Meyskens F L, Buckmeier J A, McNulty S E, Tohidian N B
Department of Medicine and the Chao Family Comprehensive Cancer Center, University of California-Irvine, Orange 92868, USA.
Clin Cancer Res. 1999 May;5(5):1197-202.
The biological basis for the general pharmacological resistance of human melanoma is unknown. A unique biochemical feature of the melanocyte is the synthesis of melanin, which leads to the generation of hydrogen peroxide and the consumption of reduced glutathione. This activity produces a state of chronic oxidative stress in these cells. We demonstrated previously that the expression of the c-jun family was dysregulated in metastatic melanoma cells compared with normal human melanocytes (D. T. Yamanishi et al., J. Invest. Dermatol., 97: 349-353, 1991). In the current investigation, we measured the levels of two major redox response transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1, in metastatic melanoma cells and normal melanocytes and their response to oxidative stress. The basal DNA-binding activity of NF-kappaB as measured by the electrophoretic mobility shift assay in metastatic melanoma cells was increased 4-fold compared with that of normal melanocytes. This level of binding was paralleled by a 1.5- to 4-fold increase in the expression of p50 (NF-kappaB1), p65 (Rel-A), and IkappaB-alpha as measured by Northern blot analysis. In contrast, the expression of p75 (c-rel) was markedly decreased (60%) in melanoma cells compared with normal melanocytes. Following oxidative stress produced by enzyme-generated H2O2, free H2O2, or incubation with buthionine sulfoximine, NF-kappaB binding activity increased 1.5- to 2.5-fold in melanoma cells (buthionine sulfoximine > H2O2), but only slightly in normal melanocytes. In contrast, activator protein-1 binding activity was unaffected or increased in normal melanocytes in response to oxidative stress, but was either unaffected or decreased in melanoma cells. These results suggest that the redox regulation of melanoma cells at the molecular level is fundamentally different from normal melanocytes and may offer a unique avenue for preventive or therapeutic intervention as well as new insights into the pathogenesis of melanocyte transformation.
人类黑色素瘤普遍存在药理抗性的生物学基础尚不清楚。黑素细胞的一个独特生化特征是黑色素的合成，这会导致过氧化氢的生成以及还原型谷胱甘肽的消耗。这种活性在这些细胞中产生了一种慢性氧化应激状态。我们之前证明，与正常人黑素细胞相比，转移性黑色素瘤细胞中c-jun家族的表达失调（D. T. 山岸等，《皮肤病学研究杂志》，97: 349 - 353, 1991）。在当前研究中，我们测量了转移性黑色素瘤细胞和正常黑素细胞中两种主要的氧化还原反应转录因子，即核因子-κB（NF-κB）和活化蛋白-1的水平，以及它们对氧化应激的反应。通过电泳迁移率变动分析测定，转移性黑色素瘤细胞中NF-κB的基础DNA结合活性比正常黑素细胞增加了4倍。通过Northern印迹分析测定，p50（NF-κB1）、p65（Rel-A）和IκB-α的表达增加了1.5至4倍，与这种结合水平平行。相比之下，与正常黑素细胞相比，黑色素瘤细胞中p75（c-rel）的表达显著降低（60%）。在由酶产生的H2O2、游离H2O2或与丁硫氨酸亚砜胺孵育产生氧化应激后，黑色素瘤细胞中NF-κB结合活性增加了1.5至2.5倍（丁硫氨酸亚砜胺 > H2O2），而在正常黑素细胞中仅略有增加。相比之下，活化蛋白-1结合活性在正常黑素细胞中对氧化应激有反应时未受影响或增加，但在黑色素瘤细胞中要么未受影响要么降低。这些结果表明，黑色素瘤细胞在分子水平上的氧化还原调节与正常黑素细胞有根本不同，可能为预防或治疗干预提供独特途径，并为黑素细胞转化的发病机制提供新的见解。
