Radiosensitization of Breast Cancer Cells by Vitamin D3 and Vitamin D Analogs

维生素 D3 和维生素 D 类似物对乳腺癌细胞的放射增敏作用

• 批准号: 8007385
• 负责人: Eden Wilson
• 金额: $ 3.87万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-11-16 至 2013-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007385
• 关键词: Acridine Orange; Adenine; Adriamycin PFS; Apoptosis; Autophagocytosis; Biological Assay; Breast Cancer Cell; Breast Cancer Treatment; Cancer Patient; Cell Death; Cell Survival; Cells; Cholecalciferol; Clinical; Disease; Electron Microscopy; Exclusion; Foundations; Genetic; Goals; Laboratories; Life; MAPK8 gene; MCF7 cell; Mammary Neoplasms; Measures; Mediator of activation protein; Methods; Mitotic; Monitor; Pathway interactions; Radiation; Radio; Radiosensitization; Recovery; Recurrence; Research; Resistance; Role; SKBR3; Signal Transduction; Staining method; Stains; Stress; System; TdT-Mediated dUTP Nick End Labeling Assay; Trypan Blue; Validation; Vitamin D; Vitamin D Analog; Western Blotting; analog; cancer radiation therapy; chemotherapy; dihydroxy-vitamin D3; human FRAP1 protein; improved; irradiation; malignant breast neoplasm; micronucleus; neoplastic cell; overexpression; prevent; small hairpin RNA

DESCRIPTION (provided by applicant): One of the primary goals of this laboratory is to improve the treatment of breast cancer in order to prevent the disease from recurring and develop strategies to overcome resistance to radiation (and chemotherapy). Our studies have demonstrated that 1,25-dihydroxy vitamin D3/1,25-D3 (the active fonn of vitamin D) or its analogs confer enhanced sensitivity to irradiation (and chemotherapy with Adriamycin) My research is focused on substantiating the role of 1,25-D3 and the vitamin D3 analog EB1089(EB) in radiosensitizing breast cancer cells and elucidating the autophagic signaling cascades involved in radiosensitization. The first specific aim is to substantiate that autophagy is the mode of radiation sensitization in breast tumor cells treated with EB or 1,25-D3. Pharmacological (3-methyl adenine, Bafilomycin, and Chloroquinone) and genetic approaches (shRNA against Beclin-1, ATG5 and ATG7) will be used to inhibit autophagy. Autophagy will be monitored via well-established methods including acridine orange staining, western blotting for LC3 cleavage, and electron microscopy. We expect that these studies may also show that suppression of proliferative recovery in irradiated cells by EB or 1,25-D3 will be reversed if autophagy is inhibited. The second specific aim is to determine if EB and 1,25 D3 can sensitize radioresistant cells, MCF-7 Her-2/neu and SKBR3 cells (that overexpress Her-2/neu) will be pretreated with 1,25-D3 or EB and cell viability will be analyzed by trypan blue exclusion and clonogenic survival assays. Cell death will be monitored by the TUNEL assay for apoptosis, acridine orange/LC3 cleavage for autophagy and micronuclei formation via DAPI staining for mitotic catastrophe. We expect that EB and 1,25- D3 will be shown to overcome resistance to radiation that occurs through overexpression of Her-2/neu. The third specific aim is to determine the involvement/modulation of the ER stress and mTOR pathways in radiosensitization of breast cancer cells. We propose to measure the impact of EB /1,25 D3 and radiation on the levels of crucial mediators involved in the ER stress (elF2a, JNK and XBP-1, PERK, IRE-1) and mTOR (4EBP-1 & S6k1) pathways in MCF-7, MCF-7 Her-2/neu and SKBR3 cells by western blot analysis. Validation of each pathway's involvement in our system will be assessed by genetic knockdown using shRNA for ER stress mediators (PERK& IRE-1) or overexpression of mTOR. Overall, we anticipate that these studies will provide an experimental foundation for the clinical use of vitamin D3 and its analogs in enhancing breast cancer radiotherapy, preventing disease recurrence, and prolonging the life of breast cancer patients.

描述（由申请人提供）：该实验室的主要目标之一是改善乳腺癌的治疗，以防止疾病复发，并制定克服放射（和化疗）耐药性的策略。我们的研究表明，1,25-二羟基维生素 D3/1,25-D3（维生素 D 的活性形式）或其类似物可增强对放射（和阿霉素化疗）的敏感性。我的研究重点是证实 1,25-D3 和维生素 D3 类似物 EB1089(EB) 在放射增敏乳腺癌细胞中的作用，并阐明 自噬信号级联涉及放射增敏。第一个具体目标是证实自噬是经 EB 或 1,25-D3 处理的乳腺肿瘤细胞的放射增敏模式。药理学（3-甲基腺嘌呤、巴弗洛霉素和氯醌）和遗传学方法（针对 Beclin-1、ATG5 和 ATG7 的 shRNA）将用于抑制自噬。将通过成熟的方法监测自噬，包括吖啶橙染色、LC3 裂解的蛋白质印迹和电子显微镜。我们预计这些研究还可能表明，如果自噬受到抑制，则 EB 或 1,25-D3 对受辐射细胞增殖恢复的抑制将会逆转。第二个具体目标是确定 EB 和 1,25 D3 是否可以使放射抗性细胞敏感，MCF-7 Her-2/neu 和 SKBR3 细胞（过度表达 Her-2/neu）将用 1,25-D3 或 EB 预处理，并通过台盼蓝排除和克隆存活测定来分析细胞活力。通过 TUNEL 测定检测细胞凋亡、吖啶橙/LC3 裂解检测自噬，通过 DAPI 染色检测微核形成检测有丝分裂灾难。我们预计 EB 和 1,25-D3 将被证明可以克服因 Her-2/neu 过度表达而产生的辐射抵抗力。第三个具体目标是确定 ER 应激和 mTOR 通路在乳腺癌细胞放射增敏中的参与/调节。我们建议通过蛋白质印迹分析来测量 EB /1,25 D3 和辐射对 MCF-7、MCF-7 Her-2/neu 和 SKBR3 细胞中涉及 ER 应激（elF2a、JNK 和 XBP-1、PERK、IRE-1）和 mTOR（4EBP-1 和 S6k1）途径的关键介质水平的影响。我们系统中每个通路参与的验证将通过使用 ER 应激介质 (PERK 和 IRE-1) 的 shRNA 进行基因敲低或 mTOR 的过表达来评估。总体而言，我们预计这些研究将为临床使用维生素D3及其类似物增强乳腺癌放疗、预防疾病复发、延长乳腺癌患者生命提供实验基础。