Enhancement of UVB-induced apoptosis by the chemopreventive bioflavonoid apigenin in multiple human keratinocyte models

Abstract

Topical application of the bioflavonoid apigenin (4',5,7-trihydroxyflavone) to mouse skin effectively reduces incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are still not fully understood. In the present study, we aimed to study the effect of apigenin treatment and UVB exposure on human keratinocytes and to investigate how apignein may alter the biological consequences of UVB exposure. The experiments described herein, employed three models of human keratinocytes: HaCaT human keratinocyte cells, normal human keratinocytes (NHK) cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures (OTKC). The ability of UVB to induce cycloxygenase-2 (COX-2) was investigated due to the role it is thought to play in photocarcinogenesis. The apoptotic response of keratinocytes to UVB is thought to be critical to the development of skin cancer, and therefore was investigated in multiple human keratinocyte models. Each keratinocyte model was exposed to a moderate dose of UVB (300-1000 J/m2), then treated with apigenin (0-50 μM) and harvested to assess apoptosis by Western blot analysis for poly-ADP-ribose polymerase (PARP) cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis more than two-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization, and increased the release of cytochrome c from the mitochondria compared to UVB exposure alone. Overexpression of the anti-apoptotic protein Bcl-2 and expression of a dominant negative form of Fas-Associated Death Domain (FADDdn) led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest enhancement of UVB-induced apoptosis by apigenin treatment involves both the stress-mediated, intrinsic pathway and the receptor-mediated, extrinsic pathway of apoptosis. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin.