干预自噬影响DPD的表达促进5-FU抗结直肠癌疗效的实验研究

背景与目的：结直肠癌作为常见的消化系统恶性肿瘤之一,其化疗与耐药一直以来备受关注,5-氟尿嘧啶（5-fluorouracil,5-FU）是结直肠癌的一线化疗药物,其疗效常因耐药或不良反应而受到影响。二氢嘧啶脱氢酶（dihydropyrimidine dehydrogenase,DPD）是5-FU代谢的关键限速酶,其表达或降解可能成为影响5-FU疗效的因素。自噬是细胞内蛋白质代谢的重要途径,其在化疗诱导的细胞死亡或增殖抑制中的作用仍存在争议。本研究旨在探讨自噬在结直肠癌化疗过程中的作用以及干预自噬影响5-FU耐药的机制。方法：体外细胞培养人结肠癌HCT-8、COLO205、LOVO和SW480细胞系,观察不同细胞系中DPD的表达对5-FU敏感性的影响,通过药物敏感性实验筛选出5-FU敏感细胞,检测5-FU敏感细胞株及其相对应的耐药细胞株中DPD的表达、自噬水平及自噬关键因子微管相关蛋白轻链3（light chain 3,LC3）、P62的表达,并通过诱导/抑制细胞自噬,观察调控自噬状态对DPD的表达变化以及肿瘤细胞生物学行为和化疗抵抗能力的影响,通过UbiBrowser数据库筛选及免疫共沉淀实验（co-inmunoprecipitation,Co-IP）实验验证DPD降解过程中的E3连接酶,探讨自噬降解DPD逆转5-FU耐药的分子机制。结果：DPD低表达的细胞系对5-FU的敏感性更强,其中COLO205细胞系在4种细胞中DPD表达量最高,并对5-FU的耐药性最强,而HCT-8细胞DPD表达最低并对5-FU最为敏感。与HCT-8细胞相比,HCT-8/FU耐药细胞表达较高水平的DPD,以及较低的基础自噬水平;雷帕霉素（rapamycin,RAPA）介导的自噬激活增强了细胞的自噬水平,降低了DPD表达,同时降低了细胞增殖、侵袭和5-FU耐药性;3甲基腺嘌呤（3-methyladenine,3-MA）及羟氯喹（hydroxychloroquine,HCQ）介导的自噬抑制减弱了细胞的自噬水平,增加了DPD表达,增强了5-FU耐药性。5-FU与自噬激活剂联合应用对细胞的抑效果要远远强于单用5-FU及与自噬抑制剂联合应用的效果;DPD的降解需要完整的自噬流的参与,其中自噬溶酶体的形成是DPD降解的关键步骤之一,而单一的自噬体无法对DPD进行降解。E3连接酶NEDD4在HCT-8/5-FU细胞中与DPD和P62蛋白免疫共沉淀,在DPD的自噬降解中发挥作用。结论：自噬参与DPD降解影响结直肠癌细胞对化疗药物的敏感性,激活自噬可促进DPD降解和抑制5-FU的分解代谢,可能成为逆转结直肠癌5-FU耐药的新途径。     

