Novel Targets of Indoles in Prostate Cancer

描述（由申请人提供）：流行病学研究表明，前列腺癌的发病率较低可能是由于环境因素，特别是饮食。食用水果和蔬菜，提供几类化合物，包括吲哚-3-甲醇（I3 C），可能对人类前列腺癌的发展具有保护作用。尽管I3 C的重要性，但缺乏关于I3 C在体外和体内作用的分子机制及其对前列腺癌的抗肿瘤活性的知识。我们的初步研究结果表明，前列腺癌细胞培养中，I3 C和其在体内的二聚体产品，二吲哚甲烷（DIM）抑制细胞增殖，伴随着增加凋亡细胞死亡的多效效应对几个细胞周期和凋亡相关蛋白。最重要的是，我们在前列腺癌细胞中观察到I3 C/DIM对Akt和NF-κ B信号通路的失活，但在非致瘤性前列腺上皮细胞中未观察到。基于这些初步结果，我们假设I3 C和DIM（I3 C/DIM）作为Akt的抑制剂发挥作用，导致NF-κ B失活，进而导致细胞生长抑制和诱导凋亡。我们进一步假设I3 C/DIM抑制SCID小鼠实验性骨转移模型中转移性前列腺癌的生长，并且暴露于DIM的动物肿瘤将导致特定基因表达的改变，这些基因将是I3 C/DIM在前列腺癌转移过程中的体内分子靶点。为了验证我们的假设，我们提出了四个具体目标。在目的-1中，我们将确定用I3 C/DIM处理非致瘤性和致瘤性前列腺上皮细胞是否使NF-κ B通路失活至相同或不同程度，以及（B）通过使用共聚焦显微镜测量Ikappa B的磷酸化和泛素化以及NF-κ B的核转位抑制来研究NF-κ B失活的机制，并将这些结果与凋亡性细胞死亡相关联。在目的-2中，我们将通过使用基因转染研究来研究作为NF-κ B的重要调节剂的细胞信号传导分子如MEKK 1、MEK、NIK和IKK来研究Akt失活导致NF-κ B下调的机制。这些研究将确定这些信号分子与I3 C/DIM诱导的前列腺癌细胞凋亡之间的因果关系。在目的3中，我们将在PC-3人前列腺癌细胞诱导的人前列腺癌Hu-SCID模型中使用DIM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-133 A Chip，Affyphase）使用DNA微阵列鉴定从我们的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I3 C/DIM的初步研究期间鉴定的其他分子靶标以及特定目标-2下描述的几种靶标相关。总的来说，这些结果将有助于确定I3 C/DIM在前列腺癌中的全面分子靶点谱，其可以进一步用于设计用于预防和/或治疗前列腺癌及其转移的相关靶向治疗。

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.