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。     

二氢嘧啶脱氢酶活性与结直肠癌患者5-FU辅助化疗毒性关系的研究

背景与目的:5-氟尿嘧啶(5-FU)应用于不同的患者中,它的毒性及疗效均不同,个体差异较大.现发现二氢嘧啶脱氢酶(Dihydropyrimidine dehydrogenase,DPD)在其中起重要作用.本研究测定结直肠癌患者血DPD活性,并评价其与结直肠癌患者5-FU辅助化疗毒性及5-FU血药浓度之间的关系.方法:入选符合标准的结直肠癌患者30例,术前采用高效液相色谱法(HPLC)法测定血内源性二氢尿嘧啶(UH2)/尿嘧啶(U)值代表血DPD活性,术后2周行甲酰四氢叶酸(CF)60 mg/m2,2 h静脉滴注,5-FU 425 mg/m2,2 h静脉滴注,连续5天方案,辅助化疗.第1天刚滴完5-FU时和第5天刚滴完5-FU时抽血查5-FU血药浓度.评价患者血DPD活性和患者5-FU化疗毒性及其与5-FU血药浓度之间的相关性.结果:30例结直肠癌患者行5-FU/CF方案辅助化疗.化疗前查血DPD活性为4.09±1.21,最低2.14,最高6.7,呈正态分布,个体差异较大.第1天刚滴完5-FU时5-FU血药浓度为(2079.12±621.41)μg/L(1200.10～3554.80 μg/L);第5天刚滴完5-FU时5-FU血药浓度为(2197.64±606.78)μg/L(1259.00～3441.03μg/L),亦呈正态分布,个体差异较大.Pearson相关分析显示,血DPD活性与第1天刚滴完5-FU时5-FU血药浓度(r=-0.773,P=0.00)及第5天刚滴完5-FU时5-FU血药浓度(r=-0.833,P=0.00)呈负相关.t检验显示第1天刚滴完5-FU时5-FU血药浓度及第5天刚滴完5-FU时5-FU血药浓度没有差异(P=0.458).Spearman相关分析统计显示,5-FU致恶心呕吐、腹泻、骨髓抑制级别与血DPD活性呈负相关,与5-FU血药浓度呈正相关(P＜0.05).结论:检测血DPD活性可以推断5-FU化疗时5-FU血药浓度及预测5-FU化疗的毒性,为结直肠癌的个体化治疗提供了理论依据.     

TS和MMR联合检测在结直肠癌预后判断中的作用

目的:对133例结直肠癌患者临床病例资料进行回顾性分析,分析胸苷酸合成酶(thymidylate synthase,TS)蛋白与错配修复(mismatch repair,MMR)状态联合检测与结直肠癌患者临床病理特征及预后之间的关系.方法:免疫组化测定TS和MMR(MLH-1/MSH-2)蛋白表达.根据TS蛋白和MMR状态差异进行相应的配对组合,将纳入患者分为四组:TS蛋白高表达/MMR蛋白高表达(HtHm)组、TS蛋白低表达/MMR蛋白高表达(LtHm)组、TS蛋白低表达/MMR蛋白低表达(LtLm)组、TS蛋白高表达/MMR蛋白低表达(HtLm)组.分析TS和MMR联合检测与患者临床病理因素及预后的关系.结果:TS和MMR联合检测生存分析显示,LtHm组患者(39例)的3年生存率为69.2%,HtLm(20例)组患者的3年生存率为40.0%,两组总体生存率具有统计学差异(P=0.012),该差异在术后辅助化疗患者中仍存在(P=0.011).结论:辅助化疗组患者中LtHm组总体生存率显著高于HtLm组,LtLm组患者较HtHm组患者更易从氟尿嘧啶类药物辅助化疗中获益.     

胃癌组织中胸苷酸合成酶、胸苷磷酸化酶mRNA表达及其临床意义

背景与目的:化学治疗在胃癌治疗中扮演了重要的作用。通过肿瘤组织中某些基因表达的水平来选择化疗药物已成为今后化疗的方向。胸苷酸合成酶(TS)、胸苷磷酸化酶(TP)是氟尿嘧啶(5-FU)体内的关键代谢酶,其表达水平影响恶性肿瘤患者接受5-FU化疗后的预后。本研究探讨胃癌组织中TS、TP mRNA表达水平与预后的关系。方法:采用实时定量RT-PCR技术检测51例胃腺癌组织TS、TP mRNA表达水平。结果:胃癌组织TS、TP mRNA表达水平的中位数分别为0.94和21.20,TS高表达组和低表达组之间无瘤生存期和总生存期差异有显著性(P<0.05),TP高表达组和低表达组之间总生存期差异有显著性(P<0.05),但无瘤生存期之间差异无显著性(P>0.05)。TS、TP mRNA表达与年龄、性别、淋巴结转移、组织学分级及临床分期均无相关性(P>0.05)。结论:检测TS、TP mRNA表达水平对接受5-FU为基础治疗方案的胃癌患者的预后有很好的预测价值。     

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.     