结直肠癌中LC3与不同表型肿瘤相关巨噬细胞的相关性及其临床意义

背景与目的：肿瘤免疫逃逸成为免疫治疗的难点，而结直肠癌中过度自噬可引起肿瘤微环境内抗原物质的增加，进而诱导抗肿瘤免疫。通过检测自噬相关因子LC3、不同表型肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）及其分泌产物在结直肠癌中的表达，探讨其相关性及临床意义。方法：采用免疫组织化学EnVision法检测滨州医学院附属医院病理科及滨州市第二人民医院病理科2013年1月—2014年12月存档的结直肠癌样本中LC3、CD16、CD163、IL-1、IL-10、TNF-α、TGF-β的表达，计数CD4 ⁺ 、CD8 ⁺ 、CD20 ⁺ 淋巴细胞及CD68 ⁺ 、CD16 ⁺ 、CD163 ⁺ 巨噬细胞，并分析其与临床病理学特征及预后的相关性。结果：自噬相关因子LC3、CD16、CD163在结直肠癌组织中的表达均高于癌旁正常黏膜组织（P<0.05）。LC3与M1型TAM及其在巨噬细胞中的占比均呈正相关，而与M2型TAM及其在巨噬细胞中的占比均呈负相关（P<0.05）。LC3、M1型TAM与IL-1、TNF-α的表达及CD4 ⁺ 、CD8 ⁺ 、CD20 ⁺ 淋巴细胞的浸润量呈正相关；而M2型TAM与IL-10、TGF-β的表达呈正相关，而与CD4 ⁺ 、CD8 ⁺ 、CD20 ⁺ 淋巴细胞的浸润量呈负相关（P<0.05）。LC3、CD16、CD163与肿瘤直径、浸润深度及淋巴结转移密切相关（P<0.05）。Kaplan-Meier及COX回归模型分析显示，LC3、CD16、CD163及淋巴结转移与结直肠癌患者预后密切相关，是结直肠癌预后的独立危险因素。结论：结直肠癌中LC3的表达增加与M1型TAM极化、肿瘤进展密切相关，提示患者预后不良。结直肠癌中自噬水平的增高可诱导巨噬细胞募集并向M1型TAM极化，进而诱导免疫细胞聚集，因此调控自噬可成为诱导结直肠癌中TAM极化、增强抗肿瘤免疫的新思路。     

莱菔硫烷对人黑色素瘤A375细胞自噬的影响

目的 探讨莱菔硫烷（sulforaphane，SFN）对人黑色素瘤A375细胞自噬的影响。方法 分别采用不同浓度（0 μmol·L^-1、5μmol·L^-1、10 μmol·L^-1、20 μmol·L^-1、40 μmol·L^-1）SFN及联合自噬抑制剂氯喹处理人黑色素瘤A375细胞后，采用MTT法检测细胞增殖情况，Real-time PCR和Western blot检测自噬相关分子LC3（LC3Ⅱ/LC3Ⅰ）、Beclin-1和p62以及凋亡相关分子Bcl-2、Bax和Caspase-3的表达水平。 结果 MTT法检测结果显示，SFN呈剂量依赖式抑制A375细胞增殖（F=21.517，P＜0.001）。与对照组比较，不同浓度（5 μmol·L^-1、10 μmol·L^-1、20 μmol·L^-1和40 μmol·L^-1） SFN均能上调自噬相关分子LC3Ⅱ、Beclin-1 mRNA和LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达，上调促凋亡蛋白Bax、Caspase-3的表达（P<0.05），同时下调自噬相关分子p62及抑凋亡蛋白Bcl-2的表达（P<0.05）。氯喹联合不同浓度SFN处理A375细胞后各组细胞增殖率均较相应剂量的单纯SFN处理组低（P<0.05）。结论 SFN可诱导黑色素瘤A375细胞自噬并促进细胞凋亡，联合自噬抑制剂氯喹能增强细胞增殖抑制作用，可能是黑色素瘤有效的治疗药物。     

二氢嘧啶脱氢酶（DPD）与胸苷酸合成酶（TS）在结直肠癌中的表达及预后价值

目的：探讨5-FU代谢酶在结直肠癌中的表达及其与预后的关系。方法：44例结直肠癌根治术后分别施以5-FU为主的辅助化疗,并通过免疫组化检测二氢嘧啶脱氢酶（DPD）和胸苷酸合成酶（TS）的表达。结果：DPD阳性表达的结直肠癌无病生存期显著缩短（P=0．047）,而总生存期也有缩短的趋势,但差异无显著意义（P=0．136）。而偈表达与预后无关（P〉0．05）,但TS在晚期肿瘤中表达较高（P〈0．05）。结论：在接受5-FU为主辅助化疗的结直肠癌患者中,DPD表达可作为预后的重要指标。TS表达与临床分期密切相关,可视为结直肠癌进展的生物学标志。     

