Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.