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

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

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

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-

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,

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

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

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

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-

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
**
**

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-

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

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

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,

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

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

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).

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

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

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.

10 Cheng Y, Qiu F, Huang J, Tashiro S, Onodera S &
Ikejima T (2008) Apoptosis-suppressing and autophagy-
promoting effects of calpain on oridonin-induced L929
cell death. Arch Biochem Biophys 475, 148–155.
11 Laurent A, Nicco C, Che
´
reau C, Goulvestre C, Alexan-
dre J, Alves A, Le
´
vy E, Goldwasser F, Panis Y, Sou-
brane O et al. (2005) Controlling tumor growth by
modulating endogenous production of reactive oxygen
species. Cancer Res 65, 948–956.
12 Pan J, She M, Xu ZX, Sun L & Yeung SC (2005)
Farnesyltransferase inhibitors induce DNA damage via
reactive oxygen species in human cancer cells. Cancer
Res 65, 3671–3681.
13 Chandra J, Samali A & Orrenius S (2000) Triggering
and modulation of apoptosis by oxidative stress. Free
Radic Biol Med 29, 323–333.
14 Ott M, Gogvadze V, Orrenius S & Zhivotovsky B
(2007) Mitochondria, oxidative stress and cell death.
Apoptosis 12, 913–922.
15 Chen Y, McMillan-Ward E, Kong J, Israels SJ &
Gibson SB (2008) Oxidative stress induces autophagic
cell death independent of apoptosis in transformed and
cancer cells. Cell Death Differ 15, 171–182.
16 Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L &
Elazar Z (2007) Reactive oxygen species are essential
for autophagy and specifically regulate the activity of

with NF-jB and IKK function. Nat Rev Mol Cell Biol
8, 49–62.
23 Javelaud D & Besancon F (2001) NF-jB activation
results in rapid inactivation of JNK in TNFa-treated
Ewing sarcoma cells: a mechanism for the anti-apopto-
tic effect of NF-jB. Oncogene 20, 4365–4372.
24 Kurland JF, Kodym R, Story MD, Spurgers KB,
McDonnell TJ & Meyn RE (2001) NF-kappaB1 (p50)
homodimers contribute to transcription of the bcl-2
oncogene. J Biol Chem 276, 45380–45386.
25 Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T,
Kojima Y, Piao JH, Yagita H, Okumura K, Doi T
et al. (2003) NF-kappa B inhibits TNF-induced
accumulation of ROS that mediate prolonged MAPK
activation and necrotic cell death. EMBO J 22,
3898–3909.
26 Djavaheri-Mergny M, Amelotti M, Mathieu J, Besan-
con F, Bauvy C, Souque
`
re S, Pierron G & Codogno P
(2006) NF-kappa B activation represses tumor necrosis
factor-alpha-induced autophagy. J Biol Chem 281,
30373–30382.
27 Davis RJ (2000) Signal transduction by the JNK group
of MAP kinases. Cell 103, 239–252.
28 Hill CS & Treisman R (1995) Transcriptional regulation
by extracellular signals: mechanisms and specificity. Cell
80, 199–211.
29 Xia Z, Dickens M, Raingeaud J, Davis RJ &
Greenberg ME (1995) Opposing effects of ERK and

schen S, Scherer G, Ussat S, Ungefroren H &
Adam-Klages S (2004) Inhibition of p38 mitogen-acti-
vated protein kinase reduces TNF-induced activation of
NF-kappaB, elicits caspase activity, and enhances cyto-
toxicity. Exp Cell Res 293, 196–206.
37 Levine B (2007) Cell biology: autophagy and cancer.
Nature 446, 745–747.
38 Wang L, Yu CF, Lu Y, He PF, Gou JH, Zhang CY,
Song QS, Ma DL, Shi TP & Chen YY (2007) TMEM
166, a novel trans-membrane protein, regulates cell
autophagy and apoptosis. Apoptosis 12, 1489–1502.
39 Gajewska M, Gajkowska B & Motyl T (2005) Apopto-
sis and autophagy induced by TGF-B1 in bovine
mammary epithelial BME-UV1 cells. J Physiol Pharma-
col 56, 143–157.
40 Espert L, Denizot M, Grimaldi M, Robert-Hebmann V,
Gay B, Varbanov M, Codogno P & Biard-Piechaczyk
M (2006) Autophagy is involved in T cell death after
binding of HIV-1 envelope proteins to CXCR4. J Clin
Invest 116, 2161–2172.
41 Boya P, Gonza
´
lez-Polo RA, Casares N, Perfettini JL,
Dessen P, Larochette N, Me
´
tivier D, Meley D,
Souquere S, Yoshimori T et al. (2005) Inhibition of
macroautophagy triggers apoptosis. Mol Cell Biol 25,
1025–1040.
42 Liu B, Cheng Y, Zhang B, Bian HJ & Bao JK (2008)

BMB Rep 41 , 369–375.
48 Chen CY, Liu TZ, Chen CH, Wu CC, Cheng JT, Yiin
SJ, Shih MK, Wu MJ & Chern CL (2007) Isoobtusilac-
tone A-induced apoptosis in human hepatoma Hep G2
cells is mediated via increased NADPH oxidase-derived
reactive oxygen species (ROS) production and the mito-
chondria-associated apoptotic mechanisms. Food Chem
Toxicol 45, 1268–1276.
Autophagy inhibits ROS-mediated apoptosis Y. Cheng et al.
1306 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS