Date of Award

2012

Degree Name

Biomedical Sciences

College

College of Science

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

W. Elaine Hardman

Second Advisor

Kinsley K. Kiningham

Third Advisor

Beverly Delidow

Fourth Advisor

Nalini Santanam

Fifth Advisor

John Wilkinson

Abstract

Neuroblastoma is the most common extracranial solid tumor in childhood, ranking third in prevalence among infant cancers. Despite intensive therapy with surgery, radiation and chemotherapy, there is a high chance of tumor recurrence. These children are therefore given retinoids in the maintenance phase to differentiate the tumor cells and to avoid/lessen this incidence. Although 13-cis retinoic acid (13-cis RA) is currently used to treat neuroblastoma, there is much evidence supporting the prevalence of its metabolite, all-trans retinoic acid (ATRA), to be the main signaling retinoid in vivo.

The SK-N-SH neuroblastoma cell line, which is composed of three sub-types: sympatheoadrenal neuroblasts (N-type), substrate-adherent non-neuronal (S-type), and intermediate (I-type), was used as a model for ATRA-mediated differentiation. Through our research, we propose that redox status (i.e. balance of reactive oxygen species (ROS) and antioxidants) plays an important role in the process of neuroblastoma differentiation. More specifically, we first investigated the effects of supplementing ATRA treatment with a known, clinically relevant antioxidant, N-acetyl-L-cysteine (NAC). As opposed to findings in many other neuronal models, we are the first to demonstrate NAC enhancement of ATRA-mediated differentiation. Furthermore, our results suggest that NAC prevents oxidation of either ATRA, or of the nuclear environment in which ATRA binds to retinoic acid receptor (RAR) to induce expression of genes involved in neuronal differentiation. In this work, we also demonstrate that ATRA stimulates superoxide (O2-•) generation, which validates its induction of manganese superoxide dismutase (MnSOD; O2-• scavenger) in this cell model (previously published).

Our subsequent studies investigate the role of MnSOD in ATRA-mediated differentiation. Interestingly, ATRA upregulated neuronal differentiation markers (neurofilament M: NF-M and N-methyl-D-aspartate receptor 1: NMDAR1) (48 hrs) prior to induction of MnSOD (72 hrs), which might suggest that this antioxidant is not essential to initiate the differentiation process. Nonetheless, by reducing MnSOD activity (at 48 hrs), we demonstrate enhanced NF-M expression either in the absence or presence of ATRA. Therefore, it is apparent that basal MnSOD activity influences at least one marker of differentiation. Subsequent analysis at 96 hrs demonstrates that by reducing MnSOD activity, there is a concomitant decrease in NF-M expression. Therefore, it is possible ATRA-induction of MnSOD promotes expression of at least one differentiation marker (neurofilament M: NF-M) at this later time point. Furthermore, we provide evidence for the prevalence of MnSOD-generated hydrogen peroxide (H2O2) at 96 hrs, which may play a role in differentiation of the SK-N-SH cells.

Our findings show enhanced NF-M expression by either supplementation with an exogenous antioxidant (NAC) or reduction of an endogenous antioxidant (MnSOD). We propose ways in which each of these can be accomplished and provide insight into the redox regulation of neuroblastoma differentiation. Additionally, our work provides a close examination of ROS and antioxidants as they are altered in this process. Because retinoid therapy is often met with unfavorable chemoresistance, there is a need to establish improved therapeutic methods, which may revolve around alteration of redox status.

Subject(s)

Neuroblastoma--Molecular aspects.

Retinoids--Therapeutic use.

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