Huang Qinwan, Wang Lin, Ran Qian, Wang Jin, Wang Chengqiang, He Hui, Li Li, Qi Hongyi
College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People's Republic of China.
College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, People's Republic of China.
Drug Des Devel Ther. 2019 Jun 6;13:1927-1940. doi: 10.2147/DDDT.S189969. eCollection 2019.
This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms. Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright-Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined. Notopterol obviously inhibited the growth of HL-60 cells with an IC value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14. This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.
本研究旨在观察羌活醇对HL-60细胞凋亡和分化的影响，并探讨其潜在的分子机制。采用磺酰罗丹明B法评估细胞活力。通过台盼蓝拒染试验测定细胞增殖。在甲基纤维素中测定集落形成单位。采用膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染、Hoechst 33342染色、线粒体膜电位检测及蛋白质印迹法进行凋亡检测。通过瑞氏-吉姆萨染色、硝基蓝四氮唑（NBT）还原试验、检测CD11b和CD14以及蛋白质印迹法检测分化诱导情况。此外，通过流式细胞术和蛋白质印迹法分析细胞周期阶段分布。检测了羌活醇与全反式维甲酸（ATRA）联合治疗对HL-60细胞的作用。羌活醇明显抑制HL-60细胞生长，IC值为40.32 μM，10、20和40 μM的羌活醇显著减少集落数量。此外，羌活醇诱导HL-60细胞凋亡百分比增加，降低线粒体膜电位，降低Bcl-2和Mcl-1蛋白表达，增加Bax表达、caspase 9、caspase 3和PARP的裂解。至于细胞分化，羌活醇明显诱导染色质凝聚；增加核质比、NBT阳性细胞、CD14和CD11b的表达以及c-Jun和Jun B的蛋白表达，并降低c-myc表达。此外，流式细胞术检测显示羌活醇诱导G0/G1期细胞周期阻滞，这可能与细胞周期相关蛋白p53、CDK2、CDK4、细胞周期蛋白D1、细胞周期蛋白E和生存素的调节有关。细胞活力试验和Hoechst 33342检测表明，羌活醇与ATRA联合使用并未增强凋亡作用。然而，与单独使用羌活醇或ATRA相比，羌活醇与ATRA联合使用增强了诱导分化的作用，核质比增加、NBT阳性细胞增加以及CD14表达增加可证明这一点。首次证明羌活醇可诱导人急性髓系白血病HL-60细胞凋亡、分化和G0/G1期阻滞，提示羌活醇对急性髓系白血病具有潜在治疗作用。羌活醇（20和40 μM）与ATRA（2 μM）联合应用可增强HL-60细胞的分化。
