Apatinib抑制套细胞淋巴瘤细胞株增殖与凋亡实验研究

Investigation of apatinib inhibiting proliferation and inducing apoptosis in mantle cell lymphoma cell lines

目的套细胞淋巴瘤(mantle cell lymphoma,MCL)的化疗效果欠佳,亟需开拓新的靶向治疗方式。本研究探讨新一代小分子血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR2)酪氨酸激酶抑制剂阿帕替尼(Apatinib)对MCL细胞株增殖与凋亡的影响及机制。方法体外培养MCL细胞株Mino和Z138;CCK8法检测不同浓度下Apatinib对Mino和Z138细胞株的增殖抑制作用;AnnexinⅤ/PI双标记流式细胞术检测不同浓度Apatinib对Mino和Z138细胞株的诱导凋亡作用;蛋白质印迹法检测药物处理后ERK1/2通路相关蛋白ras、c-raf、pMEK和pERK1/2的表达的变化。结果 Aptinib对Mino和Z138细胞株均有明显的增殖抑制作用,呈浓度依赖性。Mino细胞经过Apatinib处理48h后,2.5μmol/L的抑制率为(5.72±2.43)%、5μmol/L的抑制率为(8.19±2.2)%、10μmol/L的抑制率为(17.70±4.01)%、20μmol/L的抑制率为(44.58±7.59)%、40μmol/L的抑制率为(85.12±2.67)%,IC50为(20.63±0.18)μmol/L,经多个独立样本非参数检验Kruskal-Wallis H分析表明,各处理组与对照组多重比较,差异均有统计学意义,H=22.016,P=0.001。Z138细胞组2.5μmol/L的抑制率为(7.19±1.71)%、5μmol/L的抑制率为(10.60±1.43)%、10μmol/L的抑制率为(20.24±5.93)%、20μmol/L的抑制率为(53.46±4.91)%、40μmol/L的抑制率为(87.07±1.41)%,IC50为(18.15±0.18)μmol/L,经多个独立样本非参数检验Kruskal-Wallis H分析表明,各处理组与对照组两两比较差异有统计学意义,H=16.460,P=0.006。细胞凋亡结果显示,Apatinib处理Mino细胞48h后,对照组凋亡率为(4.03±0.99)%、10μmol/L的凋亡率为(8.10±0.98)%、20μmol/L的凋亡率为(29.46±7.18)%、30μmol/L的凋亡率为(35.06±4.17)%、40μmol/L的凋亡率为(50.01±2.14)%。经Kruskal-Wallis H分析表明,处理组与对照组两两比较差异有统计学意义,H=13.033,P=0.011。Z138细胞对照组凋亡率为(5.45±1.82)%、10μmol/L的凋亡率为(10.58±2.74)%、20μmol/L的凋亡率为(16.51±0.81)%、30μmol/L的凋亡率为(23.07±3.14)%、40μmol/L的凋亡率为(34.48±6.40)%,经Kruskal-Wallis H分析,处理组与对照组两两比较,差异有统计学意义,H=13.500,P=0.009。蛋白质印迹法结果显示,Apatinib处理Mino细胞48h后,ERK1/2通路相关蛋白ras、c-raf、pMEK和ERK1/2表达均有下降。结论 Apatinib可以抑制MCL细胞株Mino和Z138的增殖并有效诱导其发生凋亡,呈浓度依赖性其机制可能是通过干扰ERK1/2通路实现的。

OBJECTIVE The effect of chemotherapy works a poor result for the mantle cell lymphoma（MCL）and there is an urgent need for the new targeting agents.Apatinib exhibits anti-cancer activities among tumors.In this study,we investigate the effects and mechanism with regard to Apatinib,a novel VEGFR2 inhibitor,on the proliferation and apoptosis toward mantle cell lymphoma cell lines.METHODS The inhibition of proliferation in Mino and Z138 was performed by CCK8 assay in the presence of Apatinib.The induction of apoptosis was detected using AnnexinⅤ/PI staining by flow cytometry in Mino and Z138 after exposed to different concertration of Apatinib.The expression of ERK1/2 pathway-related proteins,including ras,c-raf,pmek,pERK1/2,was monitored by western blot after Apatinib treatment.RESULTS Treating with Apatinib for 48 hinduced a significant proliferation inhibition among Mino and Z138 cells in a dosedependent manner.In Mino group,the proliferation inhibition rates were respectively（5.72±2.43）% in 2.5μmol/L,（8.19±2.2）% in 5μmol/L,（17.70±4.01）% in 10μmol/L,（44.58±7.59）% in 20μmol/L,（85.12±2.67）% in40μmol/L,and IC50 was（20.63±0.18）μmol/L.All the drug groups were different significantly（check by Kruskal-Wallis H test,H=22.016,P=0.001）.In Z138 cells,the inhibition rates were（7.19±1.71）% in 2.5μmol/L,（10.60±1.43）%in 5μmol/L,（20.24±5.93）%in 10μmol/L,（53.46±4.91）%in 20μmol/L,（87.07±1.41）%in 40μmol/L with an obvious difference compared with control group（H=16.460,P=0.006）.The IC50 was（18.15±0.18）μmol/L.Apoptosis assay results showed that treatment with different concentrations（10,20,30,40μmol/L）of apatinib obviously triggered dose-dependent apoptosis against Mino and Z138.The apoptosis rates were（8.10±0.98）% in 10μmol/L,（29.46±7.18）%in 20μmol/L,（35.06±4.17）%in 30μmol/L,（50.01±2.14）%in 40μmol/L among Mino cells and（10.58±2.74）%in 10μmol/L,（16.51±0.81）%in 20μmol/L,（23.07±3.14）%in 30μmol/L,（34.48±6.40）%in40μmol/L among Z138 cells,and it was significant（H=13.033,P=0.011;H=13.500,P=0.009）compared with control group（4.03±0.99）%,（5.45±1.82）%respectively.The reduced ERK1/2 pathway was remarkably observed in Mino in the presence of Apatinib for 48 h.CONCLUSION Apatinib exerts a potential anti-tumor effect against mantle cell lymphoma cell lines by inhibiting proliferation and induction apoptosis in a dose-dependent manner via the down-regulation of ERK1/2 pathway.