谷胱甘肽过氧化物酶家族的功能及其成员作为胶质瘤免疫治疗标志物的可能性

目的：探讨谷胱甘肽过氧化物酶(GPX)家族在胶质瘤发生发展中的生物学功能和预后价值。方法：利用TCGA、GTEx和CGGA等多个数据库数据分析胶质瘤中GPX各亚型基因的表达及其相关性、蛋白之间的相互作用、基因突变、GPX表达与胶质瘤患者预后的关系以及GPX7、8的基因集富集分析；GPX8与胶质瘤中免疫细胞浸润以及免疫检查点分子表达的相关性分析，胶质瘤中GPX8表达与化疗药物IC₅₀的相关性分析。采用qPCR法、WB法和免疫荧光技术检测国人胶质瘤组织和对照组织(标本收集自2022年10月20日至12月20日间在上海奉贤区中心医院神经外科手术切除的5例胶质瘤和3例严重脑外伤的病灶组织)中GPX mRNA、蛋白以及相关免疫检查点分子的表达进行验证。结果：数据库分析显示胶质瘤中GPX各亚型蛋白之间存在相互作用、基因表达水平存在相关性(P<0.05或P<0.01)，胶质瘤组织中多个GPX亚型存在单核苷酸变异和拷贝数变异；不同类型胶质瘤组织的免疫细胞和肿瘤细胞中主要表达的GPX亚型有明显不同，在胶质瘤组织中GPX1、4、7、8均呈高表达(均P<0.05)且与...     

不同浓度的氟伐他汀对卵巢癌SKOV3细胞中GPX4蛋白表达的影响

目的:探讨不同浓度的氟伐他汀对卵巢癌SKOV3细胞中脂质过氧化物磷脂谷胱甘肽过氧化物酶4（phospholipid glutathione peroxidase 4,GPX4)蛋白表达的影响。方法:采用细胞免疫组化、qRT-PCR和Western Blot技术检测卵巢癌SKOV3细胞中GPX4 mRNA和蛋白的表达。结果:随着氟伐他汀的浓度增加（0 mmol/L、3 mmol/L、10 mmol/L、30 mmol/L）,SKOV3细胞数目减少,氟伐他汀呈浓度依赖性抑制SKOV3细胞的增殖;SKOV3细胞中GPX4蛋白和mRNA的相对表达量明显上调。结论:氟伐他汀抑制卵巢癌细胞的增殖转移与其上调卵巢癌细胞中GPX4的表达有关。     

Gal-1、GPX3及Tg水平与甲状腺癌病情严重程度的关系分析

目的 分析半乳糖凝集素-1(Gal-1)、谷胱甘肽过氧化物酶3(GPX3)及甲状腺球蛋白(Tg)水平与甲状腺癌病情严重程度的关系。方法 选取70例甲状腺癌患者,另选择同期就诊的70例甲状腺良性病变患者作为对照,两组均进行血清Gal-1、GPX3、Tg水平检测,对比其血清Gal-1、GPX3、Tg水平,评估Gal-1、GPX3、Tg对甲状腺癌的诊断价值,分析甲状腺癌患者临床参数与Gal-1、GPX3及Tg水平的关系。结果 甲状腺癌组血清Gal-1、Tg水平高于甲状腺良性病变组(P<0.05),GPX3水平低于甲状腺良性病变组(P<0.05)。当血清Gal-1、GPX3、Tg截点值分别为167.59 ng/mL、116.38μg/L、21.74 mIU/L时,约登指数最大,诊断甲状腺癌的敏感度分别为78.57%、72.86%、68.57%,特异度分别为80.00%、92.85%、84.29%。甲状腺癌TNM分期为Ⅲ～Ⅳ期者血清Gal-1、Tg水平高于TNM分期为Ⅰ～Ⅱ期者(P<0.05),GPX3水平低于TNM分期为Ⅰ～Ⅱ期者(P<0.05);淋巴结转移者血清Gal...     

