Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 2 uppsatser.

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. 15-lipoxygenase-1 (15-LO-1), an enzyme of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in CRC. In the present study, the hypothesis that the lack of 15-LO-1 expression in CRC cells may contribute to the tumorigenesis was investigated. Therefore 15-LO-1 was introduced to colon cancer cell line HT-29 that does not have detectable levels of the 15-LO-1. The HT-29 cells were transiently transfected with the eukaryotic expression vector pcDNA3.1-15-LO-1 and the effects of 15-LO-1 expression on the proliferation, apoptosis as wells as metastatic potential of the cells were investigated. Cellular proliferation was analyzed by MTT assay and the apoptotic potential of 15-LO-1 was evaluated by acridine orange, floating cell ratio and caspase-3 assays as well as expression levels of the antiapoptotic protein XIAP. Cellular migration and invasion were investigated by Boyden chamber migration and Matrigel invasion assay.The data indicates that 15-LO-1 expression significantly decreased cell proliferation and increased apoptosis. In addition, a significant reduction was observed in migratory and invasive capacity 15-LO-1 expression also significantly reduced the expression of metastasis associated 1 protein (MTA-1). Taken together we propose that 15-LO-1 expression in CRC can inhibit colon cancer cell growth through induction of apoptotic cell death and may contribute to the inhibition of metastatic capacity in vitro which may be exploited for therapeutic purposes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Die 12/15-Lipoxygenasen (12/15-LOXs) gehören zu einer heterogenen Klasse Lipid-peroxidierender Enzyme, deren biologische Rolle noch nicht vollständig geklärt ist. Eine Reihe experimenteller Daten deuten darauf hin, dass diese Enzym an Reifungs- und Differenzierungsprozessen beteiligt sind und auch für die Pathogenese verschiedener Erkrankungen (Asthma bronchiale, Entzündung, Atherosklerose) bedeutsam zu sein scheinen. Die Expression von 12/15-LOXs wird in vielen Säugetierzellen durch TH2-Zytokine reguliert und die Zytokin-induzierte Signaltransduktionskaskade verläuft über die Aktivierung van JAK-Kinasen und STAT6. Nach einer Stimulation von A549 Lungenkarzinomzellen mit Interleukin-4 (IL-4) kommt es erst nach 12 Stunden zu einer Hochregulation der 12/15-LOX mRNA Expression. Untersuchungen zum Induktionsmechanismus haben gezeigt, dass Genistein, ein Hemmstoff von Tyrosinkinasen, die Phosphorylierung von STAT6 und dessen Bindung an den Promoter des 12/15-LOX Gens verhinderte. Damit konnte die Induktion der katalytisch aktiven LOX geblockt werden. In Gegensatz dazu verhinderte Zykloheximid, ein spezifischer Hemmstoff der Proteinbiosynthese, die Expression der 12/15-LOX mRNA nicht, Dieses Ergebnis deutet darauf hin, dass die Neusynthese eines Transkriptionsfaktors im Rahmen der IL-4 induzierten Transduktionskaskade unwahrscheinlich ist. Weiterhin wurde beobachtet, dass IL-4 die zelluläre Histonacetyltransferase-Aktivität stark erhöhte und dass dieser Effekt überwiegend auf die enzymatische Aktivität des (CREB-bindenden Protein)-bindenden Proteins (CBP) zurückzuführen ist. Transfektion der Zellen mit E1A, einem viralen Protein, welches als Hemmstoff der Histonacetyltransferase-Aktivität von CBP/p300 bekannt ist, führte zu einer Unterdrückung der 12/15-LOX Expression. Andererseits stimuliert Natriumbutyrat, ein Hemmstoff der Histondeacetylase, die 12/15-LOX Synthese. Damit konnte gezeigt werden, dass die Acetylierung von Histonproteinen und von STAT6 ein essentieller Prozesse bei der IL-4 induzierten 12/15-LOX Expression in A549 Zellen ist. Weiterhin belegen diese Daten, dass sowohl die Phosphorylierung als auch die Acetylierung von STAT6 an der transkriptionellen Aktivierung des 12/15-LOX Gens