Rabdosin B对急性早幼粒白血病细胞HL-60分化的诱导作用
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摘要
目的研究螺环内酯型对映-贝壳杉烷型二萜rabdosin B(RB)诱导HL-60向成熟粒细胞分化的特征及机制。方法采用台盼蓝染色、吉姆萨染色、硝基四氮唑蓝(NBT)还原力测定及流式细胞术检测RB对HL-60细胞增殖、细胞周期、NBT还原力、细胞吞噬及细胞表面抗原CD11b表达的影响。结果 RB在1.0、3.0、5.0μmol/L下抑制HL-60细胞增殖,引起HL-60细胞G0/G1期阻滞,增加HL-60细胞肾形核和分叶核细胞比率,并伴随细胞NBT还原力和吞噬能力显著增强以及CD11b阳性细胞率明显上升。进一步检测显示,活性氧(ROS)清除剂N-乙酰-L-半胱氨酸(NAC,300μmol/L)及NADPH氧化酶抑制剂夹竹桃麻素(APO,100μmol/L)和二联苯碘锚(DPI,0.1μmol/L)不仅可显著抑制RB诱导的细胞内ROS升高,并且可显著抑制RB诱导的HL-60细胞分化的各项特征指标的改变。结论 RB可通过上调HL-60细胞NADPH氧化酶活性,升高胞内ROS浓度,诱导HL-60细胞向成熟粒细胞分化。
Objective To investigate the characteristics and mechanism of spironolactone ent-kaurane diterpene type diterpene rabdosin B(RB) induced HL-60 differentiation into mature granulocytes. Methods The effects of RB on proliferation, cell cycle, NBT reducing power, phagocytosis, and cell surface antigen CD11 b expression of HL-60 were detected by trypan blue staining, Giemsa staining, NBT reducing power assay, and flow cytometry, respectively. Results RB inhibited the proliferation of HL-60 cells at the concentration of 1.0, 3.0, and 5.0 μmol/L, which resulted in the arrest of G0/G1 phase, increased the ratio of renal nucleated and lobate nucleus, and markedly enhanced the NBT reducing power and phagocytic capacity and the significant increase of CD11 b positive cell rate. Further tests showed that the active oxygen scavenger NAC(300 μmol/L), NADPH oxidase inhibitor APO(100 μmol/L), and DPI(0.1 μmol/L) not only significantly inhibited the intracellular ROS upregulation induced by RB, but also inhibited various features of RB induced HL-60 cell differentiation. Conclusion RB can increase the activity of NADPH oxidase in HL-60 cells, increase the intracellular ROS concentration, and induce the differentiation of HL-60 cells to mature granulocytes.
Objective To investigate the characteristics and mechanism of spironolactone ent-kaurane diterpene type diterpene rabdosin B(RB) induced HL-60 differentiation into mature granulocytes. Methods The effects of RB on proliferation, cell cycle, NBT reducing power, phagocytosis, and cell surface antigen CD11 b expression of HL-60 were detected by trypan blue staining, Giemsa staining, NBT reducing power assay, and flow cytometry, respectively. Results RB inhibited the proliferation of HL-60 cells at the concentration of 1.0, 3.0, and 5.0 μmol/L, which resulted in the arrest of G0/G1 phase, increased the ratio of renal nucleated and lobate nucleus, and markedly enhanced the NBT reducing power and phagocytic capacity and the significant increase of CD11 b positive cell rate. Further tests showed that the active oxygen scavenger NAC(300 μmol/L), NADPH oxidase inhibitor APO(100 μmol/L), and DPI(0.1 μmol/L) not only significantly inhibited the intracellular ROS upregulation induced by RB, but also inhibited various features of RB induced HL-60 cell differentiation. Conclusion RB can increase the activity of NADPH oxidase in HL-60 cells, increase the intracellular ROS concentration, and induce the differentiation of HL-60 cells to mature granulocytes.
引文
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[2]Testa U,Lo-Coco F.Targeting of leukemia-initiating cells in acute promyelocytic leukemia[J].Stem cell Investig,2015,2(17):1-8.