柴胡皂苷D通过诱导自噬抑制人结直肠癌细胞SW480增殖的实验

目的:研究柴胡皂苷D（saikosaponin-D，SSD）对人结直肠癌细胞SW480自噬的影响，并探讨诱导的自噬对细胞增殖的影响。方法:Western-blot检测SSD对自噬相关蛋白LC3A/B和p62表达的影响，LC3翻转实验和GFP-RFP-LC3荧光实验验证自噬流的发生。Western-blot检测3-MA对SSD所诱导自噬的抑制作用，MTT和细胞计数实验研究3-MA抑制自噬后SSD对SW480细胞增殖的影响。结果:SSD能够诱导SW480细胞发生自噬，表现在LC3A/B II及LC3A/B II/I比值的增高、自噬经典底物蛋白p62的减少、LC3翻转试验阳性以及LC3荧光实验中黄色和红色荧光颗粒的增多（P＜0.05）。自噬抑制剂3-MA能够抑制SSD所诱导自噬的发生（P＜0.05），且3-MA抑制自噬后，SSD对SW480的增殖抑制效应减弱（P＜0.05），提示SSD诱导的自噬对细胞的增殖起抑制作用。结论:SSD通过诱导自噬抑制人结直肠癌细胞SW480的增殖。     

TRIM59在结直肠癌中的表达及其与临床病理指标的关系

目的： 探讨三结构域蛋白59（TRIM59）在结直肠癌组织中的表达及其临床意义。方法： 收集86例结直肠癌患者的癌组织及癌旁组织,分别采用实时荧光定量PCR（qPCR）和免疫组化检测TRIM59 mRNA和蛋白在结直肠癌及癌旁组织中的表达情况;分析其与患者临床病理指标和生存率的关系。结果： 结直肠癌组织中TRIM59 mRNA表达水平较配对的癌旁组织升高（t=12.86,P<0.01）。结直肠癌组织中TRIM59蛋白阳性表达率为62.79%（54/86）,高于癌旁组织的32%（37/86）,差异具有统计学意义（χ²=6.74,P=0.009）。TRIM59蛋白高表达与患者TNM分期（χ²=8.515,P=0.003）、肿瘤浸润深度（χ²=7.167,P=0.007）以及淋巴结转移情况（χ²=5.511,P=0.018）相关。Kaplan-Meier生存分析结果显示TRIM59高表达患者总生存率（OS）和无病生存期（DFS）均明显短于TRIM59低表达患者（均为P<0.05）。结论： 结直肠癌组织中TRIM59呈高表达,且与肿瘤TNM分期、肿瘤浸润深度、淋巴结转移情况及患者预后相关。     

CD10蛋白在结直肠癌组织中的表达及其临床意义

目的：研究CD10蛋白在结直肠癌组织中的表达及其与结直肠癌的各临床病理指标之间的关系，探讨其在结直肠癌发生、发展中的作用。方法：收集78例结直肠癌组织标本及22例癌旁正常组织样本，运用免疫组化EliVision法检测CD10蛋白的表达，并采用卡方检验分析其表达强度及分布比例与结直肠癌临床病理指标的关系。结果：CD10蛋白在结直肠癌组织和正常结肠黏膜组织中的阳性表达率分别为46.2%（36/78）和0，差异有统计学意义（P < 0.01）。结直肠癌组织中CD10蛋白表达比例与各临床病理指标均无明显相关（P > 0.05）。结直肠癌组织中CD10蛋白表达强度与患者性别、年龄、肿瘤部位、分化程度、浸润深度无明显相关（P > 0.05），而与肿瘤的临床分期、有无脉管侵犯及淋巴结转移有关（P < 0.05），中晚期结直肠癌CD10蛋白表达强度高于早期肿瘤（P=0.033），周围淋巴结转移阳性的结直肠癌CD10蛋白表达强度高于淋巴结阴性病例（P=0.023），存在脉管受侵犯的结直肠癌CD10蛋白表达强度高于无脉管受侵病例（P=0.004）。结论：CD10蛋白在结直肠癌组织中呈高表达，且表达强度与肿瘤的临床分期、有无脉管侵犯及淋巴结转移有关，可能有促进结直肠癌发展和浸润转移的作用。     

