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

Abstract Background Estrogen 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. Methods Female 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. Results The 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. Conclusions Our 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.Peer Reviewe

Partial Inhibition of Estrogen-Induced Mammary Carcinogenesis in Rats by Tamoxifen: Balance between Oxidant Stress and Estrogen Responsiveness

Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F2α (8-isoPGF2α), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels of tamoxifen metabolizing enzymes cytochrome P450s as well as estrogen responsive genes were also quantified. At necropsy, breast tumors were detected in 44% of rats co-treated with tamoxifen+E2. No tumors were detected in the sham or tamoxifen only treatment groups whereas in the E2 only treatment group, the tumor incidence was 82%. Co-treatment with tamoxifen decreased GPx and catalase levels; did not completely inhibit E2-mediated oxidative DNA damage and estrogen-responsive genes monoamine oxygenase B1 (MaoB1) and cell death inducing DFF45 like effector C (Cidec) but differentially affected the levels of tamoxifen metabolizing enzymes. In summary, our studies suggest that although tamoxifen treatment inhibits estrogen-induced breast tumor development and increases the latency of tumor development, it does not completely abrogate breast tumor development in a rat model of estrogen-induced breast cancer. The inability of tamoxifen to completely inhibit E2-induced breast carcinogenesis may be because of increased estrogen-mediated oxidant burden

Expression of estrogen regulated genes <i>MaoB1</i> and <i>Cidec</i> in mammary tissues.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. mRNA expression levels of <i>MaoB1</i> and <i>Cidec</i> gene in E2-, Tam- and Tam + E2-treated mammary tissues using quantitative real-time PCR are presented as fold change versus control mammary tissues. Expression of each gene was normalized using cyclophilin as internal control. These data are reported as an average of values obtained for at least 5 different animals ± SEM. ‘*’ indicates a p value<0.05 compared to controls.</p

Effects of E2 and Tam treatments on mammary tumor development in female ACI rats.

<p>Column 1 lists different treatments each group of animals received. The number of animals per group (<i>n</i>) is listed in column 2. Percent tumor incidence after 240 days of treatment period is listed in column 3, and the average number of tumors per tumor-bearing animal (tumor multiplicity) is listed in column 4. Column 5 lists the day on which the first tumor appeared in each group (appearance of first tumor). ‘*’ indicates significant difference (<i>p</i><0.05) compared to control group. ‘#’ indicates significant difference (<i>p</i><0.05) between Tam + E2 and control or Tam-treated group.</p

Expression of ER-α in mammary tissues.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. At the end of the experiment, mammary tissues were collected and used for immunohistochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. Formalin-fixed/paraffin-embedded mammary sections were immunostained with ER-α antibody. Nuclear expression of ER-α is shown in representative sections (arrow). (a) The mammary tissue of a representative control ACI rat; (b) E2-treated mammary tissue; (c) Tam-treated mammary tissue; and (d) Tam + E2-treated mammary tissue. 100× magnification.</p

Expression of antioxidant enzymes in mammary tissues.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. At the end of the experiment, mammary tissues were collected, homogenized and used for western blot analysis. In E2-treated mammary tissue, protein level of CAT is decreased while SOD2 is increased compared to age-matched control. In mammary tissues of rats treated with Tam alone or co-treated with E2, SOD2 is increased, while GPx and CAT are decreased. SOD1 protein levels remain unchanged in all the treatment groups.</p

Tamoxifen metabolizing enzymes in mammary tissues.

<p>(A) Primary enzymes involved in metabolism of Tam in mammary tissues. Regular arrows indicate the detoxication steps in the pathway, while bold arrows indicate CYP3A4-mediated bioactivation processes. (B) Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. At the end of the experiment, mammary tissues were collected, homogenized and used for western blot analysis. CYP3A4 and CYP2D6 protein levels remain unchanged in all the treatment groups but the FMO1 protein expression is decreased in E2- and Tam + E2-treated mammary tissues.</p

Activities of antioxidant enzymes in female ACI rats after 240 days of treatment.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. The activities of the antioxidant enzymes SOD1, SOD2, GPx and CAT were measured in mammary tissues from control, E2-, Tam- and Tam + E2-treated rats after 240 days of respective treatment. The data are reported as an average of values obtained for at least 8 different animals ± SEM. ‘*’ indicates a p value<0.05 compared to control mammary tissue.</p

Expression of <i>PR</i> mRNA in mammary tissues.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. At the end of the experiment, mammary tissues were collected, total RNA isolated and used for real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. <i>PR</i> mRNA expression was significantly increased in E2-treated mammary tissues and it remained unchanged in Tam- and Tam + E2-treated mammary tissues. mRNA expression data are presented as fold change versus control mammary tissue. These data are reported as an average of values obtained for at least 5 different animals ± SEM. ‘*’ indicates a p value<0.05 compared to controls.</p

8-iso-prostane F_2α (8-isoPGF_2α) formation in rat mammary tissues.

<p>Female ACI rats were treated with E2, Tam or Tam + E2 for 240 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025125#s4" target="_blank">Materials and Methods</a> section. 8-isoPGF_2α levels were measured in mammary tissues from animals in each of these groups. Fold changes were calculated for E2-treated animals relative to age-matched cholesterol-treated controls. Fold changes were calculated for Tam + E2-treated animals relative to age-matched Tam-treated controls. The data are reported as an average of fold change values obtained for at least 8 different animals ± SEM.</p