Identification of chemotherapeutic sensitizers

化疗增敏剂的鉴定

• 批准号: 8565570
• 负责人: Kyungjae Myung
• 金额: $ 23.47万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8565570
• 关键词: Adverse effects; Antineoplastic Agents; Antioxidants; B-Lymphocytes; Biochemical; Bioinformatics; Biological Assay; Biological Markers; Cardiovascular Diseases; Categories; Cell Death; Cell Line; Cells; Chemicals; Chickens; Chimeric Proteins; Chronic Hepatitis; Collaborations; Collection; DNA Damage; DNA Repair Pathway; DNA repair protein; Dose; Exhibits; Future; Gene Targeting; Genes; Genistein; Genomics; Human Cell Line; Inhibitory Concentration 50; Laboratories; Lead; Libraries; Luciferases; Malignant Neoplasms; Molecular; Molecular Bank; Multi-Drug Resistance; Mus; Mutagenesis; National Institute of Environmental Health Sciences; National Toxicology Program; Non-Insulin-Dependent Diabetes Mellitus; Osteopenia; Osteoporosis; Pathway interactions; Pharmaceutical Preparations; Production; Proliferating; Proteins; Proteolysis; Radiation; Radiation therapy; Resveratrol; Screening procedure; Signal Transduction; Small Interfering RNA; Specificity; Testing; Translational Research; Tumor Burden; United States National Institutes of Health; Xenograft procedure; anti aging; baicalein; base; cancer cell; cell killing; chemotherapeutic agent; chemotherapy; compound 30; fighting; genome-wide; high throughput screening; improved; in vivo; killings; knockout gene; mouse model; novel; response; small molecule libraries; tool; tumor

Chemotherapeutic and radiation treatments cause a variety of genotoxic insults that lead to cell death in rapidly proliferating cancer cells. To survive genotoxic insults, cancer cells depend on multiple DNA repair pathways. Depending on the types of genotoxic insult, cells use a specific DNA repair pathway. When a DNA repair pathway is compromised, cancer cells become more sensitive to certain genotoxic insults. The identification of chemotherapeutic agents acting on compromised DNA repair pathways in cancer cells would result in more efficient treatment of cancer cells. Such agents are potential sensitizers for radiation therapy. We found that ATAD5 protein is stabilized in response to almost all genotoxic insults. Thus, we hypothesized that ATAD5 would be a good biomarker to detect genotoxic insults. We generated a cell line expressing the ATAD5-luciferase fusion protein and showed that the fusion protein is also stabilized in response to genotoxic insults. Using this novel cell-based quantitative high-throughput ATAD5-luciferase assay, we screened over 4,000 compounds from commercially available compound library as well as National Toxicology Program (NTP) library in collaboration with the NIH Chemical Genomics Center (NCGC) that is now part of National Center for Advancing Translational Science (NCATS) and National Institute of Environmental Health Sciences (NIEHS). We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, and for anti-aging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anti-cancer drugs. Furthermore, resveratrol and genistein killed multi-drug resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. Furthermore, we identified 200 compounds that stabilized ATAD5-luciferase in a dose dependent-manner from 300,000 compounds in the NIH chemical library in collaboration with the NCGC. To identify DNA repair pathways targeted by the genotoxic compounds, we used 8 isogenic human cell lines as well as 10 isogenic chicken DT40 B cell lines with targeted gene knockouts in specific DNA repair pathways. Approximately 200 compounds were tested in survival assays on these cells and group into sub-categories based on their IC50 to kill these cells. We are currently confirming the killing potential of these compounds on cancer cells defective in the same DNA repair pathways. We will further investigate whether these compounds can reduce tumor burden in vivo using xenograft mice as well as gene targeted mice models. Each compound will become a good tool to dissect molecular functions of different DNA repair pathways. In collaboration with NCATS, we also used the same ATAD5-luciferase cell line to identify compounds and siRNAs that inhibit the ATAD5 stabilization in response to genotoxic insults and have identified >80 compounds and >30 siRNAs. Genes identified from these siRNA screens will unveil the unknown mechanisms that inhibit proteolysis of DNA repair proteins in response to genotoxic insults. In addition, compounds identified from the screening will be radiation and chemotherapeutic sensitizers in tumors that depend on pathways of protein stabilization in response to radiation/chemotherapy-induced DNA damage. We are currently studying to identify targets of these compounds among genes identified from siRNA screening using bioinformatic analysis, epistatic analysis, as well as biochemical interactions.

化疗和放射治疗引起各种基因毒性损伤，导致快速增殖的癌细胞死亡。为了在基因毒性损伤中存活，癌细胞依赖于多种DNA修复途径。根据基因毒性损伤的类型，细胞使用特定的DNA修复途径。当DNA修复途径受损时，癌细胞对某些基因毒性损伤变得更加敏感。确定化疗药物作用于癌细胞中受损的DNA修复途径将导致更有效的治疗癌细胞。这些药物是放射治疗的潜在致敏剂。我们发现ATAD5蛋白对几乎所有基因毒性损伤的反应都是稳定的。因此，我们假设ATAD5将是检测基因毒性损伤的良好生物标志物。我们生成了一个表达atad5 -荧光素酶融合蛋白的细胞系，并表明该融合蛋白在基因毒性损伤下也很稳定。