Original Article
Copyright ©2014 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Hepatol. Apr 27, 2014; 6(4): 230-242
Published online Apr 27, 2014. doi: 10.4254/wjh.v6.i4.230
Melatonin attenuates cisplatin-induced HepG2 cell death via the regulation of mTOR and ERCC1 expressions
Kangsadarn Bennukul, Sucha Numkliang, Vijittra Leardkamolkarn
Kangsadarn Bennukul, Sucha Numkliang, Toxicology Graduate Programme, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Vijittra Leardkamolkarn, Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Vijittra Leardkamolkarn, Center of Excellence on Environmental Health and Toxicology, Mahidol University, Bangkok 10400, Thailand
Author contributions: Bennukul K and Leardkamolkarn V contributed to experimental design, coordinate the study, interpret the data, writing and revising the manuscript; Numkliang S performed an immunocytochemistry experiment.
Supported by Center of Excellence on Environmental Health and Toxicology, Science and Technology Postgraduate Education and Research Development Office, Thailand Ministry of Education
Correspondence to: Vijittra Leardkamolkarn, PhD, Associate Professor, Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. vijittra.lea@mahidol.ac.th
Telephone: +66-2-2015402 Fax: +66-2-3547168
Received: October 25, 2013
Revised: January 9, 2014
Accepted: April 3, 2014
Published online: April 27, 2014

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: Melatonin, Cisplatin, Hepatocellular carcinoma, Excision repair cross complementary 1, Mammalian target of rapamycin, Autophagy

Core tip: Melatonin has anti-oxidative stress and anti-proliferative effects on cisplatin-treated hepatocellular carcinoma cells through a counter-balance between the roles of apoptosis and autophagy proteins. Melatonin also reduced cisplatin-induced DNA damage by decreasing the activation of excision repair cross complementary 1 in the DNA repair system. Thus, co-treatment with melatonin to ameliorate cisplatin adverse effects might be beneficial for Hepatocellular carcinoma therapy.