Synergistic effect of ethaselen and cisplatin treatment against tumor cell lines

The antitumor activities of ethaselen （BBSKE） in combination with cisplatin （CDDP） in vitro have been investigated in human stomach cancer cell line BGC 823 and human lung cancer cell line PG-BE1. MTT method was used to assess the individual effects of ethaselen and cisplatin and their combined effects on cell proliferation of BGC 823 cells and PG-BE1 cells. Additionally, we used the classic median effect theory to calculate the combination index （CI） of ethaselen and cisplatin with different dose regimes in combination, and compared the effective dosages of cisplatin in individual treatment and combination treatment. When ethaselen and cisplatin were used in combination, a synergistic effect was observed. The most remarkable synergistic effect was observed when the dose ratio of cisplatin to ethaselen was 2：3 in BGC 823 cell line and 1：3 in PG-BE1 cell line. The dose of cisplatin could be decreased markedly in combination group to reach the same inhibitory effect, and this effect was gradually raised with the increase of the concentration of drugs.

Drug efficacy and pharmacological action of an organoselenium compound ethaselen,a novel antitumor drug

Ethaselen, an organoselenium compound designed and synthesized in the School of Pharmaceutical Sciences, Peking University, has been entitled to independent intellectual property rights both at home and abroad. As one of the novel antitumor drugs, ethaselen has been extensively studied in Phase I clinical trial, and its biological target is thioredoxin reductase. In this review, we focus on the ethaselen＇s efficacy and pharmacological actions, including antitumor effects both in vitro and in vivo, and immunologic functions. These research findings not only provide the theoretical basis for the anticancer study of ethaselen, but also guide the clinical trial of ethaselen.

Investigation of the redox status in H22 hepatocellular carcinoma xenografts treated by a novel anticancer drug-ethaselen

We investigated the redox status of H22 hepatocellular carcinoma xenografts treated with various doses of ethaselen, a novel anticancer drug targeting thioredoxin reductase （TrxR）. The concentrations of low molecular weight antioxidant g!utathione （GSH） and malondialdehyde （MDA）, a product of lipid peroxidation, as well as the activities of important antioxidant enzymes were measured for elucidating the redox status of H22 tumor tissues. We found that the decreased GSH level, decreased thioredoxin reductase and superoxide dismutase （SOD） activities as well as increased MDA content were closely related to the tumor growth inhibition and ethaselen doses. Glutathione peroxidase （GPx） and glutathinne reductase （GR） activities are also affected by ethaselen treatment. However, the catalase （CAT） activity remains unchanged. Finally, we studied the relationship of tumor growth inhibition caused by ethaselen with these redox factors. This study showed that ethaselen could elevate the oxidative stress to suppress the H22 tumor growth in mice model.

Development of an HPLC-UV method for the determination of ethaselen in its injection formulation

A novel ethaselen injection formulation has been developed in our laboratory. The objective of the present study was to establish and validate a high performance liquid chromatography （HPLC） method for the determination of ethaselen in its injection formulation. Analysis was performed on an ODS column with isocratic elution at 40 ℃. Mobile phase was consisted of 0.01% phosphoric acid and methanol （60：40, v/v）. The detection wavelength was set at 320 nm and the flow rate was 1.0 mL/min. The results showed that the calibration curves of ethaselen were linear in the range of 10-50 μg/mL （r2 = 0.9999）. The limit of detection for ethaselen was 100 ng/mL. The average recovery of ethaselen was 100.14%. The labeled content of ethaselen in its injection formulation was in the range of 102%-103% of the measured content. In conclusion, this method was stable and reproducible, thus providing a useful tool for the routine analysis of this novel ethaselen injection formulation.

Study on the binding of novel antitumor drug Ethaselen to bovine serum albumin

The binding pattern of Ethaselen to bovine serum albumin （BSA） was studied by fluorescence spectroscopic technique and Autodock 3.0.5 analysis. The result showed that the binding constant K of Ethaselen and BSA is 3.5×10^4L/mol and the number of binding sites n = 0.9. The binding between Ethaselen and BSA or human serum albumin （HSA） is mainly through hydrophobic interactions.

