This is a competitive renewal application to investigate the molecular mechanisms involved in skin cancer chemoprevention by apigenin, a nontoxic and nonmutagenic bioflavonoid which inhibits UV-induced skin carcinogenesis when topically applied to mouse skin. During the previous funding period, the applicant investigated the effect of apigenin treatment on expression of the p53 tumor suppressor gene in mouse keratinocytes. In keratinocyte cell lines with wildtype p53 status, the applicant demonstrated that apigenin is extremely potent in elevating the level of wildtype p53 protein in keratinocytes (27-fold). This level of p53 induction is substantially higher than that induced in keratinocytes by UVB irradiation, for example (5-fold). The applicant further demonstrated that the increased level of p53 protein was due to protein stabilization, accompanied by increased phosphorylation of p53 at Ser15 and subsequent transcriptional activation of p53-responsive genes including p21WAF1. Interestingly, the applicant did not observe any increased accumulation of MDM2 protein in apigenin-treated cells, which is unexpected considering that the MDM2 gene is a downstream target of p53 transcriptional activation and generally responsible for feedback inhibition of p53 by promoting its degradation through ubiquitination. The applicant's results indicate that apigenin treatment of keratinocytes induces many of the same events in the p53 pathway that are normally triggered during the cellular UV DNA damage response, with the exception that the negative feedback MDM2 control loop appears to be absent. The lack of feedback inhibition by MDM2 may prolong the beneficial effects of p53 protein stabilization in apigenin-treated keratinocytes. The hypothesis to be tested in this renewal application is that apigenin's chemopreventive activity is derived from its ability to enhance the response of the normal cellular p53 pathway to UV-induced damage in keratinocytes. The applicant proposes four specific aims to test this hypothesis: (1) Investigate the mechanism(s) by which apigenin treatment induces posttranslational modification of p53, by identifying the p53 phosphorylation sites, the kinases involved in phosphorylation, and whether apigenin induces p53 acetylation in keratinocytes; (2) Having characterized p53 protein post-translational modification induced by apigenin treatment alone, investigate the combined effects of apigenin treatment plus UVB irradiation on p53 protein levels, stabilization, and post-translational modification; (3) Investigate the impact of apigenin treatment on MDM2 gene expression, on interaction of p53 and MDM2 protein, and whether apigenin treatment results in inhibition of MDM2-mediated p53 ubiquitination and degradation; (4) Investigate the combined effects of apigenin treatment plus UVB irradiation on MDM2 gene expression, MDM2/p53 protein interaction, and MDM2/p53 protein interaction, and MDM2-mediated p53 ubiquitination.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA072987-06
Application #
6513048
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Pelroy, Richard
Project Start
1996-05-01
Project End
2005-06-30
Budget Start
2002-07-09
Budget End
2003-06-30
Support Year
6
Fiscal Year
2002
Total Cost
$242,515
Indirect Cost
Name
University of Kansas
Department
Pathology
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Tong, Xin; Pelling, Jill C (2009) Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA-binding protein HuR. Mol Carcinog 48:118-29
Abu-Yousif, Adnan O; Smith, Kimberly A; Getsios, Spiro et al. (2008) Enhancement of UVB-induced apoptosis by apigenin in human keratinocytes and organotypic keratinocyte cultures. Cancer Res 68:3057-65
Kanski, Jaroslaw; Hong, Sung J; Schoneich, Christian (2005) Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry. J Biol Chem 280:24261-6
Kanski, Jaroslaw; Schoneich, Christian (2005) Protein nitration in biological aging: proteomic and tandem mass spectrometric characterization of nitrated sites. Methods Enzymol 396:160-71
Kanski, Jaroslaw; Behring, Antje; Pelling, Jill et al. (2005) Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am J Physiol Heart Circ Physiol 288:H371-81
Van Dross, Rukiyah T; Hong, Xiaoman; Pelling, Jill C (2005) Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes. Mol Carcinog 44:83-91
Nauser, Thomas; Koppenol, Willem H; Pelling, Jill et al. (2004) UV photolysis of 3-nitrotyrosine generates highly oxidizing species: a potential source of photooxidative stress. Chem Res Toxicol 17:1227-35
Nauser, Thomas; Pelling, Jill; Schoneich, Christian (2004) Thiyl radical reaction with amino acid side chains: rate constants for hydrogen transfer and relevance for posttranslational protein modification. Chem Res Toxicol 17:1323-8
Yamaguchi, Tamio; Nagao, Shizuko; Wallace, Darren P et al. (2003) Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. Kidney Int 63:1983-94
Van Dross, Rukiyah; Xue, Yue; Knudson, Alexandra et al. (2003) The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. J Nutr 133:3800S-3804S

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