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Redox regulation of metabolic and signaling pathways by thioredoxin and glutaredoxin in NOS-3 overexpressing hepatoblastoma cells.

González R, López-Grueso MJ, Muntané J, Bárcena JA, Padilla CA - Redox Biol (2015)

Bottom Line: Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO.Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and -8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells.These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new antitumoral therapies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain; Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain. Electronic address: raulangello@hotmail.com.

No MeSH data available.


Related in: MedlinePlus

Detection of nitrotyrosine levels in NOS-3 overexpressing HepG2 cells with Grx and Trx down regulation. The detection of this postranslational modification was performed by Western blot with the analysis focused on a representative 55 kDa band (arrow). The image is representative of three different experiments and densitometric data of n=3 independent experiments are presented in the histogram below as mean±SEM. The numbers 1, 2 and 3 indicate the cell treatments control, siRNAGrx and siRNATrx respectively. NOS-3 overexpression induced Tyr nitration of proteins in HepG2 cells (black bars). This effect was offset by treatment with siRNAGrx and siRNATrx as shown in the 4TO-NOS group. However, in WT and 4TO HepG2 cells siRNAGrx and siRNATrx treatment had the opposite effect, inducing Tyr nitration of proteins. Statistical significance was assessed by (p≤0.05). The values labeled with “a” were significantly different versus the corresponding internal control in the same cell lineage. The values labeled with “b” were significantly different versus the corresponding control cell lineage.
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f0030: Detection of nitrotyrosine levels in NOS-3 overexpressing HepG2 cells with Grx and Trx down regulation. The detection of this postranslational modification was performed by Western blot with the analysis focused on a representative 55 kDa band (arrow). The image is representative of three different experiments and densitometric data of n=3 independent experiments are presented in the histogram below as mean±SEM. The numbers 1, 2 and 3 indicate the cell treatments control, siRNAGrx and siRNATrx respectively. NOS-3 overexpression induced Tyr nitration of proteins in HepG2 cells (black bars). This effect was offset by treatment with siRNAGrx and siRNATrx as shown in the 4TO-NOS group. However, in WT and 4TO HepG2 cells siRNAGrx and siRNATrx treatment had the opposite effect, inducing Tyr nitration of proteins. Statistical significance was assessed by (p≤0.05). The values labeled with “a” were significantly different versus the corresponding internal control in the same cell lineage. The values labeled with “b” were significantly different versus the corresponding control cell lineage.

Mentions: Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite (ONOO−) anion and nitrogen dioxide, formed as secondary products of NO metabolism in the presence of oxidants including superoxide radicals (O2·−) and hydrogen peroxide (H2O2) [45]. For this reason, the level of Tyr nitration is indicative of the degree of nitrosative and oxidative stress and we measured it to check the effect of Trx1 and Grx1. Overexpression of NOS-3 in HepG2 cells induced protein Tyr nitration (Fig. 6, black bars). Silencing of Trx1 and Grx1 also increased protein tyrosine nitration in wild type and 4TO cells, but, interestingly, had the opposite effect on NOS-3 overexpressing cells (Fig. 6). These results uncover an apparent conflict, e.g., a decrease in antioxidant defenses, Trx1 and Grx1, results in alleviation of oxidative stress.


Redox regulation of metabolic and signaling pathways by thioredoxin and glutaredoxin in NOS-3 overexpressing hepatoblastoma cells.

González R, López-Grueso MJ, Muntané J, Bárcena JA, Padilla CA - Redox Biol (2015)

Detection of nitrotyrosine levels in NOS-3 overexpressing HepG2 cells with Grx and Trx down regulation. The detection of this postranslational modification was performed by Western blot with the analysis focused on a representative 55 kDa band (arrow). The image is representative of three different experiments and densitometric data of n=3 independent experiments are presented in the histogram below as mean±SEM. The numbers 1, 2 and 3 indicate the cell treatments control, siRNAGrx and siRNATrx respectively. NOS-3 overexpression induced Tyr nitration of proteins in HepG2 cells (black bars). This effect was offset by treatment with siRNAGrx and siRNATrx as shown in the 4TO-NOS group. However, in WT and 4TO HepG2 cells siRNAGrx and siRNATrx treatment had the opposite effect, inducing Tyr nitration of proteins. Statistical significance was assessed by (p≤0.05). The values labeled with “a” were significantly different versus the corresponding internal control in the same cell lineage. The values labeled with “b” were significantly different versus the corresponding control cell lineage.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528045&req=5

f0030: Detection of nitrotyrosine levels in NOS-3 overexpressing HepG2 cells with Grx and Trx down regulation. The detection of this postranslational modification was performed by Western blot with the analysis focused on a representative 55 kDa band (arrow). The image is representative of three different experiments and densitometric data of n=3 independent experiments are presented in the histogram below as mean±SEM. The numbers 1, 2 and 3 indicate the cell treatments control, siRNAGrx and siRNATrx respectively. NOS-3 overexpression induced Tyr nitration of proteins in HepG2 cells (black bars). This effect was offset by treatment with siRNAGrx and siRNATrx as shown in the 4TO-NOS group. However, in WT and 4TO HepG2 cells siRNAGrx and siRNATrx treatment had the opposite effect, inducing Tyr nitration of proteins. Statistical significance was assessed by (p≤0.05). The values labeled with “a” were significantly different versus the corresponding internal control in the same cell lineage. The values labeled with “b” were significantly different versus the corresponding control cell lineage.
Mentions: Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite (ONOO−) anion and nitrogen dioxide, formed as secondary products of NO metabolism in the presence of oxidants including superoxide radicals (O2·−) and hydrogen peroxide (H2O2) [45]. For this reason, the level of Tyr nitration is indicative of the degree of nitrosative and oxidative stress and we measured it to check the effect of Trx1 and Grx1. Overexpression of NOS-3 in HepG2 cells induced protein Tyr nitration (Fig. 6, black bars). Silencing of Trx1 and Grx1 also increased protein tyrosine nitration in wild type and 4TO cells, but, interestingly, had the opposite effect on NOS-3 overexpressing cells (Fig. 6). These results uncover an apparent conflict, e.g., a decrease in antioxidant defenses, Trx1 and Grx1, results in alleviation of oxidative stress.

Bottom Line: Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO.Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and -8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells.These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new antitumoral therapies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain; Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain. Electronic address: raulangello@hotmail.com.

No MeSH data available.


Related in: MedlinePlus