Prevention of mouse skin tumor promotion by dietary energy restriction requires an intact adrenal gland and glucocorticoid supplementation restores inhibition
Jeanne W. Stewart 1,
Ken Koehler 2,
William Jackson 1,
Jacqueline Hawley 1,
Weiqun Wang 1, 4,
Angela Au 1,
Ron Myers 3 and
Diane F. Birt 1, *
1 Department of Food Science and Human Nutrition, 2 Department of Statistics and 3 Department of Veterinary Pathology, Iowa State University, Ames, IA 50011, USA
4 Present address: Department of Human Nutrition, Kansas State University, Manhattan, KS 66506, USA
* To whom correspondence should be addressed Email: dbirt{at}iastate.edu
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Abstract
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Our laboratory has demonstrated in the previous studies that dietary energy restriction (DER) inhibited the promotion of skin tumorigenesis and others have found that adrenalectomy may reverse that inhibition. The purpose of the research reported here was to determine if circulating corticosterone (CCS) may be the adrenal hormone responsible for DER prevention of skin carcinogenesis. Female SENCAR mice were initiated with 7,12-dimethylbenzanthracene (DMBA) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) in either sham-operated or adrenalectomized (ADX) mice fed ad libitum (AL) or energy restricted diets. DER was 60% of the AL calorie intake with the removal of energy from fat and carbohydrate. CCS, the main glucocorticoid hormone secreted by the murine adrenal gland, was added to the drinking water of AL/ADX and DER/ADX groups to determine the role of CCS in the DER inhibition of tumor development. In sham-operated groups, DER compared with AL-fed mice experienced significantly decreased papilloma incidence and multiplicity (P < 0.0001). ADX did not alter papilloma incidence or multiplicity in AL-fed mice but ADX partially reversed the inhibition of papilloma multiplicity and incidence in DER mice. CCS supplementation to both DER/ADX and AL/ADX mice resulted in reduced papilloma incidence and multiplicity. In DER/ADX mice, CCS dramatically reduced papilloma rates while in AL/ADX mice CCS reduced the papilloma rates to those seen in the DER sham group. DER significantly reduced carcinoma multiplicity mean counts per effective animal (P < 0.0001) compared with AL-fed groups in sham and ADX/CCS groups. DER/ADX mice lost the carcinoma multiplicity protection seen in sham/DER mice. CCS treatment of ADX mice significantly decreased total carcinoma (in situ and invasive) incidence rates per effective animal (P < 0.0003). ADX followed by CCS treatment in the DER mice resulted in the lowest carcinoma incidence and multiplicity. Thus, DER-inhibition of skin tumorigenesis was mediated at least in part through CCS. However, CCS was more effective in preventing papillomas and carcinomas in DER/ADX mice than in AL/ADX mice, suggesting that other factors may also be involved in the DER prevention of tumor formation.
Abbreviations: AP-1, activator protein-1; AL, ad libitum; ADX, adrenalectomized; CCS, circulating corticosterone; DER, dietary energy restriction; DHEA, dehydroepiandrosterone; DMBA, 7,12-dimethyl-benzanthracene; ERK, extracellular signal regulated kinase; IGF-1, insulin growth factor-1; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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With the growing evidence that obesity is a risk factor for human cancer (1) and the increasing rate of obesity throughout the world (2), determining the mechanisms whereby dietary intake modulates cancer has taken on a new significance. There has long been evidence that restricting dietary intake could significantly reduce cancer rates. In particular, studies demonstrated that when mice were fed a calorie restricted (60% of control calories) diet instead of an ad libitum (AL) diet the mice exhibited
40% decreased tumor incidence in skin and mammary tissues (3,4). In addition, a more moderate restriction (80% of control intake) was also effective in skin cancer prevention (5). Since these early findings, studies have demonstrated dietary or calorie restriction are effective in the prevention of liver, lung, colon, breast and skin cancers (69).
Previous research in our laboratory that is relevant to assessing the mechanism of dietary energy restriction (DER) prevention of skin cancer, compared dietary restriction (reduction of all dietary components) and DER (reduction of calories from fat and/or carbohydrates while maintaining other nutrient intakes). The study showed that both dietary restriction and DER reduced papilloma and carcinoma incidence, and multiplicity and also delayed papilloma appearance but that DER was the most effective strategy (7). Furthermore, our laboratory has shown that the DER inhibition of tumorigenesis occurs during the promotion stage of 7,12-dimethylbenzanthracene (DMBA) initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted mouse skin tumorigenesis (10). Research in other laboratories showed that the adrenal gland was required for diet restriction prevention of skin and lung tumorigenesis (11,12). The studies of skin cancer determined that feeding less of the control diet reduced DMBA-initiated and TPA-promoted mouse skin carcinogenesis in intact mice but not in adrenalectomized (ADX) mice. These studies fed less of all dietary components (diet restriction), they did not perform DER as was used in the present investigation. Furthermore, they did not assess the key hormone from the adrenal gland that was responsible for the dependence.
The adrenal gland cortex produces three major classes of lipid soluble hormones derived from cholesterol: mineralocorticoids (controls levels of sodium and potassium), glucocorticoids (controls general cell function and especially maintains glucose homeostasis during food deprivation) and androgens (similar hormonal function as the male sex hormones) (12,13). The adrenal medulla produces the catecholamines: epinephrine and norepinephrine (13). Studies reported by Pashko and Schwartz demonstrated that adrenectomy reverses the dietary-restriction-inhibition of TPA promotion of skin papillomas (11,12). In these studies dietary restriction increased the circulating corticosterone (CCS) but there was no difference in the levels of dehydroepiandrosterone (DHEA) or aldosterone in cases where these hormones were measured (11,12,14). In our earlier studies of DER, with reduction of fat and carbohydrate calories, we observed elevated levels of corticosterone in DER mice but found no evidence of activated glucocorticoid receptors in the skin of DER mice (15).
Several lines of evidence suggest that glucocorticoid hormone may be involved in the prevention of skin carcinogenesis in DER animals. First, it is known that energy restriction elevated circulating glucocorticoid hormones as noted above (15). Second, glucocorticoid hormones were demonstrated in a number of laboratories to be potent inhibitors of mouse skin tumorigenesis (16,17). Finally, the importance of the glucocorticoid receptor in interfering with a key transcription factor, activator protein 1 (AP1), in mouse skin carcinogenesis suggests potential mechanisms for DER elevated corticosterone in preventing mouse skin tumorigenesis (18,19). Studies focused on DER prevention of mammary carcinogenesis suggested that changes in CCS metabolism contributed to, but did not explain, cancer prevention in this model (20).
Our objectives in this research were to determine if the DER (restriction of energy from fat and carbohydrate) inhibition of skin tumorigenesis in intact mice, would be lost in ADX/DER mice, whether supplementation of the ADX mice with corticosterone would restore the inhibition of skin tumorigenesis and whether the corticosterone would inhibit skin carcinogenesis when administered to ADX mice fed the control diet.
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Materials and methods
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Animals and diets
The Iowa State University Committee on Animal Care approved the procedures used for the two-stage tumorigenesis model in mice. Six-week-old SENCAR female animals were obtained from the National Institute of Health (NIH) in Frederick, MD. Animals were allowed a 1-week acclimatization period on an AL-fed, control diet (AIN-76/93) (21) as seen in Table I (custom made by Harlan Teklad, Madison, WI) before surgeries were performed: animals were then either sham operated or bilateral adrenalectomy was performed (17,22) as indicated in Figure 1. The mice were housed individually in humidity and temperature controlled rooms with a 12 h light/dark cycle. The mice were allowed a 2-week recovery period before treatment began. A dorsal patch of skin of each mouse was shaved and tumor development was initiated on mice that did not have hair regrowth 48 h later with 10 nmol DMBA/200 µl acetone. Control mice were treated with acetone alone. One half of the treated groups of animals were placed on 20% DER in the first 2 weeks and then switched to 40% DER for the rest of the experiment as seen in Table I (Harlan Teklad, Madison, WI). We pair-fed DER mice at 17:00 h since Gallo and Weinberg (23) found that mice fed on a food restricted diet (75 % of AL, control diet) at 17:00 h exhibited a plasma corticosterone pattern similar to mice fed AL, control diet. The mice were given a tumor promoter, 3.2 nmol TPA/200 µl acetone twice a week starting at week 3 after DMBA initiation and continuing for 15 weeks. Drinking water solutions were varied according to treatment: mice that were sham operated received tap water with 0.6% ethanol, while ADX mice received either 0.9% NaCl with 0.6% ethanol or 60 µg/ml corticosterone (ICN Biomedicals, Aurora, OH), dissolved in ethanol and diluted with tap water for a final concentration of 0.6% ethanol. ADX mice needed supplementation of NaCl or CCS to prevent the loss of essential electrolytes into urine owing to the lack of mineralcorticoid activity.
