Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats.

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3- (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1ß, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

The presence of Mott cells in the lymph nodes of rats with experimental autoimmune encephalomyelitis.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

Autophagy Receptor p62 Regulates SARS-CoV-2-Induced Inflammation in COVID-19.

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1ß were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1ß/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.

Reset of Inflammatory Priming of Joint Tissue and Reduction of the Severity of Arthritis Flares by Bromodomain Inhibition.

OBJECTIVE: We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model. METHODS: BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS: Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs. CONCLUSION: Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Effects of metformin and simvastatin treatment on ultrastructural features of liver macrophages in HFD mice.

Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

Downregulation of LKB1/AMPK Signaling in Blood Mononuclear Cells Is Associated with the Severity of Guillain-Barre Syndrome.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolic and immune functions mainly through the inhibition of the mechanistic target of rapamycin (mTOR)-dependent anabolic pathways and the activation of catabolic processes such as autophagy. The AMPK/mTOR signaling pathway and autophagy markers were analyzed by immunoblotting in blood mononuclear cells of 20 healthy control subjects and 23 patients with an acute demyelinating form of Guillain-Barré syndrome (GBS). The activation of the liver kinase B1 (LKB1)/AMPK/Raptor signaling axis was significantly reduced in GBS compared to control subjects. In contrast, the phosphorylated forms of mTOR activator AKT and mTOR substrate 4EBP1, as well as the levels of autophagy markers LC3-II, beclin-1, ATG5, p62/sequestosome 1, and NBR1 were similar between the two groups. The downregulation of LKB1/AMPK signaling, but not the activation status of the AKT/mTOR/4EBP1 pathway or the levels of autophagy markers, correlated with higher clinical activity and worse outcomes of GBS. A retrospective study in a diabetic cohort of GBS patients demonstrated that treatment with AMPK activator metformin was associated with milder GBS compared to insulin/sulphonylurea therapy. In conclusion, the impairment of the LKB1/AMPK pathway might contribute to the development/progression of GBS, thus representing a potential therapeutic target in this immune-mediated peripheral polyneuropathy.

Combination of Ascorbic Acid and Menadione Induces Cytotoxic Autophagy in Human Glioblastoma Cells.

We investigated the ability of the ascorbic acid (AA) and menadione (MD) combination, the well-known reactive oxidative species- (ROS-) generating system, to induce autophagy in human U251 glioblastoma cells. A combination of AA and MD (AA+MD), in contrast to single treatments, induced necrosis-like cell death mediated by mitochondrial membrane depolarization and extremely high oxidative stress. AA+MD, and to a lesser extent MD alone, prompted the appearance of autophagy markers such as autophagic vacuoles, autophagosome-associated LC3-II protein, degradation of p62, and increased expression of beclin-1. While both MD and AA+MD increased phosphorylation of AMP-activated protein kinase (AMPK), the well-known autophagy promotor, only the combined treatment affected its downstream targets, mechanistic target of rapamycin complex 1 (mTORC1), Unc 51-like kinase 1 (ULK1), and increased the expression of several autophagy-related genes. Antioxidant N-acetyl cysteine reduced both MD- and AA+MD-induced autophagy, as well as changes in AMPK/mTORC1/ULK1 activity and cell death triggered by the drug combination. Pharmacological and genetic autophagy silencing abolished the toxicity of AA+MD, while autophagy upregulation enhanced the toxicity of both AA+MD and MD. Therefore, by upregulating oxidative stress, inhibiting mTORC1, and activating ULK1, AA converts MD-induced AMPK-dependent autophagy from nontoxic to cytotoxic. These results suggest that AA+MD or MD treatment in combination with autophagy inducers could be further investigated as a novel approach for glioblastoma therapy.

MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells.

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.

Periapical lesions in two inbred strains of rats differing in immunological reactivity.

AIM: To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. METHODOLOGY: Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21 days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. RESULTS: DA rats developed significantly larger (p < .05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p < .05). In DA rats, periapical lesions had a significantly higher (p < .05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p < .05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1ß, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p < .05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p < .05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p < .05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p < .05) in the supernatant in the periapical lesions of DA rats. CONCLUSION: After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.

Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death.

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•-), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy.

Modulation of Cancer Cell Autophagic Responses by Graphene-Based Nanomaterials: Molecular Mechanisms and Therapeutic Implications.

