Role of Selenium-containing Proteins in Cancer

硒领域的许多研究人员提出，低分子量硒化合物对硒的许多健康益处负有责任，而另一种建议是硒蛋白可能是更负责任的物质。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艾滋病的进展，减缓衰老过程，并在哺乳动物发育、男性生殖和免疫功能中发挥作用。几年前，我们是第一批提出这些健康益处主要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中的作用，我们正在描述不同硒蛋白在恶性肿瘤过程中的功能。有三种硒蛋白，即硫氧还蛋白过氧化物酶1 （TR1）、硒蛋白15 （Sep15）和谷胱甘肽过氧化物酶2 (GPx2)，已经被我们和其他人证明在预防和促进癌症方面都有作用。我们的实验室已经率先研究了其中的两种硒蛋白，TR1和Sep15，如下所述。在我们对TR1促进癌症的研究中，我们使用了多种方法来阐明这种硒蛋白在促进癌症中的作用。已知亚硒酸盐对哺乳动物细胞有毒，而癌细胞对这种毒性要敏感得多。我们之前在DT细胞（一种来源于小鼠胚胎成纤维细胞NIH 3T3细胞的致癌细胞系）中靶向去除TR1和硫氧还蛋白1 (Trx1)，发现TR1缺陷细胞对亚硒酸盐暴露的敏感性明显高于Trx1缺陷细胞或对照细胞。在过去的一年里，我们完成并发表了亚硒酸盐毒性项目，证明只有tr1缺陷的细胞表现出强烈的谷胱甘肽的产生和分泌增强，这与细胞对亚硒酸盐的敏感性增加有关。这些数据揭示了TR1在癌症中的新作用，这种作用不依赖于Trx的减少，并由谷胱甘肽系统补偿。这些数据还表明，亚硒酸盐增强癌细胞毒性和同时抑制TR1可以为癌症治疗提供新的途径。就像去年提到的？根据我们的报告，TR1和Sep15在保护正常细胞免受癌症侵害方面发挥作用，我们已经在证明这两种硒蛋白也在促进癌症方面发挥了重要作用。我们之前发表了靶向去除小鼠结肠癌细胞系CT-26细胞中Sep15的表达，改变了正常细胞的一些恶性特征，并继续进行这些研究，包括靶向敲除CT-26细胞中的TR1和Sep15。就像去年提到的？在我们的报告中，Sep15或TR1的下调导致这些细胞的癌症表型逆转，但令人惊讶的是，Sep15和TR1的双重下调似乎保留了亲本细胞的癌症表型。在这一年里，我们的研究结果表明，缺乏Sep15或TR1的CT-26细胞，过度表达细胞周期调节基因（如cyclin B1相互作用蛋白1）的mRNA水平，从而导致细胞进入有丝分裂的延迟，从而减缓细胞生长。此外，微阵列分析表明，鸟苷酸结合蛋白和其他干扰素/炎症相关基因在Sep15敲低细胞中显著增加。然而，在TR1敲低的细胞或缺乏Sep15和TR1的细胞中没有观察到这种增加。因此，缺乏Sep15或TR1的细胞逆转癌细胞表型的机制似乎非常不同。我们正在继续研究这些机制之间的差异，以及同时去除结肠癌细胞中Sep15和TR1的明显代偿作用。此外，我们还研究了缺氧条件对TR1表达的调节，以进一步阐明这种硒蛋白在这些应激条件下的作用。文献中已经描述了缺氧条件会增加细胞内ROS的水平，并且这种ROS对于HIF稳定和活性的缺氧调节至关重要。由于一些硒蛋白在细胞氧化还原平衡调节中的重要性，我们开展了一个项目来研究缺氧条件对硒蛋白表达的影响。在之前的研究中，我们观察到缺氧降低了DT细胞和小鼠乳腺癌细胞系EMT6中TR1及其mRNA水平和活性。这种调节不依赖于缺氧诱导因子（hypoxia inducible factor， HIF）的活性，TR1的过表达消除了缺氧诱导的ROS水平升高，但不影响HIF活性或稳定。在去年，我们完成并发布了这个项目。作为该项目的延续，我们扩大了我们的发现，Trx1过表达增加了HeLa和EMT6细胞系中HIF的缺氧活性，而Trx1过表达后，DT、MCF-7和HT-29细胞系中HIF的活性或稳定性不受影响。Trx1对HIF活性的影响取决于被硫氧还蛋白还原酶活性还原的两种半胱氨酸残基的存在，因为Trx1突变形式的过度表达对HIF活性没有任何影响。然而，TR1的过表达并不能再现Trx1的作用。此外，Trx1在EMT6或TR1被敲低的HeLa细胞中过表达，在缺氧条件下仍然能够诱导和增加HIF活性，这表明在这一过程中，TR1以外的其他蛋白也在降低Txn1。就像去年提到的？在我们的报告中，我们报道了EMT6小鼠乳腺癌细胞中TR1敲低导致对tnf - α诱导的细胞凋亡的敏感性增加。使用siRNA技术实现Trx1的靶向去除，并检测对tnf - α诱导的细胞凋亡的敏感性，以阐明Trx系统在细胞凋亡中的作用。Trx1缺陷细胞对tnf - α的敏感性最高，与对照细胞相比，TR1缺陷细胞的敏感性更高。这些观察结果表明，Trx系统参与了抗凋亡途径。我们通过tnf - α处理检测MAPKs的激活，以检测Trx系统抗凋亡功能的途径，发现TR1和Trx1敲低细胞显示Erk和p-38的差异激活。在过去的一年里，我们发现tnf - α处理后TR1和Trx1敲低的细胞中Erk激活降低，抑制Erk激活增加了所有细胞（对照，TR1和Trx1）的caspase 3激活。这为Erk参与TR1和Trx1缺陷细胞的抗凋亡通路提供了证据。我们还观察到，TR1和Trx1敲除细胞中，tnf - α对p-Erk的核定位更高，LY294002抑制PI3K可阻断tnf - α诱导的细胞凋亡。抑制PI3K抑制了tnf - α对p-Erk的核定位，而不抑制MAPKs的整体激活，提示核p-Erk在tnf - α诱导的细胞凋亡中起作用。我们正在阐明核p-Erk在tnf - α诱导的细胞凋亡中的作用。

Numerous investigators in the selenium field have proposed that low molecular weight selenocompounds are responsible for the numerous health benefits attributed to selenium, while an alternative proposal is that selenoproteins are likely the more responsible agents. 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 were amongst the first to propose 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, we are characterizing the function of different selenoproteins in the malignancy process. There are three selenoproteins, thioredoxin peroxidase 1 (TR1), selenoprotein 15 (Sep15) and glutathione peroxidase 2 (GPx2), that have been shown by us and others to have roles in both preventing and promoting cancer. Our laboratory has taken the lead in investigating two of these selenoproteins, TR1 and Sep15, as discussed below. In our examination of TR1 in promoting cancer, we have used multiple approaches to elucidate the role of this selenoprotein in cancer promotion. Selenite is known to be toxic to mammalian cells and cancer cells are far more sensitive to this toxicity. We previously targeted the removal of both TR1 and thioredoxin 1 (Trx1) in DT cells which are an oncogenic cell line derived from the mouse embryonic fibroblast NIH 3T3 cells and found that TR1-deficient cells were significantly more sensitive to selenite exposure than Trx1-deficient cells or control cells. In the past year, we have completed and published the selenite toxicity project by demonstrating that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The data uncover a new role of TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The data also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy.As noted in last year?s report, TR1 and Sep15 have roles in protecting normal cells from cancer and, we have had a major hand in demonstrating that these two selenoproteins also have roles in promoting cancer. We previously published that the targeted removal of Sep15 expression in CT-26 cells, a mouse colon carcinoma cell line, altered several of the malignant characteristics more towards normal cells and have continued these studies to include the targeted knockdown of both TR1 and Sep15 in CT-26 cells. As noted in last year?s report, the down-regulation of either Sep15 or TR1 resulted in reversal of the cancer phenotype in these cells, but surprisingly the double-knockdown of both Sep15 and TR1 appeared to retain the cancer phenotype of the parental cells. In this year, our results indicate that CT-26 cells, lacking either Sep15 or TR1, over-express mRNA levels of cell cycle regulatory genes such as cyclin B1 interacting protein 1, thus causing a delayed entry of the cells into mitosis and, as a result, slowed cell growth. Additionally, microarray analyses indicated a strong increase in guanylate binding proteins and other interferon/inflammation-associated genes in Sep15 knockdown cells. However, this increase was not observed in TR1 knockdown cells or in the cells lacking both Sep15 and TR1. Thus, the mechanism by which cells lacking Sep15 or TR1 reverse the cancer phenotype in cells appears to be very different. We are continuing to investigate the differences between these mechanisms, and furthermore the apparent compensatory effect of simultaneously removing Sep15 and TR1 in colon cancer cells.In addition, the regulation of TR1 expression by hypoxic conditions was also examined in cancer cell lines to further elucidate the role of this selenoprotein under these stressful conditions. Hypoxic conditions have been described in the literature to increase the levels of ROS inside cells and this ROS have been described to be critical for the hypoxic-regulation of HIF stabilization and activity. Due