Mice were weighed, palpated and visually inspected every week for evidence of tumors or carcinomas and all data were recorded. The week of initiation was counted as week 0 for reporting results. Animals were continued on the experimental diets even after 15 weeks of TPA treatment to allow the development of papillomas and carcinomas. If at any time during the experiment the animal suffered pain from the progression of a tumor to a carcinoma or any other serious health problem (ulceration and scratching were observed in some groups in the present study) the mouse was killed, and visual papillomas and suspected carcinomas were collected. At week 31 all remaining mice were killed by decapitation to eliminate hormonal changes in the blood due to anesthesia or stress. Sham-treated mice were killed between 7:30 and 8:30 AM and the ADX mice were killed between 6:30 and 7:30 PM; these timings were chosen for sacrifice since the difference in corticosterone levels between AL mice and DER mice were expected to be the maximum during these time periods (21). All ADX mice were visually inspected for complete removal of adrenal tissue. If there was any evidence for the presence of adrenal tissue, the animal was removed from the statistical analysis.
Plasma analysis
The mice that survived up to the termination of the experiment were killed by decapitation. Blood was collected from the trunk into EDTA-coated tubes. The collected samples of blood were centrifuged at 825 g for 10 min and plasma collected was stored at 70°C. Corticosterone concentration in the plasma was determined by radioimmunoassay (ICN Biomedicals, Costa Mesa, CA). Insulin growth factor-1 (IGF-1) in the plasma was quantified using a radioimmunoassay kit (Nichols Institute Diagnostics, San Clemente, CA; IGF-1 IRMA 100T Kit; catalog No. 40-2250).
Tissue handling and diagnostic criteria
Papillomas were visually diagnosed on the treated dorsal skin if a papillary mass was projecting from the skin surface but there was no evidence of the lesion developing into a carcinoma. Skin lesions that were suspected of being carcinomas based on appearance, and any organ that had an abnormal appearance were collected from mice that were exsanguinated early owing to pain caused by the carcinoma or ill health and from mice that were exsanguinated at week 31. Tissues were fixed by immersion in 10% buffered neutral formalin for at least 48 h. Lesions and masses from each mouse were bisected, routinely processed, embedded in paraffin, sectioned at 6 µm thickness and stained with hematoxylin and eosin (H&E).
The skin samples collected for histology were diagnosed as papilloma if a keratinized papillary mass was projecting from the skin surface with a normal basal layer. Histologically diagnosed papillomas were added to the much larger count of visually identified papillomas for statistical analysis. In situ carcinomas were diagnosed if there was an area of atypia or dysplasia, aberrant keratinization in the basal layers, mitotic activity was excessive or suprabasilar, there was an aberrant growth of the basal layer or if the basal layer lacked reasonable polarity in a significant area of the papilloma. Invasive carcinomas were diagnosed if there was a marked pleomorphism of proliferative keratinocytes, an invasion of the dermis with neoplastic cells or significant breaching of basement membrane. The visual identification of carcinomas was 89% accurate (208 carcinomas verified histologically out of 234 lesions visually suspected of being carcinomas) in comparison with histological verification as carcinoma and there was no significant difference in accuracy amongst the DMBA/TPA treated groups. All carcinomas were histologically verified and included in the statistical analysis.
