5-LOX mRNA Research Articles

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

Purpose. Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. Methods. Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml −1) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 μm C 18 column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. Results. Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72 hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. Conclusion. These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.

5-Lipoxygenase and Leukotriene B 4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue

The 5-lipoxygenase (5-LOX) pathway is critical for pancreatic cancer cell growth and escape from apoptosis. Inhibition of 5-LOX blocks proliferation and induces apoptosis in human pancreatic cancer cells. However, the expression of 5-LOX and its downstream signaling pathway have not been investigated in human pancreatic adenocarcinoma. Reverse transcriptase-polymerase chain reaction revealed expression of 5-LOX mRNA in all pancreatic cancer cell lines tested including, PANC-1, AsPC-1, and MiaPaCa2 cells, but not in normal pancreatic ductal cells. The expression of 5-LOX protein in pancreatic cancer cell lines was demonstrated by Western blotting. Finally, 5-LOX up-regulation in human pancreatic cancer tissues was verified by intense positive staining in cancer cells by immunohistochemistry. Staining for the 5-LOX protein was particularly evident in the ductal components of the more differentiated tumors but not in ductal cells in normal pancreatic tissues from cadaver donors. Immunohistochemistry also revealed strong staining of cancer tissues with an antibody to the receptor of the downstream 5-LOX metabolite, leukotriene B(4). The current study demonstrated marked expression of 5-LOX and the leukotriene B(4) receptor in human pancreatic cancer tissues. These findings provide further evidence of up-regulation of this pathway in pancreatic cancer and that LOX inhibitors are likely to be valuable in the treatment of this dreadful disease.

Fatty acid oxidation and signaling in apoptosis.

It is well established that fatty acid metabolites of cyclooxygenase, lipoxygenase (LOX), and cytochrome P450 are implicated in essential aspects of cellular signaling including the induction of programmed cell death. Here we review the roles of enzymatic and non-enzymatic products of polyunsaturated fatty acids in controlling cell growth and apoptosis. Also, the spontaneous oxidation of polyunsaturated fatty acids yields reactive aldehydes and other products of lipid peroxidation that are potentially toxic to cells and may also signal apoptosis. Significant conflicting data in terms of the role of LOX enzymes are highlighted, prompting a re-evaluation of the relationship between LOX and prostate cancer cell survival. We include new data showing that LNCaP, PC3, and Du145 cells express much lower levels of 5-LOX mRNA and protein compared with normal prostate epithelial cells (NHP2) and primary prostate carcinoma cells (TP1). Although the 5-LOX activating protein inhibitor MK886 killed these cells, another 5-LOX inhibitor AA861 hardly showed any effect. These observations suggest that 5-LOX is unlikely to be a prostate cancer cell survival factor, implying that the mechanisms by which LOX inhibitors induce apoptosis are more complex than expected. This review also suggests several mechanisms involving peroxisome proliferator activated receptor activation, BCL proteins, thiol regulation, and mitochondrial and kinase signaling by which cell death may be produced in response to changes in non-esterified and non-protein bound fatty acid levels. Overall, this review provides a context within which the effects of fatty acids and fatty acid oxidation products on signal transduction pathways, particularly those involved in apoptosis, can be considered in terms of their overall importance relative to the much better studied protein or peptide signaling factors.

12-Lipoxygenase inhibition induced apoptosis in human gastric cancer cells.

Arachidonic acid release from membrane phospholipids is essential for tumour cell proliferation. Lipoxygenases constitute a pathway for arachidonate metabolism. The present study investigated the expression of 12-lipoxygenase and its effect on cell proliferation as well as survival in two human gastric cancer cell lines (AGS and MKN-28). RT-PCR and western blots, respectively, showed 12-LOX mRNA and protein expression in both AGS and MKN-28 cell lines. Treatment with a 12-LOX inhibitor, baicalein, significantly inhibited cancer cell proliferation, but a metabolite of 12-LOX activity, 12 hydroxyeicosatetraenoic acid (12-HETE) reversed baicalein-induced growth inhibition. Furthermore, the blockade of the 12-LOX pathway through a 12-LOX inhibitor and antisense induced apoptosis of gastric cancer cell lines. The biochemical characteristics of apoptosis were p53-independent combined with a decrease in bcl-2 expression. Caspase-7 was proteolytically activated and responsible for the apoptosis execution.

