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

项目摘要 吸烟仍然是口腔鳞状细胞癌 (OSCC) 的高危因素 强效烟草致癌物，包括多环芳烃 (PAH)，例如二苯并[a,l]芘 (DB[a,l]P)、苯并[a]芘 (B[a]P) 和烟草特有的亚硝胺 (TSNA)，例如 N'-亚硝基降烟碱 （NNN）。为了评估这些致癌物对口腔粘膜的影响，我们开发了一种新型 OSCC 小鼠模型 使用 DB[a,l]P 及其峡湾地区二醇环氧化物 (DB[a,l]PDE)。重要的是，我们表明饮食干预 冻干黑树莓 (BRB) 粉末可抑制致癌物引起的 DNA 损伤、诱变和 小鼠口腔的致癌作用。我们还确定 BRB 可以减少形成和/或增强 体外修复大体积加合物，但是，BRB 减少 DNA 损伤的机制仍有待充分研究 阐明了。考虑到DNA加合物的不同结构，我们将重点关注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 BRB 中，例如花青素，BRB 有助于保持细胞氧化还原平衡、增强氧化还原作用是合乎逻辑的 清除作用，从而防止 DNA 氧化损伤；我们的数据支持这一主张。另外 其细胞保护功能，特定的细胞还原等效 NADPH 功能在还原 生物合成反应，包括将核糖核苷酸转化为必需的脱氧核糖核苷酸 (dNTP) DNA 修复成分。根据这些数据，我们假设 BRB 可以减少 DNA 损伤（参见 方案 1，意义）以多模式方式：1）BRB 通过保留增强 NER 和 BER 功能 关键底物和辅因子（即 dNTP 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保持细胞氧化还原状态 可以抑制 DNA 修复酶，从而减少 DNA 氧化损伤。所提出的机制 研究需要两个互补但独立的具体目标：目标 1A 将调查 BRB 的影响 原代人体内共价烟草致癌物-DNA 加合物和 8-OXO-dG 的修复（NER、BER） 使用我们建立的测定法，用这些加合物转染口腔角质形成细胞。目标 1B 将确定 BRB 防止野生型氧化损伤的能力（OGG1 / ，一种成分 BER 酶）和敲除（OGG1-/-）MEF 细胞。适用于两个子目标的并行研究将 评估 BRB 对 dNTP 库和关键细胞氧化还原平衡参数的连续保存的影响 经验证的细胞范围包括原代人口腔角质形成细胞、口腔白斑和 OSCC 细胞。目标2将 首次确定局部 BRB 递送对共价 DNA 加合物形成的影响 健康吸烟者口腔细胞中的 8-OXO-dG。结果可以作为未来的框架 针对无法戒烟的成瘾吸烟者以及有戒烟史的非吸烟者或前吸烟者进行的化学预防试验 接触环境致癌物质。

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.