Mechanisms of Cytotoxicity and Radiosensitization for Antimetabolites

抗代谢物的细胞毒性和放射增敏机制

• 批准号: 8135035
• 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
• 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/8135035
• 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; 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; public health relevance; recombinational repair; repaired; response; ribonucleotide reductase M2; small hairpin RNA; tripolyphosphate; tumor; tumor xenograft

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(申请人提供)：吉西他滨是一种抗代谢药物，在患者中具有广泛的实体肿瘤活性。此外，它还是一种有效的辐射增敏剂。在之前的资助期间，我们调查了对吉西他滨的细胞毒性和放射增敏至关重要的代谢和修复途径。机制研究表明，由吉西他滨的二磷酸介导的对核糖核苷酸还原酶的抑制是抑制DNA合成的主要原因，而将类似物掺入DNA中则更有可能产生细胞毒作用。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。我们测试了这样一个假设，即脱氧核苷酸的不平衡导致核苷酸错误地结合到DNA中，如果在辐射前没有修复，就会导致放射增敏。结果表明，吉西他滨在DNA中产生错配，这种错配仅在辐射增敏浓度下发生，并仅在辐射后持续。错配修复缺陷增加了DNA的错配，抑制了p53诱导的核糖核苷酸还原酶亚单位p53R2，延长了脱氧核酸池的失衡，从而增强了吉西他滨的放射增敏作用。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产生脱氧核苷酸池改变并作为放射增敏剂的抗代谢药物，如羟基脲(核糖核苷酸还原酶抑制剂)，以及氟脱氧尿苷、甲氨蝶呤和培美曲塞(胸苷合成酶抑制剂)，从而产生了第一个关于抗代谢药物放射增敏的统一假设。我们还将评估shRNA介导的抑制这些抗代谢物靶向的酶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与电离辐射联合使用的体内抗肿瘤效果。新的数据表明，晚期发生的DNA双链断裂无法通过同源重组修复，这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抑制胸苷合成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物一样有效。机制研究将有助于开发抗代谢物和shRNAs的细胞毒性和放射增敏的一般假说。体内动物研究将探索根据药物或shRNA的量来剂量抗代谢药物或shRNA进行放射增敏，这些药物或shRNA将减少dNTPs并在肿瘤细胞DNA中产生错配的脱氧核苷酸。我们进一步假设，在体内抑制R2将产生更好的放射增敏作用，而正常组织毒性较低。这些基于机制的研究有很高的潜力转化为临床，以改进患者的放射增敏方案。 公共卫生相关性： 这些研究建议验证这样一种假设：对于以提供复制DNA所需的酶为目标的抗癌药物(吉西他滨、氟脱氧尿苷、羟基脲、甲氨蝶呤和培美曲塞)，这些化合物中药物介导的不平衡会导致DNA错误，当与放射治疗结合时，这些错误对癌细胞最有害。此外，还提出了一种新的癌症治疗方法，在这种方法中，我们将减少为DNA复制提供所需化合物的蛋白质，我们预测这将导致单独或联合放射治疗的抗癌活性。了解这些常见抗癌药物的活性机制或新的蛋白质抑制方法将有助于我们优化患者对它们的使用，最终目标是改善肿瘤控制，同时将正常组织中的不良副作用降至最低。