Autophagy inhibits reactive oxygen species-mediated apoptosis 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 China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, China Department of Natural Products Chemistry, Shenyang Pharmaceutical University, China 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 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 cellular processes are not fully understood In the present study, we demonstrated 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 reactive 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 apoptosis by Bax translocation, cytochrome c release and extracellular signalregulated kinase activation Further data confirmed that oridonin also induced L929 cell autophagy, as demonstrated by extensive autophagic vacuolization and the punctuate distribution of monodansylcadaverine staining and GFP-LC3, as well as the LC3-II ⁄ LC3-I proportion and Beclin activation Subsequently, we found that inhibition of autophagy by 3-methyladenine or small interfering RNA against LC3 and Beclin promoted oridonin-induced cell apoptosis The effects of p38 and nuclear factorkappa B in oridonin-induced apoptosis and autophagy were further examined Interruption of p38 and nuclear factor-kappa B activation by specific inhibitors or small interfering RNAs promoted apoptosis and reactive oxygen 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 oridonininduced L929 cell apoptosis is regulated by reactive oxygen species-mediated 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 Autophagy inhibits ROS-mediated apoptosis Y Cheng et al Apoptosis is a physiological cell suicide process accompanied by a series of complex biochemical events and definite morphological changes [1] Because many therapeutic agents eliminate tumor cells by inducing apoptotic 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 constituents, including lipids, proteins and DNA, thus damaging 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] Macroautophagy, the most universal form of autophagy, is the process whereby organelles and cytosolic macromolecules are sequestered into double-membrane structures known as autophagosomes, which are subsequently delivered to the lysosome for degradation [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 apoptosis (i.e a pathway that can invariably result in cell death) is quite complex Under different circumstances, autophagy may delay or promote the onset of apoptosis, and apoptosis can also induce autophagy [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 apoptosis was dependent on ROS generation and that oridonin-induced autophagy blocked apoptosis Furthermore, the roles of p38 and nuclear factor-kappa B (NF-jB) in oridonin-induced apoptosis and autophagy were demonstrated in that they inhibited 1292 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 oridonintreated L929 cells by a transmission electron microscopy As shown in Fig 1A, the control cells displayed a normal cell phenotype By contrast, oridonin-treated L929 cells showed typical apoptotic features, including chromatin condensation and margination at the nuclear periphery, as well as nuclear fragmentation To determine the involvement of ROS during oridonin-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 h to 82.36% in cells treated for 36 h Moreover, the increase was almost completely inhibited by pretreatment with ROS scavenger N-acetylcysteine (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 h At 12 h, some of these cells showed membrane blebbing and nuclei were fragmented into apoptotic bodies At 24 h, the accumulation 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 Consistent with these results, intracellular T-AOC also FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al Autophagy inhibits ROS-mediated apoptosis A Control B Oridonin Oridonin b c d e f g DCF positive cells (%) a 100 80 60 40 20 0 6h 12 h 24 h Oridonin C 0h 36 h Ori + NAC 6h 12 h 36 h 24 h Fig 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, h for oridonin; A-b,c, 24 h for oridonin) Scale bar = lm (A-a,b) and 0.5 lm (A-c) The cells were cultured with 50 lM oridonin for h (B-a), h (B-b), 12 h (B-c), 24 h (B-d) or 36 h (B-e), or co-incubated with mM NAC for 36 h (B-f) DCF, the fluorescent dye product of peroxidized DCF-DA, was measured fluorometrically at 30 post-treatment (B-g) Values are expressed as the mean ± SD (n = 4) The cells were cultured with 50 lM oridonin for 0, 6, 12 or 24 h, and cellular morphological changes were observed under a phase contrast microscope (C) Scale bar = 20 lm FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1293 12 24 Time (h) 40 30 C D 0 12 24 Time (h) 36 20 10 36 MDA (nmol·mL–1) 30 25 20 15 10 B T-AOC (U·mL–1) GSH-PX (U·mL–1) A 35 Y Cheng et al SOD(U·mL–1) Autophagy inhibits ROS-mediated apoptosis 12 24 Time (h) 36 0 12 24 Time (h) 36 Fig Changes in intracellular GSH-PX, SOD, T-AOC and MDC levels in oridonininduced L929 cells The cells were cultured with 50 lM oridonin for 0, 6, 12, 24 or 36 h, or co-incubated with mM 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 SubG1: 1.70% SubG1: 24.48% SubG1: 1.38% decreased with time (Fig 2C) A significant timedependent increase in cellular MDA content was observed after oridonin treatment (Fig 2D) Notably, these changes were completely inhibited by pretreatment with NAC These results indicate that intracellular ROS accumulation results in antioxidant system imbalance and lipid peroxidation 1294 Fig Oridonin-induced MMP collapse and cell apoptosis were rescued by NAC in L929 cells The cells were incubated with 50 lM oridonin for or 24 h, or co-incubated with mM 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) 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 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al cells, the treatment of cells with oridonin resulted in a