Kinetic and protective role of autophagy in manganese-exposed BV-2 cells

Manganese (Mn) plays an important role in many physiological processes. Nevertheless, Mn accumulation in the brain can cause a parkinsonian-like syndrome known as manganism. Unfortunately, the therapeutic options for this disease are scarce and of limited efficacy. For this reason, a great effort is being made to understand the cellular and molecular mechanisms involved in Mn toxicity in neuronal and glial cells. Even though evidence indicates that Mn activates autophagy in microglia, the consequences of this activation in cell death remain unknown. In this study, we demonstrated a key role of reactive oxygen species in Mn-induced damage in microglial cells. These species generated by Mn²⁺ induce lysosomal alterations, LMP, cathepsins release and cell death. Besides, we described for the first time the kinetic of Mn²⁺-induced autophagy in BV-2 microglial cells and its relevance to cell fate. We found that Mn promotes a time-dependent increase in LC3-II and p62 expression levels, suggesting autophagy activation. Possibly, cells trigger autophagy to neutralize the risks associated with lysosomal rupture. In addition, pre-treatment with both Rapamycin and Melatonin enhanced autophagy and retarded Mn²⁺ cytotoxicity. In summary, our results demonstrated that, despite the damage inflicted on a subset of lysosomes, the autophagic pathway plays a protective role in Mn-induced microglial cell death. We propose that 2 h Mn²⁺ exposure will not induce disturbances in the autophagic flux. However, as time passes, the accumulated damage inside the cell could trigger a dysfunction of this mechanism. These findings may represent a valuable contribution to future research concerning manganism therapies.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

A protective role for autophagy in vitiligo

A growing number of studies supports the existence of a dynamic interplay between energetic metabolism and autophagy, whose induction represents an adaptive response against several stress conditions. Autophagy is an evolutionarily conserved and a highly orchestrated catabolic recycling process that guarantees cellular homeostasis. To date, the exact role of autophagy in vitiligo pathogenesis is still not clear. Here, we provide the first evidence that autophagy occurs in melanocytes and fibroblasts from non-lesional skin of vitiligo patients, as a result of metabolic surveillance response. More precisely, this study is the first to reveal that induction of autophagy exerts a protective role against the intrinsic metabolic stress and attempts to antagonize degenerative processes in normal appearing vitiligo skin, where melanocytes and fibroblasts are already prone to premature senescence.Subject terms: Macroautophagy, Translational research     

Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy

The hemolysis of red blood cells and muscle damage results in the release of the heme proteins myoglobin, hemoglobin, and free heme into the vasculature. The mechanisms of heme toxicity are not clear but may involve lipid peroxidation, which we hypothesized would result in mitochondrial damage in endothelial cells. To test this, we used bovine aortic endothelial cells (BAEC) in culture and exposed them to hemin. Hemin led to mitochondrial dysfunction, activation of autophagy, mitophagy, and, at high concentrations, apoptosis. To detect whether hemin induced lipid peroxidation and damaged proteins, we used derivatives of arachidonic acid tagged with biotin or Bodipy (Bt-AA, BD-AA). We found that in cells treated with hemin, Bt-AA was oxidized and formed adducts with proteins, which were inhibited by α-tocopherol. Hemin-dependent mitochondrial dysfunction was also attenuated by α-tocopherol. Protein thiol modification and carbonyl formation occurred on exposure and was not inhibited by α-tocopherol. Supporting a protective role of autophagy, the inhibitor 3-methyladenine potentiated cell death. These data demonstrate that hemin mediates cytotoxicity through a mechanism which involves protein modification by oxidized lipids and other oxidants, decreased respiratory capacity, and a protective role for the autophagic process. Attenuation of lipid peroxidation may be able to preserve mitochondrial function in the endothelium and protect cells from heme-dependent toxicity.     

Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.     

Macrophage autophagy plays a protective role in advanced atherosclerosis

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Highlights? Proapoptotic oxidative stress induces autophagy in macrophages (M?s) ? Inhibition of autophagy enhances oxidative stress and apoptosis in M?s ? Atheromata of M?-Atg5^?/?Ldlr^?/? mice have increased apoptosis and plaque necrosis ? Apoptotic Atg5^?/? M?s are poorly recognized by phagocytes in vitro and in atheromata     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.     

