5-hydroxy-6-8-11-14-eicosatetraenoic-acid and zileuton

Recent studies highlight the regulatory role of arachidonate lipoxygenase5 (Alox5) and its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in cancer tumorigenesis and progression. In this study, we analyzed the expression, biological function and the downstream signaling of Alox5 in gastric cancer.. Alox5 protein levels were measured using IHC and ELISA. Growth, migration and survival assays were performed. Phosphorylation of molecules involved in growth and survival signaling were analyzed by WB. Analysis of variance and t-test were used for statistic analysis.. Alox5 and 5-HETE levels were upregulated in gastric cancer patients. ALOX5 overexpression or 5-HETE addition activates gastric cancer cells and reduces chemotherapy's efficacy. In contrast, ALOX5 inhibition via genetic and pharmacological approaches suppresses gastric cancer cells and enhances chemotherapy's efficacy. In addition, Alox5 inhibition led to suppression of ERK-mediated signaling pathways whereas ALOX5-5-HETE activates ERK-mediated signaling in gastric cancer cells.. Our work demonstrates the critical role of ALOX5-5-HETE in gastric cancer and provides pre-clinical evidence to initialize clinical trial using zileuton in combination with chemotherapy for treating gastric cancer.

Topics: Analysis of Variance; Antineoplastic Agents; Arachidonate 5-Lipoxygenase; Cell Proliferation; Cell Survival; Disease Progression; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Lipoxygenase Inhibitors; MAP Kinase Signaling System; Phosphorylation; Stomach Neoplasms; Up-Regulation

5-lipoxygenase (5-LOX), a member of the lipoxygenase gene family, is a key enzyme assisting in the conversion of arachidonic acid to 5-HETE and leukotrienes. Tumor-associated macrophages (TAMs) have a critical role in the progression and metastasis of many tumors, including ovarian tumors. Moreover, TAMs are often found in a high density in the hypoxic areas of tumors. However, the relevant mechanisms have not been studied explicitly until now. In this study, we found that the expression of 5-LOX strongly correlated with the density of TAMs in hypoxic areas of human ovarian tumor tissues. In cultured ovarian cancer cells, 5-LOX metabolites were increased under hypoxic conditons. Increased 5-LOX metabolites from hypoxic ovarian cancer cells promoted migration and invasion of macrophages, which was further demonstrated to be mediated by the upregulation of matrix metalloproteinase (MMP)-7 expression through the p38 pathway. Besides, we also showed that 5-LOX metabolites enhanced the release of tumor necrosis factor (TNF-α) and heparin-binding epidermal growth factor-like growth factor through upregulation of MMP-7. Furthermore, in animal models, Zileuton (a selective and specific 5-LOX inhibitor) reduced the MMP-7 expression and the number of macrophages infiltrating in the xenograft. Our findings suggest for the first time that increased metabolites of 5-LOX from hypoxic ovarian cancer cells promote TAM infiltration. These results of this study have immediate translational implications for the therapeutic exploitation of TAMs.

Topics: Animals; Arachidonate 5-Lipoxygenase; Cell Line; Cell Line, Tumor; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Heparin-binding EGF-like Growth Factor; Heterografts; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Leukotriene B4; Macrophages; MAP Kinase Signaling System; Matrix Metalloproteinase 7; Mice; Neoplasm Metastasis; Ovarian Neoplasms; Tumor Necrosis Factor-alpha

To investigate the expression of 5-lipoxygenase (5-LOX) in metastatic prostate cancer and whether zileuton, the inhibitor of 5-LOX, plays a role in the metastasis of prostate cancer.. An enzyme-linked immunosorbent assay was used to measure 5-hydroxyeicosatetraenoic acid (5-HETE) in patient and TRAMP mice blood samples. Kaplan-Meier analysis and the log-rank test were used to analyze the survival of the mice. Immunofluorescence and immunohistochemistry were used to assay the expression of 5-LOX in the samples. After treatment with 10 μM zileuton, cell motility and the invasion of PC-3 cells were assayed using immunofluorescence, Western blotting, and transwell. TRAMP mice were treated with zileuton (600 mg/kg and 1200 mg/kg) at 24 weeks of age. Ten weeks later, the mice were killed, and the tumors (size and number) were measured.. The levels of 5-HETE were significantly greater in the TRAMP mice with metastasis than in the tumors in situ. However, no such difference was found in the human samples. The lifespan of the mice was shorter at high levels of 5-HETE (>2.4 ng/mL). The expression of 5-LOX in the metastasis sample was notably greater than that in the tumors in situ. After treatment with zileuton, the expression of paxillin and E-cadherin in PC-3 and LNCaP cells was upregulated. In the transwell experiments, the motility of PC-3 was suppressed after treatment with zileuton. The mice treated with a high level of zileuton (1200 mg/kg) also had fewer tumors; however, the size did not show a significant difference.. The inhibitor of 5-LOX, zileuton, can suppress prostate cancer metastasis by repaired expression of E-cadherin and paxillin.

Topics: Animals; Arachidonate 5-Lipoxygenase; Cadherins; Cell Migration Assays; Enzyme-Linked Immunosorbent Assay; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Immunohistochemistry; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Paxillin; Prostatic Neoplasms; Up-Regulation

We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.5 and 29.0 +/- 8.0 microM, respectively. Recovery of cellular enzymatic activity is negligible (< 6%) following inactivation. Leukotriene A4 inactivates cellular 5-lipoxygenase without inhibiting its translocation from the cytosol to the membrane, suggesting that it impairs catalysis without impairing formation of the complex between 5-lipoxygenase and its membrane-associated activating protein. Consistent with this, leukotriene A4 inactivates purified 5-lipoxygenase from human neutrophils, via saturable, pseudo first-order kinetics with a rate constant, ki = 0.14 min-1 and a dissociation constant, Ki = 2.1 +/- 0.7 microM. Purified 5-lipoxygenase incubated with [3H]arachidonic acid incorporated a radiolabeled species that was not removed by electrophoresis under reduced denaturing conditions. Preincubation with leukotriene A4 diminished the incorporation of radiolabeled material, consistent with irreversible modification of 5-lipoxygenase by its metastable product, leukotriene A4. This unusual product inactivation mechanism may contribute to the decline in 5-lipoxygenase activity observed during catalysis.

Topics: Arachidonate 5-Lipoxygenase; Cell Differentiation; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Kinetics; Leukemia, Promyelocytic, Acute; Leukocytes; Leukotriene A4; Lipoxygenase Inhibitors; Neutrophils; Tumor Cells, Cultured

Topics: Asthma; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Leukotrienes; Lipoxygenase Inhibitors; Propionates; Quinolines