Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biologi cal processes including cell proliferation and differentiation. We have pre viously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic f raction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-appli cation of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhib its 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxyl ase (ODC) activity, as well as 7,12-dimethylbenz[a]anthracene (DMBA)-initia ted. TPA-promoted mouse skin tumorigenesis. The present studies were design ed to further characterize the effect of time and dose of application of GP F on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 m in prior to treatment with TPA resulted in a significant decrease in epider mal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet. ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were sign ificantly increased by 1.8 and 1.9-fold, respectively, compared with contro ls. A similar response was observed with the ODC inhibitor 2-difluoromethyl ornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibite d ODC activity by 62 and 68%, respectively, compared with controls (P < 0.0 5). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar tu that observed when GPF application precede d TPA. When applied to mouse skin prior to TPA, GPF was associated with a d ecrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preced ed the decrease in ODC activity. In a separate in vitro study, kinetic anal yses indicated that GPF is a competitive inhibitor of ODC activity. Collect ively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magni tude of inhibition is independent of epidermal ODC protein content. In addi tion, GPF is a competitive inhibitor of. ODC activity in vitro. The decreas e in TPA-induced ODC activity clue to GPF treatment is preceded by an inhib ition of TPA-induced PKC activity. Thus, the polyphenolic fraction from gra pes warrants further examination as a skin cancer chemopreventive agent tha t interferes with cellular, events associated with TPA promotion. (C) 2000 Elsevier Science ireland Ltd. All rights reserved.