Roszak Joanna, Smok-Pieniążek Anna, Stępnik Maciej
Nofer Institute of Occupational Medicine, Łódź, Poland.
Adv Clin Exp Med. 2017 Dec;26(9):1335-1342. doi: 10.17219/acem/65475.
Arsenic trioxide (ATO) is a well-recognized antileukemic drug used for the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL). A major drawback of therapy with ATO is the development of APL cell resistance, the mechanisms of which are still not clear.
The aim of this study was to investigate the role of the PI3K/Akt signaling pathway in ATOtreated human acute myeloid leukemia (HL-60) cells and in ATO-resistant clones.
The cytotoxicity of ATO was assessed using Trypan blue staining or a WST-1 reduction assay. The Akt phosphorylation level was measured by immunofluorescent staining and flow cytometry. Gene expression analysis was performed using real-time polymerase chain reaction (PCR).
The clones derived by culturing for 8-12 weeks in the presence of 1.75, 2.5, and 5 μM ATO were characterized by high viability but a slower growth rate compared to the parental HL-60 cells. The flow cytometry analysis showed that in the parental cells the levels of p-Akt were undetectable or very low, and that ATO had no effect on the level of p-Akt in either the ATO-treated parental cells or the clones. The gene expression analysis revealed that some of the genes involved in the Akt pathway may play a key role in the induction of resistance to ATO, e.g., genes encoding cyclin D1 (CCND1), fork head box O1 (FOXO1), Jun oncogene (JUN), protein kinase C isoform B1 (PRKCB1), because their expression profiles were predominantly changed in the clones and/or the ATO-treated parental HL-60 cells.
The overall results indicate that CCND1, FOXO1, and JUN may contribute to the induction of resistance to ATO, and that the C-Jun N-terminal kinase (JNK) signaling pathway may have greater significance than the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating the cytotoxic effects of ATO and the development of resistance to ATO in the HL-60 cell line.
三氧化二砷（ATO）是一种公认的抗白血病药物，用于治疗新诊断和复发的急性早幼粒细胞白血病（APL）。ATO治疗的一个主要缺点是APL细胞产生耐药性，其机制尚不清楚。
本研究旨在探讨PI3K/Akt信号通路在ATO处理的人急性髓系白血病（HL-60）细胞及ATO耐药克隆中的作用。
使用台盼蓝染色或WST-1还原试验评估ATO的细胞毒性。通过免疫荧光染色和流式细胞术测量Akt磷酸化水平。使用实时聚合酶链反应（PCR）进行基因表达分析。
在1.75、2.5和5 μM ATO存在下培养8-12周获得的克隆，其特点是活力高，但与亲代HL-60细胞相比生长速率较慢。流式细胞术分析表明，在亲代细胞中未检测到或p-Akt水平非常低，并且ATO对ATO处理的亲代细胞或克隆中的p-Akt水平均无影响。基因表达分析显示，一些参与Akt通路的基因可能在诱导对ATO的耐药性中起关键作用，例如编码细胞周期蛋白D1（CCND1）、叉头框O1（FOXO1）、原癌基因Jun（JUN）、蛋白激酶C同工型B1（PRKCB1）的基因，因为它们的表达谱在克隆和/或ATO处理的亲代HL-60细胞中主要发生了变化。
总体结果表明，CCND1、FOXO1和JUN可能有助于诱导对ATO的耐药性，并且在介导ATO的细胞毒性作用及HL-60细胞系中对ATO耐药性的产生方面，C-Jun氨基末端激酶（JNK）信号通路可能比磷酸肌醇3激酶（PI3K）/Akt通路具有更重要的意义。
