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Mechanisms of Cytotoxicity and Radiosensitization for Antimetabolites

抗代谢物的细胞毒性和放射增敏机制

基本信息

批准号：
8278686
负责人：
DONNA S. SHEWACH
金额：
$ 25.84万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-08 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8278686
关键词：
A549 Abbreviations Adverse effects Animals Antimetabolites Antineoplastic Agents Biological Assay Cell Cycle Progression Cell Line Cells Chinese Hamster Ovary Cell Clinic Comet Assay Cytarabine DNA DNA Damage DNA Double Strand Break DNA Mismatch Repair Protein MLH1 DNA Repair DNA Synthesis Inhibition DNA biosynthesis Data Deoxyribonucleosides Diphosphates Dose Drug Delivery Systems Enzymes Failure Floxuridine Genetic Recombination Goals Grant HCT116 Cells HT29 Cells Health High Pressure Liquid Chromatography Human In Vitro Lesion MCF7 cell MLH1 gene MSH2 gene Mediating Metabolic Methotrexate Mismatch Repair Modeling Mus Normal tissue morphology Nucleotides Pathway interactions Patients Pemetrexed Pharmaceutical Preparations Proteins Protocols documentation Radiation therapy Radiation-Sensitizing Agents Radiosensitization Ribonucleotide Reductase Ribonucleotide Reductase Inhibitor Ribonucleotide Reductase Subunit Role Schedule Solid Neoplasm Subfamily lentivirinae Testing Thymidylate Synthase Thymidylate Synthase Inhibitor Toxic effect Translations analog anticancer activity base cancer cell cancer therapy cell killing cytotoxic cytotoxicity gemcitabine homologous recombination hydroxyurea improved in vivo irradiation neoplastic cell novel novel strategies prevent recombinational repair repaired response ribonucleotide reductase M2 small hairpin RNA tripolyphosphate tumor tumor xenograft

项目摘要

DESCRIPTION (provided by applicant): Gemcitabine is an antimetabolite with broad solid tumor activity in patients. In addition, it is a potent radiation sensitizer. During the previous grant period, we investigated the metabolic and repair pathways important for cytotoxicity and radiosensitization with gemcitabine. Mechanistic studies demonstrated that inhibition of ribonucleotide reductase, mediated by the diphosphate of gemcitabine, was responsible primarily for inhibition of DNA synthesis, whereas incorporation of the analog into DNA contributed more to cytotoxicity. In addition, ribonucleotide reductase-mediated decrease in deoxynucleotides correlated strongly with radiosensitization. We tested the hypothesis that the imbalance in deoxynucleotides led to misincorporation of nucleotides into DNA which, if not repaired prior to irradiation, resulted in radiosensitization. Results demonstrated that gemcitabine produced mismatches in DNA, which occurred only at radiosensitizing concentrations and persisted only after irradiation. Radiosensitization with gemcitabine was enhanced by mismatch repair deficiency, which increased mismatches in DNA, and suppression of the p53-inducible ribonucleotide reductase subunit p53R2, which prolonged the deoxynucleotide pool imbalances. In contrast, mismatch repair deficiency decreased cytotoxicity with gemcitabine. We now propose that these mechanisms can be generalized to other antimetabolites that produce alterations in deoxynucleotide pools and function as radiosensitizers, such as hydroxyurea (ribonucleotide reductase inhibitor), and fluorodeoxyuridine, methotrexate and pemetrexed (thymidylate synthase inhibitors) to produce the first unifying hypothesis for antimetabolite radiosensitization. We will also evaluate cytotoxicity and radiosensitization of shRNA-mediated suppression of the enzymes targeted by these antimetabolites, and compare the in vivo antitumor efficacy of shRNA enzyme suppression with antimetabolites alone or with ionizing radiation. New data indicate that late- occurring DNA double strand breaks which are unable to be repaired by homologous recombination are responsible for radiosensitization with gemcitabine. The DNA damage and repair pathways, which may include ATM, ATR and homologous recombination, that produce radiosensitization will be evaluated. Preliminary results indicate that suppression of either thymidylate synthase or the R2 subunit of ribonucleotide reductase with shRNA is at least as effective as antimetabolites in producing radiosensitization. Mechanistic studies will aid in developing a general hypothesis for cytotoxicity and radiosensitization with the antimetabolites and shRNAs. In vivo animal studies will explore dosing antimetabolites or shRNA for radiosensitization based on the amount of drug or shRNA that will decrease dNTPs and produce misincorporated deoxynucleotides in tumor cell DNA. We further hypothesize that suppression of R2 in vivo will produce superior radiosensitization with lower normal tissue toxicity. These mechanism-based studies have high potential for translation to clinic to improve radiosensitization protocols for patients. PUBLIC HEALTH RELEVANCE: These studies propose to test the hypothesis that, for anticancer drugs which target enzymes required to supply compounds for replication of DNA (gemcitabine, fluorodeoxyuridine, hydroxyurea, methotrexate and pemetrexed), the drug-mediated imbalance in these compounds produces mistakes in DNA which are most harmful to the cancer cell when combined with radiotherapy. Furthermore, a novel approach to cancer therapy is proposed in which we will decrease the proteins that supply the required compounds for DNA replication, which we predict will result in anticancer activity alone or in combination with radiotherapy. Understanding the mechanisms responsible for the activity of these common anticancer drugs or the novel protein suppression approach will help us to optimize their use in patients, with the ultimate goal of improving tumor control while minimizing unwanted side effects in normal tissues.

描述（由申请人提供）：吉西他滨是一种抗代谢药，对患者具有广泛的实体瘤活性。此外，它还是一种有效的辐射增敏剂。在上一个资助期间，我们研究了对吉西他滨的细胞毒性和放射增敏作用重要的代谢和修复途径。机理研究表明，由吉西他滨二磷酸介导的核糖核苷酸还原酶的抑制主要是抑制 DNA 合成的原因，而将类似物掺入 DNA 则更能导致细胞毒性。此外，核糖核苷酸还原酶介导的脱氧核苷酸减少与放射增敏密切相关。我们测试了这样的假设：脱氧核苷酸的不平衡会导致核苷酸错误地掺入 DNA，如果在照射前不进行修复，就会导致放射增敏。结果表明，吉西他滨会在 DNA 中产生错配，这种错配仅在放射增敏浓度下发生，并且仅在辐射后持续存在。错配修复缺陷增强了吉西他滨的放射增敏作用，错配修复缺陷增加了 DNA 中的错配，并且抑制了 p53 诱导型核糖核苷酸还原酶亚基 p53R2，从而延长了脱氧核苷酸库的不平衡。相反，错配修复缺陷降低了吉西他滨的细胞毒性。我们现在提出，这些机制可以推广到其他抗代谢药物，这些抗代谢药物会导致脱氧核苷酸库发生变化并充当放射增敏剂，例如羟基脲（核糖核苷酸还原酶抑制剂）和氟脱氧尿苷、甲氨蝶呤和培美曲塞（胸苷酸合成酶抑制剂），从而提出第一个统一假设抗代谢放射增敏。我们还将评估 shRNA 介导的这些抗代谢物靶向酶抑制的细胞毒性和放射增敏作用，并比较 shRNA 酶抑制与单独的抗代谢物或电离辐射的体内抗肿瘤功效。新数据表明，晚期发生的无法通过同源重组修复的 DNA 双链断裂是吉西他滨放射增敏的原因。将评估产生放射增敏作用的 DNA 损伤和修复途径，可能包括 ATM、ATR 和同源重组。初步结果表明，用shRNA抑制胸苷酸合酶或核糖核苷酸还原酶R2亚基在产生放射增敏方面至少与抗代谢药一样有效。机制研究将有助于制定抗代谢物和 shRNA 的细胞毒性和放射增敏作用的一般假设。体内动物研究将根据药物或 shRNA 的量探索用于放射增敏的抗代谢物或 shRNA 的剂量，这些药物或 shRNA 会减少 dNTP 并在肿瘤细胞 DNA 中产生错误掺入的脱氧核苷酸。我们进一步假设，体内抑制 R2 将产生优异的放射增敏作用，同时降低正常组织毒性。这些基于机制的研究具有转化为临床以改善患者放射增敏方案的巨大潜力。公共卫生相关性：这些研究旨在检验这样的假设：对于靶向提供 DNA 复制化合物（吉西他滨、氟脱氧尿苷、羟基脲、甲氨蝶呤和培美曲塞）所需酶的抗癌药物，这些化合物中药物介导的不平衡会产生 DNA 错误，与放射治疗结合使用时，这些错误对癌细胞危害最大。此外，还提出了一种新的癌症治疗方法，其中我们将减少为 DNA 复制提供所需化合物的蛋白质，我们预测这将单独或与放射治疗相结合产生抗癌活性。了解这些常见抗癌药物的活性机制或新型蛋白质抑制方法将有助于我们优化它们在患者中的使用，最终目标是改善肿瘤控制，同时最大限度地减少对正常组织的不良副作用。