5-FU耐药免疫组化技术与MTT法敏感性比较

目的：探讨结肠癌对5-氟脲嘧啶(5-FU)产生耐药的机理，从细胞培养水平进一步论证胸苷酸合成酶(TS)对5-FU化疗产生耐药的影响，揭示不同TS蛋白表达水平的结肠癌患者对5-FU治疗的敏感性。方法：应用MTT法对体外培养的30例结肠癌细胞进行5-FU药敏试验，再应用SP免疫组化法对30例结肠癌进行TS检测，然后将两个试验结果进行比较；另采用SP法检测60例结肠癌组织切片及其正常对照组切片中TS蛋白的表达情况。结果：TS高表达组体外培养的结肠癌细胞对5-FU的敏感性明显低于TS低表达组。正常结肠粘膜组织中的TS表达水平高于结肠癌组织，结肠癌组织中TS表达水平与5-FU化疗患者的预后呈负相关，与患者的性别、有无淋巴结转移及癌组织的分化程度无关。结论：TS蛋白表达程度的高低对临床结肠癌患者化疗药物的选择有一定的指导意义；结肠癌组织中TS蛋白的表达水平可作为患者对5-FU为主的化疗疗效及预后的判断指标。     

胸苷酸合成酶对结肠癌5-Fu化疗耐药影响的前瞻性研究

 目的　探讨结肠癌对 5 Fu产生耐药的机理 ,从细胞培养水平上进一步论证胸苷酸合成酶对 5 Fu化疗产生耐药的影响。方法　应用MTT法对体外培养的 30例结肠癌细胞进行 5 Fu药敏试验 ,再应用SP免疫组化法对 30例结肠癌进行胸苷酸合成酶检测 ,然后将两个结果进行比较。结果　胸苷酸合成酶高表达组体外培养的结肠癌细胞对 5 Fu的敏感性明显低于TS低表达组。结论　胸苷酸合成酶表达程度的高低对临床结肠癌患者化疗药物的选择有一定的指导意义     

宫颈鳞状细胞癌对DDP和DDP+5-FU的体外药敏检测及与耐药蛋白表达的关系研究

目的通过对宫颈鳞状细胞癌进行顺铂(DDP)和DDP+5-氟尿嘧啶(5-FU)体外药敏检测,比较两种化疗方案的体外有效率。同时检测其耐药蛋白P糖蛋白(P-gp)、谷胱甘肽S-转移酶-π(GST-π)、DNA拓扑异构酶Ⅱ(TopoⅡ)、胸苷酸合成酶(TS)的表达,探讨DDP和DDP+5-FU体外药敏与耐药蛋白表达的关系。方法收集35例宫颈鳞状细胞癌患者的新鲜肿瘤组织,采用三磷酸腺苷生物荧光法(ATP-TCA)对DDP和DDP+5-FU进行体外药敏检测,同时采用EnVision二步法检测宫颈鳞状细胞癌组织中耐药蛋白P-gp、GST-π、TopoⅡ、TS的表达,并分析DDP和DDP+5-FU的体外药敏与耐药蛋白P-gp、GST-π、TopoⅡ、TS表达的关系。结果 35例标本均进行药敏试验,DDP的体外有效率为37.14%,与DDP+5-FU的51.43%比较,差异无统计学意义(P> 0.05)。经多因素分析发现,患者年龄、临床分期、分化程度均不为DDP、DDP+5-FU药物敏感性的影响因素(P> 0.05)。P-gp、GST-π、TopoⅡ、TS蛋白阳性表达率分别为57.14%(20/35)、51.43%(18/35)、71.43%(25/35)、57.14%(20/35)。GST-π蛋白阳性表达是宫颈鳞状细胞癌对DDP耐药的影响因素(P=0.002);TS蛋白阳性表达是宫颈鳞状细胞癌对DDP+5-FU耐药的影响因素(P=0.001)。结论 宫颈鳞状细胞癌ATP-TCA法检测DDP+5-FU与单药DDP相比,体外有效率无显著差异。GST-π、TS蛋白阳性表达可用于临床宫颈癌患者对DDP、5-FU化疗耐药的预测指标。     

lncRNA SNHG20抑制结肠癌细胞化疗敏感性的机制

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)SNHG20抑制结肠癌细胞对5-FU化疗敏感性的调控机制.方法:利用结肠癌细胞株LoVo构建对5-FU抵抗的细胞株LoVo/5-FU,通过qRT-PCR的方法检测LoVo/5-FU中lncRNA SNHG20的表达情况.进一步分别构建lncRNA SNHG20稳定过表达及表达沉默的结肠癌细胞株,利用CCK-8的方法检测lncRNA SNHG20对5-FU IC50的影响,通过平板克隆形成实验检测lncRNA SNHG20对克隆形成能力的影响.最后运用Western blot方法探讨lncRNA SNHG20抑制结肠癌对5-FU化疗敏感性的调控机制.结果:在对5-FU耐药的结肠癌细胞株LoVo/5-FU中,lncRNA SNHG20显著高表达(P<0.001).lncRNA SNHG20高表达能明显提高5-FU的IC50,而沉默lncRNA SNHG20则显著提高LoVo/5-FU对5-FU的敏感性(P<0.001).平板克隆形成实验结果显示lncRNA SNHG20显著提高细胞的克隆形成能力(P<0.001).Western blot结果表明,lncRNA SNHG20能显著抑制结肠癌细胞株PTEN蛋白的表达,而p-AKT及p-PI3K表达水平明显上调.结论:lncRNA SNHG20通过调控PTEN/PI3K/AKT信号通路抑制结肠癌细胞对5-FU的化疗敏感性.     

miR-140-5p调控Nrf2影响结肠癌细胞5-FU耐药性

目的 观察miR-140-5p对结肠癌耐药细胞株SW480/5-FU及其亲本细胞株SW480 5-FU敏感性的影响及其与核因子NF-E2相关因子2（NF-E2-related factor 2,Nrf2）的关系,探讨miR-140-5p调控结肠癌细胞5-FU耐药的可能机制。 方法 用结肠癌细胞株SW480构建结肠癌耐药细胞株SW480/5-FU,qRT-PCR法检测两株细胞中miR-140-5p的表达,CCK-8法和细胞集落形成实验评估miR-140-5p对两株细胞5-FU敏感性的影响;采用qRT-PCR法、Western blot法双荧光素酶报告基因验证miR-140-5p与Nrf2的关系,CCK-8法、细胞集落形成实验探讨miR-140-5p调控结肠癌细胞5-FU敏感性与Nrf2的关系。 结果 成功构建结肠癌耐药细胞株SW480/5-FU,其miR-140-5p表达水平显著低于亲本细胞株SW480（P＜0.05）;CCK-8法、细胞集落形成实验结果显示,miR-140-5p可增强SW480/5-FU细胞对5-FU的敏感性,而降低SW480对5-FU的耐药性;qRT-PCR法、Westren blot法、双荧光素酶报告基因实验共同证实miR-140-5p可结合并抑制Nrf2的表达;CCK-8、细胞集落形成实验表明,Nrf2过表达能逆转miR-140-5p对结肠癌耐药细胞SW480/5-FU的5-FU敏感性调控结果。结论 miR-140-5p可能通过结合并抑制Nrf2表达,增加结肠癌耐药细胞株SW480/5-FU对5-FU的敏感性。     