Regulation and role of autophagy in mammalian cells

The recent period has witnessed progress in the understanding of the lysosomal autophagic pathway. The discovery of a family of genes conserved from yeast to humans, and involved in the formation of autophagosomes, has unraveled new protein-conjugation systems and has shed light on the importance of autophagy in physiology and pathophysiology. The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of autophagy during cell death. As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) as well as intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of autophagy. The mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals. In addition, autophagy, which is characterized by a flux of membrane from the formation of the autophagosome to the fusion with the lysosome, is regulated by GTPases, similarly to the vesicular transport along the exocytic/endocytic pathway. The aim of the present review is to give an overview of autophagy and to discuss its regulation by activators and effectors of mTOR and GTPases.     

Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells

Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.     

The role of autophagy in the metabolism and differentiation of stem cells

Autophagy is a very well-coordinated intracellular process that maintains cellular homeostasis under basal conditions by removing unnecessary or dysfunctional components through orderly degradation and recycling. Under pathological conditions, defects in autophagy have been linked to various human disorders, including neurodegenerative disorders and cancer. The role of autophagy in stem cell proliferation, differentiation, self-renewal, and senescence is well documented. Additionally, cancer stem cells (CSCs) play an important role in tumorigenesis, metastasis and tumor relapse and several studies have suggested the involvement of autophagy in the maintenance and invasiveness of CSCs. Hence, considering the modulation of autophagy in normal and cancer stems cells as a therapeutic approach can lead to the development or improvement of regenerative and anti-cancer therapies. Accordingly, modulation of autophagy can be regarded as a target for stem cell-based therapy of diseases with abnormal levels of autophagy.This article is focused on understanding the role of autophagy in stem cell homeostasis with an emphasis on the therapeutic potential of targeting autophagy for future therapies.     

Sanggenon O induced apoptosis of A549 cells is counterbalanced by protective autophagy

Sanggenon O (SO) is a Diels-Alder type adduct extracted from Morus alba, which has been used for its anti-inflammatory action in the Oriental medicine. However, whether it has regulatory effect on human cancer cell proliferation and what the underlying mechanism remains unknown. Here, we found that SO could significantly inhibit the growth and proliferation of A549 cells and induce its pro-apoptotic action through a caspase-dependent pathway. It could also impair the mitochondria which can be reflected by mitochondrial membrane permeabilization. Besides, SQSTM1 up-regulation and autophagic flux measurement demonstrated that exposure to SO led to autophagosome accumulation, which plays a protective role in SO-treated cells. In addition, knocking down of LC3B increased SO triggered apoptotic cell rates. These results indicated that SO has great potential as a promising candidate combined with autophagy inhibitor for the treatment of NSCLC. In conclusion, our results identified a novel mechanism by which SO exerts potent anticancer activity.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependant degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Involvement of PKC signal pathways in oridonin-induced autophagy in HeLa cells: A protective mechanism against apoptosis

Our previous studies showed that oridonin could induce both apoptosis and autophagy in HeLa cells, and this autophagy might be a protective mechanism against apoptosis. In this study, the roles of PKC signal pathways in oridonin-induced HeLa cell autophagy and apoptosis were further investigated. We found that inhibition of PKC significantly reduced oridonin-induced autophagy whereas markedly increased apoptosis, while pretreatment with PKC activator caused opposite results. Subsequently, the oridonin-induced autophagy was also suppressed by Raf-1 or JNK inhibition accompanied by the increase of apoptosis, but it was not affected by ERK or p38 inhibition. In addition, oridonin-induced protein levels of Raf-1, JNK and p-JNK were sharply downregulated by PKC inhibitor, and they were enhanced by PKC activator. Taken together, these results demonstrate that PKC enhances oridonin-induced autophagy against apoptosis through regulating its downstream factors Raf-1 and JNK in HeLa cells.