The mechanism of cytotoxic and cytoprotective effects of fullerene (C60) nanoparticles

U ovoj doktorskoj disertaciji ispitivani su citotoksični efekti koloidnih rastvorafulerenskih nanočestica, kao i uticaj mehanohemijski pripremljenih fulerena nacitotoksičnost azot monoksida. Zbog sposobnosti da indukuju ćelijsku smrt u određenimuslovima, fulereni C60 predstavljaju potencijalne antitumorske i toksične agense.Koloidni rastvor kristalnog C60 (nC60) je izuzetno toksičan, ali mehanizmi njegovecitotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena sutri različita načina pripreme nC60 rastvora: 1. metodom izmene rastvarača utetrahidrofuranu (THF/nC60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi(H2O/nC60). Kombinovanjem eksperimentalnih analiza i matematičkog modelovanjaispitivani su uslovi pod kojim različiti rastvori nC60 ispoljavaju citotoksičnostposredovanu reaktivnim kiseoničkim vrstama (RKV). Prema sposobnosti da generišuRKV i izazovu mitohondrijalnu depolarizaciju praćenu nekrozom, rastvori nC60 surangirani sledećim redosledom: THF/nC60 > EtOH/nC60 > H2O/nC60. Matematičkomodelovanje produkcije singletnog kiseonika (1O2) pokazuje da sposobnost rastvaračaugrađenog u strukturu fulerenskog kristala da neutrališe 1O2 (THF/nC60 EtOH/nC60 > aqu/nC60. Mathematical modeling of singlet oxygen(1O2) generation indicates that the 1O2-quenching power (THF/ nC60 < EtOH/nC60<H2O/nC60) of the solvent intercalated in the fullerene crystals determines their abilityto produce ROS and cause cell damage. These results could have important implicationsfor the toxicology and biomedical application of colloidal fullerenes.Fullerenes are molecules with dual properties, able to produce ROS whenphotoexcited, but also to scavenge free radicals and exert antioxidant action. In order toassess the latter, the influence of fullerene C60 nanoparticles on the cytotoxicity of ahighly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticleswere prepared by mechanochemically assisted complexation with anionic surfactantsodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymerethylene vinyl acetate–ethylene vinyl versatate, and characterized by UV–vis andatomic force microscopy (AFM)..

Autophagy receptor p62 regulates SARS-CoV-2-induced inflammation in COVID-19

Introduction: Since the interaction between autophagy and virus-induced inflammation is complex, we investigated the interplay between autophagy and inflammation in COVID-19 patients and THP-1 cells expressing SARS-Cov2 proteins NSP5 and ORF3a. Methods: Autophagy markers in blood from 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 level of p62 in cells and its concentration in cell culture supernatants was measured by immunoblot/ELISA. The mRNA levels of proinflammatory cytokines were measured by RT-qPCR. Results: IFN-α, TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, whereas IL-10 and IL-1β were elevated at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unchanged in COVID-19. 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 cells caused an autophagy-independent decrease/autophagy-inhibition-dependent increase of intracellular and secreted p62. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, while a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. Conclusion: The autophagy receptor p62 is reduced in acute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation

The mechanism of cytotoxic and cytoprotective effects of fullerene (C60) nanoparticles

