Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine

Purpose

Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.

Methods

DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.

Results

T-dCyd markedly depleted DNMT1 in CCRF-CEM and KG1a leukemia and NCI-H23 lung carcinoma cell lines, while it was ineffective in the HCT-116 colon or IGROV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4′-thio-2′-deoxyuridine, very little was converted to 4′-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4′-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.

Conclusions

Collectively, these data show that 4′-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.     

Long intracellular retention of 4′-thio-arabinofuranosylcytosine 5′-triphosphate as a critical factor for the anti-solid tumor activity of 4′-thio-arabinofuranosylcytosine

4′-Thio-arabinofuranosylcytosine (T-araC) is a new cytosine analog, which exhibits excellent antitumor activity against various solid tumor xenografts in mice. T-araC is a structural analog of arabinofuranosylcytosine (araC), which is known to be marginally active against solid tumors. We have continued to study the biochemical pharmacology of T-araC in solid tumor cells to further characterize the mechanism of action of this new agent and to elucidate why these compounds show a profound difference in antitumor activity against solid tumors. AraC was a slightly more potent inhibitor of cell growth than T-araC when cells were continuously exposed to the drugs. However, T-araC was markedly more cytotoxic than araC when high concentrations of the compounds were given for short periods of time. Despite the fact that T-araC is a much poorer substrate, as compared to araC, for deoxycytidine kinase (the rate-limiting step in the formation of the triphosphates), similar intracellular concentrations of T-araC-5′-triphosphate (T-araCTP) and araCTP were formed in cells at these high, pharmacologically relevant concentrations due to similar V_max’s. The major difference in the metabolism of araC and T-araC was that the half-life of T-araCTP was tenfold longer than that of araCTP and much higher levels of T-araCTP were sustained in cells for long durations after exposure to T-araC. Inhibition of cytidine deaminase, deoxycytidylate deaminase, or DNA replication did not affect the half-life of either araCTP or T-araCTP. In addition, the rates of disappearance of the mono- and tri-phosphates of araC and T-araC in crude cell extracts were similar. These results indicated that these enzymes were not rate-limiting in the degradation of the respective triphosphates. However, the rate of phosphorylation of T-araC-5′-monophosphate (T-araCMP) in crude cell extracts was about tenfold greater than that of araCMP. The results of this work suggested that the longer intracellular retention of T-araCTP was responsible for the superior activity of T-araC against solid tumors in vivo, and that the greater activity of T-araCMP as a substrate of UMP/CMP kinase was responsible for the long intracellular half-life of T-araCTP. Grant Support: This work was supported by NCI Grant P01 CA 34200     

Phase II study of 4′-iodo-4′-deoxydoxorubicin in non-resectable non-small-cell lung cancer

A total of 44 patients with previously untreated, non-resectable non-small-cell lung cancer (NSCLC) were treated with 4-iodo-4-deoxydoxorubicin (IDX), which is an analogue of doxorubicin with less cardiotoxicity. Patients received 80 mg/m² i. v. every 3 weeks. Dose reductions were carried out for haematological toxicity. Response was assessed prior to each treatment according to WHO criteria. Among the 43 evaluable patients, 1 (2%; 95% confidence limits, 0–8%) achieved a partial response. Leucocytopenia of WHO grade 3 or 4 occurred in 64% of patients and corresponding thrombocytopenia grade 3 or 4 occurred in 30%. Of the 26 patients who were evaluated by measurements of the left ventricular ejection fraction (LVEF), 4 had a decline in LVEF of more than 15%, and 2 patients developed congestive heart failure. Myocardial biopsies were not done. In conclusion, IDX is not active in NSCLC at the applied dose and on the schedule used. Moreover, it does not seem possible to increase the dose intensity further due to the observed toxicity.     

Wegener′s肉芽肿的治疗

1974年5月至1989年12月共收治Wegener’s,肉芽肿患者12例,均经病理证实。局限型4例,全身播散型8例。局限型主要用放射治疗,选择深部X线、~(60)Co、高能X线、电子束混合线束,适宜剂量为40～55Gy/4～5.5周,剂量过低或射线表浅易于复发。全身播散型除用局部放疗外,需给予全身化疗,环磷酰胺4～sg,强地松30～40mg/日,持续用药1.5年后逐渐减量停药。肾脏受损时可行部分肾照射,1.5Gy/次,总量4.5Gy/3次。本组3、5、10年生存率分别为83％(10/12)、64％(7/11)和50％(2/4)。放疗加化疗、大量激素及抗炎药物的应用,是本病治疗的趋向。     

In vivo antitumor activity by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone in a solid human carcinoma xenograft model

Previously we have shown that 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone (DMC), which is isolated from the buds of Cleistocalyx operculatus, significantly inhibits the growth of human liver cancer SMMC-7721 cells and is able to induce apoptosis of SMMC-7721 cells in vitro. Here we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft mouse model using human liver cancer SMMC-7721 cells. The average tumor weights in the control group and in mice injected with 150 mg/kg DMC were 1.42±0.11 g and 0.59±0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated an aneuploid peak (representing 33.60±0.80% of the total in mice injected with 150 mg/kg DMC). To our knowledge, this is the first time that chalcone compounds have been applied to a human tumor xenograft model.     

In vivo antitumor activity by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone in a solid human carcinoma xenograft model

Previously, we showed that 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, significantly inhibited the growth of human liver cancer SMMC-7721 cells and could induce SMMC-7721 cells apoptosis in vitro. Here, we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft model with a human liver cancer SMMC-7721 cell line. Our results revealed that the average tumor weight in a control group and a 150-mg/kg DMC injection group was 1.42±0.11 g and 0.59±0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated the existence of an aneuploid peak (representing 33.60±0.80% of the total in the 150-mg/kg DMC injection group). To our knowledge, this is the first time that chalcone compounds were applied to a human tumor xenograft model.     

Metabolism of 4′-modified analogs of doxorubicin. Unique glucuronidation pathway for 4′-epidoxorubicin

The metabolism of doxorubicin (A) 4′-epidoxorubicin (E) and 4′-deoxydoxorubicin (D) was studied in vitro by incubating the analogs with rat liver subcellular fractions and in vivo by chromatographic analysis of human urine. Metabolites were identified by high-pressure liquid chromatography, fluorescence spectroscopy and enzymatic conversion. Human urine contained unchanged drug as well as the corresponding alcohol metabolites in all cases; however, urine of patients who received E also contained two glucuronides which could not be detected in the urine of patients who received A or D. We have identified these glucuronides as 4′-epidoxorubicin glucuronide (E-Glu) and 4′-epidoxorubicinol glucuronide (Eol-Glu). It was concluded that the glucuronide moiety is linked to the daunosamine sugar at the C4′-OH position. A hypothesis is proposed that this glucuronidation pathway may explain the differences in pharmacokinetics and toxicity between E and A. Rat liver microsomes were found to convert all three drugs to the 7-deoxyaglycones at the same rate. Rat liver 100,000 g supernatant was found to be capable of converting these drugs to their respective alcohol metabolites, doxorubicinol (Aol) being formed somewhat slower than 4′-epidoxorubicinol (Eol) and 4′-deoxydoxorubicinol (Dol).     

食管癌细胞 SHEEC中NGAL基因 5′-UTR和 3′-UTR的克隆与鉴定

背景与目的 : NGAL(neutrophil gelatinase-associated lipocalin)基因是 lipocalin家族的一个新成员 . 以往我们曾研究发现 NGAL基因在 TPA诱导永生化食管上皮细胞 SHEE向食管癌细胞 SHEEC恶性转化的过程中显著过表达 , 但表达调控机制不清楚 . 本研究拟从 SHEEC细胞中克隆 NGAL基因的 5′ -UTR和 3′ -UTR( untranslated region) 并进一步明确其结构特征 . 方法 : 采用 RACE方法从 SHEEC细胞中克隆 NGAL基因的 5′ -UTR和 3′ -UTR; 在测序基础上进行 BLAST分析鉴定 . 结果 : 结合以往的实验结果 , 从 SHEEC细胞中克隆了 NGAL基因的 69 bp 5′ -UTR和 147 bp 3′ -UTR, 而且序列分析未发现突变 . 结论 : SHEEC细胞中的 NGAL基因具备完整的 5′ -UTR和 3′ -UTR结构 .     

