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.
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