Tài liệu Báo cáo khoa học: Autophagy inhibits reactive oxygen species-mediated apoptosis via activating p38-nuclear factor-kappa B survival pathways in oridonin-treated murine fibrosarcoma L929 cells doc

Autophagy inhibits reactive oxygen species-mediated
apoptosis via activating p38-nuclear factor-kappa B
survival pathways in oridonin-treated murine
fibrosarcoma L929 cells
Yan Cheng
1,2
, Feng Qiu
2
, Yuan-Chao Ye
1
, Zhao-Ming Guo
1
, Shin-Ichi Tashiro
3
, Satoshi Onodera
3
and Takashi Ikejima
1
1 China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, China
2 Department of Natural Products Chemistry, Shenyang Pharmaceutical University, China
3 Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo, Japan
Keywords
apoptosis; autophagy; murine fibrosarcoma
L929 cells; oridonin; ROS
Correspondence
T. Ikejima, China-Japan Research Institute
of Medical and Pharmaceutical Sciences,
Shenyang Pharmaceutical University,
Shenyang 110016, China
Fax: +86 24 23844463
Tel: +86 24 23844463

E-mail:
(Received 4 September 2008, revised 10
December 2008, accepted 18 December
2008)
doi:10.1111/j.1742-4658.2008.06864.x
Autophagy and apoptosis have been known to be interconnected positively
or negatively; however, the molecular mechanisms mediating these two cel-
lular processes are not fully understood. In the present study, we demon-
strated that the exposure of L929 cells to oridonin led to intracellular
reactive oxygen species generation, followed by lipid peroxidation, as well
as decreases in superoxide dismutase and glutathione activities. The reac-
tive oxygen species scavenger N-acetyl-cysteine resulted in the complete
inhibition of oridonin-induced apoptosis and mitochondrial membrane
potential collapse. We showed that reactive oxygen species triggered apop-
tosis by Bax translocation, cytochrome c release and extracellular signal-
regulated kinase activation. Further data confirmed that oridonin also
induced L929 cell autophagy, as demonstrated by extensive autophagic vac-
uolization and the punctuate distribution of monodansylcadaverine staining
and GFP-LC3, as well as the LC3-II ⁄ LC3-I proportion and Beclin 1 acti-
vation. Subsequently, we found that inhibition of autophagy by 3-methy-
ladenine or small interfering RNA against LC3 and Beclin 1 promoted
oridonin-induced cell apoptosis. The effects of p38 and nuclear factor-
kappa B in oridonin-induced apoptosis and autophagy were further exam-
ined. Interruption of p38 and nuclear factor-kappa B activation by specific
inhibitors or small interfering RNAs promoted apoptosis and reactive oxy-
gen species generation, but decreased autophagy. Moreover, we showed
that inhibition of autophagy reduced oridonin-induced activation of p38.
Additionally, nuclear factor-kappa B activation was inhibited by blocking
the p38 pathway. Consequently, these findings indicate that oridonin-
induced L929 cell apoptosis is regulated by reactive oxygen species-medi-

ated signaling pathways, and that oridonin-induced autophagy may block
apoptosis by up-regulating p38 and nuclear factor-kappa B activation.
Abbreviations
3-MA, 3-methyladenine; DCF-DA, 2¢,7¢-dichlorofluorescein diacetate; ERK, extracellular signal-regulated kinase; GSH-PX, glutathione
peroxidase; I-jB, inhibitor kappa B; JNK, c-Jun N-terminal kinase; MDA, maleic dialdehyde; MDC, monodansylcadaverine; MMP,
mitochondrial membrane potential; MTT, 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acetyl-cysteine; NF-jB, nuclear
factor-kappa B; PDTC, pyrrolidine dithiocarbamate; p-ERK, phosphorylated ERK; PI, propidium iodide; p-I-jB, phosphorylated I-jB; p-p38,
phosphorylated p38; ROS, reactive oxygen species; siRNA, small interfering RNA; SOD, superoxide dismutase; T-AOC, total anti-oxidation
capability; TNF, tumor necrosis factor.
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1291
Apoptosis is a physiological cell suicide process accom-
panied by a series of complex biochemical events and
definite morphological changes [1]. Because many ther-
apeutic agents eliminate tumor cells by inducing apop-
totic cell death, the further understanding of the
apoptotic mechanisms is required for the prevention
and treatment of many diseases [2]. Reactive oxygen
species (ROS), a group of highly reactive molecules,
including singlet oxygen, hydroxyl radicals, superoxide
anion, nitric oxide and hydrogen peroxides, have been
shown to play a key role in apoptotic cell death [3].
ROS are generated from the mitochondria and other
sources, and can oxidize a wide range of cell constitu-
ents, including lipids, proteins and DNA, thus damag-
ing cell structures and compromising function [4].
When antioxidant mechanisms are overwhelmed by
ROS and subsequent oxidative stress occurs, cell
damage and cell death result [4].
As a mode of type II programmed cell death,
autophagy plays a major role in the degradation and

recycling of intracellular materials [5]. Macroauto-
phagy, the most universal form of autophagy, is the
process whereby organelles and cytosolic macromole-
cules are sequestered into double-membrane struc-
tures known as autophagosomes, which are
subsequently delivered to the lysosome for degrada-
tion [6]. During nutrient starvation or growth factor
deprivation, autophagy is a cell defense mechanism
by which intracellular nutrients are released to ensure
survival [7]. However, in certain settings, autophagy
can lead to cell death by generating a non-apoptotic
form of programmed cell death, termed autophagic
cell death [8].
The crosstalk between autophagy (i.e. a pathway
that can function primarily in cell survival) and apop-
tosis (i.e. a pathway that can invariably result in cell
death) is quite complex. Under different circum-
stances, autophagy may delay or promote the onset
of apoptosis, and apoptosis can also induce auto-
phagy [9].
A previous study reported that oridonin (an active
diterpenoid isolated from Rabdosia Rubescens)
induced L929 cell apoptosis and autophagy [10].
However, whether ROS are involved in the regulation
of apoptotic pathways and the molecular pathway of
apoptosis and autophagy in oridonin-treated L929
cells remains to be elucidated. In the present study,
we demonstrate that oridonin-induced L929 cell apop-
tosis was dependent on ROS generation and that
oridonin-induced autophagy blocked apoptosis. Fur-