Glucocorticoid receptors are required for up-regulation of neuronal 5-lipoxygenase (5LOX) expression by dexamethasone.

SPECIFIC AIM>5-lipoxygenase (5LOX) is the key enzyme in the synthesis of inflammatory leukotrienes (LTs) from arachidonic acid. We had previously found that hyperglucocorticoidemia or dexamethasone treatment increase 5LOX expression in the rat brain in vivo. The aim of this study was to characterize in detail the stimulatory action of dexamethasone on 5LOX expression in neural cultures in vitro.PRINCIPAL FINDINGS1. Dexamethasone increases 5LOX mRNA and protein content in primary cultures of rat cerebellar granule neurons (CGN) in a concentration- and time-dependent mannerExposure of CGN to dexamethasone increased the content of both 5LOX mRNA (assayed by quantitative RT-PCR) and 5LOX protein (Western blotting). These increases were concentration dependent and became statistically significant with 100 nM dexamethasone. Time course studies revealed that dexamethasone treatment increased the content of 5LOX mRNA and protein as early as 3 h and that these increases persisted for at least 24 h of dexamethasone...

Suppression of Platelet-Type 12-Lipoxygenase Activity in Human Erythroleukemia Cells by an RNA-Cleaving DNAzyme

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A “10–23” deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37°C, showing the kcat of 1 and 0.26 min−1, respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.

Lipoxygenase gene expression in baboon intrauterine tissues in late pregnancy and parturition.

Myometrium (upper and lower segment), cervix, chorion and decidua were obtained at the time of Caesarean hysterectomy in 15 baboons, 10 at varying gestational ages prior to the onset of labour in the last trimester of pregnancy, and five in spontaneous labour at term. Expression of currently recognized human lipoxygenase (LOX) genes was determined using Northern blot analysis relative to each of three housekeeping genes. Signals of similar size to human 5-LOX and human platelet 12-LOX genes were detected in all tissues. Expression of 5-LOX mRNA in the cervix decreased with advancing gestational age. In decidua, expression of 5-LOX mRNA was higher in tissues from animals in labour (compared to those not in labour), whereas in chorion, its expression was lower in tissues from animals in labour. Expression of the platelet 12-LOX gene decreased in chorion with advancing gestational age, and in cervix was lower in tissues from animals in labour. We postulate that the variation in expression of LOX genes may play a role in the onset or promotion of parturition in the baboon.

Neurogenesis and neuroprotection in the adult brain. A putative role for 5-lipoxygenase?

5-Lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) are two enzymes that are critical for the synthesis of eicosanoids, the inflammatory metabolites of arachidonic acid. Both 5-LOX and COX-2 are expressed in the brain, including in CNS neurons. The physiologic role of these proteins in neuronal functioning is not clear. In non-neuronal tissues these two enzymes often assume similar roles: in addition to their function in inflammation, 5-LOX and COX-2 appear to be associated with cell proliferation, that is, with tumor growth. High 5-LOX expression has been noticed in the proliferating brain or pancreatic tumor cells; reduction in tumor cell proliferation and/or destruction of tumor cells was achieved with 5-LOX inhibitors. Proliferation of immature neurons/neuroblasts is an important component of mitotic neurogenesis. We investigated the role of 5-LOX in proliferation using cultures of human neuronal precursor cells, NT2. We found that these cells express 5-LOX mRNA and we used 3H-thymidine incorporation as a measure of cell proliferation; this was reduced by treating the cultures with 5-LOX inhibitor AA-861. We propose that the 5-LOX pathway plays a crucial role in mitotic neurogenesis. Additional studies should explore whether 5-LOX may participate in neurogenesis related pathologies and whether it should be considered a target for procedures aimed at altering neurogenesis for therapeutic purposes.

5-Lipoxygenase and cyclooxygenase mRNA expression in rat hippocampus:early response to glutamate receptor activation by kainate.

