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
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Abstract |
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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
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Introduction |
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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.
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Materials and methods |
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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 (TrisHCl 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 (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 manufacturers 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 Students t-test comparing treatment results to those obtained without treatment (control).
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Results and discussion |
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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, either between the control and Akt MCF-7 cells or upon treatment with insulin (data not shown). Actin levels confirmed equal loading.
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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.
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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 109 M tamoxifen to >35% inhibition at 106 M tamoxifen (Figure 3B).
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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.
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Acknowledgements |
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Footnotes |
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L. A. deGraffenried and W. E. Friedrichs contributed equally to the work presented here.
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