thermore, the roles of p38 and nuclear factor-kappa
B (NF-jB) in oridonin-induced apoptosis and auto-
phagy were demonstrated in that they inhibited
apoptosis but promoted autophagy. Oridonin-induced
p38 mitogen-activated protein kinase activation was
shown to contribute to NF-jB activation, thereby
generating a survival, rather than death, pathway in
L929 cells.
Results
Oridonin-induced intracellular ROS accumulation
We first examined the ultrastructure of oridonin-
treated L929 cells by a transmission electron micros-
copy. As shown in Fig. 1A, the control cells displayed
a normal cell phenotype. By contrast, oridonin-treated
L929 cells showed typical apoptotic features, includ-
ing chromatin condensation and margination at the
nuclear periphery, as well as nuclear fragmentation.
To determine the involvement of ROS during orido-
nin-induced apoptosis in L929 cells, we measured the
intracellular ROS level by flow cytometry using the
fluorescent dye 2¢,7¢-dichlorofluorescein diacetate
(DCF-DA). The treatment of oridonin markedly
induced intracellular ROS generation. The ratio of
DCF positive cells was increased from 10.55% in cells
treated for 6 h to 82.36% in cells treated for 36 h.
Moreover, the increase was almost completely inhib-
ited by pretreatment with ROS scavenger N-acetyl-
cysteine (NAC) (Fig. 1B). These results indicate that
oridonin induced intracellular ROS generation in a
time-dependent manner. Furthermore, we observed

cellular morphological changes when the cells were
cultured with oridonin for 6, 12 or 24 h. As shown in
Fig. 1C, the majority of oridonin-treated L929 cells
became round in shape at 6 h. At 12 h, some of these
cells showed membrane blebbing and nuclei were
fragmented into apoptotic bodies. At 24 h, the accu-
mulation of apoptotic nuclei was more obvious. These
results demonstrate that the cellular morphological
changes are associated with the time-dependent
increase in cellular ROS.
Effects of oridonin on intracellular (superoxide
dismutase) SOD, glutathione peroxidase
(GSH-PX) and total anti-oxidation capability
(T-AOC) levels, as well as maleic dialdehyde
(MDA) content
ROS generation may result in changes in endogenous
GSH-PX and SOD levels. Therefore, we measured
cellular GSH-PX and SOD activities at different times.
As shown in Fig. 2A,B, GSH-PX and SOD levels were
significantly reduced after oridonin treatment. Consis-
tent with these results, intracellular T-AOC also
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1292 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
0 6 h 12 h 24 h 36 h 36 h
Oridonin
Ori. + NAC
g
0
20
40

60
80
100
B
C
0 h 6 h
12 h
24 h
A
Control
Oridonin Oridonin
a
b
c
d
e
f
DCF positive cells (%)
Fig. 1. Oridonin-induced ROS generation was blocked by NAC in L929 cells. The cells were incubated with medium or 50 lM oridonin for
24 h. The cellular ultrastructure was examined by using transmission electron microscopy (A-a, 0 h for oridonin; A-b,c, 24 h for oridonin).
Scale bar = 1 lm (A-a,b) and 0.5 lm (A-c). The cells were cultured with 50 l
M oridonin for 0 h (B-a), 6 h (B-b), 12 h (B-c), 24 h (B-d) or 36 h
(B-e), or co-incubated with 2 m
M NAC for 36 h (B-f). DCF, the fluorescent dye product of peroxidized DCF-DA, was measured fluorometrical-
ly at 30 min post-treatment (B-g). Values are expressed as the mean ± SD (n = 4). The cells were cultured with 50 l
M oridonin for 0, 6, 12
or 24 h, and cellular morphological changes were observed under a phase contrast microscope (C). Scale bar = 20 lm.
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1293
decreased with time (Fig. 2C). A significant time-

dependent increase in cellular MDA content was
observed after oridonin treatment (Fig. 2D). Notably,
these changes were completely inhibited by pretreat-
ment with NAC. These results indicate that intracellu-
lar ROS accumulation results in antioxidant system
imbalance and lipid peroxidation.
ROS scavenger NAC suppresses oridonin-induced
mitochondrial membrane potential (MMP)
collapse and apoptosis
Subsequently, we examined the integrity of the
mitochondrial membranes of cells by rhodamine 123
staining. As shown in Fig. 3A, compared to control
0 6 12 24 36
Time (h)
0
5
10
15
20
25
30
35
AB
C
D
GSH-PX (U·mL
–1
)
0 6 12 24 36
Time (h)

SOD(U·mL
–1
)
0
10
20
30
40
0 6 12 24 36
Time (h)
T-AOC (U·mL
–1
)
0
1
2
3
4
5
0 6 12 24 36
Time (h)
MDA (nmol·mL
–1
)
0
1
2
3
4
Fig. 2. Changes in intracellular GSH-PX,

SOD, T-AOC and MDC levels in oridonin-
induced L929 cells. The cells were cultured
with 50 l
M oridonin for 0, 6, 12, 24 or 36 h,
or co-incubated with 2 m
M NAC for 36 h.
The cellular levels of GSH-PX (A), SOD (B),
T-AOC (C) and MDC (D) were measured.
The symbol indicates the effect of NAC.
Values are expressed as the mean ± SD
(n = 3).
SubG1: 1.70% SubG1: 24.48% SubG1: 1.38%
A
B
C
Fig. 3. Oridonin-induced MMP collapse and
cell apoptosis were rescued by NAC in L929
cells. The cells were incubated with 50 l
M
oridonin for 0 or 24 h, or co-incubated with
2m
M NAC for 24 h. The cells were loaded
with rhodamine 123, and observed using
fluorescence microscopy (A). Scale
bar = 20 lm. The cellular morphological
changes were observed by phase contrast
microscopy (B). Scale bar = 20 lm. The
cells were stained with PI, and measured by
a flow cytometery after collection (C).
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.

