Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.     

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30 mm Hg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.     

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.     

Free fatty acids stimulate autophagy in pancreatic β-cells via JNK pathway

Recent studies have suggested that free fatty acids stimulate autophagy of pancreatic beta cells. The aim of this study was to verify the free fatty acids (FFA)-induced autophagy and investigate its molecular mechanism. As reported previously, palmitate strongly enhanced the conversion of light chain (LC)3-I to LC3-II, a marker of activation of autophagy in INS-1 beta cells. Palmitate-induced conversion of LC3-I to LC3-II was also observed in neuron-, muscle-, and liver-derived cells. In addition, palmitate induced the formation of typical autophagosomes and autolysosomes and enhanced the degradation rate of long-lived proteins. These results confirmed that palmitate activates autophagic flux in most of the cells. While FFAs reportedly activate several signal transduction pathways in beta cells, palmitate-induced autophagy was blocked by a JNK inhibitor. Although enhanced oxidative stress and endoplasmic reticulum (ER) stress are suspected to mediate FFA-induced activation of JNK1, the induction of autophagy was not associated with changes in molecular markers related to oxidative and endoplasmic reticulum stresses. On the other hand, phosphorylation of double stranded RNA-dependent protein kinase (PKR) paralleled JNK1 activation. Considered together, our study suggested that FFA stimulated functional autophagy possibly through the PKR-JNK1 pathway independent of ER or oxidative stress.     

Induction of genuine autophagy by cationic lipids in mammalian cells

Autophagy is a cellular stress response that results in the activation of a lysosomal degradation pathway. In this report, we showed that cationic lipids, a common-used class of transfection reagents, induced genuine autophagy in mammalian cells. Extensive LC3 dot formation was observed by treatment with cationic lipids (with or without DNA), but not neutral lipids, in a HeLa cell line stably expressing GFP-LC3 (HeLa-LC3). Further proofs for autophagy were obtained by the co-localization of the LC3 dots with lysosome-specific staining patterns, observation of LC3-I to LC3-II form conversion and appearance of autophagic vacuoles under TEM. The autophagic flux assay with bafilomycin A1 and degradation of p62/SQSTM1 suggested that the autophagy induced by cationic lipids was primarily due to increased formation of autophagosomes and not decreased turnover. Moreover, cationic lipids induced autophagy in an mTOR-independent manner.     

Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.     

Sirt3 activates autophagy to prevent DOX-induced senescence by inactivating PI3K/AKT/mTOR pathway in A549 cells

Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates mitochondrial redox homeostasis and autophagy and is involved in physiological and pathological processes such as aging, cellular metabolism, and tumorigenesis. We here investigate how Sirt3 regulates doxorubicin (DOX)-induced senescence in lung cancer A549 cells. Sirt3 greatly reduced DOX-induced upregulation of senescence marker proteins p53, p16, p21 and SA-β-Gal activity as well as ROS levels. Notably, Sirt3 reversed DOX-induced autophagic flux blockage, as shown by increased p62 degradation and LC3II/LC3I ratio. Importantly, the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) partially abolished the antioxidant stress and antiaging effects of Sirt3, while the autophagy activator rapamycin (Rap) potentiated these effects of Sirt3, demonstrating that autophagy mediates the anti-aging effects of Sirt3. Additionally, Sirt3 inhibited the DOX-induced activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, which in turn activated autophagy. The PI3K inhibitor LY294002 promoted the antioxidant stress and antiaging effects of Sirt3, while the AKT activator SC-79 reversed these effects of Sirt3. Taken together, Sirt3 counteracts DOX-induced senescence by improving autophagic flux.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy

Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD⁺ levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD⁺ synthesis. In addition, the mechanistic relationship of autophagic flux and NAD⁺ synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD⁺ homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD⁺ homeostasis, and it is also valuable in the development of innovative strategies to combat aging.     

Rab7b modulates autophagic flux by interacting with Atg4B

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.     

Proteasome inhibition by chronic oxidative stress in human trabecular meshwork cells

The pathophysiologic mechanisms leading to the malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) outflow pathway in glaucoma are still unclear. We hypothesize that chronic oxidative stress may contribute to the malfunction of the outflow pathway by impairing the intracellular proteasome system of the cells, decreasing the ability of the tissue to modulate outflow resistance. To study the effects of chronic oxidative stress on proteasome function, primary cultures of human TM cells were incubated under 40% oxygen and proteasome activity was analyzed by measuring the accumulation of enhanced green fluorescent protein fused to a PEST motif. Changes in proteasome content, cellular senescence, and cell viability were also monitored. After 10 days of exposure to chronic oxidative stress, TM cells showed a marked decline in proteasome activity that was associated with premature senescence and decreased cell viability. These results suggest that proteasome failure may be involved in glaucoma pathophysiology.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

Proteasome Inhibitors Activate Autophagy Involving Inhibition of PI3K-Akt-mTOR Pathway as an Anti-Oxidation Defense in Human RPE Cells

The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE), ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA) or epoxomicin (Epo), at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3) from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf). Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt), a downstream target of phosphatidylinositol-3-kinases (PI3K), and mammalian target of rapamycin (mTOR) in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3) or 4-hydroxynonenal (4-HNE). Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.     

Autophagy induction is a survival response against oxidative stress in bone marrow–derived mesenchymal stromal cells

Background aimsBone marrow–derived mesenchymal stromal cells (BMSCs) are being extensively investigated as cellular therapeutics for many diseases, including cardiovascular diseases. Although preclinical studies indicated that BMSC transplantation into infarcted hearts improved heart function, there are problems to be resolved, such as the low survival rate of BMSCs during the transplantation process and in the ischemic region with extreme oxidative stress. Autophagy plays pivotal roles in maintaining cellular homeostasis and defending against environmental stresses. However, the precise roles of autophagy in BMSCs under oxidative stress remain largely uncharacterized.MethodsBMSCs were treated with H₂O₂, and autophagic flux was examined by means of microtubule-associated protein 1A/1B-light chain 3 II/I ratio (LC3 II/I), autophagosome formation and p62 expression. Cytotoxicity and cell death assays were performed after co-treatment of BMSCs by autophagy inhibitor (3-methyladenine) or autophagy activator (rapamycin) together with H₂O₂.ResultsWe show that short exposure (1 h) of BMSCs to H₂O₂ dramatically elevates autophagic flux (2- to 4-fold), whereas 6-h prolonged oxidative treatment reduces autophagy but enhances caspase-3 and caspase-6–associated apoptosis. Furthermore, we show that pre- and co-treatment with rapamycin ameliorates H₂O₂-induced caspase-3 and caspase-6 activation and cell toxicity but that 3-methyladenine exacerbates H₂O₂-induced cell apoptotic cell death.ConclusionsOur results demonstrate that autophagy is critical for the survival of BMSCs under oxidative conditions. Importantly, we also suggest that the early induction of autophagic flux is possibly a self-defensive mechanism common in oxidant-tolerant cells.     

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl--cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.