Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells

The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-x(L) ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.     

Nitric oxide activated by p38 and NF-kappaB facilitates apoptosis and cell cycle arrest under oxidative stress in evodiamine-treated human melanoma A375-S2 cells

Nitric oxide (NO) has been identified as a fundamental molecule that interplays with reactive oxygen species (ROS) in determining cell fate. As a previous study indicated that ROS was stimulated in evodiamine-induced human melanoma A375-S2 cell apoptosis, the goal of this study was to investigate the role of NO in the cells. In this study, it was found that evodiamine has a strong inductive effect on NO production synthesized by inducible NOS (iNOS) enzyme in a positive-feedback manner. The generated NO was further showed to induce apoptosis and cell cycle arrest and linked to the activation of p53 and p21. After interruption of p38 and nuclear factor-κB (NF-κB) by pre-treatment with SB203580 and PDTC, iNOS expression, NO synthesis and cell damage were all significantly blocked. It was concluded that p38 and NF-κB were critical to the NO producing system, which contributed greatly to the apoptosis and cell cycle arrest in evodiamine-incubated cells.     

Protein tyrosine kinase pathway-derived ROS/NO productions contribute to G2/M cell cycle arrest in evodiamine-treated human cervix carcinoma HeLa cells

Abstract

A previous study indicated that reactive oxygen species (ROS) and nitric oxide (NO) played pivotal roles in mediating cytotoxicity of evodiamine in human cervix carcinoma HeLa cells. This study suggested that G2/M cell cycle arrest was triggered by ROS/NO productions with regulations of p53, p21, cell division cycle 25C (Cdc25C), Cdc2 and cyclin B1, which were able to be prevented by protein tyrosine kinase (PTK) activity inhibitor genistein or JNK inhibitor SP600125. The decreased JNK phosphorylation by addition of Ras or Raf inhibitor, as well as the increased cell viability by addition of insulin-like growth factor-1 receptor (IGF-1R), Ras, Raf or c-Jun N-terminal kinase (JNK) inhibitor, further demonstrated that the Ras-Raf-JNK pathway was responsible for this PTK-mediated signalling. These observations provide a distinct look at PTK pathway for its suppressive effect on G2/M transition by inductions of ROS/NO generations.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

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.     

Constitutive Reactive Oxygen Species Generation from Autophagosome/Lysosome in Neuronal Oxidative Toxicity

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H₂TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Abstract

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC₅₀ of approximately 80–100 and 40–60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.     

Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent

Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G₁ population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents.     

Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay

Background

Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoK_ATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoK_ATP.

Methods and Findings

In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoK_ATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.

Conclusions

Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoK_ATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.     

维生素C联合替莫唑胺对胶质瘤细胞的毒性作用及其机制*

目的:探讨维生素C(VC)联合替莫唑胺(TMZ)对胶质瘤细胞活力的毒性作用及其机制。方法:在体外条件下培养人胶质瘤细胞BMG-1和SHG44细胞,设对照组(不施加VC与TMZ)、TMZ组(0.2 mmol/L)、VC(0.5 mmol/L)+TMZ(0.2 mmol/L)组,TMZ(0.2 mmol/L TMZ)+U0126(10 μmol/L)组,每组实验重复3次。采用MTT实验检测细胞生存率;流式细胞术和Annexin V-FITC/PI染色检测细胞凋亡情况; ROS检测试剂盒检测活性氧簇(ROS)水平, Western blot检测与凋亡、自噬及ERK通路相关蛋白的表达。结果:与对照组比较,TMZ组胶质瘤细胞的存活率显著下降(P＜0.05)。与TMZ组比较,VC+TMZ组胶质细胞瘤细胞的存活率显著下降(P＜0.01),VC+TMZ组中细胞凋亡率显著升高,且Bax、Cleaved caspase-3及Cleaved PARP蛋白表达显著增加,Bcl-2表达显著降低,而ROS水平及细胞自噬率显著降低,LC3-II/LC3-1表达显著降低,p62表达显著增加(P均＜0.05)。同时,联用可降低BMG-1和SHG44细胞中的p-ERK1/2相关蛋白的表达水平,且提高细胞凋亡率(P均＜0.05)。结论:VC联合TMZ能够增强对胶质瘤细胞的毒性,而这一作用是通过ERK信号通路来促进细胞凋亡并抑制替莫唑胺所介导的自噬作用。     

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl₂ to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl₂ induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl₂-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl₂-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.     

Sonodynamic therapy induces the interplay between apoptosis and autophagy in K562 cells through ROS

Sonodynamic therapy (SDT) is a relatively new approach in the treatment of various cancers including leukemia cells. The aim of this study is to investigate the occurrence of apoptosis and autophagy after treated by protoporphyrin IX (PpIX)-mediated SDT (PpIX-SDT) on human leukemia K562 cells as well as the relationship between them. Firstly, mitochondrial-dependent apoptosis was observed through morphological observation and biochemical analysis. Meanwhile, SDT was shown to induce autophagy in K562 cells, which caused an increase in EGFP-LC3 puncta cells, a conversion of LC3 II/I, formation of acidic vesicular organelles (AVOs) and co-localization between LC3 and LAMP2 (a lysosome marker). Besides, pretreatment with autophagy inhibitor 3-MA or bafilomycin A1 was shown to provide protection against autophagy and to enhance SDT-induced apoptosis and necrosis, while the apoptosis suppressor z-VAD-fmk failed to affect formation of autophagic vacuoles or partially prevented SDT-induced cytotoxicity, which suggested that SDT-induced autophagy functioned as a survival mechanism. Additionally, this study reported apparent apoptosis and autophagy with dependence on intracellular reactive oxygen species (ROS) production. Preliminary data showed that ROS scavenger N-acetylcysteine (NAC) effectively blocked the SDT induced accumulation of ROS, reversed sono-damage, cell apoptosis and autophagy. Taken together, these data indicate that autophagy may be cytoprotective in our experimental system, and the ROS caused by PpIX-SDT treatment may play an important role in initiating apoptosis and autophagy.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

