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1.
Eur J Pharmacol ; 568(1-3): 89-98, 2007 Jul 30.
Article in English | MEDLINE | ID: mdl-17560995

ABSTRACT

Using the rat glioma cell line C6 and the human glioma cell line U251, we demonstrate the multiple mechanisms underlying the in vitro anticancer effects of the C(60) fullerene water suspension (nano-C(60) or nC(60)) produced by solvent exchange method. Nano-C(60) in a dose-dependent manner reduced the tumor cell numbers after 24 h of incubation. The observed antiglioma action of nC(60) at high concentration (1 microg/ml) was due to a reactive oxygen species-mediated necrotic cell damage that was partly dependent on oxidative stress-induced activation of extracellular signal-regulated kinase (ERK). On the other hand, low-dose nC(60) (0.25 microg/ml) did not induce either necrotic or apoptotic cell death, but caused oxidative stress/ERK-independent cell cycle block in G(2)/M phase and subsequent inhibition of tumor cell proliferation. Treatment with either high-dose or low-dose nC(60) caused the appearance of acidified intracytoplasmic vesicles indicative of autophagy, but only the antiglioma effect of low-dose nC(60) was significantly attenuated by inhibiting autophagy with bafilomycin A1. Importantly, primary rat astrocytes were less sensitive than their transformed counterparts to a cytostatic action of low-dose nC(60). These data provide grounds for further development of nC(60) as an anticancer agent.


Subject(s)
Antineoplastic Agents/pharmacology , Fullerenes/pharmacology , Glioma/drug therapy , Nanoparticles , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Glial Fibrillary Acidic Protein/metabolism , Glioma/metabolism , Humans , Lipid Peroxidation/drug effects , Mitogen-Activated Protein Kinases/metabolism , Rats , Reactive Oxygen Species/metabolism
2.
Biomaterials ; 27(29): 5049-58, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16784774

ABSTRACT

We investigated the effect of gamma-irradiation on the cytotoxicity of pure C60 solubilized in water by using tetrahydrofuran (THF/n-C60 or THF/n-C60). In contrast to THF/n-C60, its gamma-irradiated counterpart failed to generate oxygen radicals and cause extracellular signal-regulated kinase (ERK)-dependent necrotic cell death in various types of mammalian cells. Moreover, gamma-irradiated THF/n-C60 protected cells from the oxidative stress induced by native THF/n-C60 or hydrogen peroxide. The observed biological effects were associated with gamma-irradiation-mediated decomposition of THF and subsequent derivatization of the n-C60 surface. These results for the first time demonstrate gamma-irradiation-mediated changes in the physico-chemical properties of THF-prepared nanocrystalline C60, resulting in a complete loss of its cytotoxic effect and its conversion to a cytoprotective agent.


Subject(s)
Fullerenes/toxicity , Gamma Rays , Nanostructures , Animals , Cell Line, Tumor , Fullerenes/radiation effects , Furans/radiation effects , Furans/toxicity , Mice
3.
Toxicol Sci ; 91(1): 173-83, 2006 May.
Article in English | MEDLINE | ID: mdl-16476688

ABSTRACT

The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C60) and water-soluble polyhydroxylated fullerene [C60(OH)n] were investigated. Crystal violet assay for cell viability demonstrated that nano-C60 was at least three orders of magnitude more toxic than C60(OH)n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V-fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C60 caused rapid (observable after few hours), reactive oxygen species (ROS)-associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C60(OH)n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C60 toxicity, but not C60(OH)n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C60(OH)n-induced apoptosis, but not nano-C60-mediated necrosis. Finally, C60(OH)n antagonized, while nano-C60 synergized with, the cytotoxic action of oxidative stress-inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.


