斑蝥酸钠对结肠癌HCT116细胞凋亡及PUMA表达的影响

目的 探讨斑蝥酸钠对结肠癌细胞凋亡及p53上调凋亡控制因子（PUMA）表达的影响。方法 采用不同浓度斑蝥酸钠（0.1、0.25、0.5、1、2 μg／ml）处理结肠癌细胞HCT116 24 h，并设不加药对照组。测量其单层细胞跨膜电阻值（TER）以评价斑蝥酸钠对HCT116细胞凋亡的影响，同时采用实时荧光定量PCR和Western blotting分别检测HCT116细胞的PUMA mRNA和蛋白水平变化；HCT116细胞经0.05、0.1、0.2、0.4、0.8 μg／ml 抑制性κB激酶β（IKKβ）质粒转染或10、20、40、80 nmol／L 核因子（NF）-κB抑制剂（BAY 11-7082）处理后，采用Western blotting检测其PUMA蛋白水平。结果 与对照组比较，各浓度斑蝥酸钠处理后HCT116细胞的TER降低，PUMA mRNA和蛋白水平升高，且以上效应呈浓度依赖性，差异有统计学意义（P＜0.05）；与未处理的HCT116细胞相比，IKKβ质粒转染后，PUMA蛋白表达增加，而BAY 11-7082可降低斑蝥酸钠对PUMA的上调作用，差异有统计学意义（P＜0.05）。结论 斑蝥酸钠可通过NF-κB通路上调PUMA的表达，进一步诱导结肠癌细胞HCT116的凋亡。

Effect of sodium cantharidinate on apoptosis and PUMA expression of colon cancer cell line HCT116

Objective To explore the effect of sodium cantharidinate on the apoptosis and expression of p53upregulated modulator of apoptosis（PUMA）of colon cancer cell line. Methods The colon cancer HCT116 cells were treated with different concentrations of sodium cantharidinate（0.1，0.25，0.5，1，2 μg／ml） for 24 h, and control group without sodium cantharidinate was set. The transepithelial electrical resistance（TER）was analyzed to evaluate the influence of sodium cantharidinate on the apoptosis of HCT116 cell. Real-time fluorescence quantitative PCR and Western blotting were used to detect the changes of mRNA and protein level of PUMA in HCT116 cells. The protein level of PUMA was measured by Western blotting in HCT116 cells treated with inhibitor of nuclear factor（NF）-κB（BAY 11-7082）at concentrations of 10，20，40，80 nmol／L or plasmid for inhibitor of NF-κB kinase β（IKKβ）transfection at concentrations of 0.05，0.1，0.2，0.4，0.8 μg／ml. Results There were decreased TER but increased mRNA and protein level of PUMA in different concentrations of sodium cantharidinate versus control group（P＜0.05）. Compared with the untreated HCT116 cells，transfection with IKKβ plasmid increased the protein expression of PUMA，and while NF-κB inhibitor reduced the effect of sodium cantharidinate up-regulating the PUMA level with significance difference （P＜0.05）. Conclusion Sodium cantharidinate may promote the PUMA expression via NF-κB pathway，and therefore induce the apoptosis of HCT116 cells.