Reactive oxygen species mediate ethaselen-induced rapid apoptosis in A549 cells

Thioredoxin reductase （TrxR） is an important enzyme responsible for the removal of excess reactive oxygen species （ROS） in mammalian cells. This study aimed to investigate the roles of ROS in the anti-tumor effects of ethaselen, a TrxR inhibitor. In a human lung cancer cell line A549, we observed rapidly elevated ROS levels, loss of mitochondrial potential, and cell death with the treatment of ethaselen. Such changes were prevented when cells were pre-incubated with of N-acetyl-cysteine （NAC）, a ROS scavenger. The apoptosis study by Annexin V-PI assay and protein assay by Western blot suggested that mitochondrial pathway was involved in the initiation of apoptosis. In ethaselen-treated cells, cyclosporin A （CsA）, an inhibitor of mitochondrial permeability transition pores （PTPs）, inhibited the release of cytochrome c and reduced cell death in ethaselen-treated cells, while the protein levels of some Bcl-2 family proteins remained unchanged. These results suggest that ROS mediated the ethaselen-induced rapid apoptosis, at least, partially through the enhancement of mitochondrial membrane permeability, which may ultimately lead to the release of pro-apoptotic proteins. Our study, for the first time, demonstrated that the anti-tumor effects of ethaselen, including the early phase of cellular response, are associated with ROS. More studies are needed to confirm our conclusion, as well as to further explore the underlying mechanisms in the anti-tumor effects of ethaselen.

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.

乙烷硒啉对大鼠围产期毒性的研究

背景与目的:观察乙烷硒啉对大鼠妊娠晚期、分娩期、哺乳期及胚胎和胎仔出生后生长发育、学习能力以及生殖能力的影响。材料与方法:大鼠妊娠第15d至哺乳第28d,连续灌胃给予250、500和1000mg/kg剂量的乙烷硒啉,同时设1%羧甲基纤维素钠为对照组,观察各组大鼠和胎仔的生长、发育、生殖能力等项指标。结果:给药后F0代母鼠体重增长明显减慢,未见生殖、胚胎毒性反应;F1代仔鼠的出生体重和哺乳期体重增长减慢,生理达标、新生反射等发育迟缓;对停药后F1代大鼠的体重增长、摄食量、脏器系数、同笼交配率、妊娠率及胚胎发育全过程未见明显的生殖、发育等毒性变化。结论:乙烷硒啉可使母鼠和F1代仔鼠的体重增长缓慢,生理发育迟缓,但不影响F1代仔鼠器官发育、生殖能力以及停药后体重的增长和摄食量。

乙烷硒啉致突变试验研究

目的观察乙烷硒啉是否存在遗传毒性。方法应用Ames试验、中国仓鼠肺细胞体外培养染色体畸变试验和小鼠骨髓嗜多染红细胞微核试验,研究乙烷硒啉的致突变作用。结果Ames试验在<400μg/皿浓度下未见回复突变菌落数显著增加;中国仓鼠肺细胞体外培养染色体畸变试验在<3.3μg/ml浓度下未出现细胞染色体畸变率显著增高;小鼠骨髓嗜多染红细胞微核试验在<20.0g/kg剂量下未诱发微核细胞率的增高。结论乙烷硒啉在试验剂量范围内无致突变作用。

乙烷硒啉对大鼠致畸敏感期毒性试验研究

目的观察乙烷硒啉对大鼠是否存在胚胎毒性和致畸毒性。方法采用Wistar大鼠,每组妊娠鼠>15只。试验设1000mg/kgbw、500mg/kgbw、250mg/kgbw和125mg/kgbw4个剂量组,同时设1个阳性对照组和1个溶剂对照组。在大鼠胚胎器官形成期连续灌胃给药10d(孕615d),在妊娠的第20天,处死妊娠鼠,计数黄体数、着床数、活胎数、早期死胎数(包括吸收胎)和晚期死胎数;观察胎仔性别、外观、称取胎重,并检查骨骼和内脏发育情况。结果给药>250mg/kgbw剂量组的孕鼠体重增长缓慢,胎仔的活胎率降低到94.9%以下、吸收胎率增加到4.5%以上,与溶剂对照组比较差异均有统计学意义(P<0.05或<0.01);黄体数、着床数、死胎数、胎仔体重、身长、尾长和胎仔性别等与溶剂对照组基本一致。给药各剂量组未见明显的骨骼畸形和内脏器官畸形。结论乙烷硒啉在>250mg/kgbw剂量时,对大鼠具有明显的母体毒性和胚胎毒性;在<500mg/kgbw剂量时,不存在骨骼、内脏器官的致畸毒性。

