SENCAR Mice Research Articles

Metabolic pathway of dibenzoylmethane, a β-diketone analogue of curcumin, by NADPH-dependent cytochrome P450 enzymes in mouse liver microsomes

Dibenzoylmethane (DBM), a curcumin-related β-diketone analogue, has been reported to exhibit a remarkable inhibitory effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in Sencar mice. Investigation of the underlying mechanisms of DBM in the prevention of mammary tumorigenesis implied its role as an effector of Phase I enzymatic system. In this report, the metabolic fate of DBM by NADPH-dependent cytochrome P450 enzymes in mouse liver microsomes was demonstrated. Isolation of the major reductive metabolites of DBM (DBMH2), together with several minor metabolites identified by NMR, GC and LC-MS, explained the potential role of DBM as a modulator of the cytochrome P450 reductase that is required for the function of oxidase to metabolize DMBA. These might also contribute to the result of the inhibitory effect of DBM on DMBA-induced mouse mammary tumorigenesis.

7,12-dimethylbenz(a)-anthracene (DMBA) & High Fat High Sugar Diet Decrease Physical Activity in Female Mice

BACKGROUND: Regular exercise has been shown to diminish the risk of certain cancers. DMBA, (7, 12-dimethylbenz(a)-anthracene) is a complete carcinogen that is used to induce tumors in mice. It has yet to be established whether DMBA has an effect on voluntary wheel running (WR) in mice, and whether these effects may be exacerbated via consumption of a high fat high sugar (HFHS) diet. PURPOSE: Determine if DMBA treatment altered voluntary WR in female SENCAR mice, and whether a HFHS diet exacerbated treatment effects on voluntary WR. METHODS: Offspring of SENCAR breeder pairs were weaned at 3 weeks (wks) of age onto either an ad lib fed HFHS (20% protein, 45% fat/24% sucrose + 10% fructose water) or a diet restricted (DR) (12% kcal restriction, 20% protein, 10% fat, 57% cornstarch) diet. Animals were double-housed and randomly assigned to either a DMBA (n=40) treatment with HFHS (n=20) and DR (n=20) diets; or a control (CNTL) (n=18) treatment with HFHS (n=10) and DR (n=8) diets. At 4 wks of age, two plastic running wheels were mounted inside standard rat cages, and connected to a computer to record WR duration and distance. At 7-9 wks of age, mice were gavaged with DMBA dissolved in corn oil (20 μg/mouse/day) or with corn oil vehicle only (CNTL) for 5 days/wk for 6 weeks. A two-way ANOVA was employed to determine the effect of DMBA on activity with factors of treatment and diet for wks 9-20. RESULTS: Compared to CNTL, DMBA significantly decreased distance (7.41 ± 0.45 vs. 11.08 ± 0.68 km/day; p=0.0002), and duration (175.19 ± 8.24 vs. 261.23 ± 12.36 min; p<0.0001). No significant difference in speed was noted (40.31 ± 1.37 vs. 40.34 ± 2.05 m/min; p=0.77). HFHS diet compared to DR diet significantly decreased distance (5.84 ± 0.60 vs. 11.08 ± 0.55 km/day; p<0.0001), duration (168.41 ± 10.9 vs. 233.14 ± 10.1 min; p<0.0001), and speed (33.04 ± 1.81 vs. 47.12 ± 1.67 m/min; p<0.0001). No significant interactions were observed between treatment and diet groups. CONCLUSIONS: DMBA and HFHS diet decrease WR distance and duration, while only the HFHS diet decreased WR speed. Although DMBA and HFHS independently decreased WR, a lack of interaction suggest that they are not additive or synergistic. ACKNOWLEDGMENTS: Project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model.

Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model.

Protein Tyrosine Kinase 6 Regulates UVB-Induced Signaling and Tumorigenesis in Mouse Skin

Protein Tyrosine Kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and skin, where it is expressed in nondividing differentiated cells. We found PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6−/− SENCAR mice to incremental doses of UVB for thirty weeks. Wild type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of STAT3 than Ptk6−/− mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild type mice following UVB treatment. However, wild type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6−/− mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of FAK and BCAR1. Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 plays roles in normal differentiation, it also contributes to UVB induced injury and tumorigenesis in vivo.