1294 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
cells, the treatment of cells with oridonin resulted in a
decrease of fluorescence intensity due to the loss of
MMP. Oridonin induced characteristic apoptotic mor-
phological changes, such as membrane blebbing,
nuclear condensation and fragmentation (Fig. 3B). The
proportion of SubG
1
cells, a feature characteristic of
apoptosis, was also increased in oridonin-induced L929
cells (Fig. 3C). Notably, pretreatment with NAC
resulted in the complete abolition of oridonin-induced
MMP collapse and apoptosis. These results demon-
strate that ROS generation may indirectly induce
MMP loss and, eventually, apoptosis.
Oridonin-triggered Bax translocation and
cytochrome c release are suppressed by NAC
To investigate the effects of ROS on Bax translocation
and cytochrome c release, the levels of Bax and
cytochrome c in the cytosol and mitochondria were
examined by western blot analysis (Fig. 4). The
mitochondrial Bax and cytosol cytochrome c were
significantly increased after oridonin treatment. How-
ever, this augmentation was obviously blocked by
NAC employment, indicating that ROS might contrib-
ute to the translocation of Bax to the mitochondria
and subsequently cause the release of cytochrome c
into the cytosol induced by oridonin.
Oridonin-induced extracellular signal-regulated
kinase (ERK) activation is inhibited by NAC

To explore the contribution of ERK activation to
oridonin-induced cell death, L929 cells were pretreat-
ed with ERK inhibitor PD 98059. As shown in
Fig. 5A, compared to the oridonin alone-treated
group, PD 98059 pretreatment significantly decreased
oridonin-induced cytotoxity. To validate this
experiment, L929 cells were transfected with ERK
small interfering RNA (siRNA), which inhibited the
expression of ERK and oridonin-induced cell death
(Fig. 5B). To further confirm whether ERK MAPK
was activated in oridonin-treated cells, ERK and
phosphorylated ERK (p-ERK) protein levels were
determined by western blot analysis. The level of
ERK was not obviously changed and the p-ERK
level was markedly elevated after oridonin administra-
tion (Fig. 5C). Addition of NAC remarkably inhibited
the phosphorylation of ERK. Next, the effect of p38
on oridonin-induced cell death was examined.
Pretreatment of cells with p38 inhibitor SB 203580 or
transfection with p38 siRNA significantly increased
oridonin-induced cytotoxity (Fig. 5A,B). These
results show that p38 was a negative regulator of
apoptosis, whereas ERK contributed to apoptosis.
Furthermore, ROS might be involved in the
activation of ERK pathway in oridonin-induced L929
cells.
Inhibition of NF-jB and p38 activations increases
oridonin-induced apoptosis and ROS
accumulation
The transcription factor NF-jB mainly mediates cell

survival signaling pathway. In the present study, we
found that inhibition of NF-jB activation by using
NF-jB inhibitor pyrrolidine dithiocarbamate (PDTC)
or specific siRNA, which caused a reduction in
NF-jBp65 levels (Fig. 6B), significantly increased the
oridonin-induced SubG
1
cell proportion (Fig. 6A,C).
These results indicate that NF-jB suppresses oridonin-
induced apoptosis in L929 cells. We therefore aimed to
determine whether NF-jB was involved in oridonin-
induced ROS production. Accordingly, we treated
L929 cells with PDTC or p65 siRNA prior to the addi-
tion of oridonin, and the intracellular ROS level was
measured. As shown in Fig. 6D,E, the percentage of
DCF-positive cells was increased after being treated
with PDTC or transfection with p65 siRNA. The
effects of p38 on oridonin-induced apoptosis and ROS
generation were also examined. Consistent with the
above results, the inhibition of p38 activation, by using
SB 203580 or specific siRNA, sensitized L929 cells to
oridonin-induced apoptosis and ROS generation.
These results demonstrate that NF-jB and p38
blocked oridonin-induced apoptosis and ROS
generation.
Fig. 4. Effects of NAC on oridonin-induced Bax translocation and
cytochrome c release. The cells were treated with 50 l
M oridonin
in the presence or absence of 2 m
M NAC for the indicated time

periods, followed by western blot analysis for detection of Bax and
cytochrome c levels, both in the cytosol and the mitochondria.
b-Actin was used as an equal loading control.
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1295
Oridonin-induced L929 cell autophagy
Next, we investigated the effect of oridonin on cell
autophagy. We first examined the ultrastructure of ori-
donin-induced L929 cells using transmission electron
microscopy. As shown in Fig. 7A, control cells
displayed normal cell morphology. By contrast, orido-
nin-induced L929 cells showed extensive cytoplasm vac-
uolization, and some autophagic vacuoles contained
degraded organelles, such as mitochondria. The forma-
tion of autophagic vacuoles was further assessed by
monodansylcadaverine (MDC) staining and GFP-LC3
distribution. As shown in Fig. 7B,C, control cells pre-
sented diffused staining, and oridonin treatment
resulted in extensive punctuate MDC staining pattern
and GFP-LC3 localization. The MDC fluorescent
intensity of oridonin-treated cells for the indicated time
periods was analyzed by FACScan flow cytometry.
Oridonin induced L929 cell autophagy in a time-depen-
dent manner and the autophagic ratio was increased
from 10.42% at 12 h to 50.36% at 36 h (Fig. 8A).
Next, the levels of Beclin 1 and LC3, two important
proteins involved in autophagy, were examined by wes-
tern blot analysis in L929 cells treated with oridonin.
As shown in Fig. 8B, the level of Beclin 1 and conver-
sion from LC3-I to LC3-II both increased with time

