12-LOX Protein Research Articles

12-Lipoxygenase promotes epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway in gastric cancer cells.

Background12-Lipoxygenase (12-LOX) plays a major role in the progression and metastasis of various types of cancer. In gastric cancer (GC), the expression level of 12-LOX is significantly up-regulated; however, its function, and underlying mechanism of action remain unclear.MethodsThe mRNA and protein expression levels of 12-LOX were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses, respectively, in GC cell lines. 12-LOX expression was stably up-regulated using lentiviral vector in BGC823 and MGC803 cells, and cell-counting kit-8 (CCK8), colony formation, and invasion assays were performed to verify the function of 12-LOX in proliferation and metastasis. In addition, the expression levels of epithelial-mesenchymal transition (EMT) differentiation markers and downstream targets of the Wnt/β-catenin signaling pathway were examined by Western blotting. A nude mouse model of tumor growth and metastasis was established to investigate the role of 12-LOX in vivo.ResultsOur findings demonstrate that 12-LOX mRNA and protein were highly expressed in GC cell lines. 12-LOX overexpression promoted GC cell proliferation, migration, and invasion both in vitro and in vivo. In addition, up-regulation of 12-LOX promoted the EMT in GC cells, as reflected by a decrease in E-cadherin expression and an increase in N-cadherin and Snail expression. 12-LOX overexpression in GC cells also increased the expression of multiple downstream targets of the Wnt/β-catenin signaling pathway.ConclusionThese findings revealed that 12-LOX functions as an oncogene in promoting GC cell proliferation and metastasis in vitro and in vivo. In addition, 12-LOX might regulate the EMT via the Wnt/β-catenin signaling pathway, indicating a potential role for 12-LOX as a target in GC treatment.

The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma

Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

Abstract 3049: Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis

Abstract Introduction: Ionizing radiation (IR) is commonly used in the treatment of thoracic malignancies. Exposure of adjacent normal lung may result in lung injury and fibrosis. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in parynchymal depletion and precedes pulmonary fibrosis. Arachidonate 12-lipoxygenase (12-LOX) oxidizes arachidonic acid to form 12-Hydroxyeicosatetraeonic acid (12-HETE), a pro-inflammatory mediator. Increased expression of ALOX-12 has previously been associated with aging. We hypothesized that mice deficient in ALOX-12 would be resistant to IR induced fibrosis and senescence. Methods: C57/Bl6J (WT) and Alox12 homozygous knockout (Alox12-KO) mice (n>3 per group) were exposed to thoracic IR (0Gy, 5Gy, 17.5Gy, 5×5Gy, or 6×5Gy). Fibrotic foci were identified with aniline blue staining of fixed lung sections. Levels of 12-LOX mRNA and protein were assessed in WT lungs after IR with microarray, quantitative PCR, and western blotting. In vitro studies with primary murine pneumocyte cultures and A549 cells included staining for senescence associated β-galactosidase activity and western blotting for NOX4, p21, and PML following IR and 12-HETE treatment. Results: A significant increase in 12-LOX mRNA and protein was observed in murine lungs exposed to fibrogenic doses of IR (17.5 Gy, 5×5 Gy and 5×6 Gy) compared to low dose IR (5 Gy) or controls (0 Gy). 12-LOX deficiency significantly reduced fibrotic foci in murine lungs receiving fibrogenic doses of thoracic IR (6×5 Gy) at 18 weeks. Treatment of murine primary pneumocytes with 12-HETE (1 and 150 nM) increased the rate of pneumocyte senescence (2.1 fold). Treatment of murine primary pneumocytes with 12-HETE increased the expression of NOX4 (a mediator of superoxide generation), p21, and PML (senescence markers), paralleling increases observed after IR. Similarly, human type II pneumocyte A549 cells exhibited increased 12-LOX expression in β-galactosidase-stained senescent cells at 3 days after IR. Conclusion: These studies identify 12-LOX as a critical mediator in radiation lung fibrosis and type II pneumocyte senescence. 12-LOX may serve as a novel therapeutic target in mitigating IR-induced lung fibrosis. Citation Format: Eun Joo Chung, Luca F. Valle, Ayla O. White, Deborah E. Citrin. Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3049.

