Huihui Zeng

Thioredxin reductase inhibitor ethaselen increases the drug sensitivity of the colon cancer cell line LoVo towards cisplatin via regulation of G1 phase and reversal of G2/M phase arrest

SummaryWe 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.

Selenium-containing thioredoxin reductase inhibitor ethaselen sensitizes non-small cell lung cancer to radiotherapy.

It has been proposed that thioredoxin reductase (TR) is a mediator that allows non-small cell lung cancer (NSCLC) to develop resistance to irradiation; however, little is known regarding the detailed mechanisms of action. Thus, ethaselen {1, 2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)] ethane, BBSKE}, a novel organoselenium TR inhibitor, is currently being investigated in a phase I clinical trial in China. However, its radiosensitizing effect remains unexplored. In this study, we found that the activity of TR increased dramatically in both A549 and H1299 cells after radiation, and moreover, could be inhibited by pretreatment with BBSKE (5 &mgr;mol/l). As a TR inhibitor, BBSKE enhanced the efficacy of radiation therapy both in vivo and in vitro without observable toxicity. BBSKE was found to suppress irradiation-induced NF-&kgr;B activation dramatically when using A549 cells stably transfected with NF-&kgr;B luciferase reporter. These results show the critical role of TR in the radioresistance of NSCLC and suggest that BBSKE is a potentially promising agent for the treatment of patients with NSCLC clinically.

Preparation of tri-block copolymer micelles loading novel organoselenium anticancer drug BBSKE and study of tissue distribution of copolymer micelles by imaging in vivo method.

BBSKE (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)] ethane, PCT: CN02/00412) is a novel organoselenium anticancer drug that plays a role in anticancer through inhibiting TrxR (thioredoxin reductase). In this study, we prepared a tri-block copolymer micelles loading BBSKE utilizing the amphiphilic tri-block copolymers (PEG6000-PLA6000) which we synthesized. And then the characters of the copolymer micelles were investigated. When packaged in polymeric micelles, the water solubility of BBSKE was improved to 0.21 mg/ml. The IC(50) were 7.14 microM, 5.05 microM and 4.23 microM when MCF-7 breast cancer cells were treated with BBSKE after 24h, 48h and 72h. The inhibition effect of polymeric micelles to MCF-7 tumor cells was bettered when folate, whose receptor was highly expressed in various tumors, was coated on the surface of these nanoparticles. Finally, by adopting a new way of imaging in vivo, we studied the distribution of micelles in nude mice with and without MCF-7 tumor. The results demonstrated that this copolymer micelles loading BBSKE can accumulate into tumor efficiently.

A Novel Thioredoxin Reductase Inhibitor Inhibits Cell Growth and Induces Apoptosis in HL-60 and K562 Cells

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.

Novel mechanism of ethaselen in poorly differentiated colorectal RKO cell growth inhibition: Simultaneous regulation of TrxR transcription, expression and enzyme activity

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. Ethaselens 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.

Butaselen prevents hepatocarcinogenesis and progression through inhibiting thioredoxin reductase activity.

Hepatocellular carcinoma (HCC) accounts for most of primary liver cancer, of which five-year survival rate remains low and chemoprevention has become a strategy to reduce disease burden of HCC. We aim to explore the in vivo chemopreventive effect of an organoselenium-containing compound butaselen (BS) against hepatocarcinogenesis and its underlying mechanisms. Pre- and sustained BS treatment (9, 18 and 36 mg/Kg BS) could dose-dependently inhibit chronic hepatic inflammation, fibrosis, cirrhosis and HCC on murine models with 24 weeks treatment scheme. The thioredoxin reductase (TrxR), NF-κB pathway and pro-inflammatory factors were activated during hepatocarcinogenesis, while their expression were decreased by BS treatment. BS treatment could also significantly reduce tumor volume in H22-bearing models and remarkably slow tumor growth. HCC cell lines HepG2, Bel7402 and Huh7 were time- and dose-dependently inhibited by BS treatment. G2/M arrest and apoptosis were observed in HepG2 cells after BS treatment, which were mediated by TrxR/Ref-1 and NF-κB pathways inhibition. BS generated reactive oxygen species (ROS), which could be reduced by antioxidant N-acetyl-L-cysteine (NAC) and NADPH oxidase inhibitor DPI. NAC could markedly increase HepG2 cells viability. TrxR activity of HepG2 cells treated with BS were significantly decreased in parallel with proliferative inhibition. The TrxR1-knockdown HepG2 cells also exhibited low TrxR1 activity, high ROS level, relatively low proliferation rate and increased resistance to BS treatment. In conclusion, BS can prevent hepatocarcinogenesis through inhibiting chronic inflammation, cirrhosis and tumor progression. The underlying mechanisms may include TrxR activity inhibition, leading to ROS elevation, G2/M arrest and apoptosis.

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

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。

Novel thioredoxin reductase inhibitor butaselen inhibits tumorigenesis by down-regulating programmed death-ligand 1 expression

The thioredoxin system plays a role in a variety of physiological functions, including cell growth, differentiation, apoptosis, tumorigenesis, and immunity. We previously confirmed that butaselen (BS), a novel thioredoxin reductase inhibitor, can inhibit the growth of various human cancer cell lines, yet the underlying mechanism remains elusive. In this study, we investigated the anti-tumor effect of BS in vivo through regulating the immune system of KM mice. We found that BS inhibits tumor proliferation by promoting the activation of splenic lymphocytes in mice. BS can elevate the percentage of CD4−CD8+ T lymphocytes and the secretion of downstream cytokines in mice via downregulating the expression of programmed death-ligand 1 (PD-L1) on the tumor cells’ surface in vivo. Further study in HepG2 and BEL-7402 cells showed that decrease of PD-L1 level after BS treatment was achieved by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation. Taken together, our results suggest that BS has a role in promoting the immune response by reducing PD-L1 expression via the STAT3 pathway, and subsequently suppresses tumorigenesis.摘要目的评估新型硫氧还蛋白还原酶抑制剂丁烷硒啉(BS) 对小鼠免疫系统及肿瘤发生的影响,并初步探讨 其作用机制。创新点实验室自主设计的硫氧还蛋白还原酶抑制剂BS 对免疫系统的调节作用及机制探究。方法将24 只体重为18~22 g 的正常雄性KM小鼠随机 分为4 组,分别为BS 低、中、高剂量组和对照 组,每组6 只动物。于接种小鼠肝癌H22 细胞 (1×106 个/只)后第2 天开始给药,给药剂量分 别为:空白对照组(5 g/L 羧甲基纤维素钠 (CMC-Na),灌胃(i.g.),每日一次(q.d.)); BS 低剂量组(90 mg/kg,i.g.,q.d.);BS 中剂量 组(180 mg/kg,i.g.,q.d.);BS 高剂量组(360 mg/kg, i.g.,q.d.)。每天观察小鼠状态,连续8 天。结论BS 通过促进小鼠脾淋巴细胞的活化来抑制肿瘤 增殖。BS 能够通过下调体内肿瘤细胞表面的程序 性死亡配体1 ( PD-L1)的表达,提高小鼠 CD4−CD8+T 淋巴细胞百分比和下游细胞因子的 分泌。在HepG2 和BEL-7402 细胞中的进一步研 究表明,通过抑制信号传导子及转录激活子3 (STAT3)磷酸化,BS 处理后PD-L1 表达水平 降低。综上所述,BS 通过STAT3 途径降低PD-L1 表达,从而产生促进免疫应答的作用,抑制肿瘤 发生。