•BBSKE and selenite synergistically inhibited proliferation in three NSCLC cell lines.•BBSKE plus selenite induced apoptosis, necrosis and autophagy in A549 cells.•G2/M arrest was observed after BBSKE and selenite treatment.•TrxR activity, TrxR1 and Trx1 expression were remarkably attenuated.•Ref-1, AP-1 expression, and nuclear translocation of Trx and Ref-1 were inhibited.New effective treatment for human non-small cell lung cancer (NSCLC) is needed. The thioredoxin (Trx) system composes of thioredoxin reductase (TrxR), Trx and NADPH. In this study, we combined an organic selenium compound--TrxR inhibitor ethaselen (BBSKE) with low dosage sodium selenite to inhibit proliferation and induce death of NSCLC cells, and identified underlying mechanisms. Synergistic anti-proliferation effect of BBSKE and selenite was found in human NSCLC cell lines, A549, NCI-H1299 and NCI-1266. A significant increase of apoptosis, necrosis and autophagy were observed in the group of BBSKE plus selenite in A549 cells. The autophagy induced by BBSKE and selenite inhibited apoptosis and necrosis. In addition, BBSKE plus selenite induced G2/M arrest, which was verified by the alteration of gene and protein expression of cell cycle regulatory complexes. The intracellular enzyme activity of TrxR was remarkably decreased by cotreatment of BBSKE and selenite. Besides, the mRNA and protein level of TrxR1 and Trx1 were significantly inhibited by cotreatment of BBSKE and selenite. HEK 293 cells overexpressing TrxR1 were more sensitive to BBSKE plus selenite. The nuclear translocation of Trx1 and Ref-1, as well as expression of Ref-1 and AP-1 were inhibited by combination treatment. In short, BBSKE synergizes selenite in inhibiting proliferation and inducing death of NSCLC cells; BBSKE combined with selenite may be a treatment strategy for NSCLC.

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.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。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。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。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。

Thioredoxin reductase (TrxR) is critical for cellular redox regulation and is involved in tumor proliferation, apoptosis and metastasis. Its C-terminal redox-active center contains a cysteine (Cys497) and a unique selenocysteine (Sec498), which are exposed to solvent and easily accessible. Thus, it is becoming an important target for anticancer drugs. Selective inhibition of TrxR by 1,2-(bis-1,2-benzisoselenazol-3(2H)-one)ethane (4a) prevents proliferation of several cancer cell lines both in vivo and in vitro. Using the structure of 4a as a starting point, a series of novel bis-1,2-benzisoselenazol-3(2H)-ones was designed, prepared and tested to explore the structure–activity relationships (SARs) for this class of inhibitor and to improve their potency. Notably, 1,2-(5,5′-dimethoxybis(1,2-benzisoselenazol-3(2H)-one))ethane (12) was found to be more potent than 4a in both in vitro and in vivo evaluation. Its binding sites were confirmed by biotin-conjugated iodoacetamide assay and a SAR model was generated to guide further structural modification.

We evaluated the combination treatment of ethaselen (BBSKE) as a thioredoxin reductase (TrxR) inhibitor plus cisplatin (CDDP) on the human colon adenocarcinoma cell line LoVo. Therapeutic effects ranging from nearly additive to clearly synergistic demonstrated an effective combination, i.e., the cytostatic dose of CDDP could be reduced without a loss in efficacy. To further investigate the cellular response mechanisms of these favorable outcomes, we analyzed the cell-cycle profiles, mRNA expression patterns, and protein levels of several key genes after incubation with BBSKE or CDDP separately and in combination. In appropriate conditions, CDDP induced arrest at the G2/M phase accompanied by the enhanced inhibitory phosphorylation of Cdk1 and the elevated protein expression of cyclin B1. BBSKE downregulated expression of cyclin D1 by increasing mRNA and protein levels of p21, and thus induced G1 phase arrest. BBSKE returned Cdk1 to an activated state, and reduced the protein level of cyclin B1 after incubation in combination with CDDP, which was consistent with the reduction in the percentage of cells in G2/M identified by flow cytometry. By regulating the G1 phase and reversing CDDP-induced G2/M phase arrest, BBSKE increases drug sensitivity of LoVo cells toward CDDP, and probably provides a meaningful anticancer strategy for further clinical studies.

Ethaselen (1, 2-[bis (1, 2-Benzisoselenazolone-3 (2H) -ketone)] ethane, BBSKE), as a novel organoselenium compound targeting thioredoxin reductase (TrxR), has been reported to inhibit tumor growth and TrxR activity in several human tumor cell lines. It has now entered Phase I clinical trails. Here we report the effects of ethaselen and cisplatin (cis-diamminedichloroplatinum II, DDP) combination therapy (ethaselen 36 mg/kg, i.g., o.d. × 10 d and cisplatin 1 mg/kg, i.p., single at day 0) on human A549-grafted nude mouse model (female, BALB/c nude mouse, n = 5, treatment after tumor volume reached 100 mm3). Compared to single drug administration (either ethaselen: 36 mg/kg, i.g., o.d. × 10 d or cisplatin: 1.0 mg/kg, i.p., single at day 0), the combination therapy showed significantly reduced tumor size (presumably due to a synergistic effect) and no obvious toxic damage (both in terms of body weight maintenance and liver/kidney damage). These results will be significant in the development of novel anti-tumoral therapeutic strategies directed to non-small cell lung cancer (NSCLC).

Human thioredoxin reductase (TrxR) system is associated with cancer cell growth and anti-apoptosis process. Effects of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane (BBSKE), a novel TrxR inhibitor, were investigated on human leukemia cell lines HL-60 and K562. BBSKE treatment induced cell growth inhibition and apoptosis in both cell lines. Apoptosis induced by BBSKE is through Bcl-2/Bax and caspase-3 pathways. Ehrlich’s ascites carcinoma-bearing mice were used to investigate the anti-tumor effect of BBSKE in vivo. Tumor-bearing mice treated with BBSKE showed an increase of life span with a comparable effect to cyclophosphamide (CTX). These results suggest a potential usage of BBSKE as a therapeutic agent against non-solid tumors.

Thioredoxin reductase (TrxR) is a ubiquitous intracellular redox enzyme that regulates tumor growth and proliferation in various cancer cells. Ethaselen (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane), a novel anticancer agent, is designed to target mammalian TrxR1 with the aims of cancer growth inhibition and TrxR inactivation. In this study, we demonstrated that ethaselen significantly inhibits cell growth in the poorly differentiated colorectal RKO cell line, and simultaneously downregulates mammalian TrxR1 mRNA transcript levels, protein expression and enzyme activity, which differs from its actions in moderately differentiated colorectal LoVo cells. Ethaselen’s significant abatement of the Wnt/beta-catenin cell differentiation-related signaling pathway was also observed in RKO cells; this apparently leads to its strong inhibitory effect on cell growth and TrxR1 activity in this cell line. These results suggest that ethaselen has a novel mechanism affecting cell growth in the poorly differentiated RKO colorectal cell line.