Statistical analysis
The weekly food consumption levels of all the AL mice were analyzed by ANOVA using the MIXED procedure in SAS with multiple comparisons by least squares means for the first 3 weeks prior to treatment with the initiator and 5-week blocks during the rest of the experiment. All the animals on the AL diet were included in the data set until they were terminated from the experiment owing to ill health or the completion of the experimental period. Log-rank and Wilcoxon tests were used to compare KaplanMeier estimates of survivor curves for the six groups of DMBA/TPA treated mice (24). In this analysis, survival times for animals that completed the entire 31 weeks of the experiment were treated as censored at 31 weeks.
Body weights were analyzed with repeated ANOVA measures using the MIXED procedure in SAS with multiple comparisons by least squares means at each time point. The variable of surgery had three possibilities: sham/tap water + 0.6% ethanol, ADX/0.9% NaCl in 0.6% ethanol or ADX/60 µg cortiosterone in 0.6% ethanol. Since carcinoma multiplicity at the time of death exhibited Poisson distributions (variance = mean), log-linear model analysis using the GENMOD procedure in SAS was used to analyze treatment effects on carcinoma multiplicity with multiple treatment comparisons made with least squares means. Effective mice for the carcinoma analysis were defined as the mice treated with carcinogen that survived at least 23 weeks. Data for mice that survived < 23 weeks were excluded from the carcinoma analysis. Papilloma multiplicity at specific time points were also analyzed with the GENMOD procedure, using all the mice that survived to the specified time point, but greater variability necessitated the use of the negative binomial distribution (variance > mean). Fisher's exact test was used as an overall test for treatment effects on final carcinoma incidence or papilloma incidence at each time point. If the overall test showed significant differences at the 0.05 level, Fisher's exact test was used to compare each pair of treatments. Plasma corticosterone and IGF were analyzed in the surviving mice at week 31 by ANOVA, using the GLM procedure in SAS with multiple comparisons by least squares means.
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Results
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Food consumption
Consumption of diet by AL-fed mice is shown in Table II. Within the DMBA/TPA treated mice the AL/sham and AL/ADX/saline groups consumed significantly more control diet from 16 to 30 weeks than the AL/ADX/CCS mice. In comparison with acetone treated mice, DMBA/TPA treated AL/sham and AL/ADX/saline groups consumed significantly more control diet from 6 to 30 weeks. In contrast, within the AL/ADX/CCS group, the acetone treated mice consumed more control diet after week 26 than the DMBA/TPA treated mice.
Body weight
The mice on DER showed a significant loss in body weight during the first 4 weeks following the switch to this diet but maintained the reduced body weight for the rest of the experimental period (Figure 2). The AL-fed mice showed increase in body weight as the experiment progressed (Figure 2). At week 31 the DER-fed mice weighed significantly less than the AL-fed mice (P < 0.0001) as shown in Figure 2. Animals treated with acetone exhibited the same significant difference in body weights between the AL-fed mice versus the DER-fed mice (data not shown).

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Fig. 2. Body weights. Data shown are for mice treated with DMBA/TPA. Values represent mean ± SEM, N = 838. P < 0.0001 for differences by diet at each time point 0 weeks (ANOVAMIXED). Means at each time point with different superscript letters are significantly different at the P < 0.05 level (multiple comparisons by least squares means) and values with more than one letter are not significantly different from means sharing either of the letters. Body weights of mice treated with acetone were similar to the body weights of the mice treated with DMBA/TPA in all parallel groups except the acetone/DER/ADX/saline group that maintained a constant body weight throughout the experiment (data not shown).
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The changes in the body weight at the beginning of the experiment and weeks 4, 8 or 31 are provided in Table III. It is to be noted that the maximum body weight gains were in the AL/ADX/CCS treated groups in both the carcinogen (DMBA/TPA) and vehicle (acetone) administered groups. In contrast, among the DER treated groups, body weight changes did not differ between the sham, ADX/saline or ADX/CCS treated groups.
