Meanwhile, melatonin inhibited HepG2 cell viability, and the combination of the human CD95 agonistic Ab, anti-APO-1, with melatonin enhanced the growth inhibitory effect. Similarly to our results, melatonin has been shown to exhibit protective effects against doxorubicin-induced liver toxicity in rats (Oz and Ilhan, 2006), while synergistic effects on apoptosis induction of Lenalidomide 191732-72-6 melatonin and doxorubicin have been reported in hepatoma cells (Fan et al, 2010), highlighting the selectivity and beneficial properties of melatonin based on the cell type and its features. Dysregulation of apoptosis and cellular proliferation are clearly associated with the malignant HCC phenotype; therefore, advances in understanding these signalling pathways are necessary to develop an effective pharmacological therapy for this disease (M��ller et al, 1997).

We have previously demonstrated that melatonin oncostatic effects in liver cancer are partially mediated through the MT1 membrane receptor, modulation of cAMP and ERK activation (Carbajo-Pescador et al, 2011). However, the precise mechanisms whereby melatonin influences apoptosis remain unclear. FoxO transcription factors play an important role in tumour suppression by upregulation of proapoptotic genes, such as Bim (Zanella et al, 2010; Tzivion et al, 2011). While FoxO pathways have been extensively studied in different tumour cell lines (Roy et al, 2011; Hong et al, 2012), little is known about its role in HCC. The present data show for the first time that melatonin-dependent apoptosis in HepG2 cells may be mediated, at least in part, by FoxO3a activation and subsequently increased Bim expression.

Apoptotic cell death is a complex programme mainly controlled by the Bcl-2 family proteins. We have previously reported, using HepG2 cells, an extrinsic apoptosis induction after melatonin treatment, associated with upregulation of one of these proapoptotic proteins, Bax, cytochrome c release and caspases activation (Martin-Renedo et al, 2008). However, it is known that the presence of BH3-only molecules like Bid, Bim and Puma is required for direct activation of Bax at the mitochondria. In this study, we observed an increase in Bim expression, both at mRNA and at protein levels after melatonin treatment in HepG2 cells.

Upregulation and activation of Bim protein is involved in the oncostatic effect of many other chemotherapeutic drugs in liver cancer, pointing to its role in critical steps of the apoptosis initiation (Schneider-Jakob et al, 2010). It has AV-951 been reported that Bid, Bim and Puma triple-knockout mice present developmental defects associated with deficiency of Bax. Moreover, genetic deletion in neurons and T lymphocytes prevents the homo-oligomerisation of Bax and Bad, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals (Ren et al, 2010).