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Cisplatin as a highly potent cytotoxic agent was widely used in the chemotherapy of gastric cancer. It kills cancer cells by inducing apoptosis. The Extracellular signal-regulated kinase (ERK) signaling pathway plays an important role in proliferation and survival. However, its roles in apoptosis vary. This study focused on the role of ERK in cisplatin-induced apoptosis in a stomach cancer cell line, MKN-28 cells. We found that cisplatin treatment substantially activated ERK, which was prevented by MEK inhibitor U0126. Transient transfection of MKN- 28 cells with Constitutively Active-MEK1 (CA-MEK1) elevated cisplatin-induced apoptosis comparing with Dominant Negative-MEK1 (DN-MEK1). Cisplatin-induced ERK activation up-regulates pro-apoptotic gene BAK, whereas down-regulates anti-apoptotic gene BCL-2. Knocking down BCL-2 with siRNA sensitizes MKN-28 cells to the toxicity of cisplatin. These results suggested that (1) ERK activation is required for the cisplatin-induced apoptosis in MKN-28 cells; and (2) ERK mediates apoptosis by BCL-2.
Keywords: Cisplatin; Gastric cancer; Apoptosis; ERK; BCL-2; BAK
Cisplatin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy
for various types of cancers such as gastric cancer, which is one of the most malignant can- 1-5 6 cers in China.1-5 Apoptosis is an important pathogenic mechanismin solidary tumor,6 which is
characterized by morphological changes such as cell shrinkage, chromatin condensation and
fragmentation of DNA. Cisplatin treatment in vivo and in vitro results in apoptosis of cancer
cells, which usually in dose- and time-dependent manner.
Several mechanisms are believed to mediate cisplatin-induced apoptosis. The primary
mechanism of the cytotoxic effects is due to coordinative bonding between the atoms of plati-
num and DNA, leading to formation of intra-strand DNA cross-links, which induces apoptosis
by activating p53 and cell cycle arrest. Previous study has been shown that Reactive Oxygen
Species (ROS) generated by cisplatin increases lipid peroxidation, which alters enzymes and
structural proteins, and direct the cell to an apoptotic pathway.8 Also, cisplatin-induced apop- 9-11 tosis could involve in the inflammatory pathway.9-11 Moreover, previous studies revealed that
in the regulation of various cellular responses, such as cell proliferation, differentiation and
a major role in regulating cell growth and differentiation, being
highly induced in response to growth factors and cytokines. It is
also activated by some conditions of stress, particularly oxidant
injury, and such circumstances is believed to confer a survival
advantage to cells.13 Comparatively, JNK and p38 are weakly
activated by growth factors, but are highly activated in response
to stress signals including tumor necrosis factor, ionizing and
short wave length Ultraviolet irradiation (UVC), and hyperos-
motic stress. Their activation is most frequently associated with 14-16 induction of apoptosis. In cisplatin treatment, ERK was acti-
vated in a variety of cell lines. While some studies showed that
the ERK activation enhances survival of cisplatin-treated cells,13
then some other studies reported that it induces apoptosis in cis- 17-22 platin exposed cells. Gastric cancer is one of the malignant
cancers in China. The functional roles of ERK in cisplatin treat-
ment of gastric cancer remain elusive. The present study sought
to delineate the role of the ERK signaling in cisplatin-induced
apoptosis using a gastric cancer cell line, MKN-28.
Human gastric adenocarcinoma (MKN-28) cells were
obtained from the American Type Culture Collection. Cells were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hy-
clone, Logan, Utah, USA) supplemented with 10% fetal bovine
serum (Hyclone, Logan, Utah, USA) and 1% penicillin–strepto-
mycin (Gibco, Grand Island, NY, USA). Cells were cultured in
a 37 °C humidified incubator with a mixture of 95% air and 5%
CO2.
Constitutively Active-MEK1 and Domain Negative-
MEK1 was purchased from Addgene, and then sub-cloned into
pCMV vector. The MEK inhibitor U0126 and JNK inhibitor
SP600125 were purchased from Tocrics (Bristol, UK). Immuno-
blotting experiments were performed with rabbit anti-caspase-3,
anti-caspase-8, anti-caspase-9, anti-PARP, anti-Bid, anti-BAK,
anti-Bax, anti-PUMA, anti-cytochrome c and anti-BCL-2 were
from Epitomics (Burlingame, CA, USA), anti-phospho-p44/42
MAPK (p-ERK1/2) (Thr202/Tyr204), and anti-ERK1/2 antibod-
ies were purchased from Cell Signaling Technology (Beverly,
MA, USA); The Horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG polyclonal antibodies were obtained from Santa
Cruz Biotechnology (Dallas, Texas, USA).