beteiligt sind, obwohl beide Prozesse eine unterschiedliche Kinetik aufweisen. STAT6 Phosphorylierung erfolgt innerhalb der ersten Stunde nach IL-4 Stimulation, während die Acetylierungsreaktion zeitlich verzögert abläuft. Zusammenfassend kann die Signaltransduktionskaskade, die in A549 Zellen zur Expression der 12/15-LOX führt, wie folgt beschrieben werden: IL-4 induziert über die Aktivierung von JAK-Kinasen eine Phosphorylierung von STAT6, dessen Bindung an den 12/15-LOX Promotor jedoch zunächst durch nicht-acetylierte Histonproteine verhindert wird. Nach 9-11 Stunden werden Histone und der phosphorylierte STAT6 durch die Acetyltransferase-Aktivität von CBP/p300 acetyliert. Diese Reaktion führt zu einer Veränderung der Histonstruktur, wodurch die Bindung von modifizierten STAT6 und damit die Expression des 12/15-LOX Gens ermöglicht wird. Als wesentliche zellphysiologische Konsequenz der IL-4 induzierten 12/15-LOX Expression in A549 Zellen, wurde eine Apoptoseinduktion beobachtet. Das endogene 12/15-LOX Produkt 15-HETE bindet an den Kernrezeptor PPARg und induziert damit den programmierten Zelltod. Vorinkubation von A549 Zellen mit dem LOX-Hemmstoff NDGA oder der Einsatz von PPARg Dominant Negativ Vektor verhinderten die Apoptose. Mechanistische Untersuchungen zum Ablauf des durch IL-4 induzierten Zelltodes zeigten, dass der Prozess überwiegend dem extrinsischen Mechanismus folgt, bei dem Kaspasen-8 direkt zu einer Aktivierung der Effektorkaspase-3 führt. Der mitochondriale Mechanismus (Spaltung von Bid bzw. initiale Cytochrom C Freisetzung) scheint dabei nicht involviert zu sein. Die IL-4 induzierte Apoptose könnte von patho-physiologischer Bedeutung für den Verlauf von Lungenerkrankungen sein, bei denen Zellen mit hoher konstitutiver 12/15-LOX Expression, z.B. eosinophile Granulozyten, beteiligt sind. Hepoxiline sind bioaktive Mediatoren des 12/15-LOX Weges der Arachidonsäurekaskade, die durch Isomerisierung des primären Oxygenierungsproduktes 12S-HpETE gebildet werden. Zu Beginn unserer Untersuchungen war überwiegend unklar, welche Enzyme an der Isomerisierungsreaktion beteiligt sind. Bei der Suche nach geeigneten zellulären Modellen für die Untersuchung dieser Fragestellung fanden wir heraus, dass in den Ratteninsulinom-Zellen Rinm5F, die wegen ihres Mangels an Glutathionperoxidasen eine geringe Kapazität zur Reduktion von 12S-HpETE aufweisen, die Synthese von Hepoxilin A3 (HXA3) besonders hoch ist. Da wir vermuteten, dass 12/15-LOXs für die Isomerisierung von 12S-HpETE zu HXA3 verantwortlich sein könnten, verfolgten wir eine duale Forschungsstrategie um experimentelle Hinweise für unsere Arbeitshypothese zu finden. In den 12/15-LOX exprimierenden Rinm5F Zellen führte eine Immunopräzipitation mit 12/15-LOX spezifischen Antikörper zu einen vollständigen Verlust der 12/15-LOX- und der Hepoxilinsynthase-Aktivität eines Zelllysates. Beide Aktivitäten wurden fast vollständig im Immunopräzipitat wiedergefunden. 2. Transfektion von HeLa Zellen, die selbst keine 12/15-LOX exprimieren, mit 12/15-LOX und gleichzeitige Hemmung der zellulären Glutathionperoxidasen (Depletion von GSH mit Diethlmaleat) führte zu einer deutlichen zellulären Hepoxilinsynthese. Bei entsprechenden Kontrolltransfektanten wurde diese Aktivität nicht beobachtet. Weiterhin konnte festgestellt werden, dass rekombinante 12/15-LOXs (Expression in E. coli) in vitro eine intrinsische Hepoxinsynthase-Aktivität aufweisen, wenn 12S-HpETE als Substrat angeboten wird. Diese Daten belegen, dass 12/15-LOXs neben den bisher beschriebenen katalytischen Aktivitäten (Oxygenase, Hydroperoxidase, Leukotrienesynthase) auch Hepoxilinsynthase-Aktivität aufweisen. / 12/15-Lipoxygenases (human 15-LOX-1, rat 12/15-lipoxygenase) belong to family of lipid peroxidising enzymes. The enzyme has been implicated with roles in a variety of pathological conditions such as asthma, atherosclerosis, inflammation and in cellular differentiation. The enzyme expression in most human cell types is tightly regulated by Th2 cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13). Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. 15-LOX-1 mRNA expression was first observed only 12h post IL-4 stimuation and required a minimum of 11h exposure to the cytokine. The mechanism of 15-LOX-1 induction in A549 lung epithelial cells and the observed delay was studied and it was found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, it was observed that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9-11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, the results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9-11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription. The proinflammatory cytokine IL-4 is secreted in large amounts during allergic inflammatory response in asthma and plays a pivotal role in the airway inflammation. IL-4 has been shown to up-regulate 15-lipoxygenase and produce 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in A549 cells via the Janus kinase/STAT6 pathway under coactivation of CREB binding protein/p300. IL-4 has also been shown to up-regulate peroxisome proliferator-activated receptor (PPARg ) nuclear receptors in macrophages and A549 cells. In this study it is observed that 15(S)-HETE binds to PPARg nuclear receptors and induces apoptosis in A549 cells. Moreover, pre-treatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARg activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARg complex with the adapter protein Fas-associating protein with death domain and caspase-8, as shown by transfection of Fas-associating protein with death domain dominant negative vector and cleavage of caspase 8 to active subunits p41/42 and p18. Whereas IL-4 and PPARg ligands failed to induce cleavage of Bid and release of cytochrome c from mitochondria, they caused translocation of the proapoptotic protein Bax from cytoplasm to mitochondria with a concomitant decrease in the Bcl-XL level. The cells were, thereofre, observed to follow the extrinsic pathway of apoptosis where caspase-8 directly activates the effector caspase-3, bypassing the mitochondria. The data also suggests that in IL-4-stimulated cells the 15(S)-HETE/PPARg complex down-regulates Bcl-XL, and the translocation of Bax to the mitochondria commits the cell to apoptosis. The IL-4-induced apoptosis may contribute to severe loss of alveolar structures and infiltration of eosinophils, mononuclear phagocytes, etc., into the lung tissue as observed in chronic asthma patients. The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), were observed to produce solely hepoxilin A3 (HXA3). Since HXA3 synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, suggesting that a HXA3 synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption AA was incubated with HeLa cells overexpressing the rat 12/15-LOX. Neither HXA3 nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA3 synthase and 12-LOX activities. Moreover, recombinant rat 12/15-LOX produced HXA3 when incubated with 12-HpETE. Further confirmation was obtained by immunoprecipitation with 12/15-LOX specific antibodies. Immunoprecipitation of Rinm5F lysates results in the depletion of hepoxilin synthase activity. The hepoxilin synthase activity was localised in the immunoprecipitated protein. Thus, cells containing rat 12/15-LOX also possess an intrinsic HXA3 synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA3 is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as Rinm5F. Furthermore, formation of corresponding epoxyhydroxy products was observed when 15-HpETE was used as substrate, indicating a broad range of specificity for the enzyme.

Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.

Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.