15-lipoxygenase-1 metabolites down-regulate peroxisome proliferator-activated receptor gamma via the MAPK signaling pathway.

Human colon tumors have elevated levels of 15-lipoxygenase-1 (15-LO-1), suggesting that 15-LO-1 may play a role in the development of colorectal cancer. Also, 15-LO-1 metabolites can up-regulate epidermal growth factor signaling pathways, which results in an increase in mitogenesis. However, metabolites of 15-LO-1 can serve as ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), and activation of this receptor causes most colon cancer cell lines to undergo a differentiative response and reverse their malignant phenotype. Hence, the role 15-LO-1 plays in colon cancer is not clear. To clarify the role of 15-LO-1 in carcinogenesis, the effect of 15-LO-1 and its metabolites on epidermal growth factor signaling and PPARgamma was investigated. In HCT-116 cells, exogenously added 15-LO-1 metabolites, 13-(S)-hydroxyoctadecadienoic acid, 13-(R)-hydroxyoctadecadienoic acid, and 13-(S)-hydroperoxyoctadecadienoic acid, up-regulated the MAPK signaling pathway, and an increase in PPARgamma phosphorylation was observed. Furthermore, in stable overexpressing 15-LO-1 HCT-116 cells, which produce endogenous 15-LO-1 metabolites, an up-regulation in mitogen-activated protein kinase and PPARgamma phosphorylation was observed. Incubation with a MAPK inhibitor ablated MAPK and PPARgamma phosphorylation. The 15-LO-1 up-regulates MAPK activity and increases PPARgamma phosphorylation, resulting in a down-regulation of PPARgamma activity. Thus, 15-LO-1 metabolites may not only serve as ligands for PPARgamma but can down-regulate PPARgamma activity via the MAPK signaling pathway.     

Evaluation of eicosanoids and NSAIDs as PPARgamma ligands in colorectal carcinoma cells

The activation of peroxisome proliferator activated receptor gamma (PPARgamma) may play a role in the control of colorectal carcinogenesis. The expression of PPARgamma was examined by Western blotting in human colorectal tumors and matched normal adjacent tissues, as well as in various colorectal carcinoma cell lines. In the tissues, the expression of PPARgamma was elevated in tumors relative to the adjacent normal tissues. Each colorectal carcinoma cell line expressed PPARgamma. The ability of various eicosanoids to bind PPARgamma in colorectal carcinoma cells was investigated using luciferase reporter assays. The well-known PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) strongly induced PPARgamma binding activity. Products of lipoxygenases displayed moderate binding activity, while other prostaglandins and fatty acids displayed little or no reporter activation. The activation of PPARgamma by 13(S)-HODE, the major metabolite of 15-lipoxygenase-1 from linoleic acid, was concentration dependent reaching maximum at 10 micro M (35-fold activation). The endogenous production of 13(S)-HODE by expression of 15-LO-1 did not activate PPARgamma. The ability of various nonsteroidal anti-inflammatory drugs (NSAIDs) to induce PPARgamma activation was also evaluated. The conventional NSAIDs that inhibit both cyclooxygenases (COX-1 and COX-2) also induced PPARgamma binding activity. In general, however, neither COX-1- nor COX-2-specific inhibitors induced the activation of PPARgamma. Taken together, the metabolites of 15-lipoxygenase and the conventional NSAIDs were confirmed as exogenous ligands for PPARgamma in colorectal carcinoma cells.     

Evaluation of the activity and localization of 15-lipoxygenase-1 after introduction into human colorectal carcinoma Caco-2 cells.

