Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes.

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5-2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathway.

Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and nuclei corresponded temporally with significant protection against DNA photodamage. The kinetics of accumulation of the three tocopherols in whole cells and in nuclei were similar. Although only alphaTH conferred significant protection compared with irradiated controls at 2 h, the differences between individual tocopherols were not statistically significant. This work suggests that incorporation of tocopherol compounds into sunscreen products confers protection against procarcinogenic DNA photodamage and that cellular uptake and distribution of tocopherol compounds is necessary for their optimal photoprotection.

Inhibition of UVB induced DNA photodamage in mouse epidermis by topically applied alpha-tocopherol.

Ultraviolet B (UVB, 290-320 nm) exposure results in a variety of cellular insults including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions can lead to mutations in critical genes and contribute to the development of nonmelanoma skin cancer. Topically applied alpha-tocopherol (vitamin E) has previously been shown to prevent the induction of skin tumors in UVB irradiated female C3H/HeNTac mice. We hypothesized that alpha-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. To explore possible mechanisms of photoprotection, we topically applied alpha-tocopherol dispersed in a neutral cream vehicle to the dorsal epidermis of female C3H/HeNTac mice and exposed them to 2.5 J/m2/s of UVB for 60 min. Immediately after exposure, we analyzed thymine dimer levels in DNA by capillary gas chromatography with electron capture detection. Epidermal DNA from mice receiving this UVB dose contained 247 +/- 42 pmol thymine dimers/micromol thymine. Topical application of alpha-tocopherol inhibited dimer formation in a dose-dependent manner. A 1% alpha-tocopherol dispersion inhibited the formation of thymine dimers to 43% of levels in vehicle controls. Several vitamin E compounds, including alpha-tocopherol acetate, alpha-tocopherol methyl ether, gamma-tocopherol, and delta-tocopherol also inhibited thymine dimer formation, but were five- to ten-fold less potent than alpha-tocopherol. A variety of commercially available sunscreens were also less potent than alpha-tocopherol in their ability to reduce dimer formation. These results suggest that DNA photoprotection is an important mechanism by which topically applied alpha-tocopherol can inhibit UVB induced skin cancer. Alpha-Tocopherol acetate, the most common form of vitamin E in commercial skin care products, conferred less protection, perhaps due to its lower absorptivity in the UVB. Our results further underscore the importance of determining which forms of vitamin E can inhibit specific lesions involved in photocarcinogenesis.