Wolfgang Hagmann

12-lipoxygenases and 12(S)-HETE: role in cancer metastasis.

Arachidonic acid metabolites have been implicated in multiple steps of carcinogenesis. Their role in tumor cell metastasis, the ultimate challenge for the treatment of cancer patients, are however not well-documented. Arachidonic acid is primarily metabolized through three pathways, i.e., cyclooxygenase, lipoxygenase, and P450-dependent monooxygenase. In this review we focus our attention on one specific lipoxygenase, i.e., 12-lipoxygenase, and its potential role in modulating the metastatic process. In mammalian cells there exist three types of 12-lipoxygenases which differ in tissue distribution, preferential substrates, and profile of their metabolites. Most of these 12-lipoxygenases have been cloned and sequenced, and the molecular and biochemical determinants responsible for catalysis of specific substrates characterized. Solid tumor cells express 12-lipoxygenase mRNA, possess 12-lipoxygenase protein, and biosynthesize 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid], as revealed by numerous experimental approaches. The ability of tumor cells to generate 12(S)-HETE is positively correlated to their metastatic potential. A large collection of experimental data suggest that 12(S)-HETE is a crucial intracellular signaling molecule that activates protein kinase C and mediates the biological functions of many growth factors and cytokines such as bFGF, PDGF, EGF, and AMF. 12(S)-HETE plays a pivotal role in multiple steps of the metastatic ‘cascade’ encompassing tumor cell-vasculature interactions, tumor cell motility, proteolysis, invasion, and angiogenesis. The fact that 12-lipoxygenase is expressed in a wide diversity of tumor cell lines and 12(S)-HETE is a key modulatory molecule in metastasis provides the rationale for targeting these molecules in anti-cancer and anti-metastasis therapeutic protocols.

Activity and protein distribution of 12-lipoxygenase in HEL cells: Induction of membrane-association by phorbol ester TPA, modulation of activity by glutathione and 13-HPODE, and Ca2+-dependent translocation to membranes

The understanding of the intracellular regulation of 12-lipoxygenase requires a knowledge of the distribution of both enzyme protein and its activity. In human erythroleukemia cells, the membrane fraction contains about 90% of the total cellular 12-lipoxygenase activity, whereas only approximately 10% of 12-lipoxygenase activity resides in the cytosol. However, the majority of the cellular 12-lipoxygenase protein is found in the cytosol. Pretreatment of cells for 0-3 days with 160 nM TPA caused a marked, time-dependent increase in membrane-bound 12-lipoxygenase activity and protein, respectively. In contrast, the cytosolic amount of 12-lipoxygenase protein and activity, respectively, were minimally altered by this TPA treatment. Recombining the active membrane fraction with cytosol resulted in no significant inhibition of its 12-lipoxygenase activity, but the addition of GSH to the membrane fraction inhibited 12-lipoxygenase activity in a dose-dependent manner. On the other hand, the cytosolic enzyme can be rendered active in the presence of 1 microM 13-hydroperoxyoctadecadienoic acid. In HEL cell homogenates, a partial translocation of the cytosolic enzyme to the membrane takes place in a Ca(2+)-dependent manner, resulting in an increase in membrane-associated 12-lipoxygenase activity and a concomitant decrease in cytosolic 12-lipoxygenase activity above 0.1 microM Ca2+.

12-Lipoxygenase in Lewis lung carcinoma cells: Molecular identity, intracellular distribution of activity and protein, and Ca2+-dependent translocation from cytosol to membranes

Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37 degrees C. However at 24 degrees C and 10 degrees C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0-100 microM free Ca2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca(2+)-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca(2+)-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca(2+)-dependent manner, but is no longer active or becomes inactivated at the membrane site.

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 microM. After 24-hr incubation with 10 microM 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.

Eicosanoids, cancer metastasis, and gene regulation: an overview.

Eicosanoids are a group of oxygenated arachidonic acid (AA) metabolites including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), lipoxins (LXs), and various hydroperoxy and hydroxy fatty acids (1–3). AA is released from phospholipids mainly by the action of phospholipase A2 and is the substrate for lipoxygenases (LOXs), cyclooxygenase (COX) and cytochrome P-450 monooxygenase (also called epoxygenase). AA is metabolized to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-lipoxygenase (5-LOX), to 12-HPETE by 12-lipoxygenase (12-LOX; EC 1.13.11.31) and to 15-HPETE by 15-lipoxygenase (15-LOX). Then, 5-HPETE can be converted to 5-hydroxyeicosatetraenoic acid (5-HETE) and LTs, 12-HPETE to 12-HETE and hepoxilins (HXs), 15-HPETE to 15-HETE and LXs. AA can also be metabolized to PGs, prostacyclin and TXs by COX, or to epoxides and diols by cytochrome P-450. Both the LOX and COX pathways have been implicated in several aspects of cancer from carcinogenesis to metastasis (4–11).

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.

Intracellular distribution, activity, and Ca(2+)-dependent translocation of 12-lipoxygenase in Lewis lung tumor cells.

The 12-lipoxygenase enzyme (12-LOX) has been found in platelets (1) and several other cell types including leukocytes, epidermal and epithelial cells (2–4), as well as in human (5,6) and rodent tumor cells (7,8). In tumor cells, the major metabolite, 12(S)-HETE, regulates their metastatic potential (9–11) through a protein kinase C-dependent mechanism (7). The intracellular distribution of 12-LOX activity varies between different cells ranging from a predominantly cytosolic (2,4,12) to an exclusive or preferred localization in membranes (5,13,14). This distribution of 12-LOX activity can change upon stimulation of cells suggesting a translocation of the enzyme (15). However, these earlier studies on 12-LOX activity did not investigate in parallel the respective subcellular distribution of the 12-LOX protein itself. In order to obtain a more complete concept of the intracellular regulation and distribution of 12-LOX, we therefore analyzed the distribution of both 12-LOX activity and of 12-LOX protein in murine Lewis lung carcinoma cells (3LL cells) and compared their 12-LOX activity with homologous 12-LOX activity in platelets. Further we asked whether activation of the 12-LOX enzyme in 3LL cells encompasses a Ca2+-dependent translocation to membranes as is the case for 12-LOX in platelets or other enzymes of arachidonate metabolism such as phospholipase A2 and 5-LOX (16,17).