Abstract 3735: Dietary sugar induces tumorigenesis in mammary gland partially through 12 lipoxygenase pathway

Abstract Background: Compelling evidence from numerous epidemiological studies worldwide shows that high-sugar diets are associated with increased breast cancer risk, but the data is not wholly consistent and the molecular mechanisms remain unclear. One proposed mechanism for the role of sugar in cancer development involves inflammation. Bioactive lipids, especially cyclooxygenases and lipoxygenases (LOXs) metabolites of arachidonic acid, are known to be involved in inflammation and cancers. Here we examined the effect of sucrose enriched diet in the development of primary and metastatic breast tumor and relevant molecular mechanism primarily focusing on 12-LOX pathway. Methods: Mice bearing three different types of breast tumors were fed with sucrose-enriched diets, and the primary tumors and lung metastasis were measured. The levels of 12- LOX protein expression and its arachidonate metabolite 12-HETE were determined in both tumor tissues from the mice and breast cancer cells in vitro. Results: A diet with 125 g/kg sucrose, a concentration equivalent to the average sugar consumption of American population (70 lb/person/year), notably shortened the breast tumorigenesis in MMTV-ErbB2/neu mice with increase of tumor incidence by 77%. Sucrose enriched-diets (125 g/kg) promoted the development of primary breast tumors in mice bearing mouse breast cancer 4T1 cells with an average tumor weight of 389.4±64.2 mg in the control animals vs. 534.0±90.5 mg in sucrose fed animals. The tumor weight was increased by 2.2-fold in mice injected with human triple negative breast cancer MDA-MB-231 cells upon sucrose diet feeding. Further, sucrose diet (125 g/kg) potentiated lung metastasis of 4T1 mouse mammary tumor evidenced by increasing the average numbers of lung nodules per mouse from 6.6±2.2 in control group to 13.0±5.0 in 125 g/kg sucrose group. The increased tumorigenicity of sucrose appears to be consistently associated with up-regulation of 12-LOX protein expression and 12-HETE production within the tumor tissues across the three breast tumorigenesis models. Additionally, 12-LOX and 12-HETE were induced in the glucose (30 mM) treated MDA-MB-231 cells and 12-HETE promoted MDA-MB-231 cell growth, invasion and migration. Proliferation of MDA-MB-231 cells cultured in 3-D matrigel was stimulated by glucose treatment, which was abrogated with the presence of 12-LOX inhibitor, Baicalein. Conclusion: Our data provides a strong evidence that added sugar (sucrose) accelerates the development of breast cancer partially through up-regulating expression of 12-LOX and production of 12-HETE. Therefore, dietary sugar induced 12-LOX signaling may play an important role in increased incidence for breast cancer that is associated with a high-carbohydrate diet and could be a promising target for intervention and risk-reducing strategies for breast cancer. Citation Format: Yan Jiang, Yong Pan, Patrea R. Rhea, Lin Tan, Mihai Gagea-Iurascu, Lorenzo Cohen, Peiying Yang. Dietary sugar induces tumorigenesis in mammary gland partially through 12 lipoxygenase pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2015-3735

Putative biomarkers in malignant pleural mesothelioma.

e21008 Background: Malignant pleural mesothelioma (MPM) is associated with chronic inflammation due to asbestos exposure. The expression of inflammatory biomarkers associated with oncogenesis, e.g. cyclooxygenase-2 (COX2), 5-Lipoxygenase (5-LOX) and 12-LOX, may be of clinical relevance in MPM. In addition the PTEN/PI3K/AKT/MTOR signal transduction pathway may be aberrant in MPM. Loss of PTEN expression may lead to constitutive activation of AKT, resulting in cell survival and proliferation. In this study we aimed to assess the clinical relevance of 5-LOX, 12-LOX and PTEN protein expression in a large series of MPM samples. Methods: Immunohistochemical analysis was performed in 93 archival MPM tissue samples (48 epithelial, 27 biphasic, 18 sarcomatoid) to determine the expression of the PTEN, 5-LOX and 12-LOX proteins. Survival data were available for all 93 patients and prognostic factors were assessed using univariate and multivariate analyses. Results: Positive 5-LOX expression was seen in 73% (65/88) of samples and this was associated with improved survival (median overall survival 8.3 months versus 4.6 months; p=0.006). However, when histological subtype was taken into consideration, multivariate Cox regression analysis demonstrated that 5-LOX expression was not an independent prognostic variable (p=0.074). Positive 12-LOX expression was seen in 83% (69/83) of samples, but this was not associated with survival (p=0.455). Correlating this LOX expression data with our previously published COX2 expression results in the same cohort (Eur J Cancer 41:1645-8; 2005) revealed significant correlations. Positive 5-LOX expression correlated with positive COX2 expression (p=0.002). Positive 12-LOX expression correlated with 5-LOX co-expression (p=0.006). The expression of PTEN was lost in 27% (23/86) of samples and this was not associated with survival (p=0.223). Conclusions: We have demonstrated that 5-LOX and 12-LOX are expressed in a significant proportion of MPM samples and therefore may be novel therapeutic targets. Loss of PTEN expression was seen in 27% of samples. This research may lead to novel therapeutic strategies for patients with MPM.

Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival

Eicosanoid-related enzymes have been implicated in the pathogenesis of various cancers. Little is known about the relevance of lipoxygenase pathway to ovarian cancer growth. In this study, we examined the role of 12-lipoxygenase (12-LOX), the main human 12-HETE generating enzyme, in the regulation of proliferation and survival in epithelial ovarian cancer. Immunohistological analysis of 12-LOX expression in high-grade serous ovarian carcinoma and normal ovarian epithelium tissues was performed. The presence of 12-LOX-12-HETE system was confirmed in two epithelial ovarian cancer (EOC) cell lines, OVCAR-3 and SK-OV-3, using RT-PCR, Western blot and LC/MS analysis. The effects of N-benzyl-N-hydroxy-5-phenyl-pentanamide (BMD-122), a specific 12-LOX inhibitor, on cell growth, survival, apoptosis, and cell signaling were determined. We found that a significantly higher level of 12-LOX expression in high-grade serous ovarian carcinoma compared to normal ovarian epithelium. OVCAR-3 and SK-OV-3 were found to express high level of 12-LOX mRNA and protein. Both EOC increased their 12-HETE production when incubated with arachidonic acid. BMD-122 inhibited the EOC growth in a dose-dependent fashion. Purified 12-HETE significantly reversed such inhibitory effects of BMD-122. In addition, BMD-122 blocked the MAPK signaling pathway by inhibiting the phosphorylation of ERK and induced a ~20-30% increase in the EOC apoptosis. Down-regulation of the 12-LOX expression using 12-LOX siRNA also resulted in markedly reduction in cell growth. These data suggest that 12-LOX is involved in the regulation of ovarian cancer cell growth and survival and is a potential new therapeutic target.

Abstract 78: Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival

Abstract Enzymes involved in eicosanoid synthesis have previously been implicated in the progression of various types of cancer, including ovarian carcinomas. Most of the current data in ovarian cancer have focused on cyclooxygenase-2 (COX-2). It is not known whether the main human 12-hydroxyeicosatetrenoate (12-HETE) generating enzyme, 12-lipoxygenase (12-LOX), contributes to the regulation of growth of ovarian cancer cells. In the present study, we examined the function of the 12-lipoxygenase pathway in the regulation of proliferation and survival of three epithelial ovarian cancer (EOC) cell lines. We found that all three EOC cells express high level of the mRNA encoding for 12-LOX, using RT-PCR. These cells also contain significant amount of immuno-reactive 12-LOX protein as revealed by western blot. Liquid chromatography/Mass Spectrometry (LC/MS) analysis confirmed that all three EOC produce 12-HETE when challenged with arachidonic acid (AA). N-benzyl-n-hydroxy-5-phenyl-pentanamide (BMD-122) is a specific 12-LOX inhibitor. Exposure of the three EOC cell lines to an increasing dose of the 12-LOX inhibitor, BMD-122 (1 μM, 10 μM and 50 μM), resulted in a dose-dependent inhibition in cell proliferation (75±9%, 51±6% and 26±4% of untreated control, respectively), measured by MTT assay in vitro. Furthermore, treatment with 10 μM BMD-122 was associated with up to ∼30% increases in the apoptosis of these EOC cells within 24 hr, measured by Annexin-V flow cytometry. This apoptosis seems to be mediated through a mechanism independent of PI-3 kinase since the expression of phosphorylated Akt was not affected by BMD-122 treatment. Furthermore, we found that BMD-122 treatment had no effect on the three EOC cell lines ability to invasion using Matrigel assay. Taken together, these data suggest that 12-LOX is involved in regulation of ovarian cancer progression and survival, an important and novel finding. Inhibition of 12-LOX may have potential as an adjuvant therapy in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 78.

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.