Survivin在Dukes''''C期结直肠癌中的表达及其生物学意义

目的:探讨Survivin在Dukes'C期结直肠癌中的表达情况及其对辅助化疗疗效的预测作用.方法:应用免疫组化法检测85例根治性术后,行标准辅助化疗的Dukes'C期结直肠癌患者的癌组织和14例正常结直肠组织中Survivin的表达,分析与临床病理因素的相关性,并进行生存分析.结果:肿瘤组织Survivin阳性率为51.76%,正常组织中无Survivin表达,两组阳性率差异有显著性(P＜0.05).Survivin的表达与患者的年龄、性别、病程、肿瘤部位、肿瘤最大径、大体类型、组织分型、分化程度、肠壁浸润深度、淋巴结转移部位之间均无显著相关性(P均＞0.05).Survivin蛋白的表达与肿瘤的复发转移显著相关,Survivin阳性者复发转移率54.52%,显著高于Survivin阴性者(26.83%)(P=0.009).Survivin阳性患者3年、5年无病生存率和总生存率显著低于阴性患者,Cox模型多因素分析,Survivin是影响肿瘤复发转移和生存的独立的预后因素.结论:Survivin在Dukes'C期结直肠癌中表达上调,与辅助化疗的疗效相关,可作为预测辅助化疗疗效的分子指标.     

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。     

乳腺癌组织中TP和TS及DPD mRNA表达与预后的关系

目的　探讨乳腺癌组织中胸苷酸化酶 (TP)、胸苷酸合成酶 (TS)和二氢嘧啶脱氢酶(DPD)mRNA表达水平及其与预后的关系。方法　采用real time定量PCR技术检测经过微选的 9例正常乳腺组织和 86例乳腺癌组织TP、TS和DPD的mRNA表达水平。结果　肿瘤组织中TP、TS和DPDmRNA表达水平中位数分别为 16 .5 4 ,0 .38和 2 .74 ,正常乳腺组织分别为 11.75 ,0 .2 5和 8.33,差异均无显著性 (P >0 .0 5 )。除年龄与DPD表达呈负相关外 ,TP、TS和DPDmRNA表达与肿瘤体积、淋巴结转移、组织学分级、临床分期均无相关性。TP、DPD高表达组和低表达组之间 ,无瘤生存期和总生存期差异均无显著性。TS高表达组和低表达组无瘤生存期差异无显著性 (P =0 .0 6 9) ;平均总生存期分别为 5 9.0 0和 70 .30个月 ,差异有显著性 (P =0 .0 4 96 )。结论　仅检测TSmRNA对判断乳腺癌预后有参考价值 ,同时检测TP、TS和DPD具有更好的预测价值。     

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MGMT和TS的表达及其与临床病理因素的关系

目的 探讨5-FU疗效相关基因MGMT及TS蛋白在散发性结直肠癌中的表达及其与临床病理特征的关系.方法 采用EnvisionTM鼠/兔-HRP广谱检测系统检测78例原发性结直肠腺癌、26例癌旁正常黏膜、13例低级别上皮内瘤变和11例高级别上皮内瘤变标本.结果 MGMT蛋白在正常黏膜、低级别上皮内瘤变、高级别上皮内瘤变和结直肠腺癌中阳性表达率分别为26.9%、53.8%、54.5% 和62.8%,组间差异具有统计学意义(P<0.01);TS蛋白在正常黏膜、低级别上皮内瘤变、高级别上皮内瘤变与结直肠癌的阳性表达率分别为23.1%、38.5%、36.4%和61.5%,组间差异亦具有统计学意义(P<0.01);正常黏膜中(23.1%)及结直肠癌组织(61.5%)间蛋白表达差异显著(P<0.01).MGMT蛋白表达在结直肠癌中与患者的性别(P<0.05)和肿瘤组织学分级(P<0.05)相关,TS蛋白与患者的年龄(P<0.05)和性别(P<0.05)相关.在结直肠腺癌中MGMT和TS蛋白表达存在相关性(P<0.05).结论 MGMT和TS蛋白表达增高在结直肠腺癌发生、发展过程中发挥重要作用.同时检测结直肠腺癌中MGMT和TS蛋白表达水平对预测肿瘤的生物学行为和以5-FU为基础的化疗方案的疗效可能具有应用价值.     

