Oral caffeine during voluntary exercise markedly inhibits skin carcinogenesis and decreases inflammatory cytokines in UVB-treated mice

Abstract

Ultraviolet B (UVB)-pretreated SKH-1 mice were treated with water, caffeine (0.1 mg/ml), voluntary running wheel exercise (RW) or caffeine together with RW for 14 wk. Treatment of the mice with caffeine, RW, or caffeine plus RW decreased skin tumors per mouse by 27%, 35%, and 62%, respectively, and the tumor volume per mouse was decreased by 61%, 70%, and 85%, respectively. In mechanistic studies, mice were treated with water, caffeine, RW, or caffeine plus RW for 2 wk prior to a single irradiation with UVB. Caffeine plus RW increased RW activity by 22% when compared with RW alone. Caffeine ingestion was not significantly different between groups. Treatment of mice with caffeine plus RW for 2 wk decreased the weight of the parametrial fat pads and stimulated the formation of UVB-induced apoptosis to a greater extent than treatment with caffeine or RW alone. An antibody array revealed that caffeine plus RW administered to mice fed a high-fat diet and irradiated with UVB decreased the epidermal levels of lipopolysaccharide-induced CXC chemokine, soluble TNF alpha receptor-1, and macrophage inflammatory protein-1γ. Overall, caffeine during RW exerts a stronger effect than either treatment alone for decreasing tissue fat, increasing UVB-induced apoptosis, lowering the levels of cytokines associated with inflammation and for inhibiting UVB-induced carcinogenesis.

FIG 1. Effect of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination (CF+RW) on body weight, food and fluid consumption, running wheel activity and tissue fat in SKH-1 mice previously treated with UVB

One hundred and sixty female SKH-1 mice (6–7 weeks old) were exposed to UVB (30 mJ/cm²) once a day, twice a week for 20 weeks. UVB irradiation was stopped, and the mice (40/group, 10 mice/cage) were then treated with water (control), CF (0.1 mg/ml in drinking water), RW or CF plus RW for 14 weeks in the absence of further irradiation with UVB. All mice were fed Laboratory Chow 5001 diet throughout the entire study. All animals were sacrificed and body weight (A), food consumption (B), fluid consumption (C), and running wheel activity (D) was measured from the beginning to the end of the study. The weight of the parametrial fat pads (E) was measured at the end of the study. Each value is the mean ± SE from 40 mice (14 week study). *: Compared to water (control) for (B), (C), and (E), *: CF and RW compared to RW alone for (D), Dunnett’s adjusted p<0.01.

FIG 2. Effect of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination (CF+RW) on UVB-induced skin carcinogenesis in SKH-1 mice

One hundred and sixty female SKH-1 mice (6–7 weeks old) were exposed to UVB (30 mJ/cm²) once a day, twice a week for 20 weeks. UVB irradiation was stopped. The mice (40/group, 10/cage) were then treated with water (control), CF (0.1 mg/ml in drinking water), RW or CF plus RW for 14 weeks. All mice were fed Laboratory Chow 5001 diet throughout the entire study. All animals were sacrificed and body weight (A), percent mice with tumors (B), number of tumors per mouse (C), and tumor volume per mouse (D) from the beginning to the end of the study were measured every 2 weeks. Each value is the mean ± SE from 40 mice.

FIG 3. Time course for the effects of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination (CF+RW) on body weight, food consumption, fluid consumption, running wheel activity and weight of the parametrial fat pads in SKH-1 mice

Female SKH-1 mice (5 mice per time point, 4 treatments, total 220 mice) were treated with water (control), CF (0.1 mg/ml in drinking water), RW or 0.1 mg/ml of CF plus RW for 2 weeks. Laboratory Chow 5001 diet was fed to all mice ad libitum. The pretreated mice were then exposed to a single irradiation with UVB (30 mJ/cm²) and sacrificed at 0 (non-UVB), 5 min, 0.5, 1, 2, 4, 6, 10, 16, 24 and 48 hrs post UVB. Body weight (A), food consumption, (B) fluid consumption (C), and running wheel activity (D) were measured during the course of the study. The weight of the parametrial fat pads (E) was measured at the end of the study. Each value is the mean ± SE from 55 mice (2 week study). *: Compared to water (control) for (B), (C), and (E), *: CF and RW compared to RW alone for (D), Dunnett’s adjusted p<0.01.

FIG 4. Time course for the effects of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination (CF+RW) on UVB-induced apoptosis in the epidermis of SKH-1 mice

Female SKH-1 mice (5 mice per time point, 4 treatments, total of 220 mice) were treated with: water (control), CF (0.1 mg/ml in drinking water), RW or 0.1 mg/ml of CF plus RW for 2 weeks. All mice were given Laboratory Chow 5001 diet ad libitum. The pretreated mice were then exposed to a single irradiation with UVB (30 mJ/cm²) and sacrificed at 0 (non-UVB) treated mice or at 5 min, 0.5, 1, 2, 4, 6, 10, 16, 24 and 48 hrs post-UVB. Apoptotic sunburn cells were determined morphologically (A), and caspase 3 (active form) positive cells were determined immunohistochemically (B). Each value is the mean ± SE from 5 mice.

FIG 5. Time course for the effects of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination (CF+RW) on UVB-induced cell proliferation in the epidermis of SKH-1 mice

Female SKH-1 mice (5 mice per time point, 4 treatments, total 220 mice) were treated as described in Fig. 4. Bromodeoxyuridine (BrdU) was injected at 1 hr prior to sacrificing the animals, and BrdU positive cells were determined immunohistochemically. Each value is the mean ± SE from 5 mice.

FIG 6. Effect of oral administration of caffeine (CF), voluntary running wheel exercise (RW) or their combination on the levels of inflammatory cytokines in the epidermis: an antibody array study

In experiment 1, twenty-four female SKH-1 mice were given a 40% kcal high fat diet rich in omega-6 fatty acids for 4 weeks. The high-fat diet was continued, and the mice were irradiated with UVB (30 mJ/ cm²) once a day for 7 or 14 days as described in the “Materials and Methods” section. Half of the mice (3 mice per group) were treated with: water (control), CF (0.1 mg/ml drinking fluid), RW, or CF + RW for 1 week (A). The other half of the mice were treated with: water, 0.1 mg/ml of CF, RW, or CF (0.1 mg/ml) + RW for 2 weeks (B). In experiment 2, twelve male congenic hairless p53 knockout mice and twelve of their wild-type p53 littermates were given a high-fat diet rich in omega-6 fatty acids for 4 weeks as described in the “Materials and Methods” section. The high-fat diet was continued, and p53 wild-type mice (C) and p53 knockout littermates (D) (3 mice per group) were then treated with: water, CF (0.1 mg/ml), RW or CF+ RW for 1 week. In experiments 1 and 2, all mice were irradiated with UVB (30 mJ/cm²) once a day for 7 or 14 consecutive days and killed 2 hours after the last UVB irradiation. The epidermis was collected, and epidermal lysates were prepared and subjected to antibody array as described in the “Materials and Methods” section. Each value in parenthesis is the average intensity of two parallel signals quantified by densitometry. Positive controls were used to normalize the results from different membranes being compared.