Vitamin D in Prostate Cancer: Tumor Vasculature Effects

维生素 D 在前列腺癌中的作用：肿瘤脉管系统的影响

• 批准号: 7688551
• 负责人: CANDACE S JOHNSON
• 金额: $ 27.36万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7688551
• 关键词: 25-hydroxycholecalciferol-24-hydroxylase; Address; Affect; Apoptosis; Apoptotic; Binding Proteins; Biological Availability; Biological Models; Breast Carcinoma; Calcitriol; Catabolism; Cell Cycle Arrest; Cell Death; Cells; Characteristics; Conditioned Culture Media; CpG Islands; Cytotoxic agent; DNA; DNA Methylation; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; Data; Defect; Deoxycytidine; Dexamethasone; Dihydroxycholecalciferols; Endothelial Cells; Endothelium; Enzymes; Epigenetic Process; Event; Fibroblasts; G1 Arrest; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genome Scan; Glucocorticoids; Human; Incubated; Kinetics; Laboratories; Lasers; Lead; Malignant Neoplasms; Malignant neoplasm of prostate; Mammary Gland Parenchyma; Mediating; Messenger RNA; Metabolism; Methods; Methylation; Microscopy; Minerals; Mus; Normal Cell; Normal tissue morphology; Pattern; Pharmaceutical Preparations; Phase I Clinical Trials; Phase II Clinical Trials; Phosphotransferases; Population; Prostatectomy; Proteins; Resistance; Role; Screening procedure; Signal Pathway; Signal Transduction; Signaling Molecule; System; Therapeutic; Time; Tissues; Tumor-Derived; Tumor-Suppressor Gene Inactivation; Vitamin D; Vitamin D3 Receptor; androgen independent prostate cancer; angiogenesis; base; bone; enzyme activity; histone modification; in vitro activity; in vivo; matrigel; men; neoplastic cell; promoter; receptor; tumor; tumor xenograft

DESCRIPTION (provided by applicant): Calcitriol (vitamin D or 1,25 dihydroxycholecalciferol), a central factor in bone and mineral metabolism, has significant antitumor activity in vitro and in vivo. Induction of CYP24, the enzyme primarily responsible for calcitriol catabolism, may be a factor in the anti-proliferative activity pre-clinically and clinically. In addition, the endothelial cells in tumors are sensitive to calcitriol and uniquely modulate CYP24 expression through epigenetic changes. Epigenetic events affect gene expression without alteration in DNA gene sequence and lead to transcriptional gene silencing and inactivation of tumor suppressor genes in human cancer. While many studies document epigenetic changes in tumor cells, only limited data support a role for epigenetic changes in the "normal" cells found in the tumor microenvironment. Calcitriol also inhibits proliferation of endothelial cells and can inhibit angiogenesis in a number of tumor model systems. We have established a method for the isolation of fresh, tumor-derived endothelial cells (TDEC) that maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). Differences may be due to the over-expression of CYP24 in MDEC where mRNA, protein and enzymatic activity for CYP24 are markedly increased. In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5' end, which may contribute to gene silencing of CYP24 in TDEC. The extent of methylation in these two regions is significantly less in MDEC. Treatment of TDEC with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. Lastly, when MDEC are incubated with conditioned media from tumor cells for 14-21 days, these cells regain sensitivity to the antiproliferative effects of calcitriol, CYP24 induction is silenced and regions 1 and 2 of the CYP24 promoter are hypermethylated. The characteristics of these cells are similar to the endothelial cells found in tumors or TDEC. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application. Therefore, we propose 1) to elucidate the epigenetic signal transduction mechanism(s) that lead to the epigenetic silencing of CYP24; 2) to determine whether epigenetic silencing of CYP24 occurs in human endothelial cell and tumor cell populations isolated from human prostate cancer; and 3) To identify other potential targets of DNA methylation in TDEC vs. MDEC by investigating the scope of the epigenetic defect from the tumor microenvironment on the endothelial cells. We developed a model system for isolation of endothelial cells freshly from tumors and demonstrate that CYP24, the catabolic enzyme involved in vitamin D (1,25 dihydroxycholecalciferol or calcitriol) signaling, becomes epigenetically silenced selectively in tumor-derived endothelial cells. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application.

说明(申请人提供)：骨化三醇(维生素D或1，25二羟基胆钙化醇)是骨骼和矿物质代谢的中心因子，在体外和体内都具有显著的抗肿瘤活性。主要负责骨化三醇分解代谢的酶--细胞色素P24的诱导，可能是临床前和临床抗增殖活性的一个因素。此外，肿瘤中的内皮细胞对骨化三醇敏感，并通过表观遗传学变化独特地调节CYP24的表达。表观遗传事件在不改变DNA基因序列的情况下影响基因表达，并导致人类癌症中转录基因沉默和肿瘤抑制基因失活。虽然许多研究记录了肿瘤细胞的表观遗传变化，但只有有限的数据支持在肿瘤微环境中发现的“正常”细胞的表观遗传变化的作用。骨化三醇还可以抑制血管内皮细胞的增殖，并在一些肿瘤模型系统中抑制血管生成。我们已经建立了一种分离新鲜的肿瘤来源的内皮细胞(TDEC)的方法，这种细胞具有不同于从正常组织和Matrigel Plugs(MDEC)分离的内皮细胞的表型特征。在TDEC中，骨化三醇诱导G0/G1期停滞，调节p27和p21，诱导细胞凋亡，降低P-Erk和P-Akt。相反，从正常组织和MDEC分离的内皮细胞对骨化三醇介导的抗增殖作用没有反应，尽管通过维生素D受体(VDR)传递完整的信号。差异可能是由于细胞色素P24在MDEC中的过度表达，其mRNA、蛋白和酶活性显著增加。在对骨化三醇敏感的TDEC中，位于5‘端的两个CpG岛区的CYP24启动子高甲基化，这可能是导致TDEC中CYP24基因沉默的原因之一。在MDEC中，这两个区域的甲基化程度明显较低。用DNA甲基转移酶抑制剂5-氮杂-2‘-脱氧胞苷处理TDEC，可恢复骨化三醇介导的细胞色素P24的诱导和对骨化三醇的抵抗。最后，当MDEC与肿瘤细胞的条件培养液孵育14-21天时，这些细胞对骨化三醇的抗增殖作用恢复敏感，细胞色素P24的诱导被沉默，细胞色素P24启动子的1和2区域高甲基化。这些细胞的特征类似于肿瘤或TDEC中的内皮细胞。这些独特的差异从肿瘤微环境中对骨化三醇诱导的血管内皮细胞中细胞色素P24基因表达的表观遗传沉默，直接影响骨化三醇介导的信号通路，最终影响治疗应用。因此，我们建议1)阐明导致细胞色素P24表观遗传沉默的表观遗传信号转导机制(S)；2)确定在分离自人前列腺癌的人内皮细胞和肿瘤细胞中是否发生表观遗传沉默；3)通过研究肿瘤微环境对内皮细胞的表观遗传缺陷的范围，确定TDEC和MDEC中其他潜在的DNA甲基化靶点。我们开发了一个从肿瘤中分离新鲜内皮细胞的模型系统，并证明了参与维生素D(1，25二羟基胆钙化醇或骨化三醇)信号转导的分解代谢酶CYP24在肿瘤来源的内皮细胞中选择性地表观遗传沉默。这些独特的差异从肿瘤微环境中对骨化三醇诱导的血管内皮细胞中细胞色素P24基因表达的表观遗传沉默，直接影响骨化三醇介导的信号通路，最终影响治疗应用。