Octarepeat peptides of prion are essential for multidrug resistance in gastric cancer cells

OBJECTIVE: In previous studies cellular prion protein (PrPc) is confirmed to be involved in multidrug resistance (MDR) of gastric cancer. Although octarepeat peptides are important functional domains of PrPc and are closely related to the transport of Cu²⁺/Zn²⁺ and antioxidative function, the significance in MDR remains unknown. We aimed to investigate the role of octarepeat peptides in gastric cancer MDR. METHODS: Small interfering RNA (siRNA) against PrPc were transfected into adriamycin‐resistant gastric cancer cell lines to inhibit the expression of wild type PrPc, and then constructs encoding PrPc without octarepeat peptides and PrPc without the fifth repeat peptide were transfected, respectively, to establish the cell models. In vitro drug sensitivity, cell apoptosis, measurement of superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px) and glutathione (GSH), as well as changes in glutathione S‐transferase (GST) were detected. RESULTS: In vitro drug sensitivity test showed that octarepeat peptides could modulate the drug resistance of gastric cancer cells, but the deletion of the fifth repeat peptide had no effect. Specifically, the anti‐apoptotic capacity of gastric cancer cells decreased significantly when the octarepeat peptides of PrPc was absent. Moreover, the activities of total SOD, Cu²⁺/Zn²⁺‐SOD, GSH‐Px, GSH, and GST detected in different stressing periods revealed that cells lacking octarepeat peptides of PrPc exhibited weakened responses to stress. However, absence of the fifth repeat peptide did not exert any effect on stress response. CONCLUSION: The octarepeat peptides of prion is responsible for MDR in gastric cancer cells while the fifth repeat peptide is not.     

Effects of all-trans-retinoic on human gastric cancer cells BGC-823

OBJECTIVE: To determine the inhibitory effects of all‐trans‐retinoic acid (ATRA) on cell growth, cell cycle and vascular endothelial growth factor (VEGF) expression in the human gastric cancer cell line BGC‐823 in vitro. METHODS: Human gastric cancer BGC‐823 cells were treated with various concentrations of ATRA and the cell growth was then determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide viability assay. The cell cycle distribution was analyzed using a flow cytometer. The VEGF mRNA and protein expression were analyzed by semi‐quantitative RT‐PCR and Western blotting, respectively. RESULTS: ATRA at concentrations of 0.1–10 µmol/L inhibited the growth of BGC‐823 cells grown in culture; a time‐ and dose‐dependent inhibitory influence was found. ATRA arrested BGC‐823 cells at the G₀/G₁ phase in a dose‐dependent way. Both VEGF mRNA and protein were decreased by ATRA in a dose‐dependent way. CONCLUSION: The anti‐tumor effects of ATRA on human gastric cancer cells are associated with G₀/G₁ phase arrest and decreased VEGF expression.     

胃癌多药耐药相关基因的研究现状

目前化疗仍是中、晚期胃癌患者的主要治疗方法,影响疗效的关键问题之一是肿瘤的多药耐药性。对瘤组织而言,多药耐药是化疗失败的主要原因。此文就近年来有关胃癌多药耐药相关基因产物的临床研究作一综述。     

EGCG诱导人胃癌BGC-823细胞凋亡及钙稳态失衡对其的影响

目的探讨表没食子儿茶素没食子酸酯(EGCG)对人胃癌BGC-823细胞的促凋亡作用及钙稳态失衡对该作用的影响。方法对体外培养的BGC-823细胞采用EGCG干预。采用MTT比色法检测细胞活性;光镜、倒置显微镜观察细胞形态变化;DNA琼脂糖凝胶电泳检测细胞凋亡情况;流式细胞光度仪分析细胞内游离钙水平。结果 EGCG作用后BGC-823细胞核染色质固缩,核碎裂,凋亡细胞率明显升高,呈时间和剂量依赖性。EGCG作用24 h后BGC-823细胞DNA凝胶电泳出现典型梯形条带;细胞内游离钙水平呈浓度依赖性上升。用细胞内游离钙特异性阻断剂BAPT A-AM预处理细胞后,细胞内游离钙水平不再升高,且EGCG诱导的凋亡明显受抑。结论EGCG能诱导胃癌BGC-823细胞凋亡;钙稳态失衡可抑制EGCG诱导的胃癌BGC-823细胞凋亡。     

PPIs抑制空泡型质子泵逆转胃癌化疗多药耐药

肿瘤多药耐药性影响着胃癌化疗的效果,肿瘤微环境变化与多药耐药密切相关,而酸化与缺氧是微环境的两大特征.肿瘤细胞内无氧酵解生成大量H+,但却仍能维持胞内中性pH环境,推测与调节肿瘤泌酸功能的空泡型质子泵密切相关.胞外pH值的酸化可活化胞内PI3K/Akt信号通路,活化mTOR,上调HIF-1α,进而促进P-gp及MRP1...     

Extended multidrug resistance in haemopoietic cells

The development of drug resistance was studied in a series of haemopoietic cells to determine its relationship to cell lineage. Treatment of the U937 monocytic cell line with epirubicin (15 ng/ml) or vinblastine (8 ng/ml) induced drug-resistant sublines with cross-resistance to epirubicin (8- and 16-fold respectively), vinblastine (5- and 20-fold), paclitaxel (15-and 42-fold) and etoposide (19- and 13-fold). However, sublines were also 3–5-fold resistant to the alkylating agent chlorambucil, cis-platinum and methotrexate, demonstrating an extended multidrug resistance (MDR) phenotype. These cells over-expressed P-glycoprotein, but decreased drug accumulation was not restored in the presence of verapamil, suggesting that the P-glycoprotein was not functional. Similar drug treatment of the HL60 promyelocytic cell line also produced sublines exhibiting an extended MDR phenotype. The KG1a and the HEL cell lines expressed functional P-glycoprotein and were resistant to the drug concentrations used for treatment. Multidrug resistance as mediated by P-glycoprotein cannot explain the resistance of CML patients to chemotherapy, especially in blast crisis. The induction of an extended MDR phenotype specifically in myeloid cells in response to drug treatment may explain the resistance observed in the treatment of CML.     

Antineoplastic activity of 2-methoxyestradiol in human pancreatic and gastric cancer cells with different multidrug-resistant phenotypes

BACKGROUND AND AIM: Chemoresistance often leads to loss of the last treatment option for cancer. 2-Methoxyestradiol (2-ME2) has been shown to inhibit tumor growth. The aim was to examine the efficacy of 2-ME2 on multidrug-resistant human cells from pancreatic and gastric cancer. METHODS: We investigated the impact of 2-ME2 on multidrug-resistant cells derived from human pancreatic and gastric cancer cells that were positive or negative for the MDR1-gene. RESULTS: In pancreatic cancer cells, growth inhibition was 57% in parental, 72% in MDR1-negative and 87% in MDR1-positive cells after 1 micromol/L 2-ME2. In gastric cancer cells we found a growth inhibition of 75% in parental, 82% in MDR1-positive and 95% in MDR1-negative cells. Strong induction of apoptosis was induced after a low dose of 2-ME2. No significant difference in the amount of apoptotic cells was observed between parental and multidrug-resistant cells of both tumor types. The number of apoptotic cells after 2-ME2 ranged from 7.5% in parental gastric cancer cells to 20.1% in MDR1-negative gastric cancer cells. CONCLUSION: 2-ME2 may therefore have clinical application for chemoresistant cancer.     

