[1]于　建,高五集,段永彬,等.3-溴丙酮酸单独或联合牛熊去氧胆酸钠对人髓系白血病细胞存活、增殖、凋亡的影响及其机制[J].新乡医学院学报,2022,39(5):401-408.[doi:10.7683/xxyxyxb.2022.05.001]
　YU Jian,GAO Wuji,DUAN Yongbin,et al.Effects of 3-bromopyruvate alone or combination with sodium taursus deoxycholate on the survival,proliferation and apoptosis of human myeloid leukemia cells and its mechanism[J].Journal of Xinxiang Medical University,2022,39(5):401-408.[doi:10.7683/xxyxyxb.2022.05.001]
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3-溴丙酮酸单独或联合牛熊去氧胆酸钠对人髓系白血病细胞存活、增殖、凋亡的影响及其机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年5
页码:
401-408
栏目:
基础研究
出版日期:
2022-05-05

文章信息/Info

Title:
Effects of 3-bromopyruvate alone or combination with sodium taursus deoxycholate on the survival,proliferation and apoptosis of human myeloid leukemia cells and its mechanism
作者:
于　建1; 高五集2; 段永彬2; 陆枢桠2; 钟加滕1; 2
(1.新乡医学院第一附属医院病理科，河南　卫辉　453100；2.新乡医学院基础医学院病理学系，河南　新乡　453003)
Author(s):
YU Jian1; GAO Wuji2; DUAN Yongbin2; LU Shuya2; ZHONG Jiateng1; 2
（1.Department of Pathology,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China；2.Department of Pathology,School of Basic Medical Science,Xinxiang Medical University,Xinxiang 453003,Henan Province,China）
关键词:
髓性白血病; 3-溴丙酮酸; 牛熊去氧胆酸钠; 内质网应激; 凋亡
Keywords:
myelogenous leukemia; 3-bromopyruvate; sodium taursus deoxycholate; endoplasmic reticulum stress; apoptosis
分类号:
R733.71
DOI:
10.7683/xxyxyxb.2022.05.001
文献标志码:
A
摘要:
目的　探讨3-溴丙酮酸（3-BP）单独或联合牛熊去氧胆酸钠(TUDCA)对人髓系白血病细胞存活、增殖、凋亡的影响及其机制。方法　将对数生长期人髓系白血病细胞K562随机分为0 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组、80 μmol·L-1 3-BP组、120 μmol·L-1 3-BP组，分别用含终浓度0、40、80、120 μmol·L-1 3-BP的RPMI-1640 培养液培养，采用细胞计数试剂盒-8（CCK-8）检测细胞存活能力，采用流式细胞术检测细胞凋亡情况，采用Western blot法检测细胞凋亡相关蛋白B 淋巴细胞瘤-2(Bcl-2) 、 Bcl-2 相关X蛋白(Bax)、cleaved-capsase-3及内质网应激相关蛋白免疫球蛋白结合蛋白（Bip）、真核翻译起始因子-2α（EIF-2α）、磷酸化真核翻译起始因子-2α（p-EIF-2α）、转录激活因子4（ATF4）的表达。将对数生长期的人髓系白血病细胞K562随机分为0 μmol·L-1 3-BP组、5 μmol·L-1 3-BP组、10 μmol·L-1 3-BP组、20 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组，分别用含终浓度0、5、10、20、40 μmol·L-1 3-BP的RPMI-1640培养液培养，采用CCK-8法检测培养0、24、48、72、96 h时的细胞增殖能力。另将对数生长期K562细胞随机分为对照组、3-BP组、TUDCA组、3-BP+TUDCA组，分别使用含终浓度0 μmol·L-1 3-BP和 0 mmol·L-1TUDCA、40 μmol·L-1 3-BP、0.25 mmol·L-1 TUDCA、40 μmol·L-1 3-BP和0.25 mmol·L-1TUDCA的RPMI-1640培养液培养，采用CCK-8法检测各组细胞存活能力，采用流式细胞术检测各组细胞凋亡情况，采用Western blot法检测各组细胞凋亡相关蛋白Bcl-2、Bax、cleaved-capsase-3的表达。结果　3-BP 干预16 h时，随着药物浓度的升高， 0 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组、80 μmol·L-1 3-BP组、120 μmol·L-1 3-BP组细胞存活能力呈显著降低趋势（F=542.170，P<0.05）。药物干预16 h时，3-BP组、TUDCA组、3-BP+TUDCA组细胞存活能力显著低于对照组，3-BP+TUDCA组细胞存活能力显著低于3-BP组和TUDCA组(P<0.05)。3-BP干预24、48、72、96 h 时，随 3-BP干预浓度的升高，0 μmol·L-1 3-BP组、5 μmol·L-1 3-BP组、10 μmol·L-1 3-BP组、20 μmol·L-1 3-BP组、40 μmol·L-1 3-BP 组细胞增殖能力呈显著降低趋势（F=284.875、819.658、199.535、467.633，P<0.05）。