Abstract 1217: Effects of topical applications of caffeine on phospho-Chk1 (Ser317), mitotic cells with cyclin B1 and mitotic cells with caspase 3 in skin tumors from UVB pretreated SKH-1 mice

In an earlier study we irradiated female SKH-1 mice with UVB (30 mJ/cm2) twice a week for 20 weeks and UVB treatment was stopped. Three weeks later, these tumor-free high-risk mice were treated topically with 100 µl of acetone or with caffeine (6.2 µmoles) in 100 µl of acetone once a day 5 days a week for 18 weeks. The results showed that topical applications of caffeine inhibited the formation of keratoacanthomas and squamous cell carcinomas by 44% and 72%, respectively. Caffeine treatment increased the number of caspase 3 (active form) positive cells in keratoacanthomas by 87% and in squamous cell carcinomas by 92%. These effects on apoptosis were selective for tumors since treatment of the mice with caffeine had little or no effect in non-tumor areas of the epidermis (Lu, Y-P et al: PNAS 99: 12455-12460, 2002). In the present study we further analyzed these stored paraffin blocks and found that some mitotic tumor cells are undergoing apoptosis (lethal mitosis) as indicated by staining for caspase 3. Caffeine treatment significantly increased the percentage of mitotic tumor cells with caspase 3 staining by 213% in keratoacanthomas, and by 212% in squamous cell carcinomas, when compared with acetone-treated control animals. In addition, caffeine treatment caused a 66% increase in the percentage of mitotic tumor cells with cyclin B1 staining in keratoacanthomas, which is associated with an increase in the percentage of mitotic tumor cells with caspase 3 staining. Immunohistochemical analysis with these tissue sections showed multiple focal areas of phospho-Chk1 (Ser317) - staining cells in 41% of keratoacanthomas and in 64% of squamous cell carcinomas, but there was none in non-tumor areas of the epidermis. The expression of phospho-Chk1 (Ser317) was almost exclusively in the cytoplasm of the tumor cells. Caffeine administration decreased the number of phospho-Chk1 (Ser317) positive tumors by 56% and the percentage of phospho-Chk1 (Ser317) positive cells by 64% in keratoacanthomas. Although there was little or no decrease in the percentage of phospho-Chk1 (Ser317) positive cells in squamous cell carcinomas, the intensity of staining was markedly decreased. Pearson9s correlation coefficient indicated that there was a weak but statistically significant inverse relationship between the percentage of phospho-Chk1 (Ser317) positive cells and the percentage of caspase 3 positive cells in tumors. Our study suggests that topical application of caffeine to high-risk mice selectively enhances apoptosis in tumors by decreasing the level of phospho-Chk1 (Ser317), increasing the number of mitotic cells with cyclin B1 and by increasing the number of mitotic cells with apoptosis (caspase 3 staining) resulting in abrogation of the G2/M checkpoint and mitotic catastrophe (Supported by NIH Grants RO1 CA130857 and RO1 CA128997). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1217.