ABSTRACT
13-S-Hydroxyoctadecadienoic acid (13-S-HODE), the product of 15-lipoxygenase (15-LOX) metabolism of linoleic acid, enhances cellular mitogenic responses to certain growth factors. Other observations have questioned whether 13-S-HODE has tumorigenic effects. Our study evaluated the hypothesis that 15-LOX-1 is overexpressed in colon cancers resulting in an increase in intracellular 13-S-HODE. 15-LOX-1 and 13-S-HODE were quantified using western blots, ELISA and immunohistochemistry in 18 human colon cancers with paired normal colonic mucosa. Additionally, 15-LOX-1 expression was measured by western blots in three transformed colonic cell lines and in a human umbilical vein endothelial cell line. Next, we evaluated 13-S-HODE effects on cellular proliferation, cell cycle distribution and apoptosis in a transformed colonic cell line (RKO). Cell cycle distributions were measured by flow cytometry and apoptosis was assessed by phase contrast microscopy, electron microscopy, flow cytometry and DNA fragmentation assay. 15-LOX-1 immunohistochemistry staining scores were reduced in tumor tissues (P
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Colonic Neoplasms/metabolism , Linoleic Acids/metabolism , Apoptosis , Blotting, Western , Cell Cycle , Cell Division , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Tumor Cells, CulturedABSTRACT
The major lipoxygenation product derived from linoleic acid, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), has been shown to be involved in cell proliferation and differentiation in a number of systems. Rapid detection of picogram amounts of this bioactive lipid in biological samples, however, has been hindered due to lack of immunological reagents. In the current report, we have used a polyclonal antibody specific for 13-(S)-HODE to detect this bioactive lipid for the first time in human prostate adenocarcinoma specimens (PCa) and the prostate cancer cell lines LNCaP and PC-3 by enzyme immunoassay. In addition, we have verified-the quantitation of 13-HODE by chiral-phase HPLC and examined the levels of lipoxygenase expression by Western, Northern, and RT-PCR analysis. Immunohistochemically detectable 13-HODE was observed in human PCa, whereas adjacent normal tissue showed no immunoreactivity. The presence of 15-lipoxygenase was evident by Western and RT-PCR analysis in both LNCaP and PC-3 cells, while Northern blot analysis showed the presence of 15-lipoxygenase message in LNCaP cells but failed to detect any 15-lipoxygenase message in PC-3 cells. In contrast, quantitation of 13-HODE by enzyme immunoassay and chiral-phase HPLC showed significant levels of the compound in PC-3 cells but minimal enzymatically produced 13-HODE in LNCaP cells. These data provide a link between linoleic acid metabolism and the development or progression of prostate cancer.
Subject(s)
Linoleic Acids/biosynthesis , Prostatic Neoplasms/metabolism , Arachidonate 15-Lipoxygenase/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Linoleic Acids/physiology , Male , Polymerase Chain Reaction , Prostatic Intraepithelial Neoplasia , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
The oxygenated metabolite of linoleic acid, 13(S)-hydroxyoctadecadienoic acid has recently been shown to play a role in cellular regulation. To detect this molecule in biological systems, we recently developed a specific polyclonal antibody. Using this antibody, we report the presence of 13(S)-hydroxyoctadecadienoic acid in human urine, cell culture media, and untreated goat serum for the first time by a specific, sensitive, and rapid enzyme immunoassay. Furthermore, the enzyme linked immunosorbent assay data are verified by gas chromatography/mass spectrometry analysis of the same samples.
Subject(s)
Linoleic Acids/analysis , Animals , Gas Chromatography-Mass Spectrometry , Goats , Humans , Immunoenzyme Techniques , Linoleic Acids/blood , Linoleic Acids/urine , Sensitivity and Specificity , StereoisomerismABSTRACT
Linoleic acid, the predominant polyunsaturated fatty acid in the diet, can be metabolized by cyclooxygenase, lipoxygenase and P450 enzymes. The monohydroxy lipoxygenation products of linoleic acid, 9- and 13-hydroxyoctadecadienoic acids (9(S)- and 13(S)-HODEs), are the most widely distributed of the known linoleic acid metabolites. These compounds exhibit interesting biological activities, including regulation of platelet function, maintenance of vascular thromboresistance and transduction of the cellular responses to certain growth factors. In view of their biological significance, we have produced polyclonal antibodies for the first time to these bioactive lipids to develop an easy, inexpensive, sensitive, specific and rapid enzyme immunoassay method for these bioactive lipids.
