Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells.
12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.
Nie
Daotai
D
Department of Radiation Oncology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, Michigan 48202, USA.
Krishnamoorthy
Sriram
S
Jin
Rongxian
R
Tang
Keqin
K
Chen
YuChyu
Y
Qiao
Yan
Y
Zacharek
Alex
A
Guo
Yande
Y
Milanini
Julie
J
Pages
Gilles
G
Honn
Kenneth V
KV
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D002867:Chromones / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology / Q000473:pathology; D044950:Flavanones / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008297:Male; D009637:Masoprocol / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000098:blood supply* / Q000201:enzymology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D002867:Chromones; D044950:Flavanones; D016859:Lipoxygenase Inhibitors; D009637:Masoprocol; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D042461:Vascular Endothelial Growth Factor A; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C006680:baicalein