal complexity in the treated cells indicating apop-
PAPR (116-kDa), a DNA repair enzyme, is probably
best characterized caspase substrate, which is cleaved dur-
ing apoptosis to a 24-kDa and a 85-kDa fragment repre-
senting the N-terminal DNA-binding domain and the
C-terminal catalytic subunit, respectively. During apop-
tosis, PARP is selectively cleaved by several caspases,
especially by caspase-3 [19]. Detection of an 85-kDa or
24-kDa caspase cleavage fragment of PAPR was shown
to be a hallmark of apoptosis. In this study, the bands of
procaspase-3 were degraded after Farnesyl caffeate ad-
ministration, followed by the degradation of caspase-3
substrates, PARP minor 85-kDa fragment was increased.
The activation of caspase-3 and PAPR cleavage were
associated in the Farnesyl caffeate-induced apoptosis
similar to the apoptosis reported in some anti-cancer
drugs [20]. It seems that the expression of caspase-7 and
caspase-9 would be essential to further understand the
exact role of caspase’s in the Farnesyl caffeate-induced
apoptosis. These results suggest that the caspase cascade
plays a critical role in Farnesyl caffeate-mediated H157
cell line apoptosis.
The extracellular signal regulated kinase is an impor-
tant mediator of signal transduction processes that serve
to coordinate the cellular responses to a variety of ex-
tracellular stimuli [16]. We showed that the expression of
Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA 693
1.100b p DNA marker
2.H157 co ntrol
3.H157 HA 5 (2. 5 M)
4.H157 HA 5 (5 M)
5.H157 HA 5 (10 M)
1 2 3 4 5
100 bp
500 bp
100 bpDNA Ladder
(NEB, #N3231)
Base Pairs Mass (ng)
1,517 45
1,200 35
1,000 95
900 27
800 24
700 21
600 18
500/517 97
400 38
300 29
100 48
N3231_t humb
sub G0/G1G1SG2/M
cont0.8650.11 24.1625.41
HA53.4842.77 22.6531.99
BD calibur
software : WinMDI
sub G0/G1G1SG2/M
H157 cell cycle
co nt
FL2-A 1023
0Events 64
Figure 3. Farnesyl caffeate induced DNA fragmentation in H157 cell. (a) Cells were cultured with different concentrations of
Farnesyl caffeate for 24 h; (b) Cells were treated with 10 μM Farnesyl caffeate for 24 h, at which point they were fixed and
tained with prodium iodide. s
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Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA
0 24 48 72 h
Caspase 3
Caspase-3 17, 19 k Da
Caspase 7
Caspase-7 20 kDa
caspase 947 kDa
35, 3 7 kDa
35, 37 kDa
Cleaved P ARP (A sp214) : 89 kDa
Full length PARP : 116 kDa
Figure 4. Degradation of caspase-3, caspase-7 and caspase-9,
and PAPR in Farnesyl caffeate-treated H157 cell. Cells were
treated with 2.5 μM Farnesyl caffeate for the indicated
times. Caspase-3, caspase-7 and caspase-9, and PARP were
detected using Western blot analysis.
0 24 48 72 h
Figure 5. Different expression of Bax, Bcl-2 and Bcl-xL in
Farnesyl caffeate-treated H157 cell. Cells were treated with
2.5 μM Farnesyl caffeate for indicated times, and the pro-
tein bands were detected by Western blot analysis.
p44/42 MAPK
P-p44/42 M APK
(Erk1/2) 44/ 42 kDa
0 24 48 72 h
Figure 6. Expression of p53 and ERK2 in Farnesyl caffeate-
treated H157 cells. Cells were incubated with 2.5 μM Far-
nesyl caffeate for indicated times. P53 and ERK2 were de-
tected using Western blot analysis.
p53 was reduced, suggesting Farnesyl caffeate-induced
apoptosis through activation of p53 in response to DNA
Several pro-apoptotic proteins, such as Bax and Bak,
translocate to the mitochondrial membrane, and this lo-
calization is associated with their pro-apoptotic activities.
It has been reported that Bcl-2 could exert its action
through heterodimerization with Bax. Bcl-2 and Bcl-xL
are cleaved by caspase-3, and are converted to pro-
apoptotic protein similar to Bax. Therefore, the ratio be-
tween Bcl-2 and Bax or Bcl-xL and Bax is a decisive
factor to activate cell death [21-25]. H157 cell treated
with Farnesyl caffeate exhibited the elevated ration be-
tween pro-apoptotic Bax and anti-apoptotic Bcl-2 or
Bcl-xL. The oligomerization of Bax in the mitochondrial
membrane has been shown to induce cytochrome c re-
lease, meanwhile pro-apoptotic Bcl-2 cleavage product
was reported to localize on mitochondrial membrane and
caused release of cytochrome c [26-28]. In this study, the
protein expression of cytochrome c was markedly up
regulated followed by the changes of Bcl-2/Bax and Bcl-
xL/Bax ratios in H157 cell treated by Farnesyl caffeate.
These results suggested that the mitochondrial pathway
of cell death, including Bcl-2 family and cytochrome c,
might be involved in H157 cell death and orchestrates the
caspase cascades.
We demonstrated that Farnesyl caffeate-induced apop-
tosis in H157 cell via accumulation of p53, alters the
Bax/Bcl-2 ration, and activates caspases, resulting in
cytochrome c release from the mitochondria. More de-
tailed mechanism of Farnesyl caffeate-induced human
lung carcinoma cell apoptosis remains to be elucidated.
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