舒林酸对人胰腺癌细胞PANC-1增殖和凋亡的影响及机制探讨

摘要

目的：以人胰腺癌细胞系PANC-1为研究对象，利用不同浓度舒林酸处理PANC-1细胞，观察其对PANC-1细胞增殖、凋亡影响，并探讨舒林酸通过抑制Wnt/β-catenin信号通路杀伤PANC-1细胞的可能机制。方法实验分为阴性对照组（加入不含舒林酸的DMSO）和实验组（分别加入舒林酸浓度为0.25、0.5、1、1.5、2 mM的培养液，分别为：阴性对照组、0.25 mM组、0.5 mM组、1.0 mM组、1.5 mM组、2.0 mM组，总计6组）。MTT法检测PANC-1细胞生长抑制率；流式细胞术检测细胞凋亡率；RT-PCR和免疫细胞化学技术检测细胞内β-Catenin的表达。结果 MTT结果显示：PANC-1细胞经舒林酸干预后生长均受到不同程度抑制，且随着舒林酸浓度增加，抑制作用越强。流式细胞术检测凋亡结果显示PANC-1细胞早期凋亡率干预后均有不同程度增加，与对照组相比，0.5、1.0 mM组早期凋亡率差异无统计学意义，而1.5、2.0 mM组差异均有统计学意义（P＜0.05）。RT-PCR结果显示可见各组β-catenin mRNA表达量随着舒林酸浓度增加而降低，0.5、1.0 mM组与对照组相比差异无统计学意义，而1.5及2.0 mM组差异有统计学意义（P＜0.05）；应用2.0 mM的舒林酸处理PANC-1细胞0、12、24、48、72 h，随着处理时间延长，β-catenin mRNA表达量逐渐降低，各处理组与对照组相比，差异有统计学意义（P＜0.05）。ICC结果证实干预后PANC-1细胞内β-catenin表达量及核聚集降低，与对照组相比，0.25、0.5 mM组差异无统计学意义，而1.0、1.5、2.0 mM组差异有统计学意义（P＜0.05）。结论舒林酸对PANC-1细胞具有生长抑制及促凋亡作用，这种作用具有浓度-时间依赖性，舒林酸抑制Wnt/β-catenin信号通路可能是其杀伤PANC-1细胞的可能机制之一。%Objective To discuss the influence of different concentration sulindac on pancreatic cancer cell line PANC-1 cell proliferation and apoptosis,and investigate the possible mechanism that sulindac can inhibit the Wnt/beta-catenin pathway to kill pancreatic cancer cells. Methods PANC-1 cell were divided into negative control group (added containing no sulindac DMSO)and experimental group (added sulindac with concentrations of 0.25 ,0.5 ,1 ,1.5 ,2 mM medium,respectively,name as 0.25 mM group,0.5 mM group,1.0 mM group,1.5 mM group,2.0 mM group),and treated with different time,cell proliferation inhibition ratio in each group was detected by MTT assay,cell apoptosis ratio was detected by flow cytometry,the expression ofβ-catenin mRNA and protein were detected by RT-PCR and immunocytochemistry.Results MTT results showed that sulindac can inhibit the cell proliferation of PANC-1 by a dose-and time-dependent manner.Cell apoptosis increased after sulindac treatment in different degrees,and there were statistical differences between 1.5,2.0 mMgroup and control groups (P<0.05).RT-PCR results showed that the expression ofβ-catenin mRNA decreased after the treatment of sulindac,there were statistical differences between 1.5,2.0 mMgroup and control group (P<0.05). In the 2.0mM group,the expression ofβ-catenin decreased along with the time extending (P<0.05 ).ICC results showed that sulindac inhibited the expression ofβ-catenin protein and nuclear accumulation,there were no statistical differences in 0.25 ,0.5 mM group and control group,but there were statistical differences in 1.0,1.5,2.0 mMgroup.Conclusion Sulindac could inhibit cell proliferation and facilitate apoptosis of PANC-1,this effect is dose-and time-dependent.The inhibition of Wnt/beta-catenin signal pathway may be a possible mechanism of its cytotoxicity.