肿瘤坏死因子相关凋亡诱导配体联合雷公藤甲素诱导胰腺癌细胞凋亡的机制研究

江艳; 刘炜; 陈志宇

doi:10.3760/cma.j.issn.1673-9752.2015.04.013

肿瘤坏死因子相关凋亡诱导配体联合雷公藤甲素诱导胰腺癌细胞凋亡的机制研究

Mechanisms of tumor necrosis factorrelated apoptosis inducing ligandcombined with Triptolide in inducing the apoptosis of pancreatic cancer cells

摘要

摘要: 目的:探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合雷公藤甲素(Triptolide)诱导胰腺癌细胞凋亡的机制。
方法:(1) 将人胰腺癌细胞MiaPaca-2（简称MiaPaca-2细胞）分为4组：空白对照组(不加任何药物)，TRAIL+Triptolide-组(仅加TRAIL)，TRAIL-Triptolide+组(仅加Triptolide)和TRAIL+Triptolide+组(加TRAIL和Triptolide)。采用CCK-8检测各组细胞活力。采用Western blot检测各组细胞凋亡相关蛋白多聚ADP核糖聚合酶(PARP)、含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)和含半胱氨酸的天冬氨酸蛋白水解酶-8(Caspase-8)的表达。加入Caspase-8特异性抑制剂ZIETDFMK后，采用CCK-8检测细胞活力，采用CaspaseGlo荧光检测法检测Caspase-8活性。采用Western blot检测各组细胞髓样细胞白血病1(Mcl1)、额外的大B细胞淋巴瘤(BclxL)和B淋巴细胞瘤-2(Bcl-2)蛋白的表达。(2)将MiaPaca-2细胞分为8组：①TRAIL-Mcl1 siRNA-组(不加TRAIL、不转染Mcl1 siRNA)，TRAIL+Mcl1 siRNA-组(加TRAIL、不转染Mcl1 siRNA)，TRAIL-Mcl1 siRNA+组(不加TRAIL、转染Mcl1 siRNA)和TRAIL+Mcl1 siRNA+组(加TRAIL、转染Mcl1 siRNA)。②TRAIL-BclxL siRNA-组(不加TRAIL、不转染BclxL siRNA)，TRAIL+BclxL siRNA-组(加TRAIL、不转染BclxL siRNA)，TRAIL-BclxL siRNA+组(不加TRAIL、转染BclxL siRNA)，TRAIL+BclxL siRNA+组(加TRAIL、转染BclxL siRNA)。采用CCK-8检测各组细胞活力。采用Western blot检测各组细胞Caspase-3和Caspase-8蛋白的表达。符合正态分布的计量资料以±s表示，多组间比较采用单因素方差分析，两两比较采用LSDt检验。
结果:(1)空白对照组、TRAIL+Triptolide-组、TRAIL-Triptolide+组和TRAIL+Triptolide+组MiaPaca-2细胞活力分别为100.0%±1.1%、81.2%±2.3%、78.6%±3.6%、40.1%±2.5%；PARP蛋白相对表达量分别为0.510±0.028、0.720±0.072、1.250±0.023、2.560±0.220；Caspase-3蛋白相对表达量分别为0.080±0.004、0.080±0.003、0.110±0.005、2.720±0.003；Caspase-8蛋白相对表达量分别为0.070±0.003、0.080±0.005、0.120±0.003、0.990±0.006，4组比较，差异均有统计学意义(F=203.607，1 457.785，332 421.900，35 437.218，P<0.05)。TRAIL+Triptolide+组MiaPaca-2细胞活力与空白对照组、TRAIL+Triptolide-组和TRAIL-Triptolide+组分别比较，差异均有统计学意义(t=34.583，355.936，36.271，P<0.05)；TRAIL+Triptolide+组MiaPaca-2细胞中PARP蛋白相对表达量与空白对照组、TRAIL+Triptolide-组和TRAIL-Triptolide+组分别比较，差异均有统计学意义(t=591.784，63.739，2 268.987，P<0.05)；TRAIL+Triptolide+组MiaPaca-2细胞中Caspase-3蛋白相对表达量与空白对照组、TRAIL+Triptolide-组和TRAIL-Triptolide+组分别比较，差异均有统计学意义(t=3 266.153，9 145.228，1 738.713，P<0.05)；TRAIL+Triptolide+组MiaPaca-2细胞中Caspase-8蛋白相对表达量与空白对照组、TRAIL+Triptolide-组和TRAIL-Triptolide+组分别比较，差异均有统计学意义(t=663.953，1 432.878，327.584，P<0.05)。TRAIL+Triptolide+组MiaPaca-2细胞加ZIETDFMK前，Caspase-8活性为711.0%±5.1%；加入ZIETDFMK后，细胞活力为70.0%±4.8%，Caspase-8活性为73.0%±2.4%；加ZIETDFMK后细胞活力升高，Caspase-8活性降低，两者比较，差异均有统计学意义(t=17.956，55.027，P<0.05)。空白对照组、TRAIL+Triptolide-组、TRAIL-Triptolide+组和TRAIL+Triptolide+组MiaPaca-2细胞中Mcl1蛋白相对表达量分别为1.68±0.22、2.08±0.11、0.73±0.15、0.58±0.18，BclxL蛋白相对表达量分别为0.65±0.03、0.47±0.03、0.32±0.03、0.26±0.05，Bcl-2蛋白相对表达量分别为0.65±0.03、0.67±0.03、0.62±0.05、0.67±0.03，4组比较，差异均有统计学意义(F=55.178，88.683，3.411，P<0.05)。TRAIL-Triptolide+组和TRAIL+Triptolide+组MiaPaca-2细胞中Mcl1蛋白相对表达量与空白对照组分别比较，差异均有统计学意义(t=23.506，47.631，P<0.05)；上述两组与TRAIL+Triptolide-组分别比较，差异均有统计学意义(t=58.457，37.115，P<0.05)。TRAIL-Triptolide+组和TRAIL+Triptolide+组MiaPaca-2细胞中BclxL蛋白相对表达量与空白对照组分别比较，差异均有统计学意义(t=38.105，42.219，P<0.05)；上述两组与TRAIL+Triptolide-组分别比较，差异均有统计学意义(t=32.476，15.814，P<0.05)。TRAIL-Triptolide+组和TRAIL+Triptolide+组MiaPaca-2细胞中Bcl-2蛋白相对表达量与空白对照组分别比较，差异均无统计学意义(t=4.724，1.732，P>0.05)；上述两组与TRAIL+Triptolide-组分别比较，差异均无统计学意义(t=3.464，0.000，P>0.05)。(2)TRAIL-Mcl1 siRNA-组、TRAIL+Mcl1 siRNA-组、TRAIL-Mcl1 siRNA+组和TRAIL+Mcl1 siRNA+组MiaPaca-2细胞活力分别为100.0%±2.2%、79.3%±1.8%、71.2%±3.2%、37.3%±5.4%，Caspase-8蛋白相对表达量分别为0.100±0.003、0.100±0.005、0.100±0.003、0.350±0.005，Caspase-3蛋白相对表达量分别为0.020±0.003、0.060±0.003、0.020±0.003、0.590±0.004，4组比较，差异均有统计学意义(F=136.681，2 717.391，44 471.429，P<0.05)。TRAIL+Mcl1 siRNA+组MiaPaca-2细胞活力与TRAIL-Mcl1 siRNA-组、TRAIL+Mcl1 siRNA-组和TRAIL-Mcl1 siRNA+组分别比较，差异均有统计学意义(t=33.937，20.207，26.689，P<0.05)；TRAIL+Mcl1 siRNA+组MiaPaca-2细胞Caspase-8蛋白相对表达量与TRAIL-Mcl1 siRNA-组、TRAIL+Mcl1 siRNA-组和TRAIL-Mcl1 siRNA+组分别比较，差异均有统计学意义(t=216.506，433.013，144.338，P<0.05)；TRAIL+Mcl1 siRNA+组MiaPaca-2细胞Caspase-3蛋白相对表达量与TRAIL-Mcl1 siRNA-组、TRAIL+Mcl1 siRNA-组和TRAIL-Mcl1 siRNA+组分别比较，差异均有统计学意义(t=329.090，458.993，987.269，P<0.05)。TRAIL-BclxL siRNA-组、TRAIL+BclxL siRNA-组、TRAIL-BclxL siRNA+组和TRAIL+BclxL siRNA+组MiaPaca-2细胞活力分别为100.0%±2.3%、87.2%±4.1%、74.1%±3.7%、56.3±5.4%，Caspase-3蛋白相对表达量分别为0.060±0.004、0.070±0.003、0.060±0.004、0.390±0.003，4组比较，差异均有统计学意义(F=70.074，4 643.478，P<0.05)。TRAIL+BclxL siRNA+组MiaPaca-2细胞活力与TRAIL-BclxL siRNA-组、TRAIL+BclxL siRNA-组和TRAIL-BclxL siRNA+组分别比较，差异均有统计学意义(t=24.416，41.170，18.136，P<0.05)；TRAIL+BclxL siRNA+组MiaPaca-2细胞Caspase-3蛋白相对表达量与TRAIL-Mcl1 siRNA-组、TRAIL+Mcl1 siRNA-组和TRAIL-Mcl1 siRNA+组分别比较，差异均有统计学意义(t=285.788，554.256，190.526，P<0.05)。
结论:Triptolide可能通过抑制Mcl1与BclxL表达水平，致敏TRAIL，激活Caspase-8及其下游的Caspase-3，最终诱导胰腺癌细胞凋亡。

