人参皂苷Rg3对人胰腺癌细胞Pim-3及Bad凋亡蛋白表达的影响

Effect of ginsenoside Rg3 on Pim-3 and Bad proteins in human pancreatic cancer cell line PANC-1

背景与目的:人参皂苷Rg3是一种抗肿瘤中药单体,有研究表明人参皂苷Rg3对肝癌、肺癌、结肠癌、胰腺癌等肿瘤细胞的浸润具有明显抑制作用。本实验目的是研究人参皂苷Rg3对人胰腺癌细胞株PANC-1中原癌基因Pim-3及磷酸化Bad蛋白pBad(Ser112)、Bad(Ser136)表达的影响。方法:用浓度为0、10、20、40和80μmol/L的人参皂苷Rg3处理PANC-1细胞24h后,用四甲基偶氮唑盐(MTT)法检测人参皂苷Rg3对PANC-1细胞增殖的抑制作用;用倒置显微镜和流式细胞术观察人参皂苷Rg3对PANC-1细胞凋亡的诱导作用;用Western blot法检测经不同浓度人参皂苷Rg3处理后的PANC-1细胞中pim-3、Bad、pBad(Ser112)和pBad(Ser136)的表达;定向克隆Pim-3的发夹状寡核苷酸(shor thairpin RNA,shRNA)到真核表达载体形成重组质粒pSilencer 3.1-HINeo-Pim-3 shRNA,将重组质粒通过脂质体介导转染PANC-1细胞,采用Annexin V/PI流式细胞分析法检测转染后PANC-1细胞的凋亡,用Western blot法检测转染后PANC-1细胞的pim-3及pBad(Ser112)的表达。结果:10、20、40和80μmo1/L的人参皂苷Rg3对PANC-1细胞增殖的抑制率分别为20.2%、33.4%、52.8%、65.3%;经10～80μmol/L的人参皂苷Rg3处理后PANC-1细胞呈现明显的凋亡形态学改变,80μmol/L人参皂苷Rg3处理PANC-1细胞24h后,与正常对照组细胞凋亡率(3.3±2.1)%比较,处理组细胞凋亡率(12.2±1.3)%增加(P<0.05);人参皂苷Rg3处理PANC-1细胞24h后,Bad总蛋白表达没有明显变化。pim-3和pBad(Ser112)的表达均随人参皂苷Rg3浓度的增加而逐渐减弱。PANC-1细胞中pBad(Ser136)不表达;Pim-3 shRNA转染PANC-1细胞后,与正常对照组比较,转染组早期凋亡率和总凋亡率均增加[(11.5±3.7)%vs.(5.8±2.2)%,P<0.01;(20.8±2.6)%vs.(13.0±4.1)%,P<0.05],转染组pim-3及pBad(Ser112)表达均低于正常对照组。结论:人参皂苷Rg3可下调胰腺癌细胞PANC-1中Pim-3以及磷酸化Bad蛋白的表达,从而抑制PANC-1细胞增殖,诱导细胞凋亡。

Background and Objective： Ginsenoside Rg3 is a traditional Chinese medicine monomer which possesses anticancer effects. This study was to investigate the effects of ginsenoside Rg3 on Pim-3 and phosphorylated Bad （pBad） proteins, pBad （Ser112） and pBad （Ser136） in human pancreatic cancer cell line PANC-I. Methods= PANC-1 cells were exposed to 10, 20, 40 and 80 pmol/L ginsenoside Rg3 for 24 h. A short hairpin RNA （shRNA） of Pim-3 was cloned and inserted into a eukaryotic expression vector pSilencer 3.1-H1 Neo to construct pSilencer 3.1-H1 Neo- Pim-3. pSilencer 3.1-H1 Neo-Pim-3 was then transfected into PANC-1 cells. Cell proliferation was measured by MTT- assay; cell apoptosis was observed under an invert microscope and measured by flow cytometry with Annexin V/ PI staining; protein expressions of Pim-3, Bad, pBad （Ser112） and pBad （Ser136） were measured by Western blot. Results： The inhibitory rates of 10, 20, 40 and 80μmol/L ginsenoside Rg3 on PANC-1 cells were 20.2%, 33.4%, 52.8% and 65.3%, respectively. Typical morphological changes in apoptosis were induced by ginsenoside Rg3. The apoptotic rate of PANC-1 cells was significantly higher in the ginsenoside Rg3 treatment group （80 μmol/L） than in the control group （12.2% vs. 3.3%, P〈0.05）. Ginsenoside Rg3 had no influence on the total Bad protein expression, but decreased both Pim-3 and pBad （Ser112） expressions in a dose-dependent manner, pBad （Ser136） was not expressed in PANC-1 cells. Compared with the control group, the percentages of early and total apoptotic cells were significantly increased in PANC-1 cells transfected with pim-3-shRNA E （11.5±3.7）% vs. （5.8±2.2）%, P〈0.01;（20.8±2.6）% vs.（13.0±4.1）%,P〈0.05], while the expressions of pim-3 and pBad （Ser112） were both decreased. Conclusion： The anti- tumor effect of ginsenoside Rg3 may be associated with the decrease of Pim- 3 and pBad （Ser112）.