The role S‐nitrosylation in manganese‐induced autophagy dysregulation in SH‐SY5Y cells

Overexposure to manganese (Mn) has been known to induce nitrosative stress. The dysregulation of autophagy has implicated in nitric oxide (NO) bioactivity alterations. However, the mechanism of Mn‐induced autophagic dysregulation is unclear. The protein of Bcl‐2 was considered as a key role that could participate to the autophagy signaling regulation. To further explore whether S‐nitrosylation of Bcl‐2 involved in Mn‐induced autophagy dysregulation, we treated human neuroblastoma (SH‐SY5Y) cells with Mn and pretreated cells with 1400 W, a selective iNOS inhibitor. After cells were treated with 400 μM Mn for 24 h, there were significant increases in production of NO, inducible NO synthase (iNOS) activity, the mRNA and protein expressions of iNOS. Interestingly, autophagy was activated after cells were treated with Mn for 0–12 h; while the degradation process of autophagy‐lysosome pathway was blocked after cells were treated with Mn for 24 h. Moreover, S‐nitrosylated JNK and Bcl‐2 also increased and phospho‐JNK and phospho‐Bcl‐2 reduced in Mn‐treated cells. Then, the affinity between Bcl‐2 and Beclin‐1 increased significantly in Mn‐treated cells. We used the 1400 W to neutralize Mn‐induced nitrosative stress. The results showed that S‐nitrosylated JNK and Bcl‐2 reduced while their phosphorylation were recovered to some extent. The findings revealed that NO‐mediated S‐nitrosylation of Bcl‐2 directly affected the interaction between Beclin‐1 and Bcl‐2 leading to autophagy inhibition.     

ER stress‐induced autophagy in melanoma

The activation of RAF‐MEK–extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinase cascade by v‐raf murine sarcoma viral oncogene homolog B1 (BRAF)^V600E mutation is a key alteration in melanoma. Although BRAF inhibitor (BRAFi) has achieved remarkable clinical success, the positive response to BRAFi is not sustainable, and the initial clinical benefit is eventually barred by the development of resistance to BRAFi. There is growing evidence to suggest that endoplasmic reticulum (ER) stress‐induced autophagy could be a potential pro‐survival mechanism that contributes to genesis of melanoma and to the resistance to BRAFi. ER stress‐induced autophagy is an evolutionarily conserved membrane process. By degrading and recycling proteins and organelles via the formation of autophagous vesicles and their fusion with lysosomes, the autophagy plays a key role in homeostasis as well as pathological processes. In this review, we examine the autophagy phenomenon in melanocytic nevus, primary and metastatic melanoma, and its significance in BRAFi‐resistant melanoma.     

N‐acetylcysteine alleviated paraquat‐induced mitochondrial fragmentation and autophagy in primary murine neural progenitor cells

The developing brain is uniquely vulnerable to toxic chemical exposures. Studies indicate that neural stem cell (NSC) self‐renewal is susceptible to oxidative stress caused by xenobiotics. However, the impact of antioxidants on NSC self‐renewal and the potential mechanisms remain elusive. In this study, primary murine neural progenitor cells (mNPCs) from the subventricular zone were used as a research model. In addition, paraquat (PQ) was used to elicit oxidative stress and N‐acetylcysteine (NAC) was used as a powerful antioxidant. mNPCs were treated with 80 μm PQ for 24 hours with or without 4 hours of NAC pretreatment. Our results showed that PQ treatment increased intracellular reactive oxygen species production, decreased cell viability and DNA synthesis, and promoted cell apoptosis. Meanwhile, pretreatment with NAC alleviated PQ‐induced cytotoxicity in mNPCs. To elucidate the mechanisms further, we found that NAC pretreatment prevented PQ‐induced reactive oxygen species production, mitochondrial fragmentation and autophagy in mNPCs. NAC‐pretreated cells showed increased anti‐apoptotic protein Bcl‐2 and decreased pro‐apoptotic protein Bax expression. Similarly, NAC pretreatment increased p‐mTOR and decreased LC3B‐II protein expression. Moreover, NAC decreased mitophagy related mRNA Pink1 and Parkin expression. Taken together, our results suggested that the antioxidant NAC treatment significantly attenuated PQ‐induced mNPC self‐renewal disruption through decreasing autophagy and salvaging mitochondrial morphology. These findings revealed a potential mechanism for neurological treatment relating to antioxidant and suggested potentially relevant implications for PQ‐related neurodegenerative disorders. Thus, our study also provided insight into therapeutic strategies for the neurotoxic effects of oxidative stress‐associated toxicants.     