Cells were lysed in PIRA buffer (Beyongtime, China).
Following boiling for 5 min, samples were separated on 10%
SDS-polyacrylamide gels and then transferred to an Immobilon
membrane (Millipore, MA, USA). The membranes were blocked
with 5% non-fat dry milk in TBS/Tween20 (0.05% v/v) for 1 h
followed by incubation at 4 °C overnight with the indicated pri-
mary antibodies. The membranes were washed three times with
TBST buffer and then incubated for 1 h with HRP-conjugated
anti-rabbit or -mouse secondary antibodies. Visualization of
protein bands was accomplished using enhanced chemilumines-
cence (Thermo Scientific).
Cells were seeded at a density of 1.0×10 cells per well
in 24-well plates, cultured with different reagents for 48 h, and
then harvested. The cells were washed with PBS and then stained
5 μL of Propidium Iodide (PI) for 15 min followed by 5 μL of
Annexin V and at room temperature in the dark according to the
manufacturer’s instructions (Nanjing Key Gen Biotech, Nanjing,
China). The apoptosis rate (%) of the stained cells was analyzed
using FCM. The experiments were repeated three times.
Cell death was quantitively detected based on the re-
lease of LDH by using a cytotoxicity detection kit (Roche Life
Science, IN, USA) as described in manufacturer’s instruction.
All the experiments were repeated three times and the average is
shown in each figure.
To assess mitochondrial cytochrome c release, cytoso-
lic protein extracts were obtained according to instructions of
a commercial cell fractionation kit (Enzo Life Sciences, Farm-
ingdale, NY, USA). Briefly, 1 × 107 cells in a dish of 10 cm in
diameter were washed twice with cold PBS and then centrifuged
at 600g for 5 min at 4 °C. Cells were suspended in 0.1 ml of ice-
cold fractionation buffer and then incubated on ice for 10 min
before being homogenized with a syringe needle. The homoge-
nates were centrifuged at 700 g for 10 min at 4 °C, after which
the supernatants were collected and centrifuged at 10,000 g for
30 min at 4 °C. The supernatant was again collected as the cyto-
solic fraction and analyzed for cytochrome c release by western
blot assay.
SMART pool: ON-TARGET plus BCL-2 siRNA and
Transfection Reagent-1 were both purchased from Damarchon
(Lafayette, CO, USA), Cat# L-003307-00-0005. Transfection
procedure was followed the manufacturer’s instructions.
All results of bar graphs are expressed as the mean± S.D.
obtained from three independent experiments in duplicate. Sta-
tistical differences were evaluated using the Student’s t-test or
ANOVA. P < 0.05 was considered statistically significant.
Cisplatin treatment induces apoptosis in many differ-
ent cell lines. To test the effect of apoptosis in MKN-28 cells,
MKN-28 cells were treated with cisplatin in an increasing dose (0 μg/mL, 4 μg/mL, 8 μg/mL, 12 μg/mL, 16 μg/mL, and 20 μg/
mL) for different time point (12 h, 24 h, 36 h). After the treat-
ment, cells were stained by Annexin V-FITC and PI, and were
examined by Flow cytometry (FCM). As shown in figure 1A,
cisplatin treatment resulted in dose-and time-dependent man-
ner in MKN-28 cells. Most cells were dead after 48 h treatment
in dose 16 μg/mL and 20 μg/ml (Supplementary Figure 1). The
apoptotic marker such as cleaved caspase-3 and cleavage of Poly
(ADP-ribosyl) polymerase (PARP) can be detected by Western
blot analysis (Figure 1B). The LDH releasing experiment also
revealed that the LDH leaked from apoptotic cells after cisplatin
treatment (Figure 1C). Meanwhile, the cell viability was oppo-
site changed. Treatment of MKN-28 cells with cisplatin resulted
in a time-dependent increase in release of cytochrome c from
mitochondria (Figure 1D). These results suggested that cisplatin
induced MNK-28 cell apoptosis via the mitochondrial pathway.