乙烷硒啉分散片溶出度测定方法的研究

目的:建立乙烷硒啉分散片的溶出度测定方法。方法:建立溶出度紫外测定方法,分别考察乙烷硒啉分散片在不同条件下的溶出特性,运用Excel办公软件,以Weibull模型处理试验数据,用单因素方差分析及t检验进行统计学分析。结果:在优化的测定条件下,乙烷硒啉分散片60min累积溶出度达到(65.59±0.36)%,批内均一性与批间重现性良好。结论:本品溶出度优化测定条件为:溶出介质量为1000mL,溶出介质组成为0.9%盐酸水溶液-异丙醇(60∶40),采用药典附录ⅩC第二法(桨法),转速为150r.min-1。取样点为60min,限度为60%。

乙烷硒啉与顺铂或氟尿嘧啶联合应用对胃癌BGC-823的治疗作用

目的观察乙烷硒啉与顺铂或氟尿嘧啶联合应用对胃癌BGC-823的体内、外抗肿瘤作用。方法MTT法测定5、10、20、40μmol·L^(-1)乙烷硒啉单药及固定其浓度为5μmol·L^(-1)与顺铂或氟尿嘧啶联合时对BGC-823的生长抑制作用。建立裸鼠移植瘤模型,采用乙烷硒啉、顺铂、氟尿嘧啶单药及乙烷硒啉与后两者联合的不同给药方案,观察抗肿瘤作用。结果乙烷硒啉对BGC-823有明显增殖抑制作用,24、48、72h的IC_(50)值分别是30.23、19.70和11.67μmol·L^(-1),联合后效果明显增强(P<0.05)。体内实验,联合组肿瘤增长率明显降低,与单药组有非常显著差异(P<0.01)。结论乙烷硒啉单药表现明显抗肿瘤作用,与顺铂或氟尿嘧啶联合后可以增效。

乙烷硒啉对大鼠连续灌胃给药十二周的长期毒性试验研究

目的研究乙烷硒啉连续给药后对大鼠的毒副反应及其严重程度。方法采用SD种大鼠,受试物共分成3个给药组(1 000mg/kg、400 mg/kg和160 mg/kg)和1个溶剂对照组,连续口服给药12周,停药后再观察4周,进行一般症状观察,血象、生化、脏器解剖、脏器系数和组织病理学等检查。结果给药12周高剂量组的大鼠体重增长缓慢,摄食量减少,谷草转氨酶、碱性磷酸酶、总胆红素、总胆固醇增高,葡萄糖降低,心脏、肝脏、脾脏、肾脏和睾丸脏器系数值升高,肝脏存在肝细胞脂肪性变,个别已经发展到轻度肝硬变。停药4周大鼠的体重增长差值有明显的缩小,总胆固醇增高,心脏、肝脏脏器系数值仍有一定升高,肝脏轻、中度肝细胞脂肪性变和早期肝硬变仍然存在。结论乙烷硒啉在相当于临床人拟用量70倍时,出现体重增长明显缓慢、血液葡萄糖降低、肝功能和脂代谢等异常、肝脏等脏器系数值增加、肝细胞脂肪性变和早期肝硬变等的毒副反应。大鼠用药的无明显毒副反应剂量为160mg/kg。

乙烷硒啉对犬的长期毒性试验研究

目的研究乙烷硒啉连续给药后对犬的毒性反应及其严重程度。方法采用纯种Beagle犬，受试物共分成3个给药组（600mg／kg、200mg／kg和67mg／kg）和1个溶剂对照组，连续口服给药12周，停药后再观察4周，进行一般症状观察、血象、生化、心电图、尿液、骨髓象、眼睛、脏器解剖，脏器系数和组织病理学等检查。结果给药12周高剂量组个别犬在摄食过快时出现呕吐，粪便颜色变浅；谷丙转氨酶和谷草转氨酶增高，葡萄糖降低；肝脏、脾脏和肾脏脏器系数值升高；存在肝细胞脂肪性变和前列腺腺瘤样型肥大的组织病理学变化。停药恢复期基本恢复正常数值。结论乙烷硒啉在相当于临床人拟用量42倍下，连续口服给药后犬仅出现轻度消化道刺激症状，肝功能异常和血糖值降低；轻一中度肝脏、脾脏和肾脏的肿大；轻度一中度的脂肪肝和前列腺腺瘤样型肥大；毒性反应主要集中在消化道，重点在肝脏。犬用药的无明显毒性反应剂量为67mg／kg。