Resveratrol protects Leydig cells from nicotine‐induced oxidative damage through enhanced autophagy

Some studies have revealed that nicotine can damage the male reproductive system through various means including oxidative stress, which is a primary factor in the pathogenesis of male infertility. The strong anti‐oxidative capacity of resveratrol has been demonstrated previously, but its role in the context of male reproduction remains inconclusive. To explore the biological role of resveratrol in protecting male reproductive function and the potential underlying mechanism, nicotine‐induced Leydig cells were used as a cell model of oxidative damage. The data showed that resveratrol treatment increased cell viability, SOD activity and anti‐apoptotic activity in nicotine‐stressed Leydig cells. This effect was accompanied by the upregulation of autophagy, which was illustrated by MDC‐LysoTracker red staining. Moreover, pretreating with 3‐methyladenine (3‐MA), an autophagy inhibitor, attenuated resveratrol‐induced Leydig cells autophagy and promoted apoptosis. Apart from this, resveratrol enhanced AMPK phosphorylation but reduced mTOR phosphorylation. Subsequently, upon inhibiting AMPK phosphorylation by AMPK inhibitors, Leydig cell autophagy induced by resveratrol was obviously abolished. In conclusion, resveratrol may exert its cytoprotective role against oxidative injury by the activation of autophagy via AMPK/mTOR pathway.     

Angiotensin AT1 receptor activation mediates high glucose‐induced epithelial–mesenchymal transition in renal proximal tubular cells

1. Renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT) under hyperglycaemic conditions, which is associated with renal interstitial fibrosis. Activation of the renin–angiotensin system (RAS) is involved in diabetic nephropathy. The present study investigated the positive role of angiotensin AT₁ receptors in high glucose‐induced EMT in cultured tubular epithelial cells. 2. A rat kidney proximal tubular epithelial cell line (NRK‐52E) was used in the present study. Levels of EMT makers, namely E‐cadherin and vimentin, were estimated using fluorescence immunocytochemistry, mRNA levels of angiotensinogen (AGT), angiotensin‐converting enzyme (ACE) and AT₁ receptors were determined by real‐time polymerase chain reaction, protein levels of E‐cadherin, vimentin, fibronectin, matrix metallopeptidase (MMP)‐9 and phosphorylated extracellular signal‐regulated kinase (ERK) 1/2 were analysed by western blotting and the concentrations of angiotensin (Ang) II and transforming growth factor (TGF)‐β1 in the culture medium were determined by enzyme immunoassay and ELISA. 3. High glucose (30 mmol/L) induced EMT and increased the synthesis of fibronectin and MMP‐9. Furthermore, high glucose increased AGT, ACE and AT₁ receptor mRNA levels, as well as AngII and TGF‐β1 concentrations in the culture medium and ERK1/2 phosphorylation. Pretreatment of cells for 15 min with the AT₁ receptor antagonist losartan (10^?5 mol/L) attenuated high glucose‐induced increases in TGF‐β1 and ERK1/2 phosphorylation and reduced EMT, as well as the consequent synthesis of fibronectin and MMP‐9. 4. The results of the present study suggest that the activated local RAS mediates high glucose‐induced EMT. By activating AT₁ receptors and stimulating TGF‐β1 synthesis, the elevated local RAS participates in high glucose‐induced EMT and increased extracellular matrix secretion.     

Effect of the cross‐talk between autophagy and endoplasmic reticulum stress on Mn‐induced alpha‐synuclein oligomerization

Overexposure to manganese (Mn) has been known to induce alpha‐synuclein (α‐Syn) oligomerization, which is degraded mainly depending on endoplasmic reticulum stress (ER stress) and autophagy pathways. However, little data reported the cross‐talk between ER stress and autophagy on Mn‐induced α‐Syn oligomerization. To explore the relationship between ER stress and autophagy, we used 4‐phenylbutyric acid (4‐PBA, the ER stress inhibitor), rapamycin (Rap, autophagy activator) and 3‐methyladenine (3‐MA, autophagy inhibitor) in mice model of manganism. After 4 weeks of treatment with Mn, both ER stress and autophagy were activated. Exposed to Mn also resulted in α‐Syn oligomerization and neuronal cell damage in the brain tissue of mice, which could be relieved by 4‐PBA pretreatment. Moreover, when the ER stress was inhibited, the activation of autophagy was also inhibited. Rap pretreatment significantly activated autophagy and decreased α‐Syn oligomers. However, 3‐MA pretreatment inhibited autophagy resulting in increase of α‐Syn oligomers, and compensatorily activated PERK signaling pathway. Our results also demonstrated that the inhibition of autophagy by 3‐MA aggravated neuronal cell damage. The findings clearly demonstrated that the cross‐talking between autophagy and ER stress might play an important role in the α‐Syn oligomerization and neurotoxicity by Mn.     