Graphene-based nanomaterials (GNM) are plausible candidates for cancer therapeutics and drug delivery systems. Pure graphene and graphene oxide nanoparticles, as well as graphene quantum dots and graphene nanofibers, were all able to trigger autophagy in cancer cells through both transcriptional and post-transcriptional mechanisms involving oxidative/endoplasmic reticulum stress, AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and Toll-like receptor signaling. This was often coupled with lysosomal dysfunction and subsequent blockade of autophagic flux, which additionally increased the accumulation of autophagy mediators that participated in apoptotic, necrotic, or necroptotic death of cancer cells and influenced the immune response against the tumor. In this review, we analyze molecular mechanisms and structure-activity relationships of GNM-mediated autophagy modulation, its consequences for cancer cell survival/death and anti-tumor immune response, and the possible implications for the use of GNM in cancer therapy.

3-Methyladenine prevents energy stress-induced necrotic death of melanoma cells through autophagy-independent mechanisms.

We investigated the effect of 3-methyladenine (3MA), a class III phosphatidylinositol 3-kinase (PI3K)-blocking autophagy inhibitor, on cancer cell death induced by simultaneous inhibition of glycolysis by 2-deoxyglucose (2DG) and mitochondrial respiration by rotenone. 2DG/rotenone reduced ATP levels and increased mitochondrial superoxide production, causing mitochondrial swelling and necrotic death in various cancer cell lines. 2DG/rotenone failed to increase proautophagic beclin-1 and autophagic flux in melanoma cells despite the activation of AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin complex 1 (mTORC1). 3MA, but not autophagy inhibition with other PI3K and lysosomal inhibitors, attenuated 2DG/rotenone-induced mitochondrial damage, oxidative stress, ATP depletion, and cell death, while antioxidant treatment mimicked its protective action. The protection was not mediated by autophagy upregulation via class I PI3K/Akt inhibition, as it was preserved in cells with genetically inhibited autophagy. 3MA increased AMPK and mTORC1 activation in energy-stressed cells, but neither AMPK nor mTORC1 inhibition reduced its cytoprotective effect. 3MA reduced JNK activation, and JNK pharmacological/genetic suppression mimicked its mitochondria-preserving and cytoprotective activity. Therefore, 3MA prevents energy stress-triggered cancer cell death through autophagy-independent mechanisms possibly involving JNK suppression and decrease of oxidative stress. Our results warrant caution when using 3MA as an autophagy inhibitor.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Dual targeting of tumor cell energy metabolism and lysosomes as an anticancer strategy.

To sustain their proliferative and metastatic capacity, tumor cells increase the activity of energy-producing pathways and lysosomal compartment, resorting to autophagolysosomal degradation when nutrients are scarce. Consequently, large fragile lysosomes and enhanced energy metabolism may serve as targets for anticancer therapy. A simultaneous induction of energy stress (by caloric restriction and inhibition of glycolysis, oxidative phosphorylation, Krebs cycle, or amino acid/fatty acid metabolism) and lysosomal stress (by lysosomotropic detergents, vacuolar ATPase inhibitors, or cationic amphiphilic drugs) is an efficient anti-cancer strategy demonstrated in a number of studies. However, the mechanisms of lysosomal/energy stress co-amplification, apart from the protective autophagy inhibition, are poorly understood. We here summarize the established and suggest potential mechanisms and candidates for anticancer therapy based on the dual targeting of lysosomes and energy metabolism.

Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma.

We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant tissue were collected from 20 patients with ccRCC after radical nephrectomy. The mRNA levels of apoptosis (BAD, BAX, BCL2, BCLXL, BIM) and autophagy (ATG4, BECN1, GABARAP, p62, UVRAG) regulators were measured by RT-qPCR. The protein levels of autophagosome-associated LC3-II, autophagy receptor p62, apoptotic marker PARP, as well as phosphorylation of autophagy initiator Unc 51-like kinase 1 (ULK1), its activator AMP-activated protein kinase (AMPK) and 4EBP1, the substrate of ULK1 inhibitor mechanistic target of rapamycin (mTOR), were analyzed by immunoblotting. The mRNA levels of pro-apoptotic BAX, anti-apoptotic BCLXL and pro-autophagic ATG4, p62 and UVRAG were higher in ccRCC tumors. Autophagy induction was confirmed by an increase in phospho-ULK1 and degradation of the autophagic target p62, while apoptotic PARP cleavage was unaltered. AMPK phosphorylation was reduced and 4EBP1 phosphorylation was increased in ccRCC tissue. The expression of apoptosis regulators did not correlate with clinicopathological features of ccRCC. Conversely, high mRNA levels of ATG4, GABARAP and p62 were associated with lower tumor stage, as well as with smaller tumor size and better disease-specific 5-year survival (ATG4 and p62). Accordingly, low p62 protein levels, corresponding to increased autophagic flux, were associated with lower tumor stage, reduced metastasis and improved 5-year survival. These data demonstrate that transcriptional induction of autophagy in ccRCC is accompanied by AMPK/mTOR-independent increase in ULK1 activation and autophagic flux, which might slow tumor progression and metastasis independently of apoptosis.

