Moreover, further experiments focused on understanding the molecular mechanism that underlay FoxO3a activation, showed a decrease in the dephosphorylated forms of FoxO3a during at Thr32 and Ser253, complemented by an increase of total FoxO3a and Bim EL in response to melatonin treatment. FoxO3a accumulation and Bim protein expression were greatly reduced upon silencing of FoxO3a, providing evidences to suggest that FoxO3a is functioning as a transcriptional regulator of Bim expression in HepG2 cells after melatonin treatment. It has been previously demonstrated that FoxO3a pathway can induce Bim expression and subsequently cell death in several cancer models, like MCF-7 breast cancer cell line (Sharma et al, 2011), mice xenografts model of pancreas tumour (Boreddy et al, 2011) and lymphoma cells (Bhalla et al, 2011), among others.

It is generally accepted that the FoxO family is a key downstream target of the PI3K pathway (Weidinger et al, 2011). While phosphorylation of FoxO factors by Akt causes relocalisation of FoxO proteins from the nucleus to the cytoplasm, dephosphorylated FoxO forms activate target genes (Hong et al, 2012). Therefore, our work provides clear evidence of melatonin-induced activation of FoxO3 in cells, promoting changes in its phosphorylation status. Moreover, translocation of FoxO3a to the nucleus was also confirmed by fluorescence microscopy experiments as well as by FoxO3a western blot in nuclear and cytoplasmic extracts. In this study, melatonin caused an inhibition of AKT phosphorylation even after EGF stimulation, and the PI3K inhibitor LY294002, combined with melatonin, resulted in a synergic effect enhancing Bim protein expression.

Our knowledge of the mechanisms by which melatonin induces apoptosis in human HepG2 hepatoma cells are limited; however, the FoxO3a/Bim pathway has been shown to participate in apoptotic processes in response to other chemotherapeutic agents like cisplatin (Fernandez de Mattos et al, 2008; Yuan et al, 2011). Moreover, resveratrol, another antioxidant molecule, has been reported to behave as melatonin, Entinostat exerting an oncostatic and proapoptotic activity in different tumour cells, including HepG2 (Hsieh et al, 2005; Notas et al, 2006). Although little is known about the resveratrol effect on the FoxO3a pathway, several groups have reported FoxO3a dephosphorylation, nuclear translocation and Bim induction after resveratrol treatment in in vitro cancer models (Chen et al, 2010; Roy et al, 2011), helping us to support our hypothesis. Our study provides important information regarding the mechanisms by which melatonin regulates apoptosis through the activation of FoxO transcription factors.