Objective To investigate the mechanisms of tumor necrosis factor-related 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 ADP-ribose 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 Caspase-Glo assays after adding Z-IETD-FMK,a specific inhibitor of Caspase-8.The expressions of myeloid cell leukemia-1 (Mcl-1),Bcl-xL and Bcl-2 were detected by Western blot.(2) The MiaPaca-2 cells were divided into 8 groups:①TRAIL-Mcl-1 siRNA-group (no TRAIL was added and Mcl-1 siRNA cells were not transfected),TRAIL+ Mcl-1 siRNA-group (TRAIL was added and Mcl-1 siRNA cells were not transfected),TRAIL-Mcl-1 siRNA + group (TRAIL was not added and Mcl-1 siRNA cells were transfected)and TRAIL+ Mcl-1 siRNA+ group (TRAIL was added and Mcl-1 siRNA cells were transfected).②TRAIL-Bcl-xL siRNA-group (TRAIL was not added and Bcl-xL siRNA was not transfected),TRAIL+ Bcl-xL siRNA-group (TRAIL was added and Bcl-xL siRNA was not transfected),TRAIL-Bcl-xL siRNA + group (TRAIL was not added and Bcl-xL siRNA was transfected) and TRAIL+ Bcl-xL siRNA+ group (TRAIL was added and Bcl-xL 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 (x) ± s.The comparison among groups was done by ANOVA,and the pairwise comparison was done by LSD-t 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 M iaPaca-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+ Triptolidegroup 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,l 432.878,327.584,P ＜ 0.05).The vitality of caspase-8 in the TRAIL+ Triptolide+ group was 711.0％ ± 5.1％ before adding Z-IETD-FMK,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 Mcl-1 protein in the blank control group,the TRAIL + Triptolidegroup,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 Bcl-xL protein were 0.65 ± 0.03,0.47 ± 0.03,0.32 ± 0.03and 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 Mcl-1 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 Bcl-xL 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-Mcl-1 siRNA-group,TRAIL + Mcl-1 siRNA-group,the TRAIL-Mcl-1 siRNA + group and the TRAIL + Mcl-1 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 + Mcl-1 siRNA + group was significantly different from those in the TRAIL-Mcl-1 siRNA-group,the TRAIL + Mcl-1 siRNA-group and the TRAIL-Mcl-1 siRNA + group (t =33.937,20.207,26.689,P ＜ 0.05).The relative expression of Caspase-8 protein of the TRAIL + Mcl-1 siRNA +group was significantly different from those in the TRAIL-Mcl-1 siRNA-group,the TRAIL + Mcl-1 siRNA-group and the TRAIL-Mcl-1 siRNA + group (t =216.506,433.013,144.338,P ＜ 0.05).The relative expression of Caspase-3 protein of the TRAIL + Mcl-1 siRNA + group was significantly different from those in the TRAIL-Mcl-1 siRNA-group,the TRAIL + Mcl-1 siRNA-group and the TRAIL-Mcl-1 siRNA + group (t =329.09,458.993,987.269,P ＜0.05).The vitalities of MiaPaca-2 cells of the TRAIL-Bcl-xL siRNA-group,the TRAIL+ Bcl-xL siRNA-group,the TRAIL-Bcl-xL siRNA + group and the TRAIL+ Bcl-xL 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 + Bcl-xL siRNA + group was significantly different from those in the TRAIL-Bcl-xL siRNA-group,the TRAIL+ Bcl-xL siRNA-group and the TRAIL-Bcl-xL siRNA + group (t =24.416,41.170,18.136,P ＜ 0.05).The relative expression of Caspase-3 protein of the TRAIL + Bcl-xL siRNA + group was significantly different from those in the TRAIL-Mcl-1 siRNA-group,the TRAIL + Mcl-1 siRNA-group and the TRAIL-Mcl-1 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 Mcl-1 and Bcl-xL,sensitizing TRAIL and activating Caspase-8 and Caspase-3.%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Z-IETD-FMK后,采用CCK-8检测细胞活力,采用Caspase-Glo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(Mcl-1)、额外的大B细胞淋巴瘤(Bcl-xL)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-Mcl-1 siRNA-组(不加TRAIL、不转染Mcl-1 siRNA),TRAIL+ Mcl-1siRNA-组(加TRAIL、不转染Mcl-1 siRNA),TRAIL-Mcl-1 siRNA+组(不加TRAIL、转染Mcl-1 siRNA)和TRAIL+ Mcl-1 siRNA+组(加TRAIL、转染Mcl-1 siRNA).②TRAIL-Bcl-xL siRNA-组(不加TRAIL、不转染Bcl-xL siRNA),TRAIL+ Bcl-xL siRNA-组(加TRAIL、不转染Bcl-xL siRNA),TRAIL-Bcl-xL siRNA+组(不加TRAIL、转染Bcl-xL siRNA),TRAIL+ Bcl-xL siRNA+组(加TRAIL、转染Bcl-xL 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(x)±s表示,多组间比较采用单因素方差分析,两两比较采用LSD-t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(=663.953,1 432.878,327.584,P＜0.05).TRAIL+ Triptolide+组MiaPaca-2细胞加Z-IETD-FMK前,Caspase-8活性为711.0％±5.1％;加入Z-IETD-FMK后,细胞活力为70.0％±4.8％,Caspase-8活性为73.0％±2.4％;加Z-IETD-FMK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Mcl-1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,Bcl-xL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Mcl-1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(=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Bcl-xL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-Mcl-1 siRNA-组、TRAIL+ Mcl-1siRNA-组、TRAIL-Mcl-1 siRNA+组和TRAIL+ Mcl-1 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=136.681,2 717.391,44471.429,P＜ 0.05).TRAIL+ Mcl-1 siRNA+组MiaPaca-2细胞活力与TRAIL-Mcl-1 siRNA-组、TRAIL+ Mcl-1 siRNA-组和TRAIL-Mcl-1 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+ Mcl-1 siRNA+组MiaPaca-2细胞Caspase-8蛋白相对表达量与TRAIL-Mcl-1 siRNA-组、TRAIL+Mcl-1 siRNA-组和TRAIL-Mcl-1 siRNA+组分别比较,差异均有统计学意义(=216.506,433.013,144.338,P＜ 0.05);TRAIL+ Mcl-1 siRNA+组MiaPaca-2细胞Caspase-3蛋白相对表达量与TRAIL-Mcl-1 siRNA-组、TRAIL+ Mcl-1 siRNA-组和TRAIL-Mcl-1 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-Bcl-xL siRNA-组、TRAIL+ Bcl-xL siRNA-组、TRAIL-Bcl-xL siRNA+组和TRAIL+ Bcl-xL 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+ Bcl-xL siRNA+组MiaPaca-2细胞活力与TRAIL Bcl-xL siRNA-组、TRAIL+ Bcl-xL siRNA-组和TRAIL-Bcl-xL siRNA+组分别比较,差异均有统计学意义(=24.416,41.170,18.136,P ＜0.05);TRAIL+ Bcl-xL siRNA+组MiaPaca-2细胞Caspase-3蛋白相对表达量与TRAIL-Mcl-1 siRNA-组、TRAIL+ Mcl-1 siRNA-组和TRAIL-Mcl-1 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Mcl-1与Bcl-xL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.
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