Subject(s)
Fullerenes/pharmacology , Animals , Cell Line, Tumor , Drug Screening Assays, Antitumor , Flow Cytometry , Humans , Hydroxylation , Mice , Rats , Reactive Oxygen Species
4.
Ann N Y Acad Sci ; 1048: 400-5, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16154962

ABSTRACT

Treatment with 6-hydroxydopamine significantly reduced the viability of cultured rat primary astrocytes, rat astrocytoma cell line C6, and human astrocytoma cell line U251. 6-Hydroxydopamine-treated astrocytes exhibited altered nuclear morphology, DNA fragmentation, and reduced intracellular esterase activity, which indicated apoptotic cell death. Astrocytes were protected by neutralization of 6-hydroxydopamine autooxidation products H(2)O(2), O(2)(*-), and (*)OH, but not by cell-derived or chemically generated anti-apoptotic free radical nitric oxide. Finally, 6-hydroxydopamine activated extracellular signal-regulated kinase in astrocytes and selective inhibitor of extracellular signal-regulated kinase activation partially prevented astrocyte death. Taken together, these data indicate that 6-hydroxydopamine-triggered oxidative stress induces extracellular signal-regulated kinase-dependent apoptotic death of astrocytes.


Subject(s)
Astrocytes/drug effects , DNA Fragmentation/drug effects , Esterases/metabolism , Oxidative Stress/drug effects , Oxidopamine/toxicity , Adrenergic Agents/pharmacology , Animals , Astrocytes/cytology , Astrocytes/ultrastructure , Cell Death/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Free Radicals/metabolism , Humans , Nitric Oxide/metabolism , Rats , Time Factors
5.
Neuropharmacology ; 48(5): 720-31, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15814106

ABSTRACT

The role of iron in 6-hydroxydopamine (6-OHDA) toxicity towards astrocytes was investigated in vitro using rat primary astrocytes, rat astrocytoma cell line C6, and human astrocytoma cell line U251. The assessment of mitochondrial respiration or lactate dehydrogenase release has shown a dose-dependent decrease in the viability of astrocytes treated with 6-OHDA, which coincided with DNA fragmentation and the changes in cellular morphology. This was a consequence of the oxidative stress mediated by 6-OHDA autoxidation products hydrogen peroxide, superoxide anion, and hydroxyl radical. Both FeSO(4) and FeCl(3) markedly alleviated detrimental effects of 6-OHDA treatment, while MgSO(4) was without effect. The protective action of iron was neutralized by a membrane-permeable iron chelator o-phenanthroline, which also augmented astrocyte killing in the absence of exogenous iron. The mechanisms responsible for iron-mediated protection of astrocytes did not involve interference with either 6-OHDA autoxidation, hydrogen peroxide toxicity, or 6-OHDA-induced activation of extracellular signal-regulated kinase. Finally, the addition of iron potentiated and its chelation blocked 6-OHDA toxicity towards neuronal PC12 cells, suggesting the opposite roles for this transition metal in regulating the survival of astrocytes and dopaminergic neurons.


Subject(s)
Astrocytes/drug effects , Hydroxydopamines/toxicity , Iron/pharmacology , Animals , Animals, Newborn , Cell Adhesion Molecules/metabolism , Cell Survival/drug effects , Cells, Cultured , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Dose-Response Relationship, Drug , Drug Interactions , Ferric Compounds/pharmacology , Flow Cytometry/methods , Iron Chelating Agents/pharmacology , Junctional Adhesion Molecules , L-Lactate Dehydrogenase/metabolism , PC12 Cells/drug effects , PC12 Cells/metabolism , Phenanthrolines/pharmacology , Rats , Rats, Wistar , Staining and Labeling/methods , Tetrazolium Salts , Thiazoles , Time Factors
6.
J Mol Neurosci ; 22(3): 199-204, 2004.
Article in English | MEDLINE | ID: mdl-14997013

ABSTRACT

Alpha-synuclein is a small presynaptic protein associated with both normal synaptic plasticity and neurodegenerative processes. Its normal cellular function, however, remains unknown. Even though it is highly enriched in the brain, its presence was reported in other human adult tissues. In the present study, we examined tissue expression of alpha-synuclein in human and rat prenatal development. Using Western blot analysis, various peripheral tissues from 15 to 23 gestational weeks, human and E19 rat fetuses, along with human and rat adult tissues, were assayed. alpha-Synuclein expression was observed in all fetal human organs examined. In adult human tissues the high expression of alpha-synuclein was maintained in the brain, whereas in other organs the expression was greatly reduced. In contrast, both in fetal and adult rat tissues, alpha-synuclein was only detected in the brain. In addition to a 19-kDa alpha-synuclein band, 36- and 52-kDa immunoreactive bands were observed in all fetal and adult human organs, with the exception of the brain, but their identity remains to be determined. These findings suggest that apart from its function in development of the nervous system, alpha-synuclein has an important function in peripheral tissues as well during normal human prenatal development.