恶性肿瘤相关的Sweet′s综合征

Sweet's综合征是一种急性发热性、伴有嗜中性粒细胞增多的皮肤病。约20%的患者可併发各种恶性肿瘤。在伴发的恶性肿瘤中,以血液系统恶性肿瘤居多,其次为实体瘤(包括泌尿生殖系恶性肿瘤和乳癌等)。本文综述了特发性Sweet's综合征,特别是伴发肿瘤的Sweet's综合征的新近研究进展。     

Reduced cardiotoxicity and increased cytotoxicity in a novel anthracycline analogue, 4′-amino-3′-hydroxy-doxorubicin

Summary The acute and chronic cardiotoxicity and cytotoxicity of the novel doxorubicin (DXR) derivative 4-amino-3-hydroxy-DXR were compared with those of 4-deoxy-DXR and DXR. In the acute cardiotoxicity study, the ECG and hemodynamic changes recorded in anesthetized rats that had been treated i.v. with 10 mg/kg 4-amino-3-hydroxy-DXR or 8.6 mg/kg 4-deoxy-DXR were significantly less severe than those caused by 13 mg/kg DXR. In the chronic cardiotoxicity study, rats received 3 weekly i.v. injections of 3 mg/kg DXR, 3 mg/kg 4-amino-3-hydroxy-DXR, or 2 mg/kg 4-deoxy-DXR during the first 14 days of the study and were observed for an additional 35-day period. DXR induced severe cardiomyopathy that was characterized by ECG changes in vivo (ST-segment widening and T-wave flattening) and by impairment of the contractile responses (F _max,±dF/dt _max) to adrenaline of hearts isolated from treated animals. 4-Deoxy-DXR caused a progressive enlargement of the ST segment in vivo and a significant impairment of the-dF/dt _max value in vitro, which were less severe than those produced by DXR. The least cardiotoxic drug was 4-amino-3-hydroxy-DXR, which induced minor ECG changes without causing significant alterations in the contractile responses of isolated hearts to adrenaline. On the basis of the drug concentration required to inhibit 50% of the colony formation (IC₅₀) of cell lines in vitro, 4-amino-3-hydroxy-DXR was less active than 4-deoxy-DXR but at least twice as active as DXR against human cancer and murine transformed cell lines. These data indicate that 4-amino-3-hydroxy-DXR is significantly less cardiotoxic and more cytotoxic than DXR.     

Distribution of 2-chloro-2′-deoxyadenosine, 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine, Fludarabine and Cytarabine in mice: a whole-body autoradiography study

The distribution characteristics of tritiated nucleoside analogs, 2-chloro-2'-deoxyadeonosine (CdA), 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), 2-fluoroarabinosyladenine (F-ara-A) and cytosine arabinoside (ara-C) were compared in mice using whole-body autoradiography. CdA, CAFdA and F-ara-A have quite similar molecular structures, but they differ substantially in clinical activity as well as the side effects. Eight mice were injected intravenously in couples. One mouse from each pair was killed 20 min postinjection and the other mouse from each pair 4 h after the injection. The distribution of the label was then analyzed by whole-body autoradiography. The distribution of the nucleoside analogs was rapid and uniform. High concentrations were found in highly perfused organs. After 4 h the overall concentration had decreased but relatively high activities were found in the skin for CdA and CAFdA, in the thymus for ara-C and the bone marrow for CdA. Both CdA and CAFdA were found in the brain, but the concentration was surprisingly lower after 4 h for CAFdA, a lipophilic and more stable analog as compared to CdA. There was an uptake of CdA, F-ara-A and CAFdA in the skin. There were signs of retention of ara-C in parts of the thymus. The present investigations indicate that the nucleoside analog transport to the brain in mice is not primarily dependent upon passive diffusion over a lipophilic barrier, but suggestive of a specific transport mechanism.     

The role of DNA synthesis inhibition in the cytotoxicity of 2′,2′-difluoro-2′-deoxycytidine

