Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity

L. A. deGraffenried1,+,§, W. E. Friedrichs1,§, L. Fulcher1, G. Fernandes1, J. M. Silva1, J.-M. Peralba2 and M. Hidalgo2

1 Division of Medical Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX; 2 The Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, MD, USA

Received 24 January 2003; revised 18 February 2003; accepted 3 April 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Background:

Tamoxifen resistance is the underlying cause of treatment failure in a significant number of patients with breast cancer. Activation of Akt, a downstream mediator in the phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated as one of the mechanisms involved in tamoxifen resistance. Breast cancers with heightened Akt activity are frequently associated with an aggressive disease and resistance to chemo- and hormone-therapy-induced apoptosis. Inhibition of PI3K restores apoptotic response to tamoxifen in hyperactive Akt cells. Therefore, agents that demonstrate Akt inhibitory properties are attractive therapeutic agents for the treatment of hormone-resistant breast cancer. n-3 fatty acids have proven to be potent and efficacious broad-spectrum protein kinase inhibitors.

Materials and methods:

In this study we demonstrate that the n-3 fatty acid, eicosapentaenoic acid (EPA), inhibits the kinase activity of Akt. Co-treatment with EPA renders breast cancer cells that overexpress a constitutively active Akt more responsive to the growth inhibitory effects of tamoxifen by approximately 35%.

Conclusions:

These findings suggest that EPA may be useful for the treatment of tamoxifen-resistant breast cancer cells with high levels of activated Akt and provide the rationale to test this hypothesis in the clinic.

Key words: Akt, n-3 fatty acids, tamoxifen


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Tamoxifen, which functions as a cell-type-specific antiestrogen, inhibits estrogen-stimulated growth of mammary epithelial cells. However, 50% of all estrogen receptor {alpha} (ER{alpha})-positive breast cancer patients present with de novo tamoxifen resistance, and almost all initial responders eventually develop resistance. The mechanisms by which this resistance occurs remain unclear. It has been shown previously that growth factors such as epidermal growth factor, insulin-like growth factor and heregulin confer estrogen-independent growth properties to ER{alpha}-positive breast cancer cells [1, 2], perhaps through initiating a series of events which result in the phosphorylation of specific serines within the AF-1 domain of the ER{alpha}, resulting in hormone-independent growth [2]. Campbell et al. [3] recently showed that phosphatidylinositol 3-kinase (PI3K)and its downstream target Akt, a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses that are central to the process of oncogenic transformation of mammalian cells, activated ER{alpha} in the absence of estrogen. PTEN, a negative regulator of Akt activity, and a catalytically inactive Akt, decreased PI3K-induced AF-1 activity, suggesting that PI3K activation of ER{alpha} is mediated in part through Akt-dependent pathways [4]. These in vitro data, as well as emerging clinical studies, suggest that the PI3K/Akt pathway may be an attractive target for intervention in the treatment of tamoxifen-resistant breast cancer.

One such mechanism for intervention may be by the modulation of dietary components. Recent studies have demonstrated that fatty acid regulation of tumor cell growth is mediated, at least in part, via activation of PI3K and Akt [5]. The addition of exogenous arachidonic acid (AA), an n-6 fatty acid, stimulated the activity of class Ia PI3K in human myeloid and endothelial cells resulting in the phosphorylation of Akt on Thr-308 and Ser-473, while treatment with n-3 fatty acids reduced the activity of protein kinase C, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A, mitogen-activated protein kinase, and Ca2+/calmodulin-dependent protein kinase in the central nervous system [6]. Thus, n-3 fatty acids are surprisingly potent and efficacious broad-spectrum protein kinase inhibitors, suggesting that PI3K and Akt may also be targets of action for n-3 fatty acids in vivo in breast cancer cells.

