Role of Selenium-containing Proteins in Cancer and Development

含硒蛋白质在癌症和发育中的作用

• 批准号: 7965801
• 负责人: Dolph Hatfield
• 金额: $ 30.46万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965801
• 关键词: Acquired Immunodeficiency Syndrome; Aging-Related Process; Amino Acids; Anchorage-Independent Growth; Antioxidants; Apoptosis; Attention; Breast Cancer Cell; Cancer cell line; Cardiovascular system; Cell Line; Cells; Characteristics; Colon; Colon Carcinoma; Colorectal Cancer; Data; Development; Disease; Enzymes; Etiology; Excision; Fibroblasts; Goals; Growth; HIV Seropositivity; Health Benefit; Heart Diseases; Lewis Lung Carcinoma; Lipid Peroxides; Lung; Lung Neoplasms; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of lung; Membrane Lipids; Metabolism; Molecular; Molecular Weight; Mus; Myopathy; Neoplasm Metastasis; Neurodegenerative Disorders; Normal Cell; Oncogenic; Parkinson Disease; Patients; Plasmids; Proteins; RNA Interference; Reactive Oxygen Species; Reproduction; Research Personnel; Role; Selenium; Selenocysteine; Specificity; Technology; Tissues; Tumor Necrosis Factor-alpha; Tumorigenicity; Viral; base; brain tissue; cancer prevention; cell growth; disorder prevention; glutathione peroxidase; human TNF protein; immune function; in vivo; knock-down; male; malignant phenotype; mouse model; oxidized lipid; prevent; repaired; selenoprotein; subcutaneous; thioredoxin peroxidase

Numerous investigators in the selenium field have proposed that low molecular weight selenocompounds are responsible for the numerous health benefits attributed to selenium, while others have suggested that selenoproteins are likely responsible. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. We proposed several years ago that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec). Therefore, to elucidate the role of selenoproteins in cancer prevention and development, we are characterizing the function of several selenoproteins. During the past year, we have focused our attention on studying in greater detail the role of three selenoproteins, thioredoxin peroxidase 1 (TR1), glutathione peroxidase 4 (GPx4) and selenoprotein 15 (Sep15). We had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line (JBC 281: 13005, 2006) and in a cancer cell line driven by oncogenic k-ras (PLoS One 2: e1112; 1-7, 2007) resulted in several of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. This past year, we have continued to focus on the molecular basis of TR1s role in cancer development and have examined the role of TR1 in apoptosis in a breast cancer cell line in detail. The breast cancer cell line was selected since we found that TR1 knockdown in breast cancer cells showed a much higher sensitivity to TNF-alpha induced apoptosis. We are currently confirming these findings in mice in vivo as further discussed in Project 1 (Z01 BC 005317). We are also continuing our study on the intracellular role of GPx4 and have found GPx4 is essential for repairing membrane lipid hydroperoxidation. Furthermore, we have evidence that this selenoprotein may be involved in the etiology of neurodegenerative diseases such as Alzheimers and Parkinsons diseases. GPx4 has decreased levels in brain tissue of Alzheimers and Parkinsons disease mouse models. GPx4 is a selenium-containing, antioxidant enzyme whose intracellular function has not been fully resolved. To elucidate its function intracellularly, we targeted the removal of GPx4 in a mouse fibroblast cell line (NIH3T3 cells) that resulted in severe damage to the cells when the level of this enzyme was reduced below 80% of its normal level. Partial reduction in its expression led to severe growth retardation. Unexpectedly, GPx4 knockdown cells showed little change in levels of reactive oxygen species, but these cells manifested highly increased levels of oxidized lipid byproducts suggesting membrane lipid hydroperoxidation damage. The data thus far have established an essential role of GPx4 in the metabolism of membrane lipid hydroperoxides, and surprisingly, a limited role as a general antioxidant enzyme. In the past year, we have found that Sep15 appears to have a role in colorectal cancer. Our findings demonstrate that knocking down the expression of Sep15 in the murine colon carcinoma cell line, CT-26, using RNAi resulted in phenotypic changes more towards normal cells that included decreased cell growth rates and reduced anchorage-independent growth abilities. Furthermore, CT-26, Sep15 knockdown cells, when injected into mice of the same background and compared to plasmid-transfected controls, also demonstrated a significantly reduced ability to form localized subcutaneous tumors and lung metastases in vivo. Mouse Lewis lung carcinoma cells (LLC1), transfected with the same siSep15 construct, appeared to maintain their original growth characteristics, indicating that knockdown of Sep15 reduced tumorigenicity of colon (CT26), but not lung (LLC1) cells. We are continuing to investigate this apparent tissue specificity, as well as possible functions of Sep15 in colon and other tissues.