Purpose Cytotoxicity from the anticancer drug 2,2-difluoro-2-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result from inhibition of DNA synthesis, due to either (1) dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase, or (2) direct inhibition of DNA polymerases by the 5-triphosphate of dFdCyd (dFdCTP). To elucidate the role of DNA synthesis inhibition in the cytotoxicity of dFdCyd, we compared dFdCyd to hydroxyurea (HU), a ribonucleotide reductase inhibitor, and aphidicolin, an inhibitor of DNA polymerases, in the U251 and D54 human glioblastoma cell lines.Methods Sensitivity to dFdCyd, HU, and aphidicolin were determined using a colony formation assay. The effects of these drugs on DNA synthesis were measured by dual parameter flow cytometry, while the effects on nucleotide pool levels were analyzed by high-performance liquid chromatography.Results HU and aphidicolin elicited substantially less cytotoxicity than the multi-log killing with dFdCyd. When used at equitoxic concentrations (24-h IC₅₀ values), dFdCyd and HU decreased purine dNTP pools primarily, but dFdCyd was less effective than HU. dFdCyd had decreased dATP by about 80% after 4–12 h, and required 8–24 h to decrease DNA synthesis by 50%. In contrast, HU rapidly depleted dATP by >98% within 2 h, which resulted in >90% inhibition of DNA synthesis. Aphidicolin at a concentration similar to its K_i values for DNA polymerases (1 M) decreased DNA synthesis by >70% within 2 h. However, this decreased cell survival by only 10% (U251 cells) and 40% (D54 cells).Conclusions These results demonstrate that HU and aphidicolin produced a more rapid and profound inhibition of DNA synthesis than dFdCyd, but resulted in significantly less cytotoxicity. This suggests that inhibition of DNA synthesis accounted for less than one log of the multi-log cytotoxicity observed with dFdCyd, whereas incorporation of dFdCTP into DNA is a more lethal event.     

低分子肝素治疗Trousseau′s综合征

1865年,Trousseau首次报告肿瘤与血栓栓塞性疾病的关系,游走性静脉血栓形成首次被描述。而且,目前在肿瘤病人中,各种静脉和动脉其他的血栓栓塞性疾病被称为Trousseau′s综合征。尽管有争论,血栓栓塞现象一直被认为是潜隐性肿瘤,尤其是未有血栓形成的其他危险因素的癌症病人的先兆。血栓形成常可提示恶性肿瘤的诊断。 本文报告4例病人患有广泛的静脉血栓栓塞性疾病,随之诊断又患有恶性肿瘤。所有病人最后均应用enoxaparin(一种低分子肝素,low-Molecular-weight heparin,LMWH)皮下给药治疗,理想地控制了血栓栓塞。     

BCR基因5′启动子区多态性分析

背景与目的:bcr-abl融合基因被认为是慢性髓系白血病(chronic myelogenous leukemia,CML)的分子标志,该融合基因的表达受 bcr基因启动子的调控.本实验目的是研究 bcr基因启动子区的多态性,并探讨其与 CML之间可能存在的联系.方法:采用 PCR方法扩增 bcr基因 5′启动子区 1.13 kb的序列范围,通过测序获得了 30例 CML和 19例对照的启动子区序列.应用软件和在线工具对启动子序列进行转录因子结合位点分析和重复序列分析.结果:在所研究片段范围内共发现 4处新的单核苷酸多态性(single nucleotide polymorphism,SNP)和 3处与参考序列不一致的碱基变异.其中 2处 SNPs和 1处碱基插入位于转录因子结合位点中或邻近几个碱基内.各多态性基因频率在 CML和对照人群中无统计学差异.结论:bcr基因的 5′启动子区存在一定的序列多态性,主要为 SNPs,未能证实其与 CML之间存在相关性,但部分 SNPs的定位使其有可能对基因的转录和表达产生影响.     

外阴Paget′s病临床分析

目的　研究外阴Paget′s病的诊断、病理、治疗和预后。方法　回顾性总结和分析1960年~2002年期间在中国医科院肿瘤医院治疗的7例外阴Paget′s病,病例的症状、病变部位、病理、治疗方法以及随诊情况。结果　7例Paget′s病患者平均年龄67.3岁(54~81岁);外阴病变和瘙痒是主要的症状,从最初症状到确诊平均时间3.4年。所有患者初治均为手术治疗,2例行外阴单纯切除术,5例行外阴根治术。术后病理:2例浸润到真皮层;4例合并上皮下腺癌。复发5例,距初治时间平均为16.2个月,其中4例行放疗或化疗,另1例未治。平均随访时间41.1个月。3例死亡,均为浸润性病变或合并上皮下腺癌。结论　外阴Paget′s病患者的诊断较困难,手术是主要的治疗方法,外阴Paget′s病易复发,有浸润性病变或伴上皮下腺癌者预后差。     

骨髓瘤细胞5′-核苷酸酶染色研究

骨髓瘤细胞５′－核苷酸酶染色研究刘永春李彦会郝冀洪陈一丽用５′－核苷酸酶（５′－Ｎ）染色对血细胞进行观察，国外研究材料较多，国内仅李顺义等［１］用于巨核细胞研究。Ｅｌ－Ｍｏｈａｎｄｅｓ等［２］发现骨髓瘤细胞中５′－核苷酸酶活性较高，但以后报道材料较少...     