after oridonin administration. These results indicate
that oridonin induced autophagy in L929 cells.
Inhibition of autophagy up-regulates apoptosis in
oridonin-induced L929 cells
To investigate the role of autophagy in oridonin-
induced apoptosis in L929 cells, we pretreated cells
with 3-methyladenine (3-MA), a specific inhibitor of
autophagy, to inhibit the autophagy. As shown in
Fig. 9A, 3-MA completely blocked oridonin-induced
L929 cell autophagy. Apoptosis was evaluated by the
measurement of cell number in SubG
1
region. As
shown in Fig. 9B, the inhibition of autophagy
increased the oridonin-induced SubG
1
cell proportion
in L929 cells. Furthermore, we knocked down the
expressions of Beclin 1 and LC3 by using specific
siRNAs. As shown in Fig. 9C, these treatments
reduced both oridonin-induced Beclin 1 and LC3 lev-
els. In addition, transfection with Beclin 1 or LC3 siR-
NA also increased oridonin-induced cell apoptosis
(Fig. 9D). These findings demonstrate that the inhibi-
tion of autophagy increased oridonin-induced apop-
tosis in L929 cells.
0
10
20
30

40
50
60
70
80
90
A
C
B
Inhibitory ratio (%)
Inhibitory ratio (%)
+
–
–
–
+
–
+
+
–
–
–
+
+
–
+
**
**
Con p38 siRNA Control siRNA
p38

Con ERK siRNA Control siRNA
Oridonin
SB 203580
PD 98059
ERK
0 6 12 24 24
(h)
+ NAC
Oridonin
p-ERK
Oridonin
Control siRNA ERK siRNA p38 siRNA
**
**
β-actin
ERK
β-actin
β-actin
0
10
20
30
40
50
60
70
80
Fig. 5. Effects of p38 and ERK on oridonin-induced L929 cell death. L929 cell were pretreated with 10 lM SB 203580 or 10 lM PD 98059
for 1 h prior to the addition of 50 l
M oridonin and then incubated for 24 h. The inhibitory ratio was determined by MTT assay (n = 3) (A). Val-

ues are expressed as the mean ± SD. **P < 0.01 versus the group treated with oridonin alone. The cells were transfected with p38, ERK
or control siRNA for 24 h, and the p38 or ERK level was examined by western blot analysis (upper panel). The cells were transfected with
p38, ERK or control siRNA for 24 h, followed by stimulation with oridonin for 24 h, and the inhibitory ratio was determined by MTT assay
(n = 3) (lower panel) (B). Values are expressed as the mean ± SD. **P < 0.01. The cells were treated with 50 l
M oridonin in the presence
or absence of 2 m
M NAC for the indicated time periods, followed by western blot analysis for detection of ERK and p-ERK levels. b-Actin
was used as an equal loading control (C).
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1296 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
g
A
C
B
**
**
Con Ori. Ori. + PDTC PDTC Ori. + SB SB
Oridonin
Control siRNA p65 siRNA p38 siRNA
Con p65 siRNA control siRNA
p65
β-actin
Oridonin
Control siRNA p65 siRNA p38 siRNA
D
E
0
10
20
30

40
50
60
0
10
20
30
40
50
60
0
20
40
60
80
100
Con
Ori. + PDTC
Ori.
PDTC
Ori. + SB
SB
0
20
40
60
80
100
**
**

**
**
**
**
a
b
c
d
e
f
DCF positive cells (%)
DCF positive cells (%)
Percentage of sub-G1 cells (%)
Percentage of sub-G1 cells (%)
Fig. 6. Effects of NF-jB and p38 on oridonin-induced L929 cells apoptosis and OS generation. The cells were incubated in the presence of
absence of 20 l
M PDTC or 10 lM SB 203580 for 1 h prior to the addition of 50 lM oridonin and then incubated for 24 h. The proportion of
SubG
1
cells was measured by flow cytometry using PI staining (n = 3) (A). The cells were transfected with NF-jBp65 or control siRNA for
24 h, and the p65 level was examined by western blot analysis (B). The cells were transfected with p38, p65 or control siRNA for 24 h, fol-
lowed by stimulation with oridonin for 24 h, and the proportion of SubG1 cells was measured by flow cytometry using PI staining (n =3)
(C). DCF, the fluorescent dye product of peroxidized DCF-DA, was measured fluorometrically at 30 min post-treatment (D-a, 0 h for oridonin;
D-b, 24 h for oridonin; D-c, 24 h for oridonin + PDTC; D-d, 24 h PDTC; D-e, 24 h for oridonin + SB 203580; D-f, 24 h SB 203580). Values are
expressed as the mean ± SD (n = 3). **P < 0.01. The cells were transfected with p38, p65 or control siRNA for 24 h, followed by stimula-
tion with oridonin for 24 h, and ROS generation was measured by flow cytometry using DCF-DA staining (n = 3) (E).
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1297
Inhibition of p38 decreases oridonin-induced
autophagy

