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

描述(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，绿茶多酚(GTP)作为防癌药物。我们已经报道，GTP，例如(-)-表没食子儿茶素没食子酸酯或EGCG，是蛋白酶体糜蛋白酶样活性(由b5亚基介导)的有效和特异的抑制剂。GTP的甲基化是体内限制其抗癌活性的主要生物转化反应，由人类多态儿茶酚氧位甲基转移酶(COMT)介导。在-25%的美国人中发现了点突变的COMT，其酶活性(COMT-LL)降低了3-4倍。据报道，至少有一个低活性COMT等位基因(而不是COMT-HH)的女性饮茶者与不喝茶的女性相比，患乳腺癌的风险显著降低，这表明甲基化多酚的保护作用较弱。为了研究相关机制，我们假设乳腺癌细胞中COMT对GTP的O-甲基化降低了它们对蛋白酶体的抑制，从而降低了它们的生物学活性。这一假设得到了我们的初步结果的支持。为了进一步推进这项研究，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(不含邻苯二酚二醇)和多酚同系物(用-CH2OH取代-OH)，并通过计算模拟测定它们与蛋白酶体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评价这些合成化合物对蛋白酶体活性的抑制作用。(3)评价这些合成化合物对人乳腺癌细胞的诱导凋亡作用。(4)检测COMT基因在体外培养的人乳腺癌细胞中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)在荷人乳腺癌裸鼠体内，确定GTP甲基化是否为其生物学功能失活的机制。这些研究应该有助于对多态的COMT如何调节乳腺癌细胞中GTP的生物学功能以及COMT的GTP甲基化对茶的防癌效果的影响有一个基本的了解，并为设计未来单独使用GTPS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.