Toxicology and pharmacokinetics of (1,1-bis(aminomethyl)cyclohexane)oxalatoplatinum(II) (TNO-38)

Preclinical studies on toxicology and pharmacokinetics were performed for (1,1-bis(aminomethyl)cyclohexane)oxalatoplatinum(II) (TNO-38) in rats and a dog after ld₁₀ and ld₅₀ assessment in mice. In drug-treated rats, ura and creatinine concentrations were 1,4-1.9 times those in control rats. Histopathology showed necrosis of tubular epithelium of the kidneys, which was comparable to damage observed after treatment with cisplatin (CDDP), and extensive necrosis of crypt epithelium, especially in the ileum.Similar to CDDP, TNO-38 was emetic in the dog. Non-specific subacute inflammatory changes were observed in the ileum. Renal damage was much less pronounced.Half-lives of distribution and elimination were 6.2 min and 5.2 days, respectively. The cumulative excretion of Pt in urine over 1 and 7 days after drug treatment was 38.3 and 49.3% of the dose, respectively. Twelve weeks after drug administration, Pt concentrations were highest in kidneys and liver.TNO-38 is adequately water soluble. Its reported antitumour activity is consistently lower than that of CDDP. The drug's toxicity was, in general, comparable to that of CDDP. Its pharmacokinetic profile was very similar to that of CDDP. It is concluded that TNO-38 should probably not be further evaluated in clinical studies.     

Synergism of cytotoxicity between cis-diaminedichloro-platinum-(ii) and cis-diamine(1,1-cyclobutanedicarboxylate)-platinum-(ii)

In an attempts to increase the antitumor effect and to reduce normal tissue toxicity, the combined cytotoxic effect of cis-Diamminedichloroplatinum (II) (CDDP) and cis-diammine(1,1-cyclobutane dicarboxylate) platinum (II) (CBDCA) was investigated using HeLa and colon 26 cell lines and the combination index (CI). Cytotoxicity of the combination of CDDP and CBDCA on 27 surgically resected specimens of human gastric and colorectal adenocarcinomas was also evaluated using the in vitro succinate dehydrogenase inhibition (SDI) test. The CI values varied with the dose ratio examined (1:1-1:6) of CDDP and CBDCA, with findings that CI<1, synergy, was obtained at fraction affected (Fa)>0.75 for HeLa cells and at Fa<0.9 for colon 26 cells in cases of a dose ratio of 1:1 to 1:2. Of all 27 clinical human adenocarcinomas, the succinate dehydrogenase (SD) activity was significantly lower in cancer cells concomitantly exposed to both CDDP and CBDCA than in those exposed to either drug alone. These positive effects of a combination of two platinum analogues on human malignant tissues have heretofore not been reported, which would warrant the clinical application of this combination for human malignant tumors.     

Antitumor effects of three platinum complexes, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato)-platinum (II) monohydrate (DWA2114R), cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) and cis-diamminedichloroplatinum(II) (CDDP), in mice.

(-)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato++ +)platinum(II) monohydrate (DWA2114R), cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) and cis-diamminedichloroplatinum(II) (CDDP) were compared for their antitumor effects and nephrotoxicity-inducing activities at the same dosage (1/8, 1/4, 1/3, 1/2, 2/3 or 3/4 of the LD10 or LD10) on the basis of their intravenous lethal doses in mice. DWA2114R was effective against murine tumor lines, Colon 26 and Colon 38 carcinomas, M5076 ovarian sarcoma and P388 L1210 leukemias, implanted subcutaneously (s.c.). Triple injection every other day of DWA2114R was more effective than a single injection at each sublethal dose. The antitumor effects of DWA2114R against these tumors were more effective than or were similar to those of CBDCA and CDDP. The antitumor effect against CDDP-resistant L1210 leukemia implanted s.c. was only observed in the treatment of DWA2114R, but not in CBDCA and CDDP. No excellent antitumor effects of three platinum complexes were observed against Lewis lung carcinoma and B16 melanoma implanted s.c. even at triple injection every other day, and no effect was obtained against Meth-A fibrosarcoma under similar conditions. While the treatment of CDDP showed marked increases in levels of blood urea nitrogen and of urinary protein and sugar at effective doses in the antitumor evaluations, the treatment of DWA2114R as well as CBDCA showed no increase in these parameters. These results indicate that DWA2114R represents a desirable second generation antitumor platinum complex.     

Concurrent chemoradiotherapy (CRT) with S-1 and cisplatin (CDDP) in patients (pts) with locally advanced head and neck cancer (HNC)

The standard care for unresectable locally advanced head and neck cancer (HNC) is concurrent chemoradiotherapy (CRT). Although there is no standard regimen of CRT, a platinum-based regimen has shown a better survival benefit than other regimens. The control arm in a randomized trial for unresectable locally advanced HNC is radiotherapy concurrent with CDDP (100 mg/m2, every 3 weeks), which has been considered to be too toxic for clinical practice in Western countries and has required frequent dose modifications. Because the Japanese also have been considered unable to tolerate this regimen, no prospective study of it has been conducted in Japan. Most Japanese patients with locally advanced head and neck cancer have received concurrent chemoradiotherapy with 5-FU and CDDP (70-80 mg/m2). S-1 has shown high activity in HNC with a response rate of 34%. Furthermore, a combination of cisplatin and S-1 therapy for HNC has been reported to have good efficacy. With this rationale in mind, we conducted a phase I study of CRT with S-1 and CDDP for unresectable locally advanced squamous cell carcinoma of the head and neck. The CR rate was very promising, though preliminary, and warrants further investigation. The Japan Clinical Oncology Group (JCOG) is planning a multicenter phase II study of concurrent chemoradiotherapy with S-1 and CDDP for locally advanced unresectable HNC.     

In vitro evaluation of dichloro-bis(pyrazole)palladium(II) and dichloro-bis(pyrazole)platinum(II) complexes as anticancer agents

Introduction  Cisplatin (cis-diamminedichloroplatinum) was first identified for its anti-bacterial activity, and was later also shown to be an efficient anticancer agent. However, the therapeutic use of this anticancer drug is somewhat limited by its toxic side effects, which include nephrotoxicity, nausea, and vomiting. Furthermore the development of drug-resistant tumours is commonly observed following therapy with cisplatin. Hence there is a need for improved platinum derived drugs to overcome these limitations. Aims  Apoptosis contributes significantly to the cytotoxic effects of anticancer agents such as cisplatin; therefore in this study the potential anticancer properties of a series of pyrazole palladium(II) and platinum(II) complexes, [(3,5-R₂pz)₂PdCl₂] {R = H (1), R = Me (2)} and [(3,5-R₂pz)₂PtCl₂] {R = H (3), R = Me (4)}, were evaluated by assessment of their pro-apoptotic activity. Methods  The induction of apoptosis was measured in CHO cells by the detection of phosphatidylserine (PS) exposure using the annexin V and APOPercentage™ assays; DNA fragmentation using the Terminal deoxynucleotide transferase dUTP Nick End Labelling (TUNEL) assay; and the detection of activated caspase-3. Results  The platinum complexes were shown to be considerably more active than the palladium complexes, with complex 3 demonstrating the highest level of cytotoxic and pro-apoptotic activity. The LD₅₀ values for complex 3 and cisplatin were 20 and 70 μM, respectively, demonstrating that the cytotoxic activity for complex 3 was three times higher than for cisplatin. Various human cancer cell lines, including CaSki, HeLa, as well as the p53 mutant Jurkat T cell line were also shown to be susceptible to complex 3. Conclusions  Collectively, this in vitro study provides insights into action of palladium and platinum complexes and demonstrates the potential use of these compounds, and in particular complex 3, in the development of new anticancer agents.     

The interaction between protein-kinase-C (pkc) and estrogens (review)

The interactions between protein kinase C (PKC) and the steroid hormone estradiol or its receptor (ER) are reviewed. Estradiol upregulates PKC both in vitro and in vivo in the ovary, the anterior pituitary and in mammary tissue of several mammalian species. The antiestrogen tamoxifen inhibits PKC. Activation of PKC leads to a marked decrease of ER protein and ERmRNA in human breast cancer cells and some other cell lines. Inhibition or down-regulation of PKC enhances ER binding. These results indicate that there are links between the PKC signal transduction pathway and the steroid receptor family. Further studies are needed to clarify the role of PKC isoforms in normal and cancerous tissues which are known to be influenced by estradiol.