Here we demonstrate the ability of n-3 fatty acids, specifically eicosapentaenoic acid (EPA), to inhibit mitogen activation of Akt. Furthermore, we show that EPA treatment enhances the growth inhibitory response to tamoxifen in breast cancer cells with constitutively active Akt. These data suggest that n-3 fatty acids are potential therapeutic agents for the treatment of tamoxifen-resistant breast cancer in patients with high Akt activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Generation, selection and analysis of stable transfectants
The expression plasmid for the myristoylated, constitutively active Akt1 (myrAkt {Delta}4–129) was kindly provided by Richard A. Roth (Stanford University School of Medicine, Stanford, CA, USA) and has been described previously [7]. The MCF-7 cell line was transfected with either the Akt plasmid or with an empty pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, USA) as a control, using FuGene 6 (Boehringer Mannheim, Indianapolis, IN, USA). One day after transfection, cells were placed into the selection medium containing G418 1.0 mg/ml (Gibco/BRL, Gaithersburg, MD, USA). Twenty-one days after selection, individual G418-resistant colonies were subcloned. Protein expression was analyzed by Western blot using antibodies for both phosphorylated and total Akt (Cell Signaling Technology, Beverly, MA, USA), and the T7 epitope (Novagen, Madison, WI, USA).

Cell lines
The parental MCF-7 breast cancer cells were obtained from the American Type Culture Collection and maintained in Improved Minimal Essential Medium (IMEM; Gibco/BRL) supplemented with 10% fetal bovine serum (FBS; Sigma, St Louis, MO, USA) and bovine insulin 6 ng/ml (Sigma). Stable transfectant cell lines were maintained in the same IMEM treated with G418 400 mg/l.

Western blot analysis
Following a 24-h incubation in growth media, cells were washed and treated with serum-free Earles salts containing 2 mM L-glutamine as unstimulated control or Earles salts with 100 nM insulin for 24 h. For fatty acid studies, the Earles/insulin media contained EPA at 0.2, 20, 40 or 200 µM (Matreya, Inc., State College, PA, USA) or LY294002 10 µM (Sigma). Cells were harvested in 1x lysis buffer (Tris–HCl 50 mM, pH 7.5, NaCl 120 mM, 1% NP-40, EDTA 1 mM, pH 8.0, EGTA 5 mM, pH 7.5, NaF 50 mM, ß-glycerolphosphate 40 mM, sodium orthovanadate 100 µM, benzamidine 1 mM, and protease inhibitor cocktail). Protein lysates were subjected to immunodetection with antibodies to first phosphorylated and then total Akt (Cell Signaling Technology), T7 (Novagen), ER{alpha} (6F11 monoclonal antibody; Novocastra Ltd, UK) and finally actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for a loading control. Signal detection was carried out using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL, USA).

Kinase assays
Kinase activity assays were used to measure the effects of fatty acid treatment on Akt activity, as has been described previously [8]. Cells were treated as described for the Western blot analyses. For the comprehensive fatty acid analysis, cells were treated with Earles salts (EBSS) supplemented with glutamine 2 mM (control), 10% FBS, EBSS with 20 µM linoleic acid, docosahexaenoic acid, or EPA (Matreya, Inc.). After 24 h of treatment, cells were lysed in 1x lysis buffer and total Akt immunoprecipitated using total Akt antibody 1 µg (Santa Cruz Biotechnology). Immunocomplexes were incubated with Akt substrate 5 µg, a RPRAATF sequence peptide (Alpha Diagnostic International, San Antonio, TX, USA) and 32ATP (New England Nuclear). ATP incorporation was measured by scintillation counting.

Growth proliferation assay
Cell growth was assessed by MTT [3, (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazoliumbromide; Sigma] dye conversion at 570 nm following manufacturer’s instructions. Briefly, cells were seeded 5 x 103 per well in a 96-well flat-bottomed plate. Cells were allowed to grow for 24 h in the presence of 10% FBS, then placed in serum-starved conditions for 18 h. Cells were then treated with increasing concentrations of tamoxifen alone, or in combination with EPA 40 µM, as indicated, in the presence of 10% FBS. After 96 h of continuous treatment, MTT 20 µl (5 mg/ml in phosphate-buffered saline) was added to each well. After 3 h incubation at 37°C, cells were lysed by the addition of 0.1 N HCl in isopropanol.