BCR基因5′启动子区多态性分析

背景与目的：bcr-abl融合基因被认为是慢性髓系白血病(chronic myelogenous leukemia,CML)的分子标志,该融合基因的表达受bcr基因启动子的调控。本实验目的是研究bcr基因启动子区的多态性,并探讨其与CML之间可能存在的联系。方法：采用PCR方法扩增bcr基因5’启动子区1．13kb的序列范围,通过测序获得了30例CML和19例对照的启动子区序列。应用软件和在线工具对启动子序列进行转录因子结合位点分析和重复序列分析。结果：在所研究片段范围内共发现4处新的单核苷酸多态性(single nucleotide polymorphism,SNP)和3处与参考序列不一致的碱基变异。其中2处SNPs和1处碱基插入位于转录因子结合位点中或邻近几个碱基内。各多态性基因频率在CML和对照人群中无统计学差异。结论：bcr基因的5’启动子区存在一定的序列多态性,主要为SNPs,未能证实其与CML之间存在相关性,但部分SNPs的定位使其有可能对基因的转录和表达产生影响。     

Gardner′s综合征伴甲状腺癌1例

患者女 ,３６岁。于１９９３年１月１０日因右侧甲状腺肿块入院行右甲状腺摘除术。病理诊断 :甲状腺乳头状腺癌。１９９４年１０月２５日又因间歇性腹痛腹泻入院。纤维结肠镜诊断 :结肠多发性息肉 ,行全结肠切除术。肉眼见结肠９０ｃｍ ,周径８ｃｍ ,全结肠粘膜面可见较密集排列的息肉状肿物 ,０．３～０．８ｃｍ大小 ,有蒂 ,质软。病理诊断 :结肠多发性腺瘤病 ,间变Ⅱ级。术后９个月 ,患者又以腹部肿物再次入院剖腹探查 ,见中腹至盆腔腹膜后及小肠系膜弥漫分布大小不等的质硬包块 ,边界不清 ,髂内外血管及部分小肠受侵。因腹部肿物广泛 ,活…     

Antitumor activity of 3′, 5′-diesters of 5-fluoro-2′-deoxyuridine against murine leukemia L1210 cells

Summary Antitumor activity of several 3,5-diesters of 5-fluoro-2-deoxyuridine (FUdR) against L1210 leukemia cells following intraperitoneal administration was examined. Esters of FUdR with aromatic acid or aliphatic acid of longer chain length were markedly active. Their activities, with respect to ILS₃₀, were as much as 100 times that of unesterified FUdR. 3,5-ditoluoyl FUdR also had an improved therapeutic effect: its therapeutic ratio was increased to 8.1, as against 2.0 for FUdR. On the other hand, 3,5-diesters of FUdR with aliphatic acid of shorter chain length do not appear to be as active as FUdR. The relationship between the antitumor activity and plasma levels has also been examined. After 3,5-diacetyl FUdR, which is one of the drug group showing low cytotoxicity, the plasma concentration rapidly decreased to unmeasurable level 3 h after dosing. This tendency is similar to that shown in FUdR. On the other hand, with 3,5-dipalmitoyl FUdR and 3,5-dibenzoyl FUdR, each of which has a marked antitumor effect, plasma concentrations decreased slowly and were maintained for as long as 48 h after dosing. The results show that the cytotoxicity of diesters of FUdR is correlated with the duration of a high plasma level of FUdR.     

抑癌基因p53在Barrett′s食管、Barrett′s食管粘膜内癌中的表达

抑癌基因ｐ５３在Ｂａｒｒｅｔｔ′ｓ食管、Ｂａｒｒｅｔｔ′ｓ食管粘膜内癌中的表达赵立群，裘宋良，杨观瑞，郭花芹，孙豫，李珊珊，张聚真本试验应用免疫组织化学染色法，检测了抑癌基因ｐ５３在１４例Ｂａｒｒｅｔｔ′ｓ食管（简称ＢＥ）、１例ＢＥ粘膜内癌中的表达。...