Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells

Each year, hepatocellular carcinoma (HCC) is diagnosed in more than half a million people worldwide, being the most common malignant hepatobiliar disease (El-Serag, 2011; Jemal et al, 2011). Viral hepatitis B and C, as well as alcohol abuse, are the main risk factors for its development (Cornella et al, 2011). Dysregulation of cellular proliferation and apoptosis are frequent events related with malignant phenotype and poor responsiveness of HCC towards chemotherapy (Muller et al, 1997). For this reason, advances in understanding these processes are needed for developing effective pharmacological therapies for HCC. Melatonin, the hormone of the pineal gland, controls circadian rhythms, and it has been reported to exert additional functions in other organs. A large number of studies have demonstrated the protective role of melatonin in different pathophysiological situations in the liver, showing antioxidant and antiapoptotic proprieties (Pan et al, 2006; Subramanian et al, 2007; Thong-Ngam et al, 2007; Tahan et al, 2009). On the other hand, in vitro studies with different cancer cell lines have provided evidence for melatonin induction of apoptosis in tumour cells (Hill and Blask, 1988; Farriol et al, 2000; Futagami et al, 2001; Cini et al, 2005; Garcia-Santos et al, 2006; Garcia-Navarro et al, 2007; Cabrera et al, 2010; Chiu et al, 2010; Gonzalez et al, 2010). We have recently reported that melatonin administration induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cells through MT1 melatonin receptor by modulation of cAMP basal levels and ERK kinase activation (Carbajo-Pescador et al, 2009, 2011). Furthermore, melatonin-induced apoptosis was related with enhanced caspase-3 and caspase-9 activity, cytosolic cytochrome c release and upregulation of the proapoptotic protein Bax (Martin-Renedo et al, 2008). Nevertheless, the molecular pathways that underlie melatonin-induced apoptosis in human HCC are not fully elucidated. The FoxO subfamily of forkhead transcription factors (FoxO1/FKHR, FoxO3/FKHRL1 and FoxO4/AFX identified in mammals) plays an important role in tumour suppression by upregulating target genes involved in cell cycle arrest and apoptosis. Interestingly, low levels of FoxO3 have been reported to confer chemotherapy resistance in human cancers, being significantly associated with poor prognosis in cancer patients (Jin et al, 2004; Fei et al, 2009; Su et al, 2011). Moreover, enhanced activity and expression of active forms of FoxO transcription factors is required for tumour chemosensitisation (Sunters et al, 2003; Paroni et al, 2011). FoxO proteins are activated in response to a wide range of external stimuli. Regulation of its activity depends mainly on changes in the subcellular localisation, achieved via post-translational modifications, including phosphorylation, acetylation and ubiquitination (Calnan and Brunet, 2008). Several genetic and biochemical studies indicate that the FoxO family is a key downstream target of the PI3K-Akt pathway in development and longevity (Lin et al, 1997; Brunet et al, 1999). Thus, phosphorylation of FoxO factors in specific serine and/or threonine sites modulates their subcellular localisation (Rena et al, 2002; Barthel et al, 2005; Anton et al, 2007). Once placed in the nucleus, they play tumour suppressor roles through enhanced transcription of proapoptotic genes, such as BCL6, a Bcl-2-interacting mediator of cell death (Bim), and Fas ligand (Dijkers et al, 2000; Yang et al, 2006). Bim is a proapoptotic member of the Bcl-2 family, and is one of the main downstream targets of FoxO3a. After transcription, Bim mRNA undergoes an alternate splicing, giving three isoforms (BimS, BimL and BimEL) with different length (Ewings et al, 2007). Interestingly, there are in vivo and in vitro evidence demonstrating an essential role of Bim proteins in Bax activation (Ren et al, 2010). Based on this information, we focused this study on the FoxO3a regulation of Bim expression after treatment with pharmacological concentrations of melatonin, in an attempt to gain further mechanistic insights on the molecular pathways leading to melatonin-induced apoptosis in HepG2 liver cancer cells.