硒领域的许多研究人员提出，低分子量的硒化合物负责造成硒的众多健康益处，而其他人则建议硒蛋白可能是造成的。这些健康益处包括预防癌症，心脏病和其他心血管疾病和肌肉疾病，抑制病毒表达，延迟艾滋病毒阳性患者的艾滋病进展，减慢衰老过程并在哺乳动物发育，男性繁殖和免疫功能中起作用。几年前，我们建议这些健康益处主要是由于硒蛋白中硒作为氨基酸，硒代半胱氨酸（SEC）的存在。因此，为了阐明硒蛋白在癌症预防和发育中的作用，我们正在表征几种硒蛋白的功能。在过去的一年中，我们将注意力集中在更详细地研究三种硒蛋白，硫代蛋白过氧化物酶1（TR1），谷胱甘肽过氧化物酶4（GPX4）和硒蛋白15（Sep15）的作用。我们先前曾证明，在肺癌细胞系中使用RNA干扰技术（JBC 281：13005，2006）和由致癌性K-RAS驱动的癌细胞系中的TR1敲低（PLOS ONE 2：E1112; 1-7，2007）导致了几种恶性表型，这对正常细胞的效率更加逆转，这是Trum1 defor的效应。在过去的一年中，我们继续关注TR1在癌症发展中作用的分子基础，并详细研究了TR1在乳腺癌细胞系中凋亡中的作用。选择乳腺癌细胞系，因为我们发现乳腺癌细胞中的TR1敲低对TNF-Alpha诱导的细胞凋亡的敏感性更高。如项目1（Z01 BC 005317）中进一步讨论，我们目前正在体内确认这些发现。我们还继续研究GPX4的细胞内作用，发现GPX4对于修复膜脂质氢过氧化作用至关重要。此外，我们有证据表明，这种硒蛋白可能参与了神经退行性疾病的病因，例如阿尔茨海默氏病和帕金森氏病。在阿尔茨海默氏症和帕金森氏病小鼠模型的脑组织中，GPX4的水平降低。 GPX4是一种含硒的抗氧化剂酶，其细胞内功能尚未完全解决。为了细胞内阐明其功能，我们针对的是在小鼠成纤维细胞系（NIH3T3细胞）中去除GPX4，当该酶的水平降低到其正常水平的80％以下时，导致细胞造成严重损害。其表达的部分降低导致严重的生长迟钝。 出乎意料的是，GPX4敲低细胞的活性氧水平几乎没有变化，但是这些细胞表现出高度增加的氧化脂质副产物水平，表明膜脂质氢过氧化损伤。迄今为止，数据已经确定了GPX4在膜脂质氢过氧化物的代谢中的重要作用，令人惊讶的是，作为一般抗氧化剂酶的作用有限。在过去的一年中，我们发现9月15日似乎在结直肠癌中起作用。我们的发现表明，使用RNAi敲低鼠结肠癌细胞系CT-26中Sep15的表达，导致表型变化对正常细胞的表型变化更大，包括降低细胞生长速率和降低锚固依赖性生长能力。此外，当注射到相同背景的小鼠中并与质粒转染的对照相比，CT-26，SEP15敲低细胞还显示出形成局部皮下肿瘤和体内肺转移的能力。用相同的SISEP15构建体转染的小鼠刘易斯肺癌细胞（LLC1）似乎保持其原始生长特征，表明敲低Sep15降低了结肠（CT26）的肿瘤性（CT26），但没有肺（LLC1）细胞。我们正在继续研究这种明显的组织特异性，以及在结肠和其他组织中Sep15的可能功能。