Aggressive Mammary Carcinoma Progression in Nrf2
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Becks et al. BMC Cancer 2010, 10:540 http://www.biomedcentral.com/1471-2407/10/540 RESEARCH ARTICLE Open Access Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12- dimethylbenz[a]anthracene Lisa Becks1,2, Misty Prince1,2, Hannah Burson1,2, Christopher Christophe1,2, Mason Broadway1,2, Ken Itoh3, Masayuki Yamamoto4, Michael Mathis2,5, Elysse Orchard1,6, Runhua Shi2,7, Jerry McLarty2,7, Kevin Pruitt2,8, Songlin Zhang2,9, Heather E Kleiner-Hancock1,2* Abstract Background: Activation of nuclear factor erythroid 2-related factor (Nrf2), which belongs to the basic leucine zipper transcription factor family, is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1 and peroxiredoxin 1, by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE, and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis. Methods: Premalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics, and the Mann-Whitney U-test. Results: All mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice, there was a dramatic increase in mammary carcinoma growth rate, size, and weight. Although there was no difference in overall survival, the KO mice had significantly lower mammary tumor- free survival. Also, in the KO mammary carcinomas, the active forms of NF-B and b-catenin were increased ~2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed, including lymphomas. Interestingly, the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice. Conclusions: We report, for the first time, that there was no apparent difference in the formation of premalignant lesions, but rather, the KO mice exhibited rapid, aggressive mammary carcinoma progression. Background risk factors have been attributed to breast cancer occur- Breast cancer is the most common cancer among Amer- rence. Genetic susceptibility accounts for only ~10% of ican women, except for skin cancers. The chance of human breast cancer [1,2]. Known environmental risk developing invasive breast cancer at some time in a factors include radiation, obesity, and alcohol use [1,2]. woman’s life is about 1 in 8 (12%). In 2009, an estimated Nuclear factor erythroid 2-related factor (Nrf2) is an 192,370 new cases of invasive breast cancer will be diag- important regulator of genes induced by oxidative stress, nosed among women in the United States [1]. Many such as glutathione S-transferases (GSTs), heme oxyge- nase-1 (HO-1) and peroxiredoxin 1, by activating the antioxidant response element (ARE) [3]. Reactive oxygen * Correspondence: [email protected] 1Department of Pharmacology, Toxicology and Neuroscience, LSUHSC-S, species (ROS) are known to activate oncogenic tran- Shreveport, Louisiana, USA scription factors such as AP-1 and NF-Bthathave Full list of author information is available at the end of the article © 2010 Becks et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Becks et al. BMC Cancer 2010, 10:540 Page 2 of 17 http://www.biomedcentral.com/1471-2407/10/540 been shown in mouse models to be required for carci- for the first time, that there was no apparent differ- nogenesis [3]. It has also been noted that Nrf2 defi- ence in the formation of premalignant lesions, but ciency in mice shows an increased risk of chemical rather, the KO mice exhibited rapid, aggressive carcinogenesis and Nrf2 loss may contribute to tumori- mammary carcinoma progression. Many questions genesis [3]. However, the effects of Nrf2 deficiency in remain as to the mechanism, which we will explore breast cancer have not yet been explored. in future studies. Many cancer chemopreventive agents, in particular those that are naturally occurring, boost cellular antioxi- Methods dant defenses [4]. Evidence is mounting that many of Auraptene these phytochemicals activate the ARE through Nrf-2. Auraptene (AUR), a natural product isolated from Orally administered AUR induces GST activities via citrus, was initially purchased from LKT laboratories, activation of Nrf2, since these effects were significantly Inc. (West St. Paul, MN) for the previous studies and attenuated in Nrf2(-/-) knockout (KO) mice [5]. Based for the comparison study (see below). In this regard, on these studies, we hypothesized that AUR could sup- duetothelargequantityofAURneededtomixinthe press mammary carcinogenesis via activation of Nrf2/ diet for the premalignant study, it became necessary to ARE. In summary although many chemopreventive phy- synthesize AUR. tochemicals are known to activate cellular antioxidant