Role of Selenium in Cancer and Health

硒是人类和其他哺乳动物饮食中必需的微量营养素。硒对健康有许多益处，包括预防各种形式的癌症（例如结肠癌、前列腺癌、肺癌和肝癌）、心脏病和其他心血管和肌肉疾病、抑制病毒表达、延缓人类免疫缺陷病毒（艾滋病毒）阳性患者获得性免疫缺陷综合症（艾滋病）的进展、减缓衰老过程以及在哺乳动物发育中发挥作用。包括男性生殖和免疫功能。近年来，人们进行了大量的人体临床试验，以评估硒在预防癌症、延缓艾滋病进展等方面的作用，耗资数十亿美元，但对硒在哺乳动物代谢水平上如何发挥这些健康益处的机制知之甚少。我们提出硒的健康益处主要是由于它作为含硒氨基酸硒半胱氨酸（Sec）存在于硒蛋白中。因此，我们的项目重点是：1)建立小鼠模型来评估硒和硒蛋白在癌症预防和发展中的作用；2)表征和阐明各种硒蛋白的功能及其在癌症预防和发展中的作用；3)确定Sec生物合成和结合到蛋白质中的方法。本文讨论的项目探讨了我们在各种小鼠模型的开发研究，以确定硒在癌症，癌症预防和发展中的作用。在过去的一年里，我们专注于完成我们对小鼠模型的研究：1)敲除Sec tRNA基因（指定Trsp），导致硒蛋白在(a) T细胞、(b)巨噬细胞、(c)表皮皮肤组织和(d)肝脏中的表达缺失，随后用野生型或突变型Trsp转基因恢复或部分恢复硒蛋白的表达。我们在这个项目中的重点方向是转向建立小鼠模型，处理硒蛋白在癌症发生和进展中的作用，以及靶向去除单个硒蛋白，而不是通过去除Trsp来敲除所有硒蛋白。在过去的一年里，我们完成了对硒蛋白在细胞免疫中的分子机制的研究。我们在T细胞中敲除所有硒蛋白的发现为硒和生理抗氧化剂的免疫功能提供了新的见解。研究发现，巨噬细胞中硒蛋白的去除在很大程度上表现为正常的炎症反应，但硒蛋白的丢失会导致细胞外基质相关基因的异常表达和巨噬细胞在蛋白质凝胶基质中的迁移减少。我们最近开始针对T细胞和巨噬细胞中硫氧还蛋白还原酶1 （TR1）或谷胱甘肽过氧化物酶4 （GPx4）的去除。此外，我们完成了靶向去除皮肤中Trsp的研究，重点研究了单一硒蛋白GPx4的去除。GPx4的缺失几乎与所有硒蛋白的缺失相似，唯一的例外是，小鼠在第10天恢复过来，那时它们通常与Trsp基因敲除小鼠一起死亡，并且似乎过着几乎正常的生活。皮肤表皮细胞中硒蛋白的去除导致后代表型矮小和过早死亡。GPx4基因敲除小鼠出现脱发，皮肤片状脆弱，组织学研究显示表皮增生，毛囊外观改变。这些观察结果突出了硒蛋白和GPx4在皮肤发育中的未知作用。我们已经启动了体内致癌研究，涉及TR1和硒蛋白15 （Sep15）敲低的小鼠，以提供小鼠模型来检验我们的体外研究结果，阐明这些硒蛋白在癌症中的作用。显然，TR1是一把双刃剑，在保护正常细胞免受癌症侵害的同时，一旦恶性肿瘤开始，硒酶在癌症的发展中也起着作用（见Hatfield， Thioredoxin reducinase 1: a double-edged sword in cancer prevention and promotion）。科学进展，6(2),2007。我们最初只对几只老鼠进行了初步研究，以确定老鼠的遗传背景在它们对所研究的癌症的易感性方面的作用，并确保我们的方法是正确的。在我们对Sep15在结肠癌中的作用的研究中，Sep15基因敲除小鼠为C57BL/6 (B6)-SV129 （SV）背景。我们最初检测了Sep15基因敲除小鼠对偶氮甲烷处理的纯B6和B6- sv细胞系的异常隐窝形成。3个月后，我们发现Sep15基因敲除小鼠的异常隐窝数量远少于对照组小鼠（但正如预期的那样，B6基因敲除小鼠的异常隐窝数量少于混合背景基因敲除小鼠）。我们已经启动了一项正在进行的研究，涉及大量小鼠，其中对照组由具有相同遗传背景的兄弟小鼠组成。在研究TR1减少对乳腺癌和肝癌进展的作用的两种小鼠模型中，使用的转基因小鼠系是遗传敏感的FVB，并编码由我们设计的shRNA靶向载体，该载体可由多西环素诱导。shRNA的表达部分地抑制TR1的表达。我们初步检测了7,12- dmba处理的对照小鼠和TR1诱导小鼠乳腺组织中的肿瘤生成情况。4个月后，两种小鼠系均出现肿瘤，肿瘤组织中TR1的表达均显著升高。目前，我们有大量的小鼠喂食或不喂食强力霉素来检测TR1的减少是否对乳腺癌有影响。在肝癌研究中，我们有一个正在进行的试点实验，涉及一些编码TR1 shRNA靶向载体的转基因小鼠，这些小鼠已经用DEN治疗。这项初步研究已经进行了大约六个月，并将在大约六个月内完成，届时将对其进行评估，并决定如何继续进行该项目。

Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium that include preventing various forms of cancer (e.g., colon cancer, prostate cancer, lung cancer and liver cancer), heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV)-positive patients, slowing the aging process, and having roles in mammalian development, including male reproduction and immune function. Numerous human clinical trails have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of billions of dollars, but little is known about the mechanism of how selenium acts at the metabolic level in mammals to exert these many health benefits. We have proposed that the health benefits of selenium are due largely to its presence in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focuses on: 1) developing mouse models to assess the role of selenium and selenoproteins in cancer prevention and development, 2) characterizing and elucidating the function of various selenoproteins and their roles in cancer prevention and development, and 3) identifying the means by which Sec is biosynthesized and incorporated into protein. The project discussed herein examines our research on the development of various mouse models for determining the role of selenium in cancer, cancer prevention and development. During the past year, we have focused on completing our studies on characterizing mouse models that: 1) knockout of the Sec tRNA gene (designated Trsp) and consequently the loss of selenoprotein expression in (a) T cells, (b) macrophage, (c) epidermal skin tissue, and (d) liver, and subsequent rescue or partial rescue of selenoprotein expression with a wild-type or mutant Trsp transgene. Our direction of focus in this project is shifting towards generating mouse models that deal with the role of selenoproteins in cancer initiation and progression and on the targeted removal of individual selenoproteins instead of knocking out all selenoproteins through the removal of Trsp. This past year, we completed our study on examining the molecular mechanism of selenoproteins in cell immunity. Our findings with knocking out all selenoproteins in T cells provided novel insights into the immune function of selenium and physiological antioxidants. The removal of selenoproteins in macrophage was found to manifest largely normal inflammatory responses, but selenoprotein loss had abnormal expression of extracellular matrix-related genes and a reduced migration of macrophages in a protein gel matrix. We have recently begun to target the removal of only thioredoxin reductase 1 (TR1) or glutathione peroxidase 4 (GPx4) in both T cells and macrophage. In addition, we completed our study on the targeted removal of Trsp in skin and have focused on the removal of a single selenoprotein, GPx4. Loss of GPx4 virtually mimicked the loss of all selenoproteins with the exception that the mice recover after day 10, when they routinely died with the Trsp knockout mice, and appear to live a virtually normal life. Removal of selenoproteins in skin epidermal cells resulted in a runt phenotype and premature death of the progeny. The GPx4 knockout mice had alopecia along with a flaky and fragile skin and histological studies revealed epidermal hyperplasia along with changes in hair follicle appearance. These observations highlight a hitherto unknown role of selenoproteins and GPx4 in cutaneous development. We have initiated in vivo carcinogenesis studies involving TR1 and selenoprotein 15 (Sep15) knockdown mice to provide mouse models to examine our in vitro findings elucidating the role of these selenoproteins in cancer. Clearly, TR1 is a double sword in having roles in protecting normal cells from cancer and then once the malignancy in initiated, the selenoenzyme has a role in the developing cancer (see Hatfield, Thioredoxin reductase 1: A double-edged sword in cancer prevention and promotion. CCR Frontiers in Science 6: 8-10, 2007). We initially carried out pilot studies involving only a few mice to both determine the role of the genetic background of the mice in terms of their susceptibility to the cancer under study and to assure us that our approaches were correct. In our study on the role of Sep15 in colon cancer, the Sep15 knockout mice were in a C57BL/6 (B6)-SV129 (SV) background. We initially examined aberrant crypt formation in the Sep15 knockout mouse against pure B6 and B6-SV lines treated with azoxymethane. After 3 months, we found the Sep15 knockout mice had far fewer aberrant crypts than control mice (but, as expected, the B6 line had less than the mixed background line). We have initiated an ongoing study involving a larger number of mice wherein the control consists of sibling mice with the identical genetic background. In the two mouse models used to examine the role of TR1 reduction on breast and liver cancer progression, the transgenic mouse lines used are the genetically sensitive FVB and encode a shRNA targeting vector (designed by us) transgene that is inducible by doxycycline. Expression of the shRNA partially knocks down TR1 expression. We initially examined tumor production in breast tissue of control and TR1 inducible mice treated with 7,12-DMBA. After 4 months, both mouse lines had tumors and TR1 expression was highly elevated in tumor tissues. We currently have a larger number of mice either fed or not fed doxycycline for examining whether TR1 reduction has an effect on breast cancer. In the liver cancer study, we have an ongoing pilot experiment involving a few transgenic mice encoding the TR1 shRNA targeting vector that have been treated with DEN. This initial study has been ongoing for about six months and will be completed in about another six months at which time it will be evaluated and a decision will be made on how the project will be pursued.