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 m
Trang 1apoptosis via activating p38-nuclear factor-kappa B
survival pathways in oridonin-treated murine
fibrosarcoma L929 cells
Yan Cheng1,2, Feng Qiu2, Yuan-Chao Ye1, Zhao-Ming Guo1, Shin-Ichi Tashiro3, Satoshi Onodera3 and Takashi Ikejima1
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: ikejimat@vip.sina.com
(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 oxy-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.
Trang 2Apoptosis 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 compromisdamag-ing 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 autoMacroauto-phagy, 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 starvadegrada-tion 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
Trang 30 6 h 12 h 24 h 36 h 36 h
Oridonin Ori + NAC
g
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Control Oridonin Oridonin
Fig 1 Oridonin-induced ROS generation was blocked by NAC in L929 cells The cells were incubated with medium or 50 l M 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.
Trang 4decreased 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
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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).
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
2 m 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).
Trang 5cells, 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 SubG1 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) Addiadministra-tion 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 SubG1 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.
Trang 6Oridonin-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 SubG1 region As shown in Fig 9B, the inhibition of autophagy increased the oridonin-induced SubG1 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
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Oridonin
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+ NAC Oridonin
Control siRNA ERK siRNA p38 siRNA
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β-actin
ERK β-actin
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Fig 5 Effects of p38 and ERK on oridonin-induced L929 cell death L929 cell were pretreated with 10 l M SB 203580 or 10 l M 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).
Trang 7A
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
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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 l M SB 203580 for 1 h prior to the addition of 50 l M 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 generastimula-tion was measured by flow cytometry using DCF-DA staining (n = 3) (E).
Trang 8Inhibition 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-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
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).
Trang 9GSH-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, oxioxi-dative stress has been shown to induce autophagy under certain con-ditions H2O2 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).
Trang 10ation, 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 IjB
degradation and NF-jB release [22] In the present
study, we demonstrate that the inhibition of NF-jB by
PDTC or siRNA increased the oridonin-induced
SubG1 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
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.