乙烷硒啉对小鼠体内移植性肝癌(H_(22))和Lewis肺癌的抗肿瘤作用

目的观察乙烷硒啉对小鼠移植性肝癌(H22)和Lewis肺癌的抗肿瘤作用,并探讨其可能的抗肿瘤机制。方法建立小鼠体内移植性肝癌(H22)和Lewis肺癌模型,每种属60只,分别设为5组:阴性对照、阳性对照(CTX)和乙烷硒啉3个剂量组,每组12只。于接种肿瘤第2天至第8天5组分别连续腹腔注射5g/L羧甲基纤维素钠溶液、60mg/kgCTX及25、12.5、6.3mg/kg乙烷硒啉。停药3d后(第11天)每组处理一半动物,计算抑瘤率,并用流式细胞仪观察其对细胞周期的影响;另一半动物观察生命延长率。结果乙烷硒啉在25、12.5、6.3mg/kg剂量下对小鼠肝癌H22移植瘤的抑瘤率分别为52.5%、43.6%和30.2%,生命延长率分别为56.6、38.7%和14.2%;对小鼠Lewis肺癌的抑瘤率分别为43.8%、29.6%和18.9%,生命延长率分别为47.3%、17.9%和6.3%。乙烷硒啉各剂量组肿瘤细胞凋亡率明显高于对照组(P<0.05),且25.0mg/kg组出现S期阻滞(P<0.05)。结论乙烷硒啉对小鼠移植性肝癌(H22)和Lewis肺癌具有抗肿瘤作用,且随剂量的增加其抑制肿瘤作用显著增强。其抗肿瘤作用可能是通过诱导细胞凋亡,同时干扰肿瘤细胞生长周期,导致S期阻滞而实现的。

RP-HPLC法测定乙烷硒啉分散片中药物的含量

目的:建立乙烷硒啉分散片含量测定方法。方法:采用反相高效液相色谱法,使用BDS HYPERSILC18柱(250mm×4.6mm,5μm),检测波长为320nm,柱温为室温,流动相为0.01%磷酸-甲醇(60∶40),流速1.0mL.min-1。结果:溶剂与辅料对分散片中药物的含量测定无干扰,乙烷硒啉在10～400μg.mL-1范围内呈良好的线性关系(r=0.9995),精密度RSD为0.39%;日内精密度为0.57%;平均回收率为101.2%(n=9)。结论:该方法专属性强,操作方便,结果准确,重现性好,可用于乙烷硒啉分散片中药物的含量测定。

小鼠H22肝癌皮下移植瘤的生长与血硫氧还蛋白还原酶活性的相关性

背景与目的:研究表明硫氧还蛋白还原酶(thioredoxin reductase,TrxR)在肿瘤组织中的表达较正常组织中明显增多且活性升高,但关于肿瘤生长与血TrxR活性的关系报道却很少。本实验的目的是研究小鼠H22肝癌皮下移植瘤的生长与血TrxR活性的关系。方法:建立H22肝癌昆明小鼠皮下移植瘤动物模型,分为5组:空白组不接种肿瘤细胞,模型组接种肿瘤细胞,这两组小鼠均给予5‰羧甲基纤维素钠灌胃作为对照;乙烷硒啉低、中、高剂量组均接种肿瘤细胞,并分别灌胃给予36mg/kg、72mg/kg、216mg/kg乙烷硒啉。于肿瘤接种前、肿瘤生长至100mm3给药前、给药第1天、给药第4天及给药第10天5个时间点行眼眶取血,二硫代双硝基苯甲酸(dithio-bis-nitrobenzoic acid,DTNB)法测定血浆TrxR活性。处死小鼠剥离皮下肿瘤,DTNB法测定肿瘤组织TrxR活性。结果:给药结束时低、中、高剂量乙烷硒啉组小鼠的抑瘤率分别达到59.5%、74.3%、74.9%。空白组小鼠血TrxR活性稳定;接种肿瘤后小鼠血TrxR活性随肿瘤体积增大而增加,呈明显正相关;给药结束时,低、中、高剂量乙烷硒啉组小鼠血TrxR活性抑制率分别为59.2%、68.4%、68.9%,而肿瘤组织中TrxR活性抑制率分别为15.3%、25.8%、29.8%。结论:H22肝癌小鼠血TrxR活性与其肿瘤生长呈正相关;不同剂量的TrxR抑制剂乙烷硒啉干预后,血TrxR活性能够被不同程度地抑制,与肿瘤生长的抑制呈一致性。

