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首页> 外文学位 >The Role of Redox Sensitive NRF2 Transcription Factor in Melanoma Drug Resistance.
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The Role of Redox Sensitive NRF2 Transcription Factor in Melanoma Drug Resistance.

机译：氧化还原敏感性NRF2转录因子在黑色素瘤耐药中的作用。

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摘要

Human melanocytes induces cytoprotective gene expression in response to UV induced oxidative stress. NRF2, a cap'n' collar basic-region leucine zipper protein is an oxidative and electrophilic stress sensitive transcription factor responsible for the expression of antioxidants and expulsion of xenobiotic drugs. NRF2 targets a key cis-acting element called the antioxidant response elements (ARE) found at the promoter of cytoprotective genes. However, constitutive activation of NRF2 in cancer increases tumorigenicity, resistance to chemotherapy and proliferation. We hypothesized, that constitutive NRF2 activity in metastatic melanoma cells mediates resistance to chemotherapy.;The basal transcript expression of NRF2 with respect to human melanocytes is expression in all 8 melanoma cell lines. Immunoblots of NRF2 displayed abundant expression in 5 of 8 human malignant melanoma lines compared to human melanocytes. A surrogate reporter of NRF2's transcriptional activity is NQO1, a gene closely regulated by NRF2 through ARE elements at its promoter. NQO1 expression was increased in 5 of 6 metastatic melanoma; similar results in immunoblots for NQO1.;Next, to determine if NRF2 in melanoma cells leads to chemosensitivity we developed stable NRF2 knockdowns in melanoma cell lines. The shRNA targeting (mRNA)NRF2 expression, resulted in 70% reduction of NRF2 transcripts in 4 of 6 constructs in 3 metastatic melanoma cell lines, and immunoblots for NRF2 resulted in protein levels below detection in 2 transgenic lines. Loss of NRF2 in SK-MEL-28KD1 sensitized these cell lines to vemurafenib, cisplatin and DHA treatment, with a calculated fold reduction of -5.2-fold, -1.7-fold, and -2.4-fold, respectively. These results demonstrated that NRF2 loss sensitized metastatic melanoma lines to vemurafenib, cisplatin, and DHA treatment, in spite of their wide difference in mechanism that lead to apoptosis.;BRAF mutation frequency in human metastatic melanoma is about 50%, vemurafenib is an inhibitor of mutant BRAF kinase that produces an effective clinical response. However, patients eventually develop resistance to vemurafenib through a mechanism that reactivates the MAPK pathway resulting in decreased overall survival. Here, we investigated the possibility of NRF2 induction contributing to a mechanism of vemurafenib resistance in BRAF mutant human metastatic melanoma cells and found vemurafenib inhibited NRF2 expression and activity in melanoma cells. Also, MEK1/2 inhibitor induced loss of NRF2 expression. An attractive strategy to sensitize metastatic melanoma to drug treatment is to inhibit NRF2 expression, in combination with a chemotherapeutic agent. DNA damaging agent (cisplatin) and vemurafenib synergistically (CI value of 0.51 +/- .11) enhanced the cell-killing effects of the drugs. Finally, the current availability of FDA approved vemurafenib when used in combination with other drugs, offers a novel strategy for augmenting the potency of previously ineffective therapies, through the pharmacological inhibition of NRF2.;The expression of NRF2 in human melanocytes cells is highly regulated and maintains an important function in protecting cells from the oxidative effects of environmental stressors and xenobiotics. However, in human melanoma cells NRF2 is aberrantly regulated, enhancing resistance to drug treatments and overall survival. Surprisingly, vemurafenib is a novel inhibitor of NRF2, and demonstrated that NRF2 is downstream of the MAPK cascade pathway. Furthermore, acquired resistance to vemurafenib contributed to increased expression of NRF2, an effect leading to drug resistance. This study provides evidence of a therapeutic potential of a pharmacological inhibitor, which sensitized melanoma to drug treatments. Finally, the current availability of FDA approved vemurafenib when used in combination with other chemotherapies, offers a novel strategy for augmenting the potency of previously ineffective therapies, through the pharmacological inhibition of NRF2.
机译：人类黑素细胞响应紫外线诱导的氧化应激诱导细胞保护性基因表达。 NRF2是一个'cap'n'领基本区域亮氨酸拉链蛋白，是一种氧化和亲电子应激敏感的转录因子，负责表达抗氧化剂和驱逐异源药物。 NRF2靶向在细胞保护性基因启动子处发现的关键顺式作用元件，称为抗氧化反应元件（ARE）。但是，在癌症中NRF2的组成型激活会增加致瘤性，对化学疗法的抵抗力和增殖能力。我们假设转移性黑素瘤细胞中的组成型NRF2活性介导了对化学疗法的抵抗力。NRF2相对于人类黑素细胞的基础转录表达在所有8种黑素瘤细胞系中都有表达。与人类黑素细胞相比，NRF2的免疫印迹在8个人类恶性黑色素瘤细胞系中的5个中显示了丰富的表达。 NRF2转录活性的替代报道者是NQO1，它是一个由NRF2通过其启动子处的ARE元件紧密调控的基因。 6例转移性黑色素瘤中有5例NQO1表达增加。 NQO1的免疫印迹具有相似的结果。接下来，要确定黑素瘤细胞中的NRF2是否导致化学敏感性，我们在黑素瘤细胞系中开发了稳定的NRF2基因敲低。靶向（mRNA）NRF2的shRNA表达导致3个转移性黑素瘤细胞系中6个构建体中的4个构建体中的NRF2转录本减少了70％，NRF2的免疫印迹导致2个转基因系中的蛋白水平低于检测水平。 SK-MEL-28KD1中NRF2的丢失使这些细胞系对维罗非尼，顺铂和DHA处理敏感，计算出的折减分别为-5.2倍，-1.7倍和-2.4倍。这些结果表明，尽管NRF2缺失使转移性黑色素瘤细胞系对vemurafenib，顺铂和DHA治疗敏感，尽管它们导致凋亡的机制差异很大;人类转移性黑色素瘤中的BRAF突变频率约为50％，vemurafenib是VEGF的抑制剂突变BRAF激酶，可产生有效的临床反应。但是，患者最终会通过重新激活MAPK途径的机制而导致对维罗非尼的耐药性，从而导致总体生存期降低。在这里，我们调查了NRF2诱导在BRAF突变型人类转移性黑色素瘤细胞中对维拉非尼耐药的机制的可能性，并发现维拉非尼抑制了黑色素瘤细胞中NRF2的表达和活性。同样，MEK1 / 2抑制剂诱导了NRF2表达的丧失。使转移性黑素瘤对药物治疗敏感的有吸引力的策略是与化学治疗剂联合抑制NRF2表达。 DNA损伤剂（顺铂）和维罗非尼协同作用（CI值为0.51 +/- .11）增强了药物的细胞杀伤作用。最后，FDA批准的vemurafenib与其他药物组合使用时的当前可用性，通过对NRF2的药理抑制作用，为增强先前无效疗法的效力提供了一种新颖的策略; NRF2在人黑素细胞中的表达受到高度调节并在保护细胞免受环境应激物和异种生物的氧化作用方面，它起着重要的作用。但是，在人黑素瘤细胞中，NRF2受到异常调节，从而增强了对药物治疗的抵抗力和总体生存率。出人意料的是，维拉非尼是NRF2的新型抑制剂，并证明NRF2在MAPK级联途径的下游。此外，获得性的对vemurafenib的耐药性导致NRF2表达增加，从而导致耐药性。该研究提供了药理学抑制剂的治疗潜力的证据，该药理学抑制剂使黑素瘤对药物治疗敏感。最后，当与其他化学疗法联合使用时，FDA批准的vemurafenib的当前可用性提供了一种通过对NRF2进行药理学抑制来增强以前无效疗法的效力的新策略。

著录项

  • 作者

    Matundan, Harry H.;

  • 作者单位

    University of California, Irvine.;

  • 授予单位 University of California, Irvine.;
  • 学科 Biology Molecular.;Health Sciences Oncology.;Health Sciences Pharmacy.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 102 p.
  • 总页数 102
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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