to the importance of some selenoproteins in the regulation of redox balance in the cells, we undertook a project to examine the effect of hypoxic conditions on the expression of selenoproteins. In a previous study, we observed that hypoxia reduced TR1 and its mRNA levels and activity in DT cells and a mouse breast cancer cell line, EMT6. This regulation is independent of the activity of hypoxia inducible factor (HIF) and TR1 over-expression abrogates the increase in ROS levels induced by hypoxia, but does not affect HIF activity or stabilization. In the last year, we completed and published this project. As a continuation of this project, we expanded our finding that Trx1 over-expression increased HIF hypoxic activity in HeLa and EMT6 cell lines, whereas HIF activity or stabilization is not affected in DT, MCF-7 and HT-29 cell lines after Trx1 over-expression. The effect of Trx1 on HIF activity is dependent of the presence of the two cysteine residues that are reduced by thioredoxin reductase activity, as the over-expression of a mutated form of Trx1 did not have any effect on HIF activity. However, the over-expression of TR1 did not recapitulate the effects of Trx1. Furthermore, Trx1 over-expression in EMT6 or in HeLa cells in which TR1 was knocked down was still able to induce and increase in HIF activity under hypoxic conditions, suggesting that other proteins than TR1 are reducing Txn1 in this process. As noted in last year?s report, we reported that TR1 knockdown in EMT6 mouse breast cancer cells caused increased sensitivity to TNF-alpha induced apoptosis. The targeted removal of Trx1 was achieved using siRNA technology, and sensitivity to TNF-alpha induced apoptosis was examined to elucidate the role of the Trx system in apoptosis. Trx1 deficient cells had the highest sensitivity to TNF-alpha and TR1 deficient cells had a higher sensitivity compared to control cells. These observations suggested that the Trx system is involved in an anti-apoptotic pathway. We examined the activation of MAPKs by TNF-alpha treatment to examine the pathway involved in the anti-apoptotic function of the Trx system and found TR1 and Trx1 knockdown cells showed a differential activation of Erk and p-38. In the past year, we found that Erk activation was lower in TR1 and Trx1 knockdown cells after TNF-alpha treatment and inhibition of Erk activation increased caspase 3 activation in all cells (control, TR1 and Trx1). This provided evidence that Erk is involved in the anti-apoptotic pathway in TR1 and Trx1 deficient cells. We also observed that the nuclear localization of p-Erk by TNF-alpha was higher in TR1 and Trx1 knockdown cells and inhibition of PI3K with LY294002 blocked TNF-alpha induced apoptosis in each cell line. Inhibition of PI3K inhibited the nuclear localization of p-Erk by TNF-alpha, whereas it did not inhibit the overall activation of MAPKs suggesting a function of nuclear p-Erk in TNF-alpha induced apoptosis. We are in the process of elucidating the function of nuclear p-Erk in TNF-alpha induced apoptosis.