1
2
Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell
3
growth through the induction of ERK1/2-mediated apoptosis
4
Q1
Ming-Wei Lin
a,b, An-Shen Lin
d, Deng-Chyang Wu
b,c, Sophie S.W. Wang
b,c, Fang-Rong Chang
b,d,
5Yang-Chang Wu
d,e,f, Yaw-Bin Huang
a,b,⇑
6 a
Graduate Institute of Clinical Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC 7 bCancer Center, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan, ROC
8 c
Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan, ROC
9 d
Graduate Institute of Nature Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
10 e
Natural Medicinal Products Research Center and Center for Molecular Medicine, China Medical University Hospital, Taichung 402, Taiwan, ROC
11 f
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 402, Taiwan, ROC
12 13 1 5
a r t i c l e
i n f o
16 Article history: 17 Received 15 December 2011 18 Accepted 16 May 2012 19 Available online xxxx 20 Keywords: 21 Euphol22 Human gastric cancer
23 ERK1/2 24 Anti-proliferation 25 Apoptosis 26 2 7
a b s t r a c t
28 Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related29 death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with
anti-30 inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity
31 activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it
up-32 regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing
33 mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were
34 associated with the increased p27kip1 levels and decreased cyclin B1 levels. Inhibition of ERK1/2
35 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken
36 together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by
37 modulation of ERK signaling, and could thus be of value for cancer therapy.
38 Ó 2012 Published by Elsevier Ltd. 39 40 41 42
1. Introduction
43
Gastric cancer is one of the most common malignancies
world-44wide, accounting for nearly half of cancer-related mortality (
Shah
45
and Kelsen, 2010
). Chemotherapy is the treatment of choice for
46
gastric cancer, but the currently available therapeutic drugs for
47the treatment of gastric cancer have limited efficacy (
Zhang
48
et al., 2006
). Combination chemotherapy is often associated with
49
toxic side effects. Therefore, new agents that selectively target
50gastric cancer cells are urgently needed.
51
Recent studies have shown that the mitogen-activated protein
52kinase (MAPK) pathway may modulate cancer cell apoptosis and
53proliferation (
Kim and Choi, 2010
). The MAPK pathway is
well-54studied molecular targets for chemotherapeutic drug development,
55
and several related clinical trials have been completed in patients
56
with metastatic and local cancer (
Dangle et al., 2009
). Extra-cellular
57
signal-regulated kinase 1/2 (ERK1/2) belongs to one of the
sub-58
groups of MAPKs and important in a variety of signaling pathways
59
that regulate multiple cellular processes. ERK1/2 mediates gene and
60
protein expression changes in response to extracellular stimuli
61
(
Tibbles and Woodgett, 1999
). The involvement of ERK1/2 in the
62
regulation of cell proliferation has been extensively described
63
(
Ballif and Blenis, 2001
). However, in some cell models, activation
64
of ERK1/2 is associated with the induction of apoptosis (
Lu et al.,
65
2009; Wang et al., 2000
).
66
Apoptosis is a form of cell death that can be triggered by several
67
external or internal signals. The loss of mitochondrial membrane
68
potential is the hallmark of the intrinsic apoptosis pathway.
Mito-69
chondria modulate the caspase–apoptosis cascade by regulating
70
the translocation of cytochrome c from the mitochondrial
inner-71
membrane space to the cytosol. Pro-apoptotic proteins, such as
72
Bcl-2-associated X protein (BAX), can directly interact with the
73
mitochondrial permeability transition pore complex. BAX displaces
74
this complex from its inhibitory interaction with the pro-survival
75
protein, B-cell lymphoma 2 (Bcl-2), disrupting the mitochondrial
76
membrane potential and leading to the permeabilization of the
77
mitochondrial membrane and the activation of the cytochrome
0278-6915/$ - see front matter Ó 2012 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.fct.2012.05.029
Abbreviations: BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; ERK1/2, extra-cellular signal-regulated kinase 1/2; IC50, 50% inhibitory
concentra-tion; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; PBS, phosphate buffered saline; p-ERK1/2, phosphorylated extra-cellular signal-regulated kinase 1/2.
⇑
Corresponding author at: Graduate Institute of Clinical Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC. Tel.: +886 7 3121101x2166; fax: +886 7 3210683.E-mail address:yabihu@kmu.edu.tw(Y.-B. Huang).
Contents lists available at
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Food and Chemical Toxicology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x
induc-78
c-caspase-dependent apoptosis pathway (
Fulda et al., 2010; Tait
79
and Green, 2010
).
80
The latex of Euphorbia tirucalli (Euphorbiaceae), which is native
81to Madagascar, was used in indigenous medicine as a purgative and
82a remedy for rheumatism, neuralgia, and toothache in Africa and
83Asia (
Rasool et al., 1989
). In South Taiwan, its branches are boiled
84in water and used as one of ingredients of anticancer herbal drinks.
85However, the milky latex of this plant is considered to be poisonous
86(
Lin et al., 2001
) and possesses highly vesicant and irritant
proper-87ties toward the skin and mucous membranes (
Furstenberger and
88
Hecker, 1977b
). Studies have shown that the highly unsaturated
89
irritant phorbol esters were the main constituents responsible for
90the toxicity of the latex (
Furstenberger and Hecker, 1977a,b; Khan
91
et al., 1988; Lin et al., 2001; Yoshida et al., 1991
).
