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，可以提供包括吲哚-3-甲醇（I3C）在内的几种化合物，可能对人类前列腺癌的发展有保护作用。尽管I3C具有重要的作用，但对I3C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尚缺乏了解。我们的初步结果表明，对于培养的前列腺癌细胞，I3C及其体内二聚体产物，二吲哚基甲烷（DIM）通过对几种细胞周期和凋亡相关蛋白的多效性作用，抑制细胞增殖，同时增加凋亡细胞死亡。最重要的是，我们在前列腺癌细胞中观察到I3C/DIM对Akt和NF-kappaB信号通路的失活，而在非致瘤性前列腺上皮细胞中则没有。基于这些初步结果，我们假设I3C和DIM （I3C/DIM）作为Akt的抑制剂，导致NF-kappaB失活，进而导致细胞生长抑制和诱导凋亡。我们进一步假设，在SCID小鼠骨转移模型中，I3C/DIM抑制转移性前列腺肿瘤的生长，并且暴露于DIM的动物肿瘤会导致特定基因的表达改变，这将是I3C/DIM在前列腺癌转移过程中的体内分子靶点。为了验证我们的假设，我们提出了四个具体目标。在aim-1中，我们将确定用I3C/DIM治疗非致瘤性和致瘤性前列腺上皮细胞是否在相同或不同程度上使NF-kappaB通路失活，并且(b)通过共聚焦显微镜测量IkappaB的磷酸化和泛素化以及NF-kappaB核易位的抑制来研究NF-kappaB失活的机制，并将这些结果与凋亡细胞死亡联系起来。在aim-2中，我们将通过基因转染研究作为NF-kappaB重要调节因子的细胞信号分子，如MEKK1、MEK、NIK和IKK，来研究Akt失活导致NF-kappaB下调的机制。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，我们将使用DIM对PC-3人前列腺癌细胞诱导的人前列腺癌Hu-SCID模型进行饮食干预实验。最后，在aim-4中，我们将使用DNA微阵列通过询问22,215个基因（胡- 133a芯片，Affymetrix），从Aim-3中获得DIM处理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进行的初步研究中发现的其他分子靶点以及特定aim-2下描述的几个靶点相关。总的来说，这些结果将有助于确定前列腺癌中I3C/DIM的全面分子靶标谱，可以进一步开发用于预防和/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.