Independent Medical Biology Unit, Faculty of Pharmacy, Medical University of Lublin, 20-093 Lublin, Poland.
Department of Toxicology, Faculty of Pharmacy, Medical University of Lublin, 20-093 Lublin, Poland.
Molecules. 2021 Sep 17;26(18):5653. doi: 10.3390/molecules26185653.
Leflunomide, an anti-inflammatory agent, has been shown to be effective in multiple myeloma (MM) treatment; however, the mechanism of this phenomenon has not been fully elucidated. The aim of the study was to assess the role of mitochondria and dihydroorotate dehydrogenase (DHODH) inhibition in the cytotoxicity of leflunomide in relation to the MM cell line RPMI 8226. The cytotoxic effect of teriflunomide-an active metabolite of leflunomide-was determined using MTT assay, apoptosis detection, and cell cycle analysis. To evaluate DHODH-dependent toxicity, the cultures treated with teriflunomide were supplemented with uridine. Additionally, the level of cellular thiols as oxidative stress symptom was measured as well as mitochondrial membrane potential and protein tyrosine kinases (PTK) activity. The localization of the compound in cell compartments was examined using HPLC method. Teriflunomide cytotoxicity was not abolished in uridine presence. Observed apoptosis occurred in a mitochondria-independent manner, there was also no decrease in cellular thiols level. Teriflunomide arrested cell cycle in the G2/M phase which is not typical for DHODH deficiency. PTK activity was decreased only at the highest drug concentration. Interestingly, teriflunomide was not detected in the mitochondria. The aforementioned results indicate DHODH- and mitochondria-independent mechanism of leflunomide toxicity against RPMI 8226 cell line.
来氟米特是一种抗炎剂，已被证明在多发性骨髓瘤（MM）治疗中有效；然而，其作用机制尚未完全阐明。本研究旨在评估线粒体和二氢乳清酸脱氢酶（DHODH）抑制在来氟米特对 RPMI 8226 骨髓瘤细胞系的细胞毒性中的作用。使用 MTT 测定法、细胞凋亡检测和细胞周期分析来确定来氟米特的活性代谢物特立氟胺的细胞毒性作用。为了评估 DHODH 依赖性毒性，用特立氟胺处理的培养物中添加尿苷。此外，还测量了细胞硫醇水平作为氧化应激症状，以及线粒体膜电位和蛋白酪氨酸激酶（PTK）活性。使用 HPLC 方法检查了化合物在细胞区室中的定位。尿苷存在时，特立氟胺的细胞毒性未被消除。观察到的细胞凋亡是一种与线粒体无关的方式发生的，细胞硫醇水平也没有下降。特立氟胺将细胞周期阻滞在 G2/M 期，这与 DHODH 缺乏不同。仅在最高药物浓度时，PTK 活性降低。有趣的是，在线粒体中未检测到特立氟胺。上述结果表明，来氟米特对 RPMI 8226 细胞系的毒性作用具有 DHODH 和线粒体非依赖性机制。