92
Euphol is a euphane-type triterpene alcohol (
Fig. 1
). It is
iso-93lated from the dichloromethane extract of E. tirucalli, and exhibits
94a variety of biological activities, such as anti-viral (
Akihisa et al.,
95
2002
) and anti-inflammatory activities (
Akihisa et al., 1997
). In a
96recent study, a topical application of euphol was shown to
mark-97edly suppress the tumor-promoting effect in 2-stage
carcinogene-98sis in mouse skin (
Yasukawa et al., 2000
). However, the
99mechanisms underlying this effect and the potential antitumor
100properties of euphol remain to be evaluated.
101
The results of the present study indicate that euphol has
anti-102proliferative effects and selectively induces gastric cancer cell
103death in an ERK1/2-dependent manner. Moreover, euphol
modu-104lates the expression of cell cycle regulator proteins and promotes
105apoptosis by means of the mitochondrial apoptotic pathway.
106 2. Materials and methods107 2.1. Isolation of euphol
108 The fresh aerial parts of E. tirucalli (Gildenhuys, 2006; Rasool et al., 1989) were 109 collected in the Tainan County, Taiwan, in August 2002 and identified by botanist 110 Dr. Ming-Hong Yen, Kaohsiung Medical University, Kaohsiung, Taiwan. The latex 111 of the fresh plant was collected drop by drop, and the remaining aerial parts of 112 the plant (15.0 kg) were extracted with MeOH. The evaporated latex MeOH extract 113 (5.9 g) was separated by column chromatography on a silica gel (300 g) with a gra-114 dient system of n-hexane/CHCl3(3:1, 2:1, and 1:1, at 800 mL each) and CHCl3
115 (1000 mL), yielding 20 fractions. Fractions of 7–9 (4.6 g) were combined and further 116 purified by a silica gel column (200 g) with n-hexane/CHCl3(3:1, 1500 mL), yielding
117 euphol (4.2 g) as the major constituent and triterpene.
118 2.2. Cell culture
119 The novel human gastric cancer cell line, KMU-CS12 (CS12) and human gastric 120 cell line, KMU-CSN (CSN) were established in our previous studies (Yang et al.,
121 2007, 2009). CSN and CS12 cells were cultured in kerotinocyte-Serum-free medium 122 (Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum, 123 N-acetyl-L-cysteine (360
lg/mL), and
L-ascorbic acid 2-phosphate (51.2lg/mL).
124 Human gastric adenocarcinoma AGS cells were obtained from the American Type 125 Culture Collection (ATCC, Rockville, MD, USA), and MKN45 (a poorly differentiated 126 human gastric adenocarcinoma) cells were obtained from the Health Science 127 Research Resources Bank (HSRRB, Osaka, Japan). The AGS and MKN45 cells were 128 grown in RPMI-1640 medium (Invitrogen) containing 10% fetal bovine serum.129 2.3. WST-1 cell cytotoxicity assay
130 The cytotoxicity of euphol was assessed using a WST-1 cell proliferation kit
131 (Roche. Applied Science, Basel, Switzerland). The cells were seeded for 72 h at a
con-132 centration of 5 104
cells/well in culture medium containing various amounts of 133 euphol (2, 5, 10, 20, 40, and 60
lg/mL) in 96-well microplates. The reduction of
134 the tetrazolium salt of the reagent to a formazan product by cellular
dehydrogen-135 ases was detect by the generation of a yellow-color, which was measured at
136 440 nm with a microplate ELISA reader.
137 2.4. Detection of Annexin V-positive apoptotic cells
138 Apoptotic cells were detected by Annexin V staining (BioVision, Mountain View,
139 CA, USA) according to the manufacture’s instructions. Briefly, the cells were washed
140 with phosphate buffered saline (PBS) and resuspended with Annexin binding buffer
141 (Invitrogen). After treatment with annexin V-FITC (1:500) and propidium iodide
142 (PI), the cells were incubated for 15 min in the dark. Annexin V-positive apoptotic
143 cells (compared to unlabeled cells) were then analyzed by a FACScan flow
cytome-144 ter (Becton Dickinson, Mountain View, CA, USA).
145 2.5. Detection of mitochondrial transmembrane potential
146 The CS12 cells were pretreated with PD98059 (13.4
lg/mL) or vehicle (DMSO)
147 for 30 min and incubated with euphol (20
lg/mL) for 72 h. The cells were washed
148 with warm PBS and incubated with MitoTraker (Invitrogen) for 30 min at 37 °C in
149 the dark. The cells were washed with warm PBS again, and the fluorescence
inten-150 sity was determined by means of a FACScan flow cytometer (Becton Dickinson).
151 2.6. Caspase-3 activation assay
152 FITC-DEVD-FMK is cell permeable, nontoxic, and irreversibly binds to activated
153 caspase-3 in apoptotic cells. Therefore, an anti-fluorescein isothiocyanate
(FITC)-154 DEVD-FMK antibody was used to further confirm the role of the ERK1/2 MAPK
path-155 way in the euphol-induced caspase-3 activation by flow cytometry. The CS12 cells
156 were pretreated with PD98059 (13.4
lg/mL) or vehicle (DMSO) for 30 min and then
157 incubated with euphol (20
lg/mL) for 72 h. The cells were washed with PBS and
158 incubated with FITC-DEVD-FMK (BioVision) for 30 min at 37 °C in the dark. The
159 cells were washed with warm PBS, and the fluorescence intensity was determined
160 by means of a FACScan flow cytometer (Becton Dickinson) as described before
161 (Carvalho et al., 2008).
