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: 8197147
• 负责人: Eden Wilson
• 金额: $ 3.03万
• 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 2012-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197147
• 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; ERBB2 gene; 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进乳腺癌的治疗方法，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的活性因子）或其类似物可增强对照射（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）在放射致敏乳腺癌细胞中的作用，并阐明与放射致敏有关的自噬信号级联反应。第一个具体目的是证实自噬是EB或1,25- d3治疗乳腺肿瘤细胞的辐射致敏模式。药物（3-甲基腺嘌呤、巴菲霉素和氯醌）和遗传方法（shRNA对抗Beclin-1、ATG5和ATG7）将用于抑制自噬。自噬将通过完善的方法进行监测，包括吖啶橙染色，LC3切割的western blotting和电子显微镜。我们预计这些研究也可能表明，如果自噬被抑制，EB或1,25- d3对辐照细胞增殖恢复的抑制将被逆转。第二个具体目标是确定EB和1,25 D3是否可以使放射耐药细胞增敏，MCF-7 Her-2/neu和SKBR3细胞（过表达Her-2/neu）将用1,25-D3或EB预处理，细胞活力将通过台泛蓝排斥和克隆生存试验分析。细胞死亡将通过TUNEL检测凋亡，吖啶橙/LC3切割检测自噬，DAPI染色检测有丝分裂突变微核形成。我们预计EB和1,25- D3将被证明能够克服由于Her-2/neu过表达而产生的辐射抗性。第三个具体目标是确定内质网应激和mTOR通路在乳腺癌细胞放射致敏中的参与/调节。我们建议通过western blot分析来测量EB /1,25 D3和辐射对MCF-7、MCF-7 Her-2/neu和SKBR3细胞中内质网应激（elF2a、JNK和XBP-1、PERK、re -1）和mTOR （4EBP-1和S6k1）通路的影响。我们将通过使用内质网应激介质（perk和IRE-1）的shRNA基因敲低或mTOR的过表达来评估每个通路参与我们系统的有效性。总之，我们期望这些研究能为临床应用维生素D3及其类似物加强乳腺癌放疗，预防疾病复发，延长乳腺癌患者的生命提供实验基础。