Abstract: Objective:To investigate the mechanisms of tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) combined with Triptolide in inducing the apoptosis of pancreatic cancer cells.
Methods:(1) The pancreatic cancer cells (MiaPaca-2 cells) were divided into 4 groups: blank control group (no drugs were added), TRAIL+Triptolide- group (only TRAIL was added), TRAIL-Triptolide+group (only Triptolide was added) and TRAIL+Triptolide+ group (TRAIL and Triptolide were added). The vitality of cells in all the 4 groups was assessed by CCK-8. The expressions of poly ADPribose polymerase (PARP), cysteinyl aspartate specific proteinase-3 (Caspase-3) and Caspase-8 were detected by Western blot. The vitality of cells was detected by CCK-8 and the vitality of Caspase-8 was detected by CaspaseGlo assays after adding ZIETDFMK, a specific inhibitor of Caspase-8. The expressions of myeloid cell leukemia1 (Mcl1), BclxL and Bcl-2 were detected by Western blot. (2) The MiaPaca-2 cells were divided into 8 groups: ①TRAIL-Mcl1 siRNA- group (no TRAIL was added and Mcl1 siRNA cells were not transfected), TRAIL+Mcl1 siRNA- group (TRAIL was added and Mcl1 siRNA cells were not transfected), TRAIL-Mcl1 siRNA+ group (TRAIL was not added and Mcl1 siRNA cells were transfected) and TRAIL+Mcl1 siRNA+ group (TRAIL was added and Mcl1 siRNA cells were transfected). ②TRAIL-BclxL siRNA- group (TRAIL was not added and BclxL siRNA was not transfected), TRAIL+BclxL siRNA- group (TRAIL was added and BclxL siRNA was not transfected), TRAIL-BclxL siRNA+ group (TRAIL was not added and BclxL siRNA was transfected) and TRAIL+BclxL siRNA+ group (TRAIL was added and BclxL siRNA was transfected). The vitality of the cells in all the groups was detected by CCK-8. The expressions of Caspase-3 and Caspase-8 protein were detected by Western blot. The measurement data with normal distribution were presented as ±s. The comparison among groups was done by ANOVA, and the pairwise comparison was done by LSDt test.
Results:(1) The vitalities of MiaPaca-2 cells in the blank control group, TRAIL+Triptolide- group, TRAIL-Triptolide+group and TRAIL+Triptolide+ group were 100.0%±1.1%, 81.2%±2.3%, 78.6%±3.6% and 40.1%±2.5%, and the relative expressions of PARP protein were 0.510±0.028, 0.720±0.072, 1.250±0.023 and 2.560±0.220, the relative expressions of Caspase-3 were 0.080±0.004, 0.080±0.003, 0.110±0.005 and 2.720±0.003, and the relative expressions of Caspase-8 were 0.070±0.003, 0.080±0.005, 0.120±0.003 and 0.990±0.006, with significant differences among the 4 groups (F=203.607, 1 457.785, 332 421.900, 35 437.218, P<0.05). The vitality of MiaPaca-2 cells in the TRAIL+Triptolide+ group was significantly different from those in the blank control group, the TRAIL+Triptolide- group and the TRAIL-Triptolide+ group (t=34.583, 355.936, 36.271, P<0.05）. The relative expression of PARP protein of MiaPaca-2 cells in the TRAIL+Triptolide+ group was significantly different from those in the blank control group, TRAIL+Triptolide- group and TRAIL-Triptolide+ group (t=591.784, 63.739, 2 268.987, P<0.05). The relative expression of Caspase-3 protein of the MiaPaca-2 cells in the TRAIL+Triptolide+ group was significantly different from those in the blank control group, the TRAIL+Triptolide- group and theTRAIL-Triptolide+ group (t=3 266.153, 9 145.228, 1 738.713, P<0.05). The relative expression of Caspase-8 protein of the MiaPaca-2 cells in the TRAIL+Triptolide+ group was significantly different from those in the blank control group, the TRAIL+Triptolide- group and the TRAIL-Triptolide+ group (t=663.953, 1 432.878, 327.584, P<0.05). The vitality of caspase-8 in the TRAIL+Triptolide+ group was 711.0%±5.1% before adding ZIETDFMK, and then the vitality of