西奥罗尼杀伤FLT3-ITD阳性急性髓系白血病细胞的作用及机制研究

Study on Effect and Mechanism of Chiauranib on FLT3-ITD Positive Acute Myeloid Leukemia Cells

关键词:  西奥罗尼  FLT3-ITD  急性髓系白血病  VEGFR2  MEK/Erk

Key words:  chiauranib  FLT3-ITD  acute myeloid leukemia  VEGFR2  MEK/Erk

摘要:

目的 研究多靶点激酶抑制剂西奥罗尼对FLT3-ITD阳性急性髓系白血病（acute myeloid leukemia，AML）细胞的杀伤作用及其机制。方法 CCK8和集落形成试验检测不同浓度西奥罗尼对FLT3-ITD阳性AML细胞增殖和活力的影响；Annexin V/PI双染法检测西奥罗尼对FLT3-ITD阳性AML细胞株和原代细胞的凋亡诱导情况；Click-iT^® EdU试剂盒评估细胞周期的分布情况；裸鼠皮下瘤模型用于评估西奥罗尼对FLT3-ITD阳性AML细胞体内的杀伤作用；Western blotting探讨西奥罗尼杀伤FLT3-ITD阳性AML的潜在作用机制。结果 在FLT3-ITD阳性AML细胞株模型上，西奥罗尼具有降低细胞的活力、抑制细胞的集落形成、阻滞细胞周期和诱导凋亡的作用；通过8例原代FLT3-ITD阳性AML细胞研究发现，西奥罗尼对原代细胞同样具有诱导凋亡的潜能；裸鼠皮下瘤试验表明西奥罗尼降低FLT3-ITD阳性AML细胞体内增殖能力，延缓肿瘤的生长，而无明显不良反应；机制研究显示西奥罗尼降低VEGFR2的磷酸化水平，且下调其下游的MEK/Erk通路的磷酸化作用。结论 本研究明确了西奥罗尼在体外和体内具有杀伤FLT3-ITD阳性AML细胞的的潜能，抑制VEGFR2/MEK/Erk通路的活性可能是西奥罗尼发挥杀伤FLT3-ITD阳性AML细胞的潜在作用机制。

Abstract:

OBJECTIVE To investigate the injuring effect and mechanism of multikinase inhibitor chiauranib on FLT3-ITD positive acute myeloid leukemia(AML). METHODS CCK8 and colony-forming assay were separately utilized to determine cell viability and proliferation of FLT3-ITD positive AML cells exposure with various concentration of chiauranib. FLT3-ITD positive AML cell lines and primary cells treated with increasing dosage of chiauranib in designated timepoints were assessed with Annexin V/PI dual-staining assay. Click-iT^® EdU kit was used to evaluated the distribution of cell cycle. Subcutaneous xenograft model by injection with FLT3-ITD AML cells was adopted to evaluate the in vivo antileukemia efficacy of chiauranib. The underlying mechanism of chiauranib cytotoxicity was explored by Western blotting assay. RESULTS In this study, treatment FLT3-ITD positive AML cell lines with chiauranib resulted in significant reduction of cell viability, suppression of cell colony formation, blockade of cell cycle and promotion of cell apoptosis; primary FLT3-ITD AML cells obtained from 8 AML patients were separately exposed to various chiauranib doses and exhibited great sensitivity to chiauranib cytotoxicity. The anti-AML efficacy of chiauranib was validated in the FLT3-ITD AML xenograft model and had no significant safety profiles. Dephosphorylation of VEGFR2 and its downstream targets MEK and Erk was associated with the antitumor effects of chiauranib on FLT3-ITD positive AML cellular models. CONCLUSION This study demonstrates that chiauranib shows potent antileukemic effects on FLT3-ITD positive AML cells in vitro and in vivo possibly via perturbation of the activity of VEGFR2/MEK/Erk pathway.