Cancer Preventive Mechanisms by Exercise: Activation of p53 and p53‐related IGF‐1 Pathway Regulators

Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study is to investigate the effects of exercise on p53 and p53-related proteins engaged in IGF-1 pathway regulation in skin epidermis. Female SENCAR mice were fed an iso-caloric diet with or without 10-week treadmill exercise at 20 m/min for 60 min daily. Animals were treated with TPA topically 2 hours before sacrifice and the target proteins in the epidermis were assessed. All the target proteins except for MDM2 decreased under TPA treatment versus vehicle acetone as measured by immunohistochemistry and/or Western blot. A significant increase of p53 but decrease of MDM2 was observed in exercised group when compared with sedentary control. Additionally, p53-related downstream proteins such as p21, IGF-BP3, and PTEN increased significantly in response to exercise. Taken together, exercise appears to activate p53 by reducing MDM2 suppression, resulting in enhanced expression of IGF-BP3, p21, and PTEN. Both IGF-BP3 and PTEN may further reduce bioavailable IGF-1 and IGF-1-dependent signaling. A negative regulation of IGF-1 pathway through p53 activation and p53-related regulators may thus contribute to the observed cancer prevention by exercise in this mouse skin cancer model (supported by NIH CA167678).

Effect of Sodium Arsenite on Mouse Skin Carcinogenesis.

Arsenic is carcinogenic in human beings, and environmental exposure to arsenic is a public health issue that affects large populations worldwide. Thus, studies are needed to determine the mode of action of arsenic and prevent harmful effects arising from arsenic intake. The present study assessed the influence of sodium arsenite (As(3+)) on potentially carcinogenic processes that are either pre-existing or concomitant with chronic intake of water containing As(3+). Experiments using SenCar mice were designed to evaluate the effect of chronic administration of As(3+) (2, 20, or 200 mg of As(3+)/L) in drinking water that overlapped to varying degrees with a 2-stage carcinogenesis protocol carried out over 9 months. The results showed a time-dependent pattern. During early stages of carcinogenesis (6-12 weeks), animals exposed to As(3+) and the carcinogenesis protocol showed increased numbers of tumors compared to control animals. During late carcinogenesis (16-30 weeks), the number of tumors stabilized to below control values, but the tumors showed increased malignancy. These findings indicate that the outcomes of the 2-stage skin carcinogenesis protocol are modified by the presence of arsenite in drinking water, which increases the rate of carcinoma development.

Prevention of skin carcinogenesis by the β-blocker carvedilol.

The stress-related catecholamine hormones and the α- and β-adrenergic receptors (α- and β-AR) may affect carcinogenesis. The β-AR GRK/β-arrestin biased agonist carvedilol can induce β-AR-mediated transactivation of the EGFR. The initial purpose of this study was to determine whether carvedilol, through activation of EGFR, can promote cancer. Carvedilol failed to promote anchorage-independent growth of JB6 P(+) cells, a skin cell model used to study tumor promotion. However, at nontoxic concentrations, carvedilol dose dependently inhibited EGF-induced malignant transformation of JB6 P(+) cells, suggesting that carvedilol has chemopreventive activity against skin cancer. Such effect was not observed for the β-AR agonist isoproterenol and the β-AR antagonist atenolol. Gene expression, receptor binding, and functional studies indicate that JB6 P(+) cells only express β2-ARs. Carvedilol, but not atenolol, inhibited EGF-mediated activator protein-1 (AP-1) activation. A topical 7,12-dimethylbenz(α)anthracene (DMBA)-induced skin hyperplasia model in SENCAR mice was utilized to determine the in vivo cancer preventative activity of carvedilol. Both topical and oral carvedilol treatment inhibited DMBA-induced epidermal hyperplasia (P < 0.05) and reduced H-ras mutations; topical treatment being the most potent. However, in models of established cancer, carvedilol had modest to no inhibitory effect on tumor growth of human lung cancer A549 cells in vitro and in vivo. In conclusion, these results suggest that the cardiovascular drug carvedilol may be repurposed for skin cancer chemoprevention, but may not be an effective treatment of established tumors. More broadly, this study suggests that β-ARs may serve as a novel target for cancer prevention.

Abstract 1256: The chemopreventive effects of carvedilol on skin carcinogenesis

Abstract Skin cancer, the majority of which is non-melanoma skin cancer (NMSC), is the most common type of cancer in the US. The rate of NMSC is increasing mainly due to depletion of the ozone layer and increased exposure to ultraviolet radiation. To reduce the risk of skin cancer, it is recommended to all individuals to limit sun exposure and use sunscreens. Despite these efforts, the incidence of NMSC continues to rise. Novel chemopreventive targets and nontoxic agents are needed. Epidemiological studies identified an association of psychosocial factors such as chronic stress or depression with cancer onset and progression. These factors are partly mediated by activation of the sympathetic nervous system which results in release of norepinephrine and epinephrine, the effects of which are mediated through the alpha- and beta-adrenergic receptors (alpha- and beta-AR). Further, the use of beta-AR antagonists (beta-blockers) has been associated with reduced cancer incidence. However, whether beta-blockers have chemopreventive activity or whether beta-ARs contribute to carcinogenesis is unknown. To determine whether agonizing or antagonizing beta-ARs affect skin cell transformation, we tested the beta-AR agonist isoproterenol (Iso) and antagonist carvedilol on epidermal growth factor (EGF)-mediated transformation of JB6 P+ cells (a skin cell model to study tumor promotion). The cells were treated with EGF (10 ng/mL) plus 0.1 µM or 1.0 µM Iso, yet no effect was observed. However, treatment with carvedilol dose-dependently inhibited the formation of EGF-induced soft agar colonies. Such effect was not caused by growth inhibition, because cytotoxicity and inhibition of cell proliferation were not observed for tested concentrations. Next, the expression of beta1-, 2- and 3-AR was determined in JB6 cells using RT-PCR. The results showed that only beta2-AR was detectable. We also determined whether beta-ARs are expressed in other types of cancer. In tissues derived from a rat model of 7, 12-dimethylbenz[α]anthracene (DMBA)-induced mammary cancer, expression of beta1- and 2-AR was up-regulated in tumors in comparison with normal tissues. The human cancer cell lines A549 and MDA-MB-231 also express beta2-AR. To determine the in vivo chemopreventive activity of carvedilol, we used a skin hyperplasia model in SENCAR mice induced by topical treatment with 100 nM DMBA twice a week for four weeks. Carvedilol was tested topically (5 and 10 µM) or orally (5 and 20 mg/kg), beginning at two weeks before the first dose of DMBA, three times a week, for six weeks. DMBA alone increased the epidermal thickness from the average of 77.8 + 13.6 nm in normal skin to 294 + 49.7 nm in DMBA-treated skin. Both topical and oral carvedilol treatment inhibited DMBA-induced hyperplasia (P &amp;lt; 0.05). In conclusion, these results suggest that beta-blockers may have chemopreventive activity. This study also suggests that beta-ARs may serve as a novel target for cancer prevention. Citation Format: Andy Chang, Mandy Liu, Steven Yeung, Jijun Hao, Cyrus Parsa, Robert Orlando, Bradley Andresen, Ying Huang. The chemopreventive effects of carvedilol on skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2014-1256

Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity

Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

Strain-specific properties and T cells regulate the susceptibility to papilloma induction by Mus musculus papillomavirus 1.

The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×1010 MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated “MmuPV1”), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.

Comparison of dermal tumor promotion activity of cigarette smoke condensate from prototype (heated) cigarette and reference (combusted) cigarette in SENCAR mice

Test cigarette (prototype “heated” cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30mg tar/application, three times per week for 30weeks. Test groups showed a clearly longer latency at lower doses (⩽15mg), but the difference was less clear at higher doses (⩾22.5mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice

BackgroundThe major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice.MethodsWe used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey’s post hoc procedure.ResultsAnalysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of “PAM50” genes in the SK-BR-3 cell line from an ER-/Her-2+ to that resembling a “normal-like” phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin.ConclusionsThe SK-BR-3 cells and DMBA-induced tumors, both with an ER- and Her-2+ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice

The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

Bioinformatics Approaches for Weight Loss‐induced Cancer Preventive Mechanisms

Weight control by dietary calorie restriction (DCR) or exercise is associated with cancer prevention in animal models. Bioinformatics analysis of profiling changes was employed to uncover the underlying mechanisms. SENCAR mice were randomly assigned into 4 groups for 10 wks: ad lib‐fed sedentary control, ad lib‐fed exercise (AE), exercise but pair‐fed at the amount of control (PE), and 20% of DCR. Two hrs after topical TPA treatment, skin epidermis were analyzed by Affymetrix for gene expression, DIGE for proteomics, and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67, and 110 genes were significantly changed in DCR, PE, and AE, respectively. Over 600 protein spots were separated, ~120 proteins identified, and 27 proteins significantly changed by DCR. Among 338 phospholipids, five phosphatidylinositol species were decreased in PE but not AE. While gene and protein expression was not always correlated, reduced TPA‐promoted Braf in MAPK pathway and PI3K‐Akt1 in IGF‐1 signaling appeared possible targets for cancer prevention through weight loss (supported by NIH CA167678).

IGF-1 Mediates Exercise-Induced Phospholipid Alteration in the Murine Skin Tissues

We previously demonstrated that exercise with an iso-caloric diet significantly reduced body weight and skin carcinogenesis in correspondence with lower plasma IGF-1 levels and IGF-1-dependent phospholipid signaling. This study was thus designed to test the hypothesis that IGF-1 reduction plays a causal role in exercise-induced phospholipid changes. SENCAR mice were randomly assigned to one of the following three groups for 12 weeks: ad libitum-fed sedentary control, exercise but pair-fed the amount of the control (PE), and PE with IGF-1 injection (PE+IGF-1). Treadmill exercise was conducted at 13.4 m/min for 90 min/d, 5 d/week. In the last two weeks IGF1 was i.p. injected (10 μg/g B.W.) twice per week. Both body weight and plasma IGF-1 levels were significantly reduced in PE mice when compared with the control. IGF-1 injection did not affect body weight, or the plasma levels of IGF-1 at the end of the experiment due to a rapid degradation with a half-life of 3.4 hrs. Of the 338 phospholipid species detected in the skin tissues by electron spray ionization tandem mass spectrometry, 21 were significantly changed in PE mice compared to control. Fourteen of the altered 21 species in PE mice were reversed by IGF-1 injection, including the most abundant phosphatidylinositol (PI) 38:4, a substrate for lipid PI3K signaling. Western Blot Analysis further showed the reduced PI3K, but not IGF-1R, in PE mice was also reversed by IGF-1 restoration. Overall, these data provided evidence that exercise-induced reduction of IGF-1 is required in mediating the alteration of phospholipid profile and PI-related PI3K signaling.

P4-05-02: Early Developmental Exposures to a High Carbohydrate/High Fat Diet Affect Glucose Metabolism and Mammary Cancer Susceptibility.

Abstract Background The obesity epidemic in the U.S. has substantially increased the number of people with altered glucose metabolism. Alterations in metabolic programming, resulting from early exposures can affect development, metabolism as well as propensity to later diseases, including cancer. The Warburg effect describes a mechanism by which diet-induced hyperglycemia and hyperinsulinemia can contribute to cellular transformation. To investigate the effect of a hyperinsulinemia-, hyperglycemia-inducing (HI/HG) diet on metabolic programming and susceptibility to mammary cancer, we exposed developing mouse pups during three developmental stages: gestation, lactation and post-weaning. Materials and Methods: Female SENCAR mice were fed either a mildly restricted, defined, “chow like” control diet (DR) or a HI/HG, high sucrose/high fat (HS/HF) diet. At 14 weeks, both DR and HS/HF fed female mice were bred and the resulting offspring were randomized into 8 groups to model all combinations of gestational, lactational and post-weaning dietary exposures. Body weights (BW) were recorded weekly and Glucose Tolerance Tests (GTTs) were conducted on the female offspring at 10–12 weeks of age. Starting at 7–9 weeks of age, mice received 20 mg/day of DMBA or vehicle by daily gavage to induce mammary carcinogenesis. Results: Animals in the DR/DR/DR (gestational diet/nursing diet/post-weaning diet) and HS/DR/DR groups had the lowest average BW and retained normal response to glucose, while mice in the DR/HS/HS and HS/HS/HS groups had the highest average BW. Interestingly, animals born to DR-fed and nursed by HS/HF mothers and weaned onto a HS/HF diet (DR/HS/HS) were the most glucose intolerant. In response to DMBA, animals in the different dietary regimens partitioned into high, moderate or low mammary tumor incidence groups. Mice fed consistent diets throughout gestation, lactation and post-weaning (DR/DR/DR or HS/HS/HS) had intermediate tumor incidence. However, mice exposed to DR during gestation and/or lactation and then switched to a HS/HF diet at weaning had the highest tumor incidence and shortest latency. Mice exposed to a HS/HF diet during gestation and/or lactation and then switched to a DR diet at weaning had the fewest mammary tumors and longest latency. Conclusion: Our data indicate that substantial changes in the type and abundance of calories during gestation and/or lactation had long-lasting impacts on BW, glucose metabolism, and mammary tumorigenesis. Since BW did not consistently correlate with GTT results, the effects of dietary manipulation on obesity and glucose metabolism appear to be distinguishable. Results also indicate that diet-induced changes in the glucose metabolism of the mother profoundly affected tumor susceptibility in the exposed female offspring. These findings support the contention that rising levels of obesity, metabolic syndrome, and diabetes, especially in young individuals, may contribute to the increasing incidence of early onset breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-02.

Modulation of biomarkers related to tumor initiation and promotion in mouse skin by a natural β-glucuronidase inhibitor and its precursors

Carcinogen-mediated labilization of lysosomal enzymes such as β-glucuronidase (βG) is often associated with the general process of inflammation. Therefore, the primary goal of this study was to demonstrate that exposing the skin of SENCAR mice to the natural βG inhibitor D-glucaro-1,4-lactone (1,4-GL) and its precursor D-glucuronic acid-γ-lactone (GUL), prior to and during 7,12-dimethylbenz[α]anthracene (DMBA) treatment inhibits not only epidermal hyperplasia but also inflammation in the mouse skin complete carcinogenesis model, i.e., the 4-week inflammatory-hyperplasia assay. Topical administration of 1,4-GL or GUL prior to repetitive, high-dose DMBA treatment markedly and in a dose-related manner inhibited DMBA-induced epidermal hyperplasia (i.e., up to 57%). DMBA-mediated Ha-ras mutations in codon61 were reduced by up to 78% by 1,4-GL. DMBA-induced inflammation, as measured by dermal leukocyte counts and immunologically, was inhibited by up to 37% by topical 1,4-GL but not by GUL. The inhibition of cellular proliferation and inflammation coincided with the inhibition of βG expression. Thus, the present study suggests that in the DMBA-induced complete skin carcinogenesis model, 1,4-GL when applied topically had both anti-proliferative properties as well as anti-inflammatory properties, whereas GUL had only anti-proliferative when applied topically. However, the number of inflammatory cells in the dermal portion of the skin of mice was significantly reduced by dietary treatment of GUL, whereas both topical and dietary treatments with 1,4-GL were very effective.

Skin cancer chemoprevention by a-santalol

Alpha-santalol, a naturally occurring terpenoid, has been shown to have chemopreventive effects on both 7, 12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O- tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer development in CD-1 and SENCAR mice, and UVB-induced skin cancer developments in SKH-1 hairless mice in a concentration-dependent manner. Studies have demonstrated that α-santalol could be effective against skin carcinogenesis through both induction of apoptosis via caspase activation together with dissipation of mitochondria membrane potential and cytochrome c release in A431 cells, and inhibition of cell growth via induction of G2/M phase arrest in both A431 cells and melanoma UACC-62 cells by altering multiple cell cycle regulatory proteins and complexes. This review summarizes the chemopreventive effects and molecular mechanisms of α-santalol on skin cancer development in both animal models and skin cancer cell lines.

Soybean Peptide Lunasin Suppresses In Vitro and In Vivo 7,12‐Dimethylbenz[a]anthracene‐Induced Tumorigenesis

Lunasin is a novel peptide identified in soybean and other seeds. This study evaluated the anti-tumorigenic effects of lunasin on 7,12-dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene-treated (MCA) fibroblast NIH/3T3 cells. Lunasin significantly inhibited cell proliferation and cancerous foci formation in these 2 chemical carcinogens-treated cells. An in vivo SENCAR mouse model induced with DMBA was used to study the mammary cancer preventive properties of dietary lunasin contained in soy protein. Tumor incidence was 67% and 50%, and the tumor generation was 1.88 ± 0.48 and 1.17 ± 0.17, respectively, for the mice fed control diet and experimental diet obtained after AIN-93G supplementation with lunasin-enriched soy protein concentrate (containing 0.23% lunasin). However, no effects were observed in mice fed AIN-93G supplemented with soy protein concentrate (containing 0.15% lunasin). The data provided illustrate the anticancer potential of lunasin both in vitro and in vivo and supports the recommendation of soy protein as a dietary component that may aid in the prevention of mammary cancer.