Journal Article

Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis

Uddhav P. Kelavkar, Jennifer B. Nixon, Cynthia Cohen, Dirk Dillehay, Thomas E. Eling and Kamal F. Badr

in Carcinogenesis

Volume 22, issue 11, pages 1765-1773
Published in print November 2001 | ISSN: 0143-3334
Published online November 2001 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/22.11.1765
Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis

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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 [3H]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.

Keywords: AA, arachidonic acid;; COX, cyclooxygenase;; ELISA, enzyme-linked immunosorbent assay;; HETE, hydroxyeicosatetraenoic acid;; HODE, hydroxyoctadecadienoic acid; LA, linoleic acid; 15-LO-1, 15-lipoxygenase-1; LO, lipoxygenase; PBS, phosphate-buffered saline; PC-3, prostate cancer cell line-3;; VEGF, vascular endothelial growth factor.

Journal Article.  5996 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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