Survival
Mice in the DER/sham group exhibited the best survival with 30/32 (94%) of the DMBA/TPA treated mice surviving up to 31 weeks. Intermediate survival was observed in the AL/sham group with 18/31 (58%) of the DMBA/TPA treated mice surviving the experiment. All ADX mice experienced poorer survival with an average of 41/139 (29%) of the DMBA/TPA treated mice alive at week 31 (P < 0.0004). In general, mice in the ADX groups died gradually throughout the experiment. However, the DER/ADX group had 19/37 (51%) mice surviving at week 6 whereas the other three ADX groups had 91/101 (90%) mice surviving at that time in the experiment.
Papilloma incidence
Mice in the AL/sham and AL/ADX/saline groups exhibited a significantly higher papilloma incidence at 8 weeks (P < 0.0001) compared with other mice (Figure 3A). By week 10 the DER/ADX/saline treated mice were also exhibiting a significantly increased papilloma incidence compared with the mice fed the other DER diets and the AL/ADX/CCS. The mice receiving the DER/ADX/CCS protocol exhibited the most pronounced inhibition of papilloma incidence consistently throughout the study (Figure 3A). At week 31 DER significantly reduced papilloma incidence and CCS reduced papilloma incidence in both AL/ADX and DER/ADX groups (P < 0.0001).

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Fig. 3. Papilloma incidence (A) and multiplicity; mean count/mouse (B). Data shown are for mice treated with DMBA/TPA. Values represent mean ± SEM, N = 838. (A) Incidence analyzed by Fisher's exact test. (B) Analyzed by GENMOD with multiple comparisons by least squares means. P < 0.0001 for differences by diet at each time point 8 weeks. Means at each time point with different superscript letters are significantly different at the P < 0.05 level and values with more than one letter are not significantly different from means sharing either of the letters. Only one papilloma was seen in one acetone treated mice fed the AL/ADX/saline diet while 75% (40/53) of all of the acetone treated mice survived up to the end of the experiment.
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Papilloma multiplicity: mean count per animal
Papilloma multiplicity was defined as the number of papillomas per animal. Mice in the AL/sham and AL/ADX/saline groups exhibited a significant increase in papilloma multiplicity by 9 weeks of treatment (P < 0.03) and by week 11 DER/ADX/saline treated mice exhibited a significant increase in papilloma multiplicity compared with the other DER and AL/ADX/CCS treated mice (P < 0.003) (Figure 3B). At week 31 the number of papillomas on DER/ADX/saline mice was not significantly different from the number on the groups that received AL diets or the DER/sham mice (Figure 3B). DER/ADX/CCS significantly reduced papilloma multiplicity compared with all other groups (P < 0.0001).
Carcinoma incidence rates and multiplicity
Total carcinoma incidence rates include in situ and invasive carcinomas. The response to diet and CCS was similar for the total carcinoma incidence rates and the papilloma incidence rates (Table IV). There were significant differences among total carcinoma incidence rates (P < 0.0003) and also among in situ (P < 0.0001) and invasive (P < 0.0001) carcinoma incidence rates. Total carcinoma incidence rates were lowest in the DER/ADX/CCS group and highest in the AL/sham group. In situ carcinoma incidence rates were lowest in the DER/ADX/CCS group and highest in the AL/ADX/saline group. Invasive carcinoma incidence rate was significantly increased in the AL/sham diet compared with the other five groups (P < 0.0001) (Table IV). Carcinoma multiplicity was defined as the number of carcinomas per carcinoma bearing animal. Total carcinoma multiplicity was significantly lower for the DER/sham than the AL/sham group (P < 0.05) and it was lowest in the mice in the DER/ADX/CCS group (P < 0.0001). In situ carcinoma multiplicity was lowest in the DER/ADX/CCS group compared with the other five groups (P < 0.0002). Invasive carcinoma multiplicity also varied significantly across treatment groups (P < 0.0001) and was lowest in the DER/ADX/CCS group and highest in the AL/sham mice (Table IV).
Other pathology
There was a significant increase in the appearance of lymphosarcoma in the spleens of the AL/ADX/saline mice compared with the DER/sham and DER/ADX/CCS groups (P < 0.0350) (Table IV).
Gross observations
Skin ulceration in DMBA/TPA treated mice that survived at least 23 weeks differed significantly between groups (P < 0.00005). This lesion was observed in 27% (8/30) of the mice in the AL/sham, 47% (8/17) of the mice in the AL/ADX/saline and 6% (2/32) of the mice in the DER/sham groups. Skin ulcerations were not observed in other groups (0/56 mice). Casual observation of intense scratching directly correlated with the observations on skin ulcerations.
Plasma corticosterone
There was no statistical difference in plasma corticosterone concentrations between mice treated with DMBATPA or acetone for any dietarysurgery treatment. So these groups were pooled and are presented as dietarysurgery treatment group in Table V. Since the sham-operated and ADX mice were killed in the morning and evening, respectively, these groups are presented in separate columns in Table V and analyzed separately. Mice in the DER/ADX/CCS group exhibited significantly higher plasma corticosterone concentrations than any of the other ADX groups (P < 0.0001).
Plasma insulin growth factor-1 (IGF-1)
In the ADX mice there was no significant difference in plasma IGF-1 concentration between the animals receiving corticosterone or saline in the drinking water. Therefore these groups were combined in the analysis of the dietary effects. Since the sham-operated and ADX mice were killed in the morning and evening, respectively, these groups are presented in separate columns in Table VI and analyzed separately. Plasma IGF concentrations in the DER diet mice were significantly reduced to approximately one-third and one-forth of the values in mice that received the AL diet in the sham-operated groups (P < 0.0042) and the ADX-operated groups (P < 0.0015).
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Discussion
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A striking inhibition of DMBA initiated and TPA promoted papilloma development was observed in sham-operated DER mice, particularly with respect to papilloma number. However, this inhibition was partially blocked in ADX-operated DER mice. In comparison, ADX did not appreciably alter papilloma development in control mice. These observations demonstrate the dependence of DER, in the prevention of mouse skin papillomas, on an intact adrenal gland. Furthermore, daily supplementation with corticosterone in the drinking water to achieve peak corticosterone concentrations in the range of the DER mice (15) resulted in cancer prevention in both AL/ADX and DER/ADX mice. This suggests that the loss of corticosterone may have contributed to the higher tumor rate in the ADX operated DER mice.
The impact of DER and ADX on mouse skin carcinoma rates largely paralleled the effects observed on papillomas. Total carcinoma multiplicity was higher in the DER/ADX mice than in the DER/sham mice but this difference was not statistically significant. However, corticosterone was a potent inhibitor of both the total number of carcinomas and the invasive carcinomas. In contrast to the general absence of impact of ADX in the control-fed mice on papilloma and total carcinoma rates, invasive carcinomas were inhibited by ADX in control-fed mice. The reasons for the different patterns of papillomas, total carcinomas and invasive carcinomas are not clear, but these contrasts suggest that CCS in the absence of the adrenal gland may play a very different role in the conversion to invasive carcinomas than it plays in papilloma development.
It is interesting that administering corticosterone to the ADX/DER mice resulted in papilloma and carcinoma rates that were lower than those observed in the sham-operated DER mice. It is possible that the corticosterone administered in the drinking water resulted in a circulating level that was higher than was observed in the sham/DER mice. Currently, it is not possible to perfectly mimic the CCS of intact mice in ADX animals. Indeed, there are extra-adrenal sites near the thoracic and abdominal sympathetic ganglia that may produce glucocorticoid hormone (25) and these sites were not removed in our ADX mice. The data presented in this paper in Table V on plasma corticosterone in the mice surviving until the final sacrifice show that the DER/ADX/CCS group had the highest CCS of all treatment groups. Sham and ADX mice were killed at different times to allow for comparison between AL and DER mice at the times when the greatest differences in CCS would be expected. Thus, we did not compare CCS concentration in sham and ADX mice in this experiment. While our observations on CCS at the termination of the experiment are intriguing, it is important to note that data from surviving animals may be biased by uncontrolled differential survival factors. Indeed, in mice that were fed these diets and treated only with a single treatment of TPA or acetone 1 or 4 h before killing, corticosterone was generally equally elevated in both the sham-operated DER mice and in the corticosterone supplemented control-fed, and the corticosterone-supplemented DER-fed groups as described below (17,26). An alternative interpretation for the low rate of papilloma and carcinoma in the DER/ADX/CCS group is that the adrenal gland might provide both cancer-preventative (presumably elevated corticosterone in DER mice) and cancer-enhancing impacts. However, we are not aware of any data in support of this alternative hypothesis.
