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

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)是一种抗癌药物。我们已经报道了GTP，例如(-)-表儿茶素- 3-没食子酸酯或EGCG是蛋白酶体类糜蛋白酶活性的有效和特异的抑制剂(由 B5亚基)。限制其防癌作用的主要生物转化反应--GTP的甲基化 在体内的活动，是由人类多态儿茶酚氧位甲基转移酶(COMT)介导的。重点是- 突变的COMT在-25%的美国人口中被发现，具有3-4倍的酶活性降低(COMT-LL)。它 据报道，至少有一个低活性COMT等位基因(但不是COMT-HH)的女性饮茶者表现出 与不喝茶的人相比，患乳腺癌的风险显著降低，这表明 甲基化多酚。为了研究相关机制，我们假设GTP的O-甲基化 乳腺癌细胞中的COMT降低了它们的蛋白酶体抑制，从而降低了生物活性。 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)，并测定它们的结合亲和力 通过使用计算模型将蛋白酶体b5亚基。(2)评估这些项目的潜力 利用纯化的20S蛋白酶体抑制乳腺癌细胞蛋白酶体活性的合成化合物 提取物和完整的乳腺癌细胞。(3)评价这些化合物诱导细胞凋亡的能力。 人类乳腺癌细胞中的化合物。(4)检测COMT过表达和过低表达的影响 人乳腺癌细胞GTP基因甲基化及其蛋白酶体抑制作用的研究 诱导细胞凋亡的活性。(5)确定GTP的甲基化是否是使其失活的机制 荷人乳腺癌裸鼠移植瘤体内生物学功能的研究。这些研究应该 有助于对多态COMT如何调节生物 GTP在乳腺癌细胞中的作用及COMT甲基化对肿瘤的影响 饮茶的预防作用以及为设计未来癌症提供重要信息 单独使用GTPS或与COMT抑制剂联合使用的预防临床试验。

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.