Given that the efficiency of IFN-α is only marginal in treating HCC, our study aimed to evaluate the effect of NAC on IFN-α toxicity, and how the co-treatment of NAC and IFN-α modulates cell death and growth inhibition in HCC human cell lines. We showed that NAC decreased cell viability through downregulation of the NF-kB pathway and induction of apoptosis. More importantly, NAC increased the toxicity of IFN-α through an additive induction of 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 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 . 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 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 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. Data suggest that IkB simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis . It is thought that several carcinogens and tumour promoters act through the constitutive activation of NF-kB [16, 43], which induces the resistance of cancer cells to chemotherapeutic agents and radiation . The balance between proliferation and cell death is a decisive factor in the progression or inhibition of carcinogenesis, and a variety of mechanisms can be activated or inactivated to induce apoptosis . Antioxidant molecules that have a thiol group, such as NAC, have the ability to promote several of these mechanisms in different types of human tumours [13, 45]. One of these mechanisms refers to upregulation of pro-apoptotic genes together with the downregulation of inhibitors of apoptosis genes, often accompanied by increased permeability of the mitochondrial membrane and release of cytochrome c, activating the caspase cascade. And all of these events are regulated by activation or inactivation of NF-kB [24, 46, 47]. Data from the present study confirm the findings of previous studies that showed a decrease in the expression of the p65 subunit using NAC or IFN-α [31, 48–53]. More importantly, combined treatment further reduced levels of p65 in a synergistic way, again suggesting that NAC and IFN-α act in different pathways. Since several genes involved in the initiation, promotion and tumour progression are regulated by NF-kB and its activation suppresses apoptosis and promotes cell proliferation [16, 54], the rational design of treatments that decrease NF-kB activity is a good strategy to treat malignancies, as observed here.
Confirming the involvement of NF-kB on the effect of NAC, we found that cells transfected with siRNA for the p65 (KD cells) had the same response of cells treated only with NAC. Furthermore, KD cells treated with IFN-α had the same response as the combined treatment with NAC plus IFN-α while knockdown of NF-kB did not alter the sensitivity to NAC. Altogether, these data suggest that the increase in growth inhibition shown by NAC is probably due to the inhibition of NF-kB pathway. Even though it has been shown that IFN-α may have a role in blocking the NF-kB activating pathway triggered by the hepatitis B virus , this was not observed in our experiments. IFN-α treatment alone showed only a slight decrease in NF-kB activation, suggesting that IFN-α may act through different mechanisms depending on cell type and context.
In conclusion, NAC potentiates the antitumoural effect of IFN-α, decreasing cell viability, increasing apoptosis and decreasing the expression of the p65 subunit of NF-kB. Considering that NF-kB is a molecule of great importance in the initiation, promotion and progression of tumours and that NAC acts on the inactivation of this pathway with a tumoural-specific toxicity, we suggest that NAC is a promising agent for use in HCC, primarily or as an adjuvant with IFN-α.