Haifang Xu

The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea- Induced Rat Mammary Carcinogenesis

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-γ (PPARγ), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARγ receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea–induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARγ is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA–rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies. Cancer Prev Res; 3(3); 322–30

Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague‐Dawley rats were injected ip with 1‐methyl‐1‐nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO‐rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n‐3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n‐3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n‐3FA.

Fish Oil Alters Tamoxifen-Modulated Expression of mRNAs That Encode Genes Related to Differentiation, Proliferation, Metastasis, and Immune Response in Rat Mammary Tumors

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publishers online edition of Nutrition and Cancer to view the supplemental file.

Fish Oil Enhances T Cell Function and Tumor Infiltration and Is Correlated With a Cancer Prevention Effect in HER-2/neu But Not PyMT Transgenic Mice

Few studies have explored the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on immune modulation in murine models of mammary carcinogenesis. HER-2/neu and PyMT mice were randomized to 2 dietary interventions: AIN-93G-based diet with 1) 11% of diet (per gram weight) as corn oil (CO) or 2) 10% of diet as menhaden fish oil plus 1% of diet as corn oil (FO). FO significantly reduced the incidence and multiplicity of tumors (P < 0.001) in HER-2/neu, but not PyMT mice. FO-fed mice had significantly larger splenocyte counts than CO-fed mice in both the HER-2/neu and PyMT models; and in both models this was comprised of an increase in most cell types, including Gr-1+/CD11b+ cells. T cells from FO-fed HER-2/neu mice produced significantly more interleukin-2 (P = 0.004) and interferon-γ (P = 0.012) in response to in vitro stimulation with anti-CD3 (0.5 µg/ml). Lastly, FO-fed HER-2/neu mice had significantly more tumor immune infiltrates than CO-fed mice, including NK1.1+, F4/80+, and Gr-1+/CD11b+ cells (P ≤ 0.05). Greater Th1 cytokine production and significantly more tumor immune infiltrates in FO-fed Her2/neu mice may account for the cancer prevention effect of fish oil in this model.

Abstract 3657: Identification of active metabolites of docosohexaenoic acid (DHA): A first step towards unraveling the fish oil/breast cancer conundrum.

The role of fish oil (FO) rich in omega-3 fatty acid (n-3FA) in breast cancer prevention remains controversial. Based on our data showing that a high n-3:n-6 ratio (≥10) is necessary to inhibit N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis (Zhu Z, et al., Cancer Prev Res 4(10):1675, 2011), we hypothesize that this chemopreventive effect of FO may be due to one of its components (e.g., Docosohexaenoic acid, DHA, 22:6) and/or to the production of Active DHA Metabolites (ADM) which may be present in small and variable amounts in FO preparations used in different preclinical studies. To test this hypothesis, we used LC-TOF/MS followed by tandem mass spectroscopy to quantify ADM in the plasma and mammary tumors of rats fed a 20% CO diet and administered either saline or one of two doses of DHA (1.5 ml/kg and 3.0 ml/kg) by gavage twice a week for 8 weeks following ip MNU injection (50 mg/kg) at 21 days of age. The lower dose of DHA was also given in combination with Tamoxifen (Tam) (0.6 ppm) to test the hypothesis that n-3FA may inhibit the development of antiestrogen resistant tumors. We observed that both doses of DHA similarly inhibited mammary carcinogenesis and that the combination of Tam and DHA was more effective than each of the individual agents. DHA administration caused a significant dose-dependent increase in the plasma levels of several ADM including 4-OH-DHA, 7-OH-DHA, 14-OH-DHA, and 17-OH-DHA. The levels of these ADM were ∼10-fold higher in the tumors of the rats given DHA compared to control, irrespective of the dose of DHA administered. The most abundant ADM in the tumors was 14-OH-DHA followed in order by 17-OH-DHA, 4-OH-DHA, and 7-OH-DHA. In order to compare the biological activity of these ADM to that of the parent compound, we tested in vitro their ability to induce PPARγ activation using a PPARγ reporter assay since PPARγ activation is considered to be a potential mechanism by which n-3FA exert their antitumor action. We observed that every ADM was more effective than DHA by at least 2-fold in inducing PPARγ activity with the following order of magnitude of the effect, 7-OH-DHA > 14-OH-DHA > 4-OH-DHA > 17-OH-DHA. In summary, our studies identify for the first time ADM which may be responsible for the chemopreventive effect of FO. This finding may have major translational significance since compounds with well-defined chemical structure given at optimal concentrations are likely to be more effective chemopreventive agents than FO which contains an inconsistent mixture of FA with varying biological activity. In addition, since DHA inhibits carcinogenesis in Tam-treated rats, these ADM may complement the effect of Tam by inhibiting the development of hormone independent tumors. This work is supported by grant KG081632 from Susan G. Komen for the Cure. Citation Format: Andrea Manni, John P. Richie, Neil Trushin, Bogdan Prokopczyk, A Patterson, John P. Vanden Heuvel, Sharlene Washington, Haifang Xu, Cesar Aliaga, Arunangshu Das, Krishne Gowda, Shantu Amin, Jason Liao, Henry Thompson, Karam El-Bayoumy. Identification of active metabolites of docosohexaenoic acid (DHA): A first step towards unraveling the fish oil/breast cancer conundrum. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3657. doi:10.1158/1538-7445.AM2013-3657

