Novel Targets of Indoles in Prostate Cancer

描述(由申请人提供)：流行病学研究表明，前列腺癌发病率较低可能是由于环境因素，特别是饮食因素。食用水果和蔬菜提供了几类化合物，包括吲哚-3-甲醇(I3C)，可能对人类前列腺癌的发展具有保护作用。尽管I3C很重要，但对其体内外作用的分子机制(S)及其抗前列腺癌活性缺乏了解。我们的初步结果表明，对于培养中的前列腺癌细胞，I3C及其体内二聚体产物二吲哚甲烷(DIM)通过对几种细胞周期和凋亡相关蛋白的多向性作用，抑制细胞增殖，伴随着凋亡细胞死亡的增加。最重要的是，我们观察到在前列腺癌细胞中，I3C/DIM使Akt和NF-kappaB信号通路失活，但在非致癌的前列腺上皮细胞中没有。根据这些初步结果，我们假设I3C和DIM(I3C/DIM)作为Akt的抑制因子，导致NF-kappaB失活，进而导致细胞生长抑制和诱导细胞凋亡。我们进一步假设I3C/DIM能抑制SCID小鼠实验性骨转移模型中前列腺癌转移瘤的生长，并且DIM暴露会导致特定基因表达的改变，这将是I3C/DIM在前列腺癌转移过程中的体内分子靶点。为了检验我们的假设，我们提出了四个具体目标。在AIM-1中，我们将确定I3C/DIM处理非致瘤和致癌的前列腺上皮细胞是否在相同或不同程度上使NF-kappaB途径失活，以及(B)通过共聚焦显微镜检测IkappaB的磷酸化和泛素化以及对NF-kappaB核转位的抑制来探讨NF-kappaB失活的机制(S)，并将这些结果与细胞凋亡相关联。在AIM-2中，我们将通过基因转染研究作为核因子-kappaB重要调节因子的细胞信号分子，如mekk1、mek、nik和ikk来探讨Akt失活导致nf-kappaB下调的机制(S)。这些研究将确定这些信号分子与I3C/DIM诱导的前列腺癌细胞凋亡之间的因果关系。在AIM-3中，我们将利用DIM对PC-3人前列腺癌细胞诱发的人前列腺癌模型进行饮食干预实验。最后，在AIM-4中，我们将通过询问22,215个基因(HU-133a芯片，Affymetrix)，使用DNA微阵列来鉴定从我们的AIM-3获得的DIM处理的PCa的基因表达谱。这些结果将与我们在培养的PC-3细胞中使用I3C/DIM进行初步研究期间确定的其他分子靶标以及特定的AIM-2中描述的几个靶标相关联。总而言之，这些结果将有助于确定前列腺癌中I3C/DIM的全面分子靶标，可进一步利用这些靶标设计相关的靶向疗法，以预防和/或治疗前列腺癌及其转移。

DESCRIPTION (provided by applicant): Epidemiological studies have suggested that lower incidence of prostate cancer could be due to environmental factors, particularly dietary. Consumption of fruits and vegetables, which provides several classes of compounds, including indole-3- carbinol (I3C), may have a protective effect against the development of human prostate cancer. Despite the importance of I3C, knowledge on the molecular mechanism(s) of action of I3C in vitro and in vivo, and its anti-tumor activity against prostate cancer is lacking. Our preliminary results show that, for prostate cancer cells in culture, I3C and its in vivo dimeric product, Diindolylmethane (DIM) inhibits cell proliferation with a concomitant increase in apoptotic cell death by their pleitropic effects on several cell cycle and apoptosis related proteins. Most importantly, we observed inactivation of Akt and NF-kappaB signaling pathway by I3C/DIM in prostate cancer cells but not in non-tumorigenic prostate epithelial cells. Based on these preliminary results, we hypothesize that I3C and DIM (I3C/DIM) function as an inhibitor of Akt, leading to the inactivation of NF-kappaB, which in turn, causes cell growth inhibition and induction of apoptosis. We further hypothesize that I3C/DIM inhibits metastatic prostate tumor growth in experimental bone metastasis model in SCID mice, and that the animal tumors exposed to DIM will lead to alterations in the expression of specific genes, which will be the in vivo molecular targets of I3C/DIM in the processes of prostate cancer metastases. To test our hypotheses, we propose four specific aims. In aim-1, we will determine whether treatment of non-tumorigenic and tumorigenic prostate epithelial cells with I3C/DIM inactivates the NF-kappaB pathway to the same or different degrees, and (b) investigate the mechanism(s) of NF-kappaB inactivation by measuring phosphorylation and ubiquitination of IkappaB and inhibition in the nuclear translocation of NF-kappaB using confocal microscopy, and correlate these results with apoptotic cell death. In aim-2, we will investigate the mechanism(s) by which Akt inactivation leads to the down regulation of NF-kappaB by investigating cell signaling molecules that are important regulators of NF-kappaB such as MEKK1, MEK, NIK and IKK using gene transfection studies. These investigations will determine the cause and effect relationships between these signaling molecules and I3C/DIM induced apoptotic cell death in prostate cancer cells. In aim-3, we will conduct dietary intervention experiments using DIM in Hu-SCID model of human prostate cancer induced by PC-3 human prostate cancer cells. Finally, in aim-4, we will identify gene expression profiles of DIM treated PCa obtained from our Aim-3 using DNA Microarray by interrogating 22, 215 genes (Hu-133A Chip, Affymetrix). These results will be correlated with other molecular targets identified during our preliminary studies using I3C/DIM in cultured PC-3 cells, and also several targets as described under specific aim-2. Collectively, these results will help to identify a comprehensive molecular targets profile of I3C/DIM in prostate cancer that could be further exploited for devising relevant targeted therapy for the prevention and/or treatment of prostate cancer and its metastasis in the future.