核转运信号蛋白1选择性抑制剂KPT-330诱导白血病细胞凋亡的分子机制

Molecular mechanism for apoptosis of leukemia cells induced by chromosome region maintenance 1 selective inhibitor KPT - 330

目的探讨核转运信号蛋白1（CRM1）选择性抑制剂KPT－330对急性白血病细胞凋亡的诱导作用及可能的分子机制。方法白血病细胞株[人急性早幼粒白血病细胞（NB4）、人慢性粒细胞白血病细胞（K562）、人急性早幼粒白血病细胞（HL－60）]分为对照组（不予KPT－330处理）和KPT－330处理组（予终浓度0.01、0.10、0.20、0.50、1.00、5.00、10.00 μmol/L KPT－330孵育细胞）。应用细胞计数试剂盒（CCK－8）比色法观察KPT－330对白血病细胞的生长抑制作用；应用荧光显微镜观察KPT－330诱发白血病细胞形态的变化；应用Annexin V/PI标记流式细胞术分析KPT－330诱发的细胞凋亡；Western blot检测CRM1表达量及凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶－9 （Caspase－9）、半胱氨酸天冬氨酸蛋白酶3（Caspase－3）、DNA修复酶（poly ADP－ribose polymerase，PARP）的表达水平和活化状态；基因芯片分析KPT－330对急性白血病细胞基因表达谱及长链非编码RNA（lncRNA）表达谱的影响。 结果KPT－330呈剂量依赖性抑制白血病细胞的生长，并使CRM1表达下调；与对照组相比，实验组细胞出现了明显的形态异常，较低浓度的KPT－330（0.2 μmol/L和1.0 μmol/L）即可导致白血病细胞的明显凋亡，0.2 μmol/L KPT－330 24 h处理组NB4、K562、HL－60细胞凋亡率（%）分别为（4.95±0.21）%、（14.05±0.07）%、（4.95±0.63）%；0.20 μmol/L KPT－330 48 h处理组（6.10±0.56）%、（20.75±0.21）%、（5.85±0.07）%; 1.00 μmol/L KPT－330 24 h处理组（41.15±0.21）%、（35.45±0.35）%、（15.65±0.07）%；1.00 μmol/L KPT－330 48 h处理组（52.45±0.35）%、（47.45±2.47）%、（16.80±1.98）%，与对照组[24 h NB4、K562、HL－60：（3.30±0.28）%、（9.50±0.56）%、（2.00±0.14）%；48 h NB4、K562、HL－60：（3.70±0.14）%、（3.50±0.28）%、（2.15±0.21）%]比较差异均有统计学意义（均P〈0.05）。在多种白血病细胞中均出现G2期细胞显著减少，以及subG1期细胞的增加，活化型Caspase－3、Caspase－9和PARP蛋白表达水平增加。基因芯片分析表明KPT－330诱导白血病细胞凋亡可能与Toll样受体（TLR）信号及谷胱甘肽代谢信号通路等密切相关。 结论CRM1抑制剂KPT－330可显著抑制白血病细胞增殖，促进细胞凋亡。KPT－330诱导白血病细胞凋亡可能与TLR信号以及谷胱甘肽代谢信号通路等相关。

Objective To investigate the induction effect and molecular mechanisms of chromosome region maintenance 1 （ CRM1 ） selective inhibitor KPT - 330 on apoptosis of human leukemia cells. Methods Human acute promyelocytic leukemia cells （ NIM）, human chronic myelogenous leukemia cells （ K562 ） and human promyelocytic leukemia cells（ HL- 60） were divided into a control group（ without KPT- 330 treatment） and KPT- 330 treatment group （ NB4 ,K562 and HL -60 were treated with 0. 01,0.10,0. 20,0. 50,1. 00,5. 00,10. 00 μmol/L KPT - 330,respective- ly）. Cell counting kit - 8 assay was used to quantify the growth inhibition of ceils after exposure to KPT - 330. Fluores- cence microscope was used to observe the morphological change in leukemia cells when treated with KPT - 330. Annex- in V/propidium iodide staining followed by flow cytometry was used to detect the apoptosis of leukemia cells. Then, Western blot was used to detect the expression level of CRM1 and the activation of apoptosis marker cysteinyl aspartate specific proteinase - 9 （ Caspase - 9）, cysteinyl aspartate specific proteinase - 3 （ Caspase - 3 ） and poly ADP - ribose polymerase （PARP）. Long non-coding RNA （lncRNA） microarray was used to analyze the differentially expressed genes differentially by the cluster analysis. Results The inhibition of cell proliferation of leukemia cells was in a dose - dependent manner with KPT - 330 treatment. Expression of CRM1 was also down - regulated when leukemia ceils were treated with KPT - 330. Compared with the control group, abnormal cells were observed under fluorescence microscopy. Much more cells showed apoptotic feature after being treated with 0.20 μmol/L and 1.00μmol/L KPT - 330, the apoptosis rate of NB4, K562 and HL - 60 were respectively （4.95 ± 0.21 ） %, （ 14.05± 0.07 ） %, （4.95± 0.63 ） % （0.201.±mol/L24h）;（6.10±0.56）%,（20.75±0.21）%,（5.85±0.07）% （0.20 μmol/L48 h）;（41.15±0.21）%,（35.45±0.35）%,（15.65 ±0.07）% （1.00 ixmol/L24 h）;（52.45 ±0.35）%,（47.45±2.47）%, （ 16.80 ±1.98 ） % （ 1.00 ±mol/L 48 h） , respectively, and the differences were statistically significant compared to the control group[ 24 h NB4,K562,HL -60（3.30 ±0.28）%, （9.50 ±0.56） %, （2.00 ±0.14）% ;48 h NB4, K562, HL - 60 ： （ 3.70±0.14） %, （3.50 ± 0.28 ） % , （ 2.15± 0.21 ） % ] （ all P 〈 0.05 ）. G2 phase cells obviously reduced and subGl phase cells increased. The expression level of cleaved Caspase - 3, cleaved Caspase - 9 and cleaved PARP increased. Molecular function analysis showed that apoptosis induced by KPT -330 in leukemia cells was partially rela- ted to Glutathione metabolism and Toll - like receptor signaling pathway. Conclusions KPT - 330 can effectively inhibit proliferation and induce apoptosis of leukemia cells. KPT - 330 - induced apoptosis in leukemia cells is partially related to Glutathione metabolism and Toll - like receptor signaling pathway.