胃癌组织中ERCC1、TS和TUBB3与化疗药物体外药敏相关性的实验研究

目的:分析体外胃癌细胞中ERCC1与顺铂、TS与5-氟尿嘧啶及TUBB3与紫杉醇化疗药物敏感性的相关性。方法:选择经外科手术的胃癌患者的新鲜标本48例,每份癌组织标本均分成两份:一份用于原代培养细胞,ATP-TCA法检测体外化疗药物的敏感性;另一份用于免疫组化检测ERCC1、TS和TUBB3蛋白的表达。结果:45例有效的胃癌组织中,ERCC1高表达21例(46.67%),低表达24例(53.33%)。21例ERCC1高表达组敏感例数7例(33.33%),24例低表达组敏感例数17例(70.83%),ERCC1高表达者对顺铂的敏感有效率要明显低于低表达者。TS高表达19例(42.22%),低表达26例(57.78%)。19例TS高表达组敏感例数5例(26.32%),26例低表达组敏感例数18例(69.23%),TS高表达者对5-氟尿嘧啶的敏感有效率要明显低于低表达者。TUBB3高表达22例(48.89%),低表达23例(51.11%)。22例TUBB3高表达组敏感例数5例(22.73%),23例低表达组敏感例数16例(69.57%),TUBB3高表达者对紫杉醇的敏感有效率要明显低于低表达者,以上差异均具有统计学意义(P＜0.05)。结论:体外实验证实ERCC1、TS和TUBB3的表达与化疗药物耐药具有一定的相关性,低表达组的耐药发生率低于高表达组。ERCC1、TS和TUBB3有望成为判断胃癌患者化疗疗效及预后的分子标志物。     

TS mRNA表达与食管鳞癌患者5-FU化疗临床预后的相关研究

目的：探讨食管鳞癌石蜡包埋组织中胸腺嘧啶核苷酸合成酶基因（thymidylate synthase,TS）mRNA表达水平与接受氟尿嘧啶（Fluorouracil,5-FU）化疗的患者临床病理的关系及其预后意义。方法：采用实时荧光定量RT-PCR检测22例福尔马林固定-石蜡包埋食管鳞癌组织TS mRNA的表达水平,并比较其表达水平与接受5-FU化疗的患者的临床病理及生存期之间的关系。结果：TS mRNA的表达水平与患者年龄、性别、肿瘤分化程度、TNM分期、是否吸烟饮酒、浸润深度、淋巴结转移、远处转移、血管侵犯以及神经累及均无显著性相关（P〉0.05）。TS mRNA的表达水平与食管鳞癌肿瘤组织COX2（P=0.419）、p53（P=0.804）、PCNA（P=0.555）、VEGF（P=0.184）、MDR（P=0.593）、EGFR（P=0.482）、SSTR2（P=0.377）的蛋白表达情况均无显著相关性。以5-FU为基础化疗的食管鳞癌患者,TS mRNA低表达者的总生存时间较TS mRNA高表达者明显延长（P=0.017）。经COX多因素回归分析显示,食管鳞癌肿瘤组织中TS mRNA表达水平可以成为以5-FU为基础化疗的食管鳞癌患者独立的预测指标（P=0.029,95%CI：1.161-16.278）。结论：TS mRNA表达可能与食管鳞癌患者5-FU化疗后总生存时间（OS）呈负相关。食管鳞癌肿瘤组织中TS mR-NA表达水平可以作为预测以5-FU为基础化疗患者预后的独立指标。TS mRNA表达水平与食管鳞癌的恶性生物学行为无相关性。     

中晚期结直肠癌体外丝裂霉素联合5-氟尿嘧啶抗肿瘤药敏相关因素分析

目的:筛选影响中晚期结直肠癌体外联合化疗[丝裂霉素(mitomycin, MMC)+5-氟尿嘧啶(5-fluorouracil, 5-FU)]抗肿瘤药敏的相关因素.方法:2002年9月-2005年1月共169例Duke's B及C期结直肠癌(colorectal cancer,CRC)患者,分析体外联合化疗药敏相加、协同和拮抗作用以及体外联合用药药敏检测结果与患者常见肿瘤分子标志物表达水平之间的相关性,以及与患者生存状况的关系.结果:c-erbB-2的表达水平与MMC+5-FU体外联合药敏拮抗呈正相关;p53的表达水平与MMC+5-FU联合体外药敏实验结果耐药呈负相关;c-erbB-2和组织学分级与体外联合用药药敏检测结果耐药呈正相关;体外药敏拮抗与患者死亡呈正相关.结论:患者c-erbB-2为高表达,则MMC+5-FU联合化疗相互拮抗的可能性增大.c-erbB-2、p53和组织学分级与体外联合用药耐药相关,其中p53为负相关,c-erbB-2和组织学分级为正相关.MMC+5-FU体外药敏拮抗与中晚期结直肠癌患者预后不良相关.     