随着3-BP干预浓度的增加，0 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组、80 μmol·L-1 3-BP组、120 μmol·L-1 3-BP组细胞的凋亡率及Bax、cleaved-capsase-3蛋白呈显著升高趋势，Bcl-2蛋白呈显著降低趋势（F=2 148.278、1 176.838、1 350.646、6 765.482，P<0.05）。随着3-BP浓度的升高，0 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组、80 μmol·L-1 3-BP 组、120 μmol·L-1 3-BP组细胞Bip、p-EIF-2α、ATF4蛋白相对表达量呈显著升高趋势（F=1 600.447、504.672、1 949.837，P<0.05）；0 μmol·L-1 3-BP组、40 μmol·L-1 3-BP组、80 μmol·L-1 3-BP组、120 μmol·L-1 3-BP组细胞的EIF-2α蛋白相对表达量比较差异无统计学意义（F=1.034，P>0.05）。3-BP+TUDCA组细胞凋亡率及Bax、cleaved-caspase-3蛋白相对表达量显著高于对照组、3-BP组、TUDCA组（P<0.05）；3-BP+TUDCA组细胞的Bcl-2蛋白相对表达量显著低于对照组、3-BP组、TUDCA组（P<0.05）。结论　3-BP可显著抑制人髓系白血病细胞K562的增殖和存活能力，促进K562细胞凋亡，并可诱导内质网应激的发生；内质网应激抑制剂TUDCA可增加3-BP对K562细胞的促凋亡作用，抑制K562细胞存活。
Abstract:
Objective　To investigate the effects of 3-bromopyruvate (3-BP) alone or combination with sodium taursus deoxycholate (TUDCA) on the survival,proliferation and apoptosis of human myeloid leukemia cells and its mechanism.Methods　The human myeloid leukemia cells K562 in the logarithmic growth phase were randomly divided into 0 μmol·L-1 3-BP group,40 μmol·L-1 3-BP group,80 μmol·L-1 3-BP group and 120 μmol·L-1 3-BP group,which were cultured with RPMI-1640 medium containing final concentrations of 0,40,80 and 120 μmol L-1 3-BP,respectively;the cell viability was detected by the cell counting kit-8 (CCK-8) method,the cell apoptosis was detected by the flow cytometry,and the expressions of the apoptosis-related proteins B-cell lymphoma-2 (Bcl-2),Bcl-2 assaciated X protein (Bax),cleaved-capsase-3 and endoplasmic reticulum stress excitation-related protein immunoglobulin-binding protein (Bip),eukaryotic translation initiation factor-2α(EIF-2α),phosphorylated eukaryotic translation initiation factor-2α (p-EIF-2α),activating transcription factor 4 (ATF4) were detected by Western blot.Human myeloid leukemia cells K562 in the logarithmic growth phase were randomly divided into 0 μmol·L-1 3-BP group,5 μmol·L-1 3-BP group,10 μmol·L-1 3-BP group,20 μmol·L-1 3-BP group and 40 μmol·L-1 3-BP group,which were cultured with RPMI-1640 medium containing final concentrations of 0,5,10,20 and 40 μmol·L-1 3-BP,respectively;the cell proliferation ability was detected by CCK-8 method at 0、24、48、72、96 h of culture.In addition,the logarithmic growth phase K562 cells were randomly divided into control group,3-BP group,TUDCA group and 3-BP+TUDCA group,which were cultured with 100 μL of RPMI-1640 medium containing final concentrations of 0 μmol·L-1 3-BP and 0 mmol·L-1 TUDCA,40 μmol·L-1 3-BP,0.25 mmol·L-1 TUDCA,40 μmol·L-1 3-BP and 0.25 mmol·L-1 TUDCA,respectively;the cell viability was detected by CCK-8 method,the apoptosis of cells was