pEGFP-C3-hNIS重组质粒的构建及其在人胃癌BGC-823细胞的表达

目的构建人钠碘转运体（hNIS）基因真核绿色荧光蛋白融合表达载体pEGFP-C3-hNIS,并研究重组hNIS基因在人胃癌BGC-823细胞中的表达情况。方法采用Trizol一步法从甲状腺手术患者的甲状腺组织中提取总RNA,利用RT-PCR方法扩增得到hNIS的可编码区基因,并克隆到T载体上,测序后亚克隆到真核绿色荧光蛋白融合表达载体pEGFP-C3的多克隆位点,获得重组真核表达载体pEGFP-C3-hNIS。分别将pEGFP-C3-hNIS重组质粒（实验组）和pEGFP-C3（对照组）瞬时转染至人胃癌BGC-823细胞中,转染48 h后,于荧光显微镜下观察绿色荧光蛋白在细胞中的表达情况,并通过Western blot方法检测hNIS基因的蛋白表达水平。结果序列分析证实克隆片段与GenBank公布的hNIS基因序列（序列号：NM-000453）相似性达99.90%。转染48 h后,实验组与对照组细胞均可观察到较强绿色荧光信号,用hNIS基因特异性抗体检测表明实验组hNIS表达水平明显高于对照组。结论成功构建hNIS真核绿色荧光蛋白融合表达载体pEGFP-C3-hNIS,并能在人胃癌BGC-823细胞中高表达。     

TROP2 shRNA真核表达载体转染胃癌BGC-823细胞的沉默作用

目的:构建TROP2特异性短发夹环RNA(shRNA)真核表达载体, 抑制人胃癌BGC-823细胞TROP2基因的表达.方法:构建TROP2短发夹环RNA, 产生重组质粒转染胃癌BGC-823细胞, 转染24 h后用G418(浓度400 mg/L)筛选, 待细胞稳定后收集, 分别命名为W组(未处理组), HK组(随机阴性对照质粒组), KB组(空质粒组), T1组, T2组, T3组. 并运用实时荧光定量PCR和Western blot检测TROP2的表达.结果:TROP2特异性shRNA片段被成功克隆进pGensil1.1质粒中, 重组质粒shRNA编码序列与设计片断的序列完全一致. 与未转染细胞组、随机阴性对照组、空质粒组相比, 转染shRNA重组质粒的人胃癌BGC-823细胞TROP2表达在mRNA和蛋白水平都受到抑制.与T1、T2组相比, T3组对TROP2 mRNA和蛋白抑制作用最明显, 差异具统计学意义(8.79±0.23 vs 9.54±0.20, 9.57±0.23; 3.66±0.11vs 6.46±0.36, 9.31±0.11, 均P<0.05).结论:成功构建了针对TROP2 的特异性shRNA真核表达载体并抑制了TROP2的表达,为进一步研究其基因功能打下了基础.     

异欧前胡素对人胃癌BGC-823细胞的凋亡诱导作用的研究

目的探讨异欧前胡素对人胃癌BGC-823细胞的凋亡诱导作用。方法以体外培养人胃癌BGC-823细胞和裸鼠荷人胃癌模型为观察对象,采用MTT法和Annexin V-FITC/PI双染法观察异欧前胡素对人胃癌BGC-823细胞的增殖存活率及诱导凋亡能力的影响,并且通过裸鼠成瘤实验观察异欧前胡素对BGC-823细胞的增殖生长情况。结果人胃癌BGC-823细胞系的增殖存活率随着异欧前胡素浓度的提高或者处理时间的增加呈下降趋势,增殖存活率与异欧前胡素呈剂量-时间依赖性;异欧前胡素可以明显诱导BGC-823细胞发生凋亡,且在一定范围内随着异欧前胡素浓度的提高,凋亡率也随之升高;有效剂量的异欧前胡素瘤体内注射能够缩小裸鼠瘤体体积,增加瘤体抑制率,与对照组相比较,差异具有统计学意义(P0.05)。结论异欧前胡素能够有效抑制人胃癌BGC-823细胞的增殖并诱导其凋亡,抑制胃癌细胞移植瘤的生长,但对于其具体的调控机制有待进一步研究。     

Cyclooxygenase-2, multidrug resistance 1, and breast cancer resistance protein gene polymorphisms and inflammatory bowel disease in the Danish population