Subject(s)
Brain Chemistry/genetics , Brain/metabolism , Nerve Tissue Proteins/metabolism , Viscera/metabolism , Adrenal Glands/cytology , Adrenal Glands/embryology , Adrenal Glands/metabolism , Animals , Blotting, Western , Brain/cytology , Brain/embryology , Cell Differentiation/physiology , Fetus , Humans , Kidney/cytology , Kidney/embryology , Kidney/metabolism , Liver/cytology , Liver/embryology , Liver/metabolism , Lung/cytology , Lung/embryology , Lung/metabolism , Male , Myocardium/cytology , Myocardium/metabolism , Neuronal Plasticity/physiology , Organ Specificity/physiology , Rats , Species Specificity , Synucleins , Testis/cytology , Testis/embryology , Testis/metabolism , Up-Regulation/physiology , Viscera/cytology , Viscera/embryology , alpha-Synuclein
7.
Brain Res ; 996(2): 237-45, 2004 Jan 23.
Article in English | MEDLINE | ID: mdl-14697501

ABSTRACT

This study was focused on the apoptosis (programmed cell death) induction involved in the loss of dopaminergic (DA-ergic) neurons in 6-hydroxydopamine (6-OHDA) hemiparkinsonian rats. The apoptosis in the striatum and substantia nigra pars compacta (SNpc), was examined 6, 24 h and 7 days after the 6-OHDA lesions employing the TUNEL method. The changes in mRNA levels of pro-apoptotic protein tumor necrosis factor alpha (TNFalpha) and its "death receptor" TNFalphaRI and then bax mRNA, as an important regulator of apoptotic neurodegeneration were followed by RT-PCR procedure. In situ analysis revealed an increased number of TUNEL-positive neurons in 6-OHDA-treated animals in all examined time points. The highest number of apoptotic neurons was detected 24 h after the lesion, both in the ipsilateral striatum (3.41+/-0.18) and SNpc (5.8+/-0.79). A significant increase in the level of TNFalpha mRNA was observed in 6-OHDA-lesioned striatum, with maximal value after 24 h (46%) comparing to the control. In contrast, 6-OHDA did not significantly change the level of TNFalphaRI mRNA in any time point. Six and 24 h post-operatively, we observed a significant increase of bax mRNA expression (40% and 45%, respectively) in the ipsilateral striatum of treated animals in comparison with the right striatum of the controls. However, the highest level of the bax mRNA expression was reached 7 days after the surgery (94%) in the ipsilateral striatum of 6-OHDA-treated animals. These results suggest that striatal injection of 6-OHDA can induce early changes that would be an important regulator of apoptotic neurodegeneration of dopamine-producing neurons, during the first post-operative week.


Subject(s)
Adrenergic Agents/pharmacology , Apoptosis/physiology , Corpus Striatum/drug effects , Oxidopamine/pharmacology , Parkinsonian Disorders/pathology , Proto-Oncogene Proteins c-bcl-2 , Animals , Corpus Striatum/pathology , Dopamine/metabolism , Functional Laterality , Immunohistochemistry , In Situ Nick-End Labeling , Male , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Neurons/drug effects , Neurons/pathology , Neurons/physiology , Proto-Oncogene Proteins/drug effects , RNA, Messenger/analysis , RNA, Messenger/drug effects , Rats , Receptors, Tumor Necrosis Factor/drug effects , Receptors, Tumor Necrosis Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Necrosis Factor-alpha/drug effects , Tyrosine 3-Monooxygenase/metabolism , bcl-2-Associated X Protein
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