Role of Selenium-containing Proteins in Cancer

含硒蛋白质在癌症中的作用

• 批准号: 8157523
• 负责人: Dolph Hatfield
• 金额: $ 63.81万
• 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/8157523
• 关键词: Malignant Neoplasms; Proteins; Role; Selenium

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.

硒领域的许多研究人员提出，低分子量硒化合物负责造成硒的众多健康益处，而另一种建议是硒蛋白可能是负责任的药物。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，我们是第一个提出这些健康益处的人之一，这主要是由于硒蛋白中的硒作为氨基酸，硒代半胱氨酸（SEC）。因此，为了阐明硒蛋白在预防癌症中的作用，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：硫氧还蛋白过氧化物酶1（TR1）和硒蛋白15（9月15日），它们在预防和促进癌症中都起着作用。如去年报道所述，我们先前曾证明，使用RNA干扰技术在肺癌细胞系中以及由致癌K-RAS驱动的癌细胞系中使用RNA干扰技术的敲低导致几种恶性表型越来越反向正常细胞，这表明TR1缺乏症是抗肿瘤的。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，并进一步详细介绍了TR1在乳腺癌细胞系中凋亡中的作用。如前所述，选择了乳腺癌细胞系，因为我们发现乳腺癌细胞中的TR1敲低对TNF-Alpha诱导的凋亡表现出更高的敏感性。已提出TR1通过调节硫氧还蛋白（TRX）来控制各种细胞过程。我们通过将TR1和TRX1敲低细胞的响应与多种类型的刺激进行比较，建立了TRX1敲低细胞，以研究TRX作为TR1函数的主要目标。这些研究表明，两个敲低细胞均显示出与过氧化氢处理的相似反应模式（H2O2），但它们对硒诱导的毒性的反应不同。 TR1敲低细胞对硒处理更为敏感，而TRX1敲低细胞显示出与相应的对照细胞相似的敏感性。我们建立了一个TR1敲低和随后的重新表达策略，用于搜索仅在应力条件下起作用的另一种TR1结合蛋白。我们在由H2O2，TNF-Alpha和硒诱导的细胞应激期间发现了几个候选TR1结合蛋白，并正在进一步表征这些蛋白质。 TR1缺乏细胞系对硒毒性的敏感性更高的原因知之甚少，但这并不是由于氧化应激。我们正在进一步研究恶性TR1缺乏细胞对硒毒性的敏感性增强，因为TR1抑制剂和硒的组合可能会提供癌症治疗的新途径。我们还在检查恶性细胞中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，并且在许多癌细胞中确实过表达。啮齿动物中发生了四种不同的TR1剪接形式。其中三个（变体II-IV）由同一启动子驱动，而第三个（变体I）可以由其他启动子控制。我们已经确定，小鼠（正常细胞和癌细胞）中TR1最丰富的表达形式是变体II。该观察结果表明，癌细胞中TR1的过表达是由与正常细胞相同的启动子引发的。在癌细胞中阐明TR1表达的起始机制将是靶向癌症治疗中TR1上调的关键。现在，我们正在分析TR1启动子区域的甲基化状态，并在正常和癌细胞中进行比较。这将阐明DNA甲基化是否可能在TR1表达的调节中起作用。 正如去年报告所报道的那样，我们发现15KDA硒蛋白（9月15日）似乎在结直肠癌中起作用。我们证明，在鼠结肠（Adeno）癌细胞系CT26中，Sep15的靶向下调逆转了其特征，这些特性是癌细胞的典型特征，以类似于正常细胞。最近，我们扩展了对对变化剂状态的人类结肠癌细胞的研究的研究，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TR1和SEP15是双刃剑在保护正常细胞免受癌症中的作用的两侧，然后一旦开始恶性肿瘤，两种硒酶在促进癌症中都具有作用，我们已经开始研究Murine CT26 Colon Colison Cells，在这些癌症中都成功地敲破了Trrna和sep115555555555555555555555555555555年5月15日。我们计划研究这两种硒蛋白对小鼠和人类来源的其他癌细胞中恶性肿瘤的互动作用。特定感兴趣的是癌细胞系具有相对强烈增加或降低TR1和/或SEP15蛋白表达的癌细胞系。因此，我们已经开始使用具有75SE和Western印迹技术的无线电标签对各种组织的几个人类癌细胞系进行了初步筛查。