Statistical analysis
Statistical analyses for the kinase assays were carried out using two-way ANOVA and for the MTT assays by standard Student’s t-test comparing treatment results to those obtained without treatment (control).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antihormonal therapies that severely deplete estrogens or compete with estrogens for binding to the ER{alpha}, such as the antiestrogen tamoxifen, can result in tumor remissions. Tamoxifen increases overall survival and recurrence-free survival, especially in ER{alpha}-positive patients [9]. However, antiestrogen-resistant growth of ER{alpha}-positive tumors remains a significant clinical problem. It is now believed that control of breast cancer growth is comprised of an elaborate network of interacting steroid hormone- and growth factor-driven pathways that reinforce individual pathway effects on gene expression [10]. Recent studies have demonstrated that aberrations in growth factor signal transduction may circumvent the cellular requirement for estrogen stimulation, providing a significant mechanism for the development of breast disease resistant to current antihormonal therapies [10]. Current antihormonal therapies can only achieve a 50–60% objective breast tumor remission rate in ER{alpha}-positive patients, whereas these agents ultimately become ineffective in the majority of patients [11].

Constitutive Akt activity in the myr-Akt1 MCF-7 cells
In order to better understand how specific components of the PI3K/Akt signaling pathway effect development of hormone-independent breast cancer, we developed MCF-7 breast cancer cell lines that express a myristoylated, constitutively active Akt1 kinase. The src myristoylation signal confers constitutive activity by targeting Akt to the membrane, resulting in an increase in the level of Akt phosphorylation by PDK1 [7]. As seen in Figure 1, levels of phosphorylated endogenous Akt1 were very low in the MCF-7 control cells stably transfected with the pCDNA 3.1 vector (control) as well as those transfected with the myr-Akt1 plasmid (Myr-Akt1) under non-stimulated conditions (–I), but demonstrated an increase in phosphorylation of the Ser-473 residue when treated with insulin 100 nM (+I). In contrast, the Ser-473 of the myr-Akt1 was constitutively phosphorylated even under non-stimulated conditions (–I) and demonstrated an increase in phosphorylation levels with insulin treatment (+I). Total protein levels of endogenous and myr-Akt1 remained constant. No changes were observed in the expression levels of the ER{alpha}, either between the control and Akt MCF-7 cells or upon treatment with insulin (data not shown). Actin levels confirmed equal loading.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 1. Mitogen-independent activation of Akt. Protein lysates from control and Akt-transfected MCF-7 cells prior to (–I) and post (+I) stimulation with insulin 100 nM were subjected to immunodetection using antibodies to the phospho Ser-473 residue of Akt on both the endogenous (endogenous phospho-Akt) as well as the myr-Akt1 transgene product (phospho myr-Akt1), total Akt and actin.

 
n-3 fatty acids inhibit Akt kinase activity
Many studies have now demonstrated that a critical aspect of intracellular signaling is regulation of key cell functions by lipid mediators [12]. Fatty acid composition has been shown to be a key modulator of cancer cell signaling [13]. Increased breast cancer cell proliferation and prolongation of survival has been observed following treatment with the n-6 essential fatty acids, linoleic and arachidonic acid in culture, while treatment with conjugated linoleic acid was inhibitory [14]. Fatty acid composition also appears to be a modulator of cancer cell response to cytotoxic agents, since a diet rich in n-3 fatty acids increased the effectiveness of several therapeutic agents, but did not increase their toxicity to the host [15]. One mechanism by which the n-3 fatty acids may be mediating these effects is by targeting the activity of Akt, as has been described for other kinases [6].