defenses through the ARE, direct evidence that their Synthesis of Auraptene chemopreventive effects against mammary carcinogen- The synthesis of AUR was based on a literature proce- esis are due to this effect is limited. dure [10], modified as follows. Geranyl chloride (42 g, Oxidative stress is a condition of increased oxidant 0.24 mol), synthesized as reported elsewhere [11], was production in animal cells characterized by the release added to a 2-L round bottom flask along with acetone of free radicals, resulting in cellular degradation. Oxida- (800 mL), potassium carbonate (45 g, 0.32 mol), and tive stress resulting from excess ROS and/or deficiencies 7-hydroxycoumarin (35.8 g, 0.22 mol), obtained from in antioxidant capabilities may play a role in breast can- Sigma-Aldrich. The reaction mixture was allowed to stir cer etiology [6]. For example, a growing body of evi- andheatedatagentlerefluxunderanargonatmo- dence suggests that natural and synthetic estrogens, sphere for about 3 days, when thin layer chromatogra- which are oxidized to form quinones, are involved in phy (TLC) showed that no more 7-hydroxycoumarin breast cancer [7]. In a population-based, case-control was present. The reaction mixture was then cooled, fil- study (654 cases, 605 controls), African American tered, and evaporated to dryness. The residue was dis- women harboring the mitochondrial DNA G10398 poly- solved in ethyl acetate and filtered. Removal of the morphism exhibited an increased risk of invasive breast solvent gave crude product (65 g), which was heated in cancer (OR 1.60; 95% CI, 1.10-2.31, P = 0.013) [8]. hexanes along with 2 g of activated charcoal. After the MtDNA G10398A may be involved in altered structure product had dissolved, the mixture was filtered hot. The of Complex I, which could lead to increased ROS [8]. crystals that formed as the mixture cooled were filtered, Recently, a nested case-control study of postmenopausal dissolved in a toluene, hexanes solvent mixture and sub- women reported that women with genetic polymorph- jected to flash chromatography (7:1 toluene, hexanes). isms of Nrf2, NAD(P)H quinone oxidoreductase After collecting and combining the appropriate frac- (NQO1), and HO-1 that favored iron-generated oxida- tions, the solvent was removed, and the residue was tive stress were at higher risk of breast cancer [6] An crystallized from hexanes to give ~30 g of product. The epidemiological study by Santella and colleagues found 1Hand13C NMR spectra matched those of the authen- that women with higher plasma levels of oxidative pro- tic sample. The compound also co-eluted on TLC with tein damage (i.e. protein carbonyls) were at higher risk the authentic sample. The synthesis of auraptene was for breast cancer [9]. These studies suggest a role of oxi- carried out by Dr. William H. Johnson, Jr. in the labora- dative stress in breast cancer that may be attenuated by tory of Dr. Christian P. Whitman (The University of chemopreventive agents. Texas at Austin). Hence we named the synthetic AUR In order determine whether phytochemicals can pre- as “UT-AUR”. vent mammary carcinogenesis via activation of Nrf2/ ARE,firstitmustbedemonstratedthatNrf2/AREis Comparison study: LKT-AUR vs. UT-AUR involved in mammary carcinogenesis. To our knowl- We conducted a comparison study to determine if the edge, no mammary carcinogenesis studies have ever synthetic AUR (UT-AUR) possessed the same activities been done in Nrf2 knockout mice. Thus, to address this as the AUR purchased from LKT (LKT-AUR). This study critical gap in knowledge, we conducted a mammary was conducted exactly the same way as the original study carcinogenesis study in Nrf2 knockout mice. We report, in which we showed that AUR could significantly Becks et al. BMC Cancer 2010, 10:540 Page 3 of 17 http://www.biomedcentral.com/1471-2407/10/540 increase liver cytosolic GST activities in Nrf2 WT but not mammary epithelial tissue was isolated from mammary KO mice [5]. The results of the study were consistent glands using hyaluronidase and collagenase as previously with our previous study, and the synthetic AUR pos- described [13]. sessed the same activity as that from LKT (Additional Tumor Study File 1: Figure S1). UT-AUR was then shipped to Dyets, Groups of 10 mice each (WT & KO) were administered Inc. (Bethlehem, PA) where it was mixed with AIN76A vehicle for the negative controls. Groups of 34 mice and formed into pellets (formulation sheet available upon (WT) and 30 mice (KO) were dosed with medroxypro- request).