162 2.7. Western blotting
163 For ERK1/2 phosphorylation assays, CSN, CS12, AGS and MKN45 cells were
trea-164 ted with euphol (20
lg/mL) for 4, 24, 48, and 72 h. For apoptotic protein expression
165 level assays, the CS12 cells were pretreated with PD98059 (13.4
lg/mL) or vehicle
166 (DMSO) for 30 min and then incubated with euphol (10 or 20
lg/mL) for 72 h. The
167 cells were lysed using a commercially available lysis buffer, M-PER mammalian
pro-168 tein extraction reagent (Thermo Scientific, Rockford, IL, USA). Equal protein amounts
169 were loaded onto 10% SDS–PAGE gels, and the separated proteins were transferred
170 to PVDF membranes, blocked with 5% nonfat dried milk in PBST buffer, and
incu-171 bated with anti-phospho-ERK1/2 (Cell Signaling, Beverly, MA, USA), anti-ERK1/2
172 (Cell Signaling), anti-BAX (StressGen, Victoria, BC, Canada), anti-Bcl-2 (Stressgen),
173 b-actin (Sigma–Aldrich, St. Louis, MO, USA), p27 (Cell Signaling), or
anti-174 cyclin B1 (Enzo Life Sciences, Farmingdale, NY, USA) primary antibody overnight.
175 After probing with a horseradish peroxidase-conjugated secondary-antibody (GE
176 Healthcare, Piscataway, NJ, USA) and thoroughly washing the membranes, the
177 immunolabeled proteins were detected using an enhanced chemiluminescence kit
178 (GE Healthcare), followed by exposure to an X-ray film.
179 2.8. Statistical analyses
180 The results were expressed as means ± SD. Statistical comparisons were
per-181 formed with the Student t-test. The statistical significance was set at P < 0.05.
182
3. Results
183
3.1. Inhibition of gastric cancer CS12 cell proliferation by euphol
184
The antiproliferative effects of various concentrations of euphol
185
(2, 5, 10, 20, 40 and 60
l
g/mL) on CSN, CS12, AGS and MKN45 cells
186
are shown in
Fig. 2
. The results of the WST-1 assay demonstrated
187
that euphol inhibited the growth of CS12 cells and that of the
com-188
mercially available AGS and MKN45 cell lines in a dose-dependent
189
manner. To examine whether the growth inhibitory effect of euphol
190
was mediated by apoptosis induction, the gastric cancer and
Fig. 1. Chemical structure of euphol.2 M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx
FCT 6641
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induc-191
normal cells were treated with euphol (20
l
g/mL) and exposed to
192annexin V/PI staining.. As shown in
Fig. 2
E, the rate of apoptosis
193was greater in the euphol-treated gastric cancer cells than in the
194normal cells (P < 0.01). The euphol treatment significantly induced
195cell death in the gastric cancer CS12, AGS, and MKN45 cell lines
196but not in the CSN cells. The IC
50values for euphol in CSN, CS12,
197
AGS and MKN45 cells were 49.6, 12.8, 14.7 and 14.4 (
l
g/mL),
198respectively.
199
3.2. Euphol induction of ERK1/2 phosphorylation in CS12 cells
200The ERK1/2 MAPK pathway regulates many cellular activities,
201especially cell proliferation and apoptosis (
Ballif and Blenis,
202
2001; Wang et al., 2000
). To examine the role of ERK1/2 MAPK
sig-203
naling in the apoptosis of gastric cancer cells induced by euphol,
204the CS12, AGS, MKN45 and CSN cells were treated with 20
l
g/mL
205of euphol at various time points. As shown in
Fig. 3
B, the euphol
206treatment induced ERK1/2 activation in a time-dependent manner
207in the CS12 cells. Similar results were obtained in the AGS and
208MKN45 gastric cancer cell lines (
Fig. 3
C and D). In addition, the
209accumulation of phosphorylated ERK1/2 was significantly
in-210creased after 72 h in the euphol-treated gastric cancer cell lines,
211whereas no significant activation of ERK1/2 was observed in the
212CSN cells (
Fig. 3
A) under the same treatment conditions. To
con-213firm the involvement of ERK1/2 in the euphol-induced growth
214inhibition, the CS12 cells were treated with the ERK1/2 inhibitor
215PD98059. As shown in
Fig. 3
E and F, PD98059 had a mild inhibitory
216effect on the euphol-induced apoptosis in this cell line, suggesting
217that the ERK1/2 MAPK pathway may participate play a role in
eup-218hol-induced CS12 apoptotic cell death.
219
3.3. Role of ERK1/2 in the euphol-induced mitochondrial-dependent
220apoptosis pathway
221
The role of ERK1/2 in the euphol-induced apoptosis pathway
222and the expression profiles of apoptotic and prosurvival
pro-223teins in the euphol-treated CS12 cells were examined by Western
224
blotting to measure the BAX and Bcl-2 protein expression levels.
225
The treatment of the cells with euphol for 72 h markedly
upregu-226
lated the BAX expression and downregulated Bcl-2 protein
expres-227
sion in a dose-dependent manner, and PD98059 reversed the
228
effects of euphol on the expression of the apoptosis-related protein
229
(
Fig. 4
A and B). The translocation of the pro-apoptotic protein BAX
230
to mitochondria may result in the loss of mitochondrial membrane
231
potential, and the induction of the caspase-mediated apoptosis
232
pathway (
Fulda et al., 2010
). As shown in
Fig. 4
C, a shift in the
eup-233
hol-treated cells toward the left compared with the vehicle-treated
234
controls indicated that euphol (20
l
g/mL) disrupted the
mitochon-235
drial membrane potential, as assessed by flow cytometry. In
con-236
trast, the pretreatment with PD98059 (13.4
l
g/mL) resulted in a
237
right shift of the MitoTracker fluorescent curves for the
euphol-238
treated CS12 cells, indicating that the euphol-induced
mitochon-239
drial dysfunction was ERK1/2-dependent.
