ORIGINAL PAPER
Induction of autophagy in hepatocellular carcinoma cellsby SB203580 requires activation of AMPK and DAPKbut not p38 MAPK
Haitao Zhang • George G. Chen • Zhiyi Zhang •
Sukying Chun • Billy Cheuk Sing Leung •
Paul B. S. Lai
Published online: 15 December 2011
� Springer Science+Business Media, LLC 2011
Abstract SB203580 is a well-known inhibitor of p38
mitogen-activated protein kinase (MAPK). However, it can
suppress cell proliferation in a p38 MAPK independent
manner. The inhibitory mechanism remains unknown.
Here, we showed that SB203580 induced autophagy in
human hepatocellular carcinoma (HCC) cells. SB203580
increased GFP-LC3-positive cells with GFP-LC3 dots,
induced accumulation of autophagosomes, and elevated the
levels of microtubule-associated protein light chain 3 and
Beclin 1. It stimulated the phosphorylation of adenosine
monophosphate-activated protein kinase (AMPK) and p53,
but inhibited the phosphorylation of death-associated pro-
tein kinase (DAPK). Inhibition of AMPK, p53, or DAPK
attenuated SB203580-induced autophagy. AMPK activa-
tion appeared to predate the DAPK signal. The activation
of both AMPK and DAPK prompted the phosphorylation
of p53 and enhanced Beclin 1 expression. Neither the
downregulation of p38 MAPK by its siRNA or chemical
inhibitor nor the upregulation of p38 MAPK by p38 MAPK
DNA transfection affected B203580-induced autophagy.
Collectively, the findings demonstrate a novel function of
SB203580 to induce autophagy via activating AMPK and
DAPK but independent of p38 MAPK. The induction of
autophagy can thus account for the antiproliferative effect
of SB203580 in HCC cells.
Keywords SB203580 � Autophagy � Hepatocellular
carcinoma � AMPK � DAPK � p38 MAPK
Introduction
The pathway of autophagy has emerged as a promising
new target in cancer treatment. However, the relationship
of autophagy to tumors is complex. Evidence has been
shown to support both tumor-suppressive and tumor-
promoting roles [1].
SB203580, a pyridinyl imidazole, has been widely used
to assess the role of p38 mitogen-activated protein kinase
(MAPK) in a wide array of biological systems. Some
reports have demonstrated that SB203580 has antiprolif-
erative activity [2]. Interestingly, the antiproliferative
effect of SB203580 can be unrelated to p38 MAPK [2, 3].
Furthermore, Lali et al. [2] have demonstrated that
SB203580 blocked the phosphorylation of protein kinase B
(AKT)/PKB, a key protein kinase to preserve cell survival.
These results provide some mechanisms how SB203580
inhibits the cell proliferation.
AKT/PKB has been identified as a proto-oncogene that is
upregulated in various types of tumors [4]. It can activate the
mammalian target of rapamycin (mTOR) kinase [4], a
repressor of autophagy that is an important controller of cell
growth and proliferation [5]. Adenosine monophosphate-
activated protein kinase (AMPK) can induce autophagy via
suppressing mTOR. AMPK can be activated by decreasing
glucose availability level [6]. The decreased level of cell
glucose, which may result from SB203580 treatment [7], can
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10495-011-0685-y) contains supplementarymaterial, which is available to authorized users.
