Bcl-2 BH4选择性抑制剂BDA-366抑制NK/T细胞淋巴瘤细胞的机制研究  

Mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma cells

作　　者：吴佳丽 张佳慧 张萍[1] 肖昕悦 李睿 张红宇[1] Wu Jiali;Zhang Jiahui;Zhang Ping;Xiao Xinyue;Li Rui;Zhang Hongyu(Department of Hematology,Peking University Shenzhen Hospital,Shenzhen 518034,China)

机构地区：[1]北京大学深圳医院血液科,深圳518034

出　　处：《国际肿瘤学杂志》2023年第7期413-418,共6页Journal of International Oncology

基　　金：国家自然科学基金(82170191)。

摘　　要：目的研究Bcl-2 BH4选择性抑制剂BDA-366对NK/T细胞淋巴瘤(NK/TCL)的抑制作用和杀伤机制。方法用0、0.05、0.10、0.20、0.30、0.40、0.50μmol/L的BDA-366处理人NK白血病细胞株YT和人NK/TCL细胞株NK92,使用CCK-8法计算BDA-366对细胞的半抑制浓度(IC50)值;流式细胞术检测对照组和IC50浓度BDA-366处理组细胞凋亡水平;蛋白质印迹法检测对照组、1/2 IC50、IC50、2倍IC50浓度BDA-366处理组细胞凋亡相关蛋白表达水平;TMRE和Fluo-3荧光探针法检测对照组和IC50浓度BDA-366处理组细胞线粒体膜电位,以及对照组、IC50、2倍IC50浓度BDA-366处理组细胞内Ca2+浓度;对对照组和10 mg/kg BDA-366腹腔注射组NOD-SCID小鼠进行称重和小鼠组织HE染色,以评估BDA-366是否具有体内毒性。结果BDA-366对于YT和NK92细胞株的IC50分别为0.065、0.086μmol/L。流式细胞术结果显示,对照组和0.065μmol/L BDA-366组YT细胞凋亡率分别为(6.62±1.59)%、(34.60±3.06)%,对照组和0.086μmol/L BDA-366组NK92细胞凋亡率分别为(5.57±0.88)%、(29.18±0.90)%,差异均具有统计学意义(t=14.05,P<0.001;t=32.58,P<0.001)。对照组、0.043、0.086、0.172μmol/L BDA-366组NK92细胞Bax相对表达量分别为0.85±0.00、1.26±0.04、1.51±0.18、1.15±0.10(F=20.70,P<0.001),各BDA-366处理组Bax相对表达量均高于对照组(均P<0.05)。对照组和0.065μmol/L BDA-366组YT细胞TMRE荧光强度分别为8372.00±330.47、6419.67±311.34,对照组和0.086μmol/L BDA-366组NK92细胞TMRE荧光强度分别为9169.00±535.72、7311.67±295.52,差异均具有统计学意义(t=7.45,P=0.002;t=5.26,P=0.006)。在YT细胞中,0.065、0.130μmol/L BDA-366组细胞内Ca2+浓度均高于对照组(5791.67±220.45、6729.33±585.39、4874.67±112.61,F=19.16,P=0.003)(P=0.039;P=0.002);在NK92细胞中,0.086、0.172μmol/L BDA-366组细胞内Ca2+浓度均高于对照组(4553.67±17.62、4740.33±254.50、4185.67±17.67,F=10.96,P=0.010)(P=0.039;P=0.007)。BDA-366组和对照组小鼠第12天相对于�Objective To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma(NK/TCL).Methods Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0,0.05,0.10,0.20,0.30,0.40,0.50μmol/L BDA-366.CCK-8 assay was used to calculate the half inhibitory concentration(IC50)value of BDA-366 on these cells.The apoptosis levels of cells in control group and IC50 BDA-366 treated group were detected by flow cytometry.Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC50,IC50,2×IC50 BDA-366 treated groups.TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC50 BDA-366 treated group,and the intracellular Ca2+concentration of control group,IC50,2×IC50 BDA-366 treated groups.NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo.Results The IC50 of BDA-366 for YT and NK92 cells were 0.065 and 0.086μmol/L respectively.The apoptosis rates of YT cells in the control group and 0.065μmol/L BDA-366 group were(6.62±1.59)%and(34.60±3.06)%respectively.The apoptosis rates of NK92 cells in the control group and 0.086μmol/L BDA-366 group were(5.57±0.88)%and(29.18±0.90)%respectively,both with statistically significant differences(t=14.05,P<0.001;t=32.58,P<0.001).The relative expression of Bax in NK92 cells of the control group,0.043,0.086 and 0.172μmol/L BDA-366 groups were 0.85±0.00,1.26±0.04,1.51±0.18,1.15±0.10(F=20.70,P<0.001),the relative expression of Bax in BDA-366 groups were higher than that in the control group(all P<0.05).The fluorescence intensity of TMRE of YT cells in the control group and 0.065μmol/L BDA-366 group were 8372.00±330.47 and 6419.67±311.34,and that of NK92 cells in the control group and 0.086μmol/L BDA-366 group were 9169.00±535.72 and 7311.67±295.52 respectively

关 键 词：原癌基因蛋白质C-BCL-2 细胞凋亡 NK/T细胞淋巴瘤 

分 类 号：R733.1[医药卫生—肿瘤]