Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Teresy St., 91-348 Łódź, Poland.
Chem Biol Interact. 2013 Oct 5;205(3):198-211. doi: 10.1016/j.cbi.2013.07.011. Epub 2013 Aug 2.
In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a possibility of induction of different mechanisms in development of resistance to ATO depending on the drug concentration and thus involvement of different signaling mediators.
在这项研究中，研究了 PI3K/Akt 信号通路在三氧化二砷（ATO）处理的亲本 Jurkat 细胞中的作用，以及在给定 ATO 浓度存在下生长的衍生 ATO 耐药克隆中的作用。ATO 耐药克隆（在 1、2.5 和 5μM ATO 存在下培养 8-12 周）的特征是在 ATO 存在下具有高存活率，但与亲本细胞相比生长速度较慢。衍生的 ATO 耐药克隆的形态和功能特征表明，它们在细胞大小、GSH 水平、溶酶体荧光或 CD95/Fas 表面抗原表达方面与亲本 Jurkat 细胞没有根本区别。然而，与亲本 Jurkat 细胞相比，在克隆中检测到线粒体电位（JC-1 染色）略有增加。ATO 耐药克隆的侧群分析（Vybrant DyeCycle Violet™染色）并未表明任何富集的癌症干细胞。Akt1/2、AktV 或 Wortmannin 抑制剂降低了在 ATO 存在下生长的 ATO 耐药克隆的活力，但对 ATO 处理的亲本细胞没有影响。流式细胞术分析表明，ATO 降低了 ATO 处理的亲本细胞中 p-Akt 的水平，而耐药克隆表现出比亲本 Jurkat 细胞更高水平的 p-Akt 免疫染色。涉及 PI3K/Akt 通路的 84 个基因的表达分析表明，该通路在 ATO 耐药克隆中主要活跃。c-JUN 似乎在 ATO 处理的亲本 Jurkat 细胞中诱导细胞死亡中起关键作用，因为 JUN 的剂量依赖性强烈上调是 ATO 处理的亲本 Jurkat 细胞特有的。另一方面，细胞周期蛋白 D1 (CCND1)、胰岛素受体底物 1 (IRS1) 和蛋白激酶 C 同工型（PRKCZ、PRKCB 和 PRKCA）表达的变化可能导致对 ATO 的耐药性。在 ATO 耐药克隆中观察到生长因子受体结合蛋白 10 (GRB10) 的表达变化表明，在 ATO 耐药性的发展中可能诱导了不同的机制，这取决于药物浓度，从而涉及不同的信号转导介质。
