Activation of nuclear factor-kappa B in human metastatic melanomacells and the effect of oxidative stress.

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

Activation of nuclear factor-kappa B in human metastatic melanomacells and the effect of oxidative stress.

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

Melanin as a target for melanoma chemotherapy: Pro-oxidant effect of oxygen and metals on melanoma viability

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)-active 5,5'-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O-2/5% CO2 atmospheres show markedly reduced viability as compared with normal melanocytes. Cu- and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal- and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes