Abstract
Melanocytes and melanoma cells contain melanin, a complex polymer that modulates redox changes in these cells. Relative intracellular hydrogen peroxide levels measured by dichlorodihydrofluorescein are similar in the two cell types, but the levels of superoxide anion measured by dihydroethidium were markedly increased in melanoma cells. Chelator-induced oxidative stress is efficiently suppressed by melanocytes without substantial recruitment of the transcription factors NF-κB and AP-1 as measured by electrophoretic mobility shift assay and quantitated by densitometry or by a change in frequency of apoptosis as determined by annexin V binding. In contrast, NF-κB in melanoma cells is strongly recruited by changes in redox status and exhibits a correlative relationship to intracellular hydrogen peroxide (but not superoxide anion). However, the response of the NF-κB pathway to intracellular hydrogen peroxide is anomalous, including downregulation of p65 and IκBα RNA expression (Northern blot). Additionally, recruitment of AP-1 binding in melanoma cells was directly correlated with intracellular levels of superoxide anion (but not hydrogen peroxide). Neither the degree of NF-κB nor AP-1 binding in melanoma cells was related to the frequency of apoptosis. The responsiveness of NF-κB and AP-1 recruitment to intracellular levels of hydrogen peroxide and superoxide anion without concomitant control of apoptosis provides a general mechanism by which these cells can escape noxious injury (e.g., chemotherapy). The marked enhancement of apoptosis in melanoma cells by chelators indicates, however, that this alteration can be circumvented and offers a unique therapeutic window to explore.
Highlights
Metastatic melanoma is among the most therapeutically refractory of all cancers [1]
Using the molecular probes DCF and HE we measured, respectively, the basal intracellular levels of hydrogen peroxide and superoxide anion in normal melanocytes and metastatic melanoma cells cultured in their standard media (Table 1)
The ratio of superoxide anion in melanoma compared to melanocytes was increased 3to 4-fold while hydrogen peroxide was decreased as much as 43%
Summary
Metastatic melanoma is among the most therapeutically refractory of all cancers [1]. The biochemical and molecular basis of this property is largely unknown, there has been no shortage of biochemical and immunological explanations offered [2,3]. We have taken a different approach to the issue and have recently demonstrated that nuclear factor-B (NF-B) is constitutively activated in human metastatic melanoma cells compared to normal melanocytes [4]. Despite the marked increase of NF-B binding in melanoma cells under a variety of basal and culture-supplemented conditions, this property was further enhanced by oxidative stress produced by a number of experimental manipulations of these cells. As NF-B is known to enhance survival pathways in other benign and malignant cells [5,6,7,8], we proposed that a similar situation was operative in melanoma cells and that the constitutive activation of NF-B results in an ongoing and continuous stress response that affords protection against noxious agents, including chemotherapeutic and other interventions. We have previously shown that components of the AP-1 pathway are abnormally regulated in melanoma cells [10]
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