Chemoprevention by Black Raspberry of Oral Cancer Induced by Tobacco Carcinogens: Translational Studies

项目摘要 吸烟仍然是接触下列物质导致口腔鳞状细胞癌(OSCC)的高危因素 强烈的烟草致癌物质，包括多环芳烃(PAHs)，如二苯并[a，L]芘 (DB[a，L]P)，苯并[a]芘(B[a]P)和烟草特有的亚硝胺，如N‘-亚硝胺或尼古丁 (NNN)。为了评估这些致癌物对口腔粘膜的影响，我们建立了一种新的口腔鳞癌小鼠模型。 使用Db[a，L]P及其峡区二醇环氧化物(Db[a，L]PDE)。重要的是，我们证明了饮食干预 用冷冻干燥的黑覆盆子(BRB)粉抑制致癌物诱导的DNA损伤，诱变和 小鼠口腔中的致癌作用。我们还证实了BRB减少形成和/或增强 体外修复体积庞大的加合物，但BRB减少DNA损伤的机制仍未完全阐明 已澄清。考虑到DNA加合物的不同结构，我们将重点研究BRB对核苷酸的影响 切除修复(NER)和碱基切除修复(BER)酶。基于丰富的酚类化合物 在花青素等BRB中，提出BRB有助于保持细胞氧化还原平衡、增强氧化还原能力是合乎逻辑的 清除，从而起到防止DNA氧化损伤的作用；我们的数据支持这一观点。此外 对于它们的细胞保护功能，一种特定的细胞还原等价物-NADPH-在还原中发挥作用 生物合成反应，包括将核糖核苷酸转化为脱氧核糖核苷酸(DNTPs)，这是必不可少的 用于DNA修复的组件。基于这些数据，我们假设BRB可以减少DNA损伤(参见。 方案1，重要性)以多模式方式：1)BRB通过保存来增强NER和BER功能 关键底物和辅因子，即dNTPs和mg2，同时保持最佳的非氧化环境 有利于DNA修复；2)BRB通过有效清除活性物质来维持细胞的氧化还原状态 这可以抑制DNA修复酶，从而减少DNA氧化损伤。拟议的机械论 研究需要两个相辅相成但又独立的具体目标：目标1A将调查BRB的影响 共价烟草致癌物-DNA加合物和8-OXO-DG在原代人体内的修复(NER，BER) 用我们建立的检测方法将这些加合物导入口腔角质形成细胞。目标 1B将确定BRB在野生型(OGG1/)中防止氧化损伤的能力 BER酶)和基因敲除(OGG1-/-)MEF细胞。同时进行的研究，适用于两个次级目标，将 评估BRB对保存dNTP池和关键细胞氧化还原平衡参数的影响 验证的细胞范围包括原代人类口腔角质形成细胞、口腔白斑和口腔鳞癌细胞。目标2将 首次确定了局部BRB传递对共价DNA加合物形成的影响和 8-OXO-DG在健康吸烟者口腔细胞中的表达。研究结果可以作为未来研究的框架 对无法戒烟的成瘾吸烟者以及非吸烟者或曾经吸烟者进行化学预防试验 暴露在环境致癌物质中。

Project Abstract Tobacco use remains a high risk factor for oral squamous cell carcinoma (OSCC) resulting from exposure to potent tobacco carcinogens including polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[a,l]pyrene (DB[a,l]P), benzo[a]pyrene (B[a]P) and tobacco-specific nitrosamines (TSNA) such as N’-nitrosonornicotine (NNN). To assess effects of these carcinogens on oral mucosa, we developed a novel OSCC mouse model using DB[a,l]P and its fjord region diol epoxide (DB[a,l]PDE). Importantly, we showed that dietary intervention with freeze-dried black raspberries (BRB) powder inhibited carcinogen-induced DNA damage, mutagenesis and carcinogenesis in the mouse oral cavity. We have also established that BRB reduce formation and/or enhance repair of bulky adducts in vitro, but, the mechanisms by which BRB reduce DNA damage remain to be fully elucidated. Considering the varied structures of DNA adducts, we will focus on BRB effects on the nucleotide excision repair (NER) and base-excision repair (BER) enzymes. Based on the abundant phenolic compounds in BRB e.g. anthocyanins, it is logical to propose that BRB help preserve the cellular redox poise, enhance redox scavenging and thus function to prevent oxidative DNA damage; our data support this proposition. In addition to their cytoprotective functions, a specific cellular reducing equivalent-NADPH-functions in reductive biosynthetic reactions including conversion of ribonucleotides to deoxyribonucleotides (dNTPs) that are essential components for DNA repair. Based on these data, we hypothesize that BRB reduce DNA damage (cf. Scheme 1, Significance) in a multimodal fashion: 1) BRB enhance NER and BER function via preservation of key substrates and cofactors i.e. dNTPs and Mg2+, while maintaining an optimal, non-oxidized environment conducive to DNA repair; 2) BRB preserve the cellular redox status via efficient scavenging of reactive species that can inhibit DNA repair enzymes thereby reducing oxidative DNA damage. The proposed mechanistic studies entail two complementary, yet independent, Specific Aims: Aim 1A will investigate the effects of BRB on repair (NER, BER) of covalent tobacco carcinogen-DNA adducts and 8-OXO-dG in primary human oral keratinocyte cells that have been transfected with these adducts using our established assay. Aim 1B will determine the capacity of BRB to prevent oxidative damage in wild-type (OGG1+/+, a component of BER enzymes) and knockout (OGG1-/-) MEF cells. Concurrent studies, applicable to both subaims, will assess BRB effect on preservation of dNTP pools and key cellular redox poise parameters in a continuum of validated cells ranging from primary human oral keratinocytes, oral leukoplakia and OSCC cells. Aim 2 will determine for the first time the effects of local BRB delivery on formation of covalent DNA adducts and 8-OXO-dG in buccal cells of healthy smokers. The results could serve as the framework for future chemopreventive trials for addicted smokers who are unable to quit as well as non- or former-smokers who are exposed to environmental carcinogens.