Abstract 5578: Transcriptomic changes in tamoxifen-treated mammary tumors in Sprague-Dawley rats fed with omega-3 or omega-6 rich diets

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have demonstrated that administration of an omega-3 rich diet to Tamoxifen (Tam)-treated rats reduced the incidence of mammary tumors when compared to rats fed an omega-6 rich diet in the presence and in the absence of Tam treatment (A. Manni, et al. Cancer Prev Res 3:322, 2010). To understand the mechanisms of antitumor action of omega-3 fatty acids, we evaluated the transcriptomic changes in 1-Methyl-1-nitrosourea (MNU)-induced mammary tumors in Tam-treated rats fed with omega-3 (fish oil) or omega-6 (corn oil) fatty acids. Twenty one-days old Sprague Dawley rats received a single 50 mg/kg of body weight (bw) i.p. injection of MNU and were distributed into 4 groups: 1) CO group (20% corn oil diet); 2) COtam group (20% CO diet plus Tam 100 μg/Kg bw s.c., 5 days/week); 3) FO group (17% fish oil diet); 4) FOtam group (17% FO diet plus Tam). Animals were euthanized 8 weeks after MNU injection. RNA from cribiform tumors was extracted and Agilent oligonucleotide microarrays were performed in three samples per group. Tam-treated groups were compared to their respective controls (FOtam vs. FO and COtam vs. CO). Additionally, the FO-treated group was compared to the CO-treated group. Genes were considered differentially expressed when their level of expression differed by at least 3-fold with a P value <0.01. Genes of interest were validated by real time PCR. We found 32 differentially expressed genes between COtam and CO, 58 differentially expressed genes between FOtam and FO, and 19 differentially expressed genes between FO and CO. Real time PCR confirmed the differential expression of SerpinB10, Wisp2 and Apod in the FO-treated groups, which may be related to greater tumor differentiation. The down-regulation of Thrsp and Wnt5b genes in FOtam group may be related to tumor growth impairment and lower capacity of metastasis. Moreover, the up-regulation of IRF7, RT1-CE3, RT1-CE16 and RT1-Aw2 points to an improvement of the immune response against tumors in FO-treated animals. However, the up-regulation of FCER1A, HDC, MS4A2, SLP1, MCPT1 and MCTP2 suggests a shift of the immune response towards a Th2 pattern (mast cell-based immune response), which could be a mechanism of escape from the immune response caused by the combination of FO and Tam treatment. Our data suggests that an omega-3 rich diet could lead to a more differentiated tumor phenotype and an improved immune response against tumors. However, the administration of tamoxifen to FO fed rats promoted a Th2 pattern of immune response, which could represent an escape mechanism of the tumor cells from the improved immune response caused by FO per se. (Supported by the Susan G. Komen for the Cure, grant KG081632). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2011-5578

