Role of Selenium-containing Proteins in Cancer

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，而另一种说法是，硒蛋白可能是更负责任的因素。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。几年前，我们率先提出，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中的作用，我们正在研究不同的硒蛋白在恶性肿瘤过程中的功能。至少有两种硒蛋白，硫氧还蛋白过氧化物酶1(TR1)和硒蛋白15(9月15日)，同时具有预防和促进癌症的作用。正如我们在去年的报告中指出的那样，我们之前已经证明，在肺癌细胞系和致癌k-ras驱动的癌细胞系中，利用RNA干扰技术敲除TR1会导致一些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，并进一步详细地研究了TR1在乳腺癌细胞系凋亡中的作用。如前所述，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。TR1被认为通过调节硫氧还蛋白(Trx)来控制各种细胞过程。我们建立了Trx1基因敲除细胞，通过比较TR1和Trx1基因敲除细胞对多种刺激的反应，来研究TRX作为TR1s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的反应模式相似，但对硒诱导的细胞毒性的反应不同。Tr1基因敲除细胞对硒处理的敏感性要高得多，而Trx1基因敲除细胞表现出与相应对照细胞相似的敏感性水平。我们建立了TR1基因敲除和随后的再表达策略，以寻找另一种仅在应激条件下发挥作用的TR1结合蛋白。我们在过氧化氢、肿瘤坏死因子-α和硒诱导的细胞应激过程中发现了几个候选的TR1结合蛋白，并对这些蛋白进行了进一步的鉴定。TR1缺乏的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TR1缺陷细胞对硒毒性的增强敏感性，因为TR1抑制剂和硒的结合可能为癌症治疗提供一条新的途径。我们还在研究恶性细胞中TR1的上调。TR1是哺乳动物细胞中主要的抗氧化剂和氧化还原调节剂，在许多癌细胞中确实过表达。在啮齿动物中存在四种不同的TR1剪接形式。其中三个(变异体II-IV)由同一启动子驱动，而第三个(变异体I)可能由不同的启动子控制。我们已经发现TR1在小鼠(正常细胞和癌细胞)中最丰富的表达形式是变体II。这一观察表明，TR1在癌细胞中的过度表达是由与正常细胞相同的启动子启动的。阐明TR1在肿瘤细胞中表达的启动机制将是靶向上调TR1在肿瘤治疗中的关键。我们现在正在分析TR1启动子区域的甲基化状态，并将其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TR1表达的调节中发挥作用。根据去年的报道，我们发现15 kDa的硒蛋白(9月15日)似乎在结直肠癌中起作用。我们证明，在小鼠结肠(腺)癌细胞系CT26中，Sep15的靶向下调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Sep15在不同恶性状态的人结肠癌细胞系中作用的研究，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，由于我们假设TR1和Sep15是一把双刃剑的两边，在保护正常细胞免受癌症侵袭方面发挥作用，然后，一旦恶性肿瘤开始，这两种硒酶都有促进癌症的作用，我们已经开始研究小鼠CT26结肠癌细胞，在其中我们成功地使用RNAi击倒了TR1和Sep15。我们正计划研究这两种硒蛋白在这些和其他小鼠和人类来源的癌细胞中对恶性肿瘤的交互作用。特别令人感兴趣的是TR1和/或Sep15蛋白表达相对强烈增加或减少的癌细胞系。因此，我们已经开始使用75Se放射性标记和免疫印迹技术从不同组织中初步筛选几个人类癌细胞株。

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 peroxidase 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.