Redox modulation of NF-κB nuclear translocation and DNA binding in metastatic melanoma.: The role of endogenous and γ-glutamyl transferase-dependent oxidative stress
Redox modulation of NF-κB nuclear translocation and DNA binding in metastatic melanoma.: The role of endogenous and γ-glutamyl transferase-dependent oxidative stress
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DOI：
10.1177/030089160308900416
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发表时间：
2003-07-01
期刊：
影响因子：
1.9
通讯作者：
Pompella, A
中科院分区：
文献类型：
--
作者：
Dominici, S;Visvikis, A;Pompella, A
目标和背景。转录因子NF-κ B参与细胞增殖、凋亡和转移相关基因的表达。在黑色素瘤中，通常观察到高组成性水平的NF-κ B活化。NF-κ B受氧化/还原（氧化还原）过程的调节，并且已经记录了黑素瘤细胞中组成性氧化应激的发生。我们实验室最近的研究表明，膜结合γ-谷氨酰转移酶（GGT）酶活性-由许多恶性肿瘤表达，包括黑色素瘤-可以作为超氧化物，过氧化氢和其他促氧化剂的基础来源。方法：在本研究中，我们利用了Me 665/2人转移性黑素瘤的2/60克隆，其显示高水平的GGT活性，为了验证这种酶的存在-通过促进氧化还原过程-是否可能影响NF-κ B的活化状态。后者通过测定p65亚基的核转位（免疫印迹法），NF-κ B的DNA结合（电泳迁移率变动分析）和其转录活性（基因转录活性研究）进行了评估。刺激GGT活性的底物谷胱甘肽和甘氨酰甘氨酸引起额外的生产过氧化氢，高达约水平。基础水平加倍因此，在其GGT活性通过底物或抑制剂调节的Me 665/2/60细胞中研究了NF-κ B p65亚基的核转位、DNA结合和基因反式激活。刺激GGT活性导致p65核转位增加，而另一方面NF-κ B DNA结合和基因反式激活却矛盾地减少。通过用巯基还原剂二硫苏糖醇（DTT）处理细胞裂解物可以恢复NF-κ B DNA结合。用外源性过氧化氢处理细胞不影响NF-κ B激活status.Conclusions：总的来说，获得的数据表明，GGT活性可能会损害的氧化还原状态的硫醇，这是关键的NF-κ B DNA结合和基因反式激活，通过生产的促氧化剂物种据称不同于过氧化氢。因此，GGT活性似乎是调节黑色素瘤中NF-κ B转录活性的额外因素，即使在持续氧化应激将有利于NF-κ B核转位的条件下，也能够阻碍NF-κ B DNA结合。
Aims and background. The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity - expressed by a number of malignancies, including melanoma - can act as a basal source of superoxide, hydrogen peroxide and other prooxidants.Methods: In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme - through the promotion of redox processes - may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies).Results: Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreltol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status.Conclusions: Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.