TRAIL 联合喜树碱诱导胰腺癌细胞凋亡的研究

目的：探讨喜树碱（CPT）增强TRAIL 诱导胰腺癌细胞PANC-1 凋亡的机制。方法：通过MTT 检测TRAIL 组、喜树碱组、二者联合组对胰腺癌细胞PANC-1 生长抑制的影响；利用Hoechst33258 荧光染色检测各组细胞凋亡情况；应用Western-blot方法检测PANC-1 在喜树碱应用前后凋亡信号传导蛋白分子 DR4、DR5、Caspase- 8、Caspase- 3 以及c-flip 的表达情况。结果：PANC-1 细胞经不同浓度TRAIL（10、30、100、300、1 000ng/mL）作用24h 后，当浓度在100ng/mL 及以上时与对照组相比有统计学差异（P<0.01），虽然存在差异，但当TRAIL 浓度为1 000ng/mL 时，其细胞抑制率仅为（10.60± 2.36）% ，TRAIL 与不同浓度的喜树碱（10、30、100、300、1 000ng/mL）联合作用后其细胞抑制率与单独应用喜树碱相比有显著性差异（P<0.05）；Hoechst33258 荧光染色结果显示：联合组有大量细胞出现细胞核聚集、边缘化、核碎裂等典型细胞凋亡形态，而其余各组未见明显凋亡征象；Western-blot结果显示：单独TRAIL 组及单独喜树碱组仅能看到Caspase- 8 和Caspase- 3 的前体，而二者联合后不仅看到了其前体，而且还看到其裂解带，并且发现经过喜树碱预处理后，PANC-1 细胞中c-Flip 蛋白的表达被明显下调了，CPT 组及联合组与对照组及单独TRAIL 相比均有显著性差异（P<0.05），而喜树碱应用前后对DR4 和DR5 表达无显著效应。结论：TRAIL 联合喜树碱对胰腺癌细胞PANC-1 诱导的凋亡具有协同作用，其机制是喜树碱下调了c-Flip 蛋白的表达，解除了其对Caspase- 8 的抑制，激活了死亡受体通路，与DR4 和DR5 无关。

Research on TRAIL Combined with Camptothecin in Inducing Apoptosis of Pancreatic Carcinoma Cell Line PANC-1

Objective: To investigate the mechanism behind campotothecin (CPT) enhancement of TRAIL-induced apoptosis of PANC- 1 pancreatic carcinoma cell line. Methods:The growth inhibition rates of PANC- 1 cells in the TRAIL, CPT and CPT+TRAIL groups were assayed by MTT. Cellular apoptosis in each group was tested by fluorescent staining with Hoechst 33258 . The level of apoptotic signal transduction proteins, such as Caspase- 8, Caspase- 3, DR4, DR5 and c-Flip, were detected using Western blot. Results: Twenty-four hours after PANC- 1 cells were treated with TRAIL of various concentrations (10, 30, 100 , 300 , and 1000 ng/mL), there were significant differences between the treatment and control groups at 100 ng/ml and higher ( P<0.01). Despite these differences, the inhibition rate was only 10.60 ± 2.36% with a TRAIL concentration of1000ng/mL. There were significant differences between the TRAIL+CPT combination group ( 10, 30, 100 , 300 , 1000ng/mL) and the CPT alone group (P<0.05). Results of Hoechst 33258 fluorescent staining showed that typi-cal characteristics of apoptosis such as nucleus aggregation, marginalization and nuclear fragmentation occurred in the TRAIL+CPT group. However, no obvious signs of apoptosis were seen in the groups treated only with TRAIL. Western blot results showed that pro-caspase- 8 and pro-caspase- 3 could only be seen in the groups treated with TRAIL or CPT alone. However, bands for both the precursors and the cleaved products were seen in the groups treated with both drugs. In addi-tion, the expression of c-Flip protein in PANC- 1 cells was down-regulated after CPT treatment. There were significant differ-ences in the expression of c-Flip protein between the CPT and combination treatment group and the groups treated with TRAIL alone and the control groups. There were no significant differences in the expression of DR4 and DR5 before and af -ter the CPT treatment. Conclusion:CPT combined with TRAIL may have a synergistic effect on induced apoptosis in PANC-1 cells. CPT down-regulates the expression of c-Flip protein, may possibly reverse the inhibition of caspase- 8, and can activate the apoptotic signaling pathway. DR4 and DR5 do not play significant roles.