生姜提取物对胃癌HGC-27细胞增殖和凋亡影响机制探讨

目的生姜提取物活性成分如生姜醇提物与6-姜酚参与调控多种肿瘤细胞增殖、凋亡及侵袭过程。HGC-27细胞株为常见的人未分化胃腺癌细胞。本研究旨在探讨生姜醇提物与6-姜酚单体对胃腺癌HGC-27细胞增殖、周期和凋亡影响及其可能的信号通路。方法利用有机溶剂提取法和中压制备色谱技术,得到生姜醇提物以及6-姜酚单体,经HPLC分析6-姜酚纯度不低于98.0%。以不同浓度(2、3和4 mg/mL)的生姜醇提物与6-姜酚(100、200、300和400μmol/L)处理HGC-27细胞24h,CCK-8法检测其对细胞增殖的影响;流式细胞术检测细胞周期及凋亡;实时荧光定量PCR检测细胞周期调控蛋白及凋亡相关蛋白mRNA表达的变化。结果CCK-8实验结果显示,生姜醇提物及6-姜酚均可浓度依赖性地抑制HGC-27细胞增殖,F值分别为2110和40.23,均P<0.01。流式细胞术检测结果显示,当2、3和4mg/mL生姜醇提物处理细胞24h后,G1期细胞比例增加至(50.86±1.64)%、(56.78±0.60)%和(55.74±0.43)%,高于空白对照组(46.14±2.60)%,差异有统计学意义,均P<0.01;而3和4mg/mL生姜醇提物处理组凋亡率则分别增至(20.96±0.39)%和(27.36±4.48)%,与空白对照组(14.92±1.38)%相比,差异均有统计学意义,均P<0.05。当300和400μmol/L 6-姜酚处理细胞24h后,G1期细胞比例增加至(53.85±0.62)%和(51.70±1.78)%,高于空白对照组(42.57±0.19)%,差异有统计学意义,P<0.01;而其凋亡率分别增至(9.47±1.56)%和(17.04±0.60)%,与空白对照组(4.81±1.56)%相比,差异有统计学意义,P<0.01。实时荧光定量PCR检测结果显示,生姜醇提物上调促凋亡蛋白Bax mRNA表达,同时下调凋亡抑制蛋白Bcl-2 mRNA表达,而6-姜酚则增加Bax/Bcl-2比例,并下调细胞周期调控蛋白Cyclin E1 mRNA表达,上调细胞周期蛋白激酶抑制因子p21 mRNA表达。结论生姜醇提物及6-姜酚对胃癌HGC-27细胞增殖均有抑制作用,并使细胞周期发生G1期阻滞及诱导细胞凋亡,为成为治疗胃癌的天然药物提供新依据。

Effects of ginger extract on proliferation and apoptosis in human gastric cancer HGC-27cells

OBJECTIVE The active components of ginger are involved in the process of proliferation,apoptosis and metastasis of various tumor cells.HGC-27 cells line is a common human poorly differentiated adenocarcinoma of gasrtic cancer cell.The objective of this study was to explore the effects of ginger extract on proliferation,cell cycle and apoptosis of human gastric cancer HGC-27 cells and its mechanism.METHODS The ethanol extracts of ginger(EEG)and 6-gingerol were isolated from ginger,and the purity of 6-gingerol was determined to be higher than 98.0%by normalization of the peak area detected by high-performance liquid chromatography(HPLC).HGC-27 cells were cultured in vitro and treated with different concentrations of EEG and 6-gingerol for 24 h,and then the proliferation inhibitory rate was detected by CCK-8 assay;Flow cytometry was performed to assess the cell cycle distribution and cell apoptosis;Quantitative real-time quantitative PCR(qRT-PCR)assay was conducted to examine the mRNA level.RESULTS CCK-8 assay showed that both EEG and 6-gingerol inhibited the proliferation of HGC-27 cells in a concentration-dependent manner,F=2110,40.23,all P<0.01.Flow cytometry showed that the percentage of cells in the G1 phase was increased after pretreatment of EEG(2,3 and 4 mg/mL)for 24 h[(50.86±1.64)%,(56.78±0.60)%and(55.74±0.43)%,vs(46.14±2.60)%in control,P<0.01],while the apoptosis rate of EEG(3,4 mg/mL)was enhanced[(20.96±0.39)%,(27.36±4.48)%vs(14.92±1.38)%in control,all P<0.05].Pretreatment with the 6-gingerol(300,400μmol/L)also induced cell cycle arrest at G1 phase[(53.85±0.62)%,(51.70±1.78)%vs(42.57±0.19)%in control,P<0.01],while the apoptosis rate was enhanced[(9.47±1.56)%,(17.04±0.60)%vs(4.81±1.56)%in control,P<0.01].qRT-PCR analysis data further supported that EEG increased pro-apoptotic gene expression(Bax)but decreased anti-apoptotic gene expression(Bcl-2).6-gingerol increased the ratio of Bax/Bcl-2,down-regulated cyclinE1 mRNA expression,and up-regulated the expression of cyclin kinase inhibitor p21 mRNA.CONCLUSIONS EEG and 6-gingerol significantly inhibited HGC-27 cells proliferation by inducing cell cycle arrest at G1 phase and cell apoptosis.This provides new basis for the ginger extract as natural medicine for the treatment of gastric cancer.