Triptolide‐induced hepatotoxicity via apoptosis and autophagy in zebrafish

Previous research about the development of triptolide (TP) as a natural active compound has often focused on hepatotoxicity. Among its various mechanisms, autophagy and apoptosis are two important signaling pathways. In this study, we used zebrafish to establish a TP‐induced hepatotoxicity model, and investigated the roles of autophagy and apoptosis in the progress of liver injury. Zebrafish exposed to TP showed increased mortality and malformation because of the increased drug dose and duration of exposure. Meanwhile, we found that TP induced liver injury in a time‐ and dose‐dependent manner, which was observed as a reduction in liver area, slow yolk absorption, upregulation of transaminase and local neurosis. With the application of the high‐content imaging system (HCIS) technique in liver 3D imaging in vivo, clear imaging of the zebrafish liver was achieved. The results showed a decrease in volume and location of necrosis in the liver after TP exposure. Increased expression of inflammatory cytokines genes tumor necrosis factor (Tnf)α, Il1β and Il6 were shown, particularly Tnfα. The Fas‐Caspase8 signaling pathway was activated. The apoptosis‐related gene Bcl‐2 was increased, and Bax, Caspase9 and Caspase3 were increased. However, autophagy related genes Beclin1, Atg5, Atg3 and Lc3 were increased more significantly, and the changes of Beclin1 and Atg5 were the most severe. This study successfully established a TP‐induced zebrafish hepatotoxicity model and applied the HCIS technique in a zebrafish hepatotoxicity study. The result indicated Fas might be the main target of TP‐induced hepatotoxicity. Autophagy played a more important role than apoptosis and was characterized by the overexpression of Beclin1 and Atg5.     

Tea polyphenols alleviate tri‐ortho‐cresyl phosphate‐induced autophagy of mouse ovarian granulosa cells

Tri‐ortho‐cresyl phosphate (TOCP), a widely used plasticizer in industry, can cause female reproductive damage. Tea polyphenols (TPs) have multiple health effects via inhibiting oxidative stress. However, the reproductive protection of TPs in TOCP‐induced female reproductive system damage is yet to be elucidated. In the study, TOCP inhibited cell viability and induced autophagy of mouse ovarian granulosa cells; while TPs could rescue the inhibition of viability and induction of autophagy. 3‐MA, an autophagy inhibitor, could also rescue the inhibition of cell viability. These results indicated that TPs played a protective role in TOCP‐induced autophagy. Furthermore, TPs could inhibit the induction of oxidative stress of the cells by TOCP, which implying that TPs might alleviate TOCP‐induced autophagy via inhibiting oxidative stress. Furthermore, TPs could rescue TOCP‐induced autophagy and oxidative stress in the mouse ovarian tissues. Taken together, these results indicated that TPs could protect TOCP‐induced ovarian damage via inhibiting oxidative stress.     

Oleanolic acid arrests cell cycle and induces apoptosis via ROS‐mediated mitochondrial depolarization and lysosomal membrane permeabilization in human pancreatic cancer cells

Oleanolic acid (OA), a pentacyclic triterpenoid, exhibits potential anti‐tumor activity against many tumor cell lines. This study aims to examine the anti‐tumor activity of OA on pancreatic cancer cells and its potential molecular mechanism. The results showed that the proliferation of Panc‐28 cells was inhibited by OA in a concentration‐dependent manner, with an IC₅₀ (The half maximal inhibitory concentration) value of 46.35 µg ml^?1, as determined by MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. The cell cycle was arrested in S phase and G2/M phase by OA. The study also showed that OA could induce remarkable apoptosis, evidenced by an increased percentage of early/late apoptotic cells, DNA ladder and nuclear morphology change. Further study revealed that OA could induce Reactive Oxygen Species (ROS) generation, mitochondrial depolarization, release of cytochrome C, lysosomal membrane permeabilization and leakage of cathepin B. The expression of apoptosis‐correlated proteins was also affected in cells treated with OA, including activation of caspases‐3/9 and cleavage of PARP. Further study confirmed that ROS scavenger vitamin C could reverse the apoptosis induced by OA in Panc‐28 cells. Our results provide evidence that OA arrests the cell cycle and induces apoptosis, possibly via ROS‐mediated mitochondrial and a lysosomal pathway in Panc‐28 cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Lack of correlation between lysosomal enzyme activity and ascorbic acid content in brain of lead-treated animals