To investigate whether p38 plays a role in oridonin-
induced L929 cell autophagy, we first examined the
autophagic ratio in the cells treated with SB 203580 or
p38 siRNA to inhibit p38 activation. As shown in
Fig. 10A,B, compared to the oridonin treatment
group, SB 203580 or p38 siRNA treatment caused
a significant decrease in the MDC-positive cells.
Moreover, oridonin-induced Beclin 1 activation was
inhibited by p38 siRNA. Next, phosphorylated p38 (p-
p38) and p38 levels were examined by western blot
analysis (Fig. 10B). The increase in the p-p38 level was
observed in oridonin-treated cells, whereas this
increase was notably inhibited by pretreatment with
3-MA (Fig. 10C). Furthermore, pretreatment with
Beclin 1 siRNA or LC3 siRNA reduced the oridonin-
induced p-p38 level (Fig. 10D). Taken together, these
findings show that p38 contributed to oridonin-
induced autophagy and oridonin-induced autophagy
up-regulated p38 activity.
NF-jB promotes oridonin-induced autophagy
and its activation is decreased by the inhibition
of p38
To study the role of NF-jB in oridonin-induced L929
cell autophagy, the autophagic ratio was evaluated by
pretreatment cells with NF-jB inhibitor PDTC or
NF-jBp65 siRNA. As shown in Fig. 11A,B, PDTC or
p65 siRNA significantly reduced oridonin-induced
autophagy, and oridonin-induced Beclin 1 activation
was inhibited by p65 siRNA, indicating that NF-jB
promoted oridonin-induced L929 cell autophagy. Ori-

donin treatment led to a decreased inhibitor kappa B
(I-jB) level, with the increase of phosphorylated I-jB
(p-I-jB) and NF-jB levels being time-dependently
indicative of NF-jB activation. Interestingly, we found
that inhibition of p38 activation by using SB 203580
or p38 siRNA resulted in the reduced induction of
NF-jB activation (Fig. 11C,D). These results demon-
strate that NF-jB promoted oridonin-induced auto-
phagy and that oridonin-induced p38 might contribute
to NF-jB activation.
Discussion
Intracellular ROS generation plays an important role
in numerous physiological and pathological pro-
cesses, and a high level of ROS is intimately associ-
ated with apoptotic cell death [11,12]. In the present
study, we found a rapid and persistent increase in
intracellular ROS generation after oridonin exposure.
NAC pretreatment resulted in the complete inhibition
of oridonin-induced apoptosis, indicating that orido-
nin-induced apoptosis may be modulated indirectly
by the ROS-mediated signaling pathways. Under
physiological conditions, ROS generation is rapidly
eliminated by antioxidant enzymes such as SOD and
b
a
c
d
M
Oridonin
Control

GFP
GFP-LC3
Oridonin
Control
A
B
C
Fig. 7. Oridonin-induced L929 cell autophagy. The cells were incu-
bated with medium or 50 l
M oridonin for 24 h. The cellular ultra-
structure was examined by using transmission electron microscopy
(A-a, 0 h for oridonin; A-b,c,d, 24 h for oridonin). Scale bar = 1 lm
(A-a,b) and 0.5 lm (A-c,d). The arrow indicates that the autophagic
L929 cell displayed extensive cytoplasmic vacuolization, and some
autophagic vacuoles contained degraded organelles. M, mitochon-
dria. GFP or GFP-LC3 transfected cells were treated with and with-
out 50 l
M oridonin for 24 h, and then examined under a
fluorescence microscope (B). Scale bar = 20 lm. The cells were
incubated with medium or 50 l
M oridonin for 24 h. The cellular
morphological changes were observed under a fluorescence micro-
scope by MDC staining (C).
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1298 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
GSH-PX. Of note, when the antioxidant balance is
disrupted, the condition known as oxidative stress
occurs [13]. The prime damage by ROS generation
leads to lipid peroxidation, generating the lipid per-
oxide, such as MDA [14]. In the present study, we

demonstrated that SOD, GSH-PX and T-AOC acti-
vities were decreased in a time-dependent manner.
Nevertheless, MDA content was increased with time
in oridonin-induced L929 cells, indicating that
oridonin-induced apoptosis was associated with oxi-
dative stress. Besides apoptosis, oxidative stress has
been shown to induce autophagy under certain con-
ditions. H
2
O
2
and 2-methoxyestradiol treatment
induced autophagy, contributing to cell death in the
transformed cell line HEK293 and the cancer cell
lines U87 and HeLa [15]. On the other hand,
Scherz-Shouval et al. [16] reported that ROS was
involved in starvation-induced autophagy in the form
of signaling molecules in a survival pathway. In the
present study, we demonstrate that ROS generation
resulted in mitochondrial dysfunction and that auto-
phagy may be induced to remove the damaged
organelles. This is agreement with the findings of a
study conducted by Marin
˜
o and Lo
´
pez-Otı
´
n [17]
showing that many cellular stresses can cause the

induction of autophagy, such as endoplasmic reticu-
lum stress or mitochondrial dysfunction.
Many studies have demonstrated that mitochondria
work as the central executioner in apoptotic signaling
pathways. Various pro-apoptosis stimuli converge on
the mitochondria, leading to mitochondrial depolariza-
tion and cytochrome c release, which is a critical event
resulting in cell death [18]. The pro-apoptotic protein
Bax plays a vital role in the regulation of the mito-
chondrial apoptotic pathway. In particular, Bax trans-
location from the cytosol into the mitochondria was
reported to promote cytochrome c release from the
mitochondria [19]. In the present study, we show that
oridonin was able to induce MMP loss and cyto-
chrome c release, indicating that mitochondrial dys-
function occurred during oridonin-induced L929 cell
apoptosis. Moreover, Bax translocation from the cyto-
sol to the mitochondria was also observed after
oridonin treatment. These results indicate that mito-
chondrial translocation of Bax may constitute a direct
cause of cytochrome c release. ROS has also been
demonstrated to induce the depolarization of the mito-
chondrial membrane, and might function upstream of
the mitochondria [20]. In the present study, we also
demonstrate that pretreatment with the ROS scavenger
NAC completely inhibited oridonin-induced MMP col-
lapse. Moreover, Bax translocation and cytochrome c
release were also inhibited by NAC. Taken together,
these results indicate that oridonin-induced ROS gen-
eration might indirectly induce Bax translocation to