CD10蛋白在结直肠癌组织中的表达及其临床意义

目的：研究CD10蛋白在结直肠癌组织中的表达及其与结直肠癌的各临床病理指标之间的关系，探讨其在结直肠癌发生、发展中的作用。方法：收集78例结直肠癌组织标本及22例癌旁正常组织样本，运用免疫组化EliVision法检测CD10蛋白的表达，并采用卡方检验分析其表达强度及分布比例与结直肠癌临床病理指标的关系。结果：CD10蛋白在结直肠癌组织和正常结肠黏膜组织中的阳性表达率分别为46.2%（36/78）和0，差异有统计学意义（P < 0.01）。结直肠癌组织中CD10蛋白表达比例与各临床病理指标均无明显相关（P > 0.05）。结直肠癌组织中CD10蛋白表达强度与患者性别、年龄、肿瘤部位、分化程度、浸润深度无明显相关（P > 0.05），而与肿瘤的临床分期、有无脉管侵犯及淋巴结转移有关（P < 0.05），中晚期结直肠癌CD10蛋白表达强度高于早期肿瘤（P=0.033），周围淋巴结转移阳性的结直肠癌CD10蛋白表达强度高于淋巴结阴性病例（P=0.023），存在脉管受侵犯的结直肠癌CD10蛋白表达强度高于无脉管受侵病例（P=0.004）。结论：CD10蛋白在结直肠癌组织中呈高表达，且表达强度与肿瘤的临床分期、有无脉管侵犯及淋巴结转移有关，可能有促进结直肠癌发展和浸润转移的作用。     

枸杞多糖对脊髓神经细胞辐射损伤后的保护作用研究

目的：研究枸杞多糖（LBP）对辐射损伤脊髓神经（SCN）细胞的保护作用,并观察自噬相关蛋白LC3 Ⅱ/I表达的变化,探讨LBP对辐射损伤保护作用的机制。方法：体外培养SCN细胞,应用不同剂量（0、2、6、10 Gy）X射线辐照损伤后,采用四甲基噻唑蓝（MTT）法检测细胞存活率确定最佳辐射剂量,建立辐射损伤模型。用不同浓度（15、25、40 mg/L）LBP对辐射损伤的SCN细胞进行干预后,分别采用MTT法检测细胞存活率以确定最适LBP干预剂量;用最佳辐射剂量和LBP干预后,采用Western blot和免疫组化法检测自噬相关蛋白LC3 Ⅱ/I的表达。结果：X射线辐照后,SCN细胞存活率在2~10 Gy范围内随着照射剂量的增加而逐渐降低,和正常对照组比较差异均具有统计学意义（P < 0.05）;MTT实验结果显示,与辐射组相比,不同浓度LBP干预后SCN细胞存活率均明显升高,差异具有统计学意义（P < 0.05）,故确定10 Gy X射线照射和40 mg/L LBP干预进行后续造模。免疫组化和Western blot检测结果均显示,与正常对照组相比,辐射组细胞的自噬相关蛋白LC3 Ⅱ/I表达明显升高,差异具有统计学意义（P < 0.05）;与辐射组相比,LBP+辐射组细胞LC3 Ⅱ/I蛋白表达亦明显升高,差异具有统计学意义（P < 0.05）。结论：LBP对体外培养的脊髓神经元辐射损伤具有保护作用,可能与LBP促进自噬相关蛋白LC3 Ⅱ/I表达有关。     

维生素E琥珀酸酯处理人胃癌细胞过程中自噬与活性氧蓄积的交互作用

目的：探讨维生素E琥珀酸酯（VES）处理人胃癌SGC-7901细胞过程中自噬与活性氧（ROS）蓄积是否存在交互作用。方法：不同剂量（0、5、10、15、20 μg/mL）VES处理人胃癌SGC-7901细胞24 h,采用免疫荧光染色观察细胞内自噬标志蛋白微管相关蛋白l轻链3（LC3）的荧光强度和分布情况,Western blot检测自噬标志蛋白LC3和Beclin-1的表达情况,流式细胞术检测细胞内ROS水平;用ROS淬灭剂N-乙酰半胱氨酸（NAC）预处理2 h后以20 μg/mL VES处理人胃癌SGC-7901细胞24 h,免疫荧光染色观察细胞内LC3荧光强度和分布情况,Western blot检测LC3和Beclin-1的表达情况;RNA瞬时干扰阻断BECN-1基因的表达后,以20 μg/mL VES处理人胃癌SGC-7901细胞24 h,流式细胞术检测细胞内ROS水平。结果：与对照组相比,随着VES作用剂量的增加,LC3的荧光强度逐渐增强;LC3和Beclin-1蛋白表达均逐渐升高;流式细胞术检测结果显示胞内ROS水平逐渐增强;淬灭ROS之后LC3荧光强度以及LC3和Beclin-1蛋白表达水平均明显低于VES单独处理组（P<0.05）;阻断自噬关键基因BECN-1后细胞内ROS水平高于VES单独处理组（P<0.05）。结论：VES可诱导人胃癌细胞发生自噬及ROS蓄积,并且VES处理人胃癌细胞过程中自噬与ROS水平可以相互调节,具有交互作用。     

Tumor dihydropyrimidine dehydrogenase in stage II and III colorectal cancer: low level expression is a beneficial marker in oral-adjuvant chemotherapy, but is also a predictor for poor prognosis in patients treated with curative surgery alone

The aim of this study was to evaluate the prognostic significance of tumor dihydropyrimidine dehydroganase (DPD) in curatively resected colorectal cancer patients who received or did not receive oral 5-FU based-adjuvant chemotherapy. Among 182 patients with stage II-III colorectal cancers, 89 patients (adjuvant chemotherapy group) received oral 5-FU based-adjuvant chemotherapy, and 93 patients (surgery alone group) did not receive 5-FU. DPD expressions in the tumors and in the normal colonic mucosa were measured by enzyme-linked immunosorbent assays. The mean DPD expression of the tumors was significantly lower than that of the normal mucosa (54.4 +/- 40.4 versus 72.3 +/- 23.3 Unit/mg protein, P < 0.01). For survival analyses, we designated the cut-off value of tumor DPD as its median value (46.3). In the adjuvant chemotherapy group, high tumor DPD levels were associated with poor survival (HR, 5.24; P = 0.03). In the surgery alone group, high tumor DPD levels were associated with better survival (HR, 0.32; P = 0.02). In conclusion, tumor DPD level is an efficacious marker in oral 5-FU based-adjuvant chemotherapy for colorectal cancer; however, low tumor DPD predicts reduced survival in patients treated with curative surgery alone.     

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance to 5-fluorouracil in human lung cancer cells

The expressions of thymidylate synthase (TS) and intracellular metabolic enzymes have been reported to be associated with the sensitivity and/or resistance to 5-fluorouracil (5-FU). However, since the role of these enzymes in the mechanism of resistance to 5-FU has not been fully examined in lung cancer, in the present study we measured the expression levels of TS, dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT) genes in lung cancer cell lines by real-time PCR, and the sensitivity to 5-FU using the MTS assay. The expression of DPD was significantly correlated with the concentration of 5-FU for 50% cell survival in 15 non-small-cell lung cancer (NSCLC) cell lines (p<0.05), but the expressions of TS, TP, and OPRT were not. Treatment with 5-chloro-2,4-dihydroxypyridine, an inhibitor of DPD, altered the sensitivity to 5-FU in DPD-expressing RERF-LC-MT cells, indicating that modulation of DPD activity could increase the 5-FU sensitivity in lung cancer. In contrast, TS expression was dramatically higher in a 5-FU-resistant small-cell lung cancer cell line than in the parent cell line, whereas the expressions of DPD, TP, and OPRT genes were not markedly different. In order to examine the effect of other cytotoxic agents on TS and DPD expression, we compared the expressions of both genes between cisplatin-, paclitaxel-, gemcitabine-, or 7-ethyl-10-hydroxycamptothecin-resistant lung cancer cells and their respective parent cells, but found no differences between any pair of resistant subline and the corresponding parent cell line. Our results indicate that degradation of 5-FU due to DPD is an important determinant in 5-FU sensitivity, while induction of TS contributes to acquired resistance against 5-FU in lung cancer. Therefore, the expression levels of TS and DPD genes may be useful indicators of 5-FU activity in lung cancer.     

结直肠癌患者DPD水平与5-FU血药浓度、疗效及毒性的相关性研究

背景与目的:以相同的FOLFOX6方案治疗晚期结直肠癌患者,疗效和不良反应却明显不同。二氢嘧啶脱氢酶(DPD)是影响5鄄FU化疗疗效及不良反应的主要因素之一。本研究主要探讨结直肠癌患者外周血DPD水平与标准FOLFOX6方案治疗后患者5鄄FU血药浓度、不良反应及疗效的相关性。方法:应用高效液相色谱法(HPLC)检测结直肠癌患者化疗前DPD水平,按标准的FOLFOX6方案给药后,检测5鄄FU稳态血药浓度(HPLC法),同时观察化疗不良反应和疗效。结果:DPD在72例结直肠癌患者中呈正态分布,从1.55～5.94不等;5鄄FU稳态血药浓度在结直肠癌患者之间具有较大的变异,从141.1μg/L到1741.9μg/L,不呈正态分布。DPD水平和5鄄FU血药浓度呈负相关(r=-0.460,P<0.01),5鄄FU血药浓度≤600μg/L时不良反应较小,>600μg/L时不良反应明显增加,差异具有显著性(P<0.05)。不同治疗反应组的平均血药浓度之间差异具有显著性(P<0.05)。DPD水平和口腔粘膜炎、腹泻等具有相关性,DPD水平和疗效没有相关性(r=0.312,P=0.078)。结论:DPD水平和5鄄FU稳态血药浓度在结直肠癌患者之间具有较大的差异。DPD水平和5鄄FU血药浓度及毒性呈负相关,5鄄FU稳态血药浓度和不良反应以及治疗反应呈正相关。     

Gene expression in colorectal cancer and in vitro chemosensitivity to 5-fluorouracil: a study of 88 surgical specimens

To predict the sensitivity of colorectal cancer to 5-fluorouracil (5-FU), we compared the gene expression of surgically obtained colorectal cancer specimens with chemosensitivity to 5-FU as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. Eighty-eight patients with advanced and/or metastatic colorectal cancer provided written informed consent and entered the trial from September 2000 to October 2001. Fresh surgical specimens were used for the MTT assay, and sensitivity to 5-FU was evaluated at a cutoff concentration of 50 μg/ml and 48-h incubation time. Frozen samples were stored at −80°C until mRNA analysis of thymidylate synthetase (TS), dihydropyri-midine dehydrogenase (DPD), thymidine phosphorylase (TP), es-nucleoside transporter (NT), and E2F1 by real-time RT-PCR. The correlations between the variables were analyzed, and the predictive value of these mRNAs was assessed statistically using a receiver operating characteristic (ROC) curve. NT and DPD, TP and DPD, and TP and NT mRNA expression levels correlated significantly, while TS and E2F1 showed no correlations. High NT expression was associated with low sensitivity to 5-FU (P<0.013), as were high DPD and E2F1 expression ( P <0.022 for both). High TP mRNA expression correlated with low sensitivity to 5-FU ( P <0.034), although high TS mRNA expression did not. ROC curves indicated that DPD and NT mRNAs were possible predictors of sensitivity to 5-FU, with cutoff values of 0.6 and 0.4, respectively. The sensitivity of colorectal cancer to 5-FU may be regulated by DPD, the rate-limiting enzyme of catabolism, and NT, an important transmembrane transporter of nucleosides.     

