紫草素对人白血病MV4-11细胞的增殖抑制和促凋亡作用

doi:10.13481/j.1671-587x.20200117

摘要/Abstract

摘要： 目的：探讨紫草素对FMS样酪氨酸激酶3受体基因内部串联重复序列（FLT3-ITD）突变急性髓系白血病（AML）MV4-11细胞的增殖抑制和促凋亡作用，并初步阐明其分子机制。方法：生长状态良好的MV4-11细胞分为二甲基亚砜（DMSO）组和不同浓度（0.5、1.0、2.0、4.0和8.0 μmol·L^-1）紫草素组，处理细胞24和48 h，采用CCK-8法检测各组细胞增殖抑制率，并计算半数抑制浓度（IC₅₀）。MV4-11细胞分为空白对照组、DMSO组和不同浓度（0.25、0.50和1.00 μmol·L^-1）紫草素组，处理细胞48和72 h，采用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标记法检测各组细胞增殖率。MV4-11细胞分为DMSO组和不同浓度（0.702、1.404和2.808 μmol·L^-1）紫草素组，处理细胞48 h，采用流式细胞术检测各组细胞凋亡率。MV4-11细胞分为DMSO组和不同浓度（0.351、0.702和1.404 μmol·L^-1）紫草素组，处理细胞48 h，Real-time PCR法检测各组细胞中微小RNA-155（miR-155）的表达水平。结果：CCK-8法和CFSE法检测，与DMSO组比较，不同浓度紫草素组细胞增殖抑制率明显升高（P<0.05或P<0.01），增殖率明显降低（P<0.05），且呈剂量依赖性；24和48 h时IC₅₀分别为1.743和1.404 μmol·L^-1。流式细胞术检测，与DMSO组比较，不同浓度紫草素组细胞凋亡率明显升高（P<0.01），且呈剂量依赖性。Real-time PCR法检测，与DMSO组比较，不同浓度紫草素组细胞中miR-155表达水平明显降低（P<0.01），1.404 μmol·L^-1紫草素组细胞中miR-155表达水平降低超过75%。结论：紫草素对人FLT3-ITD突变AML MV4-11细胞具有增殖抑制和促凋亡作用，并可下调miR-155表达，提示紫草素可能作为FLT3-ITD突变AML的潜在治疗药物之一。

关键词: 急性髓系白血病, FMS样酪氨酸激酶3受体基因内部串联重复序列, 基因突变, 紫草素, 微小RNA-155

Abstract: Objective: To discuss the proliferation inhibition and apoptosis induction of shikonin on the FMS-like tyrosine kinase-3 receptor internal tandem duplication (FLT3-ITD) mutated acute myeloid leukemia (AML) MV4-11 cells, and to preliminarily clarify the molecular mechanisms. Methods: The MV4-11 cells were divided into DMSO group and different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 μmol·L^-1) of shikonin groups, and treated for 24 and 48 h. The inhibitory rate of proliferation was analyzed by CCK-8 assay, and half inhibitory concentration (IC₅₀) was calculated. The MV4-11 cells were divided into blank control group, DMSO group, and different concentrations (0.25, 0.50, and 1.00 μmol·L^-1) of shikonin groups, and treated for 48 and 72 h; the proliferation rate of cells was analyzed by carbox fluorescenceindiacetate succinimidyl este (CFSE). The MV4-11 cells were divided into DMSO group and different concentrations (0.702, 1.404, and 2.808 μmol·L^-1) of shikonin groups, and treated for 48 h;the apoptotic rate was determined by flow cytometry. The MV4-11 cells were divided into DMSO group and different concentrations (0.351, 0.702, and 1.404 μmol·L^-1) of shikonin groups, and treated for 48 h; the microRNA-155 (miR-155) expression level was detected by Real-time PCR. Results: The results of CCK-8 and CFSE methods indicated that the inhibitory rates of proliferation of MV4-11 cells in different concentrations of shikonin groups were increased compared with DMSO grpup(P<0.05 or P<0.01), and the proliferation rates were decreased (P<0.05 or P<0.01) in a concentration-dependent manner; the IC₅₀ of 24 and 48 h were 1.743 and 1.404 μmol·L^-1, respectively. The flow cytometry results showed that the apoptotic rates of the cells in different concentrations of shikonin groups were increased compared with DMSO group (P<0.01) in a concentration-dependent manner. The Real-time PCR results showed that the expression levels of miR-155 in the cells in different concentrations of shikonin groups were decreased significantly(P<0.01), and the expression level in 1.404 μmol·L^-1 shikonin group was decreased by more than 75%. Conclusion: Shikonin could inhibit the proliferation and promote the apoptosis of FLT3-ITD mutated AML MV4-11 cells, and down-regulate the expression of miR-155, suggesting that shikonin may be one of the potential therapeutic drugs for FLT3-ITD mututed AML.

Key words: acute myeloid leukemia, FMS-like tyrosine kinase-3 receptorinternal tandem duplication, mutation, shikonin, microRNA-155

中图分类号:

R73-361

苏龙, 张云蔚, 王卓, 宋飞, 秦田雪, 高素君. 紫草素对人白血病MV4-11细胞的增殖抑制和促凋亡作用[J]. 吉林大学学报(医学版), 2020, 46(01): 96-101.

SU Long, ZHANG Yunwei, WANG Zhuo, SONG Fei, QIN Tianxue, GAO Sujun. Proliferation inhibition and pro-apoptotic effects of shikonin on human leukemia MV4-11 cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(01): 96-101.

