High Lysyl Oxidase (LOX) in the Non-Malignant Prostate Epithelium Predicts a Poor Outcome in Prostate Cancer Patient Managed by Watchful Waiting

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.     

Inhibiting Delta-6 Desaturase Activity Suppresses Tumor Growth in Mice

Recent studies have shown that a tumor-supportive microenvironment is characterized by high levels of pro-inflammatory and pro-angiogenic eicosanoids derived from omega-6 (n−6) arachidonic acid (AA). Although the metabolic pathways (COX, LOX, and P450) that generate these n−6 AA eicosanoids have been targeted, the role of endogenous AA production in tumorigenesis remains unexplored. Delta-6 desaturase (D6D) is the rate-limiting enzyme responsible for the synthesis of n−6 AA and increased D6D activity can lead to enhanced n−6 AA production. Here, we show that D6D activity is upregulated during melanoma and lung tumor growth and that suppressing D6D activity, either by RNAi knockdown or a specific D6D inhibitor, dramatically reduces tumor growth. Accordingly, the content of AA and AA-derived tumor-promoting metabolites is significantly decreased. Angiogenesis and inflammatory status are also reduced. These results identify D6D as a key factor for tumor growth and as a potential target for cancer therapy and prevention.     

Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin‐induced apoptosis

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX‐PP) released from the secreted precursor protein (Pro‐LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her‐2/neu‐driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX‐PP on MIA PaCa‐2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa‐2 cells in culture, LOX‐PP attenuated the ERK and AKT activities and decreased the levels of the NF‐κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX‐PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF‐κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX‐PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX‐PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX‐PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities. J. Cell. Biochem. 111: 1160–1168, 2010. © 2010 Wiley‐Liss, Inc.     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.     

Molecular markers for tracking variation in lipoxygenase activity in wheat breeding

Lipoxygenase (LOX) activity is an important factor determining the color of flour and end-use products of wheat. In the present study, quantitative trait loci (QTL) for LOX activity in common wheat were mapped using 71 doubled haploid (DH) lines derived from a Zhongyou 9507 × CA9632 cross, and SSR markers. Two QTL, QLpx.caas.1AL and QLpx.caas-4B, were identified on chromosomes 1AL and 4B, closely associated with LOX activity. The SSR loci Xwmc312 and Xgwm251 proved to be diagnostic and explained 13.4–25.2% of the phenotypic variance for the 1AL locus and 14.3–27.0% for the 4B locus across four environments. The SSR markers Xgwm251 and Xwmc312 were validated across 198 Chinese wheat cultivars and advanced lines and showed highly significant (P < 0.01) association with LOX activity. We further established a multiplexed PCR with SSR marker combination Xwmc312/Xgwm251 to test these wheat cultivars and advanced lines. The results suggested that the marker combination Xwmc312/Xgwm251 is efficient and reliable for evaluating LOX activity and can be used in marker-assisted selection (MAS) for targeting flour color attributes to noodle and other wheat-based products.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3–8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9–3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.     

Lysyl oxidase 473 G>A polymorphism and breast cancer susceptibility in Chinese Han population

Lysyl oxidase (LOX) is an extracellular enzyme critical for the cross-linking of collagens and elastin. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. Recently, a single-nucleotide polymorphism (SNP) of LOX G473A (rs1800449) has been demonstrated to be associated with increased risk of breast cancer in African American women. In this hospital-based case-control study, the association of LOX polymorphism with breast cancer susceptibility in Chinese Han population was investigated. In total, 238 female patients with breast cancer and 234 age-matched healthy controls recruited were genotyped. We found a significant difference in the frequency of the LOX G473A genotype between the breast cancer and control groups. Individuals with GA genotype showed a 2.79-fold (95% confidence interval?=?1.87-4.16) increased risk of breast cancer compared with subjects carrying GG genotype (p?相似文献   

Lysyl oxidase propeptide stimulates osteoblast and osteoclast differentiation and enhances PC3 and DU145 prostate cancer cell effects on bone in vivo

Lysyl oxidase pro-enzyme is secreted by tumor cells and normal cells as a 50 kDa pro-enzyme into the extracellular environment where it is cleaved into the ~30 kDa mature enzyme (LOX) and 18 kDa pro-peptide (LOX-PP). Extracellular LOX enzyme activity is required for normal collagen and elastin extracellular cross-linking and maturation of the extracellular matrix. Extracellular LOX-PP acts as a tumor suppressor and can re-enter cells from the extracellular environment to induce its effects. The underlying hypothesis is that LOX-PP has the potential to promote bone cell differentiation, while inhibiting cancer cell effects in bone. Here we investigate the effect of LOX-PP on bone marrow cell proliferation and differentiation towards osteoblasts or osteoclasts, and LOX-PP modulation of prostate cancer cell conditioned media-induced alterations of proliferation and differentiation of bone marrow cells in vitro. Effects of overexpression of rLOX-PP in DU145 and PC3 prostate cancer cell lines on bone structure in vivo after intramedullary injections were determined. Data show that prostate cancer cell conditioned media inhibited osteoblast differentiation in bone marrow-derived cells, which was reversed by rLOX-PP treatment. Prostate cancer conditioned media stimulated osteoclast differentiation which was further enhanced by rLOX-PP treatment. rLOX-PP stimulated osteoclast differentiation by inhibiting OPG expression, up-regulating CCN2 expression, and increasing osteoclast fusion. In vivo studies indicate that rLOX-PP expression by PC3 cells implanted into the tibia of mice further enhanced PC3 cell ability to resorb bone, while rLOX-PP expression in DU145 cells resulted in non-significant increases in net bone formation. rLOX-PP enhances both osteoclast and osteoblast differentiation. rLOX-PP may serve to enhance coupling interactions between osteoclasts and osteoblasts helping to maintain a normal bone turnover in health, while contributing to bone abnormalities in disease.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

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.     

Lipoxygenase Pathway of Arachidonic Acid Metabolism in Growth Control of Tumor Cells of Different Type

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.     

Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex

We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.     

Dietary lipids modulate eicosanoid release and apoptosis of cells of a murine lung alveolar carcinoma

Dietary arachidonic acid (AA) and eicosanoids influence neoplastic cell (NC) growth, differentiation and apoptosis. Plasma membrane fatty acid and cyclooxygenase (COX) and lipoxygenase (LOX) products were investigated in lung alveolar carcinoma cells from mice fed on different diets. Two groups were fed on a basic diet plus 6% of: corn oil (rich in 18:2n-6; CO) and on olein oil (rich in 18:1n-9; O), respectively. Control group (C) received commercial diet. NC fatty acids were analyzed by GLC, and apoptosis by flow cytometry and microscopy. In NC from CO group AA levels and LOX metabolites were increased, whereas COX metabolites decreased. NC from CO compared to O group diet showed a higher count of apoptosis and increased LOX:COX ratio. High levels of AA and decreased COX eicosanoids has been involved in anti-tumoral mechanisms by increasing tumor cell apoptosis. Present data emphasizes the implications of the dietary fatty acids on the neoplastic process in this tumoral model.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.     

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down‐regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9‐hydroperoxyoctadecadienoic acid (9‐HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild‐type seeds by measuring the ultraviolet absorption of 9‐HPOD at 234 nm and by high‐performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS‐91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.