摘要
目的探讨安罗替尼对儿童T-急性淋巴细胞白血病(T-ALL)细胞的抑制作用及机制。方法给予CCRF-CEM和J.gamma1细胞不同浓度安罗替尼,使用细胞计数套件-8(CCK-8)法检测细胞增殖能力,显微镜下观察安罗替尼对细胞形态的影响,利用膜联蛋白V/碘化丙啶(Annexin V/PI)染色结合流式细胞仪检测细胞凋亡率。通过生物信息学分析预测安罗替尼作用靶标,分子对接预测作用位点。采用免疫印迹法检测安罗替尼对凋亡相关蛋白、靶标蛋白及其下游信号相关蛋白表达的影响。结果安罗替尼能够在48 h显著抑制CCRF-CEM(半抑制浓度=3.39μmol/L)和J.gamma1细胞的增殖(半抑制浓度=2.36μmol/L),引起CCRF-CEM和J.gamma1细胞皱缩;安罗替尼可诱导细胞凋亡,促进凋亡相关蛋白的表达,与对照组相比,cl-Caspase、cl-PARP及Bax的表达均升高(均P<0.05)。生物信息学分析与分子对接结果提示,安罗替尼可能靶向结合Abelson酪氨酸蛋白激酶1(ABL1);Western blot结果显示,与对照组相比,安罗替尼可降低Abelson酪氨酸蛋白激酶1(ABL1)总蛋白表达(P<0.05),从而抑制下游信号通路磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(PKB)的磷酸化水平(P<0.05),PKB激活剂则可削弱安罗替尼的抑制效应。结论安罗替尼能显著抑制T-ALL细胞增殖并诱导凋亡,其机制可能与靶向抑制ABL1表达后,削弱ABL1/PI3K/PKB信号通路有关。
Objective To investigate the inhibitory effect of anlotinib on human acute lymphatic leukemia cells and to explore its mechanism.Methods Human acute lymphatic leukemia CCRF-CEM and J.gamma1 cells were treated with anlotinib at different concentrations.Cell proliferation was detected using CCK-8 assay,cell morphology was observed under the microscope,and the apoptosis rate was detected with flow cytometry and Annexin V/PI staining.The target of action of anlotinib was predicted by bioinformatics analysis,and the site of action was predicted by molecular docking.Western blot was used to detect the expression of apoptosis-related proteins,target proteins,and their downstream signal-related proteins.Results Anlotinib significantly inhibited the proliferation of CCRF-CEM(IC50=3.39μmol/L)and J.gamma1 cells(IC50=2.36μmol/L)at 48 h.Treatment with anlotinib induced cell shrinkage.Annexin V/PI flow cytometry results suggested that anlotinib induced apoptosis of CCRF-CEM and J.gamma1 cells,and Western blot results indicated that anlotinib promoted the expression of apoptosis-related proteins of cleaved Caspase,cleaved PARP,and Bax(all P<0.05).Bioinformatics analysis and molecular docking results suggested that anlotinib may target and bind Abelson tyrosine-protein kinase 1(ABL1).Western blot confirmed that anlotinib could reduce the total protein expression of ABL1(P<0.05),thereby inhibiting the phosphorylation level of downstream signal PI3K/PKB(P<0.05).At the same time,an PKB activator could weaken the inhibitory effect of anlotinib.Conclusion Anlotinib can significantly inhibit the proliferation and induce apoptosis of human acute lymphatic leukemia cells,which may be related to the down-regulatiton of the ABL1/PI3K/PKB signaling pathway after inhibition of ABL1.
作者
杨好好
岑梦姣
王佳萍
YANG Haohao;CEN Mengjiao;WANG Jiaping(Department of Neonatology,Ningbo Medical Center,Li Huili Eastern Hospital,Ningbo 315100,China;不详)
出处
《浙江医学》
CAS
2023年第16期1723-1728,共6页
Zhejiang Medical Journal
基金
浙江省基础公益研究计划项目(LGF21H080002)。
作者简介
通信作者:杨好好,E-mail:yanghh202111@163.com。