Survivin和Bcl-2蛋白在胃癌细胞中的表达及其与化疗药物耐药性关系的研究

目的:检测Survivin和Bcl-2蛋白在胃癌组织中的表达,探讨Survivin和Bcl-2基因与胃癌化疗药物耐药性的关系。方法:取新鲜胃癌手术标本98例,免疫组化检测Survivin和Bcl-2蛋白在胃癌组织中的表达。同时进行原代胃癌细胞培养,MTT比色法检测原代培养细胞对化疗药物HCPT、CDDP、5－FU、ADM和MMC的敏感性。结果:胃癌组织中Survivin和Bcl-2蛋白的阳性率分别为74.5%和70.4%。Survivin阳性表达者对5－FU和ADM的耐药性显著高于阴性者,差异有统计学意义（P<0.05）。5-FU、ADM和MMC三种药物对Bcl-2阳性表达者的抑制率显著低于Bcl-2阴性表达者（P<0.05）,其阳性表达与5-FU、ADM及MMC的体外耐药相关。结论:Survivin和Bcl-2基因与胃癌对化疗药物敏感性相关,Survivin与胃癌细胞原发性化疗耐受有关,检测其表达对选择化疗药物有实用价值。Bcl-2可能参与介导胃癌细胞的耐药,其高表达可能是胃癌多药耐药产生的原因之一。     

氟尿嘧啶代谢酶与胃肠道肿瘤化疗敏感性关系的探讨

目的探讨氟尿嘧啶类药物代谢酶--胸苷酸合成酶(TS)、胸苷磷酸化酶(TP)和二氢嘧啶脱氢酶(DPD)表达水平与人胃肠道肿瘤细胞化疗敏感性的关系.方法MTT法分析7株人胃肠道肿瘤细胞对5-FU和FdUrd的敏感性(半数抑制浓度,IC50),RT-PCR检测3种药物代谢酶mRNA的表达,ELISA测定DPD和TP蛋白含量.结果5-FU与FdUrd对肿瘤细胞的IC50值分别为1.28～12.26μmol/L和5.02～24.21 μmol/L,DPD mRNA水平和蛋白含量与5-FU的化疗敏感性呈负相关,TSmRNA水平与FdUrd敏感性呈负相关;DPD的mRNA和蛋白表达水平之间显著相关,但TP没有相关性.结论DPD和TS表达水平分别可以作为5-FU与FdUrd化疗敏感性的预测指标.     

Thymidine phosphorylase expression correlates with malignant potential and anti-tumor effect of doxifluridine on gastric cancer: multivariate analysis for adjuvant chemotherapy doxifluridine vs. 5-fluorouracil

Doxifluridine (5'-DFUR) is an anticancer drug converted to 5-fluorouracil (5-FU) by thymidine phosphorylase (TP). TP is an angiogenetic and platelet-derived endothelial cell growth factor. We evaluated the relation between TP expression and chemotherapeutic efficacy and prognosis for gastric cancer. Advanced gastric cancer patients given oral adjuvant chemotherapeutics either 5'-DFUR; 163 patients or 5-FU; 162 patients were examined. TP expression was assessed with immunohistochemical staining. Multivariate analysis for influencing survival was done, employing variables such as gender, age, procedure, tumor size, location, Borrmann type, histologic factors [type, depth of invasion, lymph node metastasis (n), lymphatic invasion (ly), and venous invasion (v)], drug administered, and TP expression. In the patients with serosal invasion, 5'-DFUR in TP positive was an independent prognostic factor (risk ratio, 4.450; 95% confidence limit, 2.099-9.436), indicating significantly improved prognosis over the 5-FU group. In TP negative, n and ly were independent prognostic factors, but the survival curves of the two chemotherapeutic groups were not significantly different. TP expression was not prognostic factor in 5'-DFUR group, while, in 5-FU group, TP expression was an independent prognostic factor (2.834, 1.467-5.476). In conclusion, it was suggested that TP positive gastric cancer with serosal invasion increased malignant potential of the tumor and 5'-DFUR efficacy.     

