JNK protects Drosophila from oxidative stress by trancriptionally activating autophagy

JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, we investigated the role of autophagy as such a JNK-regulated protective mechanism. We show that oxidative stress can induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms.     

Erythropoietin alleviates hepatic steatosis by activating SIRT1-mediated autophagy

Erythropoietin (EPO), besides its stimulatory effect on erythropoiesis, is beneficial to insulin resistance and obesity. However, its role in hepatic steatosis remains unexplored. Activating autophagy seems a promising mechanism for improving fatty liver disease. The present study investigated the role of EPO in alleviating hepatic steatosis and sought to determine whether its function is mediated by the activation of autophagy. Here, we show that EPO decreased hepatic lipid content significantly in vivo and in vitro. Furthermore, EPO/EPO receptor (EPOR) signalling induced autophagy activation in hepatocytes as indicated by western blot assay, transmission electron microscopy, and confocal microscopy. In addition, EPO increased the co-localization of autophagosomes and cellular lipids as shown by double labelling of the autophagy marker light chain microtubule-associated protein 3 (LC3) and lipids. Importantly, suppression of autophagy by an inhibitor or small interfering RNA (siRNA) abolished the EPO-mediated alleviation hepatic steatosis in vitro. Furthermore, EPO up-regulated sirtuin 1 (SIRT1) expression, and siRNA-mediated SIRT1 silencing abrogated the EPO-induced increases in LC3 protein and deacetylation levels, thereby preventing the alleviation of hepatic steatosis. Taken together, this study revealed a new mechanism wherein EPO alleviates hepatic steatosis by activating autophagy via SIRT1-dependent deacetylation of LC3. This finding might have therapeutic value in the treatment of hepatic steatosis.     

Ginsenoside F2 induces apoptosis in humor gastric carcinoma cells through reactive oxygen species-mitochondria pathway and modulation of ASK-1/JNK signaling cascade in vitro and in vivo

Ginsenoside F₂ (F₂) is a potential bioactive metabolite of major ginsenosides. The potential anti-cancer effect of F₂ in gastric cancer cells has not been appraised. This study investigated the effects of F₂ on the production of reactive oxygen species (ROS). We also investigated the in vitro and in vivo effects of F₂ on the downstream signaling pathways leading to apoptosis in human gastric cancer cells. The in vitro data revealed that F₂ induces ROS accumulation followed by a decrease in mitochondrial transmembrane potential (MTP), and the release of cytochrome c (cyto c), which induced the caspase-dependent apoptosis. Further assay indicated that modulation of ASK-1/JNK pathway contributes to apoptosis. In vivo, F₂ exhibits the obvious anti-cancer effect compared with cisplatin with no obvious toxicity. Jointly, these results suggest that F₂ induces apoptosis by causing an accumulation of ROS and activating the ASK-1/JNK signaling pathway. This provides further support for the use of F₂ as a novel anticancer therapeutic candidate.     

mIGF-1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD(+) -dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.     

DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.     

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

Rhopaloic acid A induces apoptosis,autophagy and MAPK activation through ROS-mediated signaling in bladder cancer

BackgroundBladder cancer (BC) is a very common type of malignant cancer in men and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that Rhopaloic acid A (RA), a compound isolated from marine sponges, fights cancer but its potential anti-tumor effect on BC is still unknown.PurposeThe present study was aimed to explore the potential anti-tumor effects of RA against human BC cells and the underlying molecular mechanism.MethodsCell cytotoxicity was determined using the MTT and colony formation assays. Cell cycle distribution, apoptosis induction and generation of mitochondrial reactive oxygen species (ROS) were analyzed by flow cytometry. Mitochondrial membrane potential, acridine orange staining and intracellular ROS levels were observed using fluorescence microscopy. Levels of various signaling proteins were assessed using Western blotting. Furthermore, a zebrafish BC xenotransplantation model was used to confirm the anti-tumor effect of RA in vivo.ResultsTreatment with RA significantly suppressed the proliferation of BC cells that resulted from G2/M cycle arrest. Additionally, RA induced mitochondrial-mediated apoptosis and autophagy in BC cells. The death of BC cells induced by RA was rescued by treatment with inhibitors of apoptosis (Z-VAD-FMA) or autophagy (3-MA). RA activated the MAPK pathway and increased the production of cellular and mitochondrial ROS. Treatment with the ROS scavenger N-acetyl cysteine, effectively reversed the induction of apoptosis, autophagy, JNK activation and DNA damage elicited by RA. Finally, RA significantly inhibited tumor growth in a zebrafish BC xenotransplantation model.ConclusionTaken together, our findings indicate that RA induces apoptosis and autophagy and activates the MAPK pathway through ROS-mediated signaling in human BC cells. This RA-induced pathway offers insights into the molecular mechanism of its antitumor effect and shows that RA is a promising candidate for the treatment of BC.     

Resveratrol protects cardiomyocytes from oxidative stress through SIRT1 and mitochondrial biogenesis signaling pathways

Reactive oxygen species (ROS) is generated by oxidative stress and plays an important role in various cardiac pathologies. The SIRT1 signaling pathway and mitochondrial biogenesis play essential roles in mediating the production of ROS. SIRT1 activated by resveratrol protects cardiomyocytes from oxidative stress, but the exact mechanisms by which SIRT1 prevents oxidative stress, and its relationship with mitochondrial biogenesis, remain unclear. In this study, it was observed that after stimulation with 50 μM H₂O₂ for 6 h, H9C2 cells produced excessive ROS and downregulated SIRT1. The mitochondrial protein NDUFA13 was also downregulated by ROS mediated by SIRT1. Resveratrol induced the expression of SIRT1 and mitochondrial genes NDUFA1, NDUFA2, NDUFA13 and Mn-SOD. However, the production of these genes was reversed by SIRT1 inhibitor nicotinamide. These results suggest that resveratrol inhibits ROS generation in cardiomyocytes via SIRT1 and mitochondrial biogenesis signaling pathways.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.     

Notoginsenoside R1 attenuates oxidative stress-induced osteoblast dysfunction through JNK signalling pathway

Oxidative stress (OS)-induced mitochondrial damage and the subsequent osteoblast dysfunction contributes to the initiation and progression of osteoporosis. Notoginsenoside R1 (NGR1), isolated from Panax notoginseng, has potent antioxidant effects and has been widely used in traditional Chinese medicine. This study aimed to investigate the protective property and mechanism of NGR1 on oxidative-damaged osteoblast. Osteoblastic MC3T3-E1 cells were pretreated with NGR1 24 h before hydrogen peroxide administration simulating OS attack. Cell viability, apoptosis rate, osteogenic activity and markers of mitochondrial function were examined. The role of C-Jun N-terminal kinase (JNK) signalling pathway on oxidative injured osteoblast and mitochondrial function was also detected. Our data indicate that NGR1 (25 μM) could reduce apoptosis as well as restore osteoblast viability and osteogenic differentiation. NGR1 also reduced OS-induced mitochondrial ROS and restored mitochondrial membrane potential, adenosine triphosphate production and mitochondrial DNA copy number. NGR1 could block JNK pathway and antagonize the destructive effects of OS. JNK inhibitor (SP600125) mimicked the protective effects of NGR1while JNK agonist (Anisomycin) abolished it. These data indicated that NGR1 could significantly attenuate OS-induced mitochondrial damage and restore osteogenic differentiation of osteoblast via suppressing JNK signalling pathway activation, thus becoming a promising agent in treating osteoporosis.     

EV71 infection induces cell apoptosis through ROS generation and SIRT1 activation

Several studies have substantiated the correlation between reactive oxygen species (ROS) and Sirtuin 1 (SIRT1). Normally, enterovirus 71 (EV71) is associated with severe clinical manifestations and death. However, the effect of EV71 on the induction of cellular death and the interplay between ROS/SIRT1 in cell death has not been confirmed yet. In the current study, an increase in the number of apoptotic cells was observed as soon as the EV71 infection was initiated in cells and mice. Furthermore, EV71 infection also promoted a rise in the levels of three commonly known proinflammatory cytokines, interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-α. During EV71-induced apoptosis in the different cell lines, ROS generation and SIRT1 downregulation were observed. Further investigations showed that the administration of ROS inhibitor, N-acetyl- l -cysteine (NAC), reduced the level of apoptosis and inflammation, reduced EV71 propagation, and increased SIRT1 expression in EV71-infected cells. In addition, combined administration of NAC and EX527 (SIRT1 inhibitor) restored apoptosis in the EV71-infected cells, which was reduced due to NAC. This data demonstrated that ROS generation is positively associated with EV71-induced apoptosis and inflammation, while this effect could be reversed by SIRT1 inhibition. Collectively, we have shown that EV71 induces apoptosis and inflammation by promoting ROS generation and reducing SIRT1 expression.     

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.     

Cigarette smoke exposure induces ROS-mediated autophagy by regulating sestrin,AMPK, and mTOR level in mice

Many pathological conditions linked to cigarette smoking are caused by the production of reactive oxygen species (ROS). The present study was conducted to analyze the effect of ROS on the lungs of Swiss mice exposed to cigarette smoking, focusing on autophagy-mediated mechanisms, and investigate the involvement of SESN2, AMPK, and mTOR signaling. Mice were exposed to cigarette smoke (CS) for 7, 15, 30, 45, and 60 days; the control group was not exposed to CS. Only mice exposed to CS for 45 days were selected for subsequent N-acetylcysteine (NAC) supplementation and smoke cessation analyses. Exposure to CS increased the production of ROS and induced molecular changes in the autophagy pathway, including an increase in phosphorylated AMPK and ULK1, reduction in phosphorylated mTOR, and increases in SESN2, ATG12, and LC3B levels. NAC supplementation reduced ROS levels and reversed all molecular changes observed upon CS treatment, suggesting the involvement of oxidative stress in inducing autophagy upon CS exposure. When exposure to CS was stopped, there were decreases in the levels of oxidative stress, AMPK and ULK1 phosphorylation, and autophagy-initiating molecules and increase in mTOR phosphorylation. In conclusion, these results suggest the involvement of ROS, SESN2, AMPK, and mTOR in the CS-induced autophagic process in the lung.     

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer.