N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine, a novel compound with antitumor activity, induces radicals and dissipation of mitochondrial membrane potential

The novel compound N-benzoxazol-2-yl-N′-1-(isoquinolin-3-yl-ethylidene)-hydrazine (EPH136) has beenshown to exhibit antitumor activity in vitro and in vivo. ACOMPARE analysis showed that the patterns of cellulareffects of EPH136 are not related to any of 175 standardantitumor agents with a known mechanism of action. Inorder to help identify the mechanism of action weemployed a bioinformatics approach called partial leastsquares modelling in latent variables in which the expressionlevels of ~8,000 genes in each of 56 untreated NCIpanel cell lines were correlated with the respective IC50values of each cell line following treatment with EPH136.The 60 genes found to be most important for theantiproliferative effect of EPH136 are involved in nucleoside,nucleotide, nucleic acid binding and metabolism,developmental processes, protein modification and metabolism.In addition, using a DNA microarray we measuredthe expression of ~5,000 known genes following treatmentof HT-29 colon carcinoma cells with a two-fold IC50concentration of EPH136. The genes that were up-regulatedmore than two-fold compared to untreated controls belongto the same classes as found by the bioinformatic approach.Many of these proteins are regulated by oxidation/reductionand so we concluded that formation of radicals may beinvolved in the mechanism of action. We show here thatEPH136 leads to generation of oxygen radicals, swelling ofmitochondria and dissipation of the mitochondrial membranepotential. The antiproliferative activity of EPH136 wasprevented by the radical scavenger N-acetylcysteine. Cellswith elevated glutathione exhibited resistance to EPH136.In summary, the mechanism of the novel experimentalanticancer drug EPH136 is generation of radicals anddissipation of the mitochondrial membrane potential.