August Copenhagen Irish Rats Research Articles

Less Carcinogenic Chlorinated Estrogens Applicable to Hormone Replacement Therapy.

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17β-estradiol (2-ClE2) or 4-chloro-17β-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17β-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.

Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal death through p53/SLC7A11-mediated ferroptosis

Cerebral ischemia is the most common cause of hippocampal neuronal death and the most prevalent cause of stroke with high mortality rate. Ferroptosis has been suggested to affect the role of hippocampal neurons. This study explores the influence of lentivirus infection-induced ferritin overexpression in hippocampal neuronal injury and death through simulations in August Copenhagen Irish rat models. Twenty-four-hour cerebral ischemia–reperfusion injury was induced in the rats after 90-min middle cerebral artery occlusion (MCAO). Ferritin overexpression was induced through lentivirus infection. The Morris Water Maze (MWM) test and tau hyperphosphorylation test were performed on hippocampal neurons to establish a MCAO model. The effect of ferritin overexpression on hippocampal neuronal death was evaluated using hematoxylin–eosin staining and annexin V/propidium iodide flow cytometry. The MWM test revealed that MCAO modeling decreased the cognitive and locomotor capacity of the rats, whereas ferritin overexpression partially reversed the effect of MCAO. In addition, the hyperphosphorylation of tau caused by MCAO was reduced by ferritin. Pathogenic changes, impaired viability, increased apoptosis, and elevated caspase-9 cleavage in hippocampal neurons were clearly recovered by ferritin. Moreover, robust reactive oxygen species production and glutathione consumption, which was induced by MCAO modeling, were ameliorated by ferritin. Furthermore, two key modulators of ferroptosis, p53 and SLC7A11, were demonstrated to be upregulated by MCAO modeling and downregulated by ferritin. Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal ferroptosis mediated via p53 and SLC7A11.

Sulforaphane Diminishes the Formation of Mammary Tumors in Rats Exposed to 17β-Estradiol.

Elevated levels of estrogen are a risk factor for breast cancer. In addition to inducing DNA damage, estrogens can enhance cell proliferation as well as modulate fatty acid metabolism that collectively contributes to mammary tumorigenesis. Sulforaphane (SFN) is an isothiocyanate derived from broccoli that is currently under evaluation in multiple clinical trials for prevention of several diseases, including cancer. Previous studies showed that SFN suppressed DNA damage and lipogenesis pathways. Therefore, we hypothesized that administering SFN to animals that are co-exposed to 17β-estradiol (E2) would prevent mammary tumor formation. In our study, 4–6 week old female August Copenhagen Irish rats were implanted with slow-release E2 pellets (3 mg x 3 times) and gavaged 3x/week with either vehicle or 100 μmol/kg SFN for 56 weeks. SFN-treated rats were protected significantly against mammary tumor formation compared to vehicle controls. Mammary glands of SFN-treated rats showed decreased DNA damage while serum free fatty acids and triglyceride species were 1.5 to 2-fold lower in SFN-treated rats. Further characterization also showed that SFN diminished expression of enzymes involved in mammary gland lipogenesis. This study indicated that SFN protects against breast cancer development through multiple potential mechanisms in a clinically relevant hormonal carcinogenesis model.

Carcinogenic potential of fluorinated estrogens in mammary tumorigenesis

Fluorination preventing metabolic hydroxylation of 17β-estradiol (E2) was applied to investigate the mechanisms underlying estrogen-induced carcinogenesis. Either 2-fluoro-17β-estradiol (2-FE2) or 4-fluoro-17β-estradiol (4-FE2) was administered subcutaneously for 52 weeks to August Copenhagen Irish (ACI) rats, the preferred animal model for human breast cancer. 4-FE2 induced frequent mammary tumors whereas 2-FE2 did not. The cumulative incidence of mammary tumors in rats treated with 4-FE2 was comparable to that observed with E2. The carcinogenic results were supported by histological examination of mammary glands of fluorinated estrogen-treated ACI rats. To evaluate the estrogenic potential of the fluorinated estrogens, 2-FE2 or 4-FE2 was administrated subcutaneously to ovariectomized rats. Both 4-FE2 and 2-FE2 showed high uterotrophic potency. Our results indicate that estrogenic potential may not be the sole factor driving mammary tumorigenesis. Since fluorination inhibits metabolic hydroxylation of E2 at the substituted position, the carcinogenic effect may occur through the metabolic activation of 2-hydroxylated E2, in combination with the compound’s estrogenic potency.