GPX1在结直肠癌组织中的表达及其对癌细胞增殖、侵袭及迁移的影响

目的:探讨谷胱甘肽过氧化物酶1 (glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响.方法:收集2015年6月至2016年3月间海口市人民医院滨江分院普外科手术切除的60例临床CRC组织及癌旁组织,应用实时荧光定量PCR(qPCR)技术检测CRC组织及癌旁组织中GPX1 mRNA水平.在高表达GPX1 mRNA的HT29细胞株用慢病毒携带shRNA感染的方法构建敲除GPX1的细胞株,瞬时转染法在低表达GPX1 mRNA的LOVO细胞系构建过表达GPX1的细胞株,并在GPX1 mRNA及蛋白水平进行验证.通过MTS法检测CRC细胞的增殖能力的变化,用划痕实验、Transwell侵袭实验分别检测CRC细胞迁移侵袭能力的变化,同时用Western blotting检测E-钙黏蛋白和波形蛋白表达的变化.结果:CRC组织中GPX1 mRNA表达水平显著低于癌旁组织(0.051±0.024 vs 0.142±0.051,P＜0.01).敲除GPX1后,HT29细胞的增殖能力显著增强(12.901±2.790 vs 6.617±2.462,P＜0.01)、侵袭能力显著增强[(384.7±37.9) vs (209.2±31.2)个,P＜0.01]、迁移能力显著增强[(0.139±0.025)vs (0.251±0.038)mm,P＜0.01]、E-钙黏蛋白表达下调(P＜0.01)、波形蛋白表达上调(P＜0.05).过表达GPX1的LOVO细胞的增殖能力显著降低(P＜0.01)、迁移和侵袭能力下降(均P＜0.05)、E-钙黏蛋白上调(P＜0.01)、波形蛋白表达下调(P＜0.01).结论:GPX1mRNA在人CRC组织中低表达,GPX1负向调控CRC细胞的增殖、迁移和侵袭,其在CRC中可能发挥抑癌作用.     

GPX1过表达对肺癌BERP35T1细胞DNA氧化损伤和恶性表型的影响

目的: 探讨谷胱甘肽过氧化物酶1(GPX1)过表达对α粒子辐射致肺癌BERP35T1细胞DNA氧化损伤和恶性表型的影响。方法: 构建pEGFP-GPX1真核表达载体,经PCR、双酶切和测序鉴定后,采用脂质体法将重组质粒及其对照空载体分别转染至BERP35T1细胞中,筛选出具有G418抗性的细胞株,经Western blot法测定GPX1蛋白在细胞株中的表达情况;通过免疫细胞化学法、四甲基噻唑蓝(MTT)法、划痕愈合试验和锚着独立生长试验分别检测GPX1过表达对BERP35T1细胞内DNA氧化损伤产物8-羟化脱氧鸟苷(8-OHdG)水平,细胞增殖、迁移以及锚着独立生长能力的影响。结果: pEGFP-GPX1经PCR、双酶切鉴定和DNA测序证实,GPX1片段的序列完全正确;并获得GPX1稳定表达BERP35T1细胞株BERP35T1-GPX1-6,其GPX1蛋白水平是对照空载体转染细胞的4.01倍(P<0.05);与BERP35T1细胞和对照空载体转染细胞相比,BERP35T1-GPX1-6细胞内8-OHdG水平降低,细胞的增殖、迁移和锚着独立生长能力均减弱(P<0.05)。结论: 过表达GPX1可能通过清除细胞内活性氧降低DNA氧化损伤水平,从而抑制BERP35T1细胞增殖、迁移和锚着独立生长能力等恶性表型。     

谷胱甘肽过氧化物酶GPX4在铁死亡中的作用与机制研究进展

谷胱甘肽过氧化物酶4(GPX4)是一种能够特异性催化谷胱甘肽将脂质过氧化物转化为类脂醇的硒蛋白,它在调节细胞铁死亡方面发挥着重要的作用。本文将对GPX4的蛋白结构、分子作用机制及其表达调控进行介绍,对其在铁死亡中的功能以及在癌症等疾病中的应用进行系统综述,对未来存在的问题进行了分析与展望。     

重楼皂苷通过JNK/p53诱导肺癌细胞铁死亡的作用机制研究

目的 分析重楼皂苷对肺癌细胞铁死亡的诱导作用及可能机制。方法 取对数期A549细胞，分为对照组、重楼皂苷组、SP600125组、联合组，对照组常规培养，重楼皂苷组加入重楼皂苷(4 g/L),SP600125组加入SP600125(10μmol/L),联合组加入重楼皂苷(4 g/L)及SP600125(10μmol/L)。MTT实验检测细胞铁死亡抗性；荧光探针法检测细胞内活性氧(ROS)堆积；检测细胞丙二醛(MDA)水平；流式细胞术检测细胞凋亡率；Western blot法检测溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶(GPX4)、c-Jun氨基末端激酶(JNK)、p-JNK、p53蛋白表达。结果 与对照组比较，重楼皂苷组铁死亡抗性，SLC7A11、GPX4蛋白表达降低，ROS阳性细胞数增加，细胞内MDA水平、细胞凋亡率、p-JNK/JNK及p53蛋白表达升高(P<0.05),SP600125组铁死亡抗性，SLC7A11、GPX4蛋白表达升高，ROS阳性细胞数减少，细胞内MDA水平、细胞凋亡率、p-JNK/JNK及p53蛋白表达降低(P<0.05);与重楼皂...     