Supplementary Figure 1: Annexin V/PI apoptosis assay used to detected apoptosis of MKN- 28 cells treated with Cisplatin in different doses for 48 hours. Most cells died at 16 and 20 μg/ mL concentration.
Cells were exposed in cisplatin at various time points
in a dose of 20 μg/ml. The activation of MAPK pathway was
then detected by Western blot, including ERK1/2 (p42/44), p38,
and JNK. In the treatment, JNK was activated moderately while
ERK was activated remarkably. (Figure 2) This result showed a
relationship between cisplatin-mediated apoptosis and ERK sig-
naling cascade.
Pharmaceutical inhibition was applied to test if the were cultured for 6 h, 12 h, 24 h, 36 h and 48 h in the presence of Cisplatin (20
MAPK/ERK pathway involved in cisplatin-induced apoptosis.
The effect of MAPK inhibitors was detected with Western blot
(Figure 3A). Apoptosis assay analysis showed that cisplatin-
induced apoptosis of MKN-28 cells was blocked by the MEK
inhibitor U0126, but not c-Jun N-terminal Kinase (JNK) inhibi-
tor SP600125 at the 24 hr and 36 hr. (Figure 3B). These results
demonstrated that the cisplatin-induced apoptosis with MAPK
pathway activation. It suggested that cisplatin-induced MKN-28
apoptosis was abolished by the MEK inhibitor U0126, but not
by JNK inhibitor SP600125.
In addition, we test the role of ERK by ectopically ex-
pressing constitutively active and dominant-negative ERK mu-
tant. Cells were transfected with pCMV-CA-MEK1 and pCMV-
DN- MEK1 (Figure 3C). Highly activation of phospho-ERK
was detected by Western blot after transiently transfected CA-
MEK1, but not for the DN-MEK1 transfected cells. When these
cells were treated with cisplatin, CA-MEK1 transfected cells
presented higher activation than control group while a reduced
level of ERK activation appeared in dominant-negative mutant
expressing cells (Figure 3D). The apoptosis assay suggested that
CA-MEK1 group acquired higher ERK1 activation and apopto-
sis (Figure 3E and 3F). These data indicate that ERK activation
induces the cisplatin-induced MKN-28 cells apoptosis.
pathway. Immunoblot analysis of the MAPK actiwere untreated or were cultured for 6 h, 12 h, 24 which incubated with or without cisplatin, were calculated by Annexin V/PI FCM
To examine the relationship between ERK activation
and BCL-2 family, we investigated the BCL-2 family by West-
ern Blot. After cisplatin treatment, the BCL-2 expression level
was down-regulated. Meanwhile, the BAK level was up-regu-
lated (Figure 4A). To test if BCL-2 is the downstream transduc-
ers of the activated ERK-induced apoptosis, MKN-28 cells were
transient transfected with CA-MEK. Dramatically, we found
that BCL-2 was down-regulated concurrently with BAK up-
regulation after CA-MEK1 transfection. The opposite result can
be found after DN-MEK1 transfection (Figure 4B). This result
suggested that BCL-2 and BAK are both downstream of acti-
vated ERK. Furthermore, when BCL-2 was knocked down by
smart pool siRNA, cisplatin-induced apoptosis was remarkably
increased (Figure 4C and 4D). Taken together, the results sug-
gested that, with cisplatin treatment, (1) ERK activation regulate
BCL-2 and BAK, (2) BCL-2 down-regulation facilitate apopto-
sis.
Two major distinct apoptosis pathways have been de-
scribed for mammalian cells: (1) Mitochondrial independent
pathway, which is recruited by the adapter molecule Fas/APO-1-
-associated death domain protein to death receptors upon extra- 23,24 cellular ligand binding; (2) cytochrome c release-dependent
activation of caspase-9 through Apaf-1. We did, however,
observe an increase of cytochrome c in the cytoplasm of cispla-
tin-treated cells relative to untreated cells, suggesting that cyto-
chrome c release is involved in cisplatin-induced apoptosis.
