1582

The effect of Kelch-like ECH associating protein 1 (Keap1) knockdown in human keratinocytes was studied to determine the potential of Keap1 as a target for chemoprevention against Non Melanoma Skin Cancer (NMSC). UVA-induced Activator Protein-1 (AP-1) activity, which is involved in skin tumor promotion, served as a marker for stress responses. During homeostatic conditions, Keap1 binds Nuclear factor-erythroid 2 p45-related factor-2 (Nrf2) in the cytoplasm, effectively inactivating it. Dissociation of Nrf2 from Keap1 naturally occurs under oxidative stress. To induce Keap1 knockdown, HCl-14 cells (HaCaTs, human keratinocytes, stably transfected with an AP-1 driven luciferase reporter construct) were transfected with Keap1 siRNA. Controls were transfected with either the scrambled sequence of Keap1 siRNA or the transfection reagent alone. Real-time RT-PCR was performed to show that the siRNA sufficiently reduced Keap1 mRNA levels. Keap1 mRNA levels were significantly decreased in relation to their scrambled controls between 24 and 72 hours post-transfection with 50nmol of Keap1 siRNA. Increased Nrf2 activity was examined by quantification of a common target of Nrf2, NAD(P)H-quinone oxidoreductase-1 (NQO1). Real-time RT-PCR showed that NQO1 mRNA levels were significantly increased in comparison to their scrambled controls between 48 and 72 hours post transfection with 50nmol Keap1 siRNA. To study the effects of Keap1 knockdown on UVA-induced AP-1 activation, the cells were irradiated with 250,000 J/m2 of UVA light. AP-1 activity was inhibited by Keap1 siRNA treatment in a time and dose-dependent manner. At the 50nmol siRNA dose, there was a 48% inhibition in UVA-induced AP-1 activity compared to scrambled controls 72 hours post transfection. At the 100nmol siRNA dose, there was a 68% inhibition of UVA-induced AP-1 activity compared to scrambled controls 48 hours post transfection. Based on these results, we conclude that the increase of active Nrf2 by elimination of its regulatory protein, Keap1, has potential as a strategy against UVA-induced skin carcinogenesis. This work was supported in part by NIH grant CA 27502 and MARC grant T34 GM08718.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA