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Oxid Med Cell Longev ; 2020: 9260748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32377313


Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 µM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.

Anal Biochem ; 597: 113698, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32222539


Nitroxides and nitroxide-containing nanoparticles (RNP) are excellent antioxidants. However, they have relatively high reduction potentials, which make them behave like oxidants or show little activity in some antioxidant assays. We found that stable nitroxyl radicals (TEMPO and 4-amino-TEMPO) has low reactivity in the test of scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS•). As a result, supplementation of blood plasma with nitroxides may decrease its total antioxidant capacity assayed with ABTS•. Nitroxides oxidize Fe2+ and in this way interfere with the ferric-Xylenol Orange assay of peroxides. Nitroxides as well as RNP directly oxidize glutathione and fluorogenic probes used for estimation of reactive oxygen species (ROS) (dihydro-2'7'-dichlorofluorescein diacetate, dihydroethidine and dihydrorhodamine 123) and thus produce artefacts in assays of glutathione and ROS in cell-free and cellular systems. These results point to the necessity of careful interpretation of antioxidant assays concerning nitroxides and RNP or performed in their presence.

Aging (Albany NY) ; 12(2): 1910-1927, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31962290


The study was aimed at evaluation of the role of secondary oxidative stress in the stress-induced premature senescence (SIPS) of human fibroblasts induced by H2O2. Two fibroblast lines were used: lung MRC-5 and ear H8F2p25LM fibroblasts. The lines differed considerably in sensitivity to H2O2 (IC50 of 528 and 33.5 µM, respectively). The cells were exposed to H2O2 concentrations corresponding to IC50 and after 24 h supplemented with a range of antioxidants. Most of antioxidants studied slightly augmented the survival of fibroblasts at single concentrations or in a narrow concentration range, but the results were not consistent among the cell lines. Chosen antioxidants (4-amino-TEMPO, curcumin, caffeic acid and p-coumaric acid) did not restore the level of glutathione decreased by H2O2. Hydrogen peroxide treatment did not induce secondary production of H2O2 and even decreased it, decreased mitochondrial potential in both cell lines and induced changes in the mitochondrial mass inconsistent between the lines. Antioxidant protected mitochondrial potential only in H8F2p25LM cells, but attenuated changes in mitochondrial mass. These results speak against the intermediacy of secondary oxidative stress in the SIPS induced by H2O2 and suggest that the small protective action of antioxidants is due to their effects on mitochondria.

Molecules ; 24(20)2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31652556


Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.

Antineoplásicos/farmacologia , Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dendrímeros , Glioblastoma/tratamento farmacológico , Leucina/análogos & derivados , PPAR gama/agonistas , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biotinilação , Celecoxib/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Dendrímeros/metabolismo , Dinoprostona/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Leucina/farmacologia , Leucina/uso terapêutico , Necrose/tratamento farmacológico , PPAR gama/metabolismo , Proteína Supressora de Tumor p53/metabolismo
Life Sci ; 227: 212-223, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928407


AIMS: 3-Bromopyruvate (3-BP), an alkylating agent and a glycolytic inhibitor, is a promising anticancer agent, which can be efficient also against multidrug-resistant cancer cells. The aim of this study was to examine how 3-BP affects the survival and mobility of rat (MAT-LyLu and AT-2) and human (DU-145 and PC-3) metastatic prostate cancer cell lines. MAIN METHODS: Cytotoxicity was estimated with Neutral Red. Cell mobility was analyzed by time-lapse microscopic monitoring of trajectories of individual cells at 5-min intervals for 6h. ATP was estimated with luciferin/luciferase and glutathione (GSH) with o-phthalaldehyde. Actin cytoskeleton was visualized with phalloidin conjugated with Atto-488. KEY FINDINGS: All metastatic prostate cell lines studied were very sensitive to 3-BP (IC50 of 4-26µM). 3-Bromopyruvate drastically reduced cell movement even at concentrations of 5-10µM after 1h treatment. This compound depleted also cellular ATP and GSH, and disrupted actin cytoskeleton. SIGNIFICANCE: The data obtained suggest that 3-BP can potentially be useful for treatment of metastatic prostate cancer and, especially, be efficient in limiting metastasis.

Neoplasias da Próstata/tratamento farmacológico , Piruvatos/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Trifosfato de Adenosina/análise , Animais , Linhagem Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glutationa/análise , Humanos , Masculino , Invasividade Neoplásica , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ratos
Food Chem ; 278: 692-699, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30583431


Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H2O2) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H2O2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H2O2 production. Green tea also generated H2O2; tea prepared on tap water generated significantly more H2O2 than tea prepared on deionized water. Ascorbic acid decreased H2O2 production although it generated H2O2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H2O2. Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro.

Antioxidantes/química , Peróxido de Hidrogênio/química , Catalase/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Polifenóis/química , Galato de Propila/química , Galato de Propila/farmacologia , Quercetina/química , Quercetina/farmacologia , Chá/química , Chá/metabolismo , Elementos de Transição/química