Fig. 4
D shows a shift of
240
the euphol-induced FITC fluorescence to the right, which was
241
inhibited by PD98059, suggesting that euphol-induced apoptosis
242
in gastric cancer CS12 cells may be mediated by ERK1/2 regulation
243
of the mitochondrial apoptotic pathway.
244
3.4. ERK1/2 contributes to the antiproliferative effect of euphol
245
To examine the mechanisms underlying the antiproliferative
ef-246
fects of euphol in gastric cancer CS12 cells, the expressions of
247
p27
kip1and cyclin B1 were assessed by Western blotting. As shown
248
in
Fig. 5
, euphol altered the expression of these cell cycle
regula-249
tory proteins by inducing p27
kip1expression and inhibiting cyclin
250
B1 expression. Furthermore, pretreatment with PD98059 markedly
251
abolished the upregulation of p27
kip1and downregulation of cyclin
252
B1 in response to the euphol treatment.
253
4. Discussion
254
The present results demonstrate that euphol has
antiprolifera-255
tive activity against CS12 gastric cancer cells and its mechanism
256
of action involves the alteration of the expression of cell cycle
Fig. 2. Inhibitory effect of euphol on gastric cancer cell growth. The results of the WST-1 assays showing that euphol inhibited (A) CSN, (B) CS12, (C) AGS, and (D) MKN45 proliferation in a dose-dependent manner after 72 h of exposure to the drug. (E) The CSN, CS12, AGS, and MKN45 cells were treated with euphol (20lg/mL) for 48 h. Euphol
selectively induced apoptosis in 3 gastric cancer cells (CS12, AGS, and MKN45). The bars represent the mean ± SD of the 3 independent experiments (⁄P < 0.01, compared with the CSN cells).
M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx 3
induc-257
regulatory proteins and the induction of apoptosis. The
pretreat-258ment with the ERK1/2 inhibitor PD98059 suppressed
euphol-259induced apoptosis, suggesting that the effect of euphol is
participat-260ing mediated by an ERK1/2-associated pathway. Although ERK1/2
261activation is generally related to cell proliferation and survival (
Bal-262
lif and Blenis, 2001
), increasing evidence indicates that ERK1/2 also
263
transmits death signals. Its role in the promotion of apoptosis
264induced by anticancer drugs has been reported. The sustained
265activation of ERK1/2 for a period of 1–72 h has been reported to
pro-266mote cell death in different cell types (
Cagnol and Chambard, 2010
).
267Long-term activation of the ERK1/2 pathway has been detected in
268association with cisplatin-, apiginin-, gemcitabine-, and
adriamy-269cin-induced apoptosis in HeLa, prostate, and pancreatic cancer cells
270
(
Wang et al., 2000; Zhao et al., 2006
). Prolonged ERK1/2 activation
271
has been associated with cell growth arrest and cell death (
Martin
272
et al., 2006; Martin and Pognonec, 2010; Tong et al., 2011
). Previous
273
studies have shown that the activities of platinum-based
chemo-274therapeutic drugs are ERK1/2 dependent (
Sheridan et al., 2010;
275
Wang et al., 2000
). However, the sustained ERK1/2 activation
276
resulting in cell death remains poorly understood.
Lu et al. (2009)
277
demonstrated that ERK1/2 mediated the ubiquitination of the
278proto-oncogene MDM2, induced by the medical plant hispolon,
279indicating that it could be useful for the treatment of tumors with
280constitutive ERK1/2 activation. In the present study, enhanced
281ERK1/2 activation was observed in gastric CS12, AGS, and MKN45
282
cancer cells, but not in gastric CSN nontumorigenic cells, 72 h after
283
the addition of 20-
l
g/mL euphol. The sustained activation of the
284
ERK1/2 pathway in gastric cancer cells may play a significant role
285
in the induction of apoptosis and growth arrest by euphol. ERK1/2
286
activation is tightly regulated in normal cells by ERK-specific
phos-287
phatases that ensure cellular homeostasis (
Murphy and Blenis,
288
2006
). However, the sustained activation of ERK1/2 triggers the
289production of ROS, which further inhibit ERK-specific phosphatases
290
(
Levinthal and Defranco, 2005
). The dysregulation of ERK1/2
activa-291
tion thus induces the progressive accumulation of death-promoting
292
factors and cell death by apoptosis or necrosis.
293
Euphol is a cholesterol-like compound and therefore may
pos-294
sess toxic properties through its interaction with the plasma
mem-295
brane and replacement of cholesterol. These effects should be
296
investigated in future studies. Cholesterol is a key molecule in
297
the cell membrane and is the main component of specialized lipid
298
microdomains called lipid rafts, which are involved in the
regula-299
tion of phosphorylation cascades (
George and Wu, 2012
).
Deple-300
tion of cholesterol from the cell membrane alters signal
301
transduction cascades and induces cancer cell death (
Bionda
302
et al., 2008
). Cholesterol was reported to accumulate in a variety
303of tumor types (
Freeman and Solomon, 2004
), and high cholesterol
304
levels in the cell membrane induced tumor cell proliferation
305
through the lipid raft-AKT pathway (
Zhuang et al., 2005
). In
addi-306
tion, elevated levels of membrane cholesterol in cancer cells were
Fig. 3. Euphol-induced sustained ERK1/2 phosphorylation in gastric cancer cells. (A–D) The Western blot analyses of ERK1/2 phosphorylation. ERK1/2 level was used as internal control for phospho-ERK1/2. The euphol-induced ERK1/2 phosphorylation in the CS12, AGS, and MKN45 gastric cancer cells in a time-dependent manner. The sustained ERK1/2 activation was observed in the CS12, AGS, and MKN45 cells but not in the CNS cells. (E) The CS12 cells were pretreated with PD98059 (13.4lg/mL) or
vehicle (DMSO) for 30 min and then incubated with euphol (20lg/mL) for 72 h. The percentage of apoptotic cells was quantified by flow cytometry. (F) Pretreatment with
PD98059 reduced euphol-induced apoptosis. The bars represent the mean ± SD of the 3 independent experiments (⁄⁄P < 0.01, compared with the euphol-treated cells).
4 M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx
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induc-307
correlated with apoptosis sensitivity induced by
methyl-b-cyclo-308dextrin, a cholesterol-depleting agent (
Li et al., 2006
). These
find-309ings suggest that differences in the potency of euphol between
310cancer and noncancer cells may be related to the membrane
cho-311lesterol content, lipid raft-related signal transduction and
phos-312phatase regulation.
313
Euphol induced apoptosis in CS12 cells, as evidenced by
annex-314in V-binding assays, flow cytometric detection, and Western
blot-315ting. Because gastric cancer cells show higher phosphatidylserine
316levels in the outer leaflet of the plasma membrane (
Woehlecke
317
et al., 2003
), inhibition of ERK1/2 only slightly reduced annexing
318
V binding in our results. However, the pretreatment with
319PD98059 markedly inhibited the downregulation of Bcl-2 in
re-320sponse to the euphol treatment, indicating that the ERK1/2
path-321way may be involved in the antiproliferative effect of euphol.
322Moreover, euphol-induced apoptosis was associated with the
323upregulation of BAX, loss of mitochondrial membrane potential,
324and increased caspase-3 activity. BAX plays a critical role in the
325breakdown of the mitochondrial potential by translocating to the
326mitochondria in response to death stimuli (
Tait and Green,
327
2010
). The loss of mitochondrial membrane potential is associated
328with mitochondrial dysfunction, which is linked to apoptosis
329
(
Green and Reed, 1998
). Therefore, euphol may play a critical role
330
in the induction of apoptosis by altering the BAX/Bcl-2 ratio and
331activating caspase signaling, resulting in apoptotic cell death.
Tong
332
et al. (2011)
demonstrated that the sustained activity of the ERK1/2
333
pathway modulates apoptosis by regulating the BAX/Bcl-2 ratio
334and caspase activation. Euphol-induced gastric cancer cell
apopto-335sis may be mediated by a similar pathway leading to the activation
336of the caspase cascade.
337
In the present study, the inhibition of gastric cancer cell
prolif-338eration by euphol was found to be mediated by ERK1/2-dependent
339p27
kip1upregulation and cyclin B1 inhibition. These results were in
340
agreement with those of previous studies on gastric, breast, and
341colon cancers (
Guo et al., 2011; Lin et al., 2010; Ollinger et al.,
342
2007; Park et al., 2011
). Icaritin, a prenyl-flavonoid derivative from
343
the genus Epimedium, induced sustained ERK1/2 phosphorylation
344
and the subsequent downregulation of Bcl-2 and cyclin B1 protein
345
expressions in MDA-MB-453 and MCF7 breast cancer cells. It is
346
interesting that an inhibitor of ERK1/2 activity abrogated
icaritin-347
induced G2/M cell cycle arrest and cell apoptosis (
Guo et al.,
348
2011
). Cannabinoids were reported to reduce cancer cell
prolifera-349tion by activating ERK1/2 signaling, inhibiting the survival AKT
350
pathway and inducing p27
kip1expression, leading to gastric cancer
351
cell cycle arrest (
Park et al., 2011
). P27
kip1, an important cell cycle
352
regulatory protein and tumor suppressor, has been implicated in a
353
variety of cellular processes, including the induction of cell cycle
354
arrest and apoptosis (
Said et al., 2001
). Most important is that
355
p27
kip1has been reported to promote apoptosis in gastric cancer
356
(
Zheng et al., 2005
), and low p27
kip1levels may promote
carcino-357
genesis associated with the Helicobacter pylori infection (
Eguchi
358
et al., 2004
).
359
The cyclin B1 protein level has been shown to be a critical factor
360
affecting survival, and cyclin B1 overexpression is correlated with
361
the aggressiveness and metastatic potential of gastric cancer.
Cy-362
clin B1 overexpression was found in approximately 49% of gastric
363
carcinomas (
Begnami et al., 2010
). Knockdown of cyclin B1 was
364
shown to inhibit cancer cell proliferation in vitro and in vivo (
And-365
roic et al., 2008
). A recent study provided evidence that the growth
366inhibitory and apoptosis induction effects of betulinic acid are
367
mediated by targeting cyclin B1 protein downregulation in human
368
gastric AGS cancer cells (
Yang et al., 2010
). Furthermore, null or
369
low expression of p27
kip1in tumor cells in diffuse large B-cell
lym-370
phomas was reported to be strongly associated with increased
371
expression of cyclin B1 (
Bai et al., 2001
). Knockdown of the tumor
372
suppressor FHL1 in lung cancer cells also suppressed p27
kip1373
expression and elevated the expression of cyclin B1 simultaneously
374
(
Niu et al., 2011
). In contrast, overexpression of cyclin B1 and
375
downregulation of p27
kip1protein were suggested to result in
tu-376
mor progression and development (
Begnami et al., 2010; Kim,
377
2007
).
378
Our results suggest that euphol may inhibit cancer cell growth
379
and tumor development by inhibition of cyclin B1 expression and
380
elevation of p27
kip1protein levels.
Fig. 4. Role of ERK1/2 in the euphol-induced mitochondrial membrane potential loss and apoptotic-related protein expression. (A) The Western blot analyses of the expressions of BAX and Bcl-2 in the euphol-treated CS12 cells. Beta-actin was used as an internal control. (B) Quantification of the Bcl-2 and BAX protein expressions from the Western blot analyses. Euphol increased the BAX/Bcl-2 ratio in a dose-dependent manner; however, PD98059 decreased the ratio in the euphol-treated cells. (C) The mitochondria membrane potential was analyzed by flow cytometry. PD98059 reversed the euphol-induced mitochondrial membrane potential loss in the CS12 cells. (D) Caspase-3 activation was determined by flow cytometry, using an anti-FITC-DEVD-FMK antibody. Each bar is the mean ± SD of the 3 independent experiments (⁄⁄
P < 0.01, compared with the control group).
M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx 5
induc-381
5. Conclusions
382
The present study demonstrated that euphol has
antiprolifera-383tive effects and selectively promotes apoptosis in human gastric
384cancer cells. The mechanism underlying the effect of euphol
in-385volves mitochondrial-dependent caspase-3 activation and growth
386arrest through induction of p27
kip1and inhibition of cyclin B1 in
387
human gastric CS12 cancer cells. ERK1/2 participated in the
eup-388hol-induced apoptosis and growth inhibition. This study provides
389a mechanistic insight and supports the premise that euphol is a
390potentially promising agent for development as chemotherapy
391against gastric cancer in humans. The specificity of euphol in
tar-392geting cancer cells may lead to the reduction of toxic side effects
393in cancer patients.
394
Conflict of Interest
395
Authors declare that there is no conflict of interest.
396
Acknowledgments
397
This work was supported by Grant from the Department of
398
Health, Executive Yuan, ROC (Taiwan) (DOH100-TD-C-111-002).
399
References
400 Akihisa, T., Kithsiri Wijeratne, E.M., Tokuda, H., Enjo, F., Toriumi, M., Kimura, Y., Koike,
401 K., Nikaido, T., Tezuka, Y., Nishino, H., 2002. Eupha-7,9(11),24-trien-3beta-ol
402 (‘‘antiquol C’’) and other triterpenes from Euphorbia antiquorum latex and their
403 inhibitory effects on Epstein-Barr virus activation. J. Nat. Prod. 65, 158–162.
404 Akihisa, T., Yasukawa, K., Kimura, Y., Takase, S., Yamanouchi, S., Tamura, T., 1997.
405 Triterpene alcohols from camellia and sasanqua oils and their
anti-406 inflammatory effects. Chem. Pharm. Bull. (Tokyo) 45, 2016–2023.
407 Androic, I., Kramer, A., Yan, R., Rodel, F., Gatje, R., Kaufmann, M., Strebhardt, K.,
408 Yuan, J., 2008. Targeting cyclin B1 inhibits proliferation and sensitizes breast
409 cancer cells to taxol. BMC Cancer 8, 391.
410 Bai, M., Vlachonikolis, J., Agnantis, N.J., Tsanou, E., Dimou, S., Nicolaides, C.,
411 Stefanaki, S., Pavlidis, N., Kanavaros, P., 2001. Low expression of p27 protein
412 combined with altered p53 and Rb/p16 expression status is associated with
413 increased expression of cyclin A and cyclin B1 in diffuse large B-cell
414 lymphomas. Mod. Pathol. 14, 1105–1113.
415 Ballif, B.A., Blenis, J., 2001. Molecular mechanisms mediating mammalian
mitogen-416 activated protein kinase (MAPK) kinase (MEK)-MAPK cell survival signals. Cell
417 Growth Differ. 12, 397–408.
418 Begnami, M.D., Fregnani, J.H., Nonogaki, S., Soares, F.A., 2010. Evaluation of cell cycle
419 protein expression in gastric cancer: cyclin B1 expression and its prognostic
420 implication. Hum. Pathol. 41, 1120–1127.
421 Bionda, C., Athias, A., Poncet, D., Alphonse, G., Guezguez, A., Gambert, P.,
Rodriguez-422 Lafrasse, C., Ardail, D., 2008. Differential regulation of cell death in head and
423 neck cell carcinoma through alteration of cholesterol levels in lipid rafts
424 microdomains. Biochem. Pharmacol. 75, 761–772.
425 Cagnol, S., Chambard, J.C., 2010. ERK and cell death: mechanisms of ERK-induced
426 cell death–apoptosis, autophagy and senescence. FEBS J. 277, 2–21.
427 Carvalho, M.A., Zecchin, K.G., Seguin, F., Bastos, D.C., Agostini, M., Rangel, A.L., Veiga,
428 S.S., Raposo, H.F., Oliveira, H.C., Loda, M., Coletta, R.D., Graner, E., 2008. Fatty
429 acid synthase inhibition with Orlistat promotes apoptosis and reduces cell
430 growth and lymph node metastasis in a mouse melanoma model. International
431 journal of cancer. J. Int. Cancer 123, 2557–2565.