Thymidine phosphorylase expression is useful in selecting adjuvant chemotherapy for stage III gastric cancer

Thymidine phosphorylase (TP) is an enzyme which converts doxifluridine (5'-DFUR) to 5-fluorouracil (5-FU). To assess whether TP expression is useful for selecting adjuvant chemotherapy in advanced gastric cancer, we compared effects of oral 5'-DFUR and 5-FU and assessed correlation between drug efficacy and TP expression level. We examined TP expression in 286 patients. TP expression was assessed with immunohistochemical staining. When we compared prognosis in two chemotherapy groups with high TP expression, better survival was observed in 5'-DFUR than in 5-FU group (p=0.0413). Especially in stage III, patients with high TP had better survival in 5'-DFUR than in 5-FU group.     

Thymidine phosphorylase expression and efficacy of adjuvant doxifluridine in advanced colorectal cancer patients

To clarify the correlation between the expression level of thymidine phosphorylase (TP) and efficacy of doxifluridine (5'-DFUR) and 5-fluorouracil (5-FU), samples from 177 colorectal cancer patients who underwent curative resection were evaluated by immunohistochemical staining using a newly developed monoclonal antibody 1C6-203. Patients were randomly given either oral 5'-DFUR or 5-FU as postoperative adjuvant chemotherapy. In Dukes' C staged colon cancer patients treated with 5'-DFUR, better survival was observed in the high TP patients than the low TP patients (P=0.025 by the log-rank test). The observed 5-year survival rates were 91.2 and 74.8%, respectively. No correlation between TP expression and patient prognosis was detected in the 5-FU group. In Dukes' C stage colon patients with high TP expression, the 5'-DFUR group had slightly better survival than the 5-FU group. These findings suggest that TP may be a chemosensitive marker for 5'-DFUR as postoperative adjuvant chemotherapy for advanced colon cancer patients.     

Role for alpha1,2-fucosyltransferase and histo-blood group antigen H type 2 in resistance of rat colon carcinoma cells to 5-fluorouracil

5-Fluorouracil (5-FU) is a drug of standard use in chemotherapy of colon carcinoma. However, its efficacy is limited by inherent and acquired cell resistance. Major changes in histo-blood group antigenic expression, at times associated with poor prognosis, occur on colon cancer cells. To assess whether these antigens might play a role in the resistance to 5-FU, a rat model of colon carcinoma was used. We observed that in vivo treatment of tumors with the drug increased expression of antigen H type 2. The increase was also observed after in vitro short-term exposure to 5-FU, as well as on a cell-resistant variant selected by continuous exposure to the drug, and was accompanied by an increase in alpha1,2-fucosyltransferase activity, the key enzyme involved in synthesis of H antigens. Transfection of cells devoid of this enzymatic activity by an alpha1, 2-fucosyltransferase cDNA allowed expression of H type 2 antigen and increased resistance to 5-FU. Inversely, transfection of cells which possess enzymatic activity by a cDNA in anti-sense orientation reduced both H type 2 cell-surface antigen and resistance to the drug. These results demonstrate that, in this experimental model, alpha1,2-fucosyltransferase and H type 2 antigen are involved in cellular resistance to 5-FU.     

High hRFI expression correlates with resistance to fluoropyrimidines in human colon cancer cell lines and in xenografts

We previously reported that the over-expression of hRFI, a protein preferentially expressed in the digestive tract regions of several cancers, exhibited a tendency to inhibit TNF-alpha induced apoptosis. In this study, we sought to determine the potential effect of hRFI expression on the sensitivity to 5-fluorouracil (5-FU) and/or other fluoropyrimidines. For the whole lysates of 8 colon cancer cell lines, we performed Western blotting with anti-hRFI antibody and analyzed the correlations between the expression level of hRFI and the cell lines' sensitivity to 5-FU induced apoptosis. Furthermore, for a tissue microarray consisting of 32 xenograft derived human cancer cell lines, we examined the expression levels of hRFI and survivin by immunohistochemical staining, and analyzed the correlations between the expression of each protein and the sensitivity to several chemotherapeutic agents in the xenografts examined. Both in colon cancer cell lines and in xenografts, the expression level of hRFI was correlated with resistance to 5-FU and its derivatives. This evidence suggests that hRFI may be a marker predicting the response to fluorouracil derived chemotherapeutic agents and that the reduction of the expression level of hRFI might improve the outcome of chemotherapy.     

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.