Abstract 3710: Individual and combined effects of Tamoxifen and fish oil on mammary carcinogenesis in polyoma middle T transgenic mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL There are currently no known effective regiments able to prevent the development of estrogen-receptor (ER) negative breast cancer in humans. We hypothesize that ER, frequently upregulated in preneoplastic mammary lesions, may contribute to the development of hormone-independent tumors through crosstalk with multiple cellular oncogenic pathways. Therefore, a multi-targeted approach is likely to be necessary to prevent ER negative tumors. In our previous study, we tested this hypothesis in CD rats and we showed that omega-3 rich fish oil (FO) potentiated the suppressive effect of Tamoxifen (Tam) on mammary carcinogenesis (A. Manni, et al. Cancer Prev Res 3:322, 2010). In the present investigations, we tested this hypothesis in polyoma middle T transgenic mice, since in this experimental system the development of ER negative tumors is preceded by preneoplastic lesions overexpressing functional ER, as evidenced by the concomitant upregulation of progesterone receptors which are products of estrogen action. Administration of increasing concentrations of Tam (1 ppm, 10 ppm, and 100 ppm) admixed with 20% corn oil (CO) modified AIN-76A diet, starting at 3 weeks of age, significantly delayed mammary carcinogenesis, inhibited tumor multiplicity by ∼50%, tumor volume by ∼90%, and tumor weight by ∼70% in a dose dependent fashion. At termination (week 12), however, tumor incidence was nearly 100% in all experimental groups. When Tam treatment was started after tumors had developed, no inhibition of tumor growth was observed, a finding consistent with the absence of ER in invasive adenocarcinomas in this experimental model. Administration of increasing concentrations of FO in the diet (5% FO + 15% CO; 10% FO + 10% CO, 17% FO + 3% CO) started at 3 weeks of age did not affect any of the tumor parameters compared to the control 20% CO diet. Combined administration of suboptimal (1 ppm) and optimal (100 ppm) doses of Tam with 17% FO and 10% FO respectively, delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as observed with Tam alone. Therefore, the results indicate that, under these experimental conditions, FO failed to potentiate the chemopreventive action of Tam. Our results, however, provide support for the role of ER expression by the preneoplastic lesions in the development of hormone independent tumors and, consequently, for the importance of including ER targeting in combined chemoprevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2011-3710

Abstract 5571: Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Our laboratories are interested in testing the value of combining omega-3 fatty acids and antiestrogens as a safe and effective chemopreventive strategy for breast cancer. We have shown that a fish oil rich diet (FO) (17% FO + 3% corn oil [CO]) enhances the chemopreventive action of Tamoxifen (Tam) in a prepubertal model of N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis (A. Manni, et al., Cancer Prev Res 3:322, 2010). To gain insight into mechanisms of prevention and the importance of different ratios of n-3:n-6 fatty acids in inhibiting carcinogenesis, we tested the effects of two FO rich diets (17% FO + 3% CO and 10% FO + 10% CO) individually and in combination with Tam on the histologic progression of preneoplastic lesions, indices of proliferation and apoptosis, as well as expression of multiple mammary gland specific and systemic biomarkers of oxidative stress in carcinogen treated rats. Following MNU administration at 21 days of age, separate groups of rats within each experimental arm were sacrificed 24 hours, 21 days, 28 days and 35 days prior to the development of palpable tumors. Increasing the amount of FO in the diet progressively increased the n-3:n-6 ratio in the plasma from 0.13 (20% CO) to 0.38 (10% FO) and 0.96 (17% FO). Although the FO rich diets significantly reduced Ki67 expression in hyperplastic lesions in a dose-dependent fashion (17% FO > 10% FO), neither the diets nor Tam decreased the development of preneoplastic lesions indicating that the inhibitory effect on carcinogenesis results from blocking the transition from hyperplasia to adenocarcinoma, consistent with our recent published study. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers (plasma protein carbonyls and blood protein bound GSH) or mammary specific biomarkers of oxidative stress, based on oxidative damage to DNA measured as 8-hydroxy-2’-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric-acid reactive substances (TBARS). On the other hand, tissue levels of 8-isoprostanes, prostaglandin-like compounds formed by non-enzymatic oxidation of arachidonic acid in the cell membrane, were markedly reduced by both FO rich diets at all time points (p<0.0001). In conclusion, these results, together with our previous findings, suggest that Tam, either alone or in combination with FO, exerts its protective effects on mammary carcinogenesis by blocking the transition from hyperplasia to carcinoma. The effects of FO may be due to changes in arachidonic acid specific pathways but do not involve early changes in levels of select oxidative stress markers examined in this experimental system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2011-5571

