Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs

Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance.

Cloning, characterization and expression analysis of a microsomal glutathione S-transferase gene from the seagrass Zostera marina

The response of glutathione S-transferase(GST)in Zostera marina to temperature variation was analyzed at molecular level by cloning the microsomal GST gene and texting the microsomal GST expression regularity under different temperature.Specific speaking,express ZmGST in Escherichia coli,then purify the recombinant protein and make the thermal stability analysis.Therefore,the experiments were carried out to provide a theoretical basis for the further elaboration to the population degradation mechanisms of Z.marina.In conclusion,the thermostability and the response of ZmGST gene to temperature changes can determine its temperature tolerance range,and affect its resilience in turn.

Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis(Thunberg)(Orthoptera:Acrididae)

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.

A systemic review of glutathione S-transferase P1 Ile105Val polymorphism and colorectal cancer risk

Objectives： To investigate the correlation between glutathione S-transferase P1 （GSTP1） Ilel05Val polymorphism and colorectal cancer （CRC） risk. Methods： Studies were identified to investigate the association between GSTP1 Ilel05Val polymorphism and CRC risk. Systematic computerized searches of the PubMed, Chinese National Knowledge Infrastructure, WANFANG and SinoMed were performed. Summary odds ratios （OR） and 95% confidence intervals （95 % CI） were used to measure GSTP 1 Ile 105Val polymorphisms and CRC risk. Results： A total of 23 retrospective studies were included in the meta-analysis. During all studies including 6,981 cases and 8,977 controls, sample sizes ranged from 146 to 2,144. Overall, the pooled results revealed that lie 105Val polymorphism was not associated with CRC risk and confused results were found in subgroup analyses. Further meta-analyses were conducted after excluding low-quality studies. GSTP1 Ilel05Val is associated with increased risk of CRC limited in studies with matched control. There was no significant heterogeneity in all genetic comparisons, but heterogeneity existed in subgroup analyses of heterozygous and dominant comparisons. The meta-regression analyses indicated that matched controls were the significant factor influencing between-study heterogeneity in all possible influential factors including published year, ethnicity, source of control, sample size, Hardy-Weinberg equilibrium （HWE） in control and matched controls. Sensitivity analysis revealed the pooled ORs were not changed before and after removal of each single study in all genetic comparisons, indicating the robustness of the results. Conclusions： GSTP1 Ilel05Val might be associated with increased risk of CRC. However, more high- quality case-control studies should be performed to confirm the authenticity of our conclusion.

Yeast One-hybrid System Used to Identify the Binding Proteins for Rat Glutathione S-transferase P Enhancer I

Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.

Expression and Characterization of a Sigma-Class Glutathione S-transferase of the Oriental Migratory Locust, Locusta migratoria manilensis (Meyen)

A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame （204 amino acid polypeptide）, which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein （LmGSTs1） was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue （RB） was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid （ECA）, LmGSTs1 showed （81±3）% of the original activities.

ENZYME-LINKED IMMUNOSORBENT ASSAY OF HUMAN PLACENTA TYPE GLUTATHIONE S-TRANSFERASE AND ITS APPLICATION IN THE DIAGNOSIS OF HEPATOCARCINOMA

GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.

Frequency of Null Phenotypes of Glutathione S-Transferase M1 and T1 among the Populations of Tabuk (Northwestern Part of Saudi Arabia)

Background: The variability in the distribution of the null phenotypes of GSTM1 and GSTT1, due to total or partial gene deletion resulting in the lack of the active enzyme, has been reported in different populations, especially in ethnically well-defined groups but not in Tabuk. This study investigated the variability in the distribution of the null phenotypes of GSTM1 and GSTT1 in the population of Tabuk (northwestern part of Saudi Arabia). Method: This study was conducted on 200 subjects of Tabuk—northwestern part of Saudi Arabia among which 100 were chronic smokers and 100 were nonsmokers. The subjects were reporting to hospital for routine checkup. All were without past history of any chronic disease and no significant abnormality. GST genotyping was done by multiplex PCR-based methods. The smoker and control groups were compared using a chi-square test with P GSTM1 deletion homozygosity of 14% and 1% was reported among non smokers and smokers, respectively whereas GSTT1 deletion homozygosity of 28% and 6% was reported among non smokers and smokers, respectively. Our results indicate that there are major differences in allelic distribution of GSTM1 and GSTT1 genes between the two groups investigated. Combined analysis of both genes revealed that 15% of smokers and non smokers harbor the deleted genotype of GSTM1 and 34% of smokers and non smokers harbor the deleted genotype of GSTT1 with significant differences. Conclusion: This study enables selecting subgroups among the general population who are more susceptible to DNA damage and will help genetic studies on the association of GST polymorphisms with disease risks and drug effects in Arab population. Studies with a larger sample size are needed to evaluate and confirm the validity of our results.