U ovoj doktorskoj disertaciji ispitivani su citotoksični efekti koloidnih rastvora fulerenskih nanočestica, kao i uticaj mehanohemijski pripremljenih fulerena na citotoksičnost azot monoksida. Zbog sposobnosti da indukuju ćelijsku smrt u određenim uslovima, fulereni C60 predstavljaju potencijalne antitumorske i toksične agense. Koloidni rastvor kristalnog C60 (nC60) je izuzetno toksičan, ali mehanizmi njegove citotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena su tri različita načina pripreme nC60 rastvora: 1. metodom izmene rastvarača u tetrahidrofuranu (THF/nC60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi (H2O/nC60). Kombinovanjem eksperimentalnih analiza i matematičkog modelovanja ispitivani su uslovi pod kojim različiti rastvori nC60 ispoljavaju citotoksičnost posredovanu reaktivnim kiseoničkim vrstama (RKV). Prema sposobnosti da generišu RKV i izazovu mitohondrijalnu depolarizaciju praćenu nekrozom, rastvori nC60 su rangirani sledećim redosledom: THF/nC60 > EtOH/nC60 > H2O/nC60. Matematičko modelovanje produkcije singletnog kiseonika (1O2) pokazuje da sposobnost rastvarača ugrađenog u strukturu fulerenskog kristala da neutrališe 1O2 (THF/nC60 < EtOH/nC60 < H2O/nC60) predstavlja presudni faktor koji određuje sposobnost nanočestice da proizvodi RKV i izaziva ćelijsko oštećenje. Ovi rezultati skreću pažnju na toksikološki aspekt koloidnih fulerena u njihovoj potencijalnoj upotrebi u biomedicini. Fulereni imaju dvojnu prirodu tako da osim što proizvode RKV kada su pobuđeni vidljivom svetlošću, oni deluju i kao ,,skupljači“ slobodnih radikala i ispoljavaju značajna antioksidativna svojstva. Iz tog razloga je, u drugom delu studije, ispitivan uticaj fulerenskih nanočestica na citotoksičnost azot monoksida (NO), veoma reaktivnog slobodnog radikala. Fulerenske nanočestice su pripremljene mehanohemijski potpomognutom kompleksacijom uz korišćenje anjonskog surfaktanta natrijum dodecil sulfata (SDS), makrocikličnog oligosaharidγa -ciklodekstrina (γCDX) ili kopolimera acetat-etilen vinil versatata (EVA-EVV) i karakterisane metodama UV-vis spektroskopije i mikroskopijom atomskih sila (AFM)...In this doctoral dissertation we investigated cytotoxic effects of fullerene colloidal suspensions, as well as the influence of mechanochemically synthesized fullerene C60 nanoparticles on the cytotoxicity of nitric oxide (NO). Because of their ability to induce cell death in certain conditions, fullerenes are potential toxic and anticancer agents. Colloidal suspension of crystalline C60 (nC60) is very toxic, but the mechanisms of its cytotoxicity are not completely explained. By combining experimental analysis and mathematical modeling, the requirements for the reactive oxygen species (ROS)- mediated cytotoxicity of different nC60 suspensions were investigated. Colloidal suspensions of nC60 were prepared by solvent exchange method in tetrahydrofuran (THF/nC60) and ethanol (EtOH/nC60), or by extended mixing in water (H2O/nC60). With regard to their capacity to generate RKV and cause mitochondrial depolarization followed by necrotic cell death, the nC60 suspensions were ranked in the following order: THF/nC60 > EtOH/nC60 > aqu/nC60. Mathematical modeling of singlet oxygen (1O2) generation indicates that the 1O2-quenching power (THF/ nC60 < EtOH/nC6 0<H2O/nC60) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These results could have important implications for the toxicology and biomedical application of colloidal fullerenes. Fullerenes are molecules with dual properties, able to produce ROS when photoexcited, but also to scavenge free radicals and exert antioxidant action. In order to assess the latter, the influence of fullerene C60 nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymer ethylene vinyl acetate–ethylene vinyl versatate, and characterized by UV–vis and atomic force microscopy (AFM)..

The mechanism of cytotoxic and cytoprotective effects of fullerene (C60) nanoparticles