Zyflamend®-mediated inhibition of human prostate cancer PC3 cell proliferation: Effects on 12-LOX and Rb protein phosphorylation

The multiherb anti-inflammatory product Zyflamend was investigated for its antiproliferative effects on PC3 human prostate cancer cells and eicosanoid metabolism in this prostate cancer cell line. Zyflamend produced a concentration-dependent inhibition of cloned COX-1, COX-2, and 5-LOX enzyme activities, with inhibition of 5-HETE production being greater than that of PGE2 formation. Applied to intact PC3 cells, Zyflamend was found to be most potent against 12-LOX, followed by 5-LOX and then COX activities. The concentration-dependent inhibition of PC3 cell proliferation was associated with a selective G2/M arrest of the cell cycle and induction of apoptosis, as evidenced by flow cytometric staining of PC3 cells with annexin V. Zyflamend also produced a concentration-dependent down-regulation of 5-LOX and 12-LOX expression. Determination of cell signal transduction proteins demonstrated that Zyflamend produced an increase in p21 phosphorylation but down-regulated phosphorylation of retinoblastoma (Rb) protein. The decrease in pRb protein was shown to be due to 12-LOX inhibition and a decline in 12-HETE levels in the cells. Replenishing 12-HETE in Zyflamend-treated cells overcame the ability of this multiple herb product to inhibit cell proliferation, and concordantly, 12-HETE blocked Zyflamend’s ability to down-regulate phosphorylation of Rb protein. We conclude that the effective control of human prostate cancer cell proliferation with Zyflamend is multi-mechanistic but, in part, involves regulation of aberrant tumor cell eicosanoid metabolism, especially on 5- and 12-LOX, as well as restoration of Rb tumor suppressor protein function through regulation of its phosphorylation status.

Molecular identification, localization and function of platelet-type 12-lipoxygenase in human melanoma progression, under experimental and clinical conditions

As previous studies suggested the expression of a 12-LOX enzyme in murine and human melanoma cell lines, the primary aim of this project was to genetically identify the 12-LOX enzyme (platelet-, leukocyte- or epithelial form). By using reverse transcriptase-polymerase chain reaction, sequencing and various immunological techniques we have demonstrated conclusively the expression of the platelet-type 12-LOX in human melanoma cells of different origin, in their transplanted xenografts and in fresh human skin tumors. Furthermore, we found that p12-LOX is able to provide a survival signal for melanoma cells since inhibition of the enzyme by general LOX or selective 12-LOX inhibitors induced apoptosis in vitro. p12-LOX of human melanoma has been shown to be involved in the control of the metastatic phenotype, since we have detected the upregulation of the 12-LOX protein expression in spontaneously metastasizing xenografts and in thick human skin tumors (> 3.0 mm) characterized by high risk for the development of metastasis. Co-expression of two megakaryocytic genes, p12-LOX and alphaIIb integrin chains, was found to be a frequent phenomenon in human melanoma (approximately 70%) suggesting a common regulatory defect in this tumor.

Expression of lipoxygenase in human prostate cancer and growth reduction by its inhibitors

The metabolism of arachidonic acid by either the cyclooxygenase (COX) or lipoxygenase (LOX) pathway generates eicosanoids, which have been implicated in the pathogenesis of a variety of human diseases, including cancer. They are believed to play important roles in tumor promotion, progression, and metastasis. Involvement of LOXs expression and function in tumor growth and metastasis has been reported in human tumor cell lines. Expressions of 5- and 12-LOX in prostate cancer (PC) patients, prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and normal prostate (NP) tissues were examined, as well as effects of their inhibitors on cell proliferation in 2 PC cell lines (PC3, DU-145). Expression of 5- and 12-LOX protein was detected by immunohistochemistry. Effects of LOX inhibitors on prostate cancer cell growth were examined by MTT assay, and Hoechst staining was used to determine whether or not the LOX inhibitors induce apoptosis. While 5- and 12-LOX expressions were slightly detected in BPH and NP tissues, marked expressions of 5- and 12-lipoxygenase were detected in PIN and PC tissues. The LOX inhibitors caused marked reduction of prostate cancer cells in a concentration- and time-dependent manner. The LOX inhibitors caused marked inhibition of PC cells through apoptosis. LOX is induced in prostate cancer, and our results suggest that LOX inhibitors may mediate potent antiproliferative effects against prostate cancer cells. Thus, LOX may become a new target in treatment of prostate cancer.

Expression of 12-lipoxygenase in human renal cell carcinoma and growth prevention by its inhibitor

