Chemopreventive effects of sarcophine-diol on skin tumor development .

The objective of this study was to study the effects of sarcophine-diol (SD) on both chemical- and UVB- induced skin tumor development in mice and to elucidate possible mechanism of action of SD in human epidermoid carcinoma A431 cells. For chemical-induced skin tumor protocol, four to five weeks old female CD-1 mice were used. Tumorigenesis in mice was initiated with topical application of DMBA (200 nmol/100 muL acetone). Beginning 1 week after initiation, mice in all groups were treated topically with TPA (5 nmol/100 muL acetone) twice a week throughout the duration of the experiment. The effects of SD treatment on the TPA-induced 3H-thymidine incorporation in epidermal DNA of CD-1 mice were also investigated. For UVB-initiated and promoted skin tumor in SKH-1 hairless mice, four to five week old female SKH-1 mice were used. Tumorigenesis in mice was initiated with UVB exposure for two weeks (180 mJ/cm2). After two weeks, tumorigenesis in mice in all groups was promoted by UVB exposure (180 mJ/cm2) twice per week throughout the duration of the experiment (30 weeks). Effects of SD on the expression of caspases were investigated after 19 weeks of tumorgenesis. The proteins from epidermal homogenates of experimental mice were used for SDS-PAGE and Western blotting using specific antibodies against caspase-3, caspase-8, caspase-9 and COX-2 respectively. For the in vitro studies, effects of SD on cell proliferation and apoptosis in human epidermoid carcinoma A431 cells were determined to elucidate possible mechanism of action. MTT assay was used for cell viability; BrdU incorporation assay was used for cell proliferation; fluorescence-activated cell sorting (FACS) analysis of Annexin V/propidium iodide staining and TUNEL assay were used for determining apoptotic cells; Western blotting was used for the expression of caspase-3 and colorimetric caspase activity assays were used for determination of caspase-3, -8, and -9 activity. For the chemical-induced skin tumor development in CD-1 mice, the results showed that tumor incidence in control, initiation and promotion groups was 100%; the mean number of tumors in control, initiation and promotion groups was 19.7, 16.5 and 10.8 respectively. The caspase-3 levels in initiation and promotion groups were increased by 128% and 170% when compared to control group; the caspase-8 levels in initiation and promotion groups were increased by 103% and 166% when compared to control group; the COX-2 levels in initiation and promotion groups were decreased by 64% and 42% when compared to control group. Furthermore, SD resulted in a 95% reduction in the TPA-induced incorporation of 3H-thymidine in epidermal DNA. For the UVB-induced skin tumor development in SKH-1 hairless mice, the results showed that tumor incidence in control and treatment group was 100%; tumor multiplicity in control and treated groups was 25.8 and 16.5 tumors per mouse respectively. SD treatment (P < 0.05) significantly increased the expression of caspase-3 and caspase-8. For the in vitro studies, the results showed that SD treatment at concentration of 200 muM-600 muM resulted in concentration-dependent decrease in cell viability and cell proliferation in A431 cells, which largely inhibited cell growth. SD treatment induced a strong apoptosis and significantly (P < 0.05) increased DNA fragmentation in A431 cells. Furthermore, SD treatment significantly (P < 0.05) increased the activity and expression of caspase-3 through activation of upstream caspase-8 in A431 cells rather than the activation of caspase-9. The results from this study provided data on the chemopreventive effects of SD on skin cancer development and the...