Bennukul Kangsadarn, Numkliang Sucha, Leardkamolkarn Vijittra
Kangsadarn Bennukul, Sucha Numkliang, Toxicology Graduate Programme, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
World J Hepatol. 2014 Apr 27;6(4):230-42. doi: 10.4254/wjh.v6.i4.230.
To elucidate the effects of melatonin on cisplatin-induced hepatocellular carcinoma (HepG2) cell death and to identify potential cross-talk pathways.
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.
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.
Melatonin attenuated cisplatin-induced cell death in HepG2 cells via a counter-balance between the roles of apoptotic- and autophagy-related proteins.
阐明褪黑素对顺铂诱导的肝癌（HepG2）细胞死亡的影响，并确定潜在的相互作用途径。
用褪黑素和/或顺铂处理肝癌HepG2细胞24至48小时。通过MTT法计算细胞活力和50%细胞毒性浓度（CC50）。研究了选定浓度的褪黑素（1 mmol/L）和顺铂（20 μmol/L）诱导的效应和细胞内事件。细胞死亡和存活检测主要使用荧光显微镜评估4',6-二脒基-2-苯基吲哚DNA染色和吖啶橙溶酶体染色，然后使用抗LC3抗体通过免疫细胞化学进一步分析。通过逆转录-聚合酶链反应以及对与细胞存活和死亡相关的基因和蛋白质进行蛋白质印迹分析，研究联合处理后褪黑素对顺铂介导的潜在分子反应。使用流式细胞术进行细胞周期分析。
褪黑素对HepG2细胞活力具有浓度依赖性作用。在1 mmol/L时，褪黑素显著提高细胞活力百分比，并降低顺铂诱导的活性氧产生。褪黑素减少顺铂诱导的细胞死亡，降低磷酸化p53凋亡蛋白、裂解的半胱天冬酶3和Bax水平，但增加抗凋亡Bcl-2基因和蛋白质表达。与顺铂联合使用时，褪黑素诱导HepG2细胞S期（DNA合成）细胞周期停滞并促进自噬事件。褪黑素对Beclin-1及其自噬调节因子雷帕霉素哺乳动物靶标（mTOR）以及DNA切除修复交叉互补1（ERCC1）蛋白也具有浓度依赖性作用。在单独使用顺铂或褪黑素处理期间，这些蛋白的表达水平在HepG2细胞中发生改变。在联合处理中，褪黑素通过抑制mTOR和ERCC 1的过度表达并提高Beclin-1和微管相关蛋白轻链3-II的表达水平来逆转顺铂的作用，导致细胞内自噬体进展。
褪黑素通过凋亡相关蛋白和自噬相关蛋白作用之间的平衡来减轻顺铂诱导的HepG2细胞死亡。