In human colorectal carcinoma Caco-2 cells, sodium butyrate (NaBT) induces the expression of the reticulocyte, 15-lipoxygenase-1 (15-LO-1) and causes these cells to undergo differentiation and apoptosis. 15-LO-1 is also expressed in human colorectal epithelium with a significant higher expression observed in colorectal tumors. In this study, we have prepared stable Caco-2 cells that expressed 15-LO-1 under control of an inducible promoter. These cells provide a model system to study regulation of 15-LO-1 activity in colorectal cells without the interfering presence of NaBT and are useful to study the biological function of 15-LO-1. The expressed 15-LO-1 was highly active as measured in cell lysates, but we were unable to detect metabolism in intact cells. The addition of calcium to the media for the Caco-2 cells was required for 15-LO-1 to translocate from the cytosol to the membrane which is frequently a requirement for lipoxygenase activity. Despite the addition of calcium and translocation, little lipoxygenase activity was detected with intact cells. However, after removal of phenol red, a common constituent of cell culture media, we were able to detect 15-LO-1 activity in the transfected Caco-2 cultured cells. Thus the presence of calcium and the absence of antioxidants present in commonly used culture media are required for expressed 15-LO-1 to be catalytically active and to permit an examination of its biological effects.     

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.     

Opposing effects of 15-lipoxygenase-1 and -2 metabolites on MAPK signaling in prostate. Alteration in peroxisome proliferator-activated receptor gamma

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARgamma in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARgamma phosphorylation while a subsequent decrease in PPARgamma phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARgamma. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.     

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.     

Orthotopic expression of human 15-lipoxygenase (LO)-1 in the dorsolateral prostate of normal wild-type C57BL/6 mouse causes PIN-like lesions

The lipid-peroxidating enzyme, 15-lipoxygenase (LO)-1 and its metabolite, 13-S-hydroxyoctadecadienoic acid (13-S-HODE), likely contribute to prostate tumorigenesis. Thus, this study evaluated adenovirus-mediated overexpression of 15-LO-1 on normal mouse prostate. Adenovirus expressing either human 15-LO-1 tagged with green fluorescent protein (GFP) or GFP alone was orthotopically injected into the dorsolateral prostates of C57BL/6 mice, three times over the course of 60 days. On day 90, pathological changes in prostate tissue were assessed by hematoxylin and eosin (H&E) staining. Expression of the proliferation marker Ki-67 was evaluated by immunohistochemistry and expression of angiogenesis markers were analyzed by an antibody array. Based on the latter study, immunoprecipitation analysis was used to measure the effect of 13-S-HODE, with or without conditioned media, on fibroblast growth factor-a and b (FGF-a and FGF-b) expression in human PrEC (normal prostate epithelial), PrSMC (normal prostate smooth muscle) and PrSC (normal prostate stromal) lines. Expression of viral 15-LO-1-GFP, but not GFP alone, resulted in the development of a prostate intraepithelial neoplasia (PIN)-like phenotype with increased expression of Ki-67. Aberrant 15-LO-1 expression also induced the angiogenic markers FGF-a and FGF-b. Human PrEC, PrSMC and PrSC lines demonstrated an increase in FGF-b expression upon stimulation with 13-S-HODE, which was further increased by the addition of conditioned media from the epithelial or smooth muscle cells. Using adenoviral mediated 15-LO-1 gene delivery, this study suggests that aberrant 15-LO-1 overexpression in normal prostate can trigger events leading to prostate epithelial and stromal cell proliferation. Thus, our findings demonstrate the effectiveness of this viral system for 15-LO-1 expression studies in tissues.     

Expression of 15-lipoxygenase type-1 in human mast cells

Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.     

Differential expression of sialic acid and N-acetylgalactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential.

