Roles of GST-π and polβ Genes in Chemoresistance of Esophageal Carcinoma Cells

The main aim of this study was to investigate the roles of GST-π and polβ genes in the chemoresistance ofesophageal carcinoma cells. Eukaryotic expression vectors containing each gene were constructed and transfectedinto EC9706 cells, and the biological effects of the two genes assessed based on a resistance index. We additionallyinvestigated the in vitro and in vivo anti-resistance effects of GST-π and polβ genes using recombinant lentivirusescarrying siRNAs against the two genes. Our results showed that upregulation of GST-π and polβ genes suppresseschemosensitivity of esophageal carcinoma cells to cisplatin, while downregulation of these two genes with RNAitechnology reverses this chemoresistance. Multi-site injection of recombinant lentivirus targeting the GST-πgene into transplanted cDDP tumors effectively reversed their chemoresistant phenotype. However, the sametreatment against the polβ gene did not lead to significant efficacy against chemoresistance.     

重组 GST-β-GT 融合蛋白的原核表达、纯化及活性鉴定

目的：构建噬菌体β‐GT蛋白的原核重组体系,诱导表达、纯化GST‐β‐GT 融合蛋白并对其进行酶活性测定。方法利用PCR技术从T4噬菌体中扩增β‐GT基因;经T‐A克隆,获得β‐GT基因片段,经酶切连接,构建原核表达载体pGEX‐6P‐1‐β‐GT ;经测序鉴定序列正确后,将所构建的载体转化进入大肠埃希菌BL21（DE3）中进行表达。利用IPTG诱导,经10％ SDS‐PAGE鉴定目的蛋白表达后,采用GST柱纯化目的蛋白;通过酶切和qPCR鉴定其活性。结果成功扩增了噬菌体β‐GT基因;重组质粒在大肠埃希菌BL21（DE3）中诱导表达出GST‐β‐GT融合蛋白;通过GST柱纯化后获得了GST‐β‐GT融合蛋白,纯度达95％;通过酶切和qPCR验证,此GST‐β‐GT融合蛋白具有T4‐β‐GT酶的活性。结论。原核表达GST‐β‐GT融合蛋白具有糖基转移酶活性。     

Lifespan extension by n-butanol extract from seed of Platycladus orientalis in Caenorhabditis elegans

Aim of the study

As a traditional Chinese medicine, seed of Platycladus orientalis(Linnaeus) Franco has been extensively used as a tonic and sedative remedy. The present study was conducted to investigate whether lifespan was extended and the mechanisms of n-butanol extract from seed of Platycladus orientalis (BSPO) in Caenorhabditis elegans. The findings could provide the pharmacological basis for a treatment in traditional medicine.

Materials and methods

Lifespan extension by BSPO was evaluated under normal culture conditions and in a stress test. A possible mechanism of the anti-aging effect of BSPO, a change in the stress-resistance of related proteins, was also investigated in C. elegans.

Results

It has been shown that BSPO could significantly extend lifespan of C. elegans in a concentration dependent manner under normal culture conditions and stress. Further studies demonstrated that BSPO treatment significantly decreased reactive oxygen species (ROS) accumulation, up-regulated resistance to stress of related proteins, including glutathione S-transferase-4 (GST-4) and heat shock protein-16.2 (HSP-16.2), and reduced the amount of lipofuscin in transgenic C. elegans.

Conclusion

These results indicated that BSPO extended the lifespan, which could be attributed to its direct ROS scavenging activity, reducing the amount of lipofuscin and increasing the expression of gens associated with resistance to stress. These obtained data provided valuable support for traditional clinical practice to extend lifespan and to provide tonic remedy.     

PgP、TP、GST-π、MRP在食管癌组织中的表达及与化疗的关系

目的:检测食管癌组织中P-糖蛋白(P-glycopro-tein,PgP)、胸苷酸磷酸化酶(thymidine phosp-horylase,TP)、谷胱甘肽-S-转移酶(glutathione S-transferase,GST-π)、多药耐药相关蛋白(multidrug resistance-associated protein,MRP)表达情况,探讨其与食管癌化疗敏感性的关系.方法:选取我院食管癌患者58例,用免疫组织化学方法检测食管癌组织中多药耐药相关蛋白PgP、TP、GST-π、MRP蛋白的表达情况;行双铂方案新辅助化疗,分析化疗疗效与PgP、TP、GST-π、MRP蛋白表达差异的关系.结果:PgP阳性颗粒分布于胞膜和胞浆;TP阳性颗粒分布于细胞胞浆和胞核;GST-π阳性颗粒分布于胞浆;MRP阳性颗粒分布于胞膜和胞浆,他们在食管癌组织中都存在一定的表达率,表达率分别为41.37%、32.76%、20.69%和37.93%.化疗有效组患者与化疗无效组患者相比较,食管癌组织中PgP、GSH和MRP的阳性表达率低,TP阳性表达率高,两组之间有显著性差异.结论:食管癌组织中PgP、TP、GST-π、MRP存在一定的表达率;PgP、GST-π、MRP表达率低而TP表达率高,化疗治疗的效果好.     