432 Dangle, P.P., Zaharieva, B., Jia, H., Pohar, K.S., 2009. Ras-MAPK pathway as a
433 therapeutic target in cancer–emphasis on bladder cancer. Recent Pat Anticancer
434 Drug Discov. 4, 125–136.
435 Eguchi, H., Carpentier, S., Kim, S.S., Moss, S.F., 2004. P27kip1 regulates the apoptotic
436 response of gastric epithelial cells to Helicobacter pylori. Gut 53, 797–804.
437 Freeman, M.R., Solomon, K.R., 2004. Cholesterol and prostate cancer. J. Cell.
438 Biochem. 91, 54–69.
439 Fulda, S., Galluzzi, L., Kroemer, G., 2010. Targeting mitochondria for cancer therapy.
440 Nat. Rev. Drug Discov. 9, 447–464.
441 Furstenberger, G., Hecker, E., 1977a. The new diterpene 4-deoxyphorbol and its
442 highly unsaturated irritant diesters. Tetrahedron Lett. 18, 925–928.
443 Furstenberger, G., Hecker, E., 1977b. New highly irritant euphorbia factors from
444 latex of Euphorbia tirucalli L. Experientia 33, 986–988.
445 George, K.S., Wu, S., 2012. Lipid raft: a floating island of death or survival. Toxicol.
446 Appl. Pharmacol. 259, 311–319.
447 Gildenhuys, S., 2006. The three most abundant tree Euphorbia species of the
448 Transvaal (South Africa). Euphorbia World 2, 9–14.
449 Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312.
450 Guo, Y., Zhang, X., Meng, J., Wang, Z.Y., 2011. An anticancer agent icaritin induces
451 sustained activation of the extracellular signal-regulated kinase (ERK) pathway
452 and inhibits growth of breast cancer cells. Eur. J. Pharmacol. 658, 114–122.
453 Khan, A.Q., Rasheed, T., Malik, A., 1988. Tirucaline—a new macrocyclic diterpene
454 from Euphorbia tirucalli. Heterocycles 27, 2851–2856.
455 Kim, D.H., 2007. Prognostic implications of cyclin B1, p34cdc2, p27(Kip1) and p53
456 expression in gastric cancer. Yonsei Med. J. 48, 694–700.
457 Kim, E.K., Choi, E.J., 2010. Pathological roles of MAPK signaling pathways in human
458 diseases. Biochim. Biophys. Acta 1802, 396–405.
459 Levinthal, D.J., Defranco, D.B., 2005. Reversible oxidation of ERK-directed protein
460 phosphatases drives oxidative toxicity in neurons. J. Biol. Chem. 280, 5875–5883.
461 Li, Y.C., Park, M.J., Ye, S.K., Kim, C.W., Kim, Y.N., 2006. Elevated levels of
cholesterol-462 rich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced
463 by cholesterol-depleting agents. Am. J. Pathol. 168, 1107–1118, quiz 1404–1105.
464 Lin, K.L., Su, J.C., Chien, C.M., Tseng, C.H., Chen, Y.L., Chang, L.S., Lin, S.R., 2010.
465 Naphtho[1,2-b]furan-4,5-dione induces apoptosis and S-phase arrest of
MDA-466 MB-231 cells through JNK and ERK signaling activation. Toxicol. In Vitro 24,
467 61–70.
468 Lin, S.J., Yeh, C.H., Yang, L.M., Liu, P.C., Hsu, F.L., 2001. Phenolic compounds from
469 Formosan Euphorbia tirucalli. J. Chin. Chem. Soc. 48, 105–108.
470 Lu, T.L., Huang, G.J., Lu, T.J., Wu, J.B., Wu, C.H., Yang, T.C., Iizuka, A., Chen, Y.F., 2009.
471 Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited
472 ERK1/2 activity in breast and bladder cancer cells. Food Chem. Toxicol. 47,
473 2013–2021.
474 Martin, P., Poggi, M.C., Chambard, J.C., Boulukos, K.E., Pognonec, P., 2006. Low dose
475 cadmium poisoning results in sustained ERK phosphorylation and caspase
476 activation. Biochem. Biophys. Res. Commun. 350, 803–807.
Fig. 5. Role of ERK1/2 in the expressions of the cell cycle regulatory proteins in the euphol-treated cells. (A) PD98058 suppressed euphol-induced p27kip1
protein expression but elevated cyclin B1 expression. Beta-actin was used as an internal control. (B) Quantification of the cyclin B1 protein expression from the Western blot analyses. Euphol inhibited cyclin B1 expression in a dose-dependent manner, and PD98059 reversed this effect in the euphol-treated cells. (C) Quantification of p27kip1protein expression from the Western blot analyses. Euphol increased the
p27kip1
expression in a dose-dependent manner, and PD98059 reversed this effect in the euphol-treated cells. Each bar is the mean ± SD of the 3 independent experiments (⁄⁄
P < 0.01, compared with the control group).
6 M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx
FCT 6641
No. of Pages 7, Model 5G
29 May 2012
induc-477 Martin, P., Pognonec, P., 2010. ERK and cell death: cadmium toxicity, sustained ERK 478 activation and cell death. FEBS J. 277, 39–46.
479 Murphy, L.O., Blenis, J., 2006. MAPK signal specificity: the right place at the right 480 time. Trends Biochem. Sci. 31, 268–275.
481 Niu, C., Liang, C., Guo, J., Cheng, L., Zhang, H., Qin, X., Zhang, Q., Ding, L., Yuan, B., Xu, 482 X., Li, J., Lin, J., Ye, Q., 2011. Downregulation and growth inhibitory role of FHL1 483
Q2
in lung cancer. Int. J. Cancer.484 Ollinger, R., Kogler, P., Troppmair, J., Hermann, M., Wurm, M., Drasche, A., 485 Konigsrainer, I., Amberger, A., Weiss, H., Ofner, D., Bach, F.H., Margreiter, R., 486 2007. Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle 6,
487 3078–3085.
