人参皂苷单体Rh2对人鼻咽癌CNE2细胞株增殖、凋亡的影响及其机制

Effect of ginsenoside Rh2 on proliferation and apoptosis of human nasopharyngeal carcinoma-2 cell strain and relevant mechanism

目的研究人参皂苷单体Rh2对人鼻咽癌CNE2细胞株增殖、凋亡的影响及其可能的机制。方法取对数生长期的CNE2细胞,用不同浓度的Rh2(20、40、60、80、100μmol/L)处理不同时间(24、48、72 h),并设对照组(正常培养),采用MTT法检测各组细胞的增殖活力;用最适作用浓度的Rh2分别处理CNE2细胞24、48、72 h,采用Hoechst荧光染色法观察细胞凋亡形态;流式细胞术检测细胞凋亡率、周期分布以及膜电位的变化;Western blot法检测细胞凋亡相关蛋白Bax、Bcl-2、Caspase-3、CyclinD1、P53的表达水平。结果 Rh2对CNE2细胞的增殖具有明显的抑制作用,且呈时间和剂量依赖性,60μmol/L Rh2作用72 h为最适作用浓度和时间。经60μmol/L的Rh2作用48、72 h后,凋亡细胞数目增多,凋亡细胞体积变小,细胞核出现染色质不均匀,核浓缩聚集、碎裂,边集程度增大等现象。与对照组比较,经60μmol/L的Rh2作用24、48、72 h,CNE2细胞的凋亡率逐渐增加(P<0.01);G0/G1期细胞比例逐渐升高(P<0.01),S期(P<0.01)和G2/M期细胞比例逐渐下降;细胞的膜电位逐渐降低(P<0.05);促凋亡蛋白Bax、激活型Caspase-3和P53蛋白表达上调(P<0.05),抗凋亡蛋白Bcl-2和细胞周期相关蛋白CyclinD1表达下调(P<0.05)。结论人参皂苷单体Rh2具有抑制CNE2细胞增殖,使细胞周期阻滞在G0/G1期,诱导细胞凋亡的作用,其机制可能是通过Caspase/CyclinD1信号通路实现的。

Objective To investigate the effect of ginsenoside Rh2 on proliferation and apoptosis of human nasopharyngeal carcinoma CNE2 cell strain as well as the relevant mechanism. Methods CNE2 cells at logarithmic phase were treated with Rh2 at various concentrations （20, 40, 60, 80 and 100 μmol/L） for various （24, 48 and 72） hours, using those in routine culture as control, and determined for proliferative activity by MTT assay. CNE2 cells were treated with Rh2 at the optimal concentration for 24, 48 and 72 h respectively, then observed for apoptotic morphology by Hoechst staining, observed for apoptotic rate, distribution of cell cycles and change of membrane potential by flow cytometry, and determined by expressions of Bax, Bcl-2, Caspase-3, Cyclin D1 and p53 proteins by Western blot. Results Rh2 showed significantly inhibitory effect on the proliferation of CNE2 cells significantly, in dose- and time-dependent manners, while the optimal concentration and time for treatment were 60 μmol/L and 72 h respectively. After treatment with Rh2 for 48 and 72 h, the number of apoptotic CNE2 cells increased, while the volume decreased, and the phenomena such as uneven nuclear chromatin,nuclear condensation, nuclear fragmentation and increased aggregation at edge appeared. As compared with those in control group, the apoptosis rate of CNE2 cells after treatment with 60 p, mol/L Rh2 for 24, 48 and 72 h increased gradually （P 〈 0. 01 ）, while the percentage in G0/G1 phase increased gradually （P 〈 0. 01 ） and those in S （P 〈 0. 01 ） and G2/M phases decreased gradually, and the membrane potential decreased gradually （P 〈 0. 05）; the expression of proapoptosis protein Bax, cleaved Caspase-3 and P53 increased （P 〈 0. 05）, while those of antiapoptosis protein Bcl-2 and cell cycle-associated protein Cyclin D1 decreased （P 〈 0. 05 ）. Conclusion Ginsenoside Rh2 inhibited the proliferation of CNE2 cells, arrested the cell cycle at G0/G1 phase and induced the cell apoptosis by a possible mechanism associated with Caspase / CyclinD1 signaling pathway.