The metabolism of arachidonic acid (AA) by either the cyclooxygenase (COX) or lipoxygenase (LOX) pathway generates eicosanoids, has been implicated in the pathogenesis of a variety of human diseases, including cancer, and may play important roles in tumor promotion, progression, and metastasis. The involvement of 12-LOX expression and function in tumor growth and metastasis has been reported in both murine and human tumor cell lines. The expression of 12-LOX in human renal cell carcinoma (RCC), normal kidney (NK) tissues and 3 kinds of RCC cell lines (Caki-1, A498, RC-1), and its effects on cell proliferation in 3 RCC cell lines were examined. Expression of 12-LOX protein was detected by immunohistochemistry and 12-LOX mRNA in RCC cell lines was detected by nested RT-PCR. Effects of 12-LOX inhibitor on RCC cell growth was examined by MTT assay, and to determine whether or not 12-LOX inhibitors induce apoptosis, we used Hoechst staining. While 12-LOX expression was detected slightly in NK tissues, a marked expression of 12-LOX was detected in RCC tissues. All human RCC cell lines expressed 12-LOX mRNA. The 12-LOX inhibitor baicalein caused marked inhibition of all three kinds of RCC cells in a concentration- and time-dependent manner. Cells treated with 12-LOX inhibitor showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These results suggest 12-LOX may play a role in the progression of RCC in human tissue, and its inhibitors may become anticancer agents in human RCC.

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.

Expression of 12-lipoxygenase as a biomarker for melanoma carcinogenesis.

12-Lipoxygenase (12-LOX), through its metabolite 12( )-hydroxyeicosatetraenoic acid [12( )-HETE], has been demonstrated to play a pivotal role in experimental melanoma invasion and metastasis, and 12-LOX expression may be important in early human melanoma carcinogenesis. We have studied the differences in 12-LOX protein expression during the progression of melanoma from human melanocytic cells to benign and dysplastic naevi to malignant metastatic disease. 12-LOX expression was determined in normal human skin melanocytes and in melanocytes found in compound naevi, dysplastic naevi and melanomas using a platelet-type 12-LOX antibody with a diaminobenzidine immunoperoxidase system detection system and was quantified using the analysis software NIH Image 1.62. Mean cellular pixel densities for 12-LOX staining ( = 50 cells/histological type) were unchanged in compound naevi ( = 0.14) and were increased in dysplastic naevi and melanomas compared with normal skin melanocytes ( = 0.03 and = 0.01, respectively). Similarly, melanomas had higher levels of expression compared with dysplastic naevi ( = 0.03). 12-LOX expression was significantly different between compound naevus and dysplastic naevus melanocytes ( = 0.01). These data suggest that 12-LOX may be an important novel marker for cancer progression within the melanoma system, and therefore could be a useful biomarker and therapeutic target for melanoma chemoprevention.

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.

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.

Role of autocrine motility factor in a 12-lipoxygenase dependent anti-apoptotic pathway.

Apoptosis or programmed cell death, plays an indispensable role in embryonic development, maturation of the immune system and maintenance of tissue and organ homeostasis (1–2). A wide spectrum of molecular entities including oncogenes, tumor suppressor genes, signal transducers, cell cycle proteins, free radicals, cations and proteases have been implicated in apoptosis. Recent work from our laboratory (3) demonstrated that 12-lipoxygenase (12-LOX) may play an essential role in regulating apoptosis in rat Walker carcinosarcoma (W256) cells. Down regulation of 12-LOX gene expression by antisense oligonucleotides triggered time and dose dependent apoptosis. The W256 cell death, triggered by antisense oligos could be partially blocked by exogenous 12(S)-HETE. The antisense oligo treatment also resulted in a significant decrease in the ratio of Bcl-2/Bax proteins which are critical determinants of apoptosis. As expected, over expression of Bcl-2 protein provided partial death sparing effects (3). It also was demonstrated that exogenous arachidonic acid (AA) protected against apoptosis (4). Exogenous AA in a time and dose dependent fashion suppressed NDGA induced W256 apoptosis as well as DNA fragmentation. Serum withdrawal also caused W256 cells to undergo typical apoptosis which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation induced W256 cell apoptosis (4). Further studies demonstrated that the factor responsible for AA induced protection against apoptosis was the 12-LOX metabolite of AA 12(S)-HETE. 12-LOX has been demonstrated by our laboratory to be expressed in numerous tumor cells from human, rat and mouse (5). Factors controlling the regulation of 12-LOX are largely unknown. Several growth factors have been shown to stimulate 12-LOX activity in tumor cells. For example, in A431, human epidermoid carcinoma cells platelet type 12-LOX is expressed (6). Serum starvation induces a time and dose dependent reduction in the expression of 12-LOX protein (8). Another cytokine which has been demonstrated to regulate 12-LOX levels in tumor cells is autocrine motility factor (AMF). AMF stimulates cell motility and invasion via receptor mediated signaling pathways. AMF is also a potent growth factor. Our laboratory demonstrated that AMF induced cell motility was mediated by the lipoxygenase generation of 12(S)-HETE (7). In a subsequent report, we also demonstrated that AMF was able to increase the cellular protein levels of 12-LOX in high but not in low metastatic clones (8).

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.