, 2001). In 1995, (Nobel et al. (1995)) demonstrated that another DC, pyrrolidine dithiocarbamate (PDTC), induces apoptosis in thymocytes by increasing the intracellular level of copper and, consequently, changing the redox activity in the medium, a similar result obtained here, but with relation with time and concentration of the DC. (Viquez et al. (2008)) suggested that DEDTC had the ability to accumulate copper, leading to lipid oxidation and damage with myelin inflammation in rats. Our studies showed that the cellular response was influenced by the DEDTC concentration with a non-linear accumulation of copper

Apoptosis inhibitor inside the cell. The effect of a higher DEDTC concentration that did not cause cell toxicity – as 25 μM – is a lower intracellular

copper accumulation than DEDTC at 5 μM, proving that copper content inside the cell can be responsible for the effects of DEDTC toxicity. Consequently, the intracellular accumulation of copper could generate oxidative stress with the formation of reactive oxygen species (ROS); many studies have reported the influence of copper on the formation of ROS in neuroblastoma cells that causes DNA damage (Arciello et al., 2005, Marengo et al., 2005, Gabbianelli et al., 1999 and Filomeni et al., 2007). Our results of the cell cycle studies (Fig. 2C) showed an increase in number of cells in the sub-G1 phase upon DEDTC treatment, confirming that the chelation caused some damage to cellular DNA. This result was confirmed by Annexin V/FITC and PI flow cytometry studies that showed an increase in the sub-G1 population selleck kinase inhibitor during the incubation with DEDTC, clearly indicating an apoptotic process (Fig. 3B). The tumor suppressor protein p53 is a transcription factor that regulates cell cycle progression and DNA repair in cells exposed to genotoxic

stress (Culmsee and Mattson, 2005). In many types of cells, the accumulation of p53 triggers apoptosis (Morrison et al., 2003 and Meulmeester and Jochemsen, 2008). Our results suggested that DEDTC induced the accumulation of p53 (Fig. 4) and, thus, triggered apoptosis in the SH-SY5Y cells. Apoptosis is regulated and executed by two main families of proteins, the Bcl-2 family and caspases (Aravind et al., 1999 and Hacker PRKD3 et al., 2011), and their activation can be initiated in two different ways, the extrinsic pathway (cytoplasmic) or the intrinsic pathway (mitochondrial). Our intention was to clarify the role of caspase 8 in p53-dependent apoptosis induced by DEDTC. Caspase 8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via the cleavage of proapoptotic Bid. In our studies, the observed activation of caspases 8 and 3 (Fig. 3A) and the unchanged levels of Bcl-2 (data not shown) suggested that the mechanism of DEDTC-induced apoptosis mainly involved the extrinsic pathway.