We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in ...human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.
In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC
60) on tumour cell growth in vitro and in vivo. NanoC
60 suspension was prepared by solvent exchange using ...tetrahydrofuran to dissolve C
60. In vitro, nanoC
60 caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (
125I) nanoC
60 readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC
60 over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC
60 was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC
60-injected mice. These data demonstrate that nanoC
60, in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.
In vitro and in vivo anti-melanoma action of metformin Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Misirkic-Marjanovic, Maja ...
European journal of pharmacology,
10/2011, Letnik:
668, Številka:
3
Journal Article
Recenzirano
The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G
2/M cell cycle arrest associated with apoptotic ...death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. Treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21- and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.
Display omitted
Purpose
The fullerene (C
60
/C
70
mixture—C
60/70
) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/
n
C
60/70
) and polyhydroxylated C
60/70
C
60/70
(OH)
n
...were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF).
Materials and Methods
TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes.
Results
THF/
n
C
60/70
augmented, while C
60/70
(OH)
n
reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/
n
C
60/70
or C
60/70
(OH)
n
. The antioxidant
N
-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/
n
C
60/70
or C
60/70
(OH)
n
potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization.
Conclusion
The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.
Abstract
We explored the interplay between the intracellular energy sensor
AMP
‐activated protein kinase (
AMPK
), extracellular signal‐regulated kinase (
ERK
), and autophagy in phorbol myristate ...acetate (
PMA
)‐induced neuronal differentiation of
SH
‐
SY
5Y human neuroblastoma cells.
PMA
‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (
LC
3)‐I to autophagosome‐bound
LC
3‐
II
, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of
AMPK
and sustained activation of
ERK
. Pharmacological inhibition or
RNA
interference‐mediated silencing of
AMPK
suppressed
PMA
‐induced expression of neuronal markers, as well as
ERK
activation and autophagy. A selective pharmacological blockade of
ERK
prevented
PMA
‐induced neuronal differentiation and autophagy induction without affecting
AMPK
phosphorylation. Conversely, the inhibition of autophagy downstream of
AMPK
/
ERK
, either by pharmacological agents or
LC
3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of
PMA
‐induced differentiation of
SH
‐
SY
5Y cells. Therefore,
PMA
‐induced neuronal differentiation of
SH
‐
SY
5Y cells depends on a complex interplay between
AMPK
,
ERK
, and autophagy, in which the stimulatory effects of
AMPK
/
ERK
signaling are counteracted by the coinciding autophagic response.
image
Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule‐associated protein 2, and β‐tubulin, and subsequent neuronal differentiation of SH‐SY5Y neuroblastoma cells through AMP‐activated protein kinase (AMPK)‐dependent activation of extracellular signal‐regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin‐1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.
In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC(60)) on tumour cell growth in vitro and in vivo. NanoC(60) suspension was prepared by solvent exchange ...using tetrahydrofuran to dissolve C(60). In vitro, nanoC(60) caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (125I) nanoC(60) readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC(60) over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC(60) was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC(60)-injected mice. These data demonstrate that nanoC(60), in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.
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 C 60 predstavljaju potencijalne antitumorske i toksične agense. Koloidni rastvor kristalnog C 60 (nC60) je izuzetno toksičan, ali mehanizmi njegove citotoksičnosti još uvek nisu dovoljno ispitani ni razjašnjeni. U ovoj studiji korišćena su tri različita načina pripreme nC 60 rastvora: 1. metodom izmene rastvarača u tetrahidrofuranu (THF/nC 60), 2. etanolu (EtOH/nC60) ili 3. produženim mešanjem u vodi (H2O/nC 60). 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 nC 60 su rangirani sledećim redosledom: THF/nC 60 > EtOH/nC 60 > H2O/nC 60. Matematičko modelovanje produkcije singletnog kiseonika (1 O 2) pokazuje da sposobnost rastvarača ugrađenog u strukturu fulerenskog kristala da neutrališe 1 O 2 (THF/nC 60 < EtOH/nC 60 < H2O/nC 60) 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).Iako je pokazano da mišjifibroblasti L929 internalizuju C 60 nanočestice, one nisu bile citotoksične, već su idelimično štitile L929 ćelije od citotoksičnog efekta NO donora kao što su natrijumnitroprusid (SNP), S nitrozo-N-acetilpenicilamin (SNAP), S –nitrosoglutation (GSNO) i3-morfolino-sidonimin (SIN-1). C 60 nanočestice su smanjile apoptotsku smrt ćelijatretiranih NO donorom inhibicijom depolarizacije mitohondrijalne membrane, aktivacijekaspaza i posledične fragmentacije DNK. Protektivno dejstvo nanočestica C 60 nijezavisilo od direktne interakcije sa NO-om, nego od neutralizacije superoksid anjonanastalog u mitohondrijama ćelija tretiranih NO donorom, što je pokazano korišćenjemviše različitih redoks senzitivnih fluorescentnih boja. Ovi podaci ukazuju na mogućuupotrebu kompleksa C 60sa odgovarajućim molekulima u sprečavanju ćelijskihoštećenja izazvanih azot monoksidom u zapaljenskim i autoimunskim oboljenjima.
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