Aim: To elucidate the effects of melatonin on cisplatin-induced hepatocellular carcinoma (HepG2) cell death and to identify potential cross-talk pathways.
Methods: Hepatocellular carcinoma HepG2 cells were treated with melatonin and/or cisplatin for 24 to 48 h. Cell viability and the 50% cytotoxic concentration (CC50) were calculated by MTT assays. The effects and intracellular events induced by the selected concentrations of melatonin (1 mmol/L) and cisplatin (20 μmol/L) were investigated. Cell death and survival detection were primarily evaluated using a fluorescence microscope to assess 4',6 diamideno-2-phenylindol DNA staining and acridine orange lysosome staining and then further analyzed with immunocytochemistry using an anti-LC3 antibody. The potential molecular responses mediated by melatonin against cisplatin after the combined treatment were investigated by reverse transcription-polymerase chains reaction and Western blot analyses of the genes and proteins associated with cell survival and death. A cell cycle analysis was performed using a flow cytometry assay.
Results: Melatonin had a concentration-dependent effect on HepG2 cell viability. At 1 mmol/L, melatonin significantly increased the cell viability percentage and decreased reactive oxygen species production due to cisplatin. Melatonin reduced cisplatin-induced cell death, decreasing phosphorylated p53 apoptotic protein, cleaved caspase 3 and Bax levels but increasing anti-apoptotic Bcl-2 gene and protein expression. When combined with cisplatin, melatonin induced S phase (DNA synthesis) cell cycle arrest and promoted autophagic events in HepG2 cells. Melatonin also had a concentration-dependent effect on Beclin-1 and its autophagic regulator mammalian target of rapamycin (mTOR) as well as the DNA excision repair cross complementary 1 (ERCC1) protein. The expression levels of these proteins were altered in HepG2 cells during cisplatin or melatonin treatment alone. In the combination treatment, melatonin reversed the effects of cisplatin by suppressing the over-expression of mTOR and ERCC 1 and enhancing the expression levels of Beclin-1 and microtubule-associated protein-light chain3-II, leading to intracellular autophagosome progression.
Conclusion: Melatonin attenuated cisplatin-induced cell death in HepG2 cells via a counter-balance between the roles of apoptotic- and autophagy-related proteins.
Keywords: Autophagy; Cisplatin; Excision repair cross complementary 1; Hepatocellular carcinoma; Mammalian target of rapamycin; Melatonin.