参考文献

[1] PATEL J P, GÖNEN M, FIGUEROA M E, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia[J]. N Engl J Med, 2012, 366(12):1079-1089.
[2] PAPAEMMANUIL E, GERSTUNG M, BULLINGER L, et al. Genomic classification and prognosis in acute myeloid leukemia[J]. N Engl J Med, 2016, 374(23):2209-2221.
[3] PASIC I,DA'NA W,LAM W,et al. Influence of FLT3-ITD and NPM1 status on allogeneic hematopoietic cell transplant outcomes in patients with cytogenetically normal AML[J]. Eur J Haematol,2019,102(4):368-374.
[4] MARCUCCI G, MAHARRY K S, METZELER K H, et al. Clinical role of microRNAs in cytogenetically normal acute myeloid leukemia:miR-155 upregulation independently identifies high-risk patients[J]. J Clin Oncol, 2013, 31(17):2086-2093.
[5] GERLOFF D,GRUNDLER R,WURM A A,et al. NF-κB/STAT5/miR-155 network targets PU.1 in FLT3-ITD-driven acute myeloid leukemia[J]. Leukemia,2015,29(3):535-547.
[6] KHALIFE J,RADOMSKA H S,SANTHANAM R,et al. Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924(Pevonedistat) in FLT3-ITD acute myeloid leukemia[J]. Leukemia,2015,29(10):1981-1992.
[7] YOON Y,KIM YO,LIM NY,et al. Shikonin, an ingredient of Lithospermum erythrorhizon induced apoptosis in HL60 human premyelocytic leukemia cell line[J]. Planta Med,1999,65(6):532-535.
[8] MAO X,YU C R,LI W H,et al. Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells[J]. Cell Res,2008,18(8):879-888.
[9] ZHAO Q L, ASSIMOPOULOU A N, KLAUCK S M, et al. Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells[J]. Oncotarget, 2015, 6(36):38934-38951.
[10] KOTTARIDIS P D,GALE R E,FREW M E,et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy:analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials[J]. Blood,2001,98(6):1752-1759.
[11] THIEDE C,STEUDEL C,MOHR B,et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia:association with FAB subtypes and identification of subgroups with poor prognosis[J]. Blood,2002,99(12):4326-4335.
[12] FRÖHLING S, SCHLENK R F, BREITRUCK J, et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics:A study of the AML Study Group Ulm[J]. Blood, 2002, 100(13):4372-4380.
[13] SCHMID C,LABOPIN M,SOCIÉ G,et al. Outcome of patients with distinct molecular genotypes and cytogenetically normal AML after allogeneic transplantation[J]. Blood,2015,126(17):2062-2069.
[14] SONG Y, MAGENAU J, LI Y M, et al. FLT3 mutational status is an independent risk factor for adverse outcomes after allogeneic transplantation in AML[J]. Bone Marrow Transplant, 2016, 51(4):511-520.
[15] DEOL A,SENGSAYADETH S,AHN K W,et al. Does FLT3 mutation impact survival after hematopoietic stem cell transplantation for acute myeloid leukemia? A Center for International Blood and Marrow Transplant Research (CIBMTR) analysis[J]. Cancer,2016,122(19):3005-3014.
[16] CANAANI J,LABOPIN M,HUANG X J,et al. T-cell replete haploidentical stem cell transplantation attenuates the prognostic impact of FLT3-ITD in acute myeloid leukemia:A report from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation[J]. Am J Hematol,2018,93(6):736-744.
[17] ZHANG Y Y,MO X D,ZHANG X H,et al. FLT3 internal tandem duplication does not impact prognosis after haploidentical allogeneic hematopoietic stem cell transplantation in AML patients[J]. Bone Marrow Transplant,2019,54(9):1462-1470.
[18] LARROSA-GARCIA M, BAER M R. FLT3 inhibitors in acute myeloid leukemia:current status and future directions[J]. Mol Cancer Ther, 2017, 16(6):991-1001.
[19] STONE R M, MANDREKAR S J, SANFORD B L, et al. Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation[J]. N Engl J Med, 2017, 377(5):454-464.
[20] XU L H,GUO Y,CEN J N,et al. Overexpressed miR-155 is associated with initial presentation and poor outcome in Chinese pediatric acute myeloid leukemia[J]. Eur Rev Med Pharmacol Sci, 2015,19(24):4841-4850.
[21] RAMAMURTHY R, HUGHES M, MORRIS V, et al. MiR-155 expression and correlation with clinical outcome in pediatric AML:A report from Children's Oncology Group[J]. Pediatr Blood Cancer, 2016, 63(12):2096-2103.
[22] ANDUJAR I,RECIO M C,BACELLI T,et al. Shikonin reduces oedema induced by phorbol ester by interfering with IkappaBalpha degradation thus inhibiting translocation of NF-kappaB to the nucleus[J]. Br J Pharmacol,2010,160(2):376-388.
[23] CHENG Y W,CHANG C Y,LIN K L,et al. Shikonin derivatives inhibited LPS-induced NOS in RAW 264.7 cells via downregulation of MAPK/NF-kappaB signaling[J]. J Ethnopharmacol,2008, 120(2):264-271.
[24] ANDUJAR I,RÍOS J L,GINER R M,et al. Pharmacological properties of shikonin-a review of literature since 2002[J]. Planta Med,2013,79(18):1685-1697.
[25] SU L,LIU L H,WANG Y L,et al. Long-term systemic toxicity of shikonin derivatives in Wistar rats[J]. Pharm Biol,2013,52(4):486-490.