P-gp、GST-л、TopoⅡ在胃癌组织中的表达及临床意义

目的 探讨P gp、GST л、TopoⅡ在胃癌组织中的表达及临床意义。 方法 采用免疫组化方法研究了P gp、GST л、TopoⅡ在胃癌组织中的表达情况。 结果 P gp、GST л、TopoⅡ在胃癌组织中的表达率分别为 5 9.74 %、6 7.5 3%、83.12 %。除TopoⅡ的表达与组织学类型有关外 (P <0 .0 0 1) ,其余与各项临床病理参数均无关 (P >0 .0 5 )。结论 胃癌组织中存在着P gp、GST л、TopoⅡ表达水平的变化 ,临床检测P gp、GST л、TopoⅡ对胃癌化疗方案的制定具有重要的指导意义。

GST-π和P-gp在胃癌中的表达及其临床意义

目的探讨耐药蛋白GST-π和P-gp在胃癌中的表达及其与胃癌临床病理特征的关系。方法用免疫组化法检测209例胃癌组织GST-π和P-gp的表达情况,并收集临床病理相关资料作回顾性分析。结果 GST-π表达阳性率为81.82%(171/209),与淋巴结转移数目有关(P<0.05);P-gp表达阳性率为79.90%(167/209),在印戒细胞癌中表达率较高(P<0.05);GST-π和P-gp表达具有相关性(r=0.241,P<0.05)。结论 GST-π和P-gp的表达与胃癌的生物学行为有一定的相关性。

GST Pull-down实验鉴定NF-κB相互作用多肽

目的体外鉴定NF-κB相互作用多肽与p50和p65间的相互作用。方法首先构建GST-作用多肽融合蛋白的原核表达载体pET-42a/polypeptide及NF-κB p50和p65亚基保守结构域的原核表达载体pET22b/p50和pET22b/p65,并在大肠杆菌E.coliBL-21中诱导表达,后进行GST pull-down实验验证多肽与NF-κB p50和p65的结合效应。结果经诱导表达获得了可溶性的GST-多肽融合蛋白和具有DNA结合活性的p50p、65蛋白,GST pull-down实验证实3条多肽与p50发生特异地相互作用。结论证实3条多肽在体外能与p50发生物理性的相互作用,这为获得靶向NF-κB的功能拮抗多肽工作奠定了基础。

P-gp,MRP,LRP和GST-π等多药耐药蛋白在肝癌中的协同表达研究

目的:探讨多药耐药相关标志物P-gp,MRP,LRP和GST-π在肝癌细胞中的共表达情况.方法:以69例肝癌患者为本次研究对象,应用免疫组化SP法检测多药耐药相关标志物P-gp,MRP,LRP和GST-π在肝癌组织的共表达情况.结果:不同分型、不同分期肝癌组织P-gp,MRP,LRP和GST-π阳性表达率及共表达差异无显著性(P>0.05);分化程度不同的肝癌组织多药耐药相关标志物P-gp,MRP,LRP和GST-π阳性表达率及共表达有明显差异(P<0.05).结论:多药耐药相关标志物P-gp,MRP,LRP和GST-π在不同分型、不同分期和不同分化程度的肝癌组织中均有明显共表达,在不同分化程度的肝癌组织中阳性表达率及共表达更为明显.

