NRF2 Mediates Neuroblastoma Proliferation and Resistance to Retinoic Acid Cytotoxicity in a Model of In Vitro Neuronal Differentiation

Abstract

Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.

Inhibition of Human Neuroblastoma Cell Proliferation by N-acetyl-L-cysteine as a Result of Increased Sulfane Sulfur Level

“…In this study we showed that NAC administered to the SH-SY5Y cells in non-cytotoxic concentrations -1.0, 1.5, and 2 mM [cytotoxicity lower than 5% (Figure 1)] affects the inhibition of their proliferation (Figure 2). De Miranda Ramos et al (24) have also demonstrated that NAC significantly decreased the proliferation of the control SH-SY5Y cells after 72 h of culture, but the concentrations of NAC were much higher (2.5, 5, 10 mM) than those used in the present study. In our results, 5 mM NAC had the highest cytotoxic effects on the SH-SY5Y cells (about 30% cytotoxicity) during 48 h of culture (Figure 1).…”

Pterostilbene Inhibits the Growth of Human Esophageal Cancer Cells by Regulating Endoplasmic Reticulum Stress

“…In particular, increased ROS originating from mitochondria plays important roles in mitochondrial-induced apoptosis, whereby potential collapse of the mitochondrial outer membrane contributes to the release of the pro-apoptotic factor cytochrome c from the inter-membrane space to the cytosol and activates caspases involved in the mitochondrial apoptotic pathway [16,45,46]. Conversely, inhibition of ROS desensitizes cancer cells to anti-cancer treatments [28,29]. In our study, PTE treatment of EC109 cells significantly increased cytochrome c translocation from mitochondria to cytosol and activated Caspase 9 and Caspase 12, further promoting ROS generation.…”

Differentiating Neuroblastoma: A Systematic Review of the Retinoic Acid, Its Derivatives, and Synergistic Interactions

“…While vitamin E does not share a signalling pathway with vitamin A, it is still implicated in cancer prevention due to its antioxidative action. Nevertheless, Trolox, a vitamin E analogue, did not affect ATRA-treated non-MNA SH-SY-5Y cells [41]. On the other hand, n-acetyl cysteine, another antioxidant, has decreased cellular proliferation and induced cellular apoptosis when used with ATRA.…”

“…These findings suggest that glutathione synthesis inhibition potentiates RA-mediated cell death and might become an addition to the NB treatment protocol. However, the results of this study might be limited by the absence of traditional NB differentiation markers [41].…”