Arsenic trioxide induces the differentiation of retinoic acid-resistant neuroblastoma cells via upregulation of HoxC9.

Abstract

Neuroblastoma (NB) is one of the most common extracranial tumors with limited therapeutic options. Retinoic acid (RA) has been identified to play anticancer role against NB cells by inducing the differentiation and apoptosis of immature neuroblasts. However, silencing HoxC9 promoter by EZH2-induced H3K27me3 hypermethylation can lead to RA resistance. Previous studies have suggested that arsenic trioxide (ATO), an inhibitor of DNA methylation, could downregulate the expression of EZH2 in breast cancer cells. In our study, we attempted to obtain some insight into the mechanisms of differentiation of RA-resistant NB cells by detecting the expressions of HoxC9 and EZH2 in NB cells treated with ATO, so as to provide a basis for the subsequent treatment of RA-resistant NB by ATO. Two NB cell lines, SK-N-AS (retinoic acid-resistant neuroblastoma cells) and SK-N-SH (retinoic acid-sensitive neuroblastoma cells), were used in our experiments. Cell proliferation and apoptosis were respectively determined with Cell Counting Kit-8 (CCK-8) assay kit and Annexin V staining. The inverted phase contrast microscope was used to observe cell growth and measure the total length of nerve synapses. We employed label-free quantitative proteomic analysis to profile ATO-dependent changes in the proteome of NB cells. Western blot was used to detect the expressions of HoxC9, HoxD8 and EZH2. Arsenic trioxide inhibited the cell proliferation and increased apoptosis and total length of synapses in two NB cell lines. The expressions of HoxC9 and HoxD8 were upregulated, while the expression of EZH2 was downregulated in the SK-N-AS cell line. No significant changes in the 3 proteins mentioned above were observed in the SK-N-SH cell line after ATO treatment. Arsenic trioxide may reactivate the expression of HoxC9 by downregulating EZH2, which leads to restoring RA sensitivity and promoting the differentiation and apoptosis of RA-resistant NB cells.