磷酸酶PHLPP2抑制GPX4活性增强非小细胞肺癌铁死亡诱导剂RSL3敏感性的作用分析

摘 要：［目的］ 探讨磷酸酶PHLPP2在铁死亡诱导剂RSL3诱导非小细胞肺癌细胞铁死亡发生中的作用及机制。［方法］ 体外培养人肺癌细胞株，GPX4抑制剂RSL3处理后MTT法检测RSL3抑制非小细胞肺癌细胞增殖作用，实时荧光定量PCR检测mRNA水平，Western blot法检测蛋白表达。流式细胞术检测脂质ROS水平，分别采用逆转录病毒/腺病毒感染上调/下调PHLPP2表达，两组间比较采用独立样本t检验。［结果］ RSL3有效抑制非小细胞肺癌细胞的活力，RSL3处理后HCC827、H1975、H1650、A549和H1299细胞的增殖率分别为15.22%±2.35%、 25.05%±5.92%、 4.40%±1.61%、41.48%±10.01%和13.17%±2.54%。 PHLPP2表达水平最高的H1650细胞对RSL3抑制增殖作用最显著，RSL3抑制增殖作用与 PHLPP2表达水平呈正比。上调PHLPP2表达可以增加RSL3对A549细胞增殖抑制作用，并增加铁死亡关键标志脂质ROS的累积，A549-vector和A549-PHLPP2组的脂质ROS水平分别为 4.34%±0.39%和15.36%±0.80%（P<0.001）；相反，下调PHLPP2表达H1650细胞中，RSL3对H1650细胞增殖抑制作用减弱，减少了其诱导的脂质ROS的累积，H1650-shControl和H1650-shPHLPP2脂质ROS水平分别为14.76%±1.22%和4.89%±1.81%（P=0.0 015）。公共数据库挖掘分析PHLPP2与铁死亡关键蛋白GPX4、SLC7A11和ASCL4的相关性，发现PHLPP2与GPX4表达呈负相关（r=-0.336，P<0.001）。Western blot进一步验证了上调PHLPP2表达可增强RSL3对GPX4蛋白的抑制作用。［结论］ PHLPP2促进GPX4抑制剂RLS3诱导铁死亡发生，其机制主要通过协同抑制GPX4活性。     

铁死亡相关蛋白SLC7A11及GPX4在肾细胞癌中的表达及意义

目的:探讨SLC7A11及GPX4在肾细胞癌及正常肾组织中的表达差异,初步分析SLC7A11及GPX4的表达情况与肾癌预后之间的关系。方法:采用免疫组化方法检测SLC7A11及GPX4在肾癌及正常肾组织中的表达,同时分析SLC7A11及GPX4表达水平与肾癌患者临床病理参数的相关性。采用Spearman法分析SLC7A11及GPX4之间表达的相关性,利用Kaplan-Meier生存曲线及Cox回归方法分析SLC7A11,GPX4表达的高低及临床因素对肾癌患者预后产生的影响。结果:SLC7A11及GPX4在肾癌中为高表达,而在正常肾组织为低表达（P<0.05）,SLC7A11及GPX4蛋白表达与肾癌大小、远处转移及临床分期密切相关,SLC7A11与GPX4之间呈正相关(r=0.583,P<0.01)。在肾癌组织中,SLC7A11,GPX4表达阳性的患者与阴性表达的患者相比预后较差（P<0.05）。多因素分析提示肾癌大小、远处转移、临床分期、SLC7A11及GPX4是影响肾癌预后的独立因素。结论:SLC7A11及GPX4在肾癌中高表达,可能参与了肾癌的发生,SLC7A11或GPX4高表达的肾癌患者预后不良,未来可能成为肾癌治疗新靶点。     