Novel insight in estrogen homeostasis and bioactivity in the ACI rat model of estrogen-induced mammary gland carcinogenesis.

Despite being widely used to investigate 17β-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28-42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.

Long-term Functioning of Allogeneic Islets in Subcutaneous Tissue Pretreated With a Novel Cyclic Peptide Without Immunosuppressive Medication.

There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes. In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets. Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts. These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Differential Gene Regulation and Tumor-Inhibitory Activities of Alpha-, Delta-, and Gamma-Tocopherols in Estrogen-Mediated Mammary Carcinogenesis.

Despite experimental evidence elucidating the antitumor activities of tocopherols, clinical trials with α-tocopherol (α-T) have failed to demonstrate its beneficial effects in cancer prevention. This study compared the chemopreventive efficacy of individual tocopherols (α-, δ-, and γ-T) and a γ-T-rich tocopherol mixture (γ-TmT) in the August-Copenhagen Irish (ACI) rat model of estrogen-mediated mammary cancer. Female ACI rats receiving 17β-estradiol (E2) implants were administered with 0.2% α-T, δ-T, γ-T, or γ-TmT for 30 weeks. Although α-T had no significant effects on mammary tumor growth in ACI rats, δ-T, γ-T, and γ-TmT reduced mammary tumor volume by 51% (P < 0.05), 60% (P < 0.01), and 59% (P < 0.01), respectively. Immunohistochemical analysis revealed that δ-T, γ-T, and γ-TmT reduced levels of the cell proliferation marker, proliferating cell nuclear antigen, in the rat mammary tumors. To gain further insight into the biological functions of different forms of tocopherols, RNA-seq analysis of the tumors was performed. Treatment with γ-T induced robust gene expression changes in the mammary tumors of ACI rats. Ingenuity Pathway Analysis identified "Cancer" as a top disease pathway and "Tumor growth" and "Metastasis" as the top signaling pathways modulated by γ-T. Although the results need further functional validation, this study presents an unbiased attempt to understand the differences between biological activities of individual forms of tocopherols at the whole transcriptome level. In conclusion, δ-T and γ-T have superior cancer preventive properties compared to α-T in the prevention of estrogen-mediated mammary carcinogenesis. Cancer Prev Res; 10(12); 694-703. ©2017 AACR.

Chemoprevention of Rat Mammary Carcinogenesis by Apiaceae Spices.

Scientific evidence suggests that many herbs and spices have medicinal properties that alleviate symptoms or prevent disease. In this study, we examined the chemopreventive effects of the Apiaceae spices, anise, caraway, and celery seeds against 17β-estrogen (E2)-mediated mammary tumorigenesis in an ACI (August-Copenhagen Irish) rat model. Female ACI rats were given either control diet (AIN 93M) or diet supplemented with 7.5% (w/w) of anise, caraway, or celery seed powder. Two weeks later, one half of the animals in each group received subcutaneous silastic implants of E2. Diet intake and body weight were recorded weekly, and animals were euthanized after 3 and 12 weeks. E2-treatment showed significantly (2.1- and 3.4-fold) enhanced growth of pituitary gland at 3 and 12 weeks, respectively. All test spices significantly offset the pituitary growth by 12 weeks, except celery which was effective as early as three weeks. Immunohistochemical analysis for proliferative cell nuclear antigen (PCNA) in mammary tissues showed significant reduction in E2-mediated mammary cell proliferation. Test spices reduced the circulating levels of both E2 and prolactin at three weeks. This protection was more pronounced at 12 weeks, with celery eliciting the highest effect. RT-PCR and western blot analysis were performed to determine the potential molecular targets of the spices. Anise and caraway diets significantly offset estrogen-mediated overexpression of both cyclin D1 and estrogen receptor α (ERα). The effect of anise was modest. Likewise, expression of CYP1B1 and CYP1A1 was inhibited by all test spices. Based on short-term molecular markers, caraway was selected over other spices based on its enhanced effect on estrogen-associated pathway. Therefore, a tumor-end point study in ACI rats was conducted with dietary caraway. Tumor palpation from 12 weeks onwards revealed tumor latency of 29 days in caraway-treated animals compared with first tumor appearance at 92 days in control group. At the end of the study (25 weeks), the tumor incidence was 96% in the control group compared with only 70% in the caraway group. A significant reduction in tumor volume (661 ± 123 vs. 313 ± 81 mm3) and tumor multiplicity (4.2 ± 0.4 vs. 2.5 ± 0.5 tumors/animal) was also observed in the caraway group compared with the control group. Together, our data show dietary caraway can significantly delay and prevent the hormonal mammary tumorigenesis by modulating different cellular and molecular targets.

Abstract 5235: Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats

Abstract Tocopherols (T), members of the vitamin E family, occur in four forms designated as α, β, δ and γ. Epidemiological studies suggest that tocopherols could reduce the risk of cancer. α-T is known as the classical vitamin E, being the predominant form of tocopherol found in human blood and tissues. However, clinical studies with α-T to substantiate the role of tocopherols in cancer prevention have provided inconclusive results. Such outcomes indicate that the cancer preventive activity of tocopherols might be attributed to the other forms of tocopherols, but not α-T. γ-T, which is the most abundant tocopherol in vegetable oils, is our major dietary source of tocopherols. Recent studies have demonstrated that δ-T and γ-T have higher anti-cancer activity than α-T in several animal models of cancer. The present study investigated the cancer preventive activity of individual purified forms of tocopherols α-T, δ-T, γ-T as well as a naturally occurring γ-T rich mixture of tocopherols (γ-TmT) in an in vivo model of estrogen receptor (ER)-positive breast cancer, namely estrogen supplemented August-Copenhagen Irish (ACI) rats. ACI rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and administered with diets containing 0.2% α-T, δ-T, γ-T or γ-TmT. Rats were euthanized at 30 weeks of mammary carcinogenesis when the E2 control group exhibited 100% tumor incidence. α-T had no effect on estrogen-induced mammary tumors. However, treatment with 0.2% δ-T, γ-T and γ-TmT reduced the tumor volume by 51% (p&amp;lt;0.05), 60% (p&amp;lt;0.01) and 59% (p&amp;lt;0.01), respectively. Administration of α-T, δ-T and γ-T resulted in a 2.1-, 80- and 62-fold increase of these tocopherols, respectively, in the serum of ACI rats. Supplementation with the individual tocopherols also increased the levels of their respective short chain metabolite, carboxyethyl hydroxychromans (CEHCs) in the serum. Administration of δ-T and γ-T was associated with 185- and 99-fold increase in their CEHC metabolites, respectively. The high serum levels of δ- and γ-CEHC metabolites could contribute to the enhanced chemopreventive activity of δ-T and γ-T. In conclusion, δ-T, γ-T and γ-TmT could be potential natural agents for the prevention of estrogen-induced mammary tumorigenesis. (This work was supported by the NIH grant R01 AT007036 and the New Jersey Commission on Cancer Research Postdoctoral Fellowship to Soumyasri Das Gupta). Citation Format: Soumyasri Das Gupta, Joseph Wahler, Min Ji Bak, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5235.

Prevention of hormonal breast cancer by dietary jamun.