Nitric oxide (•NO) generation but not ROS plays a major role in silibinin-induced autophagic and apoptotic death in human epidermoid carcinoma A431 cells

Abstract

Silibinin, a major active constituent of silymarin, is clinically used as a hepatoprotectant, and in recent years, it has been used for the treatment of cancer in China. Because the mechanism of silibinin action on cancer cells was still unclear, we investigated the contribution of silibinin to the induction of apoptosis and autophagy via generation of reactive oxygen species (ROS) and nitric oxide (?NO) in human epidermoid carcinoma A431 cells. Silibinin inhibited the cell growth in a dose‐and time-dependent manner. Obvious autophagy was observed after treatment with different doses of silibinin. At a high dose (400 μM), silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. Loss of mitochondrial membrane potential by silibinin led to mitochondrial dysfunction and decreased ROS levels, suggesting that silibinin might act as an antioxidant in this process. Furthermore, silibinin induced ?NO generation in a time‐and dose-dependent manner. The ?NO scavenger PTIO could effectively clear ?NO and exerted a minor cell protection effect through partial inhibition of silibinin-induced apoptosis and autophagy.     

The role of nitric oxide signaling in renoprotective effects of hydrogen sulfide against chronic kidney disease in rats: Involvement of oxidative stress,autophagy and apoptosis

The interplay between H₂S and nitric oxide (NO) is thought to contribute to renal functions. The current study was designed to assess the role of NO in mediating the renoprotective effects of hydrogen sulfide in the 5/6 nephrectomy (5/6 Nx) animal model. Forty rats were randomly assigned to 5 experimental groups: (a) Sham; (b) 5/6 Nx; (c) 5/6Nx+sodium hydrosulfide-a donor of H ₂S, (5/6Nx+sodium hydrosulfide [NaHS]); (d) 5/6Nx+NaHS+ L -NAME (a nonspecific nitric oxide synthase [NOS] inhibitor); (e) 5/6Nx+NaHS+aminoguanidine (a selective inhibitor of inducible NOS [iNOS]). Twelve weeks after 5/6 Nx, we assessed the expressions of iNOS and endothelial NOS (eNOS), oxidative/antioxidant status, renal fibrosis, urine N-acetyl-b-glucosaminidase (NAG) activity as the markers of kidney injury and various markers of apoptosis, inflammation, remodeling, and autophagy. NaHS treatment protected the animals against chronic kidney injury as depicted by improved oxidative/antioxidant status, reduced apoptosis, and autophagy and attenuated messenger RNA (mRNA) expression of genes associated with inflammation, remodeling, and NAG activity. Eight weeks Nω-nitro-l-arginine methyl ester ( L -NAME) administration reduced the protective effects of hydrogen sulfide. In contrast, aminoguanidine augmented the beneficial effects of hydrogen sulfide. Our finding revealed some fascinating interactions between NO and H ₂S in the kidney. Moreover, the study suggests that NO, in an isoform-dependent manner, can exert renoprotective effects in 5/6 Nx model of CKD.     

Autophagy protects auditory hair cells against neomycin-induced damage

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.     

AGEs induces apoptosis and autophagy via reactive oxygen species in human periodontal ligament cells

The apoptosis of human periodontal ligament cells (HPDLCs) may be an important factor of the negative effect of advanced glycation end products (AGEs) on the periodontal tissue of diabetic patients. However, the pathways or potential effects of apoptosis in AGEs-treated HPDLCs have not been fully elucidated. Autophagy is closely related to apoptosis. Herein, we investigated the potential mechanism of apoptosis and autophagy in HPDLCs treated with AGEs via an in vitro model. We found that AGEs-treated HPDLCs showed a time- and concentration-dependent reduction in the cell survival rate. The mitochondrial-dependent apoptosis was induced in AGEs-treated HPDLCs, as confirmed by the mitochondrial membrane potential depolarization, decreased Bcl-2 expression, increased Bax expression, and increased caspase-3 and PARP cleavage. Autophagy was also induced in AGEs-treated HPDLCs, as indicated by the conversion of LC3-II/LC3-I and the presence of autophagosomes. Interestingly, our study results suggested that apoptosis and autophagy were related to reactive oxygen species (ROS) production. In addition, AGEs-induced autophagy acted as a latent factor in decreasing the generation of ROS in HPDLCs and protecting against the AGEs-induced apoptosis. In summary, our study shows that ROS are essential in AGEs-induced HPDLCs apoptosis and autophagy, which may be a molecular mechanism for the repairment of ROS-induced damage in HPDLCs treated with AGEs to promote cell survival. The present study might provide new insights into the therapeutic targeting of HPDLCs autophagy, which could be an additional strategy for periodontitis in patients with diabetes mellitus.     

Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC(50) of approximately 80-100 and 40-60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.