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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 |
| TITLE： | |
| 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 |
| 阅读数： | 387 次 |
| 本月下载数： | 4 次 |
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