detected by flow cytometry,the expressions of apoptosis-related proteins Bcl-2,Bax and cleaved-capsase-3 were detected by Western blot.Results　When 3-BP intervened for 16 h,with the increase of drug concentration,the viability of cells in the 0 μmol·L-1 3-BP group,40 μmol·L-1 3-BP group,80 μmol·L-1 3-BP group,120 μmol·L-1 3-BP group was significantly decreased (F=542.170,P<0.05).After drug intervention for 16 h,the viability of cells in the 3-BP group,TUDCA group and 3-BP+TUDCA group was significantly lower than that in the control group,and the viability of cells in the 3-BP+TUDCA group was significantly lower than that in the 3-BP group and TUDCA group (P<0.05).When 3-BP intervened for 24,48,72 and 96 h,with the increase of 3-BP intervention concentration,the proliferation ability of cells in the 0 μmol·L-1 3-BP group,5 μmol·L-1 3-BP group,10 μmol·L-1 3-BP group,20 μmol·L-1 3-BP group and 40 μmol·L-1 3-BP group showed a significant decrease trend (F=284.875,819.658,199.535,467.633;P<0.05).With the increase of 3-BP intervention concentration,the apoptosis rate and the relative expression level of Bax and cleaved-capsase-3 protein of cells were significantly increased,and the relative expression level of Bcl-2 protein was significantly decreased in the 0 μmol·L-1 3-BP group,40 μmol·L-1 3-BP group,80 μmol·L-1 3-BP group and 120 μmol·L-1 3-BP group (F=2 148.278,1 176.838,1 350.646,6 765.482;P<0.05).With the increase of 3-BP concentration,the relative expression of Bip,p-EIF-2α and ATF4 protein of cells in the 0 μmol·L-1 3-BP group,40 μmol·L-1 3-BP group,80 μmol·L-1 3-BP group,120 μmol·L-1 3-BP group showed a significant increase trend (F=1 600.447,504.672,1 949.837;P<0.05);there was no significant difference in the relative expression level of EIF-2α protein of cells among the 0 μmol·L-1 3-BP group,40 μmol·L-1 3-BP group,80 μmol·L-1 3-BP group,120 μmol·L-1 3-BP group (F=1.034,P>0.05).The apoptosis rate and the relative expression of Bax and cleaved-caspase-3 proteins in the 3-BP+TUDCA group were significantly higher than those in the control group,3-BP group and TUDCA group (P<0.05);the relative expression of Bcl-2 protein in the 3-BP+TUDCA group was significantly lower than that in the control group,the 3-BP group and the TUDCA group (P<0.05).Conclusion　3-BP can significantly inhibit the proliferation and survival of human myeloid leukemia cells K562,promote the apoptosis of K562 cells,and induce the occurrence of endoplasmic reticulum stress.The endoplasmic reticulum stress inhibitor TUDCA can increase the pro-apoptotic effect of 3-BP on K562 cells and inhibite the survival of K562 cells.

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更新日期/Last Update: 2022-05-05