阿的平诱导结外NK/T淋巴瘤细胞凋亡的实验研究

Experimental study on quinacrine inducing apoptosis in extranodal natural killer(NK)/T-cell lymphoma cells

背景与目的:随着含有门冬酰胺酶化疗方案以及局部放疗和免疫检查点抑制剂的成功应用,结外NK/T细胞淋巴瘤[extranodal natural killer(NK)/T-cell lymphoma,ENKTCL]患者缓解率和无病生存率较以往有显著提高,然而仍有相当一部分进展期ENKTCL患者成为复发/难治性病例。前期研究发现,抗寄生虫小分子化合物阿的平可以抑制包括血液肿瘤在内多种肿瘤细胞增殖并诱导其凋亡,可能成为新型抗肿瘤药物。进一步探究阿的平对ENKTCL细胞的影响及其可能作用机制。方法:以不同浓度阿的平处理ENKTCL细胞系SNK6和NK92(分别由上海交通大学医学院附属新华医院血液内科和上海血液学研究所提供),观察细胞生长和形态并采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测其增殖,同时应用流式细胞术检测细胞周期、凋亡率以及细胞内线粒体跨膜电位和活性氧水平,进一步利用蛋白[质]印迹法(Western blot)检测细胞自噬相关蛋白LC3B和P70表达的变化。结果:SNK6和NK92细胞经阿的平处理48 h后,细胞增殖抑制率分别为(47.08±2.19)%和(30.46±7.95)%,明显高于对照组[(11.85±1.89)%和(10.08±2.01)%,P值均<0.05],阿的平处理24 h后处理组SNK6和NK92细胞凋亡率分别达到(86.45±6.54)%和(76.5±10.8)%;显著高于对照组[(3.3±3.24)%和(2.64±1.67)%,P值均<0.05]。同时阿的平处理组SNK6和NK92细胞周期均发生明显S期阻滞,但细胞内线粒体跨膜电位均无显著变化。进一步研究还发现,阿的平能够促使SNK6细胞内活性氧水平升高并抑制SNK6和NK92细胞内P70蛋白磷酸化,同时上调LC3B蛋白表达。结论:阿的平能够诱导ENKTCL细胞增殖阻滞和凋亡,其机制可能与阿的平介导细胞内活性氧水平升高以及抑制mTOR信号通路进而激活细胞自噬有关。

Background and purpose:With the clinical application of sequential L-asparaginase-containing chemotherapy and radiotherapy as well as immune checkpoint inhibitors,both rates of response and progression-free survival of patients with extranodal natural killer(NK)/T-cell lymphoma(ENKTCL)have dramatically improved.However,a considerable number of advanced-stage patients become relapsed/refractory cases.It has been found that quinacrine,as an antiparasitic small molecular compound,could induce cell cycle arrest and apoptosis in different types of tumor cells in vitro including hematologic malignancies,which might be a promising novel antineoplastic pharmacotherapy.The present study aimed to explore the possible effects of quinacrine on ENKTCL cells and its potential mechanisms.Methods:The ENKTCL cell line SNK6 and NK92 cells(provided by Department of Hematology,Xinhua Hospital Affiliated to School of Medicine,Shanghai Jiao Tong University and Shanghai Institute of Hematology)were treated with different concentrations of quinacrine.The proliferations of SNK6 and NK92 cells were evaluated through cellular morphology and cell counting kit(CCK-8)assay.Meanwhile,flow cytometry was used to analyze changes of cell cycle distribution and apoptotic rate as well as mitochondrial transmembrane potential(ΔΨm)and intracellular reactive oxygen species(ROS)level.Furthermore,Western blot assay was applied to detect expression levels of autophagy modulator LC3B and P70.Results:After treating with quinacrine for 48 h,the growth inhibition rates of SNK6 and NK92 cells reached to(47.08±2.19)%and(30.46±7.95)%respectively,which were significantly higher compared with control group[(11.85±1.89)%and(10.08±2.01)%;P<0.05].The apoptotic rates of SNK6 and NK92 cells in quinacrine groups after 24 h were(86.45±6.54)%and(76.5±10.8)%respectively,which were significantly higher compared with control group[(3.3±3.24)%and(2.64±1.67)%;P<0.05].It was also shown that quinacrine could induce S-phase cell cycle arrest in SNK6 and NK92 cells,though little influence on mitochondrialΔΨm was observed.Furthermore,quinacrine could trigger elevation of ROS level and inhibition of P70 phosphorylation as well as up-regulate expression of LC3B in these cells.Conclusion:Quinacrine could induce proliferation inhibition and apoptosis in ENKTCL cells,which might be mediated by intracellular ROS and simultaneous suppression of mTOR signaling pathway to trigger cell autophagy.