Abstract 5360: Differential induction of the anti-oxidant response by arsenic trioxide and darinaparsin in leukemic cells
Authors: Garnier Nicolas M. , Kourelis Maria , Flores Manuel , Mann Koren K. , Miller Wilson H. 
Published: June 2012

Darinaparsin (DAR) is a significantly more potent arsenical than arsenic trioxide (ATO) at mediating apoptosis in various malignant cell lines and is highly active against Acute Promyelocytic Leukemia (APL) cells (NB4). The mechanisms behind DAR's increased efficacy versus ATO remain to be elucidated. We previously reported that DAR triggers apoptosis by inducing cellular responses that do not completely overlap with ATO (Diaz et al. Leukemia). Oxidative damage has been postulated to be a key mechanism by which arsenicals initiate the apoptotic process. Consistent with its increased cytotoxicity, we observed that DAR induces superoxide (O2·−) and hydrogen peroxide (H2O2), as shown respectively by hydorethidine and dichloro-dihydro-fluorescein staining quantified by flow cytometry. In response to oxidative stress, cells induce an arsenal of protective proteins of which HO-1 (heme oxygenase-1), NQO1 (NAD(P)H:quinone oxidoreductase), TrxR1 (thioredoxin-reductase-1) and γGCS (glutamyl cysteine synthetase) are well recognized members. We therefore studied the expression of these stress response proteins after ATO and DAR treatment. As expected, we observed a significantly higher increase in NQO1, TrxR1 and γGCS mRNA and protein levels as assessed by Western Blot and quantitative real PCR, with DAR than with ATO. However, HO-1 expression was not induced by DAR. Nrf2 is a transcription factor that mediates the expression of key protective enzymes through antioxidant response elements (AREs). AREs have been identified in the regulatory regions of genes encoding detoxification enzymes and other cytoprotective proteins, including NQO1, TrxR1, γGCS, and HO-1. Nucleo-cytoplasmic fractionation followed by Western Blot show that both compounds induce Nrf2 translocation to the nucleus. Transcriptional regulation of these detoxifying enzymes involves a competition between the activator Nrf2 and the repressor Bach1 for interaction with their AREs. Accordingly, ChIP assays demonstrate that Bach1 is released and Nrf2 is recruited to the HO-1 ARE in ATO treated cells but not in cells treated with DAR.

HO-1's widely reported cytoprotective effects in vitro and in vivo have been related to the generation of the antioxidants biliverdin and bilirubin. In order to elucidate whether the lack of HO-1 expression contributes to the increased efficacy of DAR, we examined the response to ATO and DAR in co-treatment with OB-24, a recently developed HO-1 inhibitor. As indicated by Propidium Iodide staining followed by flow cytometry analysis, pharmacological inhibition of HO-1 increased ATO toxicity but has no significant effect on DAR induced apoptosis. We therefore hypothesize that the increased efficacy of DAR versus ATO might be partially due to the absence of HO-1 up-regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2011-5360