MiaPaca-2 cells and caspase-8 changed to 70.0%±4.8% and 73.0%±2.4%, with significant differences (t=17.956, 55.027, P<0.05). The relative expressions of Mcl1 protein in the blank control group, the TRAIL+Triptolide- group, the TRAIL-Triptolide+ group and the TRAIL+Triptolide+ group were 1.68±0.22, 2.08±0.11, 0.73±0.15 and 0.58±0.18, the relative expressions of BclxL protein were 0.65±0.03, 0.47±0.03, 0.32±0.03 and 0.26±0.05, the relative expressions of Bcl-2 protein were 0.65±0.03, 0.67±0.03, 0.62±0.05 and 0.67±0.03, with significant difference among the 4 groups (F=55.178, 88.683, 3.411, P<0.05). The relative expressions of Mcl1 protein of the MiaPaca-2 cells in the TRAIL-Triptolide+ group and the TRAIL+Triptolide+ group were significantly different from those of the blank control group (t=23.506, 47.631, P<0.05）and the TRAIL+Triptolide- group (t=58.457, 37.115, P<0.05). The relative expressions of BclxL protein of the MiaPaca-2 cells in the TRAIL-Triptolide+ group and the TRAIL+Triptolide+ group were significantly different from those of the blank control group (t=38.105, 42.219, P<0.05) and the TRAIL+Triptolide- group (t=32.476,15.814, P<0.05）. The relative expressions of Bcl-2 protein in the TRAIL-Triptolide+ group and the TRAIL+Triptolide+ group were not significantly different from those of the blank control group (t=4.724, 1.732, P>0.05) and the TRAIL+Triptolide- group (t=3.464, 0.000, P>0.05). (2) The vitalities of MiaPaca-2 cells of the TRAIL-Mcl1 siRNA- group, TRAIL+Mcl1 siRNA- group , the TRAIL-Mcl1 siRNA+group and the TRAIL+Mcl1 siRNA+group were 100.0%±2.2%, 79.3%±1.8%, 71.2%±3.2% and 37.3%±5.4%, the relative expressions of Caspase-8 protein were 0.100±0.003, 0.100±0.005, 0.100±0.003 and 0.350±0.005, and the relative expressions of Caspase-3 protein were 0.020±0.003, 0.060±0.003, 0.020±0.003 and 0.590±0.004, with significant differences among the 4 groups (F=136.681, 2 717.391, 44 471.429, P<0.05). The vitality of MiaPaca-2 cells of the TRAIL+Mcl1 siRNA+group was significantly different from those in the TRAIL-Mcl1 siRNA- group, the TRAIL+Mcl1 siRNA- group and the TRAIL-Mcl1 siRNA+group (t=33.937, 20.207, 26.689, P<0.05). The relative expression of Caspase-8 protein of the TRAIL+Mcl1 siRNA+ group was significantly different from those in the TRAIL-Mcl1 siRNA- group, the TRAIL+Mcl1 siRNA- group and the TRAIL-Mcl1 siRNA+group (t=216.506, 433.013, 144.338, P<0.05). The relative expression of Caspase-3 protein of the TRAIL+Mcl1 siRNA+ group was significantly different from those in the TRAIL-Mcl1 siRNA- group, the TRAIL+Mcl1 siRNA- group and the TRAIL-Mcl1 siRNA+ group (t=329.09, 458.993, 987.269, P<0.05). The vitalities of MiaPaca-2 cells of the TRAIL-BclxL siRNA- group, the TRAIL+BclxL siRNA- group , the TRAIL-BclxL siRNA+ group and the TRAIL+BclxL siRNA+group were 100.0%±2.3%, 87.2%±4.1%, 74.1±3.7% and 56.3%±5.4%, and the relative expressions of Caspase-3 protein were 0.060±0.004, 0.070±0.003, 0.060±0.004 and 0.390±0.003, with significant differences among the 4 groups (F=70.074, 4 643.478, P<0.05). The vitality of MiaPaca-2 cells of the TRAIL+BclxL siRNA+group was significantly different from those in the TRAIL-BclxL siRNA- group, the TRAIL+BclxL siRNA- group and the TRAIL-BclxL siRNA+group (t=24.416, 41.170, 18.136, P<0.05). The relative expression of Caspase-3 protein of the TRAIL+BclxL siRNA+ group was significantly different from those in the TRAIL-Mcl1 siRNA- group, the TRAIL+Mcl1 siRNA- group and the TRAIL-Mcl1 siRNA+group (t=285.788, 554.256, 190.526, P<0.05).
Conclusion:Triptolide could induce the apoptosis of MiaPaca-2 cells by inhibiting the expressions of Mcl1 and BclxL, sensitizing TRAIL and activating Caspase-8 and Caspase-3.

HTML全文

参考文献(0)

施引文献

资源附件(0)