Department of Cell and Molecular biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
Cancer Med. 2020 May;9(10):3537-3550. doi: 10.1002/cam4.2982. Epub 2020 Mar 16.
Drug resistance is a fundamental clinical concern in pediatric acute lymphoblastic leukemia (pALL), and methotrexate (MTX) is an essential chemotherapy drug administered for the treatment. In the current study, the effect of iron in response to methotrexate and its underlying mechanisms were investigated in pALL cells. CCRF-CEM and Nalm6 cell lines were selected as T and B-ALL subtypes. Cells were pretreated with ferric ammonium citrate, exposed to the IC50 concentration of MTX and cell viability was assessed using MTT, colony formation, and flow cytometry assays. Iron-loaded cells were strongly resistant to MTX cytotoxicity. The inhibitory effect of N-acetyl cysteine to reverse the acquired MTX resistance was greater than that of the iron chelator, deferasirox, highlighting the importance of iron-mediated ROS in MTX resistance. Subsequently, the upregulation of BCL2, SOD2, NRF2, and MRP1 was confirmed using quantitative RT-PCR. Moreover, a positive correlation was demonstrated between the MRP1 expression levels and bone marrow iron storage in pALL patients. Further supporting our findings were the hematoxylin and eosin-stained histological sections showing that iron-treated nude mice xenografts demonstrated significantly more liver damage than those unexposed to iron. Overall, iron is introduced as a player with a novel role contributing to methotrexate resistance in pALL. Our findings suggest that the patients' bone marrow iron stores are necessary to be assessed during the chemotherapy, and transfusions should be carefully administrated.
耐药性是小儿急性淋巴细胞白血病（pALL）的一个重要临床关注点，甲氨蝶呤（MTX）是一种用于治疗的重要化疗药物。在本研究中，研究了铁对甲氨蝶呤的反应及其潜在机制在 pALL 细胞中的作用。选择 CCRF-CEM 和 Nalm6 细胞系作为 T 和 B-ALL 亚型。用柠檬酸铁铵预处理细胞，使其暴露于 MTX 的 IC50 浓度下，并用 MTT、集落形成和流式细胞术检测细胞活力。负载铁的细胞对 MTX 的细胞毒性具有很强的抗性。N-乙酰半胱氨酸逆转获得性 MTX 耐药的抑制作用大于铁螯合剂地拉罗司，这突出了铁介导的 ROS 在 MTX 耐药中的重要性。随后，通过定量 RT-PCR 证实了 BCL2、SOD2、NRF2 和 MRP1 的上调。此外，在 pALL 患者中，还证明了 MRP1 表达水平与骨髓铁储存之间存在正相关。进一步支持我们的研究结果的是苏木精和伊红染色的组织学切片，显示接受铁处理的裸鼠异种移植物的肝损伤明显多于未暴露于铁的移植物。总的来说，铁被引入作为一个具有新作用的参与者，导致 pALL 中甲氨蝶呤耐药。我们的研究结果表明，在化疗期间有必要评估患者的骨髓铁储存，并且应谨慎进行输血。
