Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis.
The effect of overexpression of 15-lipoxygenase-1 (15-LO-1) was studied in the human prostate cancer cell line, PC-3. Stable PC-3 cell lines were generated by transfection with 15-LO-1-sense (15-LOS), 15-LO-1-antisense (15-LOAS) or vector (Zeo) and selection with Zeocin. After characterization by RT-PCR, western and HPLC, a PC3-15LOS clone was selected that possessed 10-fold 15-LO-1 enzyme activity compared with parental PC-3 cells. The PC3-15LOAS clone displayed little or no 15-LO-1 activity. These PC-3 cell lines were characterized for properties of tumorigenesis. The proliferation rates of the cell lines were as follows: PC3-15LOS > PC-3 = PC3-Zeo > PC3-15LOAS. Addition of a specific 15-LO-1 inhibitor, PD146176, caused a dose-dependent inhibition of proliferation in vitro. Overexpression of 15-LO-1 also caused [(3)H]thymidine incorporation to increase by 4.0-fold (P < 0.01). Compared with parental and PC-3-Zeo cells, PC3-15LOS enhanced whereas PC3-15LOAS reduced the ability of PC-3 cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. These data suggested a pro-tumorigenic role for 15-LO-1 in PC-3 cells in vitro. Therefore, to clarify the role of 15-LO-1 in vivo, the effect of 15-LO-1 expression on the growth of tumors in nude mice was investigated. The PC-3 cell lines were inoculated subcutaneously into athymic nude mice. The frequency of tumor formation was increased and the sizes of the tumors formed were much larger in the PC3-15LOS compared with PC3-15LOAS, parental PC-3 and PC-3-Zeo cells. Immunohistochemistry for 15-LO-1 confirmed expression throughout the duration of the experiment. The expression of factor VIII, an angiogenesis marker, in tumor sections was increased in tumors derived from PC3-15LOS cells and decreased in those from PC3-15LOAS cells compared with tumors from parental or Zeo cells. These data further supported the evaluation by ELISA of vascular endothelial growth factor (VEGF) secretion by PC-3 cells in culture. Secretion of this angiogenic factor was elevated in PC3-15LOS cells compared with the other cell lines. These results support a role for 15-LO-1 in a novel growth-promoting pathway in the prostate.
Kelavkar
U P
UP
Department of Anatomic Pathology, Emory University, 1639 Pierce Drive, Atlanta, GA 30322, USA. kelavkar@emory.edu
Nixon
J B
JB
Cohen
C
C
Dillehay
D
D
Eling
T E
TE
Badr
K F
KF
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid; D003114:Colony-Forming Units Assay; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000378:metabolism; D004790:Enzyme Induction; D005169:Factor VIII / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D019787:Linoleic Acid / Q000378:metabolism; D008041:Linoleic Acids / Q000378:metabolism; D008222:Lymphokines / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008041:Linoleic Acids; D008222:Lymphokines; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C024348:13-hydroxy-9,11-octadecadienoic acid; D005169:Factor VIII; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase