Combined use of green tea and quercetin in prostate cancer

描述（由申请人提供）： 绿色茶的抗癌活性已在许多流行病学、动物和细胞培养研究中得到证实。冲泡绿色茶（GT）中负责健康益处的主要绿色茶多酚（GTP）是表没食子儿茶素没食子酸酯（EGCG）和表没食子儿茶素（EGC），以及少量的表儿茶素（EC）和表儿茶素没食子酸酯（ECG）。它们有限的生物利用度和高代谢（结合和甲基化）可能会降低GT在化学预防中的潜力。我们确定，在人类前列腺组织和尿液中，50%或更多的GTP是以甲基化形式存在的，甲基化显著降低了EGCG的抗癌活性。儿茶酚-O-甲基转移酶（COMT）是负责具有儿茶酚结构的多酚的甲基化的酶。儿茶酚类化合物，如EGCG和槲皮素，一种存在于许多水果和蔬菜中的天然类黄酮，抑制前列腺癌细胞（LNCaP）中的COMT活性。槲皮素和EGCG联合处理可显著增强COMT抑制作用，降低EGCG甲基化程度，提高EGCG生物利用度。因此，我们假设槲皮素和绿色茶的共同治疗将通过抑制EGCG甲基化和增加前列腺癌（CaP）中的生物利用度来增加GT的化学预防活性。我们将使用两种不同的小鼠模型来验证我们的假设并研究其机制：A）用单独的绿色茶（GT）、GT和0.2%槲皮素（Q）-饮食、GT+0.4%Q-饮食、单独的0.2%Q-饮食、单独的0.4%Q-饮食或水对照治疗接种LNCaP异种移植瘤的严重联合免疫缺陷（SCID）小鼠;和B）通过用单独的GT、单独的Q和GT+ Q一起处理10号染色体上缺失的转基因磷酸酶和张力蛋白同源物（PTEN）敲除小鼠，使用显示对SCID小鼠最有效的浓度。小鼠前列腺中肿瘤抑制因子PTEN的缺乏概括了人类CaP发展的许多病理特征。为了确定化学预防活性，我们将通过免疫组化测量肿瘤重量和体积、肿瘤分期以及细胞增殖和凋亡。肿瘤组织将用于使用Western印迹和定量实时PCR测量COMT和5-胞嘧啶DNA甲基转移酶1（DNMT 1）酶活性以及蛋白质和基因表达。多药耐药蛋白（MRP）可能在与槲皮素共同治疗增加表没食子儿茶素的生物利用度中发挥作用。因此，我们还将确定肿瘤组织中MRP 1和MRP 2的蛋白和基因表达。通过高效液相色谱法和Coularray电化学检测法分析肿瘤组织、肝、肺和肾中的茶多酚、槲皮素及其代谢产物，确定对生物利用度的影响。这种天然补充剂和替代剂的新型组合利用槲皮素降低GTP甲基化的能力，同时增加其生物利用度。这种新的治疗方法将保留绿色茶的无毒特性，同时增加其在CaP化学预防中的活性。

DESCRIPTION (provided by applicant): The anticarcinogenic activity of green tea has been demonstrated in many epidemiological, animal and cell culture studies. The main green tea polyphenols (GTPs) in brewed green tea (GT) responsible for the health benefits are epigallocatechin gallate (EGCG) and epigallocatechin (EGC), with smaller amounts of epicatechin (EC) and epicatechin gallate (ECG). Their limited bioavailability and high metabolism (conjugation and methylation) may decrease the potential of GT in chemoprevention. We determined that 50 percent or more of GTPs in human prostate tissue and urine is found in methylated form and that methylation significantly decreased the anticarcinogenic activity of EGCG. Catechol-O-methyltransferase (COMT) is the enzyme responsible for methylation of polyphenols with a catechol structure. Catechol-compounds such as EGCG and quercetin, a natural flavonoid occurring in many fruits and vegetable, inhibit COMT activity in prostate cancer cells (LNCaP). Co-treatment of quercetin and EGCG enhanced the COMT inhibitory effect, decreased EGCG methylation and increased EGCG bioavailability significantly. Therefore it is our hypothesis that co-treatment with quercetin and green tea will increase the chemopreventive activity of GT by inhibiting EGCG methylation and increasing the bioavailability in prostate cancer (CaP). We will test our hypothesis and investigate the mechanism using two different mouse models: A) by treating severe combined immunodeficient (SCID) mice inoculated with LNCaP xenograft tumors with green tea (GT) alone, GT and 0.2% quercetin (Q)-diet, GT+0.4%Q-diet, 0.2%Q-diet alone, 0.4%Q-diet alone or water control; and B) by treating transgenic phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knock out mice with GT alone, Q alone and GT+ Q together using the concentration shown to be most effective for SCID mice. Lack of the tumor suppressor PTEN in the mouse prostate recapitulates many of the pathological features of CaP development in humans. To determine the chemopreventive activity we will measure tumor weight and volume, tumor stage, and cell proliferation, apoptosis by immunohistochemistry. Tumor tissue will be used to measure COMT and 5- cytosine DNA methyltransferase 1 (DNMT1) enzyme activity as well as protein and gene expression using Western blot and quantitative real-time PCR. Multidrug resistance proteins (MRP) may play a role in the increase in bioavailability of EGCG by co-treatment with quercetin. Therefore we will also determine protein and gene expression of MRP1 and MRP2 in tumor tissue. The effect on bioavailability will be determined by analysis of tea polyphenols, quercetin and their metabolites in tumor tissue, liver, lung, and kidney by high performance liquid chromatography and coularray electrochemical detection. This novel combination of natural complementary and alternative agents utilizes the ability of quercetin to reduce the methylation of GTPs and increase their bioavailability at the same time. This novel treatment will retain the non-toxic character of green tea and at the same time increase its activity in chemoprevention of CaP.