Current Development of Metal Complexes with Diamine Ligands as Potential Anticancer Agents.

BACKGROUND: The discovery of cisplatin and the subsequent research revealed the importance of dinitrogen-containing moiety for the anticancer action of metal complexes. Moreover, certain diamine ligands alone display cytotoxicity that contributes to the overall activity of corresponding complexes. OBJECTIVE: To summarize the current knowledge on the anticancer efficacy, selectivity, and the mechanisms of action of metal complexes with various types of diamine ligands. METHODS: The contribution of aliphatic acyclic, aliphatic cyclic, and aromatic diamine ligands to the anticancer activity and selectivity/toxicity of metal complexes with different metal ions were analyzed by comparison with organic ligand alone and/or conventional platinum-based chemotherapeutics. RESULTS: The aliphatic acyclic diamine ligands are present mostly in complexes with platinum. Aliphatic cyclic diamines are part of Pt(II), Ru(II) and Au(III) complexes, while aromatic diamine ligands are found in Pt(II), Ru(II), Pd(II) and Ir(III) complexes. The type and oxidation state of metal ions greatly influences the cytotoxicity of metal complexes with aliphatic acyclic diamine ligands. Lipophilicity of organic ligands, dependent on alkyl-side chain length and structure, determines their cellular uptake, with edda and eddp/eddip ligands being most useful in this regard. Aliphatic cyclic diamine ligands improved the activity/toxicity ratio of oxaliplatin-type complexes. The complexes with aromatic diamine ligands remain unexplored regarding their anticancer mechanism. The investigated complexes mainly caused apoptotic or necrotic cell death. CONCLUSION: Metal complexes with diamine ligands are promising candidates for efficient and more selective alternatives to conventional platinum-based chemotherapeutics. Further research is required to reveal the chemico-physical properties and molecular mechanisms underlying their biological activity.

Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells.

We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

AMP-activated protein kinase inhibits MPP+-induced oxidative stress and apoptotic death of SH-SY5Y cells through sequential stimulation of Akt and autophagy.

We investigated the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), prosurvival kinase Akt, oxidative stress, and autophagy in the cytotoxicity of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) towards SH-SY5Y human neuroblastoma cells. MPP+-mediated oxidative stress, mitochondrial depolarization, and apoptotic cell death were associated with rapid (within 2 h) activation of AMPK, its target Raptor, and prosurvival kinase Akt. Antioxidants N-acetylcysteine and butylated hydroxyanisole suppressed MPP+-induced cytotoxicity, AMPK, and Akt activation. A genetic or pharmacological inhibition of AMPK increased MPP+-triggered production of reactive oxygen species and cell death, and diminished Akt phosphorylation, while AMPK activation protected SH-SY5Y cells from MPP+. On the other hand, genetic or pharmacological inactivation of Akt stimulated MPP+-triggered oxidative stress and neurotoxicity, but did not affect AMPK activation. At later time-points (16-24 h), MPP+ inhibited the main autophagy repressor mammalian target of rapamycin, which coincided with the increase in the levels of autophagy marker microtubule-associated protein 1 light-chain 3B. MPP+ also increased the concentration of a selective autophagic target sequestosome-1/p62 and reduced the levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification, suggesting that MPP+-induced autophagy was coupled with a decrease in autophagic flux. Nevertheless, further pharmacological inhibition of autophagy sensitized SH-SY5Y cells to MPP+-induced death. Antioxidants and AMPK knockdown reduced, whereas genetic inactivation of Akt potentiated neurotoxin-triggered autophagy. These results suggest that MPP+-induced oxidative stress stimulates AMPK, which protects SH-SY5Y cells through early activation of antioxidative Akt and late induction of cytoprotective autophagy.

Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors.

We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling.