Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H2O2)-mediated oxidative stress

Abstract Non-steroidal anti-inflammatory drugs (NSAID) have shown promise as anticancer agents by inducing cell death apart from their antipyretic, anti-inflammatory and anti-thrombogenic effects. In our current study, we investigated the oxidative stress mediated cell death mechanism of a NSAID derivative NCX4040 (a nitric oxide releasing form of aspirin) in castration-resistant prostate cancer (CRPC) PC3 cell line. Our data revealed that NCX4040 is more potent than its parent compound aspirin or nitric oxide (NO) releasing compound DETA NONOate. NCX4040 significantly induced hydrogen peroxide formation with ensuing oxidative stress and mitochondrial depolarization resulting in lipid peroxidation, cell cycle arrest, inhibition of colony growth and induction of apoptosis in PC3 cells. Moreover, NCX4040 inhibited migration potential of PC3 cells by depolymerizing F-actin and promoting anoikis. Interestingly, elevated levels of NADPH oxidase 1 (NOX1), superoxide dismutase (SOD) 1 and 2 were observed upon NCX4040 treatment. However, down regulation of anti-apoptotic markers B-cell lymphoma 2 (BCl2) and anti-oxidant thioredoxin reductase 1 (TXDR) expression were observed. In addition, NCX4040 down regulated cyclin D1 expression in PC3 cells further supporting the anticancer effect of NCX4040. Western blot analysis revealed that significant down regulation of key anti-apoptotic markers such as cellular inhibitor of apoptosis protein-1 (cIAP1), X-linked inhibitor of apoptosis (XIAP), survivin, and c-Myc. On the other hand, NCX4040-treated cells showed upregulation of pH2AX, cleaved caspase3 and cleaved PARP1. Taken together, our data demonstrate that NCX4040 treatment enhances free radical formation which in turn induces oxidative stress leading to mitochondrial mediated cell death in metastatic PC3 cells.