, 2007). The introduction of the meta-nitrophenylamine group in C-3 of nor-beta, leading to QPhNO2, could increase its growth inhibitory effects toward HL-60 cells, regardless of the incubation period. It is interesting to note that while QPhNO2 presents a higher Androgen Receptor Antagonist price IC50 value (0.91 μM)
after 72 h incubation, the other quinones, nor-beta and doxorubicin, presented a clear time-dependency, increasing their activity after 72 h. Quinones are redox active molecules that form semiquinones and hydroquinones that can redox cycle in the presence of oxygen, leading to the formation of reactive oxygen species (ROS) (Asche, 2005, de Abreu et al., 2002a, Hillard et al., 2008 and Ferreira et al., 2009). In fact, it is postulated that ROS generation Protein Tyrosine Kinase inhibitor and the alkylation of cellular nucleophiles, including DNA and enzymes with a –SH group, account for the mechanism of cytotoxic action of drugs containing a quinone moiety (Asche, 2005, de Abreu et al., 2002a, Hillard et al., 2008 and Ferreira et al., 2009). In view of this fact, we measured the cytotoxic effect of QPhNO2 in the presence of NAC, an antioxidant that acts as a ROS scavenger (Zafarullaha et al., 2003). The IC50 value for QPhNO2 increased from 0.32 to 1.03 μM and that for nor-beta increased from 2.01 to 2.72 μM (Table 1, column 3). While these data suggest the participation of ROS in QPhNO2 cytotoxic effects,
they do not exclude other direct targets. Doxorubicin effects, in contrast, were not affected by NAC treatment (Table 1). ROS production was also evaluated in HL-60 cells by flow cytometry using the oxidation-sensitive fluorescent dye H2-DCF-DA after 1 h of incubation. QPhNO2 and nor-beta stimulated ROS generation, while doxorubicin was inactive (Fig. 2). ROS generation was higher in the first hour than after 3 h of incubation (data not shown). The pre-incubation with
NAC protected the cells from oxidative stress, reducing intracellular ROS generation. Cell death can be classified according to its morphological appearance, which may be apoptotic, necrotic, Sitaxentan autophagic or associated with mitosis catastrophe (Melino, 2001 and Okada and Mak, 2004). Cells undergoing apoptosis show typical well-defined morphological changes, including plasma membrane blebbing, chromatin condensation with margination of chromatin to the nuclear membrane, karyorrhexis (nuclear fragmentation), and formation of apoptotic bodies (Kerr et al., 1972), as already described. Cells treated with QPhNO2 displayed those features, suggesting that this compound induces apoptosis in HL-60 leukemia cells (data not shown). Considering the observed morphological features, we conducted a flow cytometry analysis of several cellular and biochemical events to assess the mechanisms involved in cell death induced by the tested compounds.