Topical applications of caffeine or (-)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice

Abstract

SKH-1 hairless mice were irradiated with ultraviolet B (UVB) twice weekly for 20 weeks. These tumor-free mice, which had a high risk of developing skin tumors during the next several months, were then treated topically with caffeine (6.2 micromol) or (-)-epigallocatechin gallate (EGCG; 6.5 micromol) once a day 5 days a week for 18 weeks in the absence of further treatment with UVB. Topical applications of caffeine to these mice decreased the number of nonmalignant and malignant skin tumors per mouse by 44% and 72%, respectively. Topical applications of EGCG decreased the number of nonmalignant and malignant tumors per mouse by 55% and 66%, respectively. Immunohistochemical analysis showed that topical applications of caffeine or EGCG increased apoptosis as measured by the number of caspase 3-positive cells in nonmalignant skin tumors by 87% or 72%, respectively, and in squamous cell carcinomas by 92% or 56%, respectively, but there was no effect on apoptosis in nontumor areas of the epidermis. Topical applications of caffeine or EGCG had a small inhibitory effect on proliferation in nonmalignant tumors as measured by BrdUrd labeling (16-22%), and there was also a similar, but nonsignificant, inhibitory effect on proliferation in malignant tumors. The results suggest a need for further studies to determine whether topical applications of caffeine or EGCG can inhibit sunlight-induced skin cancer in humans.

Fig 1.

Effect of topical applications of caffeine or EGCG on tumorigenesis in UVB-pretreated high-risk mice. SKH-1 hairless mice were irradiated with UVB (30 mJ/cm²) twice weekly for 20 weeks to obtain tumor-free high-risk mice. The mice were then treated topically with 100 μl of acetone, caffeine (6.2 μmol in 100 μl of acetone), or EGCG (6.5 μmol in 100 μl of acetone) once a day 5 days a week for 18 weeks. Mean values ± SE are indicated. (A) a, P < 0.01; b, P < 0.05; c, P < 0.10. (B and C) All points from the groups treated with caffeine or EGCG are statistically different from the comparable points from the acetone-treated control group (P < 0.01).

Fig 2.

Morphological characteristics of caspase 3-positive cell(s) in tumors. SKH-1 hairless mice were irradiated with UVB (30 mJ/cm²) twice weekly for 20 weeks to obtain tumor-free high-risk mice. The mice were then treated topically with 100 μl of acetone, caffeine (6.2 μmol in 100 μl of acetone), or EGCG (6.5 μmol in 100 μl of acetone) once a day 5 days a week for 18 weeks. Most of the cells with caspase 3 (active form) immunoreactive staining had the morphological characteristics of apoptotic cells. The kinds of cells observed are described below. (Left) A cell with strong cytoplasmic caspase-3 (active form) immunoreactive positive staining and the characteristics of an apoptotic cell (condensed nuclei and slight cell membrane shrinkage). We hypothesize this represents an intermediate stage of an apoptotic cell. (Right) Cells with weak cytoplasmic caspase-3 (active form) immunoreactive positive staining and the characteristics of apoptotic cells (condensed nuclei and obvious cell membrane shrinkage). We hypothesize these cells represent a late stage of apoptosis. (Magnification: ×1,000.)