[3]Ge D,Sheng Y,Cai X.Combined staurosporine and retinoic acid induces differentiation in retinoic acid resistant acute promyelocytic leukemia cell lines[J].SciRep,2014,4(4821):1-8.
[4]Mc Culloch D,Brown C,Iland H.Retinoic acid and arsenic trioxide in the treatment of acute promyelocytic leukemia:current perspectives[J].Onco Targets Ther,2017,14(10):1585-1601.
[5]Brigger D,Schl?fli A M,Garattini E,et al.Activation of RARαinduces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity[J].Cell Death Disease,2015,6(8):1861-1870.
[6]Ng C H,Chng W J.Recent advances in acute promyelocytic leukaemia[J].F1000 Res,2017,doi:10.12688/f1000research.10736.1.
[7]Zhou G B,Kang H,Wang L,et al.Oridonin,a diterpenoid extracted from medicinal herbs,targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21)leukemia in vitro and in vivo[J].Blood,2007,109(8):3441-3450.
[8]Zhen T,Wu C F,Liu P,et al.Targeting of AML1-ETO in t(8;21)leukemia by oridonin generates a tumor suppressor-like protein[J].Sci Transl Med,2012,4(127):127-138.
[9]Liu C X,Zhou H C,Yin Q Q,et al.Targeting peroxiredoxins against leukemia[J].Exper Cell Res,2013,319(2):170-176.
[10]Hou J K,Huang Y,He W,et al.Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells[J].Cell Death Dis,2014,5(9):1400-1407.
[11]丁兰,陈祎平,刘国安,等.Wangzaozin A调节NADPH氧化酶源性活性氧诱导HL-60细胞分化[J].华中师范大学学报:自然科学版,2015,49(2):252-260.
[12]丁兰,李佩蔚,孔花青,等.对映-贝壳杉烷型二萜Leukamenin E对人早幼粒白血病HL-60细胞分化的诱导作用[J].中国药房,2016,27(13):1771-1774.
[13]丁兰,陈宗儒,陈平,等.NADPH氧化酶源性活性氧介导epinodosin诱导HL-60细胞分化及细胞骨架重组[J].中国细胞生物学学报,2016,38(3):273-284.
[14]Sun H D,Huang S X,Han Q B.Diterpenoids from Isodon species and their biological activities[J].Nat Product Rep,2006,23(5):673-698.
[15]吴炎鹏,许卫兵,孔花青,等.P(HPMA-FMA)荧光探针制备,细胞毒性评估及细胞吞噬示踪检测[J].功能高分子学报,2014,27(1):92-98.
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[17]韩锐.抗癌药物研究与实验技术[M].北京:北京医科大学中国协和医科大学联合出版社,1997.
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[19]Chen H,Zhang B,Yao Y,et al.NADPH oxidase-derived reactive oxygen species are involved in the HL-60 cell monocytic differentiation induced by isoliquiritigenin[J].Molecules,2012,17(11):13424-13438.
[20]孙汉董,许云龙,姜北.番茶菜属植物二萜化合物[M].北京:科学出版社,2001.
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[29]Huang J,Wu L,Tashiro S,et al.Reactive oxygen species mediate oridonin-induced Hep G2 apoptosis through p53,MAPK,and mitochondrial signaling pathways[J].J Pharmacol Sci,2008,107(4):370-379.
[30]Guiso G,Rambaldi A,Dimitrova B,et al.Determination of orally administered all-trans-retinoic acid in human plasma by high-performance liquid chromatography[J].JChromatogr B:Biomed Sci Appl,1994,656(1):239-244.
[31]Li D,Wang Z,Chen H,et al.Isoliquiritigenin induces monocytic differentiation of HL-60 cells[J].Free Rad Biol Med,2009,46(6):731-736.
[32]Chen H,Zhang B,Yuan X,et al.Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation[J].Cell Biol Intern,2013,37(11):1215-1224.
[33]Wang C Y,Yang T T,Chen C L,et al.Reactive oxygen species-regulated glycogen synthase kinase-3βactivation contributes to all-trans retinoic acid-induced apoptosis in granulocyte-differentiated HL60 cells[J].Biochem Pharmacol,2014,88(1):86-94.
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