the mitochondria and subsequently alter membrane
permeability, allowing mitochondrial cytochrome c
release into the cytosol.
The transcription factor NF-jB pivotally controls
the inflammatory and immune response, as well as
other genetic programs that are central to cell prolifer-
B
A
Fig. 8. Oridonin-induced L929 cell autophagy. The cells were trea-
ted with 50 l
M oridonin for 0, 12, 24 or 36 h. The MDC fluorescent
intensity of oridonin-treated cells was analyzed by flow cytometery
(A). Values are expressed as the mean ± SD (n = 3). The cells
were treated with 50 l
M oridonin for the indicated time periods, fol-
lowed by western blot analysis for detection of Beclin-1 and LC3
levels. b-Actin was used as an equal loading control (B).
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1299
ation, survival and decreasing the sensitivity of cancer
cells to apoptosis [21]. Typically, in most unstimulated
cells, NF-jB is sequestered in the cytoplasm by bind-
ing to the inhibitor of NF-jB (I-jB). In response to a
variety of stimuli, activation of NF-jB typically
involves the phosphorylation of I-jB, resulting in I jB
degradation and NF-jB release [22]. In the present
study, we demonstrate that the inhibition of NF-jBby
PDTC or siRNA increased the oridonin-induced
SubG
1

cell proportion. Additionally, oridonin induced
a decrease of I-jB levels but an increase of p-I-jB and
NF-jB levels. These results indicate that oridonin
activated the NF-jB pathway, which was a negative
regular of apoptosis. However, the survival signaling
elicited by NF-jB remains to be discovered. Some
studies that focused on identifying the anti-apoptotic
mechanism of NF-jB have demonstrated that the acti-
vation of NF-jB impaired c-Jun N-terminal kinase
(JNK) activation [23], or resulted in an increase of
Bcl-2 family protein levels [24]. In the present study,
we found that NF-jB significantly inhibited oridonin-
induced ROS production, which was essential for cell
apoptosis. Similarly, NF-jB activation has been
reported to suppress the ROS accumulation in tumor
necrosis factor (TNF)-induced murine embryonic fibro-
blasts [25]. Recently, a role for NF-jB in the autopha-
gic signaling pathway has been reported in that NF-jB
activation mediates the repression of autophagy, which
is a cell death mechanism in TNF-treated Ewing
sarcoma cells [26]. In the present study, we show that
inhibition of NF-jB decreased oridonin-induced auto-
phagy, which inhibited apoptosis. Our findings, when
taken together with these results, support the idea that
the anti-apoptotic function of NF-jB activation might
consist of the promotion or repression of autophagy,
depending on whether autophagy is a survival or death
process.
In addition to the aforementioned signaling path-
ways, mitogen-activated protein kinase pathways,

which include ERK1 ⁄ 2, JNK and p38, are involved
in various biological responses, such as differentia-
tion, proliferation and cell death [27]. The functional
roles of the activation of these kinases are often con-
troversially discussed. The ERK1 ⁄ 2 cascade is
regarded to be the main method of transmission for
cell proliferative and survival signaling pathways [28],
whereas the JNK or p38 pathway mediates stress sig-
nals and apoptosis [29]. By contrast, in the present
SubG1: 1.70% SubG1: 24.48% SubG1: 36.81%
A
B
C
D
Fig. 9. The relationship between apoptosis
and autophagy. The cells were incubated
with 50 l
M oridonin for 0 or 24 h, or
co-incubated with 3-MA for 24 h. The cellu-
lar morphological changes were observed
under a fluorescence microscope by MDC
staining (A). Scale bar = 20 lm. The cells
were stained with PI at 37 °C for 30 min,
and measured by flow cytometery after col-
lection (B). The cells were transfected with
Beclin 1, LC3 or control siRNA for 24 h, and
the Beclin 1 or LC3 levels were examined
by western blot analysis (C). The cells were
transfected with Beclin 1, LC3 or control
siRNA for 24 h, followed by stimulation with

oridonin for 24 h. The cells were stained
with PI, and measured by flow cytometery
after collection (D). Values are expressed as
the mean ± SD (n = 3). **P < 0.01.
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1300 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
study, we report that inhibition of ERK activation
decreased oridonin-induced L929 cell apoptosis, indi-
cating that ERK contributes to cell death under this
situation. The positive regulation of apoptotic activity
by ERK in H
2
O
2
-mediated L929 cell apoptosis has
also been reported by Yoon-Jin Lee et al. [30], who
showed that oxidative damage-induced apoptosis is
mediated by ERK1 ⁄ 2 phosphorylation. In the present
study, we also found that pretreatment with ROS
scavenger NAC inhibited oridonin-induced p-ERK
activation. The present results, together with these
previous studies, indicate that the pro-apoptotic role
of ERK activation might be regulated by ROS gener-
ation. Previously, the role of p38 MAPK has mostly
been considered to cause cell apoptosis. For example,
the inhibition of p38 activity enhanced cell viability
and prevented apoptosis induced by cadmium in a
human nonsmall lung carcinoma cell line (CL3), sug-
gesting that persistently-activated p38 participates in
apoptosis [31]. Another study demonstrated that H