Syzygium cumini (jamun) is perhaps the only berry that has the diversity of anthocyanidins of blueberry and bilberry and the abundance of ellagitannins/ellagic acid of black raspberry. Here, we report the potential of jamun against 17β-estrogen-mediated breast cancer and the role of miRNAs and other targets in disease inhibition. Female August-Copenhagen Irish rats were given AIN-93M diet or diet supplemented with jamun. Two weeks later, animals received 17β-estradiol and were palpated weekly for the mammary tumors. At the end of 26 weeks, the jamun-diet significantly delayed the first tumor appearance by 21 days, and reduced the tumor incidence (65% versus 96%), tumor burden (313 ± 95 versus 661 ± 123 mm(3) ) and tumor multiplicity (1.8 ± 0.3 versus 4.2 ± 0.4 tumors/rat) compared to control. The experimental diet significantly reduced the estrogen-associated growth of pituitary prolactinomas, circulating prolactin and estradiol levels and offset estrogen-associated increases in mammary cell-proliferation, estrogen receptor-alpha (ER-α), and cyclinD1. miRNAs that were either overexpressed (miR-182 and miR-375) or underexpressed (miR-127 and miR-206) following estrogen-treatment were significantly protected by jamun diet. Together, our data show that jamun significantly offset estrogen-mediated alterations in mammary cell-proliferation, ER-α, cyclinD1, and candidate miRNAs, and that the modulation of these biomarkers correlated with a reduction in mammary carcinogenicity.

Soy isoflavone exposure through all life stages accelerates 17β-estradiol-induced mammary tumor onset and growth, yet reduces tumor burden, in ACI rats.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17β-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56%, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.

Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in Ih activation curve, and an altered responsiveness of Ih to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.

Dietary γ-Tocopherol-Rich Mixture Inhibits Estrogen-Induced Mammary Tumorigenesis by Modulating Estrogen Metabolism, Antioxidant Response, and PPARγ.

This study evaluated the anticancer activity and mechanism of action of a γ-tocopherol-rich tocopherol mixture, γ-TmT, in two different animal models of estrogen-induced breast cancer. The chemopreventive effect of γ-TmT at early (6 weeks), intermediate (18 weeks), and late (31 weeks) stages of mammary tumorigenesis was determined using the August-Copenhagen Irish rat model. Female rats receiving 17β-estradiol (E2) implants were administered with different doses (0%, 0.05%, 0.1%, 0.3%, and 0.5%) of γ-TmT diet. Treatment with 0.3% and 0.5% γ-TmT decreased tumor volume and multiplicity. At 31 weeks, serum concentrations of E2 were significantly decreased by γ-TmT. γ-TmT preferentially induced expression of the E2-metabolizing enzyme CYP1A1, over CYP1B1 in the rat mammary tissues. Nrf2-dependent antioxidant response was stimulated by γ-TmT, as evident from enhanced expression of its downstream targets, NQO1, GCLM, and HMOX1. Serum concentrations of the oxidative stress marker, 8-isoprostane, were also decreased in the γ-TmT-treated groups. Treatment with γ-TmT increased expression of PPARγ and its downstream genes, PTEN and p27, whereas the cell proliferation marker, PCNA, was significantly reduced in γ-TmT-treated mammary tumors. In an orthotopic model in which human MCF-7 breast cancer cells were injected into the mammary fat pad of immunodeficient mice, γ-TmT inhibited E2-dependent tumor growth at all the doses tested. In conclusion, γ-TmT reduced mammary tumor development, in part through decreased E2 availability and reduced oxidative stress in mammary tissues; γ-TmT could thus be an effective agent for the prevention and treatment of E2-induced breast cancer.

Abstract 2797: Dietary administration of a γ-tocopherol rich mixture of tocopherols inhibits estrogen-mediated mammary tumorigenesis

Abstract Tocopherols (T), members of the vitamin E family, occur in four forms designated as α, β, δ and γ. Tocopherols are suggested to reduce the risk of cancer. α-T is the predominant form of tocopherols found in human tissues. However, clinical studies with α-T have provided inconclusive results. γ-T is our major dietary source of tocopherols. Recent studies have demonstrated that γ-T has higher anti-cancer activity than α-T in animal models of cancer. This study investigated the cancer preventive activity of a naturally occurring γ-T rich mixture of tocopherols (γ-TmT) in two different in vivo models of estrogen receptor (ER)-positive breast cancer, namely estrogen supplemented August-Copenhagen Irish (ACI) rats and MCF-7 xenograft tumor mouse models. ACI rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and administered with diets containing 0.05%, 0.1%, 0.3% and 0.5% γ-TmT. Rats were euthanized at early (6 weeks), intermediate (18 weeks) and late (31 weeks) stages of E2-induced mammary carcinogenesis. Treatment with 0.3% and 0.5% γ-TmT reduced the tumor volume by 51% (p&amp;lt;0.01) and 41% (p&amp;lt;0.01), respectively while the lower doses of γ-TmT (0.05% and 0.1%) were not effective. Tumor multiplicity was also reduced by 38% (p&amp;lt;0.02) and 32% (p&amp;lt;0.02) in the 0.3% and 0.5% γ-TmT treatment groups, respectively. Dietary γ-TmT increased the levels of tocopherols and their metabolites in the serum. Serum levels of E2 were significantly lowered in the 0.3% and 0.5% γ-TmT administered groups. Analysis of mammary glands and tumors revealed that γ-TmT led to induced mRNA expression of the estrogen metabolizing enzyme, CYP1A1 at each experimental time point. CYP1A1 metabolizes E2 to its non-carcinogenic metabolite, 2-hydroxyestradiol. Gene expression of the Nrf2 mediated phase II detoxification enzymes NQO1, GCLM, UGT1A1, COMT and the antioxidant enzyme heme oxygenase were also upregulated in the γ-TmT treated groups. Thus, γ-TmT exerts its protective activity in E2-induced mammary carcinogenesis by lowering the levels of available E2 through increased metabolism of E2 and simultaneous removal of E2 metabolites through induction of the antioxidant response pathway. mRNA levels of PPARγ, a nuclear receptor which inhibits cell proliferation was also induced in the 0.3% and 0.5% γ-TmT treated groups. In a second in vivo model, the ER positive breast cancer cell line, MCF-7 was utilized. Mice were injected with MCF-7 cells after implantation of E2 pellets. Effect of the different doses of dietary γ-TmT (0.05%, 0.1%, 0.3% and 0.5%) treatment on the mammary xenograft tumors was studied. A significant reduction in the volume and weight of mammary tumors was observed in each of the γ-TmT treatment groups. In conclusion, γ-TmT inhibits mammary tumorigenesis in two different in vivo models of E2-induced breast cancer and could be a potential safe, natural agent for the chemoprevention of ER-positive breast cancer. Citation Format: Soumyasri Das Gupta, Sudathip Sae-tan, Joseph Wahler, Jae Young So, Larry C. Cheng, Min Ji Bak, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. Dietary administration of a γ-tocopherol rich mixture of tocopherols inhibits estrogen-mediated mammary tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2797. doi:10.1158/1538-7445.AM2015-2797

Gene expression and epigenetic profiles of mammary gland tissue: Insight into the differential predisposition of four rat strains to mammary gland cancer

Rats are excellent experimental models for studying breast cancer, but rat strains differ in susceptibility. Among the four strains used in this study, Fischer rats are less susceptible to spontaneous breast cancer, yet they are highly prone to extremely severe metastatic and drug-resistant tumors, in those case where they actually develop the disease. In contrast, Sprague Dawley rats are the most susceptible to spontaneous breast cancer among the strains. ACI rats are highly prone to estrogen-induced cancer. Long–Evans rats are commonly used in mammary gland carcinogenesis studies. The molecular mechanisms of differential breast cancer susceptibility among rat strains are not well understood. Here, gene expression analysis was conducted in the mammary gland tissue of four rat strains – August×Copenhagen Irish (ACI), Long Evans, Fischer-344 and Sprague Dawley – to evaluate possible explanations for the differing breast cancer predispositions. According to the DAVID functional annotation analysis, there were at least eleven, five, and one significantly different pathways, respectively, in Fischer-344, Long–Evans and Sprague Dawley rats, in comparison to ACI rats. Two strains, Fischer-344 and Long–Evans, displayed differential expression in the complement and coagulation cascades, chemokine signaling, PPAR signaling, renin–angiotensin system, ECM-receptor interaction, focal adhesion and glutathione metabolism pathways. The only pathway that was significantly different between the Sprague Dawley and the ACI rats was the ribosome pathway. Our data indicate that general cancer susceptibility and predisposition to the development of aggressive and metastatic cancer are independent genetic conditions. Moreover, we have identified several important differences in the basal epigenetic profile of four rat strains with varying degrees of susceptibility to spontaneous and induced mammary carcinogenesis.

Tacrolimus promotes hepatocellular carcinoma and enhances CXCR4/SDF-1α expression in vivo

The aim of our study was to elucidate the effect of tacrolimus (FK506) and of C-X-C chemokine receptor type 4 (CXCR4), which is a receptor specific to the stromal cell-derived factor-1α (SDF-1α), on growth and metastasis of hepatocellular carcinoma (HCC). Following treatment with different concentrations of FK506, AMD3100 or normal saline (NS), the proliferation of Morris rat hepatoma 3924A (MH3924A) cells was measured by the MTT assay, the expression of CXCR4 was analyzed with immunohistochemistry, and the morphological changes and the invasiveness of cells were studied with a transwell assay and under a scanning electron microscope, respectively. In addition, August Copenhagen Irish rat models implanted with tumor were used to examine the pathological changes and invasiveness of tumor in vivo, the expression of CXCR4 in tumor tissues and the expression of SDF-1α in the adjacent tissues to the HCC ones, using immunohistochemistry. In vitro, FK506 (100–1,000 μg/l) significantly promoted the proliferation of MH3924A cells (P<0.01), and increased the expression of CXCR4 in MH3924A cells, albeit with no significance (P>0.05). By contrast, AMD3100 had no effect on the proliferation of MH3924A cells, but significantly reduced the expression of CXCR4 (P<0.05). The invasiveness of MH3924A cells was significantly (P<0.01) enhanced following treatment with FK506, SDF-1α, FK506 + AMD3100, FK506 + SDF-1α or FK506 + AMD3100 + SDF-1α. In vivo, tumor weight (P=0.041), lymph node metastasis (P=0.002), the number of pulmonary nodules (P=0.012), the expression of CXCR4 in tumor tissues (P=0.048) and that of SDF-1α in adjacent tissues (P=0.026) were significantly different between the FK506-treated and the NS group. Our results suggest that FK506 promotes the proliferation of MH3924A cells and the expression of CXCR4 and SDF-1α in vivo. Therefore, inhibiting the formation of the CXCR4/SDF-1α complex may partly reduce the promoting effect of FK506 on HCC.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways.

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17β-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways.

Abstract P4-09-03: A diet enriched by soy-derived isoflavones impacts on the development of premalignant stages of breast cancer in an estrogen driven carcinogenesis animal model