Recent research has identified in central nervous system neurons the expression of two enzymes from the inflammatory pathway of the metabolism of arachidonic acid, the 5-lipoxygenase (5LOX) and the cyclooxygenase-2 (COX2). Expression of both enzymes appears to be upregulated during aging; upregulated 5LOX/COX2 expression in neurons may be responsible for the increased neuronal vulnerability to degeneration. Involvement of the excitatory neurotransmitter glutamate in aging-associated neurodegeneration has also been suggested. Stimulation of glutamate receptors by kainic acid (kainate) has been shown independently to affect the brain expression of 5LOX or COX2. Using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to measure the contents of mRNAs we found 3h after kainate injection (intraperitoneally; 10 mg/kg) increased mRNA levels of 5LOX and COX2, but not that of COX1 in the hippocampus of rats. Pretreatment with the COX2 inhibitor NS-398 (9 mg/kg, 1h prior to kainate) inhibited the kainate-stimulated increase of 5LOX and COX2 mRNA levels. Our results indicate that hippocampal expression of both 5LOX and COX2 increases rather promptly when glutamate receptors are stimulated by kainate. The mechanism of how NS-398 inhibits this action of kainate should be further investigated.

Inflammatory 5-LOX mRNA and protein are increased in brain of aging rats

5-Lipoxygenase (5-LOX) is the key enzyme in the synthesis of leukotrienes, inflammatory mediators of arachidonic acid. 5-LOX is also expressed in neurons (in particular in the hippocampus and the cerebellum), and it seems to be capable of promoting neurodegeneration. Recently, we observed greater 5-LOX mRNA content in the hippocampus of older (24 months) than younger (2 months) rats. In this study, we measured in the hippocampus and the cerebellum of younger and older male F344 rats the contents of: 5-LOX mRNA, FLAP (5-LOX activating protein) mRNA, and 5-LOX protein. By using a quantitative reverse transcription/polymerase chain reaction (PCR) (RT-PCR) with internal standards we found that 5-LOX but not FLAP mRNA content is greater (both in hippocampus and cerebellum) of older than younger rats. By using quantitative Western immunoblotting, we found a greater content of 5-LOX protein in the hippocampus and the cerebellum of older rats; we also established that the membrane/cytosol 5-LOX content ratio is larger in the brains of older than younger rats (statistically significant in the cerebellum). The latter can be considered an indication of 5-LOX translocation/activation during aging. Together these results suggest that aging increases both neuronal 5-LOX expression and protein translocation, and indicate that the 5-LOX system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

Ribozyme-mediated suppression of platelet type 12 lipoxygenase in human erythroleukemia cells.

The platelet type 12 lipoxygenase (12-LOX) adds molecular oxygen to C-12 arachidonic acid to yield 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. It has been suggested that 12-LOX and its metabolites play an important role in tumor angiogenesis. A hammerhead ribozyme (Rz) targeted to the first GUC site within the 12-LOX mRNA was designed and cloned into an in vitro transcriptional or mammalian expression vector. In vitro, the Rz was able to cleave its substrate efficiently in a time-dependent manner. Under multiple turnover conditions, the Rz performed well at 37 degrees C, with a further improvement at 50 degrees C. When cloned into a mammalian expression vector, pSV2neo, the Rz construct efficiently decreased the level of 12-LOX mRNA in stably transfected human erythroleukemia cells to levels that were undetectable by Northern blot analyses. 12-LOX enzyme activity assays showed that Rz significantly reduced the 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid production in human erythroleukemia cells; this effect was sustained for up to 6 months in cell culture. The Rz developed in this study may represent a powerful tool for potential applications, ranging from an understanding of tumor angiogenesis to cancer gene therapy.