miR-338通过靶向GPX4抑制非小细胞肺癌细胞增殖

目的:探讨miRNA-338（miR-338）通过靶向调控谷胱甘肽过氧化物酶4（GPX4）表达在非小细胞肺癌(non-small cell lung cancer,NSCLC)增殖中的作用及机制研究。方法:通过实时定量聚合酶链式反应（qRT-PCR）检测非小细胞肺癌组织、癌旁、细胞系及对照细胞系中miR-338及GPX4 mRNA的表达水平;通过Western Blot检测非小细胞肺癌组织、癌旁、细胞系及对照细胞系中GPX4蛋白水平;通过荧光素酶报告基因时间验证miR-338直接靶向调节GPX4的表达;通过CCK-8探索miR-338是否通过调节铁死亡影响肿瘤细胞增殖;通过试剂盒检测细胞脂质氧化和活性氧水平。结果:NSCLC组织和细胞系中miR-338表达水平低于癌旁组织和对照细胞系;NSCLC组织和细胞系中GPX4 mRNA及蛋白表达水平高于癌旁组织和对照细胞系;Starbase软件分析发现GPX4 mRNA序列中含有miR-338特异作用位点,荧光素酶报告基因实验结果证实miR-338直接靶向调节GPX4表达;过表达miR-338提高肿瘤细胞中脂质氧化及活性氧水平,并抑制肿瘤细胞增殖;而铁死亡抑制剂预处理可以逆转miR-338的抑癌作用。结论:miR-338通过负向调控GPX4表达进而促进肿瘤细胞铁死亡,最终抑制NSCLC细胞增殖。     

GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

Hypermethylation of GPX3 (glutathione peroxidase 3) promoter has been identified in various solid tumors. However, the pattern of GPX3 promoter methylation in acute myeloid leukemia (AML) remains unknown. The current study was intended to investigate the clinical significance of GPX3 promoter methylation in de novo AML patients and further determine its role in regulating GPX3 expression. GPX3 promoter methylation status was detected in 181 de novo AML patients and 44 normal controls by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Real-time quantitative PCR was carried out to assess GPX3 expression. GPX3 promoter was significantly methylated in AML patients compared with normal controls (P=0.022). The patients with GPX3 methylation presented significantly older age than those with GPX3 unmethylation (P=0.011). GPX3 methylated patients had significantly lower frequency of C/EBPA mutation and higher incidence of FLT3-ITD mutation (P=0.037 and 0.030, respectively). The non-M3 patients with GPX3 methylation had significantly lower overall survival than those with GPX3 unmethylation (P=0.036). No significant correlation was observed between GPX3 expression and its promoter methylation (R=0.110, P=0.284). However, GPX3 mRNA level was significantly increased after 5-aza-2’-deoxycytidine treatment in leukemic cell line THP1. Our data suggest that GPX3 methylation predicts adverse clinical outcome in non-M3 AML patients. Moreover, GPX3 expression is regulated by its promoter methylation in leukemic cell line THP1.     

GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

Hypermethylation of GPX3 (glutathione peroxidase 3) promoter has been identified in various solid tumors. However, the pattern of GPX3 promoter methylation in acute myeloid leukemia (AML) remains poorly known. The current study was intended to investigate the clinical significance of GPX3 promoter methylation in de novo AML patients and further determine its role in regulating GPX3 expression. GPX3 promoter methylation status in 181 de novo AML patients and 44 normal controls was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Real-time quantitative PCR was carried out to assess GPX3 expression. GPX3 promoter was significantly methylated in 181 AML patients compared with normal controls (P=0.022). The patients with GPX3 methylation presented significantly older age than those with GPX3 unmethylation (P=0.011). GPX3 methylated patients had significantly lower frequency of C/EBPA mutation and higher incidence of FLT3-ITD mutation (P=0.037 and 0.030). The non-M3 patients with GPX3 methylation had significantly lower overall survival than thoes with GPX3 unmethylation (P=0.036). No significant correlation was observed between GPX3 expression and its promoter methylation (R=0.110, P=0.284). However, GPX3 mRNA level was significantly increased after 5-aza-2’-deoxycytidine treatment in leukemic cell line THP1. GPX3 methylation predicts adverse clinical outcome in non-M3 AML patients. Moreover, GPX3 expression is regulated by its promoter methylation in leukemic cell line THP1.     