Predictive role of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer patients receiving adjuvant 5-fluorouracil

OBJECTIVE: The combined assessment of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) gene expressions in metastatic colorectal cancer has been reported to be able to predict the efficacy of fluoropyrimidine-based chemotherapy. In order to evaluate the prognostic role in the adjuvant setting, we investigated the TS, DPD and TP expression in primary tumors of colorectal cancer patients treated with 5-fluorouracil (5-FU). METHODS: TS, DPD and TP expression levels were determined by immunohistochemistry in paraffin-embedded primary tumor tissues from 62 patients with Dukes' stage B and C colorectal cancers who underwent surgery and received adjuvant systemic chemotherapy with 5-FU. The median follow-up was 90 months (range 17-127). RESULTS: Dukes' stage C cancer and high TS expression were independent markers of poor prognosis for disease-free survival (DFS; p = 0.0009 and p = 0.007, respectively) and overall survival (OS; p = 0.0005 and p = 0.011, respectively). By multivariate analysis, patients with high DPD expression had significantly shorter DFS (p = 0.007) and OS (p = 0.005) compared to patients with low DPD expression. In the combined analysis of 2 markers, patients with low TS and low DPD had the best outcome in terms of DFS (p = 0.007) and OS (p = 0.03). The analysis of all 3 proteins showed that the patients with low expression of all 3 markers had significantly longer DFS (p = 0.04) and OS (p = 0.01) than patients with a high value of any one of the protein expressions. However, the joint analysis of 3 markers (group with TS-/DPD-/TP-) could not identify a subgroup of patients with a better prognosis compared to the analysis of 2 markers (group with TS-/DPD-). The analysis of Dukes' stage C cancer patients confirmed a significant benefit in terms of DFS and OS (p = 0.001 and p = 0.006, respectively) when all 3 markers had low expression. We also found a positive significant correlation between TS and TP protein expression (p = 0.033). CONCLUSIONS: This retrospective investigation suggests that the combined assessment of TS and DPD may be useful to evaluate the prognosis of patients with Dukes' B and C colon carcinoma receiving 5-FU adjuvant chemotherapy. The role of TP as a predictor for 5-FU-based therapy needs further investigations.     

Thymidylate synthase and dihydropyrimidine dehydrogenase gene expression in breast cancer predicts 5-FU sensitivity by a histocultural drug sensitivity test

Thymidylate synthase (TS), Dihydropyrimidine dehydrogenase (DPD) and Thymidine Phosphorylase (TP) gene expressions are reported to be predictive markers for 5-fluorouracil (5-FU) sensitivity in gastrointestinal cancer. However, in breast cancer, it is still controversial whether those molecular markers predict 5-FU sensitivity or not. One possible reason for the difficulty may be the histological heterogeneity in breast cancer specimens. In this study, TS, DPD and TP mRNA expression in 40 breast cancer tumors were semi-quantified separately in cancer cells (Ca), cancerous stroma (Str) and normal glands (Nor) using laser capture microdissection and real time RT-PCR (LCM+RT-PCR). The histoculture drug response assay (HDRA) for 5-FU sensitivity was performed for 22 tumors. TS and TP mRNA expressions were higher in Ca than Str, although DPD gene expression was lower in Ca than Str. The group of high TS and high DPD gene expression in Ca was resistant to 5-FU, and the group of low TS and low DPD gene expression in Ca was sensitive to 5-FU (P=0.048 chi-square test). TS and DPD mRNA expressions measured using LCM+RT-PCR might be useful predictive markers for 5-FU sensitivity in human breast cancer.