The administration of lead acetate increased acid phosphatase activities in the rat brain and four regions (cerebral cortex, diencephalon plus mesencephalon, pons plus medulla, and cerebellum) of the guinea pig brain, whereas ascorbic acid content in the brain of these animals remained unchanged following lead administration. Acid phosphatase activity also showed an increase in scorbutic guinea pig brain. Lipid peroxidation facilitated by ascorbic acid in the cerebral lysosome fraction of rat was not affected by lead. These results suggest that ascorbic acid is not directly concerned with the alteration in acid phosphatase activity induced by lead treatment.     

Overexpression of miR‐138‐5p suppresses MnCl2‐induced autophagy by targeting SIRT1 in SH‐SY5Y cells

The mechanism of manganism caused by manganese (Mn), an important environmental risk factor for Parkinson's disease, is still unclear. Recent evidence suggested that autophagy participated in neurodegenerative diseases, in which microRNA played a crucial role. However, roles of microRNA in the aberrant autophagy that occurs in neurodegenerative diseases remains controversial. In nervous system, miRNA‐138‐5p is highly expressed and plays a key role in regulating memory and axon regeneration. Importantly, we also found that miR‐138‐5p expression decreased significantly after SH‐SY5Y cells exposed to manganese chloride (MnCl₂) in previous study. To explore the role of miR‐138‐5p in Mn‐induced autophagy, autophagy associated indicators were detected. And we found that MnCl₂ could induce autophagic dysregulation and inhibit expression of miR‐138‐5p. While the levels of LC3‐II/LC3‐I, Beclin1, and p62, the number of autophagosome formation significantly decreased after miR‐138‐5p over‐expression, which demonstrated that miR‐138‐5p could clearly retard Mn‐induced autophagy. In additional, we found there were classical and evolutionarily conserved miR‐138‐5p binding sites in 3′‐UTR region of SIRT1, which was inhibited when overexpression of miR‐138‐5p. Therefore, it was speculated that elevated expression of SIRT1 may be resulted from inhibition of miR‐138‐5p after cells exposed to MnCl₂. Finally, we found that SIRT1 inhibitor EX‐527 suppressed Mn‐induced autophagy as well as miR‐138‐5p, while the suppression was reversed by SIRT1‐specific activator SRT1720. These results indicated that overexpression of miR‐138‐5p suppressed Mn‐induced autophagy by targeting SIRT1.     

The anti-tumourigenic effect of ellagic acid in SKOV-3 ovarian cancer cells entails activation of autophagy mediated by inhibiting Akt and activating AMPK

This study investigated the effect of ellagic acid (EA) on SKOV-3 cell growth and invasiveness and tested if the underlying mechanism involves modulating autophagy. Cells were treated with EA in the presence or absence of chloroquine (CQ), an autophagy inhibitor, compound C (CC), an AMPK inhibitor, or an insulin-like growth factor-1 (IGF-1), a PI3K/Akt activator. EA, at an IC₅₀ of 36.6 µmol/L, inhibited cell proliferation, migration, and invasion and induced cell apoptosis in SKOV-3 cells. These events were prevented by CQ. Also, EA increased levels of Beclin-1, ATG-5, LC3I/II, Bax, cleaved caspase-3/8 and reduced those of p62 and Bcl-2 in these cancer cells. Mechanistically, EA decreased levels of p-S6K1 (Thr³⁸⁹) and 4EBP-1 (Thr^37/46), two downstream targets of mTORC1, and p-Akt (Thr³⁰⁸) but increased levels of AMPK (Thr¹⁷²) and p-raptor (Ser⁷⁹²), a natural inhibitor of mTORC1. CC or IGF-1 alone partially prevented the effect of EA on cell survival, cell invasions, and levels of LDH, Beclin-1, and cleaved caspase-3. In conclusion, EA can inhibit SKOV-3 growth, migration, and invasion by activating cytotoxic autophagy mediated by inhibition of mTORC1 and Akt and activation of AMPK.     