Comparison of the mRNA expression of factors related to drug resistance in lung tumors and adjacent normal tissue

Drug resistance related proteins P-glycoprotein (P170), multidrug resistance associated protein (MRP), glutathione S-transferase-pi (GST-pi), glutathione peroxidase (GPX), topoisomerase II (Topo II), thymidylate synthase (TS), O-6-methylguanine-DNA-methyltransferase (MGMT), the heat shock proteins HSP27 and HSP70 and the protooncogenes Fos, Jun and EGFR were investigated in human lung carcinomas and matched normal tissues. We found that the mRNA expression of Topo II and TS were elevated in tumor tissue versus corresponding normal tissue. Additionally Topo II and TS correlated with the proliferating activity determined by expression of histone 3. P170, MRP, HSP70 and also EGFR mRNA were elevated in some tumor probes, but not GST-pi, GPX, MGMT and HSP27 mRNA. Additionally, we determined various values of Fos and Jun mRNA expression but there was no uniform pattern. The finding that some proteins were abnormally expressed in lung tumors compared to the adjacent normal tissue is an important finding for further investigations on the development of individualized chemotherapy but more samples should be examined to extend these observations.     

直肠癌与GPX2表达关系的研究进展

直肠癌是严重危害人类健康的一类疾病,大多数直肠癌患者诊断时为局部进展期直肠癌,新辅助放化疗是这类患者的标准治疗,新辅助放化疗的疗效直接影响患者预后。GPX2（胃肠道谷胱甘肽过氧化物酶）是一类参与氧化还原反应的蛋白质,GPX2的表达水平与患者放疗敏感性直接相关,除此以外,GPX2也在结直肠炎症性疾病中发挥重要作用,本文对直肠癌中GPX2的表达与治疗的研究进展进行系统的综述。     

Implications of Glutathione Peroxidase 3 Expression in a Cohort of Egyptian Patients with Acute Myeloid Leukemia

Background: The impact of low expression of Glutathione peroxidase 3 (GPX3) on the clinical course of acute myeloid leukemia (AML) is poorly investigated. Aims: To explore the status of GPX3 expression and analyze its clinical characteristics and prognosis in a cohort of Egyptian patients with AML. Methods: GPX3 mRNA level was assessed by RT-q PCR in 40 newly diagnosed AML patients and 10 healthy controls. Results: The gene expression level was significantly lower in AML patients than the control group (P < 0.001). A cut off value (0.1223) for the discrimination between AML and controls was obtained by ROC curve. According to this cutoff value; the patients were reassigned into 2 groups; 28 patients with lower GPX3 expression and 12 patients with high GPX3 expression. GPX3low expression was significantly associated with higher incidence of induction death (P= 0.037) and lower CR rate (P=0.048). Moreover, GPX3low expression was significantly associated with shorter cumulative 1-year overall survival (OS) (P = 0.001) and disease-free survival (DFS) (P=0.028). Conclusion: GPX3low expression status is considered a poor prognostic factor in AML predicting shorter OS and DFS. The study highlights the importance of targeting glutathione metabolism as a central component of the anti-leukemia therapy.     

Loss of heterozygosity of the human cytosolic glutathione peroxidase I gene in lung cancer

The consistent deletion of 3p21 in lung cancer has led to intensiveefforts to identify a lung tumor suppressor gene at this locus.We recently mapped the gene for the seleniumdependent drug-detoxifyingenzyme glutathione peroxidase 1 (GPX1) to this location by insitu hybridization. We developed a polymerase chain reaction-basedassay which demonstrated the existence of three GPX1 allelescharacterized by the number of alanines in a polyalanine codingsequence in exon 1. These three alleles produced a heterozygotefrequency of 70% in two separate populations: normal tissueDNA taken from Centre d'Etude du Polmorphisme Humain (CEPH)parents and normal tissue taken from cancer patients. In contrast,10 heterozygote tumors were detected out of 64 lung cancer specimens.Linkage analysis of GPX1 to Genethon 3p markers in CEPH pedigreesdemonstrated that GPX1 was located between the two microsatellitemarkers believed to flank the lung cancer deletion site. Nucleotidesequence analysis of GPX1 alleles did not reveal any mutationsof this gene in lung tumors. However, sequence analysis didreveal that the three GPX1 alleles were characterized by threenucleotide substitutions in addition to the polyalanine polymorphism,including a substitution at codon 198 which results in eithera proline or leucine at that position. Therefore, the differentGPX1 alleles encode structurally different hGPx1 subunits. Inaddition, analysis of allele frequency suggests that the GPX1*ALA7allele may occur less frequently in tumors with 3p21 deletions.