decrease of fluorescence intensity due to the loss of MMP Oridonin induced characteristic apoptotic morphological 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 demonstrate 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 However, this augmentation was obviously blocked by NAC employment, indicating that ROS might contribute 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- Fig Effects of NAC on oridonin-induced Bax translocation and cytochrome c release The cells were treated with 50 lM oridonin in the presence or absence of mM 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 Autophagy inhibits ROS-mediated apoptosis 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 administration (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 SubG1 cell proportion (Fig 6A,C) These results indicate that NF-jB suppresses oridonininduced apoptosis in L929 cells We therefore aimed to determine whether NF-jB was involved in oridonininduced ROS production Accordingly, we treated L929 cells with PDTC or p65 siRNA prior to the addition 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 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1295 Autophagy inhibits ROS-mediated apoptosis A 90 Y Cheng et al B ** Con Inhibitory ratio (%) 80 p38 siRNA Control siRNA p38 70 β-actin 60 50 ** 40 Con 30 ERK siRNA Control siRNA ERK 20 10 β-actin – + – + + – C – – + + – + Oridonin + NAC 12 24 24 (h) ERK Inhibitory ratio (%) Oridonin + SB 203580 – PD 98059 – 80 70 60 50 40 30 20 10 ** ** Oridonin p-ERK Control siRNA ERK siRNA p38 siRNA β-actin Fig 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 h prior to the addition of 50 lM oridonin and then incubated for 24 h The inhibitory ratio was determined by MTT assay (n = 3) (A) Values 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 lM oridonin in the presence or absence of mM 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) Oridonin-induced L929 cell autophagy Next, we investigated the effect of oridonin on cell autophagy We first examined the ultrastructure of oridonin-induced L929 cells using transmission electron microscopy As shown in Fig 7A, control cells displayed normal cell morphology By contrast, oridonin-induced L929 cells showed extensive cytoplasm vacuolization, and some autophagic vacuoles contained degraded organelles, such as mitochondria The formation of autophagic vacuoles was further assessed by monodansylcadaverine (MDC) staining and GFP-LC3 distribution As shown in Fig 7B,C, control cells presented 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-dependent 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 and LC3, two important proteins involved in autophagy, were examined by western blot analysis in L929 cells treated with oridonin As shown in Fig 8B, the level of Beclin and conver1296 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 oridonininduced 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 and LC3 by using specific siRNAs As shown in Fig 9C, these treatments reduced both oridonin-induced Beclin and LC3 levels In addition, transfection with Beclin or LC3 siRNA also increased oridonin-induced cell apoptosis (Fig 9D) These findings demonstrate that the inhibition of autophagy increased oridonin-induced apoptosis in L929 cells FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al Autophagy inhibits ROS-mediated apoptosis Percentage of sub-G1 cells (%) A 60 ** B 50 Con ** 40 p65 siRNA control siRNA p65 30 β-actin 20 10 D a d e Ori Ori + PDTC PDTC Ori + SB SB b c Con f Percentage of sub-G1 cells (%) C 60 ** 50 ** 40 30 20 10 Oridonin Control siRNA p65 siRNA p38 siRNA E 80 ** ** g DCF positive cells (%) 60 40 20 Oridonin 100 ** 80 ** 60 40 20 SB SB + Or i TC PD TC PD p38 siRNA i + p65 siRNA Or Control siRNA Co n Or i DCF positive cells (%) 100 Fig 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 lM PDTC or 10 lM SB 203580 for h prior to the addition of 50 lM oridonin and then incubated for 24 h The proportion of SubG1 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, followed 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 post-treatment (D-a, 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 stimulation with oridonin for 24 h, and ROS generation was measured by flow cytometry using DCF-DA staining (n = 3) (E) FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1297 Autophagy inhibits ROS-mediated apoptosis A a 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 activation was inhibited by p38 siRNA Next, phosphorylated p38 (pp38) 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 siRNA or LC3 siRNA reduced the oridonininduced p-p38 level (Fig 10D) Taken together, these findings show that p38 contributed to oridonininduced autophagy and oridonin-induced autophagy up-regulated p38 activity b c Y Cheng et al d M B Control NF-jB promotes oridonin-induced autophagy and its activation is decreased by the inhibition of p38 Oridonin GFP GFP-LC3 C Control Oridonin Fig Oridonin-induced L929 cell autophagy 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, h for oridonin; A-b,c,d, 24 h for oridonin) Scale bar = 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, mitochondria GFP or GFP-LC3 transfected cells were treated with and without 50 lM oridonin for 24 h, and then examined under a fluorescence microscope (B) Scale bar = 20 lm The cells were incubated with medium or 50 lM oridonin for 24 h The cellular morphological changes were observed under a fluorescence microscope by MDC staining (C) Inhibition of p38 decreases oridonin-induced autophagy To investigate whether p38 plays a role in oridonininduced 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 1298 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 activation was inhibited by p65 siRNA, indicating that NF-jB promoted oridonin-induced L929 cell autophagy Oridonin 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 demonstrate that NF-jB promoted oridonin-induced autophagy and that oridonin-induced p38 might contribute to NF-jB activation Discussion Intracellular ROS generation plays an important role in numerous physiological and pathological processes, and a high level of ROS is intimately associated 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 oridonin-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 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al A B Fig Oridonin-induced L929 cell autophagy The cells were treated with 50 lM 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 lM oridonin for the indicated time periods, followed by western blot analysis for detection of Beclin-1 and LC3 levels b-Actin was used as an equal loading control (B) 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 peroxide, such as MDA [14] In the present study, we demonstrated that SOD, GSH-PX and T-AOC activities were decreased in a time-dependent manner Nevertheless, MDA content was increased with time Autophagy inhibits ROS-mediated apoptosis in oridonin-induced L929 cells, indicating that oridonin-induced apoptosis was associated with oxidative stress Besides apoptosis, oxidative stress has been shown to induce autophagy under certain conditions 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 autophagy may be induced to remove the damaged organelles This is agreement with the findings of a ´ study conducted by Marino and Lopez-Otı´ n [17] ˜ showing that many cellular stresses can cause the induction of autophagy, such as endoplasmic reticulum 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 depolarization 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 mitochondrial apoptotic pathway In particular, Bax translocation 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 cytochrome c release, indicating that mitochondrial dysfunction occurred during oridonin-induced L929 cell apoptosis Moreover, Bax translocation from the cytosol to the mitochondria was also observed after oridonin treatment These results indicate that mitochondrial translocation of Bax may constitute a direct cause of cytochrome c release ROS has also been demonstrated to induce the depolarization of the mitochondrial 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 collapse Moreover, Bax translocation and cytochrome c release were also inhibited by NAC Taken together, these results indicate that oridonin-induced ROS generation 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- FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1299 Autophagy inhibits ROS-mediated apoptosis Y Cheng et al A B SubG1: 1.70% SubG1: 24.48% SubG1: 36.81% Fig The relationship between apoptosis and autophagy The cells were incubated with 50 lM oridonin for or 24 h, or co-incubated with 3-MA for 24 h The cellular 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 collection (B) The cells were transfected with Beclin 1, LC3 or control siRNA for 24 h, and the Beclin 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 D C 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 binding 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 activation 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 oridonininduced ROS production, which was essential for cell apoptosis Similarly, NF-jB activation has been reported to suppress the ROS accumulation in tumor 1300 necrosis factor (TNF)-induced murine embryonic fibroblasts [25] Recently, a role for NF-jB in the autophagic 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 autophagy, 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 pathways, mitogen-activated protein kinase pathways, which include ERK1 ⁄ 2, JNK and p38, are involved in various biological responses, such as differentiation, proliferation and cell death [27] The functional roles of the activation of these kinases are often controversially discussed The ERK1 ⁄ 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 signals and apoptosis [29] By contrast, in the present FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al Autophagy inhibits ROS-mediated apoptosis A Fig 10 Effect of p38 on oridonin-induced autophagy The cells were incubated with 50 lM oridonin for or 24 h, or co-incubated with SB 203580 for 24 h The MDC fluorescent 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 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 lM oridonin in the presence or absence of mM 3-MA for the indicated time periods, followed by western blot analysis for p-p38 and p38 levels b-Actin was used as an equal loading control 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) B D C study, we report that inhibition of ERK activation decreased oridonin-induced L929 cell apoptosis, indicating that ERK contributes to cell death under this situation The positive regulation of apoptotic activity by ERK in H2O2-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 ⁄ 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 generation 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), suggesting that persistently-activated p38 participates in apoptosis [31] Another study demonstrated that H2O2 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 markedly enhanced oridonin-induced cell apoptosis and ROS generation, indicating that the p38 MAPK pathway has a protective function against oridonininduced 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 demonstrated 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 oridonininduced autophagic process was positively regulated by p38 in human cervical carcinoma HeLa cells [35] In the present study, we demonstrate that the inhibition of p38 activation blocked oridonin-mediated stimulation of autophagy in L929 cells and that oridonin-induced p-p38 activation was inhibited by 3-MA, FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1301 Autophagy inhibits ROS-mediated apoptosis A C Y Cheng et al B Fig 11 Effect of NF-jB on oridonin-induced autophagy The cells were incubated with 50 lM oridonin for or 24 h, or co-incubated with 10 lM PDTC for 24 h The MDC fluorescent 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 transfected 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 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 lM oridonin in the presence or absence of 10 lM SB 203580 for the indicated 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) D Beclin 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, indicating that oridonin-induced p38 activation contributes to NF-jB activation, generating a survival signaling pathway A recent study demonstrated that NF-jBdependent gene expression is reduced by p38 inhibition in TNF-induced L929 and NIH3T3 cells [36] Apoptosis and autophagy have long been classified as different forms of programmed cell death However, apoptosis invariably contributes to cell death and autophagy commonly is associated with cell survival [37] Several studies have demonstrated that both apoptosis and autophagy can occur concomitantly 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 con1302 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, depending on the cellular context, autophagy may have proapoptotic or anti-apoptotic functions Nevertheless, the molecular mechanism that controls the crosstalk between apoptosis and autophagy remains to be elucidated The results of the present study suggest that the L929 cells induced by oridonin exhibit both autophagic 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 apoptosis but promoted autophagy We demonstrate that p38 contributed to NF-jB activation, which inhibited ROS generation Therefore, we have provided possible molecular mechanisms for the crosstalk between FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS Y Cheng et al apoptosis and autophagy and suggest that autophagy may protect cells from apoptosis by activating p38NF-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 oridonin 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 survival signal pathway These inspiring findings provide new evidence for the further understanding of more significant molecular mechanisms between apoptosis and autophagy Autophagy inhibits ROS-mediated apoptosis ROS formation After treatment with 50 lm oridonin for the indicated time periods, the cells were incubated with 10 lm DCF-DA for 30 at 37 °C Next, the cells were harvested and the pellets were suspended in mL of NaCl ⁄ Pi The samples were analyzed at an excitation wavelength 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 mitochondrial dye rhodamine 123 [43] After incubation with oridonin for the indicated time periods, the cells were stained with lgỈmL)1 rhodamine 123 for 30 at 37 °C The fluorescence intensity of cells was observed under a fluorescence microscope (Olympus, Tokyo, Japan) 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-(4,5dimetrylthiazol-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 ⁄ 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 purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 lgỈmL)1 streptomycin, 100 mL)1 penicillin and 0.03% l-glutamine and maintained at 37 °C with 5% CO2 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 DCFDA, which is a relatively specific probe for intracellular Measurement of SubG1 cells Cell cycle and SubG1 distribution were determined by staining 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 ⁄ Pi and 10 mL of 70% ethanol at °C overnight; then, after washing twice with NaCl ⁄ Pi, the cells were incubated with mL of PI staining solution (50 mgỈL)1 of PI and gỈL)1 of RNase A) at °C for 30 The percentage of cells at different phases of the cell cycle or having Sub-G1 DNA content was measured by FACScan flow cytometry Transmission electron microscopy L929 cells were treated with 50 lm oridonin for the indicated time periods The collected cells were fixed with NaCl ⁄ Pi containing 3% glutaraldehyde, postfixed with NaCl ⁄ Pi containing 1% OsO4 The samples were dehydrated in graded alcohol, embedded and sectioned Ultrathin 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 The cellular fluorescent changes were observed under a fluorescence microscope (Olympus) The fluorescence 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- FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS 1303 Autophagy inhibits ROS-mediated apoptosis Y Cheng et al 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 previously [47] Preparation of mitochondrial and cytosolic extracts L929 cells were collected and then washed twice with icecold NaCl ⁄ Pi The cell pellets were resuspended in ice-cold HMKEE buffer (250 mm sucrose, 20 mm Hepes, 10 mm KCl, 1.5 mm MgCl2, mm EDTA, mm EGTA, mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride, 10 lgỈmL)1 pepstatin and 10 lgỈmL)1 leupeptin) The cells were homogenized and centrifuged at 14 000 g at °C for 60 The supernatant was used as the cytosol fraction and the pellet was resuspended in lysis buffer as the membrane 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 independent experiments and are expressed as the mean ± SD Statistical 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mechanisms Food Chem Toxicol 45, 1268–1276 FEBS Journal 276 (2009) 1291–1306 ª 2009 The Authors Journal compilation ª 2009 FEBS ... 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 oridonininduced apoptosis. .. with Beclin or LC3 siRNA also increased oridonin-induced cell apoptosis (Fig 9D) These findings demonstrate that the inhibition of autophagy increased oridonin-induced apoptosis in L929 cells FEBS... was increased with time Autophagy inhibits ROS-mediated apoptosis in oridonin-induced L929 cells, indicating that oridonin-induced apoptosis was associated with oxidative stress Besides apoptosis,