褪黑素对小鼠肝癌H22细胞抗增殖和促凋亡的p53依赖性机制研究

p53-dependent Antiproliferation and Apoptosis of H22 Cell Induced by Melatonin

背景与目的本室已经证明了褪黑素(melatonin,MLT)对小鼠肝癌H22细胞的生长具有抑制作用,本研究旨在探讨其机理。方法(1)建立荷瘤小鼠模型并给予褪黑素处理,取肿瘤组织进行p53、cyclinE水平的免疫组化分析;(2)不同浓度褪黑素体外培养小鼠肝癌H22细胞,采用流式细胞技术(flowcytometry,FCM)检测细胞周期分布和凋亡率;免疫组化法分析培养细胞的p53和cyclinE的蛋白水平;采用RT-PCR方法检测FasmRNA的水平。结果(1)FCM显示褪黑素可引起H22细胞G0/G1期比例升高,从75.24%上升至85.46%,同时细胞凋亡增多,从5.07%升至12.77%;(2)褪黑素作用后p53水平较对照组增高42.5%(培养细胞)和19.5%(肿瘤组织),而cyclinE的水平则降低31.7%(培养细胞)和39.9%(肿瘤组织);(3)RT-PCR结果显示FasmRNA水平增高44.2%。结论MLT通过阻滞细胞周期和诱导细胞凋亡抑制H22细胞增殖。其机理可能是通过p53抑制下游的cyclinE的表达,阻止细胞进入S期;同时又通过p53上调Fas表达,从而诱导细胞凋亡。

BACKGROUND & OBJECTIVE: It has been shown that melatonin has a direct inhibitory effect on the proliferation of H22 mouse hepatoma cells in our research. This study was designed to investigate its molecular mechanism. METHODS:(1) Animal models were established by transplanting H22 cells and treated with melatonin, and then the p53 and cyclin E of the tumor tissue were determined by immunohistochemical analysis. (2) After treatment of H22 cells with melatonin in vitro, the percentage of cells in each cell cycle phase and apoptosis rate were analyzed by flow cytometry. p53 and cyclin E were determined again by immunohistochemical analysis. The level of Fas mRNA was examined by real time polymerase chain reaction (RT PCR). RESULTS: (1) After treated with melatonin (1×10-6 mol/L), the number of the H22 cells in phase G0/G1 were elevated from 75.24% to 85.46%, while which in phase S almost decreased from 10.32% to 0, and at the same time, the number of apoptotic cells increased from 5.07% to 12.77%. (2) Compared with the control, the level of p53 elevated 42.5% (in vitro) and 19.5% (in vivo), however, the level of cyclin E decreased 31.7% (in vitro) and 39.9%(in vivo). (3) Fas mRNA increased about 44.2% after melatonin treatment (P< 0.01). CONCLUSION: Melatonin inhibits the proliferation of H22 cells by arrest and apoptosis, and the mechanism perhaps interferes with increasing p53 that results in down regulation of cyclin E indirectly and stimulates the expression of Fas gene.