Role of Selenium-containing Proteins in Cancer

硒领域的许多研究人员提出，低分子量的硒化合物是硒对健康的许多益处的原因，而另一种建议是硒蛋白可能是更负责任的试剂。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，延缓艾滋病病毒阳性患者的艾滋病进展，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的人之一，这些健康益处主要是由于硒蛋白中存在硒作为氨基酸，硒代半胱氨酸（Sec）。因此，为了阐明硒蛋白在癌症预防中的作用，我们正在表征不同硒蛋白在恶性过程中的功能。至少有两种硒蛋白，硫氧还蛋白还原酶1（TR 1）和硒蛋白15（Sep 15），具有预防和促进癌症的作用。正如去年的报告中所指出的，我们以前已经表明，在肺癌细胞系和由致癌k-ras驱动的癌细胞系中使用RNA干扰技术敲低TR 1导致几种恶性表型向正常细胞的恶性表型逆转，这表明TR 1缺陷是抗肿瘤的。在过去的一年中，我们继续关注TR 1在癌症发展中作用的分子基础，并进一步详细研究了TR 1在乳腺癌细胞系凋亡中的作用。如前所述，选择乳腺癌细胞系是因为我们发现乳腺癌细胞中TR 1敲低对TNF-α诱导的细胞凋亡显示出高得多的敏感性。TR 1被认为通过调节硫氧还蛋白（Trx）来控制多种细胞过程。我们建立了Trx 1敲低细胞，通过比较TR 1和Trx 1敲低细胞对多种类型刺激的反应，来检查Trx作为TR 1 s功能的主要靶点的参与。这些研究表明，两种敲除细胞对过氧化氢处理（H2 O2）的反应模式相似，但对硒诱导的细胞毒性反应不同。TR 1敲除细胞对硒处理更敏感，而Trx 1敲除细胞显示出与相应对照细胞相似的敏感性水平。我们建立了一个TR 1敲低和随后的再表达策略，以寻找另一个TR 1结合蛋白，该蛋白仅在应激条件下起作用。我们发现了几个候选人，TR 1结合蛋白在细胞应激诱导H2 O2，TNF-α和硒，并进一步表征这些蛋白。TR 1缺陷细胞系对硒毒性更敏感的原因尚不清楚，但这不是由于氧化应激。我们正在进一步研究恶性TR 1缺陷细胞对硒毒性的敏感性增强，因为TR 1抑制剂和硒的组合可能提供癌症治疗的新途径。我们还在研究恶性细胞中TR 1的上调。TR 1是哺乳动物细胞中的主要抗氧化剂和氧化还原调节剂，并且它确实在许多癌细胞中过度表达。在啮齿类动物中有四种不同的TR 1剪接形式。其中三个（变体II-IV）由相同的启动子驱动，而第三个（变体I）可能由不同的启动子控制。我们已经确定，小鼠（正常细胞和癌细胞）中TR 1最丰富的表达形式是变体II。这一观察结果表明，TR 1在癌细胞中的过表达是由与正常细胞相同的启动子启动的。阐明TR 1在癌细胞中表达的起始机制将是靶向上调TR 1在癌症治疗中的关键。我们现在正在分析TR 1启动子区域的甲基化状态，并在正常细胞和癌细胞中进行比较。这将阐明DNA甲基化是否可能在TR 1表达的调节中发挥作用。 近年来的研究发现，15 kDa硒蛋白（Sep 15）在大肠癌的发生发展中起一定的作用。我们证明，在小鼠结肠癌细胞系CT 26中靶向下调Sep 15，逆转了其典型的癌细胞特征，使其更像正常细胞。最近，我们扩大了对Sep 15在不同恶性状态的人结肠癌细胞系中的作用的研究，并继续研究这种硒蛋白的可能功能及其在结肠癌中的作用。此外，因为我们假设TR 1和Sep 15是一把双刃剑的两面，在保护正常细胞免受癌症侵害方面具有作用，然后，一旦恶性肿瘤开始，这两种硒酶在促进癌症方面具有作用，我们已经开始研究小鼠CT 26结肠癌细胞，其中我们成功地使用RNAi敲除TR 1和Sep 15。我们正计划研究这两种硒蛋白在小鼠和人类起源的这些和其他癌细胞中对恶性肿瘤的相互作用。特别感兴趣的是TR 1和/或Sep 15蛋白表达相对强烈增加或降低的癌细胞系。因此，我们已经开始使用放射性标记与75硒以及蛋白质印迹技术从各种组织的几个人癌细胞系的初步筛选。

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 at least two selenoproteins, thioredoxin reductase 1 (TR1) and selenoprotein 15 (Sep15), that have roles in both preventing and promoting cancer. As noted in last years report, we had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line and in a cancer cell line driven by oncogenic k-ras resulted in several of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. In 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 further detail. As noted previously, 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. TR1 has been proposed to control various cellular processes by regulating thioredoxin (Trx). We established Trx1 knockdown cells to examine the involvement of Trx as the major target of TR1s function by comparing responses of TR1 and Trx1 knockdown cells to numerous types of stimuli. These studies showed that both knockdown cells showed similar response patterns to hydrogen peroxide treatment (H2O2), but they responded differently to selenium induced-cytotoxicity. TR1 knockdown cells were much more sensitive to selenium treatment whereas Trx1 knockdown cells showed a similar level of sensitivity as the corresponding control cells. We established a TR1 knockdown and subsequent re-expression strategy for searching for another