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

描述(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，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的活性成分)或其类似物可增强对放射(和阿霉素化疗)的敏感性。我的研究集中在证实1，25-D3和维生素D3类似物EB1089(EB)在辐射增敏乳腺癌细胞中的作用，并阐明参与辐射增敏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处理的乳腺肿瘤细胞的辐射增敏模式。药理学方法(3-甲基腺嘌呤、巴菲霉素和氯醌)和遗传方法(针对Beclin-1、ATG5和ATG7的shRNA)将用于抑制自噬。自噬将通过成熟的方法进行监测，包括吖啶橙染色、Western blotting检测LC3裂解，以及电子显微镜。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对受照射细胞增殖恢复的抑制将被逆转。第二个特异点是确定EB和1，25-D3是否能增敏放射抵抗细胞，用1，25-D3或EB处理高表达HER-2/neu的MCF-7 HER-2/neu和SKBR3细胞，用台盼蓝拒染法和克隆形成法检测细胞存活率。细胞死亡将通过TUNEL法检测细胞凋亡，吖啶橙/LC3裂解检测自噬，微核形成通过DAPI染色检测有丝分裂突变。我们预计EB和1，25-D3将被证明可以克服通过过度表达HER-2/neu而产生的辐射抗性。第三个具体目的是确定ER应激和mTOR通路在乳腺癌细胞放射增敏中的参与/调制。我们建议通过蛋白质印迹分析来检测EB/1，25 D3和辐射对MCF-7、MCF-7 HER-2/neu和SKBR3细胞中参与ER应激的关键介质(elF2a、JNK和XBP-1、PERK、IRE-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&ire-1)的shRNA或mTOR的过表达来评估每条途径在我们系统中的参与情况。总体而言，我们预计这些研究将为维生素D3及其类似物在加强乳腺癌放射治疗、防止疾病复发和延长乳腺癌患者生命方面的临床应用提供实验基础。

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.