447. In-vivo and in-vitro action of glucocorticoids on neuronal 5-lipoxygenase

5-lipoxygenase (5-LOX), the enzyme responsible for the synthesis of inflammatory leukotrienes, is expressed in the brain, including in neurons. The presence of hormone response elements for several hormones, among them, melatonin, glucocorticoids, estrogens, and thyroid hormone, in the promoter of the 5-LOX gene suggests possible regulation of this gene by the above-mentioned hormones. Hormonal changes that occur during aging might participate in the aging-associated up-regulation of 5-LOX mRNA and protein in the brain, which we observed in our previous work. In human monocytes, 5-LOX mRNA content increases on exposure to the synthetic glucocorticoid dexamethasone, which prompted us to hypothesize that glucocorticoids (which are up-regulated during aging) might increase 5-LOX expression in the brain as well. After treatment of rats for 10 days either with corticosterone (implanted subcutaneously) or with dexamethasone (injected daily) we found increased levels of 5-LOX mRNA and protein in hippocampus and cerebellum. To understand better whether the stimulatory effect of glucocorticoids on 5-LOX mRNA/protein requires the involvement of glucocorticoid receptors, we studied 5-LOX expression in primary cultures of rat cerebellar granule cells. The cultures were grown in serum-free chemically defined medium (B-27) and treated for 24 hours either with corticosterone or with dexamethasone in the presence or absence of the glucocorticoid receptor antagonist mifepristone (RU486). Corticosterone and dexamethasone both increased 5-LOX mRNA and protein levels; RU486 decreased the dexamethasonestimulated 5-LOX expression, which suggests that the effect of glucocorticoids is achieved via a receptor-mediated mechanism. Thus, increased glucocorticoid levels, which occur during aging and/or in stress, may up-regulate the neuronal 5-LOX pathway and thereby contribute to the development of neurodegenerative pathologies.

446. Effect of aging on 5-lipoxygenase (5-LOX) mRNA and protein in the rat brain

446. Effect of aging on 5-lipoxygenase (5-LOX) mRNA and protein in the rat brain

Melatonin: an endogenous negative modulator of 12-lipoxygenation in the rat pineal gland

Major biochemical activities of the pineal gland include melatonin biosynthesis and 12-lipoxygenation. In this paper, we provide evidence in vivo that melatonin regulates 12-lipoxygenation via 12-lipoxygenase (LOX) expression. The relationship between these two biochemical activities was established by monitoring levels of endogenous melatonin and a 12-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), in the rat pineal gland both during the light-dark cycle and after isoproterenol injection using GC/MS with negative ion chemical ionization. As pineal melatonin production reflected a typical diurnal variation, 12-HETE levels showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, decreased the 12-HETE level significantly. The reduction of 12-HETE levels during the dark phase and after isoproterenol injection was accompanied by decreases in 12-LOX mRNA and protein levels. Direct administration of melatonin to rats by intravenous injection decreased pineal 12-LOX protein levels significantly, indicating that melatonin plays a role in down-regulating 12-LOX expression. When pineal glands were incubated with exogenous melatonin in culture, time-dependent reduction of 12-LOX protein levels was observed. The melatonin-induced reduction in 12-LOX protein was abolished in the presence of the melatonin receptor antagonist luzindole, establishing further the role of melatonin in this process. Incubation of pineal homogenates with exogenous melatonin partially inhibited 12-LOX activity. Taken together, an inverse relationship exists in the endogenous production of 12-HETE, 12-LOX mRNA and protein with respect to melatonin production in the rat pineal gland. Melatonin decreased both 12-LOX mRNA and protein levels in addition to 12-LOX enzyme activity, indicating that melatonin is an endogenous modulator of pineal 12-lipoxygenation.

Melatonin: an endogenous negative modulator of 12-lipoxygenation in the rat pineal gland

Major biochemical activities of the pineal gland include melatonin biosynthesis and 12-lipoxygenation. In this paper, we provide evidence in vivo that melatonin regulates 12-lipoxygenation via 12-lipoxygenase (LOX) expression. The relationship between these two biochemical activities was established by monitoring levels of endogenous melatonin and a 12-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), in the rat pineal gland both during the light-dark cycle and after isoproterenol injection using GC/MS with negative ion chemical ionization. As pineal melatonin production reflected a typical diurnal variation, 12-HETE levels showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, decreased the 12-HETE level significantly. The reduction of 12-HETE levels during the dark phase and after isoproterenol injection was accompanied by decreases in 12-LOX mRNA and protein levels. Direct administration of melatonin to rats by intravenous injection decreased pineal 12-LOX protein levels significantly, indicating that melatonin plays a role in down-regulating 12-LOX expression. When pineal glands were incubated with exogenous melatonin in culture, time-dependent reduction of 12-LOX protein levels was observed. The melatonin-induced reduction in 12-LOX protein was abolished in the presence of the melatonin receptor antagonist luzindole, establishing further the role of melatonin in this process. Incubation of pineal homogenates with exogenous melatonin partially inhibited 12-LOX activity. Taken together, an inverse relationship exists in the endogenous production of 12-HETE, 12-LOX mRNA and protein with respect to melatonin production in the rat pineal gland. Melatonin decreased both 12-LOX mRNA and protein levels in addition to 12-LOX enzyme activity, indicating that melatonin is an endogenous modulator of pineal 12-lipoxygenation.

Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

Lipoxygenase Inhibitors Abolish Proliferation of Human Pancreatic Cancer Cells

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by 3H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.

Expression of arachidonate platelet-type 12-lipoxygenase in human rheumatoid arthritis type B synoviocytes

In the present study, we have demonstrated platelet-type 12-lipoxygenase (12-LOX) expression in human rheumatoid arthritis (RA) type B synoviocytes by reverse-transcription polymerase chain reaction (RT-PCR). The presence of 12-LOX mRNA in these cells was revealed by classical RT-PCR analysis using platelet-type 12-LOX cDNA primers and the PCR fragment (246 bp) was purified, amplified and sequenced. By sequence analysis, this fragment was determined to be 100% identical to that from platelet-type 12-LOX cDNA. Immunofluorescence data demonstrate that interleukin-1β (IL-1β) increases cellular 12-LOX protein. Other results associate specific inflammatory cytokines with the activity of 12-LOX in human RA type B synoviocytes. IL-1β increased 12 S-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) production (4-fold) and we also observed an increase in 12-HETE production (2.5-fold) after incubation of human RA type B synoviocytes with TNFα. In contrast to the action of IL-1β on 12-HETE synthesis, IL-4 and IL-6 did not enhance 12-HETE production. This is the first demonstration of platelet-type 12-LOX cDNA derived from the mRNA of cultured human RA type B synoviocytes.

Requirement for epidermal growth factor receptor tyrosine kinase and for 12-lipoxygenase activity in the expression of 12-lipoxygenase in human epidermoid carcinoma cells

We studied the dependency of basal 12-lipoxygenase (12-LOX; arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) expression and activity on functional protein tyrosine kinase of the epidermal growth factor receptor (EGF-R) and on 12-LOX activity in human A431 epidermoid carcinoma cells. Treatment of cells with inhibitors of high specificity for EGF-R tyrosine kinase, namely PD 153035 and 4,5-dianilinophthalimide (DAPH1), decreased cellular 12-LOX at mRNA, protein, and activity levels in a time- and dose-dependent manner, with PD 153035 being effective at concentrations below 1 μM. After 24-hr incubation with 10 μM PD 153035 or DAPH1, 12-LOX activity dropped to 14% (39%), and 12-LOX protein to 25% (24%) of control level. Inhibition of 12-LOX activity by the compound N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP) also resulted in a substantial decrease in 12-LOX protein expression. 12-LOX mRNA levels were diminished or undetectable by reverse transcription-polymerase chain reaction after cell treatment with these inhibitors. Our results suggest that basal 12-LOX expression in A431 tumor cells largely depends on functional EGF-R tyrosine kinase, and that 12-LOX activity is required in the EGF-elicited intracellular signaling maintaining the expression of 12-LOX.

EGF-receptor tyrosine kinase and 12-lipoxygenase activity regulate expression of 12-lipoxygenase in human tumor cells.

Various human tumor cells including the epidermoid carcinoma A431 cells express the platelet-type isoform of 12-lipoxygenase (12-LOX)1–3. Tumor cell 12-LOX and the arachidonate metabolite generated via 12-LOX activity, 12(S)-HETE, have been shown to contribute to the metastatic potential of tumor cells in vivo and in vitro 4. Most recently, elevated expression of 12-LOX mRNA in prostate epithelial cells was demonstrated to correlate with poor prognosis in human prostate cancer5.KeywordsEpidermal Growth FactorA431 CellA431 Tumor CellEpidermoid Carcinoma A431 CellArachidonate MetaboliteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.