Roles of polymorphic COMT, tea polyphenols and proteasome in cancer prevention

描述（由申请人提供）：研究支持蛋白酶体抑制剂作为潜在的新型抗癌药物和绿茶多酚（gtp）作为癌症预防剂。我们已经报道了gtp，例如(-)-表没食子儿茶素- 3-没食子酸酯或EGCG，是蛋白酶体凝乳胰蛋白酶样活性（由b5亚基介导）的有效和特异性抑制剂。人类多态儿茶酚- o -甲基转移酶（COMT）介导了gtp的甲基化，这是限制其体内抗癌活性的主要生物转化反应。点突变的COMT，在-25%的美国人群中发现，酶活性降低3-4倍（COMT- ll）。据报道，至少有一个低活性COMT等位基因（但不是COMT- hh）的女性饮茶者与不饮茶者相比，患乳腺癌的风险显着降低，这表明甲基化多酚的保护作用较小。为了研究其机制，我们假设乳腺癌细胞中COMT对gtp的o -甲基化降低了它们的蛋白酶体抑制作用，从而降低了它们的生物活性。我们的初步结果支持这一假设。为了进一步研究，我们提出了5个具体目标。(1)合成o -甲基化GTP类似物（用-OCH3取代-OH）、脱氧化合物（不含儿茶酚二醇）和多酚同源物（用-CH2OH取代-OH），并通过计算模型确定它们与蛋白酶体b5亚基的结合亲和力。(2)用纯化的20S蛋白酶体、乳腺癌细胞提取物和完整的乳腺癌细胞来评估这些合成化合物抑制蛋白酶体活性的能力。(3)评价这些合成化合物对人乳腺癌细胞的诱导凋亡能力。(4)测定培养的人乳腺癌细胞中COMT基因过表达和过低表达对gtp甲基化及其蛋白酶体抑制和诱导凋亡活性的影响。(5)通过移植人乳腺肿瘤的裸鼠实验，确定GTPs甲基化是否是使其生物功能失活的机制。这些研究将有助于了解多态COMT如何调节乳腺癌细胞中GTP的生物学功能，以及COMT对GTP甲基化对饮茶预防癌症作用的影响，并为设计未来使用GTP单独或与COMT抑制剂联合使用的癌症预防临床试验提供重要信息。

DESCRIPTION (provided by applicant): Research has supported proteasome inhibitors as potential novel anticancer drugs and green tea polyphenols (GTPs) as a cancer-preventative agent. We have reported that GTPs, e.g. (-)-epigallocatechin- 3-gallate or EGCG, are potent and specific inhibitors of proteasomal chymotrypsin-like activity (mediated by b5 subunit). Methylation of GTPs, a major biotransformation reaction limiting their cancer-preventative activities in vivo, is mediated by human polymorphic catechol-O-methyltransferase (COMT). The point- mutated COMT, found in -25% of US population, has 3-4-fold decreased enzymatic activity (COMT-LL). It was reported that women tea-drinkers with at least one low activity COMT allele (but not COMT-HH) showed a significantly reduced risk of breast cancer compared with non-tea drinkers, suggesting less protection by methylated polyphenols. To study the responsible mechanism, we hypothesize that O-methylation of GTPs by COMT in breast cancer cells decreases their proteasome-inhibitory and consequently biological activities. This hypothesis is supported by our preliminary results. To further this study, we propose 5 specific Aims. (1) Synthesize O-methylated GTP analogs (by replacing -OH with -OCH3), the deoxy compounds (without the catechol diol), and polyphenol homologs (replacing -OH with -CH2OH) and determine their binding affinity to the proteasomal b5 subunit by employing computational modeling. (2) Evaluate potencies of these synthetic compounds to inhibit the proteasome activity using purified 20S proteasome, breast cancer cell extracts, and intact breast cancer cells. (3) Evaluate the apoptosis-inducing potencies of these synthetic compounds in human breast cancer cells. (4) Determine the effects of over- and under-expression of COMT gene in cultured human breast cancer cells on methylation of GTPs and their proteasome-inhibitory and apoptosis-inducing activities. (5) Determine whether methylation of GTPs is a mechanism to inactivate their biological functions in vivo using nude mice bearing human breast tumor xenografts. These studies should help develop a fundamental understanding about how the polymorphic COMT regulates the biological functions of GTPs in breast cancer cells and the impact of GTP methylation by COMT on the cancer- preventative effects of tea consumption as well as generate important information for designing future cancer prevention clinical trials using GTPs alone or in combination with a COMT inhibitor.