The activity of NADPH oxidase is increased in malignant skin keratinocytes. We demonstrated that inhibition of NADPH oxidase activity by diphenyleneiodonium (DPI) suppressed free radical production, inhibited cell growth and promoted cell differentiation of B16 melanoma cells, as indicated by cell morphology, increased production of melanin, and increased expression of microphthalmia-associated transcription factor (MITF). siRNA to NADPH oxidase subunit Rac1 or p47 induced the expression of MITF, verifying that the pro-differentiation effects are due to the inhibition of NADPH oxidase. Biochemical studies suggest that ERK plays a positive role whereas PKCα plays a negative role during this differentiation event. In addition, the protein levels of the tumor suppressor p53 were suppressed by DPI, suggesting that p53 is activated by oxidative stress and may negatively regulate differentiation in melanoma cells. Taken together, these results suggest that inhibiting NADPH oxidase activity promotes cell differentiation of B16 melanoma cells.

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