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

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天相对于第0天体重变化差异无统计学意义[(3.18±0.01)g比(2.73±0.58)g,t=0.60,P=0.570],HE染色结果未见BDA-366组小鼠心、肝、脾、肺、肾形态异常。结论BDA-366在体外可促进NK/TCL细胞发生凋亡,在体内不引起小鼠体重减轻和器官HE染色形态改变。BDA-366对NK/TCL细胞的抑制作用可能是通过提高Bax表达、诱导Ca2+释放和降低线粒体膜电位实现的。

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,and there were statistically significant differences(t=7.45,P=0.002;t=5.26,P=0.006).In YT cells,the intracellular Ca2+concentrations of 0.065 and 0.130μmol/L BDA-366 groups were significantly higher than that of the control group(5791.67±220.45,6729.33±585.39,4874.67±112.61,F=19.16,P=0.003)(P=0.039;P=0.002).In NK92 cells,the intracellular Ca2+concentrations of 0.086 and 0.172μmol/L BDA-366 groups were significantly higher than that of the control group(4553.67±17.62,4740.33±254.50,4185.67±17.67,F=10.96,P=0.010)(P=0.039;P=0.007).There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group[(3.18±0.01)g vs.(2.73±0.58)g,t=0.60,P=0.570],and HE staining showed no abnormal morphology of heart,liver,spleen,lung and kidney in BDA-366 group.Conclusion BDA-366 promotes NK/TCL cells apoptosis in vitro,but does not cause weight loss and morphological changes of organs by HE staining in vivo.The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression,inducing Ca2+release and reducing mitochondrial membrane potential.