H. Zhang � G. G. Chen (&) � Z. Zhang � S. Chun �B. C. S. Leung � P. B. S. Lai
Department of Surgery, Faculty of Medicine, The Chinese
University of Hong Kong, Prince of Wales Hospital, Shatin,
New Territories, Hong Kong, China
e-mail: [email protected]
H. Zhang
Department of Biochemistry and Molecular Biology, Guangdong
Medical College, Zhanjiang, Guangdong, China
123
Apoptosis (2012) 17:325–334
DOI 10.1007/s10495-011-0685-y
lead to an increase in AMP/ATP ratio that activates AMPK
[8]. Therefore SB203580 may possess a novel function to
induce autophagy via activating AMPK and death-
associated protein kinase (DAPK). DAPK, an apoptosis
modulator, can induce cell autophagy via phosphorylating
Beclin 1 [9, 10]. The activity of DAPK can be negatively
regulated by auto-phosphorylation on S308. The phosphory-
lated DAPK (pDAPK) has lower activity than its unphos-
phorylated counterpart [11]. There are other signal
transduction pathways may regulate autophagy in a cell spe-
cific manner. Such pathways include those associated with
p53 [12], insulin-like growth factor I [12, 13], ras/mitogen-
activated protein kinase/extracellular signal-regulated kinase
(ERK) 1/2 [14], receptor-interacting protein 1 (RIP1) kinase
[15], and c-Jun NH2-terminal kinase [16–18]. In this study,
our data demonstrated that SB203580 induced autophagy in
hepatocellular carcinoma (HCC) cells in a pathway involved
in AMPK, DAPK and p53 but independent of p38 MAPK.
Materials and methods
Cell culture
Five human liver cancer cell lines (HepG2, Hep3B, PLC/PRF/
5, Huh-7, SK-Hep-1) and Chang liver cell line were obtained
from American Type Culture Collection (Rockville, MD), and
cultured in the Dulbecco’s modified Eagle medium (Gibco
BRL, Grand Island, NY).
Agents and chemicals
3-(4, 5-Dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium
bromide (MTT), ethidium bromide, propidium iodide,
Hoechst 33342, compound C, pifithrin-a (PFT-a),
DAPK(C-55) and S308-phosphorylated DAPK monoclonal
antibodies were purchased from Sigma-Aldrich (St. Louis,
MO). T308-phosphorylated AKT, T183-phosphorylated
T183-Jun N-terminal kinase (JNK), T202-phosphorylated
ERK, S15, S20-phosphorylated p53 antibodies were from
Cell Signaling (Danvers, MA). SB203580, p53, AMPK,
phosphorylated AMPK (pAMPK), acetyl-CoA carboxylase
a(ACCa), S78/80-phosphorylated ACCa (S78/80-ACCa),
PARP, procaspase-3, microtubule-associated protein light
chain 3I/II (LC3I/II), GAPDH, Beclin 1 antibodies, siRNA
products and BIRB0796 were from Santa Cruz Biotech-
nology (Santa Cruz, CA). pcDNA3.1-p38 MAPK were
constructed using pMT3 p38 from Addgene (Cambridge,
MA). SB203580 was dissolved in water.
MTT assay
MTT assay was performed as the previous report [19].
Flow cytometry analysis of cell cycle
The cells were collected by centrifugation after treatments,
washed twice in PBS and fixed with ice-cold 70% ethanol
for overnight. Prior to flow cytometry analysis, the fixed
cells were washed once with PBS and incubated with
100 lg/ml propidium iodide plus 200 lg/ml RNase. Cell
cycle was analyzed by flow cytometry [20].
Cell apoptosis analysis by fluorescence staining
Apoptosis was evaluated by in situ uptake of propidium
iodide and Hoechst 33342 [21]. Apoptotic index was
determined by calculating the number of apoptotic nuclei
versus total number of nuclei at each visual field 9 100%.
Detection of autophagy by pEGFP-LC3
HCC cells were transfected with pEGFP-LC3 to measure
autophagy level. Lipofectamine 2000 (Invitrogen, Carlsbad,
CA) was employed to transfect HCC cells. Following the
induction of autophagy by SB203580, the cellular localization
pattern of GFP-LC3 was photographed using the Zeiss fluo-
rescence microscope. GFP-LC3 is a highly specific fluores-
cence marker of autophagy and can be used to measure
autophagy [22]. When autophagy occurs, the percentage of
GFP-LC3-positive cells with GFP-LC3 punctate dots
increases and the dots redistribute from a diffuse pattern to a
punctate cytoplasmic pattern (GFP-LC3 dots) that specifically
labels preautophagosomal and autophagosomal membranes.
Down-regulation of DAPK and p53 by siRNA
Cells were transfected with different siRNAs and control
siRNA using lipofectamine 2000. Before transfection, cells
were seeded in 6-well plates or 60 mm culture dishes
containing DMEM medium without antibiotics for 24 h.
Cells were transfected with 100 pmol siRNA in each well.
The transfected cells were treated with SB203580 for 24 h.
The target proteins were measured by Western blot 24 h
post-transfection.
Western blot
Total protein was isolated for Western blot analysis and the
experimental procedures were performed according to the
previous report [23].
Transmission electron microscope
Cells were harvested by trypsinization, washed twice with
PBS, fixed with ice-cold 3% glutaraldehyde–0.1 mol/l
cacodylate buffer, re-fixed in osmium tetroxide. Cells were
326 Apoptosis (2012) 17:325–334
123
embedded in Epong and cut to a 1.0-lm-thin section.
Before being viewed with a Philips electron microscope,
sections were stained with methylene buffer ArumeII [24].
Statistical analysis
The values given are presented as mean ± SD. Statistical
analysis was performed using one-way analysis of variance
(ANOVA) followed by Student’s t test. P \ 0.05 was
considered as significant.
Results
Induction of autophagy by SB203580
The autophagy was first assessed by observing the change in
the cell morphology. After incubation with SB203580 for
24 h, the morphological evaluation under visible light
revealed a significant increase in the number of HepG2 cells
with autophagosomes (Fig. 1a) which were recognized as
characteristic double-membrane vacuolar structures contain-
ing various kinds of cytoplasmic contents [24]. Similar results
were also observed in other HCC cells and Chang cells
(supplemental Fig. 1). The occurrence of authophage was
confirmed by the detection of GFP-LC3 dots. Compared with
the control cells, SB203580-treated cells displayed more
GFP-LC3 dots (Fig. 1b, c). The percentage of GFP-LC3-
positive cells with GFP-LC3 punctate dots was increased in a
dose-dependent manner after SB203580 treatment (Fig. 1b,
c). In addition to autophagosomes and GFP-LC3 dots, the
increased expression of LC3-II protein is another marker for
autophagy [5, 6]. The level of LC3-II was increased by
SB203580 in a dose-dependent manner (Fig. 1c). In order to
check whether the apoptosis also occurred in SB203580-
treated HCC cells, HepG2 cells were stained with Hoechst
33342 to detect apoptosis. The result did not show any typical
characteristic of apoptosis in cells treated with SB203580.
Moreover, PARP was not cleavaged in HepG2 cells treated
with SB203580 (Fig. 1e), confirming the absence of apopto-
sis. As a positive control, we used etoposide, a known apop-
totic inducer, to treat HepG2 cells, and the cells showed DNA
condensation and cleavaged PARP (Fig. 1d, e), both of which
are the characteristics of apoptosis.
The effect of SB203580 on the expression of proteins
involved in cell survival or death was determined. These
proteins included p38 MAPK, JNK, ERK, AKT, procaspase-
3, p53, DAPK, AMPK, Beclin 1, LC3-I and LC3-II. The
result showed that the levels of phosphorylated p38 MAPK
(pp38 MAPK), JNK, ERK, AKT, and DPAK were decreased
in SB203580-treated HepG2 cells (Fig. 1f). However, the
levels of pAMPK, S78/80-ACCa, S15- and S20-phosphor-
ylated p53 (S15-p53, S20-p53), Beclin 1 and LC3-II were
increased in SB203580-treated cells. Pro-caspase-3 was not
changed by SB203580. The levels of AMPK, DAPK and
AKT were not different between the treated and non-treated
cells (Fig. 1f and supplemental Fig. 2).
We also determined the effect of SB203580 on the cell
proliferation, reflected by cell survival rate measured by
MTT assay. The result showed that SB203580 at
25–100 lM significantly inhibited the cell proliferation
(supplemental Fig. 3A).In three out of five HCC cell lines
tested (HepG2, Hep3B and Huh-7), the survival rate was
about 20–25% less than the control when they were treated
with 50 lM SB203580 for 24 h. Cell cycle analysis
showed that SB203580 arrested the cells at G0/G1 phase
(supplemental Fig. 2B).