Objective. Crohn's disease (CD) and ulcerative colitis (UC) are characterized by an impaired mucosal defence to normal constituents of the intestinal flora and a dysregulated inflammatory response. The purpose of the study was to investigate whether single nucleotide polymorphisms (SNPs) in genes involved in these processes were associated with CD and UC. Material and methods. Allele frequencies of the cyclooxygenase 2 (COX-2/PTGS2/PGHS2) G-765C and breast cancer resistance protein (BCRP/ABCG2) C421A as well as allele and haplotype frequencies of multidrug resistance 1 (MDR1, ABCB1) SNPs G2677T/A, C3435T and G-rs3789243-A (intron 3) were assessed in a Danish case-control study comprising 373 CD and 541 UC patients and 796 healthy controls. Results. Carriers of the homozygous COX-2 and MDR1 intron 3 variant had a relatively high risk of CD, odds ratio (95% CI) (OR (95% CI))=2.86 ((1.34–5.88) p=0.006) and 1.39 ((0.99–1.92) p=0.054), respectively, and for UC of 2.63 ((1.33–5.26) p=0.005) and 1.28 ((0.96–1.51) p=0.093), respectively, assuming complete dominance. No association was found for BCRP or other MDR1 SNPs, or for selected MDR1 haplotypes. No effect-modification of smoking habit at the time of diagnosis was found. Conclusions. An effect of the COX-2 polymorphism on both CD and UC was shown which is compatible with the presence of a recessive allele in linkage equilibrium with the SNP marker in the COX-2 gene. The polymorphism located in intron 3 of the MDR1 gene showed a weak association with CD, and a marginally suggestive association with UC.     

胃癌相关基因GCRG213真核表达载体的构建及其对胃癌细胞MKN45的影响

目的 研究胃癌相关基因GCRG213正义、反义转染对胃癌细胞MKN45的影响.方法 采用分子克隆及基因转染技术将GCRG213基因转入哺乳动物细胞,并结合反义转染技术研究GCRG213基因对胃癌细胞恶性生物学行为的影响.结果结果 GCRG213正向和反向克隆正确插人真核表达载体pcDNA3.1(+);重组子pcDNA3.1-a、pcDNA3.1-b和空载体转染人胃癌细胞系MKN45细胞;正义转染其mRNA的表达上调,而反义转染下调;正义转染加快MKN45细胞生长增殖速度、降低细胞凋亡率,反义转染减慢MKN45细胞生长增殖速度,增加细胞凋亡率.结论 胃癌相关基因GCRG213可促进肿瘤细胞生长、分裂和转移,抑制肿瘤细胞的凋亡,可能是一个新发现的恶性肿瘤癌变的促进因素.     

Bax基因对人胃癌耐药细胞多药耐药性的逆转作用

目的 研究凋亡相关基因Ｂax对胃癌多药耐药细胞的多药耐药性的逆转作用。方法 构建含有人Ｂax cＤＮＡ全长的真核表达载体pＢＫ－Ｂax,经脂质体导入缺乏Ｂax蛋白表达的人 胃癌多药耐药细胞系ＳＧＣ７９０１/ＶＣＲ中。Ｗestern印迹观察Ｂax基因在转导细胞中的表达,ＭＴＴ法检测Ｂax转导细胞与空载体转导细胞对化疗药物的敏感性。结果 成功构建了Ｂax的真核表达载体,Ｂav转导细胞能稳定表达Ｂax蛋白,与空载体转导细胞相比。Ｂ     

幽门螺杆菌对胃癌细胞BGC-823形态及凋亡相关基因表达的影响

目的:探讨H.pylori 刺激人胃癌细胞BGC-823 后,对细胞形态以及凋亡抑制基因Survivin和凋亡基因caspase-3 mRNA表达的影响.方法:分别用东亚型H.pylor i 菌株和西方型H.pylor i 菌株超声提取物,刺激胃癌细胞BGC-823,显微镜下直接观察细胞形态的变化.同时利用荧光定量聚合...     

Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells

Summary. In an attempt to mimic clinical conditions for the treatment of leukaemia, the HL60 promyelocytic cell line was treated for 18 h with low, clinically relevant, levels of the anthracycline epirubicin and the Vinca alkaloid vinblastine. The resulting drug-resistant sublines not only expressed P-glycoprotein and the MDR phenotype but were also cross-resistant to chlorambucil, methotrexate but were also cross-resistant to chlorambucil, methotrexate and cisplatinum, and had increased resistance to radiation. Development of resistance was associatted with an aberrant differentiation phenotype with decreased expression of myeloid antigens and expression of glycophorin A. an antigen normally associated with erythroid differentiation. The ability of HL60 cells to terminally differentiate in response to all- trans -retinoic acid (vitamin A acid) was lost in the sublines. These results suggest that either a single novel mechanism is responsible for multiple drug resistance or the initial response to drug treatment is the co-induction of multiple mechanisms. These cells and the method by which they were generated therefore provide a clinically relevant model for the study of the initial events in the development of not only multidrug resistance but also the extended multiple drug resistance usually encountered in the treatment of leukaemia.     

胃癌多药耐药蛋白的相互作用蛋白鉴定及分子机制

目的:研究多药耐药蛋白(multidrug resistance protein,MRP)的相互作用蛋白及其对肿瘤耐药性的影响.方法:采用免疫沉淀结合质谱方法分离鉴定MRP的相互作用蛋白,并对其中的耐药相关蛋白之一Annexin A5进行功能研究,Western blot及siRNA干扰技术分析MRP、Annexin A5蛋白表达的变化、相关性及胃癌SGC-7901细胞的耐药性变化.结果:经免疫沉淀结合质谱方法分离鉴定发现了14个MRP的相互作用蛋白,其中AnnexinA5为其中得分最高的蛋白;Westernblot检测显示耐药细胞株SGC-7901/DDP中MRP、AnnexinA5蛋白表达均高于SGC-7901和siRNA-SGC-7901/DDP,siRNA干扰AnnexinA5表达后,siRNA-SGC-7901/DDP中MRP蛋白表达下调,MRP、AnnexinA5蛋白在SGC-7901和siRNA-SGC-7901/DDP之间表达无明显差异;MTT法结果显示经siRNA干扰SGC-7901/DDP后,顺铂、5-Fu和紫杉醇作用后的IC50值明显减少,对其的敏感性分别增加了36倍、17倍和4倍.结论...     

Retrovirus-mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells

Summary. We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34⁺ cells as a target for human multidrug resistance (MDR1) gene transfer, Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, effciency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 μg/ml taxol. In uninfected control, 1–2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14–31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometiric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotien, was found on 17–25% of viable CD34⁺ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.     

肺癌患者外周血中MDR1的表达及临床意义

目的 检测多药耐药基因-1（MDR1）在肺癌患者外周血中的表达,探讨MDR1的表达水平与肺癌病理类型及临床化疗进程之间的关系.方法 应用荧光定量PCR（qRT-PCR）技术,动态监测45例肺癌患者化疗前后外周血中MDR1的表达水平,并与48例健康者进行对照比较.结果 肺癌组化疗前MDR1的阳性率28.89%（13/45）,明显高于健康对照组2.08%（1/48）,差异显著（P〈0.05）;各种病理类型的肺癌随着化疗次数的增多MDR1的表达均增强;化疗前后各期MDR1的表达程度：非小细胞肺癌（NSCLC,肺鳞癌和肺腺癌）明显高于小细胞肺癌（SCLC）,差异具有统计学意义（P〈0.05）,而肺鳞癌和肺腺癌之间MDR1的表达程度相当,无显著差异,（P＞0.05）.结论 化疗可诱导各种病理类型肺癌MDR1表达增加;病理类型不同诱导化疗耐药的机制可能不同：NSCLC可能为原发性MDR,SCLC可能为获得性MDR;动态检测肺癌患者外周血中MDR1的表达水平,有利于进行肺癌耐药的监测与评价.