In previous short term studies we assessed CCS in the diet and hormonal protocols used in the experiment reported in the present paper. In both studies after 1012 weeks of treatment, the plasma corticosterone concentration in 17- to 22-week-old mice was elevated (3- to 5-fold) in sham/DER mice compared with sham/AL mice (17,26). ADX mice had lower corticosterone and did not differ by diet. An elevation in corticosterone in hormone supplemented groups to the level in the sham/DER mice was observed in blood collected late in the day (26). When the blood was collected in the morning, the supplemented groups had only a minor increase in corticosterone (17). However, we would not anticipate observing an elevation in CCS in the corticosterone-supplemented groups in the morning because mice consumed more drinking water later in the day when they had food available. Administration of corticosterone in the drinking water was done in the current experiment instead of implanting corticosterone pellets because drinking water administration mimicked the CCS levels of DER mice in earlier studies (21) more closely. ADX and corticosterone supplementation did not change the glucocorticoid receptor activity in our earlier work (15).
Another hormone that merits being studied for contributing to the DER prevention of skin carcinogenesis is IGF-1. IGF-1 is a cell survival factor that inhibits apoptosis and increases cell proliferation (27). Diet restriction was shown to reduce circulating IGF-1 in our laboratory (26) and by others (28). In addition, IGF-binding proteins, which appear to control the release of IGF-1, were also modulated by diet restriction in a manner that would be expected to reduce IGF-1 activation (29). The most compelling study to suggest that reduction in circulating IGF-1 plays a key role in DER prevention of cancer was conducted by Dunn et al. (28). Urinary bladder cancer was induced by p-cressidine and after pre-neoplasia was confirmed, mice were fed 20% DER with or without recombinant IGF-1 administered with mini-pumps. Mice fed ad libitum were included in a parallel group. The circulating IGF-1 was reduced by 24% after 5 weeks of feeding DER diet and the levels were restored to the AL values in the DER plus IGF-1 group. The progression of tumors was inhibited in the DER group but it was not reduced in the DER plus IGF-1 group (28). Studies by Zhu et al. (20) used dietary corticosterone supplements to assess the role of CCS in DER prevention of mammary cancer. In the SpragueDawley rat model used for these studies they observed elevated CCS and decreased IGF-1 in response to the dietary corticosterone supplement.
IGF-I was measured in the mice in our tumor study is presented in this paper and in previous 12 week, short term studies of SENCAR mice (26). In the short term studies DER decreased circulating IGF-I (231 ± 14 ng/ml, mean ± SEM) (P < 0.05) in comparison with AL mice (402 ± 18 ng/ml), but TPA treatment, ADX and corticosterone supplementation did not alter these values (26). In the present tumor study circulating IGF-I was reduced in the DER mice irrespective of the other experimental treatments. These were important observations since considerable research has suggested that reduced circulating IGF-I or elevated IGFBPs in the DER mice contributes to cancer prevention by DER (30). Thus, since the hormonal treatments that we used to modulate CCS did not mediate circulating IGF-1, the effects that we attribute to CCS were probably not owing to IGF-1 in the present investigation.
It is unlikely that aldosterone, epinephrine or norepinephrine were responsible for the adrenal dependence of DER inhibition of mouse skin carcinogenesis. Earlier studies in our laboratory indicated that there was no difference in aldosterone levels between the AL-fed mice or the mice on DER diet (15). Furthermore, we did not find reports on the role of epinephrine or norepinephrine in skin carcinogenesis.
