More importantly, NAC increased the toxicity of IFN-α through an additive induction of PF-04929113 mouse apoptosis and a synergistic decrease of NF-kB expression in HCC cells, pointing to different targets being modulated by IFN-α and NAC. IFN-α has been shown to reduce the incidence of pre-neoplastic foci and cancer in liver cancer models [28, 29]. Our results in vitro using 2.5 x 104 U/mL showed a GSK3326595 decrease in cell viability of around 30%, which could be considered a poor response. These results are in agreement with the poor response observed clinically, in which only around 30% of the patients respond to treatment [30]. These data confirmed that development

of alternative compounds to treat HCC, such as NAC tested here, is necessary. The selective induction of apoptosis in cancer cells is an exciting possibility

for the selective development of future therapies to treat HCC [31–33]. Knowing that one of the IFN-α mechanisms of action involves apoptosis through p53 induction and the activation of caspases [34–36], here we used cell lines with a different p53 status in order to establish the mechanisms involved in the toxicity of IFN-α and NAC in HCC cells. Some studies indicated that the presence of p53 would facilitate apoptosis induction [22, 37]. In our study we demonstrated that, despite leading to apoptosis in a p53-independent way, NAC triggered apoptosis in HepG2 p53 functional cells after 24 NVP-LDE225 in vivo h of treatment, while in p53-deficient cells (Huh7) this effect was observed only after 48 hours of treatment. Furthermore, in HepG2 cells, NAC not only potentiated the effect of IFN-α in reducing cell viability, but also increased labelling with annexin V after 24 h without increasing the overall amount of apoptosis. More interestingly, after 48 h and 72 h of treatment

with NAC, we did not observe any more annexin-positive cells in the HepG2 cells, while in IFN-α and NAC plus IFN-α treatments, we still observed annexin-positive cells after 48 h and 72 h. This suggests that NAC triggered apoptosis in some of the HepG2 cells, and those that Endonuclease remained were resistant to treatment, while co-treatment surpassed this resistance. This finding is an important point to be considered in clinical approaches using NAC or co-treatment with IFN-α. High expression of pro-angiogenic factors such as hypoxia-inducible factor-1α and cell growth/survival factors such as CD24 and activation of inflammatory signalling pathways such as Wnt/β-catenin, nuclear factor-kappa B and signal transducer and activator of transcription 3 predict early recurrence of HCC [4, 38]. Wnt/B-Catenin signaling is one of many pathways that are also altered in HCC, but it is also known that it responds to both NAC and INF used alone. It is conceivable that the use of both drugs could also have a synergistic effect on this pathway as well [39–41]. p53 and other transcription factors have been closely linked to cancer and related therapies.