鸦胆子素D联合紫杉醇对胰腺癌Capan-2细胞增殖的抑制作用及机制研究

Study on Inhibitory Effects and Mechanism of Bruceanine D Combined with Taxol on the Proliferation of Human Pancreatic Cancer Capan-2 Cells

目的:探讨鸦胆子素D(BD)联合紫杉醇(Taxol)对人胰腺癌Capan-2细胞增殖的抑制作用及可能机制。方法:以Capan-2细胞为对象,采用磺酰罗丹明B法检测不同剂量BD(5、10、15、20μmol/L)、Taxol(10、20、30、40 nmol/L)、BD+Taxol(5μmol/L+10nmol/L、10μmol/L+20 nmol/L、15μmol/L+30 nmol/L、20μmol/L+40 nmol/L)作用48 h后的细胞增殖情况,并计算细胞存活率和药物合用指数(CI)。采用克隆形成试验检测BD(20μmol/L,下同)、Taxol(40 nmol/L,下同)、BD+Taxol(20μmol/L+40 nmol/L,下同)作用24 h后的细胞克隆集落形成情况,并计算克隆形成率;采用DAPI染色法观察BD、Taxol、BD+Taxol作用24 h后的细胞凋亡情况,采用Western blotting法检测的BD、Taxol、BD+Taxol作用后凋亡相关蛋白[Bcl-2、多聚二磷酸腺苷核糖聚合酶(PARP)、胱天蛋白酶3(Caspase-3)、切割胱天蛋白酶3(Cleaved-caspase-3);药物作用48 h]以及c-Jun氨基末端激酶(JNK)、磷酸化c-Jun氨基末端激酶(p-JNK)蛋白(药物作用4、6、12 h)的表达情况。结果:经10、15、20μmol/L BD,20、30、40 nmol/L Taxol以及两药上述剂量联合作用48 h后,细胞的存活率均显著降低,且联用组显著低于BD、Taxol同剂量单药组(P<0.05),各联用组(BD 5μmol/L+Taxol10 nmol/L、BD 10μmol/L+Taxol 20 nmol/L、BD 15μmol/L+Taxol 30 nmol/L、BD 20μmol/L+Taxol 40 nmol/L)的CI值分别为0.63±0.04、0.68±0.08、0.89±0.12、0.84±0.05。经20μmol/L BD、40 nmol/L Taxol以及两药上述剂量联合作用后,细胞的克隆集落形成有所减少,并伴有不同程度的染色质浓聚和细胞核皱缩,细胞的克隆形成率(24 h)以及Bcl-2(48 h)、RAPR(48 h)、Caspase-3(48h)、JNK(4、6 h,Taxol单药组除外)的相对表达量均显著降低,Cleaved-caspase-3(48 h)、p-JNK(4、6、12 h)的相对表达量均显著升高,且BD+Taxol联用组均显著优于同时间点BD、Taxol同剂量单药组[JNK(4、6、12 h)、p-JNK(4 h)除外](P<0.05或P<0.01)。结论:BD、Taxol均可抑制人胰腺癌Capan-2细胞的增殖并促进其凋亡,且两者联合具有一定的协同作用,效果优于任一药物单用。上述作用可能与激活Caspase通路以及JNK磷酸化等途径有关。

OBJECTIVE:To investigate the inhibitory effect and potential mechanism of Brucein D(BD)combined with Taxol on the proliferation of human pancreatic cancer Capan-2 cells.METHODS:Using Capan-2 cells as object,the proliferations after treated with BD(5,10,15,20μmol/L),Taxol(10,20,30,40 nmol/L)and BD+Taxol(5μmol/L+10 nmol/L,10μmol/L+20 nmol/L,15μmol/L+30 nmol/L,20μmol/L+40 nmol/L)for 48 h were determined by sulfonyl rhodamine B method.Survival rate of cells and combination index(CI)were calculated.The clone formation assay was performed to detect the formation of clonal colonies after treated with BD(20μmol/L,hereinafter),Taxol(40 nmol/L,hereinafter)、BD+Taxol(20μmol/L+40 nmol/L,hereinafter)for 24 h.The rate of clone formation was calculated.DAPI method was used to observe the apoptosis of cells after treated with BD,Taxol and BD+Taxol for 24 h.Western blotting was used to detect the expression of apoptosis-related protein(Bcl-2,PARP,Caspase-3,Cleaved-caspase-3)after treated by BD,Taxol,BD+Taxol for 48 h and the expression of JNK and p-JNK after treated by BD,Taxol,BD+Taxol for 4,6,12 h.RESULTS:After treated with 10,15 and 20μmol/L BD,20,30 and 40 nmol/L Taxol or two-drug combination for 48 h,survival rates of cells were decreased significantly;the survival rate of drug combination group was significantly lower than the same dose of BD group and Taxol group(P<0.05).CI values of drug combination groups(BD 5μmol/L+Taxol 10 nmol/L,BD 10μmol/L+Taxol 20 nmol/L,BD 15μmol/L+Taxol 30 nmol/L,BD 20μmol/L+Taxol 40 nmol/L)were 0.63±0.04,0.68±0.08,0.89±0.12 and 0.84±0.05.After treated with 20μmol/L BD,40 nmol/L Taxol and two-drug combination,the formation of clonal colonies was decreased with different degrees of chromatin concentration and nuclear shrinkage;the rate of clone formation(24 h),the expression of Bcl-2(48 h),PARP(48 h),Caspase-3(48 h)and JNK(4,6 h,except for Taxol group)were decreased significantly,while the relative expression of Cleaved-caspase-3(48 h)and p-JNK(4,6,12 h)were increased significantly.Those of BD+Taxol group were significantly better than those of BD group and Taxol group[except for JNK(4,6,12 h),p-JNK(4 h)](P<0.05 or P<0.01).CONCLUSIONS:Both BD and Taxol can inhibit the proliferation and promote apoptosis of human pancreatic cancer Capan-2 cells,and the combination have a certain synergistic effect,which is better than any single drug.It may be associated with activating Caspase pathway and JNK phosphorylation.