Ursolic acid induces apoptosis and autophagy in oral cancer cells

Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose‐ and time‐dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase‐dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF‐κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP‐2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B‐II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA‐mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.     

The length‐dependent activation of contraction is equally impaired in impuberal male and female rats in monocrotaline‐induced right ventricular failure

The length‐dependent activation of contraction is attenuated in the failing myocardium of adult male rats. This pathological change is not seen in adult female rats, possibly because of a protective effect of sex hormones. The present study evaluated length‐dependent changes in isometric twitch, Ca²⁺ transient (CaT) and action potential (AP) in the right ventricular myocardium of impuberal healthy male and female rats (control) and in rats treated with a single injection of 50 mg/kg monocrotaline (MCT). Compared with sex‐matched control rats, MCT‐treated male and female rats exhibited increased right ventricular weight (134% and 142% of control, respectively), decreased left ventricular weight (72% and 79%), twitch attenuation (48.8 ± 2.7% and 57.5 ± 1.2%) and prolongation (125 ± 3% and 127 ± 2%), CaT attenuation (37.8 ± 0.4% and 39.1 ± 1.1%) and prolongation (114 ± 1% and 116 ± 1%) and AP prolongation at 90% repolarization (195 ± 2% and 203 ± 1%). The MCT‐treated male rats exhibited a 50% lower integral magnitude and an approximately 25% larger time‐to‐peak ‘bump’ compared with control male rats. These parameters in MCT‐treated female rats tended to show similar changes to those seen in the control female rats, with no significant difference between the two groups. In all groups, integral magnitude and time‐to‐peak ‘bump’ increased with length. In conclusion, the length‐dependent activation of contraction was equally blunted in the failing right ventricular myocardium of impuberal male and female rats. This was related to changes in CaT and AP, which were similar between male and female rats. Therefore, puberty is necessary for manifestation of the protective effects of sex hormones on this remodelling.     

3‐Methyladenine alleviates excessive iodine‐induced cognitive impairment via suppression of autophagy in rat hippocampus

Drinking water with high levels of iodine has been identified as the key contributor to iodine excess, but the mechanisms of neurotoxicity induced by excessive iodine remain elusive. The present study aimed to explore the role of autophagy in the neurotoxic effect induced by excessive iodine in vivo. The Morris water maze test results demonstrated that excessive iodine impaired the learning and memory capabilities of rats, which were associated with marked body weight and brain weight abnormalities. In addition, iodine treatment increased malondialdehyde accumulation, decreased superoxide dismutase activity and glutathione (GSH) level, and enhanced levels of autophagy markers in the hippocampus. Notably, inhibition of autophagy with 3‐methyladenine (3‐MA) could significantly alleviate excessive iodine‐induced cognitive impairment. These data imply that autophagy is involved in the cognitive impairment elicited by excessive iodine as a pathway of cell death, and inhibition of autophagy via 3‐MA may significantly alleviate the above damage.     

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G ₀/G₁‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)?1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.     

Lithium limits trimethyltin‐induced cytotoxicity and proinflammatory response in microglia without affecting the concurrent autophagy impairment

Trimethyltin (TMT) is a highly toxic molecule present as an environmental contaminant causing neurodegeneration particularly of the limbic system both in humans and in rodents. We recently described the occurrence of impairment in the late stages of autophagy in TMT‐intoxicated astrocytes. Here we show that similarly to astrocytes also in microglia, TMT induces the precocious block of autophagy indicated by the accumulation of the autophagosome marker, microtubule associated protein light chain 3. Consistent with autophagy impairment we observe in TMT‐treated microglia the accumulation of p62/SQSTM1, a protein specifically degraded through this pathway. Lithium has been proved effective in limiting neurodegenerations and, in particular, in ameliorating symptoms of TMT intoxication in rodents. In our in vitro model, lithium displays a pro‐survival and anti‐inflammatory action reducing both cell death and the proinflammatory response of TMT‐treated microglia. In particular, lithium exerts these activities without reducing TMT‐induced accumulation of light chain 3 protein. In fact, the autophagic block imposed by TMT is unaffected by lithium administration. These results are of interest as defects in the execution of autophagy are frequently observed in neurodegenerative diseases and lithium is considered a promising therapeutic agent for these pathologies. Thus, it is relevant that this cation can still maintain its pro‐survival and anti‐inflammatory role in conditions of autophagy block. Copyright © 2016 John Wiley & Sons, Ltd.