MELK抑制剂OTSSP167抗弥漫性大B细胞淋巴瘤的实验研究

Effect of MELK Inhibitor OTSSP167 on Diffuse Large B-Cell Lymphoma

目的:探讨MELK抑制剂OTSSP167在抗弥漫性大B细胞淋巴瘤(DLBCL)中的作用。方法:采用CCK-8法检测OTSSP167对细胞生长活性的影响;EdU染色后流式检测OTSSP167对DLBCL细胞增殖能力的影响;Annexin V-FITC/PI双染色法检测OTSSP167对DLBCL细胞凋亡的影响;DLBCL细胞接种裸鼠后给予OTSSP167治疗,4周后检测OTSSP167对DLBCL体内生长的影响;Caspase-Glo^(TM)3/7酶活性试剂盒检测OTSSP167对DLBCL细胞Caspase 3/7酶活性的影响;Western blot检测凋亡和周期相关蛋白的表达水平。结果:OTSSP167显著抑制了SUDHL2、HBL1细胞的活性并呈一定的剂量依赖性(r=-0.61,r=-0.52);EdU染色后流式检测结果表明,OTSSP167能够显著抑制SUDHL2、HBL1细胞的增殖;Annexin V-FITC/PI结果显示,OTSSP167均能够显著促进SUDHL2、HBL1细胞的凋亡(P<0.001);体内实验结果表明,OTSSP167能够抑制SUDHL2细胞在裸鼠体内的生长,肿瘤组织TUNEL染色结果进一步证实OTSSP167能够促进SUDHL2细胞的凋亡;Caspase 3/7酶活性检测结果表明,OTSSP167均能够显著提高SUDHL2、HBL1细胞中Caspase活性(r=0.98,r=0.87);Western blot结果表明,OTSSP167能够剂量依赖性抑制凋亡信号通路中PARP、Bcl-xL、Bcl-2的表达(r=-0.93,r=-0.66,r=-0.87),而p53则显著上调(r=0.82),同时与细胞周期相关的蛋白cdc2、Cyclin E1、Cyclin A2及Cyclin B1也呈现剂量依赖性下调(r=-0.89,r=-0.83,r=-0.61,r=-0.93)。结论:MELK抑制剂OTSSP167能够通过抑制周期相关蛋白和抗凋亡相关蛋白的表达进而抑制DLBCL细胞增殖和促进DLBCL细胞的凋亡。

Objective:To investigate the effect of MELK inhibitor OTSSP167 against diffuse large B-cell lymphoma(DLBCL).Methods:The effect of OTSSP167 on activity,proliferation,and apoptosis of DLBCL cell line(SUDHL2 and HBL1)was detected by CCK-8 assay,5-ethynyl-2'-deoxyuridine(EdU)staining,and Annexin V-FITC/PI double staining,respectively.DLBCL cells were inoculated into nude mice,after 4 weeks of OTSSP167 treatment,the effect of OTSSP167 on DLBCL growth in vivo was detected.Caspase-Glo^(TM)3/7 enzyme activity assay kit was used to detect the effect of OTSSP167 on Caspase 3/7 enzyme activity of DLBCL cells.The expression levels of apoptosis and cycle-related proteins were detected by Western blot.Results:OTSSP167 significantly inhibited the activity of SUDHL2 and HBL1 cells in a dose-dependent manner(r=-0.61,r=-0.52).EdU staining showed that OTSSP167 could significantly inhibit the proliferation of SUDHL2 and HBL1 cells.Annexin V-FITC/PI result showed that OTSSP167 could significantly promote the apoptosis of SUDHL2 and HBL1 cells(P0.001).The result of in vivo experiment showed that OTSSP167 could inhibit the growth of SUDHL2 cells in nude mice.The result of TUNEL staining of tumor further confirmed that OTSSP167 could promote the apoptosis of SUDHL2 cells.Caspase 3/7 enzyme activity test demonstrated that OTSSP167 could significantly increase caspase activity in SUDHL2 and HBL1 cells(r=0.98,r=0.87).Western blot showed that OTSSP167 could dose-dependently inhibit the expression of PARP,Bcl-xL,and Bcl-2 in apoptosis signaling pathway(r=-0.93,r=-0.66,r=-0.87),while p53 protein was significantly up-regulated(r=0.82).The expression of cell cycle-related proteins cdc2,Cyclin E1,Cyclin A2,and Cyclin B1 also showed a dose-dependent down-regulation(r=-0.89,r=-0.83,r=-0.61,r=-0.93).Conclusion:The MELK inhibitor OTSSP167 can inhibit the proliferation and promote the apoptosis of DLBCL cells by inhibiting the expression of cyclerelated proteins and anti-apoptosis-related proteins.