Abstract 5657: Ursolic acid prevents activation of oncogenic factors in UV-treated HaCaT cells

Abstract

Abstract The aim of this study was to determine the anti-cancer potential of the phytonutrient ursolic acid (UA) in a UV-treated HaCaT human keratinocyte cell line. Earlier studies have shown UA strongly inhibits DMBA/TPA-mediated carcinogenesis in SENCAR mice, but effects of UA on UV-induced skin cancer are unknown. Our hypothesis was that treating cells with UA, found in plant sources including rosemary and apple, may inhibit UV-induced cell death, ROS production, and transcription and activation of oncogenes and oncoproteins. These in vitro studies would suggest UA may inhibit UV-mediated skin carcinogenesis. HaCaT cells were seeded at 2,000 cells/well for MTT and DHR assays, and at 1.6 × 104 cells/cm2 for RT-PCR and Western blotting. 24 hours after seeding, cells were treated with concentrations of UA up to 25 μM. Cells were UV-treated with various doses using a transilluminator emitting 36% of its energy in the UVA range and 64% in the UVB range with a peak of 312 nm. For cell death assays, cells were incubated for 4 hours in media with MTT, UV treated, then dissolved in DMSO after various timepoints prior to measuring the absorbance at 570 nm. For quantification of intracellular H2O2, cells were incubated in media containing 5 µM DHR 123 for 1 hour prior to UV-treatment, followed by reading fluorescence at 530 nm with an excitation of 485 nm. RT-PCR was performed for bcl-2 and c-jun using SYBR green probe. Western blotting for phospho-p38 MAPK was done with whole cell lysates, isolated using RIPA buffer. Phospho-p65 and phospho-c-jun were quantified by blotting nuclear and cytosolic fractions, which were isolated using a hypotonic/hypertonic nuclear extraction procedure. We find that treating cells for 24 hours before UV and 12 hours post-UV with non-toxic doses of UA does not protect cells from UV-death. Also, pretreating this cell line with these doses of UA does not inhibit a UV-induced rise in intracellular H2O2 within an hour post-UV. However, RT-PCR results show UA treatment exacerbates a UV-induced rise in bcl-2 RNA levels and inhibits a UV-mediated increase in c-jun RNA 1 hour post-UV. Also, Western blotting reveals that 25 μM UA inhibits a 500 mJ/cm2-induced increase in phospho-p38 MAPK by 85%, and that treatment with 10 μM UA reduces 100 mJ/cm2-initiated nuclear accumulation of oncogenic phospho-c-jun by 47.6%, 1 hour after UV. Finally, treatment with 10 and 25 μM UA dose-dependently contributes to a decrease in nuclear phospho-p65 seen 1 hour post-UV treatment. These in vitro results show UA may prevent UV-mediated skin cancer by modulating the activities of the oncogenic transcription factors AP-1 and NFκB. Supported by NIH grants CA 102747 and P30 CA 54174-16S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5657.