Anti-tumor properties of curcumin in oral cancers: role of thioredoxin reductase

描述（由申请人提供）：硫氧还蛋白还原酶1 （TxRd1）是一种必需的硒蛋白，在细胞生长、死亡（凋亡）、转录、DNA修复和血管生成的氧化还原调节中起着明确的作用。TxRd1在许多人类癌症中过度表达，由于细胞对化疗和放疗的耐药性增强，TxRd1水平升高与预后不良有关。TxRd的化学抑制剂作为癌症治疗和放射增敏剂在临床前和临床试验中取得了可喜的结果。姜黄素是一种天然存在的植物多酚类抗氧化剂，在临床前动物模型中具有有效的化学预防和抗肿瘤特性，在I期临床试验中显示出低毒性。最近的数据表明，姜黄素与TxRd1中保守的c-末端cys-x- secs -cys基序之间存在共价相互作用。从而抑制TxRd和硫氧还蛋白之间的催化相互作用。NADPH提供的电子被转移到O2上，形成超氧化物，最终形成过氧化氢。在本研究中，我们将在体外和体内研究TxRd1作为姜黄素单独或与电离辐射联合在口咽癌细胞系中抗肿瘤作用的关键介质的作用。我们的初步数据显示，与口腔白斑细胞系MSK-Leuk1或未转化的角化细胞相比，人鳞状癌细胞系FaDu中TxRd1的水平高出50倍。在具体的目标1中，我们计划使用shRNA将人类TxRd1基因的3'UTR克隆到pSilencer表达载体中，从而降低FaDu细胞中的TxRd1水平。相反，通过转染全长野生型TxRd1基因（包括来自3'UTR的硒代半胱氨酸插入元件），TxRd1将在MSK-leuk1细胞中过表达。以上选择的稳定克隆将用于确定TxRd1是否介导姜黄素的体外抗增殖和促凋亡作用。在具体目标2中，我们将使用上述选择的稳定克隆来确定姜黄素作为辐射增敏剂的功效是否也取决于对TxRd1的抑制。在特异性目的3中，TxRd1敲低和敲低细胞系以及亲代细胞系将在胸腺裸鼠颈部皮下组织中生长为肿瘤。将测量姜黄素单独或与电离辐射联合使用对原位肿瘤生长和肿瘤细胞死亡的影响。由于姜黄素是一种天然的生物活性食品成分，没有明显的全身毒性，这些临床前实验的有希望的结果可以很容易地转化为化学预防方案或化疗-放疗联合治疗头颈癌的方案。公共卫生相关性：头颈癌（HNC）是全球第六大最常见的癌症形式，每年新诊断病例超过50万例。尽管手术技术的进步以及放化疗的联合治疗降低了发病率，但初步预后后5年生存率仍< 50%。此外，由于吸烟和饮酒是与HNSCC相关的两个最普遍的危险因素，因此在20%的HNC患者中，现场癌变导致第二原发癌症。黄瓜是一种从植物姜黄根中提取的天然产物，在临床前和1期临床试验中显示出作为癌症化学预防、化疗和放射增敏剂的前景。使用姜黄素的主要缺点是由于到达循环系统的药物浓度低，生物利用度有限。然而，姜黄素对结肠癌和HNC等肿瘤有效，在这些肿瘤中可以直接使用这种药物。本研究的主要目的是开发姜黄素作为治疗HNC的化疗和放射增敏剂。

DESCRIPTION (provided by applicant): Thioredoxin reductase 1 (TxRd1), is an essential selenoprotein with defined roles in redox regulation of cell growth, death (apoptosis), transcription, DNA repair, and angiogenesis. TxRd1 is overexpressed in many human cancers and these elevated levels of TxRd1 have been associated with poor prognosis due to enhanced cellular resistance to chemo- and radiation therapy. Chemical inhibitors of TxRd have yielded promising results in preclinical and clinical trials as cancer therapeutics and radiation sensitizers. Curcumin is a naturally occurring plant polyphenolic antioxidant with potent chemopreventive and antintumorigenic properties in preclinical animal models and a demonstrated low toxicity profile in Phase I clinical trials Recent data demonstrate a covalent interaction between curcumin and the conserved c-terminal cys-x-sec-cys motif in TxRd1. resulting in inhibition ofthe catalytic interaction between TxRd and thioredoxin. The electrons donated by NADPH are instead diverted to O2, to form superoxide and ultimately hydrogen peroxide. In this proposal we will investigate the role of TxRd1 as a critical mediator of the antitumorigeniceffects of curcumin alone and in combination with ionizing radiation in oraopharyngeal cancer cell lines, in vitro and in vivo. . Our preliminary data demonstrated > 50 fold higher level of TxRd1 in the human squamous carcinoma cell line FaDu compared to either an oral leukoplakia cell line MSK-Leuk1 or nontransformed keratinocytes. In specific aim 1, we plan to knockdown TxRd1 levels in FaDu cells using shRNA to the 3'UTR of the human TxRd1 gene cloned into the pSilencer expression vector. Conversely, TxRd1 will be overexpressed in MSK-leuk1 cells by transfection with the full length wild type TxRd1 gene, including the selenocysteine insertion element, from the 3'UTR. The stable clones selected above will be used to determine whether TxRd1 mediates the anti-proliferative and pro-apoptotic actions of curcumin, in vitro. In specific aim 2, we will use the stable clones selected above, to determine whether curcumin's efficacy as a radiation sensitizer also depends on inhibition of TxRd1. In specific aim 3, the TxRd1 knockdown and knock in cell lines, and the parental cell lines, will be grown as tumors in the cervical subcutaneous tissue of athymic nude mice. The efficacy of curcumin administered in the diet alone or in combination with ionizing radiation on orthotopic tumor growth and tumor cell death will be measured. Since curcumin is a natural bioactive food component with no overt systemic toxicities, promising results from these preclinical experiments can be readily translated into a chemoprevention schemes or combined chemo-radiation therapy protocol for treating head and neck cancers. PUBLIC HEALTH RELEVANCE: Head and neck cancers (HNC) are the sixth most common form of cancer worldwide, with over 500,000 new cases diagnosed annually. Although advances in surgery, and combined of radio and chemotherapies, have decreased morbidity, the survival rate at 5 years after initial prognosis remains at < 50%. Moreover, since smoking and alcohol use are the two most prevalent risk factors associated with HNSCC, field cancerization leads to second primary cancers in 20% of HNC patients. Cucumin, a natural product derived from the root of the plant Curcuma longa, has shown promise as a cancer chemopreventive, chemotherapeutic and radiation sensitizer, in preclinical and phase 1 clinical trials. The primary drawback to the use of curcumin is limited bioavailability due to low concentrations of the drug reaching the circulation. However, curcumin has been effective in tumors, such as colon and HNC, where direct access to the drug is possible. The primary aim of this study is to develop curcumin as a chemotherapeutic and radiation sensitizer for treating HNC..