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Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions ¹

Intervention Section, Department of Cell and Cancer Biology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, NIH Clinical Center, Bethesda, Maryland 20892-1906, USA; and
^* Walter Reed Army Institute of Research, Division of Pathology, Washington D.C. 20307-5100, USA

²Correspondence: Intervention Section, Department of Cell and Cancer Biology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, NIH Clinical Center, 9000 Rockville Pike, Bethesda, MD 20892-1906, USA.

SPECIFIC AIMS

In recent studies we have described the function of various arachidonic acid (AA) metabolites in the growth regulation of aerodigestive cancers demonstrating overexpression of IGF-R and its ligand as conserved features of lung cancer and breast cancer. In lung cancer, IGF-1-dependent growth stimulation and a survival effect can be neutralized by blocking five-lipoxygenase (5-LO). We now address the hypothesis that products of 5-LO activity participate in the growth stimulation of breast cancer cells and reciprocally how other specific AA metabolites arising as a consequence of blockage of 5-LO activity may be relevant in the inhibition of breast cancer growth.

PRINCIPAL FINDINGS

1. Production of the 5-LO metabolite 5-HETE occurs in vitro and mediates growth stimulation
Insulin-like growth factor 1 (IGF-1) stimulation of breast cancer cells induces the activity of the 5-LO enzyme as demonstrated by a specific RIA for 5-HETE. Four breast cancer cell lines were exposed to IGF-1 or transferrin, resulting in increased production of 5-HETE two- to fourfold above control levels in all of the lines. Exogenous addition of 5-HETE and cysteinyl leukotrienes enhanced tumor cell proliferation 20–45% above control in several breast cancer cell lines.

2. Biochemical inhibition of AA metabolism
The products of 5-LO activity can be blocked in a variety of ways using various selective biochemical inhibitors. AA metabolites generated by IGF-1 stimulation of the cell line MCF-7 were evaluated by HPLC with or without pre-exposure to the FLAP inhibitor MK 886 (Fig. 1 ). In control cells not exposed to the inhibitor (open bars), the metabolites derived from 5-LO activation were increased by 5- to 30-fold relative to control values after exposure to IGF-1. This pattern changed upon exposure to MK 886 relative to cells exposed to IGF-1 alone, as shown in Fig. 1 : downstream metabolites (including 5-HETE and its products) were reduced to below baseline levels (filled bars). In contrast, 15-LO products were elevated from 6- to 13-fold (15-HPETE), from 11- to 24-fold (15-HETE and its metabolites), and from 5- to 8-fold (lipoxin A4, a tri HETE) in a fashion consistent with endoperoxide shunting. Production of prostanoids was elevated from 21- to 34-fold by the inhibitors, which is also consistent with a diversion from the 5-LO metabolism to the other arachidonate pathways. The functional consequence of exposing breast cancer cells to 5-LO or 5-LO-activating protein (FLAP) -directed inhibitors was growth inhibition, but this was not seen at comparable drug doses with a cyclooxygenase (COX) inhibitor. The growth inhibition of the FLAP-directed inhibitor was reversible 12 h after drug exposure by incubating ZR-75 cells in 1.5 nM 5-HETE.

View larger version (23K): [in this window] [in a new window]   Figure 1. HPLC characterization of AA metabolism in MCF-7 cells exposed to 2 ng/ml IGF-1 (open bars) and IGF-1 with the addition of 5 µM MK 886 (filled bars). The various metabolites were separated by RT-HPLC and quantitated using an online radioactivity detector. The values shown are major measurable products released and are represented as fold increases over control. The data shown were obtained at 90 s, a period showing maximal production of many metabolites of interest. The data represent the average of at least 3 experiments and the SE of the values is shown as error bars. Statistical analysis was performed comparing fold change induced by IGF-1. The differences between cells exposed to MK 886 vs. control were statistically significant (P<0.05) for all of the metabolites shown except for 12-HETE. NS: not statistically significant.

3. Inhibitors of the 5-LO pathway induce apoptosis in vitro and in vivo
To test the hypothesis that an apoptotic pathway was involved in response to the LO-inhibitors, we examined breast cancer cell lines in vitro after exposure to 5-LO inhibitors. In contrast to the result with a COX inhibitor, which had no effect on growth, application of 5-LO or FLAP-directed inhibitors reduced growth, increased apoptosis, down regulated bcl-2, up regulated bax, and resulted in cell cycle arrest in G1. Using immunocytochemical analysis, we observed down-regulation of bcl-2 in MCF-7 cells treated with a FLAP inhibitor (MK 886) compared to untreated cells (Fig. 2A , B ). In addition, bax expression was increased by exposure of the breast cancer cells to MK 886 vs. untreated cells (Fig. 2C , D ). To confirm these observations, we examined the levels of bcl-2 and bax by Western blot analysis after treatment of the cell line MCF-7 with NDGA and a FLAP inhibitor. As shown in Fig. 2E , treatment of MCF-7 with the inhibitors resulted in a decrease of bcl-2 and a concomitant increase in bax immunoreactivity.

View larger version (100K): [in this window] [in a new window]   Figure 2. A–D) Immunocytochemical analysis of cell line MCF-7 with antibodies against Bcl-2 (A, B) and Bax (C, D). A, C) Untreated control cells; B, D) treated for 24 h with 2 µM MK 886. Cytospins were performed and the specimens were analyzed using a Zymed Histostain kit (A–D, x1600). Western blot analysis of the effect of LO inhibitors on levels of Bcl-2 and Bax protein (Fig. 2E ). MCF-7 cells were treated with either 4 µM NDGA or 2 µM MK 886 for the times indicated. Western blot analysis was performed using anti-Bcl-2 or anti-Bax antibodies using chemiluminescent detection.

In a pilot in vivo study, an increased number of apoptotic cells was observed in xenografts from nu/nu mice injected with MCF-7 tumor cells and treated with 5-LO inhibitors. This finding correlated with a reduction in tumor size.

4. PPAR induction and effect of PPAR ligands on tumor cell growth
In light of recent reports regarding the mechanistic basis of the antiproliferative effect of the FLAP inhibitor, we explored alternative mechanisms for growth effects on 5-LO inhibition. After exposure of the breast tumor cell line ZR-75 to 5 µM MK 886 or NDGA, mRNA was up-regulated at 1, 6, 12, 24, and 48 h for PPAR and PPAR. To further evaluate the possible involvement of PPAR activation in the growth regulation of breast cancer cell lines, we tested a range of selective PPAR agonists for their effect on breast cancer cell lines. This panel included ligands for PPAR (WY-14643, clofibrate, fenofibrate) and PPAR (LY 171883). When breast cancer cell lines T47D and ZR-75 were incubated with each of the four PPAR ligands, a dose-dependent growth reduction was observed with all the compounds for both cell lines compared with media-only control. To evaluate whether the PPAR up-regulation was contributing to the growth inhibitory effect of the 5-LO inhibitors, we exposed T47D cells for 12 h to MK 886. After washing the cells, we incubated them with the PPAR ligand LY171883. This sequential drug exposure was significantly more antiproliferative and apoptotic than were the parallel exposures to either FLAP inhibitor or PPAR ligand alone. These results suggest that the enhanced inhibitory effect of sequential drug exposure could be due to the interaction of PPAR induction in the face of increased production related to endoperoxide shunting of alternative eicosanoid products capable of activating PPAR.

CONCLUSIONS AND SIGNIFICANCE

Our results suggest that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. This may involve the interplay of loss of growth stimulation by 5-LO products and/or potential recruitment of the inhibitory effect of PPARs, especially PPAR. FLAP inhibitors and NDGA both mediated PPAR induction. Endoperoxide shunting after exposure to FLAP inhibitors resulted in the buildup of eiscosanoids known to activate at least PPAR.

In considering the emerging literature about the nature of fatty acid-based growth regulation, there is growing awareness of the inherent measure of promiscuity in the function of this class of molecules. The specificity of the enzymes acting on AA and related substrates is relative; interaction of the resultant AA metabolites with binding partners also is only relatively specific and downstream transcriptional regulation of relevant recognition elements is subject to considerable modulation, depending on the context. Dissecting the precise nature of these interactions will continue to be challenging, as all analytical approaches will be subject to this same inherent biological promiscuity. This situation mirrors the recent discussion as to the complexity of counterbalancing forces affecting angiogenesis when considered from a heterotypic perspective, where the biology is complicated by the dynamic interactions of mixed cell populations. We recently provided experimental evidence suggesting that the dominant effect of COX inhibition in another cancer system could be mediated through the heterotypic effect of inflammatory cell products, such as interleukin 6 mediating a promotional effect on neighboring cancer cell populations. For breast cancer, the possibility of interactions of AA metabolites generated from inflammatory cells, epithelial cells, and stromal cells are highly likely, as these cells all have enzymatic machinery to metabolize AA. Elucidating the effects of FLAP inhibitors on breast cancer cells has also added dimension to the biology of PPAR regulation. This PPAR family of molecules has been described as being much more promiscuous in terms of their binding interactions with ligands than the previously characterized members of nuclear receptor families. Investigators from Glaxo have proposed that this situation may relate to the physical characteristics of the ligand binding domain. However, the transcriptional regulation of AA metabolism is further complicated by the large number of proteins that interact as binding partners with relevant response element ligands. Given this complexity in delineating the consequences of blocking aspects of AA metabolism, a major concern is whether it is possible to exploit this biology for therapeutic application.

Our major interest in studying the contribution of 5-LO activities to breast cancer growth was to develop a sound basis for translational research in controlling the progression of early breast cancer, so we have a pragmatic perspective. Fundamentally, the problem with clinical cancer is the loss of apoptotic regulation; therefore, the focus on whether a particular antagonists of the 5-LO pathway consistently induces apoptosis is of central importance. Our data suggest that targeting 5-LO, especially with the FLAP inhibitors, does mediate a predictable apoptotic effect. This interaction potentially involves loss of 5-HETE and related growth signals as well as both up-regulation of PPAR and activation due to binding with products formed by alternative AA metabolite utilization. This discussion relative to breast cancer biology may be particularly important in light of the broader epidemiological concern about the etiology of breast cancer relative to the contribution of dysregulation of fatty acid biochemistry. For this reason, despite the daunting complexity, targeting AA metabolism may represent a successful intervention strategy for breast cancer; this area merits further research.

View larger version (121K): [in this window] [in a new window]   Figure 3. Schema of effects of AA metabolites on breast cancer growth. We propose a snapshot model of the dynamic interactions with AA metabolites that may influence growth regulation in breast cancer cell lines. For an epithelial cell, the functional extremes from cell growth resulting in clonal expansion to differentiation that results in apoptotic cell death are a result of layers of complex, highly modulated interactions, including the action of various AA metabolizing enzymes on substrates, binding interactions of AA metabolites with receptors forming transcription factors, and assembly of transcription factors with relevant binding partners at various locations within the response elements of the genes regulating cell growth or apoptosis. The PPARs are shown together as growth regulators for simplicity; however, our understanding of their functions is incomplete. Emerging data suggest that more complex interactions (even divergent effects) can occur in distinct settings.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0866fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.00-0866fje

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