The MAPK pathways, including ERK, were typically
studied previously. However, the roles of ERK involved in can- 28-30 cer protection are controversial. Most investigations revealed
the importance of MAPK signaling pathways in regulating
apoptosis during conditions of stress. But majority of the studies
supported the general hypothesis that ERK activation delivers
a survival signal and inhibition of ERK signaling leads to in- 16,31,32 creased sensitivity of some cell lines to cisplatin. Although
more often associated with survival, a pro-apoptotic function for
the ERK pathway has also been suggested in several other model 21,22 systems. In our study, however, we provided evidence that ac-
tivation of ERK is important for the inducting cisplatin-induced
apoptosis in MKN-28 cells. By modulating ERK activity, we
found that both Domain-Negative MEK transfection and MEK
specific inhibitor U0126 led to an inhibition of cisplatin-induced
apoptosis. Meanwhile, enhancement of ERK activity by over-
expressing Constitutively Active MEK in MKN-28 cells accen-
tuated cell apoptosis. ERK activity-dependent cisplatin-induced
apoptosis was not limited to MKN-28 cells, but also occurred in 17,21 human lung cancer A549 cells and Hela cells.
Although several studies have shown that JNK is ac-
tivated in cisplatin treatment, their role in determining survival
is unclear. A number of studies have provided evidence indi- 25-27 cating that the JNK pathway contributes to cisplatin-induced 15,16 apoptosis. However, others have suggested that JNK signalwere cultured for 6 h, 12 h, 24 h, 36 h and 48 h in the presence of cisplatin (20
ing is important for cell survival. Such differential effects
observed from one study to another could indicate cell type
specificity. In our own studies using MKN-28 cells, we found
that JNK was indeed activated by cisplatin treatment. Howev-
er, down-regulation of this pathway by JNK specific inhibitor
SP600125 did not made any changes of the sensitivity to cispla-
tin. Thus, only ERK appears to play a major role in influencing
the survival of cisplatin-treated MKN-28 cells.
How does ERK activation lead to apoptosis of cispla-
tin-treated cells? What are the downstream transducers involved
in mediating the apoptosis? Based on previous and our study,
ERK activation is necessary, but not sufficient to induce apopto-
sis, as TPA treatment (Supplementary Figure 2) or constitutively
active-MEK over expression did not result in cell death. With
cisplatin, no significant activation occurs until 6 h, but the activ-
ity remains highly elevated through the remaining time period
examined (48 h).
Supplementary Figure 2: Immunoblot assay was used to detect the ERK phosphorylation in MKN-28 cells that transfected with CA-MEK1or treated with TPA without cisplatin treatment. The apoptosis of MKN-28 control cells, CA-MEK1transfected cells and TPA treated cells were calculated by Annexin V/PI FCM apoptosis assay. Each experiment was repeated for at least three times.
BCL-2 is known as an anti-apoptotic BCL-2 fam-
ily member, for which amplification has been associated with
a more aggressive malignant phenotype and drug resistance to
various categories of chemotherapeutic drugs in malignancies.35
Moreover, BCL-2 was reported as a target gene for many sig-
naling cascades, such as Phosphatidylinositol 3 kinase (PI3K)/
nuclear factor-κB (NF-κB) signaling pathway, the cyclic AMP
(cAMP)/protein kinase A (PKA) cascade, MAPK cascade and 36,37 so on. In this study, we found that treatment of cisplatin in-
creased phosphorylation of ERK proteins, which intervened
downstream anti-apoptosis molecular BCL-2 and up-regulation
of pro-apoptosis molecular BAK in gastric cancer MKN-28
cells. With BCL-2 and BAK change, cisplatin promoted MKN-
28 cells apoptosis.
Increasing amount of evidence indicated that the ERK
pathway is important in malignant disease. One recent study indicated that ERK activation by TGF-beta plays a key oncogenic
role in Colorectal cancer (CRC) with SMAD4 inactivation mu-
tations. The MEK inhibitor U0126 can surprisingly reduce CT26
cells tumor genesis and liver metastasis in vivo and in vitro,
suggesting that MEK inhibitors can be a primary approach for
treatment of human malignancies. However, our findings indi-
11,33,34 21,22 approach. On the other hand, coupled with previous studies,
our findings supported a role of the ERK signaling pathway in
enhancing cisplatin-induced apoptosis, further suggesting that
strategies targeting activation of this cascade could be employed
to enhance the therapeutic effectiveness of cisplatin. In addition,
evaluation of ERK activity could be useful marker for predict-
ing which tumors would present favorable response to cisplatin
therapy.
The authors declare no conflict of interest.
We should thank all the staffs in my lab for discussing the
project. This work was supported by the State Key Project on
Infection Disease of China (Nos. 2012ZX10002016-004 and
2012ZX10002010-001-004). The National Natural Science
Foundation of China (81202300 and 81372327).