新型小分子激酶抑制剂Ibr-7对人胰腺癌Capan-2细胞的抑制作用及机制研究
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篇名: 新型小分子激酶抑制剂Ibr-7对人胰腺癌Capan-2细胞的抑制作用及机制研究
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摘要: 目的:观察新型小分子激酶抑制剂Ibr-7[依鲁替尼(Ibr)衍生物]对人胰腺癌Capan-2 细胞的抑制作用及其可能机制。方法:以Capan-2细胞为对象,采用CCK-8法检测1、2、4、8 μmol/L Ibr、Ibr-7作用48 h后的细胞增殖情况,计算细胞存活率;同法检测1 μmol/L Ibr、Ibr-7对不同剂量吉西他滨/紫杉醇(均分别为0.062 5、0.125、0.25、0.5、1 μmol/L)的增敏作用。采用克隆形成试验检测1、2、4 μmol/L Ibr、Ibr-7作用48 h后的细胞克隆形成情况,并记录细胞集落形成数量。采用流式细胞术或JC-1法检测2、4、8 μmol/L Ibr-7作用24或16 h后细胞凋亡情况和线粒体膜电位变化情况,并计算总凋亡率及细胞线粒体膜电位下降比例。采用Western blotting法检测细胞中相关凋亡蛋白[多聚二磷酸腺苷核糖聚合酶(PARP)、Noxa、Bcl-2、Bax、髓样细胞白血病1(Mcl-1)、B淋巴细胞瘤xL(Bcl-xL)]的表达情况。结果:1、2、4、8 μmol/L Ibr、Ibr-7作用48 h后,细胞的存活率均显著下降,各剂量Ibr-7组均显著低于同剂量Ibr组,且Ibr-7的半数抑制浓度显著低于Ibr(P<0.05或P<0.01)。联用Ibr、Ibr-7后,细胞的存活率均显著低于同剂量吉西他滨/紫杉醇单用组,且Ibr-7联用组显著低于同剂量Ibr联用组(P<0.05或P<0.01)。经2、4 μmol/L Ibr以及1、2、4 μmol/L Ibr-7作用48 h后,细胞集落形成数量均显著减少,且各剂量Ibr-7组均显著低于同剂量Ibr组(P<0.01)。经不同剂量Ibr-7作用24或16 h后,Capan-2细胞的总凋亡率(2、4、8 μmol/L组)、细胞线粒体膜电位下降比例(8 μmol/L组)以及Noxa(2、4、8 μmol/L组)、Bax(8     μmol/L组)蛋白的相对表达量均显著上升,PARP(8 μmol/L组)、Bcl-2(4 μmol/L组)、Mcl-1(2、4、8 μmol/L组)蛋白的相对表达量均显著下降,且8 μmol/L Ibr-7组上述指标(PARP、Bcl-2相对表达量除外)均显著优于同剂量Ibr组(P<0.05或P<0.01)。而各组细胞Bcl-xL蛋白的相对表达量差异均无统计学意义(P>0.05)。结论:与Ibr相比,Ibr-7对人胰腺癌Capan-2细胞具有更强的体外增殖抑制作用和促凋亡作用,且具有更强的化疗药物增敏活性;其作用机制可能与降低细胞线粒体膜电位,下调细胞中PARP、Bcl-2、Mcl-1蛋白的表达,上调Noxa、Bax蛋白的表达有关。
ABSTRACT: OBJECTIVE: To observe the inhibitory effects and possible mechanism of new small molecular kinase inhibitors Ibr-7 [Irutinil(Ibr) derivatives] on human pancreatic cancer Capan-2 cells. METHODS: Taking Capan-2 cells as objects, CCK-8 method was used to determine the proliferation of cells after treated with 1, 2, 4, 8 μmol/L Ibr/Ibr-7 for 48 h. The survival rates of cells were calculated. Sensitization effects of 1 μmol/L Ibr/Ibr-7 on different doses of gemcitabine/paclitaxel (0.062 5, 0.125, 0.25, 0.5, 1 μmol/L) were detected. Clone formation test was used to detect the situation of cell clone formation after treated with 1, 2, 4 μmol/L Ibr/Ibr-7 for 48 h. The number of cell colony formation was recorded. Flow cytometry or JC-1 method was used to detect the apoptosis of cells after treated with 2, 4, 8 μmol/L Ibr-7 for 24 or 16 h and the changes of mitochondrial transmembrane potential; total apoptotic rate and the percentage of mitochondrial membrane potential decrease were calculated. Western blotting was used to detect the expression of related apoptotic protein (PARP, Noxa, Bcl-2, Bax, Mcl-1, Bcl-xL). RESULTS: After treated with 1, 2, 4, 8 μmol/L Ibr/Ibr-7 for 48 h, the survival rates of cells were decreased significantly; those of Ibr-7 groups were significantly lower than those of same-dose Ibr groups; IC50 of Ibr-7 was significantly lower than that of Ibr (P<0.05 or P<0.01). After combined with Ibr/Ibr-7, the survival rate of cells was significantly lower than that of same-dose gemcitabine/paclitaxel alone group, and the Ibr-7 combination group was significantly lower than same-dose Ibr combination group (P<0.05 or P<0.01). After treated with 2, 4 μmol/L Ibr and 1, 2, 4 μmol/L Ibr-7 for 48 h, the number of cell clone formation was decreased significantly, while Ibr-7 groups were significantly lower than same-dose Ibr groups (P<0.01). After treated with different doses of Ibr-7 for 24 or 16 h, total apoptosis rate of cells (2, 4, 8 μmol/L), the proportion of cell mitochondrial membrane potential decrease (8 μmol/L), the relative protein expression of Noxa (2, 4, 8 μmol/L) and Bax (8 μmol/L) were increased significantly, while the protein expression of PARP (8 μmol/L), Bcl-2 (4 μmol/L), Mcl-1 (2, 4, 8 μmol/L) were decreased significantly; above indexes (except for relative expression of PARP and Bcl-2) of 8 μmol/L Ibr-7 group were significantly better than same-dose Ibr group (P<0.05 or P<0.01). There was no statistical significance in protein expression of Bcl-xL among those groups (P>0.05). CONCLUSIONS: Compared with Ibr, Ibr-7 has better inhibitory and apoptotic effects on human pancreatic cancer Capan-2 cells in vitro, and has stronger chemotherapeutic drug sensitization activity, the mechanism of which may be associated with reducing mitochondrial transmembrane potential, down-regulating the protein expression of PARP, Bcl-2 and Mcl-1 and up-regulating the protein expression of Noxa and Bax.
期刊: 2019年第30卷第4期
作者: 阎优优,张博,张琪,周冬梅,林能明
AUTHORS: YAN Youyou,ZHANG Bo,ZHANG Qi,ZHOU Dongmei,LIN Nengming
关键字: 依鲁替尼;依鲁替尼衍生物;人胰腺癌Capan-2 细胞;增殖;凋亡;线粒体膜电位;凋亡相关蛋白
KEYWORDS: Ibrutinib; Irutinil derivatives; Human pancreatic cancer Capan-2 cells; Proliferation; Apoptosis; Mitochondrial transmembrane potential; Apoptosis-related protein
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