PDF(Pubmed)
Abstract：
Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.
摘要：
急性髓性白血病（AML）的特征是骨髓祖细胞的增殖失控和成熟受损，导致骨髓和血液中的未成熟细胞积聚，导致造血功能障碍。化学抗性，生存途径的过度活跃，和miRNA的改变是导致AML患者治疗失败和不良预后的主要因素。本研究旨在探讨p38丝裂原活化蛋白激酶（MAPK）抑制剂SB203580对AML干细胞KG1a对治疗药物柔红霉素（DNR）的化学耐药潜能的影响。用增加浓度的DNR处理KG1a和化学敏感性白血病HL60细胞。CellTiter-Glo®,流式细胞术,磷酸激酶和蛋白质阵列，Westernblot技术,和逆转录定量聚合酶链反应（RT-qPCR）用于评估细胞活力，半最大抑制浓度（IC50）测定，凋亡状态检测，细胞周期分析,凋亡相关蛋白和基因表达监测。使用共聚焦显微镜观察caspase和线粒体通透性转换孔(mPTP)活性。暴露在不同的孵育时间，KG1a细胞的DNRIC50值高于HL60细胞，确认KG1a细胞化学抗性电位。暴露于DNR，SB203580预处理后KG1a细胞的晚期凋亡诱导增强，定义为联合治疗。聚（ADP-核糖）聚合酶的裂解增加证实了这种增强，联合处理后，KG1a细胞中的caspase-9，caspase-3和caspase-3/-7和mPTP活性增强，与DNR相比。使用磷酸激酶和凋亡蛋白阵列，与DNR相比,联合治疗降低了KG1a细胞中存活Akt磷酸化和抗凋亡Bcl-2表达水平,同时增加了肿瘤抑制因子p53和细胞周期蛋白依赖性激酶抑制剂p21的表达水平.细胞周期分析显示DNR导致KG1a细胞生长停滞在G2/M期，虽然联合治疗导致细胞生长停滞在S期，主要与细胞周期蛋白B1表达水平有关。值得注意的是,SB203580预处理后，KG1a细胞对DNR的敏感性增强与miR-328-3p的上调增加和miR-26b-5p的轻微下调有关，与DNR效应相比。总之,这些发现可能有助于通过靶向p38MAPK通路开发一种新的治疗策略,以改善难治性或复发性AML患者的治疗结果.
