The present study investigated the hemolytic properties of fullerene (C(60)) nanoparticles prepared by solvent exchange using tetrahydrofuran (nC(60)THF), or by mechanochemically assisted ...complexation with macrocyclic oligosaccharide gamma-cyclodextrin (nC(60)CDX) or the copolymer ethylene vinyl acetate-ethylene vinyl versatate (nC(60)EVA-EVV). The spectrophotometrical analysis of hemoglobin release revealed that only nC(60)THF, but not nC(60)CDX or nC(60)EVA-EVV, was able to cause lysis of human erythrocytes in a dose- and time-dependent manner. Atomic force microscopy revealed that nC(60)THF-mediated hemolysis was preceded by erythrocyte shrinkage and increase in cell surface roughness. A flow cytometric analysis confirmed a decrease in erythrocyte size and demonstrated a significant increase in reactive oxygen species production in red blood cells exposed to nC(60)THF. The nC(60)THF-triggered hemolytic activity was efficiently reduced by the antioxidants N-acetylcysteine and butylated hydroxyanisole, as well as by serum albumin, the most abundant protein in human blood plasma. These data indicate that nC(60)THF can cause serum albumin-preventable hemolysis through oxidative stress-mediated damage of the erythrocyte membrane.
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.
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.
We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant ...hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. In vitro, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect.
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.
The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible ...nitric oxide (NO) synthase (iNOS; for gene
Nos2
) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 μl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO,
l
-arginine (
l
-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of
Nos2
mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of
l
-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased
Nos2
gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.
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.
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