Chemoprevention of Bladder CA by soybean bioactive comp.

描述(申请人提供)：这项提案的重点是大豆活性成分对膀胱癌的化学预防活性。这一假说认为，增加膳食中大豆活性成分的摄入量可能是一种有效的营养方案，通过调节膀胱癌血管生成、凋亡和/或增殖来预防膀胱癌的进展和转移。支持这一假说的理论基础是：(1)大豆活性成分通过血液循环和与膀胱粘膜的直接接触发挥抗膀胱癌的作用；(2)我们的动物实验和体内机制研究表明，大豆植物化学物质通过诱导肿瘤细胞凋亡、抑制肿瘤增殖和抑制肿瘤血管生成来抑制膀胱癌的进展。在这项提案中，将通过临床相关的原位膀胱模型来设计三个特定的目标来检验我们的假设。具体目的1是确定大豆成分对高分化、低转移(RT4)和低分化、高转移(253J B-V)人膀胱肿瘤原位生长和/或转移的影响。将评估大豆成分(大豆异黄酮缺失的大豆蛋白、大豆植物化学浓缩物和大豆染料木素)对体内RT4和253J-B-V肿瘤生长和转移的影响。具体目的2是确定大豆成分对与肿瘤细胞凋亡、增殖和肿瘤血管生成相关的肿瘤生物标志物表达的影响。这些细胞凋亡和增殖的生物标志物包括：(A)凋亡指数和相关的凋亡诱导因子(P53、p21/WAFL、Bax、TNF-α)和凋亡抑制因子(bcl2、bclxl)；(B)增殖指数和细胞周期相关的细胞周期蛋白(A、D、E)和细胞周期蛋白依赖蛋白(CDKs2、4、6)。我们的体外微阵列分析表明，与血管生成网络相关的标记是受大豆成分显著调控的重要基因之一。这些血管生成生物标志物包括肿瘤血管生成因子(血管内皮生长因子、碱性成纤维细胞生长因子、血管生成素-1)和抗血管生成因子血管生成素-2。采用逆转录聚合酶链式反应(RT-PCR)和免疫组织化学结合图像分析的方法检测上述分子标志物的表达。目的3利用微阵列基因芯片技术，进一步阐明基因和大豆活性成分在体内外对膀胱癌调控作用的相互作用机制。通过使用包含与大约12,000个全长人类cDNA互补的序列的寡核苷酸阵列，将在体外和体内样本中确定剂量依赖和时间依赖的mRNA表达模式。其中一些显著不同的基因转录本将通过RT-PCR进行确认。通过将营养学、分子生物学和遗传学整合到膀胱癌预防研究中，拟议的研究结果将有望为饮食大豆在膀胱癌预防中的应用提供重要的见解。

DESCRIPTION (provided by applicant): This proposal is focused on the chemopreventive activity of soybean bioactive components on bladder cancer. The hypothesis is that increased dietary consumption of soybean bioactive components may serve as an effective nutritional regimen for the prevention of bladder cancer progression and metastasis by modulating bladder tumor angiogenesis, apoptosis and/or proliferation. The rationale for supporting this hypothesis is based on the followings: (1) Soy bioactive components exert their effects on anti-bladder cancer via both blood circulation and direct contact with the mucosa of the bladder, and (2) Our animal studies and in vivo mechanistic studies indicated that soy phytochemicals inhibited bladder tumor progression by inducing tumor cell apoptosis, inhibiting tumor proliferation and inhibiting tumor angiogenesis. In this proposal, three specific aims will be designed to test our hypothesis by using a clinically relevant orthotopic bladder model. Specific Aim 1 is to determine the effect of soybean components on orthotopic growth and/or metastasis of both well-differentiated, low metastatic (RT4) and poorly differentiated, highly metastatic (253J B-V) human bladder tumors. Soybean components (soy isoflavone-depleted soy protein, soy phytochemical concentrate, and soy genistin) will be evaluated for their effects on the growth and metastasis of both RT4 and 253J-B-V tumors in vivo. Specific aim 2 is to determine the effect of soybean components on the expression of tumor biomarkers that are related to tumor cell apoptosis and proliferation and tumor angiogenesis. These apoptotic and proliferation biomarkers include (a) apoptotic index and related apoptosis inducers (p53, p21/wafl, bax, TNF-alpha) and apoptosis repressors (bcl-2, bcl-xl), (b) proliferation index and cell cycle related cyclins (A, D, E) and cyclin-dependent kinases (cdks 2, 4, 6). Our in vitro microarray assays have suggested that markers that are related to angiogenesis network are among important genes that are significantly modulated by soy components. These angiogenic biomarkers include tumor angiogenic factors (VEGF, bFGF, angiopoietin-1) and anti-angiogenic factor angiopoietin-2. RT-PCR and immunohistochemistry followed by image analysis will be applied to determine the expression of above molecular markers. Specific aim 3 is to further elucidate mechanisms involved in the interactive affect of genes and soy bioactive components on bladder cancer modulation in vitro and in vivo by employing microarrayed cDNA chip technology. By using an oligonucleotide array containing sequences complementary to about 12,000 full-length human cDNAs, both dose-dependent and time-dependent mRNA expression patterns will be determined in both in vitro and in vivo samples. Some of these significantly different gene transcripts will be confirmed by RT-PCR. Results derived from the proposed studies by integrating nutrition, molecular biology and genetics in bladder cancer prevention research will be expected to provide significant insight into the utilization of dietary soy in the prevention of bladder cancer.