P-糖蛋白,GST-π,TopoⅡ在食管癌组织中的表达及其临床意义

目的：探讨P－gp、GST－π、TopoⅡ在食管癌组织中的表达及意义。方法：采用免疫组织化学方法研究了P－gp、GST－π、TopoⅡ在食管癌组织中的表达情况。结果：P－gp、GST－π在食管癌组织中的表达升高，表达率分别为58．02％和72．84％，P－gp 的高表达与淋巴结转移、癌肿浸润深度有关P＜0．05。GST－π的高表达与细胞分化程度有关P＜0．05。TopoⅡ在食管癌组织中的表达降低，表达率为61．73％，TopoⅡ的表达降低与临床病理参数无关P＞0．05。结论：食管癌组织中存在着P－gp、GST－π、TopoⅡ表达水平的变化，临床检测P－gp、GST－π、TopoⅡ对食管癌化疗方案的制定、判断预后有重要的指导意义。

TopoⅡα、GST-π、P-gp在卵巢癌化疗耐药中的作用

目的探讨TopoⅡα、GSTπ、Pgp在卵巢癌化疗耐药中的作用。方法采用免疫组化SP法、计算机图像分析技术对80例卵巢癌、20例良性上皮性卵巢肿瘤、20例正常卵巢组织中TopoⅡα、GSTπ、Pgp的表达进行检测。结果卵巢癌中TopoⅡα、GSTπ、Pgp的表达显著高于正常组及良性肿瘤组,P<0.05。TopoⅡα、GSTπ的表达与肿瘤分化程度有关,分化越差表达越高,P<0.05;Pgp的表达与多种病理因素无关,P>0.05。术前化疗组GSTπ、Pgp的阳性表达率显著高于术前未化疗组,P<0.05。结论TopoⅡα、GSTπ、Pgp在卵巢癌耐药中发挥重要作用,这三项指标的联合检测对制定合理的化疗方案具有积极的指导意义。

乳腺癌中耐药蛋白P-gp、GST-π、Topo-Ⅱ的表达

目的明确乳腺癌中是否存在P-gp、GST-π、TopoⅡ三种原发耐药蛋白;三种耐药蛋白的表达与乳腺癌的组织类型、细胞分化程度、TNM分期是否相关;是否影响乳腺癌患者的预后。方法采用S-P免疫组化方法,检测260例乳腺癌患者中P-gp、TopoⅡ、GST-π的表达情况,同时取10例乳腺病及10例癌旁乳腺组织作对照。结果乳腺癌中三种耐药蛋白呈异质性表达。P-gp、GST-π和TopoⅡ在良性病变中弱表达,在伴有早期浸润的乳腺导管内癌和乳腺浸润性导管癌中的表达同样明显高于乳腺导管内癌;其中P-gp和GST-π表达和预后相关,差异显著(P<0.01,P<0.05)。结论乳腺癌在发生早期浸润的同时就伴随着耐药的发生;P-gp和GST-π是乳腺癌患者预后的影响因素,综合检测优于单一检测。

p53、Ki-67、TopoⅡ、GST、P170对三阴乳腺癌预后的相关性分析

下载PDF阅读器目的 探讨p53、Ki-67、DNA拓扑异构酶Ⅱ（DNA topoisomeraseII,TopoII）、谷胱甘肽转移酶（glutathione-S-transfreases,GST）、P-糖蛋白（P-glycoprotein,P170）免疫组化的表达在三阴乳腺癌发生发展中的生物学特征及其对预后评估的意义.方法 回顾性分析1993年1月-2003 年12月我院初诊乳腺癌病人共515 例进行筛选,共得出88例三阴乳腺癌（ER、PR、HER-2 均为阴性）.根据ER、PR、HER-2的表达,将其分为3组：三阴组（ER-、PR-、HER-2-）、HR组（ER＋ /PR＋HER-2-）和HER-2组（ER-、PR-、HER-2＋）.统计这3组三阴乳腺癌标本p53、Ki-67、 TopoⅡ、GST、P170 的表达,并调查这3组病人5 年无病生存率与5 年内死亡率.结果 与非＂三阴＂乳腺癌相比,＂三阴＂乳腺癌患者p53、P170、Ki-67、GST表达水平均为最高（59.5%、78.6%、19.0%、66.7%）,在术后5年内三阴组的复发率为23.9%,死亡率为22.7%,复发风险（HR 2.46,CI：1.04-5.84;P＜0.05）和死亡风险（HR 2.77,CI：1.09-7.02;P＜0.05）,之后明显降低.结论 p53、P170、Ki-67、GST 在三阴乳腺癌的高表达与其不良预后具有相关性.