488 Park, J.M., Xian, X.S., Choi, M.G., Park, H., Cho, Y.K., Lee, I.S., Kim, S.W., Chung, I.S., 489 2011. Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest 490 in human gastric cancer cells. J. Cell. Biochem. 112, 1192–1205.
491 Rasool, N., Khan, A.Q., Malik, A., 1989. A taraxerane type triterpene from Euphorbia 492 tirucalli. Phytochemistry 28, 1193–1195.
493 Said, T.K., Moraes, R.C., Singh, U., Kittrell, F.S., Medina, D., 2001. Cyclin-dependent 494 kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse 495 mammary preneoplasia. Cell Growth Differ. 12, 285–295.
496 Shah, M.A., Kelsen, D.P., 2010. Gastric cancer: a primer on the epidemiology and 497 biology of the disease and an overview of the medical management of advanced 498 disease. J. Natl. Compr. Canc. Netw. 8, 437–447.
499 Sheridan, C., Brumatti, G., Elgendy, M., Brunet, M., Martin, S.J., 2010. An ERK-500 dependent pathway to Noxa expression regulates apoptosis by platinum-based 501 chemotherapeutic drugs. Oncogene 29, 6428–6441.
502 Tait, S.W., Green, D.R., 2010. Mitochondria and cell death: outer membrane 503 permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11, 621–632. 504 Tibbles, L.A., Woodgett, J.R., 1999. The stress-activated protein kinase pathways. 505 Cell. Mol. Life Sci. 55, 1230–1254.
506 Tong, J.S., Zhang, Q.H., Huang, X., Fu, X.Q., Qi, S.T., Wang, Y.P., Hou, Y., Sheng, J., Sun, 507 Q.Y., 2011. Icaritin causes sustained ERK1/2 activation and induces apoptosis in 508 human endometrial cancer cells. PLoS ONE 6, e16781.
509 Wang, X., Martindale, J.L., Holbrook, N.J., 2000. Requirement for ERK activation in 510 cisplatin-induced apoptosis. J. Biol. Chem. 275, 39435–39443.
511 Woehlecke, H., Pohl, A., Alder-Baerens, N., Lage, H., Herrmann, A., 2003. Enhanced
512 exposure of phosphatidylserine in human gastric carcinoma cells overexpressing
513 the half-size ABC transporter BCRP (ABCG2). Biochem. J. 376, 489–495.
514 Yang, L.J., Chen, Y., Ma, Q., Fang, J., He, J., Cheng, Y.Q., Wu, Q.L., 2010. Effect of
515 betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric
516 adenocarcinoma AGS cells. Acta Pharmacol. Sin. 31, 66–72.
517 Yang, Y.C., Wang, S.W., Hung, H.Y., Chang, C.C., Wu, I.C., Huang, Y.L., Lin, T.M., Tsai,
518 J.L., Chen, A., Kuo, F.C., Wang, W.M., Wu, D.C., 2007. Isolation and
519 characterization of human gastric cell lines with stem cell phenotypes. J.
520 Gastroenterol. Hepatol. 22, 1460–1468.
521 Yang, Y.C., Wang, S.W., Wu, I.C., Chang, C.C., Huang, Y.L., Lee, O.K., Chang, J.G., Chen,
522 A., Kuo, F.C., Wang, W.M., Wu, D.C., 2009. A tumorigenic homeobox (HOX) gene
523 expressing human gastric cell line derived from putative gastric stem cell. Eur. J.
524 Gastroenterol. Hepatol. 21, 1016–1023.
525 Yasukawa, K., Akihisa, T., Yoshida, Z.Y., Takido, M., 2000. Inhibitory effect of euphol,
526 a triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion by
527 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse
528 skin. J. Pharm. Pharmacol. 52, 119–124.
529 Yoshida, T., Yokoyama, K.I., Namba, O., Okuda, T., 1991. Tannins and related
530 polyphenols of euphorbiaceous plants. VII. Tirucallins A, B and euphorbin F,
531 monomeric and dimeric ellagitannins from Euphorbia tirucalli L. Chem. Pharm.
532 Bull. 39, 1137–1143.
533 Zhang, X., Yashiro, M., Ren, J., Hirakawa, K., 2006. Histone deacetylase inhibitor,
534 trichostatin A, increases the chemosensitivity of anticancer drugs in gastric
535 cancer cell lines. Oncol. Rep. 16, 563–568.
536 Zhao, Y., Shen, S., Guo, J., Chen, H., Greenblatt, D.Y., Kleeff, J., Liao, Q., Chen, G., Friess,
537 H., Leung, P.S., 2006. Mitogen-activated protein kinases and chemoresistance in
538 pancreatic cancer cells. J. Surg. Res. 136, 325–335.
539 Zheng, J.Y., Wang, W.Z., Li, K.Z., Guan, W.X., Yan, W., 2005. Effect of p27(KIP1) on cell
540 cycle and apoptosis in gastric cancer cells. World J. Gastroenterol. 11, 7072–
541 7077.
542 Zhuang, L., Kim, J., Adam, R.M., Solomon, K.R., Freeman, M.R., 2005. Cholesterol
543 targeting alters lipid raft composition and cell survival in prostate cancer cells
544 and xenografts. J. Clin. Invest. 115, 959–968.
545
M.-W. Lin et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx 7
