There was no significant difference in weight gain between control and SD treatment groups throughout the entire experiment (data are not shown). SD pre-treatment did not show any apparent skin toxicity to SKH-1 mice and also did not influence the normal growth and development of the mice during the whole experimental period.

The effects of SD treatment on the incidence of skin tumors in SKH-1 mice are shown in Figure 2. Skin tumors appeared in the 10^th week of UVB-promotion phase in both control and SD treatment groups. Skin tumor incidence was 100% in both control and SD treatment groups respectively by 23 weeks of UVB-promotion. Results showed that SD pre-treatment did not have significant (P < 0.05) effect on the incidence of tumor development throughout the experiment.

The effects of SD treatment on tumor multiplicity are shown in Figure 3. The mean number of tumors per mouse was 25.8 and 16.5 in control and SD treatment groups respectively accounting for 36% inhibition in SD treated group at the end of the experiment. Overall, SD pre-treatment resulted in a significant (P < 0.05) reduction in tumor multiplicity from 15^th week in the promotion phase to the end of experiment (the 30^th week of UVB-promotion).

The effects of SD treatment on tumor area are presented in Figure 4. As shown in Figure 4, the mean ratio of total tumor area to total back area was 18.0% and 5.0% in control and SD treatment groups respectively accounting for 73% inhibition in tumor area in SD treated group at the end of the experiment (P < 0.05).

The histopathological examination of tumor progression was investigated after 30 weeks of the promotion. Results suggested that both control and SD treatment group were showing squamous cell carcinoma in the skin (pictures not shown).

Epidermal lysates were collected from mice of both control and SD treatment group with 19 weeks of UVB promotion (shortened protocol), when first tumor appeared in both groups. The effects of SD treatment on caspase-3 and caspase-8 expressions are shown in Figure 5. According to the density of bands, SD treatment increased 1.5 times of the expression of cleaved caspase-3 at 17 KDa. Moreover, as shown in Figure 5, the cleaved caspase-8 band at 18 KDa for control group is very faint and can be hardly observed, whereas, the band for SD Treated group is very intense and dark. However, the bands for cleaved caspase-9 in both control and SD treated group were not observed meaning that SD treatment did not increase the expression of caspase-9 (data not shown).

DNA fragmentation is the biochemical hallmark of apoptosis, an irreversible event that commits the cell to die [22,23]. TUNEL staining was used to localize apoptotic cells with DNA fragmentation in the mouse skin samples from mechanistic study protocol in situ. As shown in Figure 6A, all the cells from control group expressed green color, which were normal cells without DNA fragmentation. However, SD treatment for 19 weeks of the promotion phase modestly increased some DNA fragmented apoptotic cells with brown color nuclear staining as shown in Figure 6B. Thus, SD treatment induced DNA fragmentation of cells in SKH-1 mice.

Sodium chloride, SDS and phenylmethylsulphonylfluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tris(hydroxymethyl)-aminomethane and glycine were purchased from USB corporation (Cleveland, OH, USA). Leupepth and pepstatin were from Roche Diagnostics GmbH (Mannheim, Germany). Acrylamide was purchased from Bio Rad Laboratories (Hercules, CA, CA) and nitrocellulose membrane from Bioexpress (Kaysville, UT, USA). Primary antibody against cleaved caspase-3 was purchased from Cell Signaling Technology, Inc., (Beverly, MA, USA) and primary antibodies against caspase-8, caspase-9 and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from BD Biosciences (Rockville, MD, USA). ECL Kit was bought from Amersham Biosciences (Piscataway, NJ, USA). TACS TdT in situ apoptosis detection kit was from R&D systems (Minneapolis, MN, USA). Other reagents were obtained in their highest purity grade available commercially.

Sarcophine was isolated from the soft coral Sarcophyton glaucum by multiple extractions with petroleum ether at room temperature as reported [14, 20] at the laboratories of Faculty of Pharmacy, Misr International University, Cairo, Egypt. The dried extract was evaporated under reduced pressure and chromatographed on silica gel column using hexane: ethyl acetate (1:2) as eluent. Pure sarcophine was obtained by crystallization from ethanol. SD was synthesized according to the following procedure: sarcophine was reduced to its lactone opened ring analog (1 mmol) to which selenium dioxide (98%, 1 mmol) in dry 1,4-dioxane (30 mL) was added and the reaction mixture was stirred at room temperature for 15 min and followed by TLC to check for completion of reaction. Water was then added to the reaction mixture, and the product was extracted with CH₂Cl₂. Saturated NaHCO₃ solution was used to wash the CH₂Cl₂ layer which was dried over anhydrous Na₂SO₄. The solvent was evaporated and the residue was chromatographed on silica gel using hexane: ethyl acetate (1:2) as an eluent to obtain SD (90% yield) [14].

The structure of SD was fully characterized as shown in Figure 1 by spectroscopic methods and was identical to analytical sample prepared according to previous reported method of synthesis [14, 20]. Purity was confirmed by HPLC.

Female SKH-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All mice were housed in the College of Pharmacy animal facility under climate-controlled environment with a 12 hours light/dark cycle. Mice were allowed free access to food pellets and water placed inside the food chamber on top of the cage cover. The experimental protocol was approved by the Institutional Animal Care and Use Committee.