结直肠癌组织中P-gp、GST-π、Topo-Ⅱ和p53的协同表达

目的 :探讨结直肠癌组织中P 糖蛋白 (P gp)、拓扑异构酶 (Topo Ⅱ )、谷甘胱肽 S 转移酶 (GST π)和 p5 3的表达与病理组织学分级、临床分期、淋巴结转移和生存率的相关性及其协同表达的意义。方法 :用免疫组化方法检测 136例术前未进行化疗的结直肠癌组织中P gp、GST π、Topo Ⅱ和p5 3的表达。 结果 :P gp、GST π和 p5 3的表达与病理组织学分级、淋巴结转移和临床分期无关 (P >0 0 5 ) ,与生存率明显相关 (P <0 0 1)。Topo Ⅱ的表达与本组临床病理参数均无相关性。 结论 :应用免疫组化法检测结直肠癌组织中P gp、GST π和 p5 3的表达 。

GST-π、P-gp和Ts在消化道肿瘤中的表达及临床意义

目的:探讨GST-π、P-gp及Ts在消化道肿瘤中的表达及其与临床病理特征的关系。方法:应用免疫组化法检测127例消化道肿瘤(32例食管癌、53例胃癌、42例结直肠癌)组织中GST-π、P-gp及Ts的表达情况,与临床病理相关资料进行回顾性分析。结果:GST-π表达阳性率80.95%、P-gp表达阳性率44.86%及Ts表达阳性率18.10%。GST-π表达、P-gp表达与年龄、性别、淋巴结转移、脉管癌栓及分化程度无关。Ts表达也与年龄、性别、淋巴结转移及分化程度无关,但与脉管癌栓有关(P<0.05)。P-gp与GST-π、Ts表达具有相关性(P<0.05)。结论:消化道肿瘤组织可表达P-gp与GST-π、Ts等耐药因子,三者表达具有相关性,GST-π、P-gp与Ts在消化道肿瘤组织中联合检测有重要的临床意义。

P-gp、GST-π、Topo-Ⅱ在宫颈癌组织中的表达及意义

目的探讨P-糖蛋白(P-gp)、谷胱甘肽S-转移酶-π(GST-π)和DNA拓扑异构酶Ⅱ(TopoⅡ)在宫颈癌组织中的表达及其临床意义。方法采用免疫组化方法检测20例正常宫颈、30例宫颈上皮内瘤变(CIN)、60例初治宫颈癌及10例复发宫颈癌组织中P-gp、GST-π、TopoⅡ的表达,分析P-gp、GST-π、TopoⅡ的表达与宫颈癌患者临床病理特征及3年生存率之间的关系。结果①P-gp、GST-π、TopoⅡ在正常宫颈、CIN、初治宫颈癌及复发宫颈癌组织中均有表达,表达强弱顺序依次为:复发宫颈癌>初治宫颈癌>CIN>正常宫颈。②宫颈癌组织中存在3种耐药蛋白的共表达,P-gp与GST-π、TopoⅡ的表达呈正相关(P<0.05);GST-π与TopoⅡ的表达无显著性相关(P>0.05)。③P-gp、GST-π与初治宫颈癌临床分期,组织学类型和病理分级无关(P>0.05);TopoⅡ在宫颈腺癌中的表达水平明显低于鳞癌(P<0.05)。④P-gp、GST-π低表达者3年生存率优于高表达者,差别有统计学意义(P<0.05);TopoⅡ低表达者与高表达者3年生存率差别无统计学意义(P>0.05)。结论P-gp、GST-π、TopoⅡ可能与宫颈癌的发生发展相关。P-gp、GST-π表达强度的增加不仅可为宫颈癌化疗用药提供参考,而且可作为宫颈癌判断预后的指标。