南瓜蛋白对胰腺癌PANC-1细胞增殖和凋亡的影响

Effects of Cucurmosin on the Cell Proliferation and Apoptosis in Human Pancreatic PANC-1 Cells

目的观察南瓜蛋白对胰腺癌PANC-1细胞增殖和凋亡的影响。方法采用MTT法观察南瓜蛋白对PANC-1细胞生长抑制情况;非肥胖型糖尿病/重症联合免疫缺陷性小鼠(NOD/SCID)小鼠体内观察南瓜蛋白对胰腺原位移植瘤的抑瘤率;电镜观察南瓜蛋白作用后PANC-1细胞超微结构的改变;流式细胞仪定量检测其细胞周期和凋亡发生率的影响;ELISA法定量检测南瓜蛋白作用后PANC-1细胞Caspase-3活性变化。结果 0.125、0.25、0.5mg/kg的南瓜蛋白对小鼠移植瘤抑瘤率(%)分别为45.2、50.0、59.7(P<0.05)。南瓜蛋白10μg/mL作用PANC-1细胞24h,部分细胞开始凋亡,表现为核内染色质浓集,出现核碎裂,作用72h,凋亡细胞增多,表现为染色质聚集,部分核膜消失,凋亡小体出现;南瓜蛋白(0、2.5、10、40μg/mL)作用PANC-1细胞72h,流式细胞周期分析显示G0/G1期的比例(%)分别为46.56±5.08、53.33±5.05、67.50±6.50和77.00±6.73(P<0.05),annexin V/PI分析显示PANC-1细胞凋亡率(%)分别为2.50±0.13、8.30±1.23、23.40±2.45和48.50±3.65(P<0.05);Caspase-3活性分别为0.009±0.002、0.011±0.003、0.035±0.009和0.065±0.009酶活力单位(P<0.05),提示南瓜蛋白以浓度依赖性诱导PANC-1细胞凋亡;40μg/mL南瓜蛋白作用PANC-1细胞24、48、72h,G0/G1期的比例(%)分别为56.60±6.65、67.83±6.76和77.00±6.73(P<0.05),annexin V/PI分析显示PANC-1细胞凋亡率(%)分别为16.51±2.97、38.51±2.38和48.50±3.65(P<0.05),提示南瓜蛋白以时间依赖性诱导PANC-1细胞凋亡。结论南瓜蛋白对PANC-1细胞具有明显的抑制作用,诱导G0/G1周期阻滞和细胞凋亡可能是其主要机制。

Objective To observe the effects of cucurmosin（CUS） on the cell proliferation and apoptosis in pancreatic PANC-1 cells.Methods The inhibition of CUS on the PANC-1 cell growth was observed using MTT assay.The inhibition ratio of CUS on the pancreatic orthotopic transplantation was in vivo observed in the NOD/SCID mouse model.The changes of microstructure of the apoptosis-inducing effect of CUS on PANC-1 was observed under electron microscope.The cell cycle and apoptosis after CUS intervention was detected using flow cytometry.The Caspase-3 activity after CUS treatment was detected using enzyme linked immunospecific assay（ELISA）.Results Treatment with CUS at the dose of 0.125,0.25,and 0.5 mg/kg inhibited the growth of pancreatic carcinoma PANC-1 xenografs with the ratio of 45.2%,50.0%,and 59.7%,respectively（P0.05）.After exposure to 10 μg/mL CUS for 24 h,most cells presented typical morphologic changes of apoptosis such as chromatin condensation and shrunken nucleus.The apoptotic cells increased.Some nuclear shrinkage and fragmentation,as well as the apoptotic body were observed when cells were exposed to CUS for 72 h.Being exposed to 0,2.5,10.0,and 40.0 μg/mL of the CUS for 72 h,the percentage of G0/G1 phase cells was 46.56%±5.08%,53.33%±5.05%,67.50%±6.50%,and 77.00%±6.73%,respectively（P0.05）.The apoptosis ratio was 2.50%±0.13%,8.30%±1.23%,23.40%±2.45%,and 48.50%±3.65% shown by Annexin Ⅴ/PI（P0.05）.The Caspase-3 activity（unit） was 0.009±0.002,0.011±0.003,0.035±0.009,and 0.065±0.009,respectively（P0.05）.These data showed that CUS induced the apoptosis of PANC-1 cells in a dose-dependent maner.Being exposed to 40.0 μg/mL of the CUS for 24,48,and 72 h,the percentage of G0/G1 phase cells was 56.60%± 6.65%,67.83%±6.76%,and 77.00%±6.73%,respectively（P0.05）,the apoptosis ratio was 16.51%±2.97%,38.51%±2.38%,and 48.50%±3.65% shown by Annexin Ⅴ/PI（P0.05）.These data showed that CUS induced apoptosis of PANC-1 cells in the G0/G1 phase of the cell cycle in a time-dependent maner.Conclusion CUS significantly inhibited the growth of PANC-1 cells possibly through the G0/G1 cell cycle arrest and apoptosis.