Platelet-type 12-lipoxygenase activates NF-κB in prostate cancer cells
Introduction
Oxidative metabolism of arachidonic acid by lipoxygenases, especially 12-lipoxygenase (12-LOX), plays an important role in cancer pathophysiology [1], [2], [3]. While 12-LOX exists in three isoforms, viz. platelet-type, leukocyte-type, and epithelial-type [4]; it is the platelet-type enzyme that is widely expressed in tumor cells and its level of expression was correlated with the tumor stage and grade of human prostate carcinoma [5], [6]. 12(S)-Hydroxy eicosatetraenoic acid (12(S)-HETE), the sole and stable end product of arachidonic acid metabolism by the platelet-type 12-LOX, has been shown to protect tumor cells from apoptosis and induce invasion, motility, and angiogenesis [3], [7], [8], [9]. Promotion of such divergent biological functions by 12(S)-HETE is indicative of a complex signaling mechanism leading to metastasis and survival of tumor cells. Attempts at understanding the 12(S)-HETE signaling mechanisms revealed the involvement of PLC/PKC pathway as well as ERK cascade and Src family kinases [10], [11]. The natural convergence point for this vast array of mitogenic signaling mechanisms is the extremely variable transcriptional machinery. One possible integrator of these 12(S)-HETE signaling mechanisms in tumor cells is the pleiotropic transcription factors NF-κB, which play an important role in the control of cell proliferation and apoptosis.
NF-κB is a family of five DNA binding proteins (viz. c-Rel, RelB, p65/RelA, p50/p105, p52/p100) that regulate the expression of a variety of genes involved in host immune responses and inflammation [12]. This family of transcription factors is induced in response to a wide range of signals that lead to cell proliferation, differentiation, regulation of apoptosis, and neoplastic transformation [13], [14], [15]. These transcription factors bind to DNA target sites as homo- or heterodimers and the prototypical NF-κB is a heterodimer of p65 and p50 proteins [16], [17], [18]. In the cytosol these dimeric transcription factors exist in an inactive state where they are bound to any one of several inhibitor proteins collectively known as IκB [19]. The vertebrate IκB family consists of IκBα, IκBβ, IκBε, IκBζ, p105, p100, and Bcl-3 [20], [21]. Activation of NF-κB involves the degradation of IκB by the ubiquitin–proteasome pathway following signal-induced phosphorylation [22], [23]. While normally involved in immune responses and inflammation, a disregulated NF-κB activation can cause undue cell proliferation and inhibition of apoptosis [24], [25]. Indeed, constitutive activation of NF-κB was observed in several different tumors and inhibition of such activation using mutated (degradation resistant) IκBα promoted apoptosis in prostate cancer cells upon TNFα treatment [26]. Given the established role of the platelet-type 12-LOX, hence 12(S)-HETE, to inhibit apoptosis and promote mitogenesis in cancer cells, it is highly probable that NF-κB is involved in the downstream signaling cascade of 12(S)-HETE.
We report here a direct relationship between platelet-type 12-LOX overexpression and NF-κB activation in prostate cancer cells. In addition 12(S)-HETE was shown to induce proteasomal degradation of IκBα, nuclear translocation of NF-κB, and transcriptional activation. Evidence is also presented to directly link the enzymatic activity of platelet-type 12-LOX to NF-κB activation and increased cell viability.
Section snippets
Cell cultures and reagents
PC-3 and Du145 cells were obtained from ATCC and were grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 °C in a 5% CO2 atmosphere. 12-LOX-transfected (nL-8 and nL-12) and mock-transfected (neo) PC-3 cells have previously been developed and characterized in our laboratory [27]. 13(S)-HODE, 12(S)-HETE, 5(S)-HETE, 15(S)-HETE, and arachidonic acid were obtained from Cayman Chemical Company, Ann Arbor, MI. MG-132 was obtained from Biomol Research
Results
Wherever 12-LOX overexpressing cells were used in experiments, both nL-8 and nL-12 clones were used. Since the results obtained were similar for the two clones, only data from either one of the clones was reported.
Discussion
Apart from cyclooxygenase-2, platelet-type 12-LOX is the most commonly expressed arachidonic acid metabolizing enzyme present in a variety of human cancer cells [2], [4], [7] Enhanced expression of platelet-type 12-LOX was observed in many cancers [4] and, in a clinical study, its expression was positively correlated with stage and grade of tumors from prostate cancer patients [5]. 12-LOX-driven metabolism of arachidonic acid produces 12(S)-HETE and this lipid molecule is known for promoting
Acknowledgements
This work was supported by National Institutes of Health Grant CA 29997 and Department of Defense Grant DAMD17-98-1-8502 (both to K.V.H.).
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