Hii et al. [5] demonstrated that arachidonic acid, an n-6 fatty acid, transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473 in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. To investigate the effects of n-6 and n-3 fatty acids on Akt activity in our breast cancer cells, we assessed Akt activity in control MCF-7 cells treated for 24 h with Earles salts without FBS (control), 10% FBS, or Earles salts without FBS with n-6 linoleic acid (LA), n-3 docosahexaenoic acid (DHA), or n-3 EPA (Figure 2A). Akt1 activity under serum-starved conditions was 40% less than that observed under 10% FBS conditions. Treatment with LA increased Akt activity levels to close to those observed with 10% FBS treatment. Treatment with n-3 DHA decreased Akt1 activity by almost 20% compared with that in the control treatment, and n-3 EPA decreased Akt1 activity by about 67%. These results are consistent with findings by Mirnikjoo et al. [6], who demonstrated that EPA is a more effective kinase inhibitor than DHA.



View larger version (44K):
[in this window]
[in a new window]
 
Figure 2. Akt kinase activity inhibited by n-3 fatty acids. (A) Kinase assay results measuring Akt activity in control MCF-7 cells treated for 24 h with Earles salts without FBS (control), 10% FBS, or Earles salts without FBS with n-6 linoleic acid (LA), n-3 docosahexaenoic acid (DHA), or n-3 eicosapentaenoic acid (EPA). Results are presented as percentage of activity obtained from 10% FBS-treated cells and are a combination of three independent experiments. (B) Kinase assay results measuring Akt activity in control (left panel) and Akt (right panel) MCF-7 breast cancer cells treated for 24 h with Earles salts [1], Earles salts with insulin 100 nM [2], or Earles salts with insulin 100 nM with n-3 eicosapentaenoic acid (EPA) 0.2 µM [3], 20 µM [4], 40 µM [5], or 200 µM [6], or with the PI3K inhibitor, LY294002 10 µM [7]. Results are the combination of three independent experiments and are presented as the level of Akt activity compared with that of the control treatment (1, left panel). Bars represent standard deviation, and all differences were statistically relevant (P <0.05) except between control 2 and 3. (C) Protein lysates used for the kinase assays were analyzed by immunoblot detection for the presence of phospho Ser-473 on both the endogenous Akt (pAkt) as well as the myr-Akt1 transgene product (pmyr-Akt1). Actin levels were measured to confirm equal loading.

 
Since we and others found EPA to be the more effective kinase inhibitor, we used EPA for all subsequent studies. We treated both the control as well as the Akt MCF-7 cells with increasing concentrations of EPA (Figure 2B and C). As demonstrated in Figure 2B, the control MCF-7 cells demonstrated low basal Akt kinase activity compared to the level exhibited in the Akt MCF-7 cells (Figure 2B, 1). Addition of insulin 100 nM (Figure 2B, 2) increased Akt activity three-fold in the control cells and two-fold in the Akt cells. Addition of increasing concentrations of EPA from 0.2 µM to 200 µM (Figure 2B, 3–6) resulted in an initial increase (which in the control cells was not statistically different from the activity with insulin alone) (Figure 2B, 3), but then a dose-dependent decrease in insulin-stimulated Akt activity in both the control as well as the Akt MCF-7 cells by 15% to >80% in the Akt MCF-7 cells. In the Akt MCF-7 cells, treatment with EPA 200 µM (Figure 2B, 6) inhibited Akt kinase activity to levels observed with treatment with the PI3K inhibitor, LY294002 (Figure 2B, 7). Kinase activity results were confirmed by immunoblot analysis of the phosphorylation status of Akt in the cells after treatment (Figure 2C). The endogenous Akt (pAkt) displayed no or little expression of phosphorylated Akt under unstimulated conditions, in contrast to the myr-Akt1 protein (pmyr-Akt), which remained constitutively phosphorylated under mitogen-free conditions (Figure 2C, 1). Levels of endogenous pAkt were increased upon treatment with insulin 100 nM (Figure 2C, 2), and were higher in the myr-Akt1 cells compared with the control cells. The levels of Akt phosphorylation remained high with the addition of EPA 0.2 µM (Figure 2C, 3), but subsequently decreased in a dose-dependent manner with increasing concentrations of EPA (20–200 µM; Figure 2C, 4–6). Surprisingly, EPA had no observable effect on myr-Akt1 phosphorylation, suggesting first that the observed effects on kinase activity by EPA were reflective of the inhibition of endogenous Akt phosphorylation and/or EPA interfered with Akt kinase enzymatic activity, and secondly, that the EPA was interfering with the recruitment of Akt to the plasma membrane rather than the ability of PDK1 to phosphorylate Akt. The higher levels of phosphorylated Akt in the myr-Akt1 cells were reflected by the ~10-fold greater level of Akt kinase activity in the myr-Akt1 cells compared with the control cells at every concentration of EPA. Treatment with LY294002 (Figure 2C, 7) inhibited insulin-stimulated phosphorylation of the endogenous Akt completely in both cell lines, and even decreased phosphorylation of the myr-Akt1. These data demonstrate that at higher concentrations, n-3 fatty acids are effective inhibitors of Akt.

Co-treatment with n-3 fatty acids increases sensitivity to tamoxifen
Previous studies have demonstrated that inhibition of PI3K restores tamoxifen response in breast cancer cells with high Akt activity [3, 16]. In order to determine whether inhibition of Akt with EPA also restores tamoxifen response, we treated both the control as well as the Akt MCF-7 cells with EPA 40 µM either alone (Figure 3A) or with increasing concentrations of tamoxifen, again in the presence of 10% FBS and insulin 6 ng/ml (Figure 3B). Treatment with EPA alone had limited effect on growth in either the control or the Akt cells, decreasing the rate of growth by only 15% in the control cells, and increasing the rate of growth of the Akt cells by 10%. Tamoxifen, when administered as a single agent, also had little effect on the growth rate of the Akt cells, while inhibiting growth of the control MCF-7 cells in a dose-dependent manner. However, when administered in conjunction with EPA 40 µM, the sensitivity to the effects of tamoxifen of the Akt MCF-7 cells was increased, resulting in dose-dependent growth inhibition, from 0% inhibition at 10–9 M tamoxifen to >35% inhibition at 10–6 M tamoxifen (Figure 3B).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 3. Co-treatment with n-3 fatty acids increases sensitivity to tamoxifen. (A) Growth of both the control and Akt MCF-7 cells after treatment with EPA 40 µM in 10% FBS was measured by MTT analysis and is presented as the relative percentage growth compared with treatment with 10% FBS alone. (B) Growth of the control (white bars) and the Akt (black bars) MCF-7 cell lines in the presence of increasing concentrations of tamoxifen (10–9 M to 10–6 M) and the Akt MCF-7 cells co-treated with EPA 40 µM (striped bars). Results are presented as the relative percentage growth compared with growth in 10% FBS alone and is the combination of three independent experiments.

 
These data show that the n-3 fatty acid, EPA, significantly inhibited insulin-mediated activation of Akt kinase activity. Previous studies have demonstrated that fish oil diets, and more specifically n-3 fatty acids, are effective antitumor agents [17]. However, the mechanisms by which these compounds mediate their effects remain elusive and are probably multifocal. These results suggest that one significant mechanism may be the ability of the n-3 fatty acids to act as efficacious protein kinase inhibitors. Recent studies from other groups indicate that mitogen-induced ER{alpha} activation leads to tamoxifen resistance [3, 18], perhaps through ER{alpha} phosphorylation at Ser-167 by Akt [3]. In addition, it was recently shown that activated Akt2 phosphorylates ER{alpha} at Ser-167 in vitro and in vivo and that this Akt2-induced ER{alpha} activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, suggesting that ER{alpha} phosphorylation at Ser-167 contributes to tamoxifen resistance [19]. Additionally, Akt signaling contributes significantly to both survival and proliferation in breast cancer cells (reviewed in [10]). High Akt activity confers resistance to non-hormonal, cytotoxic agents such as doxorubicin and paclitaxel [16, 20], suggesting that perhaps non-ER{alpha} pathways are more important for the development of hormone therapy resistance in cells with aberrant regulation of Akt. Additional studies are essential to distinguish the mechanism(s) that are responsible for the resistance to tamoxifen observed with high Akt activity, and the subsequent restoration of sensitivity to tamoxifen growth inhibition by treatment with n-3 fatty acids.

Taken together, these data suggest that exogenous inhibitors of the Akt signaling network and other mitogenic pathways can abrogate or delay the emergence of antiestrogen resistance, thus providing an evaluable therapeutic strategy in human breast carcinoma.


    Acknowledgements
 
This work was supported in part by a grant to L. A. deGraffenried by the Susan G. Komen Foundation, PDF 2000 655.


    Footnotes
 
+ Correspondence to: L. A. deGraffenried, Division of Medical Oncology, UT Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Tel: +1-210-567-4777; Fax: +1-210-567-6687; E-mail: degraffenri{at}uthscsa.edu Back

§ L. A. deGraffenried and W. E. Friedrichs contributed equally to the work presented here. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Lupu R, Cardillo M, Cho C et al. The significance of heregulin in breast cancer tumor progression and drug resistance. Breast Cancer Res Treat 1996; 38: 57–66.[ISI][Medline]

2. Kato S, Endoh H, Masuhiro Y et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995; 270: 1491–1494.[Abstract]

3. Campbell RA, Bhat-Nakshatri P, Patel NM et al. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 2001; 276: 9817–9824.[Abstract/Free Full Text]

4. Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 1999; 286: 1741–1744.[Abstract/Free Full Text]

5. Hii CS, Moghadammi N, Dunbar A, Ferrante A. Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family. J Biol Chem 2001; 276: 27246–27255.[Abstract/Free Full Text]

6. Mirnikjoo B, Brown SE, Kim HF et al. Protein kinase inhibition by omega-3 fatty acids. J Biol Chem 2001; 276: 10888–10896.[Abstract/Free Full Text]

7. Kohn AD, Takeuchi F, Roth RA. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 1996; 271: 21920–21926.[Abstract/Free Full Text]

8. Franke TF. Assays for Akt. Methods Enzymol 2000; 322: 400–410.[ISI][Medline]

9. Early Breast Cancer Trialists’ Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Lancet 1992; 339: 1–15.[ISI][Medline]

10. Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 2002; 14: 381–395.[CrossRef][ISI][Medline]

11. Robertson JF. Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer 1996; 73: 5–12.[ISI][Medline]

12. Triboulot C, Hichami A, Denys A, Khan NA. Dietary (n-3) polyunsaturated fatty acids exert antihypertensive effects by modulating calcium signaling in T cells of rats. J Nutr 2001; 131: 2364–2369.[Abstract/Free Full Text]

13. Yu C, Chen Y, Zong H et al. Mechanism by which fatty acids inhibit insulin activation of IRS-1 associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002; 277: 50230–50236.[Abstract/Free Full Text]

14. Park Y, Allen KG, Shultz TD. Modulation of MCF-7 breast cancer cell signal transduction by linoleic acid and conjugated linoleic acid in culture. Anticancer Res 2000; 20: 669–676.[ISI][Medline]

15. Hardman WE, Avula CP, Fernandes G, Cameron IL. Three percent dietary fish oil concentrate increased efficacy of doxorubicin against MDA-MB 231 breast cancer xenografts. Clin Cancer Res 2001; 7: 2041–2049.[Abstract/Free Full Text]

16. Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther 2002; 1: 707–717.[Abstract/Free Full Text]

17. Calviello G, Palozza P, Piccioni E et al. Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis. Int J Cancer 1998; 75: 699–705.[CrossRef][ISI][Medline]

18. Kurokawa H, Lenferink AE, Simpson JF et al. Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res 2000; 60: 5887–5894.[Abstract/Free Full Text]

19. Sun M, Paciga JE, Feldman RI et al. Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor {alpha} (ER{alpha}) via interaction between ER{alpha} and PI3K. Cancer Res 2001; 61: 5985–5991.[Abstract/Free Full Text]

20. Tanaka M, Koul D, Davies MA et al. MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells. Oncogene 2000; 19: 5406–5412.[CrossRef][ISI][Medline]