UVB-induced apoptosis in keratinocytes is enhanced by the chemopreventive bioflavonoid apigenin.

B125

Chronic exposure to Ultraviolet B (UVB, 290-320nm) is thought to be the major cause of squamous cell carcinoma and basal cell carcinoma. Therefore, the need for an agent that is able to reverse, suppress, or prevent UVB-induced carcinogenic progression in the skin is clearly warranted. Previous studies have demonstrated the bioflavonoid apigenin topically applied to mouse skin reduces tumor formation and multiplicity in UVB-treated SKH-1 hairless mice. In our current study we are evaluating the ability of apigenin to exert its chemopreventive effects by inducing apoptosis in human keratinocytes alone and in combination with UVB. We are employing three different cell-based models: HaCaT keratinocytes, primary keratinocytes derived from human neonatal foreskin, and epidermal equivalent cultures. Western blot analysis for PARP cleavage demonstrated the induction of apoptosis in HaCaT cells and primary keratinocyte cultures treated with apigenin (10-25 uM) for 4-24 hours. Treatment of epidermal equivalent cultures with 50 uM apigenin resulted in PARP cleavage as assessed by Western blot analysis. HaCaT keratinocytes and primary human keratinocytes were exposed to UVB (300-1000 J/m2) and harvested 24 hours later. Apoptosis was assessed in monolayer cultures by caspase-9 activation, Annexin-V staining, and Western blot analysis for PARP cleavage. In epidermal equivalent cultures apoptosis was determined by Western blot analysis, as well as histochemical evaluation for sunburn cells and active caspase-3. When HaCaT cells and primary keratinocytes were treated with both UVB and apigenin, the level of UVB-induced apoptosis was enhanced compared to UVB treatment alone. Our results demonstrate that UVB induces apoptosis and furthermore, that apigenin treatment can enhance the apoptotic response initiated by UVB. The chemopreventive action of apigenin may be explained by its ability to enhance UV-induced apoptosis.