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: gchen@cuhk.edu.hk

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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