Role of Selenium-containing Proteins in Cancer and Development

许多硒领域的研究人员提出，低分子硒化合物对硒的众多健康益处负有责任，而另一些人则认为硒蛋白可能起作用。这些健康益处包括预防癌症、心脏病和其他心血管和肌肉疾病，抑制病毒表达，延缓艾滋病毒阳性患者的艾滋病进程，减缓衰老过程，并在哺乳动物发育、男性生殖和免疫功能方面发挥作用。我们几年前提出，这些健康益处很大程度上是由于硒蛋白中硒作为氨基酸--硒半胱氨酸(SEC)的存在。因此，为了阐明硒蛋白在癌症预防和发展中的作用，我们正在研究几种硒蛋白的功能。在过去的一年里，我们专注于更详细地研究三种硒蛋白的作用，硫氧还蛋白过氧化物酶1(TR1)、谷胱甘肽过氧化物酶4(Gpx4)和硒蛋白15(9月15日)。我们之前已经证明，在肺癌细胞系(JBC281：13005,2006年)和致癌k-ras驱动的癌细胞系(PLoS one 2：E1112；1-7,2007年)中，使用RNA干扰技术敲除TR1导致了几种恶性表型更多地逆转为正常细胞的表型，这表明TR1缺乏是抗肿瘤形成的。在过去的一年里，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在乳腺癌细胞系凋亡中的作用。之所以选择乳腺癌细胞系，是因为我们发现乳腺癌细胞中的TR1基因被敲除后，对肿瘤坏死因子-α诱导的细胞凋亡表现出更高的敏感性。我们目前正在小鼠体内证实这些发现，项目1(Z01 BC 005317)进一步讨论了这一点。我们还在继续研究Gpx4在细胞内的作用，并发现Gpx4对于修复膜脂过氧化是必不可少的。此外，我们有证据表明，这种硒蛋白可能参与了阿尔茨海默病和帕金森氏病等神经退行性疾病的病因学。Gpx4降低了阿尔茨海默病和帕金森病小鼠模型脑组织中的水平。Gpx4是一种含硒的抗氧化酶，其细胞内功能尚未完全解决。为了阐明Gpx4在细胞内的功能，我们以去除小鼠成纤维细胞系(NIH3T3细胞)中的Gpx4为靶点，当Gpx4的水平降至正常水平的80%以下时，该酶会对细胞造成严重的损伤。其表达的部分下调会导致严重的生长迟缓。出乎意料的是，Gpx4基因敲除细胞的活性氧水平几乎没有变化，但这些细胞表现出氧化脂质副产物水平的高度增加，表明膜脂过氧化损伤。到目前为止，这些数据已经确定了Gpx4在膜脂过氧化代谢中的重要作用，但令人惊讶的是，作为一般抗氧化酶的作用是有限的。在过去的一年里，我们发现9月15似乎在结直肠癌中起到了作用。我们的发现表明，使用RNAi下调小鼠结肠癌细胞系CT-26中Sep15的表达会导致更接近正常细胞的表型变化，包括细胞生长速度下降和锚定非依赖性生长能力降低。此外，当将CT-26、Sep15基因敲除细胞注射到相同背景的小鼠体内时，与质粒转染组相比，在体内形成局部皮下肿瘤和肺转移的能力也显著降低。转染相同siSep15基因的小鼠Lewis肺癌细胞(LLC1)似乎保持了其原有的生长特性，这表明Sep15基因的敲除降低了结肠癌(CT26)细胞的致瘤性，但不降低肺(LLC1)细胞的致瘤性。我们正在继续研究这种明显的组织特异性，以及Sep15在结肠和其他组织中可能的功能。

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.