Redox balance influences differentiation status of neuroblastoma in the presence of all-trans retinoic acid

Neuroblastoma is the most common extra-cranial solid tumor in childhood; and patients in stage IV of the disease have a high propensity for tumor recurrence. Retinoid therapy has been utilized as a means to induce differentiation of tumor cells and to inhibit relapse. In this study, the expression of a common neuronal differentiation marker [neurofilament M (NF-M)] in human SK-N-SH neuroblastoma cells treated with 10 μM all-trans retinoic acid (ATRA) showed significantly increased expression in accordance with reduced cell number. This was accompanied by an increase in MitoSOX and DCFH2 oxidation that could be indicative of increased steady-state levels of reactive oxygen species (ROS) such as O2•− and H2O2, which correlated with increased levels of MnSOD activity and immuno-reactive protein. Furthermore PEG-catalase inhibited the DCFH2 oxidation signal to a greater extent in the ATRA-treated cells (relative to controls) at 96 h indicating that as the cells became more differentiated, steady-state levels of H2O2 increased in the absence of increases in peroxide-scavenging antioxidants (i.e., glutathione, glutathione peroxidase, and catalase). In addition, ATRA-induced stimulation of NF-M at 48 and 72 h was enhanced by decreasing SOD activity using siRNA directed at MnSOD. Finally, treatment with ATRA for 96 h in the presence of MnSOD siRNA or PEG-catalase inhibited ATRA induced increases in NF-M expression. These results provide strong support for the hypothesis that changes in steady-state levels of O2•− and H2O2 significantly contribute to the process of ATRA-induced differentiation in neuroblastoma, and suggest that retinoid therapy for neuroblastoma could potentially be enhanced by redox-based manipulations of superoxide metabolism to improve patient outcome

Nuclear interaction between ADR-induced p65 and p53 mediates cardiac injury in iNOS (-/-) mice.

Adriamycin (ADR) treatment causes an imbalance in the levels of nitric oxide ((•)NO) and superoxide (O2(•-)) production leading to cardiac injury. Previously we demonstrated that mice lacking inducible nitric oxide synthase (iNOS) have increased oxidative stress and mitochondrial injury. The molecular events leading to increased mitochondrial injury in iNOS deficient mice is unknown. ADR in the absence of iNOS preferentially activates a proapoptotic pathway without a concurrent increase in prosurvival pathways. Treatment with ADR leads to an increase in DNA binding activity of nuclear factor kappa B (NFκB) and p53 in wildtype mice. Following ADR treatment, p53, but not NFκB DNA binding activity, as well as the level of Bax, a p53 target gene, was increased in iNOS (-/-) mice. This apoptotic signaling effect in iNOS (-/-) is alleviated by overexpression of manganese superoxide dismutase (MnSOD). Increases in NFκB and p53 in ADR-treated wildtype mice did not lead to increases in target genes such as MnSOD, bcl-xL, or Bax. Moreover, co-immunoprecipitation analysis revealed that p65, a prominent member of the NFκB family, interacts with p53 in the nucleus. These results suggest that NFκB and p53 may counter act one another's actions in ADR-treated wildtype (WT) mice. Further, these results identify a novel mechanism by which oxidative stress may regulate transcription of proapoptotic genes

Transgenic mice overexpressing MnSOD have increased enzyme activity and protein expression.

<p>(a) Superoxide dismutase activity of heart tissue homogenate in four genotypes: WT, iNOS (−/−), TgM (++), and iNOS (−/−)-TgM (++). Data are expressed as mean ± SEM. *p<0.05 for MnSOD compared to WT and iNOS (−/−). (b) Protein extracts were analyzed by Western blot using an antibody detecting MnSOD. Protein extracts were co-hybridized with an antibody detecting CuZnSOD to assess equal loading of the samples.</p

MnSOD and bcl-xL mRNA expression are not changed following treatment with ADR.

<p>Real time PCR was used to evaluate (a) MnSOD and (b) Bcl-xL mRNA levels (6 h) following ADR treatment in WT, iNOS (−/−), TgM (++), and iNOS (−/−)-TgM (++) mice.</p

Serum nitrate is significantly increased in WT but not iNOS (−/−), TgM (+/+), or iNOS (−/−)-TgM (+/+) mice following treatment with ADR.

<p>Serum nitrate concentration (µM) following time course treatment with ADR (n = 6–8 mice for each time point) was determined. Values are expressed as mean ± SEM. *p<0.0001 versus saline for WT mice.</p

Induction and interaction of p65 with p53 in nuclear extract following treatment with ADR.

<p>Expression of p65 increases following treatment with ADR (A). Immunoprecipitation with p65 demonstrating an interaction with p53 in the nucleus after treatment with ADR. Anti-p65 was used to reprobe membrane to verify presence of p65 (B).</p

p53 DNA binding activity is significantly increased in WT and mice lacking iNOS.

<p>Nuclear extract (12 µg) from heart tissue was isolated, pooled (N = 4–6 animals per lane) from (a) WT and iNOS (−/−), (b) TgM (++), and (c) iNOS (−/−)-TgM (++) mice, and assayed for p53 DNA binding activity. Specificity of DNA binding activity was determined using an anti-p53 antibody, as well as competition with unlabeled p53 oligonucleotide. Densitometric quantitation as fold increase from respective saline control using both individual animals and pooled samples is shown in (d). *p<0.025 versus saline for WT and iNOS (−/−) mice.</p

NFκB DNA binding activity is increased in WT mice.

<p>Nuclear extract (12 µg) from heart tissue of WT, iNOS (−/−), TgM (++), and iNOS (−/−)-TgM (++) mice was isolated and assayed for NFκB DNA binding activity. Densitometric quantitation as fold increase from respective saline control is shown (with gel shift inset). *p<0.001 versus saline for WT.</p

Bax mRNA expression is significantly increased in iNOS (−/−) mice following treatment with ADR.

<p>Real time PCR was used to evaluate Bax mRNA expression (6 h) following ADR treatment in WT, iNOS (−/−), TgM (++), and iNOS (−/−)-TgM (++) mice. Data are expressed as fold increase from respective saline control. <b>*</b>p<0.025 versus saline for iNOS (−/−) mice.</p