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

doi:10.3760/cma.j.cn371439-20230227-00080

摘要/Abstract

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、0.50 μmol/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、0.086 μmol/L。流式细胞术结果显示，对照组和0.065 μmol/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0.086 μmol/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、0.172 μmol/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0.065 μmol/L BDA-366组YT细胞TMRE荧光强度分别为8 372.00±330.47、6 419.67±311.34，对照组和0.086 μmol/L BDA-366组NK92细胞TMRE荧光强度分别为9 169.00±535.72、7 311.67±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、0.130 μmol/L BDA-366组细胞内Ca²⁺浓度均高于对照组（5 791.67±220.45、6 729.33±585.39、4 874.67±112.61，F=19.16，P=0.003）（P=0.039；P=0.002）；在NK92细胞中，0.086、0.172 μmol/L BDA-366组细胞内Ca²⁺浓度均高于对照组（4 553.67±17.62、4 740.33±254.50、4 185.67±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。

关键词: 原癌基因蛋白质c-bcl-2, 细胞凋亡

Abstract:

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 0，0.05，0.10，0.20，0.30，0.40，0.50 μmol/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 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/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 0.086 μmol/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 0.172 μmol/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 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34，and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±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 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group （5 791.67±220.45，6 729.33±585.39，4 874.67±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 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group （4 553.67±17.62，4 740.33±254.50，4 185.67±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.

Key words: Proto-oncogene proteins c-bcl-2, Apoptosis

吴佳丽, 张佳慧, 张萍, 肖昕悦, 李睿, 张红宇. 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[J]. 国际肿瘤学杂志, 2023, 50(7): 413-418.

Wu Jiali, Zhang Jiahui, Zhang Ping, Xiao Xinyue, Li Rui, Zhang Hongyu. Mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma cells[J]. Journal of International Oncology, 2023, 50(7): 413-418.

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组别	Bcl-2	pBcl-2	Bax
对照组	0.85±0.04	0.41±0.29	0.85±0.00
0.043 μmol/L BDA-366组	1.30±0.08^a	0.64±0.52	1.26±0.04^a
0.086 μmol/L BDA-366组	1.25±0.14^a	0.85±0.51	1.51±0.18^a
0.172 μmol/L BDA-366组	1.05±0.10	0.68±0.39	1.15±0.10^a
F值	14.02	0.53	20.70
P值	0.002	0.670	<0.001

距第1次给药时间（d）	对照组（g）	BDA-366组（g）	t值	P值
0	20.58±0.93	20.58±1.09	<0.01	>0.999
2	21.45±1.06	21.58±1.09	0.16	0.875
4	22.48±1.27	22.65±0.70	0.24	0.817
6	22.78±1.65	23.35±0.65	0.65	0.540
8	23.00±1.35	23.45±1.04	0.52	0.619
10	22.93±1.21	23.53±1.28	0.68	0.523
12	23.30±1.52	23.75±1.10	0.48	0.648