TR1 binding protein which functions only in the stressed condition. We found several candidate, TR1 binding proteins during cellular stress induced by H2O2, TNF-alpha and selenium and are further characterizing these proteins. The reason for the greater sensitivity of the TR1 deficient cell line to selenium toxicity is poorly understood but this was not due to oxidative stress. We are further examining the enhanced sensitivity of malignant TR1 deficient cells to selenium toxicity since a combination of a TR1 inhibitor and selenium may provide a novel avenue in cancer therapy. We are also examining the up-regulation of TR1 in malignant cells. TR1 is a major antioxidant and redox regulator in mammalian cells and it is indeed over-expressed in many cancer cells. There are four different splicing forms of TR1 that occur in rodents. Three of them (variants II-IV) are driven by the same promoter while the third (variant I) may be controlled by a different promoter. We have identified that the most abundant expression form of TR1 in mice (both normal cells and cancer cells) is variant II. This observation indicates that over-expression of TR1 in cancer cells is initiated by the same promoter as normal cells. Elucidation of the initiation mechanism of TR1 expression in cancer cells will be the key to targeting the up-regulation of TR1 in cancer therapy. We are now analyzing the methylation status of the TR1 promoter region and comparing it in normal and cancer cells. This will elucidate whether DNA methylation may play a role in the regulation of TR1 expression. As reported in last years report, we found that the 15kDa selenoprotein (Sep15) appears to play a role in colorectal cancer. We demonstrated that the targeted down-regulation of Sep15 in the murine colon (adeno)carcinoma cell line, CT26, reversed its characteristics that were typical of a cancer cell to resemble those more like normal cells. Recently, we expanded our studies on the investigation of the role of Sep15 into human colon cancer cell lines of varying malignant statuses, and continue to examine the possible function of this selenoprotein and its role in colon cancer. Furthermore, because we hypothesize TR1 and Sep15 to be the two sides of a double-edged sword in having roles in protecting normal cells from cancer and then, once the malignancy is initiated, both selenoenzymes have roles in the promoting cancer, we have begun studying murine CT26 colon cancer cells in which we successfully knocked down both TR1 and Sep15 using RNAi. We are planning on investigating the interactive effects of these two selenoproteins on malignancy in these and other cancer cells of both mouse and human origin. Of specific interest are cancer cell lines with comparatively strongly increased or decreased TR1 and/or Sep15 protein expression. Thus, we have begun an initial screening of several human cancer cell lines from various tissues using both radio-labeling with 75Se as well as western blotting techniques.