Abstract 5722: Effects of dietary omega-3 and omega-6 in the prevention of rat mammary cancer with tamoxifen

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC There is evidence that omega 3-rich diet strengthened the inhibitory effect of tamoxifen on the development of estrogen dependent tumors, pointing to omega 3 as a potential adjuvant to standard therapy (Manni A. et al. Cancer Prev Res, in press). However, the mechanistic basis of this observation is unknown. The objective of the present work is to analyze the genomic changes in chemically induced mammary tumors that developed in tamoxifen treated rats fed omega 3 or omega 6 fatty acids. Sprague Dawley rats, 21 days-old, were administered an i.p. injection of 1-Methyl-1-nitrosourea (MNU) 50 mg/kg body weight. The animals were distributed into 4 groups (G), and maintained under standard conditions and were given the following treatments for 8 weeks: G1 received a diet of 20% of corn oil (CO); G2 received a diet with 17% of fish oil (FO) and 3% CO; G3 received the CO diet and tamoxifen; G4 received FO diet and tamoxifen. Tamoxifen was administered s.c 100 μg/Kg b.w., 5 days/week. The animals were euthanized after 8 weeks. Mammary tumors were frozen in liquid nitrogen and stored at −80C. RNA from cribiform tumors was extracted and one-color Agilent 44K whole rat genome oligonucleotide microarray was performed in three tumor samples per group. Tamoxifen-treated groups (G3 and G4) were compared with their respective controls (G1 and G2). The genes were considered differentially expressed when the p value was lower or equal to 0.01 and had at least 1.50 fold change. A total of 488 genes were considered statistically different comparing G3 to G1 (G3/G1), whereas 206 genes were considered statistically different comparing G4 to G2 (G4/G2). Gene ontology analysis showed that 125 genes of G3/G1 comparison and 58 of G4/G2 comparison were related to cancer, apoptosis, cellular movement, cellular proliferation, inflammatory response or angiogenesis. In the G3 /G1 comparison, the expression pattern of genes related to cell proliferation and apoptosis was found to be divergent, as well as the genes related to cell proliferation in the G4/G2 comparison. In contrast, G4/G2 did not showed relevant genes expressed related to apoptosis. CO treatment in presence of tamoxifen (G3/G1) induced an up-regulation of C5r1, Hck, Il6Ra, Myo1f_predicted and Tlr6, and down-regulation of Mif, indicating an increase of the inflammatory host response. These changes were accompanied by up-regulation of Ets1, Tlr6 and Wdr44 genes controlling angiogenesis and cell migration, which can lead to metastasis. These changes were not observed in the FO and tamoxifen treated group. We conclude that an omega 6 rich-diet could modify the genomic profile of tamoxifen treated tumors making them more prone to an aggressive behavior or result in tamoxifen resistance. (Supported by the Susan G. Komen for the Cure, grant KG081632). The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5722.