宫颈癌中MRP、GST-π的表达及意义

目的:检测多药耐药相关蛋白(multi-drug resistance-associated protein,MRP)、胎盘型谷胱甘肽S-转移酶(glutathione S-transferase-π,GST-π)在宫颈癌(cervical cancer,CC)中的表达,探讨其在宫颈癌多药耐药(multi-drug resistance,MDR)形成中的作用和相互关系。方法:运用荧光定量PCR(flurescence quantitative-PCR,FQ-PCR)检测47例宫颈癌组织及12例正常宫颈组织中耐药基因MRP、GST-πmRNA的表达情况,并分析其与临床病理参数之间的关系。结果:MRP、GST-π在宫颈癌中的表达分别为0.51±0.28、0.44±0.24,正常宫颈组织中的表达分别为0.20±0.11、0.18±0.10,耐药基因在宫颈癌中的表达高于正常宫颈组织,差异具有统计学意义(P<0.05)。MRP、GST-π表达与病人的淋巴转移、肿瘤分化程度、临床分期之间差异无统计学意义(P>0.05)。MRP与GST-π之间的表达具有统计学意义。结论:MRP、GST-π基因高表达可能在宫颈癌原发性耐药中起重要作用,检测MRP、GST-π基因的表达情况对预测宫颈癌的化疗效果、协助临床选择化疗方案可能有一定意义。     

Terminal modification of polymeric micelles with π-conjugated moieties for efficient anticancer drug delivery

High drug loading content is the critical factor to polymeric micelles for efficient chemotherapy. Small molecules of cinnamic acid, 7-carboxymethoxy coumarin and chrysin with different π-conjugated moieties were immobilized on the terminal hydroxyl groups of PCL segments in mPEG-PCL micelles to improve drug loading content via the evocation of π-π stacking interaction between doxorubicin (DOX) and polymeric micelles. The modification of π-conjugated moieties enhanced the capability of crystallization of mPEG-PCL block copolymers. The drug loading content increased dramatically from 12.9% to 25.5% after modification. All the three modified mPEG-PCL micelles were nontoxic to cells. Chrysin modified polymeric micelles exhibited the most efficient anticancer activity. The in vivo anticancer activity of 10 mg/kg DOX dose of chrysin modified micelle formulation for twice injections was comparable to that of 5 mg/kg dose of free DOX·HCl for four injections under the circumstance of same total DOX amount. The systemic toxicity of DOX loaded chrysin modified micelles was significantly reduced. This research provided a facile strategy to achieve polymeric micelles with high drug loading content and efficient anticancer activity both in vitro and in vivo.     

GST-π、TopoⅡ在涎腺肿瘤中的表达及意义

目的：探讨谷胱苷肽S转移酶-π（GST-π）和DNA拓扑异构酶Ⅱ（TopoⅡ）在涎腺肿瘤组织中的表达及临床意义。方法：采用免疫组化方法检测10例正常涎腺组织、16例涎腺良性肿瘤、27例涎腺恶性肿瘤组织中GST-π、TopoⅡ的表达，分析GST-π、TopoⅡ的表达与涎腺肿瘤临床病理特征。结果：涎腺恶性肿瘤GST-π的阳性率为77．78％，明显高于涎腺良性肿瘤GST-π的阳性率12．50％（P〈0．001）。涎腺肿瘤中GST-π表达与TopoⅡ表达之间无明显相关性（Ｐ〉0．05）。27例涎腺恶性肿瘤中GST-π、TopoⅡ的表达与肿瘤病理类型有关（P〈0．05），而与患者的年龄、性别、有无淋巴结转移及临床分期无关。结论：GST-π、TopoⅡ可能与涎腺肿瘤的发生发展相关。检测GST-π、TopoⅡ的表达情况不仅可为涎腺恶性肿瘤化疗用药提供参考，而且可作为涎腺恶性肿瘤判断预后的指标。     

Measurement of glutathione S-transferase and its class-pi in plasma and tissue biopsies obtained after laparoscopy and endoscopy from subjects with esophagus and gastric cancer

OBJECTIVES: To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients. METHODS: GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies. RESULTS: Plasma GST was significantly higher (p < 0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, p 相似文献   

人胎盘型谷胱甘肽S转移酶在胃癌组织中的表达与基因甲基化关系的研究

目的：研究胃癌组织中ＧＳＴ－π的表达与其基因甲基化状态的关系。方法：应用Ｓ－Ｐ法检测１１６例胃癌和５３例胃癌前病变的ＧＳＴ－π表达和限制性内切酶及ＰＣＲ、Ｓｏｕｔｈｅｒｎ印迹方法检测１４例胃癌及其相应正常胃粘膜ＧＳＴ－πＤＮＡ５′端调控区ＣＣＧＧ特定位点的甲基化水平。结果：ＧＳＴ－π阳性率,正常胃粘膜１０％（４／３９）,胃癌７７％（８９／１１６）,肠上皮化生７６％（１９／２５）,不典型增生８９％（２５／２８）。ＧＳＴ－π在胃癌及癌前病变中的表达较正常胃粘膜增高（Ｐ＜００１）。胃癌组织中ＧＳＴ－π基因较正常胃粘膜呈现高度去甲基化（Ｐ＜００１）,胃癌ＧＳＴ－π基因５′端调控区低甲基化与其ＧＳＴ－π表达增加呈相关性（Ｐ＜００５）。结论：ＧＳＴ－π在胃癌及癌前病变中的表达与胃癌的发生发展密切相关,ＧＳＴ－π可作为胃癌的肿瘤相关抗原;ＧＳＴ－π基因５′端调控区低甲基化可能是ＧＳＴ－π高表达的分子机理。     

不同食管上皮组织中GST—Pi的表达及意义

目的 探讨食管不同上皮组织中GST—Pi表达以了解其在癌发生发展过程中的作用。方法 应用免疫组化SP法观察40例“正常”食管鳞状上皮、10倒轻度不典型增生、20例重度不典型增生和40倒食管鳞状上皮癌组织中GST—Pi蛋白阳性表达率。结果由正常食管鳞状上皮-轻度不典型增生-重度不典型增生食管癌组织中GST—Pi的表达呈逐渐降低的趋势看出，癌组织的表达明显低于正常上皮和轻度增生组，差异均有显著意义（P〈0．01）。结论 GST—Pi可能与食管癌的发生、发展密切相关。