Ethaselen: a novel organoselenium anticancer agent targeting thioredoxin reductase 1 reverses cisplatin resistance in drug-resistant K562 cells by inducing apoptosis

Abstract

It has been reported that Ethaselen shows inhibitory effects on thioredoxin reductase (TrxR) activity and human tumor cell growth. In order to find an efficient way to reverse cisplatin resistance, we investigated the reversal effects of Ethaselen on cisplatin resistance in K562/cisplatin (CDDP) cells that were established by pulse-inducing human erythrocyte leukemic cell line K562, which are fivefold more resistant to cisplatin compared to K562 cells. The morphology and growth showed that the adhesion of K562/CDDP further decreased while the cell volume increased. The proliferation of K562/CDDP is strengthened. The antitumor activities in vitro were assessed by MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and combination index (CI), showing the significant synergic effects of cisplatin and Ethaselen. Focusing on apoptosis, a series of comparisons was made between K562 and K562/CDDP. Cisplatin induced higher reactive oxygen species (ROS) generation in K562 and subsequently induced the formation of mitochondrial permeability transition pores (PTPs). In addition, cisplatin increased the ratio of Bax to Bcl-2 in K562, which can influence the mitochondrial membrane permeability. PTP formation and mitochondrial membrane permeabilization eventually resulted in the release of cytochrome c and activation of the Caspase pathway. However, these effects were not clearly seen in K562/CDDP, which may be the reason for the acquired CDDP resistance. However, Ethaselen can induce a high level of ROS in K562/CDDP by TrxR activity inhibition and increased ratio of Bax to Bcl-2 in K562/CDDP by nuclear factor κB (NF-κB) suppression, which subsequently induces the release of cytochrome c in K562/CDDP. This response is partly responsible for the reversal of the cisplatin resistance in K562/CDDP cells.

摘 要

目 的

研究凋亡调控相关蛋白来了解顺铂耐药成因, 同 时考察乙烷硒啉(Ethaselen)在K562 耐药细胞 中逆转顺铂耐药的作用, 并初步探讨其作用机 制。

创新点

首次研究乙烷硒啉在逆转顺铂耐药中的作用, 且 此作用与乙烷硒啉诱导细胞凋亡相关。

方 法

通过长时间脉冲诱导得到顺铂耐药K562 细胞, 并观察耐药细胞形态及倍增时间。采用MTT 法 考察乙烷硒啉、顺铂及其联用组在不同细胞株间 的生长抑制作用。流式细胞术分析细胞凋亡情况 以及细胞内活性氧(ROS)水平。最后, 通过蛋 白质免疫印迹(Western blot)考察凋亡调控相关 蛋白水平的变化。

结 论

脉冲诱导得到的K562 耐药细胞对顺铂的耐受性 是原K562 细胞的5.34 倍。形态学观察发现, 耐 药细胞体积增大, 粘附性进一步降低。乙烷硒啉 与顺铂联用表现出协同效应。当加入少量的乙烷 硒啉(顺铂与乙烷硒啉的摩尔比率为10:1), 顺 铂作用K562 耐药细胞的半抑制浓度(IC50)值可 以减少21 倍。流式细胞术及Western blot 表明, 乙烷硒啉能够诱导耐药细胞凋亡。其逆转顺铂耐 药主要是通过调控Bcl-2 及Bax 蛋白比例以及通 过提高细胞内活性氧水平引起线粒体通透转运 孔道(PTP)蛋白孔道的形成来促使释放细胞色 素c, 进而引起Caspase 凋亡途径。