Our tumor study determined that DER inhibition of skin tumor promotion was in large measure mediated through increasing CCS. However, it is noteworthy that CCS did not explain all the difference between the AL and DER mice since supplementation with corticosterone was more effective in preventing papillomas and carcinomas in DER/ADX mice than in AL/ADX mice. These results support the hypothesis that, while CCS contributes to cancer prevention by DER, other factors are also involved. It is important to note that changes in circulating IGF-I did not appear to contribute to the DER effects that were attributed to CCS in our study as noted above. However, the elevated glucocorticoid hormone may interact with other hormonal effects, such as the lowered IFG-1 to prevent skin tumorigenesis.
The changes in the body weight over time showed that the DMBA/TPA initiated, AL/ADX/CCS treated mice actually gained considerably more weight (more or less double) than the AL/sham operated mice (Table III). This observation was paralleled by data showing a lower food intake in these mice in comparison with the AL/sham and AL/ADX/saline groups from weeks 1030 (Table II). These results suggest that the dose of corticosterone in these mice may have caused salt and water retention, known impacts of excessive mineralocorticoid in humans (25). This may have been owing to changes in potassium balance in these mice since there is evidence in humans that excessive glucocorticoid therapy can cause potassium depletion and associated water retention, and body weight gain with prolonged mineralocorticoid treatment (25). Intracellular potassium is replaced with sodium ions and water is retained with the sodium ions (25). Interestingly, the DMBA/TPA initiated, AL/ADX/CCS mice generally did not show elevated food intake in comparison with all of the acetone initiated groups (Table II).
An intriguing observation was the elevated lymphosarcoma incidence in the AL/ADX mice in comparison with the DER/sham and DER/ADX/CCS groups. This lesion appears to have been reduced by both DER and CCS treatments, and elevated by ADX in mice not supplemented with CCS. There is no evidence that this lesion interfered with skin tumor development in the current experiment since the results did not correlate clearly with any of the skin lesions. The other lesion observed was the high rate of skin ulceration in the AL/sham and AL/ADX/saline groups and a low rate in the DER/sham group. Skin ulceration was reduced either by CCS treatment or by DER. These lesions may have reduced survival in the two AL groups and thus may have blunted the papilloma and carcinoma rates in these groups.
In separate studies we are assessing the mechanism for DER and corticosterone prevention of mouse skin carcinogenesis. These studies suggest that DER and corticosteroid hormone prevention of mouse carcinogenesis occurs through inhibiting signaling down the extracellular signal regulated kinase (ERK)1,2, reducing activator protein 1 (AP1): DNA binding and inhibiting transcriptional regulation through AP1 (26,31). This inhibition appeared to be downstream from the inhibition of specific isoforms of protein kinase CPKC
and PKC
(32,33). These isoforms were also reduced by corticosterone supplementation in ADX mice in short-term studies (21). The inhibition of ERK1,2 (17) and the reduction in AP1:DNA (26) binding by DER were eliminated in ADX mice demonstrating the dependence of these DER effects on an intact adrenal gland. Supplementation with CCS in the ADX animals restored the inhibition of TPA-induced ERK1,2 and AP1:DNA binding (17,26). Studies of AP1-regulated genes indicate that DER inhibits TPA-induced transcriptional activation of an AP1-luciferase reporter gene in a transgenic mouse model (34). The known importance of TPA-induced ERK1,2 signaling and AP1 transcriptional regulation in mouse skin carcinogenesis suggests that DER blockage of these events causally contributes to DER prevention of skin carcinogenesis. The data presented in this paper show that skin tumor development in mice in protocols with DER, ADX and CCS treatment can be explained, at least in part, by these molecular effects of DER and CCS.
In summary, DER significantly inhibited tumorigenesis in intact mice as was previously observed. This inhibition was dependent upon an intact adrenal gland and corticosterone supplementation to DER/ADX mice restored the tumorigenesis inhibition. Corticosterone supplementation itself inhibited tumor progression when given to ADX mice fed the control diet and had an additive inhibitory impact when given to ADX mice fed the DER diet.
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Acknowledgments
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This research was supported by National Institute of Health/National Cancer Institute grant CA77451, the Iowa Agriculture and Home Economics Experiment Station and the Center for Designing Foods to Improve Nutrition (Iowa Nutrition Special Grant from the USDA).
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Received October 4, 2004;
revised February 1, 2005;
accepted February 8, 2005.