不同载体材料改善难溶性药物乙烷硒啉溶出性能的实验研究

目的:改善水不溶性药物乙烷硒啉的溶出性能,以提高乙烷硒啉固体制剂的药物溶出度。方法:以聚维酮(polyvinylpyrrolidone,简称PVP)、枸橼酸、酒石酸、羟丙基-β-环糊精(hydroxylpropyl-β-cyclodex-trin,简称HP-β-CD)等辅料为载体材料,以溶剂溶解与分散法制备乙烷硒啉固体颗粒及其物理混合物,并进行体外溶出度试验。利用差示扫描量热法(Differential Scanning Calorimetry,简称DSC)和X-射线衍射法来验证药物的存在状态。结果:以PVP为载体材料所制固体颗粒的体外溶出行为显著优于其他载体材料,且载体材料的比例越大,药物溶出越快。结论:DSC和X-射线衍射图实验证明,PVP载体材料改变了乙烷硒啉的存在状态与亲水性,溶出性能得到显著改善。

抗肿瘤新药乙烷硒啉对人舌鳞状细胞癌细胞增殖和迁移的影响

目的:探讨新型有机硒化合物乙烷硒啉(BBSKE)对人舌癌CAL27细胞株生长和增殖的影响。方法:分别采用体外细胞实验MTT、Hoechst 33258免疫荧光染色、Annexin V-FITC/PI染色流式细胞术、Transwell小室实验检测2、5、10、20μmol/L(含1‰DMSO)的BBSKE对CAL27细胞增殖、细胞周期及迁移的影响,并设置溶剂对照组和正常对照组。结果:5、10、20μmol/L的BBSKE作用CAL27细胞后,其增殖抑制率较对照组明显增加(P<0.01),且随作用时间的延长而增加。Hoechst 33258免疫荧光染色结果显示5、10μmol/L BBSKE组相比对照组细胞出现凋亡形态改变。Annexin V-FITC/PI染色流式细胞术分析结果显示与对照组相比,2、5、10、20μmol/L BBSKE组CAL27细胞G2/M期比率呈梯度增加(P<0.01),细胞周期被阻滞于G2/M期(P<0.01)。Transwell小室实验结果显示,CAL27细胞的迁移数量在2、5、10、20μmol/L BBSKE组相比对照组明显减少(P<0.01),且迁移细胞数与药物浓度呈反比。结论:BBSKE对CAL27细胞具有增殖抑制作用,其机制可能是通过引起细胞周期阻滞于G2/M期而诱导细胞凋亡,并且抑制细胞迁移,提示BBSKE可作为舌鳞状细胞癌患者化疗治疗的备选药物。

有机硒化合物乙烷硒啉抑制腺样囊性癌细胞生长的体外研究

目的探讨乙烷硒啉(BBSKE)对腺样囊性癌细胞的抑制作用及机制,为相关研究提供参考。方法用3(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测BBSKE对腺样囊性癌细胞系(SACC-83、SACC-LM)的敏感性。BBSKE作用SACC 48 h后,用赫斯特(Hoechst)荧光染色法检测细胞凋亡形态;BBSKE作用SACC 48 h后,检测BBSKE对腺样囊性癌细胞半胱氨蛋白水解酶-3(Caspase-3)活性的影响。用[5,5-二硫双(2-硝基苯甲酸)](DTNB)法检测BBSKE对腺样囊性癌细胞中硫氧还蛋白还原酶(TrxR)活性的影响。结果BBSKE可以明显抑制腺样囊性癌细胞的增殖,作用于SACC-83和SACC-LM腺样囊性癌细胞72 h,抑制肿瘤细胞生长达到50%时的药物浓度(IC50)分别为12μmol/L和10μmol/L。BBSKE作用SACC 48 h后,细胞发生核固缩、核破碎等形态变化;随着BBSKE浓度增大,SACC-83细胞中Caspase-3活性显著增高,SACC-LM细胞中Caspase-3活性增加更明显;TrxR活性随BBSKE浓度增大而降低。结论新型含硒化合物BBSKE可以抑制腺样囊性癌细胞的增殖。其作用可能是通过抑制TrxR活性,增加半胱氨蛋白水解酶-3活性而实现的。