The UVB irradiation unit, manufactured by Daavlin Corporation (Bryan, OH, USA) consists of four UVB lamps. The exposure dose can be controlled by using two Daavlin flex control integrating dosimeters. The dose of UVB exposure is expressed in millijoules/cm² (mJ/cm²).

The tumorigenesis protocol as described by Dwivedi et al. [21] was used. Female SKH-1 mice were randomly divided into two groups having 27 mice per group, control and SD treatment. Both initiation as well as promotion was induced by UVB radiation (180 mJ/cm²). During the initiation phase, control group was treated with 100 μL of acetone and SD treatment group was treated with 100 μL of SD (30 μg/100 μL of acetone) 1 h prior to UVB exposure. The treatment and UVB exposure were done every day at the same time and was continued for 14 days. During the promotion phase, both control and SD treatment groups were treated in the same way as they were treated during the initiation phase. However, the treatment was done only twice a week (Tuesday and Friday) and was continued until the next 30 weeks for the promotion phase. Tumor counts and group weights were taken once every week.

Pictures of the mice were taken at the end of tumorigenesis protocol (the 30^th week of UVB-promotion). These digital photographs of the mice were used to calculate the visual surface area for each mouse and the tumor associated with each mouse. The boundaries of each tumor was clarified using Photoshop CS3 (Adobe Systems, San Jose, CA, USA), and the areas were determined with the measure-area feature of Image-Pro Plus 5.1 (Media Cybernetics, Inc., Bethesda, MD, USA).

Mice were sacrificed by cervical dislocation at the termination of above protocol. Skin collected from mice was prepared for histopathological examination by immersion fixation in 10 % neutral buffered formalin for several days at room temperature. Fixed tissues were processed for paraffin-wax embedding, sectioned 4 to 6 micrometers thick, stained with hematoxylin and eosin (HE), and evaluated under light microscope.

Female SKH-1 mice were randomly divided into two groups, control and SD treatment. Both initiation as well as promotion was induced by UVB radiation. During the initiation phase, control group was treated with 100 μL of acetone and SD treatment was treated with 100 μL of SD (30 μg/100 μL of acetone) 1 h prior to UVB treatment (180 mJ/cm²). This was done every day and was continued for seven days. One week after the initiation phase, promotion phase was started. During the promotion phase, control was treated with 100 μl of acetone and SD treated with 100 μL of SD (30 μg/100 μL of acetone) 1 h prior to UVB treatment (100 mJ/cm²) twice a week (Tuesday and Friday) for 19 weeks.

Mice from the mechanistic study protocol described above were sacrificed by cervical dislocation and epidermis of mice from both control and SD treated groups was collected. Cell lysate for Western blotting and analysis of caspase-3, -8, and -9 expressions were prepared as described earlier by Zhang et al. and Kundoor et al. [16–18]. Briefly, the fat and tumors in the skin of these two groups were removed and then epidermis was homogenized in 0.1 mM Tris-HCl (pH 7.4) containing 0.15 M sodium chloride. The epidermal homogenate was filtered by cheesecloth and then filtrate was centrifuged at 10,000 g for 20 min in the Beckman J2-21 Centrifuge. This pellet combined with 5% SDS containing 100 mM PMSF, 0.5% leupepth and 0.5% pepstatin was allowed to pass through 25G needle, centrifuged at 13,000 g for 20 min and heated in heating block (100 °C) for 5 min. Finally proteins were collected and used for determining expressions of caspase-3, -8, and -9 by SDS-PAGE and Western blotting.

Protein concentration was measured in each cell lysate by the protein assay (Pierce, Illinois) with albumin as a standard. Equal amount of protein lysates (60 μg) were resolved on SDS-poly-acrylamide gels and transferred to nitrocellulose membrane. Membranes were blocked for 1 h in 5% skim milk in TBS (10 mM Tris, 100 mM NaCl), and then probed with primary antibodies against caspase-3, -8, and -9. The secondary antibodies conjugated to horseradish peroxidase were used for development with the enhanced chemiluminescence detection (ECL) kit. Western blots were quantified using a UVP Biochem Gel Documentation system (UVP, Inc., Upland, California). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

To identify DNA fragmentation of cells on the skin samples of mice from mechanistic study procedure, TACS TdT in situ apoptosis detection kit was used according to the procedure of manufacturer (R&D systems, Minneapolis, MN, USA). Briefly, skin samples were first fixed to prevent the loss of low molecular weight DNA fragments. To make the DNA accessible to the labeling enzyme, the cell membranes are permeabilized with proteinase K reagent. Endogenous peroxidase activity was quenched using hydrogen peroxide. Next, biotinylated nucleotides were incorporated into the 3′-OH ends of the DNA fragments by terminal deoxynucleotidyl transferase (TdT). The biotinylated nucleotides were detected by using streptavidin-horseradish peroxidase conjugate followed by the substrate, diaminobenzidine (DAB). DAB-stained samples were examined using a light microscope. The enzyme reaction generated an insoluble colored precipitate where DNA fragmentation occurred.

The software INSTAT (Graph Pad, San Diego, CA, USA) was used to analyze the data. Chi Square was used for analyzing the data on tumor incidence. Student t-test was applied to compare the tumor multiplicity, weight gain, percent of tumor area, caspase-3, and caspase-8 expressions. Significance in all the cases was considered at P < 0.05.