, 2003). Concomitantly with the loss of mitochondrial membrane potential observed in our study, the mitochondrial permeability transition induction by ROS releases several factors relevant to apoptosis, such as cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (EndoGIA) ( Cai and Jones, 1998 and van Gurp et al., 2003). In previous studies from our laboratory, it was

also demonstrated that G8 and G12 decrease the reduced/oxidized glutathione ratio ( Locatelli et al., 2008 and Locatelli et al., 2009). Changes in the GSH level and in the redox state of mitochondria are associated Thiazovivin manufacturer with oxidative stress induced by various oxidizing agents ( Brodie and Reed, 1992 and Mckernan et al., 1991). At the cellular level, the gallic acid esters, are hydrolyzed enzymatically by cytoplasmic esterases to gallic acid and alcohols (Kubo et al., 2002 and Nakagawa et al., 1995). Studies on the carcinogenicity of propyl gallate in human leukemia cell line suggest that its hydrolysis product, gallic acid, plays an important role in this effect since it is more easily oxidized than propyl gallate, resulting in redox activity enhancement, and consequently in increasing the reactive oxygen species production (Kobayashi et al., 2004). On the other hand, when rat hepatocytes were incubated with the

esterase inhibitor diazinon the cytotoxic effects of propyl gallate was enhanced suggesting GSK2118436 that the hepatotoxicity induced by propyl gallate was not mediated by its metabolites (Nakagawa et al., 1995). In our study, gallic acid did not alter cell viability, mitochondrial potential nor cellular redox status in mouse melanoma B16F10 cells suggesting that unlike the experiment with propyl gallate mentioned above, these effects do not depend on gallic acid formation by esterases

PDK4 hydrolysis of G8 and G12. In conclusion, to increase the reliability of our results, we used more than one assay to determine the effects of G8 and G12 on the viability of B16F10 cells. G8 and G12 induced lysosomal damage and a significant LDH release in lower concentrations than those necessary to obtain the same effect on mitochondria. The interaction of the compounds with the plasma membrane probably triggered the cascade of cell death. Additionally, it has been shown evidences that, at least in particular conditions, the release into the cytosol of lysosomal constituents may initiate the events related to apoptosis. The triggering of the apoptotic cascade may involve early release of lysosomal constituents leading to an increase in mitochondrial oxidant production, additional lysosomal rupture followed by mitochondrial cytochrome c release. The induced apoptotic cell death by G8 and G12 that was demonstrated by our previous studies was confirmed here by caspase-3 activation.