In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions of the cells. Fluorescence and electron microscopic analyses revealed differences in the expression profile of carbohydrates recognized by the lectins used on the cell surface of IEC-6, Caco-2, and HCT-116 cells. Lectin blotting identified a range of eight HPA-binding glycoprotein bands with molecular weights of 16-66 kD in Caco-2 cells, six glycoproteins of 16-36 kD, and three protein bands of 35, 24, and 21 kD in IEC-6 cells. A minor band of 66 kD and a major one of 50 kD for WGA-binding glycoproteins were observed in IEC-6 cells and seven glycoproteins of 18-97 kD in Caco-2 and HCT-116 cells but with a visible higher expression of these glycoproteins in the latter. Furthermore, significant quantitative difference in levels of expression of WGA- but not of HPA-binding glycoconjugates was noted, as analyzed by high-resolution scanning electron microscopy using backscattered electron images of cells incubated with gold-labeled lectins.     

Metabolism of oxidized linoleic acid by glutathione transferases: peroxidase activity toward 13-hydroperoxyoctadecadienoic acid

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.     

Expression of 15-lipoxygenase-1 in human nasal epithelium: its implication in mucociliary differentiation

15-lipoxygenase-1 (15-LO-1) is involved in the differentiation of human tracheobronchial epithelial cells. Here, we investigated the relation between 15-LO-1 expression and the differentiation of human nasal epithelium. In retinoic acid (RA)-sufficient culture media, 15-LO-1 expression in normal human nasal epithelial cell time-dependently increased, but its expression was undetectable in RA-deficient culture media. Moreover, in RA-deficient culture media, IL-4 at 1 ng/ml concentration time-dependently induced 15-LO-1 expression. In addition, MUC8 gene expression, a marker of mucociliary differentiation, was up-regulated by 15-LO-1, which was itself induced by IL-4. In murine nasal mucosa, the expression of leukocyte type-12-LO, a functional equivalent of 15-LO-1, reduced after postnatal day 7. Our findings suggest that 15-LO-1 is related to the differentiation of human nasal epithelium, and that it may mediate the mucociliary differentiation of human nasal epithelium.     

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.     

Association between E-cadherin expression by human colon,bladder and breast cancer cells and the 13-HODE:15-HETE ratio. A possible role of their metastatic potential

The relationship between 15(S)-HETE and 13(S)-HODE from different human tumor cells exposed to n-6 and n-3 essential fatty acids (EFAs) and E-cadherin expression was studied. Colon cancer cells (HRT-18) exposed to gamma linoleic acid (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) (50microM) showed an increased expression of E-cadherin. Breast cancer (MCF-7) exposed to EPA showed an increment whereas GLA had no effect on E-cadherin expression. No expression of E-cadherin was observed for urothelial cancer (T-24) after GLA or EPA treatment. Significant levels of 15(S)-HETE and 13(S)-HODE were detected after GLA or EPA treatment for all tumor lines. E-cadherin expression was inversely proportional to the 13(S)-HODE:15(S)-HETE ratio when cells were pretreated with GLA or EPA. Nevertheless, the liberation of these metabolites seems to be independent of the E-cadherin expression. The increase in the13(S)-HODE:15(S)-HETE correlates to a decrease in the expression of E-cadherin. Both factors may play a role in metastasis development.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

Anticancerogenic effect of a novel chiroinositol-containing polysaccharide from Bifidobacterium bifidum BGN4

Strains of bifidobacteria have many health-promotion effects. Whole cells or cytoplasm extracts of Bifidobacterium bifidum BGN4, isolated from human feces, inhibited the growth of several cancer cell lines. The polysaccharide fraction (BB-pol) extracted from B. bifidum BGN4 had a novel composition, comprising chiroinositol, rhamnose, glucose, galactose, and ribose. Three human colon cancer cell lines were treated with BB-pol: HT-29, HCT-116, and Caco-2. Trypan blue exclusion assay and BrdU incorporation assay showed that BB-pol inhibited the growth of HT-29 and HCT-116 cells but did not inhibit the growth of Caco-2 cells.     

Changes in Subcellular Localization of Visfatin in Human Colorectal HCT-116 Carcinoma cell Line After Cytochalasin-B Treatment

The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB) and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS) level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.Key words: Visfatin, cytochalasin B, immunogold labeling, TEM, adipocytokines