SB203580-induced autophagy was independent of p38
MAPK
In order to test the role of p38 MAPK in the SB203580-
induced autophagy, we used siRNA to block the expression
of p38 MAPK in HCC cells. It showed that siRNA treat-
ment did not change the SB203580-induced autophagy
(Fig. 2), suggesting that SB203580-induced autophagy was
independent of p38 MAPK. Similar results were obtained
when p38 MAPK activity was inhibited by BIRB0796, a
special p38 MAPK inhibitor [25] (supplemental Fig. 4).
The p38 MAPK-independent autophagy induced by
SB203580 was further confirmed by the over-expression
experiment, in which cells were transfected with
pcDNA3.1-p38 MAPK to enhance the level of p38 MAPK.
p38 MAPK over-expression also failed to affect
SB203580-induced autophagy (Supplemental Fig. 5).
AMPK inhibitor compound C prevented SB203580-
induced autophagy
It is well known that AMPK activation is involved in
autophagy induction [6]. We thus investigated whether
AMPK could influence autophagic process induced by
SB203580. The result demonstrated that SB203580 treat-
ment increased the levels of pAMPK, S78/80-ACCa, LC3-
II (Fig. 3d) and autophagosome (Fig. 3a), indicating the
occurrence of autophagy. However, pre-treated with com-
pound C, a cell-permeable pyrrazolopyrimidine derivative
that functions as a potent ATP-competitive inhibitor of
AMPK, significantly decreased the number of cells with
autophagosome (Fig. 3a), and suppressed the levels of
LC3-II, pAMPK and S78/80-ACCa (Fig. 3d). Furthermore,
SB203580-induced GFP-LC3-positive cells with GFP-LC3
punctate dots were also reduced by compound C (Fig. 3b,
c). We also showed that the level of pDAPK was increased
but the expression of phosphorylated p53 (pp53) was
decreased in presence of compound C (Fig. 3d).
Apoptosis (2012) 17:325–334 327
123
DAPK siRNA prevented SB203580-induced autophagy
Since DAPK plays a critical role in autophagy [9, 10] and
this protein was activated (dephosphorylated) in
SB203580-induced autophagy, we investigated the func-
tional role of DAPK in SB203580-induced autophagy
using DAPK siRNA. Our data showed that the decrease of
DAPK alleviated SB203580-induced autophagy, evident
by the changes in the cell morphology (Fig. 4a), the
decreased percentage of GFP-LC3-positive cells with GFP-
LC3 punctate dots (Fig. 4b, c), and the reduction of auto-
phagosome (Fig. 4a) and LC3-II (Fig. 4d). These results
Fig. 1 Autophagy induced by SB203580. HepG2 cells were treated
with SB203580 for 24 h. a Morphology of HepG2 cells was recorded.
Images of fluorescence microscope and phase contrast microscope
9200; images of transmission electron microscope, 95,800; image of
partial enlarged detail, 925,000. b Cells transfected with GFP-LC3
were treated with 50 lM SB203580 for 24 h. c GFP-LC3-positive
cells with GFP-LC3 dots. The data represent the mean ± SD, n = 4.aP \ 0.05, versus control group; bP \ 0.01, versus Control. d, e Cells
were treated by 50 lM SB203580 or 50 lM etoposide. Apoptosis
detected by Hoechst 33342 and propidium iodide staining, 9200.
Cells treated by 50 lM SB203580 did not show typical characteristics
of apoptosis. However, after cells were treated with 50 lM etoposide
for 24 h, they showed DNA condensation, the typical characteristic of
apoptosis. Apoptotic index was shown as bar figure. Apoptotic index
was determined by calculating the number of apoptotic nuclei versus
total number of nuclei at each visual field 9100%. f Influence of
SB203580 on the expression of cell survival or death proteins. HepG2
cells were treated with 50 lM SB203580 for 6, 12 and 24 h. After the
treatments, total protein was isolated and subjected to Western blot
328 Apoptosis (2012) 17:325–334
123
confirm that DAPK plays a positive role in SB203580-
induced HCC cell autophagy. Our data also showed that
DAPK siRNA decreased the level of S20-p53 but did not
affect the expression of S15-p53. These findings indicate
that DAPK may help to phosphate p53 at S20 but not at
S15. DAPK siRNA affected neither AMPK nor LC3-II
(Fig. 4d). It was also noted that DAPK siRNA could not
100% prevent SB203580-induced autophagy (Fig. 4b, c),
suggesting that molecules other than DAPK are also sig-
nificant in the SB203580-induced autophagy.
PFT-a and p53 siRNA inhibited SB203580-induced
autophagy
Although SB203580 did not up-regulate total p53 level in
HCC cells, the levels of S15-p53 and S20-p53 were
increased (Fig. 1f). We examined how the downregulation
of p53 affected SB203580-induced autophagy by inhibiting
p53 with chemical and siRNA methods. Our result showed
that inhibition of p53 by p53 siRNA significantly blocked
SB203580-induced autophagy in HepG2 cells (Fig. 5).
However, it did not inhibit SB203580-mediated dephos-
phorylated DAPK and pAMPK (Fig. 5d). The similar
results were obtained when PFT-a, a well-known chemical
inhibitor for p53 [26], was used (Supplemental Fig. 6).
These data suggest that p53 is involved in the autophagy
induced by SB203580 and that AMPK and DAPK are
likely to function upstream of p53 in SB203580-induced
autophagy (Fig. 6).
Discussion
In the present study, we demonstrated that SB203580
induced autophagy in HCC cells. SB203580 is traditionally
used as a p38 MAPK inhibitor, and it can function as an
anti-apoptotic or pro-survival agent to regulate cell death
[27, 28]. In our model tested, SB203580 inhibit the
Fig. 2 p38MAPK knocked-
down by siRNA unaffected
SB203580-induced autophagy.
HepG2 cells were treated with
p38 MAPK siRNA and 50 lM
SB203580 or either agent alone
for 24 h. After the treatment, the
cell morphology was observed
and total protein was isolated
for Western blot analysis.
a Morphology 9200. b Cells
transfected with GFP-LC3 were
treated with p38 MAPK siRNA
and SB203580 or either agent
alone for 24 h. c The expression
of proteins was determined by
Western blot. d GFP-LC3-
positive cells with GFP-LC3
dots. The data represent
mean ± SD, n = 4. aP \ 0.01
versus control group; bP \ 0.01
versus Control siRNA group;cP \ 0.01 versus p38 MAPK
siRNA-treatment group
Apoptosis (2012) 17:325–334 329
123
proliferation of HCC cells via inducing autophagy. The
induction of autophagy by SB203580 was confirmed by the
formation of autophagosomes, the detection of GFP-LC3
dots and the expression of LC3-II protein. The apoptosis
does not appear to be involved in SB203580-mediated anti-
proliferation since neither DNA condensation nor the
cleaved PARP was observed. SB203580-induced autoph-
agy was also found to be independent of caspase-3.
Caspase-3 is frequently found being non-related to
autophagy [29]. The involvement of caspase-3 has been
reported to constitute a switch between autophagic or
apoptotic cell death pathways [29]. Therefore, disassocia-
tion between SB203580-induced cell death and caspase-3
may indicate that the inhibition of cell proliferation in our
model is mainly caused by autophagy rather than apoptosis,
which is consistent with the morphological observations
and biochemical tests in this study.
The signaling pathway constituted by PI3K, Akt, and
mTOR plays key roles in the regulation of cell prolifera-
tion, differentiation, and survival [6, 8]. The level of Akt is
frequently up-regulated in various cancer cells to maintain
tumor cell survival and growth [4, 30]. The increase of Akt
usually results from the mutational activation of PI3 K. In
the present experiments, SB203580 treatment causes a
significant reduction of the phosphorylated Akt (pAkt),
suggesting that SB203580 may induce autophagy by sup-
pressing the activation of Akt. The involvement of the
reduced Akt in SB203580-induced autophagy is further
supported by the following observations. First, disruption
of the Akt pathway by inhibiting its activity is associated
with autophagy induced by a variety of antineoplastic
agents in cancer cells [31]. Second, Akt positively regu-
lates the mTOR signaling pathway to inhibit autophagy
[4, 5]. Finally, Akt activation promotes glucose utilization
Fig. 3 Effect of AMPK
inhibitor compound C on
SB203580-induced autophagy.
HepG2 cells were pre-incubated
with 20 lM compound C for
2 h, followed by 50 lM
SB203580 for 24 h. Total
protein was isolated for Western
blot analysis. a Morphology
9200. b Cells transfected with
GFP-LC3 were treated with
50 lM SB203580 for 24 h.
c GFP-LC3-positive cells with
GFP-LC3 dots. The data
represent mean ± SD, n = 4.aP \ 0.001 versus DMSO
control group; bP \ 0.01 versus
SB203580-treatment group.
d The expression of proteins
was determined by Western blot
330 Apoptosis (2012) 17:325–334
123
whereas disruption of Akt pathway inhibits cell glycolysis
[32, 33]. The inhibition of cell glycolysis may reduce
intracellular AMP/ATP ratio, which induces autophagy [6].
In addition to Akt, SB203580 activated AMPK by stimu-
lating its phosphorylation at T172. The role of AMPK in
SB203580-induced autophagy was verified using its
inhibitor compound C since compound C prevented AMPK
from SB203580-mediated phosphorylation and signifi-
cantly suppressed SB203580-induced autophagy. Indeed,
our experiments have shown that the inhibition of AMPK
affected several molecules that are proved to be altered by
SB203580 in this study and also known to be involved in
autophagy. For example, AMPK inhibition reduced the
levels of Beclin 1 and the pp53 (at S15 and S20) but
increased the pDAPK. It is noted that the inhibition of
DAPK by its siRNA does not change the pAMPK in our
study, suggesting that DAPK is down-stream of AMPK in
SB203580-induced HCC cell autophagy. DAPK is capable
of initiating both apoptotic and autophagic cell death,
depending on the cell type and the specific stimulus [9–11].
Our study showed that block of DAPK by its siRNA
counteracted the SB203580-mediated elevation of GFP-
LC3 punctate dots, Beclin and LC3-II, S20-p53 but not
S15-p53. These findings appear to suggest a positive role of
DAPK in autophagy in HCC cells tested, in which DAPK
was activated by SB203580 via decreasing the level of
pDAPK. Therefore, this study support an active role of
DAPK in SB203580-induced autophagy, which is in line
with the fact that DAPK activation promotes autophagy [9,
10, 34].
SB203580-mediated AMPK-dependent p53 phosphory-
lation should have a role in the induction of autophagy. The
phosphorylation of p53 at S15 is essential for p53-depen-
dent cell-cycle arrest [35]. We found that SB203580
arrested HepG2 cells at G0/G1 phase, at which autophagy
occurred. Therefore, the phosphorylation of p53 at S15
may promote autophagy by arresting cells at G0/G1 phase,
though it is unknown how the arrest contributes to
Fig. 4 The down-regulation of
DAPK prevented SB203580-
induced autophagy. HepG2 cells
were treated with DAPK siRNA
and 50 lM SB203580 or either
agent alone for 24 h. After the
treatment, the cell morphology
was observed and total protein
was isolated for Western blot
analysis. a Morphology 9200.
b Cells transfected with GFP-
LC3 were treated with DAPK
siRNA and SB203580 or either
agent alone for 24 h. c GFP-
LC3-positive cells with GFP-
LC3 dots. The data represent
mean ± SD, n = 4. aP \ 0.01
versus control group; bP \ 0.05
versus control
siRNA?SB203580-treatment
group; cP \ 0.01 versus control
siRNA-treatment group. d The
expression of proteins was
determined by Western blot
Apoptosis (2012) 17:325–334 331
123
SB203580-induced autophagy. In addition to the increase
of S15-p53, the S20-p53 was also enhanced by SB203580.
The S20-p53 can increase the transcription factor activity
of p53 [36]. p53 transcriptionally activates the damage-
regulated autophagy modulator (DRAM) gene to induce
autophagy in a DRAM-dependent manner [37]. Further-
more, the activation of p53 inhibits mTOR activity to
induce autophagy [13]. The positive role of p53 in
SB203580-induced autophagy was supported by p53 inhi-
bition experiments in which p53 was suppressed by siRNA
or PFT-a that modulates the nuclear import or export (or
both) of p53 to decrease the stability of nuclear p53,
leading to the inhibition of p53-dependent transcription
[26]. Both approaches significantly inhibited SB203580-
induced autophagy. Although p53 may inhibit autophagy in
some types of cells [38], our result supports a positive role
of p53 in SB203580-induced autophagy in HCC cells.
However, autophagy can be also induced by SB203580 in
Fig. 5 The down-regulation of
p53 by siRNA inhibited
SB203580-induced autophagy.
HepG2 cells were treated with
p53 siRNA and 50 lM
SB203580 or either agent alone
for 24 h. After the treatment, the
cell morphology was observed,
GFP-LC3-positive cells with
GFP-LC3 dots was measured,
and total protein was isolated
for Western blot analysis.
a Morphology, 9200. b Cells
transfected with GFP-LC3 were
treated with p53 siRNA and
SB203580 or either agent alone
for 24 h. c GFP-LC3-positive
cells with GFP-LC3 dots. The
data represent mean ± SD,
n = 4. aP \ 0.001 versus
control group; bP \ 0.05 versus
control siRNA?SB203580-
treatment group; cP \ 0.001
versus control siRNA -treatment
group. d The expression of
proteins was determined by
Western blot
Fig. 6 The proposed pathway for SB203580-indued autophagy in
HCC cells
332 Apoptosis (2012) 17:325–334
123
p53-mutant HCC cells (supplemental Fig. 1), suggesting
that p53 is not an absolute requirement for SB203580-
induced autophagy.
One of the most significant findings in this study is that
SB203580-induced autophagy in HCC cells is not depen-
dent on p38 MAPK though SB203580 is often used as a
p38 MAPK inhibitor. The p38 MAPK-independent
autophagy induced by SB203580 was verified by three
different experiments. First, the block of p38 MAPK by its
siRNA failed to have an impact on SB203580-induced
autophagy. Second, the suppression of p38 MAPK activity
by its chemical inhibitor BIRB0796 also did not affect
SB203580-induced autophagy. Finally, the over-expression
of p38 MAPK using a p38 MAPK expressing vector had no
effect on SB203580-induced autophagy. These findings
have convincingly demonstrated that SB203580-induced
autophagy in HCC was independent of p38 MAPK. In
addition, we have also tested the roles of JNK, ERK and
MAPK in SB203580-induced autophagy using the inhibi-
tors of these kinases. It appears that SP600125 (JNK
inhibitor), U0126 (ERK inhibitor) and PD98059 (MAPK)
did not significantly induce autophagy in HCC cells (data
no show).
It is noted that SB203580 may inhibit drug-induced
autophagy in some cases [16, 39, 40], which is inconsistent
with our present findings. The exact reason for the differ-
ence is unknown. However, this conflict may be due to the
different types of cells tested. In our experiments, HCC
cells were used but non-HCC cells were employed in the
previous studies. The different doses of SB203580 used
may also contribute to the conflicting result. In our study,
SB203580 was used at relatively high concentrations
(12.5–50 lM). However, the use of SB203580 at concen-
trations higher than 10 lM has also been reported in other
studies [16, 41, 42].
Nevertheless, our results appear to indicate that
SB203580 induces HCC cell autophagy independent of p38
MAPK and caspase-3 via multiple channels, and none of
these channels can be 100% responsible for the induction
of autophagy. For example, the suppression of AMPK by
compound C does not completely inhibit SB203580-
induced autophagy, neither does the inhibition of DAPK
nor p53. SB203580 is a well-known p38 MAPK inhibitor
to block apoptosis induced by various agents. The induc-
tion of autophagy by SB203580 may provide us with some
novel concepts when dealing with cancer cell death. For
example, in some situations, though the apoptosis has been
inhibited by SB203580, cell survival may continue to
decrease [43–45]. The autophagy induced by SB203580
should give a reasonable answer to this scientific puzzle,
thereby helping the development of more effective treat-
ments for cancers.
Conflict of interest All authors do not have any conflict of interest
to disclose.
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