2
O
2
induced apoptosis through a p38-dependent mitochon-
drial pathway in HeLa cells [32]. In the present study,
we show that inhibition of p38 MAPK activity mark-
edly enhanced oridonin-induced cell apoptosis and
ROS generation, indicating that the p38 MAPK path-
way has a protective function against oridonin-
induced cell death in L929 cells. This conclusion is
supported by the fact that inhibition of p38 MAPK
increased cell death after stimulation with TNF in the
human myelomonocytic cell line U937 and in primary
murine splenic macrophages [33]. The role of p38 in
the modulation of autophagy activity has been dem-
onstrated in recent studies. For example, it was shown
that pharmacological blockade of the p38 pathway in
colorectal cancer cells causes cell cycle arrest and
autophagic cell death [34]. Furthermore, the oridonin-
induced autophagic process was positively regulated
by p38 in human cervical carcinoma HeLa cells [35].
In the present study, we demonstrate that the inhibi-
tion of p38 activation blocked oridonin-mediated
stimulation of autophagy in L929 cells and that orido-
nin-induced p-p38 activation was inhibited by 3-MA,
A
C
D
B
Fig. 10. Effect of p38 on oridonin-induced

autophagy. The cells were incubated with
50 l
M oridonin for 0 or 24 h, or co-incubated
with SB 203580 for 24 h. The MDC fluores-
cent intensity of oridonin-treated cells was
analysed by FACScan (A). Values are
expressed as the mean ± SD (n = 4).
**P < 0.01. The cells were transfected with
p38 or control siRNA for 24 h, followed by
stimulation with oridonin for 24 h. The MDC
fluorescent intensity of oridonin-treated cells
was analyzed by flow cytometery, and the
Beclin 1 level was examined by western
blot analysis. b-Actin was used as an equal
loading control (B). Values are expressed as
the mean ± SD (n = 3). **P < 0.01. The
cells were treated with 50 l
M oridonin in
the presence or absence of 2 m
M 3-MA for
the indicated time periods, followed by wes-
tern blot analysis for p-p38 and p38 levels.
b-Actin was used as an equal loading con-
trol. The cells were transfected with
Beclin 1, LC3 or control siRNA for 24 h, and
the p-p38 level was examined by western
blot analysis. b-Actin was used as an equal
loading control (C,D).
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1301

Beclin 1 siRNA or LC3 siRNA. These results suggest
that p38 promoted autophagy and that autophagy
up-regulated p38 activation in oridonin-induced L929
cells. Interestingly, we found that inhibition of p38
decreased oridonin-induced NF-jB activation, indicat-
ing that oridonin-induced p38 activation contributes
to NF-jB activation, generating a survival signaling
pathway. A recent study demonstrated that NF-jB-
dependent gene expression is reduced by p38 inhibi-
tion in TNF-induced L929 and NIH3T3 cells [36].
Apoptosis and autophagy have long been classified
as different forms of programmed cell death. How-
ever, apoptosis invariably contributes to cell death
and autophagy commonly is associated with cell sur-
vival [37]. Several studies have demonstrated that
both apoptosis and autophagy can occur concomi-
tantly in the same cells under some circumstances
[38,39]. Accumulating evidences suggests that complex
interrelationships exist between the autophagic and
the apoptotic cell pathway. Under certain stress con-
ditions, autophagy is a cell death pathway in which
activation can lead to apoptosis [40]. Conversely, it
has also been suggested that autophagy activation
may function to prevent apoptosis [41]. Thus, depend-
ing on the cellular context, autophagy may have pro-
apoptotic or anti-apoptotic functions. Nevertheless,
the molecular mechanism that controls the crosstalk
between apoptosis and autophagy remains to be eluci-
dated. The results of the present study suggest that
the L929 cells induced by oridonin exhibit both auto-

phagic and apoptotic characteristics. Inhibition of
autophagy increased apoptotic cell death, suggesting
that autophagy has an anti-apoptotic function.
Importantly, we simultaneously investigated the roles
of p38 and NF-jB in oridonin-induced apoptosis and
autophagy, and found that they both inhibited apop-
tosis but promoted autophagy. We demonstrate that
p38 contributed to NF-jB activation, which inhibited
ROS generation. Therefore, we have provided possi-
ble molecular mechanisms for the crosstalk between
AB
C
D
Fig. 11. Effect of NF-jB on oridonin-induced
autophagy. The cells were incubated with
50 l
M oridonin for 0 or 24 h, or co-incubated
with 10 l
M PDTC for 24 h. The MDC fluo-
rescent intensity of oridonin-treated cells
was analyzed by flow cytometery (A).
Values are expressed as the mean ± SD
(n = 4). **P < 0.01. The cells were trans-
fected with NF-jB or control siRNA for
24 h, followed by stimulation with oridonin
for 24 h. The MDC fluorescent intensity of
oridonin-treated cells was analyzed by flow
cytometery, and the Beclin 1 level was
examined by western blot analysis. b-Actin
was used as an equal loading control (B).

Values are expressed as the mean ± SD
(n = 4). **P < 0.01. The cells were treated
with 50 l
M oridonin in the presence or
absence of 10 l
M SB 203580 for the indi-
cated time periods, followed by western
blot analysis for p-I-jB, I-jB and NF-jB
levels (C). The cells were transfected with
p38 or control siRNA for 24 h, and the
p-I-jB, I-jB and NF-jB levels were
examined by western blot analysis. b-Actin
was used as an equal loading control (D).
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1302 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS
apoptosis and autophagy and suggest that autophagy
may protect cells from apoptosis by activating p38-
NF-jB survival signaling pathways. Thus, it would be
important to identify more biochemical switches
between apoptosis and autophagy in future studies.
In summary, the results obtained in the present
study indicate that ROS generation induced by orido-
nin contributes to the activation of mitochondrial and
ERK signaling pathways in oridonin-induced L929 cell
apoptosis. Moreover, oridonin-induced autophagy can
block apoptosis by up-regulating the p38-NF-jB sur-
vival signal pathway. These inspiring findings provide
new evidence for the further understanding of more
significant molecular mechanisms between apoptosis
and autophagy.

Experimental procedures
Reagents
Oridonin was obtained from the Kunming Institute of
Botany, Chinese Academy of Sciences (Kunming, China);
and its purity was determined to be 99.4% by HPLC
measurement. SOD, GSH-PX, T-AOC and MDA kits were
purchased from Institute of Jiancheng Biological Engineering
(Nanjing, China). Fetal bovine serum was purchased from
TBD Biotechnology Development (Tianjin, China); 3-(4,5-
dimetrylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT), 3,3-diaminobenzidine tetrahydrochloride, MDC,
3-MA, NAC, DCF-DA, rhodamine 123, propidium iodide
(PI), RNase A, p38 inhibitor SB 203580, ERK1 ⁄ 2 inhibitor
PD98059 and NF-jB inhibitor PDTC were purchased from
Sigma Chemical (St Louis, MO, USA). Rabbit polyclonal
antibodies against Bax, Beclin 1, LC3, p38, p-p38, ERK,
I-jB, p-I-jB, NF-jB and b-actin; mouse polyclonal
antibodies against cytochrome c and p-ERK; and horseradish
peroxidase-conjugated secondary antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture
Murine fibrosarcoma L929 cells (#CRL-2148) were pur-
chased from American Type Culture Collection (ATCC,
Manassas, VA, USA). The cells were cultured in RPMI-
1640 medium supplemented with 10% fetal bovine serum,
100 lgÆmL
)1
streptomycin, 100 UÆmL
)1
penicillin and

0.03% l-glutamine and maintained at 37 °C with 5% CO
2
in a humidified atmosphere. All the experiments were
performed on logarithmically growing cells.
Measurement of intracellular ROS generation
Generation of intracellular ROS was examined by DCF-
DA, which is a relatively specific probe for intracellular
ROS formation. After treatment with 50 lm oridonin for
the indicated time periods, the cells were incubated with
10 lm DCF-DA for 30 min at 37 °C. Next, the cells were
harvested and the pellets were suspended in 1 mL of
NaCl ⁄ P
i
. The samples were analyzed at an excitation wave-
length of 480 nm and an emission wavelength of 525 nm by
FACScan flow cytometry (Becton Dickinson, Franklin
Lakes, NJ, USA) [42].
Measurement of MMP
Alterations in the MMP were investigated with the mito-
chondrial dye rhodamine 123 [43]. After incubation with
oridonin for the indicated time periods, the cells were
stained with 1 lgÆmL
)1
rhodamine 123 for 30 min at 37 °C.
The fluorescence intensity of cells was observed under a
fluorescence microscope (Olympus, Tokyo, Japan).
Measurement of SubG
1
cells
Cell cycle and SubG

1
distribution were determined by stain-
ing DNA with PI [44]. L929 cells were treated with 50 lm
oridonin for the indicated time periods. The collected cells
were fixed with 500 lL of NaCl ⁄ P
i
and 10 mL of 70% eth-
anol at 4 °C overnight; then, after washing twice with
NaCl ⁄ P
i
, the cells were incubated with 1 mL of PI staining
solution (50 mgÆL
)1
of PI and 1 gÆL
)1
of RNase A) at 4 °C
for 30 min. The percentage of cells at different phases of
the cell cycle or having Sub-G
1
DNA content was measured
by FACScan flow cytometry.
Transmission electron microscopy
L929 cells were treated with 50 lm oridonin for the indi-
cated time periods. The collected cells were fixed with
NaCl ⁄ P
i
containing 3% glutaraldehyde, postfixed with
NaCl ⁄ P
i
containing 1% OsO

4
. The samples were dehy-
drated in graded alcohol, embedded and sectioned. Ultra-
thin sections were stained with uranyl acetate and lead
citrate, and examined using a JEM-1200 transmission
electron microscope (JEOL, Tokyo, Japan) [45].
Measurement of autophagy
After incubation with 50 lm oridonin for the fixed times,
cells were cultured with 0.05 mm MDC at 37 °C for
60 min. The cellular fluorescent changes were observed
under a fluorescence microscope (Olympus). The fluores-
cence intensity of cells was analyzed by FACScan flow
cytometry [46].
Cells were transfected with GFP-LC3 plasmid (kindly
provided by Y. Chen, Peking University Center for Human
Disease Genomics) using the Lipofectamine 2000 reagent
(Invitrogen, Carlsbad, CA, USA) according to the manu-
Y. Cheng et al. Autophagy inhibits ROS-mediated apoptosis
FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1303
facturer’s instructions. The fluorescence of GFP-LC3 was
observed under a fluorescence microscope.
Western blot analysis
L929 cells were treated with 50 lm oridonin for 0, 6, 12
and 24 h, or co-incubated with the given inhibitors for
24 h. Equivalent amounts of total protein were separated
by SDS ⁄ PAGE and transferred to nitrocellulose membrane.
Immunoblot analyses were performed as described previ-
ously [47].
Preparation of mitochondrial and cytosolic
extracts

L929 cells were collected and then washed twice with ice-
cold NaCl ⁄ P
i
. The cell pellets were resuspended in ice-cold
HMKEE buffer (250 mm sucrose, 20 mm Hepes, 10 mm
KCl, 1.5 mm MgCl
2
,1mm EDTA, 1 mm EGTA, 1 mm
dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride,
10 lgÆmL
)1
pepstatin and 10 lgÆmL
)1
leupeptin). The cells
were homogenized and centrifuged at 14 000 g at 4 °C for
60 min. The supernatant was used as the cytosol fraction
and the pellet was resuspended in lysis buffer as the mem-
brane fraction [48].
siRNA transfection
siRNAs against mouse ERK, NF-jBp65, p38, Beclin 1,
LC3 and control siRNA were purchased from Invitrogen.
Cells were transfected with siRNAs at a final concentration
of 33 nm using Lipofectamine 2000 (Invitrogen) according
to the manufacturer’s instructions. The transfected cells
were used for subsequent experiments 24 h later.
Statistical analysis
All the presented data were confirmed in at least three inde-
pendent experiments and are expressed as the mean ± SD.
Statistical comparisons were made by Student’s t-test.
P < 0.05 was considered statistically significant.

Acknowledgements
We are grateful to Dr Bo Liu (Sichuan University) for
his critical review of the manuscript.
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