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、8μmol/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1μmol/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、1μmol/L)的增敏作用。采用克隆形成试验检测1、2、4μmol/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、8μmol/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Westernblotting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x L(Bcl-xL)]的表达情况。结果:1、2、4、8μmol/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、4μmol/L Ibr以及1、2、4μmol/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16h后,Capan-2细胞的总凋亡率(2、4、8μmol/L组)、细胞线粒体膜电位下降比例(8μmol/L组)以及Noxa(2、4、8μmol/L组)、Bax(8μmol/L组)蛋白的相对表达量均显著上升,PARP(8μmol/L组)、Bcl-2(4μmol/L组)、Mcl-1(2、4、8μmol/L组)蛋白的相对表达量均显著下降,且8μmol/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。
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.
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.
引文
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[2]SIEGEL RL,MILLER KD,JEMAL A.Cancer statistics:2016[J].CA Cancer J Clin,2016,66(1):7-30.
[3]CONROY T,DESSEIGNE F,YCHOU M,et al.FOLFIRI-NOX versus gemcitabine for metastatic pancreatic cancer[J].N Engl J Med,2011,364(19):1817-1825.
[4]OETTLE H,NEUHAUS P,HOCHHAUS A,et al.Adjuvant chemotherapy with gemcitabine and long-term outcomes among patients with resected pancreatic cancer:the CONKO-001 randomized trial[J].JAMA,2013,310(14):1473-1481.
[5]SIROHI B,SINGH A,DAWOOD S,et al.Advances in chemotherapy for pancreatic cancer[J].Indian J Surg Oncol,2015,6(1):47-56.
[6]KOKABEE L,WANG X,SEVINSKY CJ,et al.Bruton’s tyrosine kinase is a potential therapeutic target in prostate cancer[J].Cancer Biol Ther,2015,16(11):1604-1615.
[7]GRASSILLI E,PISANO F,CIALDELLA A,et al.A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation[J].Oncogene,2016,35(33):4368-4378.
[8]HAMACHER-BRADY A,BRADY NR.Bax/Bak-dependent,Drp1-independent targeting of X-linked inhibitor of apoptosis protein(XIAP)into inner mitochondrial compartments counteracts Smac/DIABLO-dependent effector caspase activation[J].J Biol Chem,2015,290(36):22005-22018.
[9]MASSó-VALLéS D,JAUSET T,SOUCEK L.Ibrutinib repurposing:from B-cell malignancies to solid tumors[J].Oncoscience,2016,3(5/6):147-148.
[10]YU K,LUCAS J,ZHU T,et al.PWT-458,a novel pegylated-17-hydroxywortmannin,inhibits phosphatidylinositol3-kinase signaling and suppresses growth of solid tumors[J].Cancer Biol Ther,2005,4(5):538-545.
[11]KIM KY,SEOL JY,JEON GA,et al.The combined treatment of aspirin and radiation induces apoptosis by the regulation of Bcl-2 and caspase-3 in human cervical cancer cell[J].Cancer Lett,2003,189(2):157-166.
[12]ALBERSHARDT TC,SALERNI BL,SODERQUIST RS,et al.Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA[J].J Biol Chem,2011,286(28):24882-24895.
[13]KOTSCHY A,SZLAVIK Z,MURRAY J,et al.The MCL1inhibitor S63845 is tolerable and effective in diverse cancer models[J].Nature,2016,538(7626):477-482.
[14]HUANG J,NAKAMURA K,ITO Y,et al.Bcl-xL gene transfer inhibits Bax translocation and prolongs cardiac cold preservation time in rats[J].Circulation,2005,112(1):76-83.
[15] LOOR G,KONDAPALLI J,SCHRIEWER JM,et al.Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis[J]. Free Radic Biol Med,2010,49(12):1925-1936.