U ovoj doktorskoj disertaciji ispitivani su citotoksični efekti koloidnih rastvorafulerenskih nanočestica, kao i uticaj mehanohemijski pripremljenih fulerena nacitotoksičnost azot monoksida. Zbog sposobnosti da indukuju ćelijsku smrt u određenimuslovima, fulereni C60 predstavljaju potencijalne antitumorske i toksične agense.Koloidni rastvor kristalnog C60 (nC60) je izuzetno toksičan, ali mehanizmi njegovecitotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena sutri različita načina pripreme nC60 rastvora: 1. metodom izmene rastvarača utetrahidrofuranu (THF/nC60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi(H2O/nC60). Kombinovanjem eksperimentalnih analiza i matematičkog modelovanjaispitivani su uslovi pod kojim različiti rastvori nC60 ispoljavaju citotoksičnostposredovanu reaktivnim kiseoničkim vrstama (RKV). Prema sposobnosti da generišuRKV i izazovu mitohondrijalnu depolarizaciju praćenu nekrozom, rastvori nC60 surangirani sledećim redosledom: THF/nC60 > EtOH/nC60 > H2O/nC60. Matematičkomodelovanje produkcije singletnog kiseonika (1O2) pokazuje da sposobnost rastvaračaugrađenog u strukturu fulerenskog kristala da neutrališe 1O2 (THF/nC60 EtOH/nC60 > aqu/nC60. Mathematical modeling of singlet oxygen(1O2) generation indicates that the 1O2-quenching power (THF/ nC60 < EtOH/nC60<H2O/nC60) of the solvent intercalated in the fullerene crystals determines their abilityto produce ROS and cause cell damage. These results could have important implicationsfor the toxicology and biomedical application of colloidal fullerenes.Fullerenes are molecules with dual properties, able to produce ROS whenphotoexcited, but also to scavenge free radicals and exert antioxidant action. In order toassess the latter, the influence of fullerene C60 nanoparticles on the cytotoxicity of ahighly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticleswere prepared by mechanochemically assisted complexation with anionic surfactantsodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymerethylene vinyl acetate–ethylene vinyl versatate, and characterized by UV–vis andatomic force microscopy (AFM)..

The mechanism of cytotoxic and cytoprotective effects of fullerene (C60) nanoparticles

U ovoj doktorskoj disertaciji ispitivani su citotoksični efekti koloidnih rastvora fulerenskih nanočestica, kao i uticaj mehanohemijski pripremljenih fulerena na citotoksičnost azot monoksida. Zbog sposobnosti da indukuju ćelijsku smrt u određenim uslovima, fulereni C60 predstavljaju potencijalne antitumorske i toksične agense. Koloidni rastvor kristalnog C60 (nC60) je izuzetno toksičan, ali mehanizmi njegove citotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena su tri različita načina pripreme nC60 rastvora: 1. metodom izmene rastvarača u tetrahidrofuranu (THF/nC60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi (H2O/nC60). Kombinovanjem eksperimentalnih analiza i matematičkog modelovanja ispitivani su uslovi pod kojim različiti rastvori nC60 ispoljavaju citotoksičnost posredovanu reaktivnim kiseoničkim vrstama (RKV). Prema sposobnosti da generišu RKV i izazovu mitohondrijalnu depolarizaciju praćenu nekrozom, rastvori nC60 su rangirani sledećim redosledom: THF/nC60 > EtOH/nC60 > H2O/nC60. Matematičko modelovanje produkcije singletnog kiseonika (1O2) pokazuje da sposobnost rastvarača ugrađenog u strukturu fulerenskog kristala da neutrališe 1O2 (THF/nC60 < EtOH/nC60 < H2O/nC60) predstavlja presudni faktor koji određuje sposobnost nanočestice da proizvodi RKV i izaziva ćelijsko oštećenje. Ovi rezultati skreću pažnju na toksikološki aspekt koloidnih fulerena u njihovoj potencijalnoj upotrebi u biomedicini. Fulereni imaju dvojnu prirodu tako da osim što proizvode RKV kada su pobuđeni vidljivom svetlošću, oni deluju i kao ,,skupljači“ slobodnih radikala i ispoljavaju značajna antioksidativna svojstva. Iz tog razloga je, u drugom delu studije, ispitivan uticaj fulerenskih nanočestica na citotoksičnost azot monoksida (NO), veoma reaktivnog slobodnog radikala. Fulerenske nanočestice su pripremljene mehanohemijski potpomognutom kompleksacijom uz korišćenje anjonskog surfaktanta natrijum dodecil sulfata (SDS), makrocikličnog oligosaharidγa -ciklodekstrina (γCDX) ili kopolimera acetat-etilen vinil versatata (EVA-EVV) i karakterisane metodama UV-vis spektroskopije i mikroskopijom atomskih sila (AFM)...In this doctoral dissertation we investigated cytotoxic effects of fullerene colloidal suspensions, as well as the influence of mechanochemically synthesized fullerene C60 nanoparticles on the cytotoxicity of nitric oxide (NO). Because of their ability to induce cell death in certain conditions, fullerenes are potential toxic and anticancer agents. Colloidal suspension of crystalline C60 (nC60) is very toxic, but the mechanisms of its cytotoxicity are not completely explained. By combining experimental analysis and mathematical modeling, the requirements for the reactive oxygen species (ROS)- mediated cytotoxicity of different nC60 suspensions were investigated. Colloidal suspensions of nC60 were prepared by solvent exchange method in tetrahydrofuran (THF/nC60) and ethanol (EtOH/nC60), or by extended mixing in water (H2O/nC60). With regard to their capacity to generate RKV and cause mitochondrial depolarization followed by necrotic cell death, the nC60 suspensions were ranked in the following order: THF/nC60 > EtOH/nC60 > aqu/nC60. Mathematical modeling of singlet oxygen (1O2) generation indicates that the 1O2-quenching power (THF/ nC60 < EtOH/nC6 0<H2O/nC60) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These results could have important implications for the toxicology and biomedical application of colloidal fullerenes. Fullerenes are molecules with dual properties, able to produce ROS when photoexcited, but also to scavenge free radicals and exert antioxidant action. In order to assess the latter, the influence of fullerene C60 nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymer ethylene vinyl acetate–ethylene vinyl versatate, and characterized by UV–vis and atomic force microscopy (AFM)..

The mechanism of cytotoxic and cytoprotective effects of fullerene (C60) nanoparticles

U ovoj doktorskoj disertaciji ispitivani su citotoksični efekti koloidnih rastvora fulerenskih nanočestica, kao i uticaj mehanohemijski pripremljenih fulerena na citotoksičnost azot monoksida. Zbog sposobnosti da indukuju ćelijsku smrt u određenim uslovima, fulereni C60 predstavljaju potencijalne antitumorske i toksične agense. Koloidni rastvor kristalnog C60 (nC60) je izuzetno toksičan, ali mehanizmi njegove citotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena su tri različita načina pripreme nC60 rastvora: 1. metodom izmene rastvarača u tetrahidrofuranu (THF/nC60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi (H2O/nC60). Kombinovanjem eksperimentalnih analiza i matematičkog modelovanja ispitivani su uslovi pod kojim različiti rastvori nC60 ispoljavaju citotoksičnost posredovanu reaktivnim kiseoničkim vrstama (RKV). Prema sposobnosti da generišu RKV i izazovu mitohondrijalnu depolarizaciju praćenu nekrozom, rastvori nC60 su rangirani sledećim redosledom: THF/nC60 > EtOH/nC60 > H2O/nC60. Matematičko modelovanje produkcije singletnog kiseonika (1O2) pokazuje da sposobnost rastvarača ugrađenog u strukturu fulerenskog kristala da neutrališe 1O2 (THF/nC60 < EtOH/nC60 < H2O/nC60) predstavlja presudni faktor koji određuje sposobnost nanočestice da proizvodi RKV i izaziva ćelijsko oštećenje. Ovi rezultati skreću pažnju na toksikološki aspekt koloidnih fulerena u njihovoj potencijalnoj upotrebi u biomedicini. Fulereni imaju dvojnu prirodu tako da osim što proizvode RKV kada su pobuđeni vidljivom svetlošću, oni deluju i kao ,,skupljači“ slobodnih radikala i ispoljavaju značajna antioksidativna svojstva. Iz tog razloga je, u drugom delu studije, ispitivan uticaj fulerenskih nanočestica na citotoksičnost azot monoksida (NO), veoma reaktivnog slobodnog radikala. Fulerenske nanočestice su pripremljene mehanohemijski potpomognutom kompleksacijom uz korišćenje anjonskog surfaktanta natrijum dodecil sulfata (SDS), makrocikličnog oligosaharidγa -ciklodekstrina (γCDX) ili kopolimera acetat-etilen vinil versatata (EVA-EVV) i karakterisane metodama UV-vis spektroskopije i mikroskopijom atomskih sila (AFM)...In this doctoral dissertation we investigated cytotoxic effects of fullerene colloidal suspensions, as well as the influence of mechanochemically synthesized fullerene C60 nanoparticles on the cytotoxicity of nitric oxide (NO). Because of their ability to induce cell death in certain conditions, fullerenes are potential toxic and anticancer agents. Colloidal suspension of crystalline C60 (nC60) is very toxic, but the mechanisms of its cytotoxicity are not completely explained. By combining experimental analysis and mathematical modeling, the requirements for the reactive oxygen species (ROS)- mediated cytotoxicity of different nC60 suspensions were investigated. Colloidal suspensions of nC60 were prepared by solvent exchange method in tetrahydrofuran (THF/nC60) and ethanol (EtOH/nC60), or by extended mixing in water (H2O/nC60). With regard to their capacity to generate RKV and cause mitochondrial depolarization followed by necrotic cell death, the nC60 suspensions were ranked in the following order: THF/nC60 > EtOH/nC60 > aqu/nC60. Mathematical modeling of singlet oxygen (1O2) generation indicates that the 1O2-quenching power (THF/ nC60 < EtOH/nC6 0<H2O/nC60) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These results could have important implications for the toxicology and biomedical application of colloidal fullerenes. Fullerenes are molecules with dual properties, able to produce ROS when photoexcited, but also to scavenge free radicals and exert antioxidant action. In order to assess the latter, the influence of fullerene C60 nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymer ethylene vinyl acetate–ethylene vinyl versatate, and characterized by UV–vis and atomic force microscopy (AFM)..

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C-60) nanoparticles

The influence of fullerene (C-60) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide gamma-cyclodextrin or the copolymer ethylene vinyl acetate-ethylene vinyl versatate. C-60 nanoparticles were characterized by UV-vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C-60 nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C-60 nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C-60 nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C-60 complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders. (C) 2009 Elsevier Ltd. All rights reserved

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 mu g), metformin (50, 100 or 200 mu g), 5-amino-imidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR, 25 mu g) and L-Ieucine (1 mu g) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, 56K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting 56K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus. Copyright (c) 2012 S. Karger AG, BaselMinistry of Science and Technological Development of the Republic of Serbia [41025, 175067

Arylpiperazine-mediated activation of Akt protects SH-SY5Y neuroblastoma cells from 6-hydroxydopamine-induced apoptotic and autophagic death

We investigated the ability of 19 recently synthesized arylpiperazine compounds to protect human SH-SY5Y neuroblastoma cells from the neurotoxin 6-hydroxydopamine (6-OHDA). The compound with the most potent neuroprotective action was N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), which reduced 6-OHDA-induced apoptotic death through stabilization of mitochondrial membrane and subsequent prevention of superoxide production, caspase activation and DNA fragmentation. 6-OHDA-triggered autophagic response was also reduced by 6b, which prevented inactivation of the main autophagy repressor mTOR, upregulation of proautophagic beclin-1, conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to autophagosome-associAed LC3-II, as well as intracytoplasmic acidification induced by 6-OHDA. The inhibition of autophagy using LC3 beta gene silencing or pharmacological autophagy blockers 3-methyladenine or bafilomycin A1, mimicked the cytoprotective effect of 6b. While the treatment with 6b had no effect on the phosphorylation of proapoptotic MAP kinases ERR and JNK, it markedly increased the phosphorylation of the prosurvival kinase Akt in 6-OHDA-treated cells. Akt inhibitor DEBC or RNA interference-mediated Akt silencing reduced the ability of 6b to block 6-0HDA-triggered apoptotic and autophagic responses, thus confirming their dependency on Akt activation. The cytoprotective effect of 6b was also observed in 6-OHDA-treated neuronal PC12 cells, but not in SH-SY5Y or PC12 cells exposed to 1-methyl-4-phenylpyridinium, indicating that the observed neuroprotection was dependent on the cytotoxic stimulus. Because of the ability to prevent 6-OHDA induced apoptotic/autophagic cell death through activation of Akt, the investigated arylpiperazines could be potential candidates for treatment of neurodegenerative diseases