Abstract Background: Hundred thousands of mainly postmenopausal women are using dietary supplements enriched with soy-derived isoflavones (sISO), such as the Novasoy® extract, to relief from climacteric complaints. At the same time, hundreds of studies controversially discuss the impact of sISO on the development of breast cancer. Whether sISO positively or negatively affect the tumorigenesis of the mammary gland presumably is dependent on the time point of the sISO exposure. An exposure during gestation, lactation as well as prepubertal is believed to be preventive whereas an exposure later in life might be adverse. Objective: Aim of the study was to assess whether and how sISO influence the development of breast cancer. Therefore, August-Copenhagen-Irish (ACI) rats, a model for estrogen driven carcinogenesis, were exposed towards sISO. Dietary exposure was started preconceptional and maintained in offspring animals continuously throughout the whole individual life. Experimentals: All ACI rats were kept under controlled conditions and had ad libitum access either to a sISO-free control diet or a diet supplemented with 500ppm of the Novasoy®650 extract. Pregnant females were kept under the established dietary regimen throughout gestation and lactation period and the female offspring was investigated in the following experimental procedures. On postnatal day 60 (PND60) the animals of each dietary group randomly were implanted a 17beta-estradiol (E2) releasing (approximately 2mg release in 90 days) or a placebo pellet to induce tumorigenesis of the mammary gland. The rats were weekly palpated for mammary tumors and weighed weekly for the duration of the experiment (until PND240). Finally, we assessed physiological (e.g. organ weights, sISO exposition, mammary gland areas), (patho-)histological (HE staining), immunohistological (PCNA expression) as well as molecular end points (gene expression) of the resulting mammary gland phenotypes. Results: Control diet animals had a daily sISO exposure of ∼0.5mg/kg body weight, whereas animals of the Novasoy®650 group had a daily exposure of ∼30mg/kg body weight. Independent of the dietary background, none of the animals with an implanted placebo pellet showed tumor precursors. Ductual carcinomas in situ (DCIS) were detected in 83.3% of the animals of the control diet and in 100% of the animals of the Novasoy®650 diet, both following implantation of an E2-release pellet. Moreover, control diet animals exposed to E2 had significantly reduced mammary gland areas compared to the respective placebo group. This difference was not detectable in the Novasoy®650 dietary group. Additionally, no differences in the uterus as well as liver wet weights were noticed. On a molecular level, the expression of several genes responsible for proliferation (Ki67), estrogenicity (Tff1) and cell cycle control (Cdk4) were modified in animals lifelong exposed towards sISO. Conclusions: In ACI rats, a prenatally started and lifelong maintained sISO exposure clearly affects the estradiol induced malignant transformation of the mammary gland. This is evidenced by a higher number of DCIS as well as increased proliferation levels which were found in animals chronically exposed to sISO. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-09-03.

Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer

BackgroundEstrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.MethodsFemale ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2.ResultsThe expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage.ConclusionsOur studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.

(4S)-4-(3-18F-Fluoropropyl)-l-Glutamate for Imaging of xC¯ Transporter Activity in Hepatocellular Carcinoma Using PET: Preclinical and Exploratory Clinical Studies

(4S)-4-(3-(18)F-fluoropropyl)-l-glutamate ((18)F-FSPG, or BAY 94-9392) is a new tracer to assess system x(C)(¯) transporter activity with PET. The aim of this study was to explore the tumor detection rate of (18)F-FSPG, compared with that of (18)F-FDG, in patients with hepatocellular carcinoma (HCC). Preclinically, in vivo HCC models of orthotopically implanted Huh7 and MH3924a cancer cells were studied with (18)F-FSPG in Naval Medical Research Institute nude mice (n = 3) and August-Copenhagen Irish rats (n = 4), respectively. Clinically, 5 patients with HCC who had hyper- or isometabolic lesions on (18)F-FDG PET were enrolled for evaluation of the tracer. Dynamic whole-body PET images with (18)F-FSPG were acquired for up to 120 min after injection of approximately 300 MBq of (18)F-FSPG. Immunohistochemical expression levels of the xCT subunit of the system x(C)(¯) and CD44 of HCC were studied in 4 patients with HCC. Strong tumor uptake and low background from nontarget tissue allowed excellent tumor visualization in animal models with orthotopically implanted liver tumors. (18)F-FSPG PET procedures were well tolerated in all patients. (18)F-FSPG PET and (18)F-FDG detected lesions in 5 of 5 and 3 of 5 patients, respectively. The maximal standardized uptake values (SUV) were comparable ((18)F-FSPG, 4.7 ± 3.2; (18)F-FDG, 6.1 ± 2.9). The ratios of maximal SUV of the tumor to mean SUV of normal liver were also comparable ((18)F-FSPG, 3.6 ± 2.2; (18)F-FDG, 2.7 ± 1.3), but the mean SUV of normal liver of (18)F-FSPG was significantly lower than that of (18)F-FDG (P < 0.05). Two patients with HCC who showed both xCT and CD44 expression had moderate or intense accumulation of (18)F-FSPG, but the remaining 2 patients with negative CD44 expression showed mild uptake. (18)F-FSPG was successfully translated from preclinical evaluation into patients with HCC. (18)F-FSPG may be a promising tumor PET agent with a high cancer detection rate in patients with HCC.