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Carcinogenesis, Vol. 22, No. 11, 1765-1773, November 2001
© 2001 Oxford University Press

CANCER BIOLOGY

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

Uddhav P. Kelavkar,4, Jennifer B. Nixon2, Cynthia Cohen1, Dirk Dillehay1, Thomas E. Eling2 and Kamal F. Badr3

Renal Division and
1 Department of Anatomic Pathology, Emory University, 1639 Pierce Drive, Atlanta, GA 30322, USA,
2 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health, Research Triangle Park, NC 27709, USA and
3 Department of Internal Medicine, American University of Beirut, Lebanon


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.

Abbreviations: 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.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Cancer of the prostate (PCa) is the most commonly diagnosed malignancy among men in the United States and Europe, killing thousands every year (1). In year 2001, approximately 31 900 men will die of the disease, accounting for over 35% of all cancers affecting men. Metastatic prostate cancer responds initially to androgen withdrawal therapy, but hormone resistance always develops (2). Chemotherapeutic agents currently available have little or no impact on the survival of the patients with hormone-refractory prostate cancer. For this reason, metastatic prostate cancer almost always has a fatal outcome. The underlying molecular mechanism involved and therapies to ameliorate the progression phase of the disease is an active area of current research.

Aberrant expression of the enzymes that convert unsaturated fatty acid arachidonic acid (AA) and linoleic acid (LA) to bioactive lipid metabolites appears to significantly contribute to the development of PCa. Many studies relating the development of cancer to lipid metabolizing enzymes have focused on the increased expression of COX-2 in tumor tissue (2–6). COX-2 is expressed in human prostate cancer (4), but the lipoxygenase enzymes that convert unsaturated fatty acids to hydroxylated metabolites also appear to play a role in prostate cancer. For example, 12-LO (7,11–15) promotes tumor cell adhesion and endothelial cell transmigration, and may contribute to metastasis (12–14) and tumor growth via angiogenesis (15). In addition, Ghosh and Myers (8,16) reported selective 5-LO inhibitors but not 12-LO or COX inhibitors suppress cell growth of PC-3 cells. Inhibitors of 5-LO and 5-LO-activating protein (FLAP) (17) can also induce apoptosis in PC-3 and LNCaP cell lines.

Recently, we have reported high expression of 15-LO-1 in human prostate tumors (18). The levels of expression of 15-LO-1 appeared to correlate with the Gleason score of the cancer. The higher the Gleason score is, the higher the expression of 15-LO-1. Others have reported that 15-LO-2 is expressed in normal prostate tissue, but poorly expressed in prostate tumors. The reduced 15-LO-2 expression is inversely correlated with the Gleason score of the tumor. Thus 15-LO-1 is highly expressed in prostate tumors while 15-LO-2 is highly expressed in normal tissue (10). 15-LO-1 in prostate cancer tumors converts LA, its preferred substrate to 13-S-hydroxyoctadecadienoic acid (13-(S)-HODE) and other metabolites. These metabolites appear to alter cellular signaling pathways (19–26), and thus the inappropriate expression might alter biological events and contribute to tumor development. For example, vascular homeostasis, cell growth and differentiation are altered by LA metabolites (24,27). The high expression of 15-LO-1 in PCa epithelium makes 15-LO-1 an attractive candidate as a key player in tumor development (18).

The goal of this study was to determine if the increase in 15-LO-1 expression might contribute to the malignant phenotype in prostate cancer. For this purpose, PC-3 cells were developed that overexpress 15-LO-1, termed PC3-15LOS (sense). Our data suggests that the 15-LO-1 and metabolic product, 13-(S)-HODE enhances cellular proliferation, increases growth in soft agar and increases tumor growth in the nude mouse model. In addition, vascular endothelial growth factor (VEGF) expression appears to be enhanced by 15-LO-1 expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
PC-3 parental prostate cancer epithelial cells (CRL-1435) were obtained from American Type Culture (ATCC; Manassas, VA) and cultured in complete RPMI medium (Gibco-BRL, Bethesda, MA) without phenol red, containing 10% fetal calf serum (FBS) per ml in 5% CO2 at 37°C. The cells were split every 3 days. The stable transfectants as described below, PC3-15LOS, PC3-15LOAS and PC3-Zeo (mock-transfected), were grown in medium containing 50 µg/ml Zeocin (Invitrogen, Carlsbad, CA).

Construction of engineered PC-3 cell lines
To obtain stable transfection in PC-3 cells, 15-LO-1 cDNA was inserted into the plasmid mammalian expression vector pcDNA3.1 (Invitrogen) (pcDNA3/15-LO-1S-sense; pcDNA3/15-LO-1AS-antisense) was performed. Briefly, a 1.7 kb full-length 15-LO-1 cDNA fragment was inserted into the EcoRI site of pcDNA3.1 and plasmids from clones were screened by sequencing for sense and antisense orientation plasmids. The pcDNA3.1 vector contains a constitutive cytomegalovirus enhancer-promoter and a Zeocin (antibiotic) resistance gene as a marker for selecting mammalian cells grown in the presence of Zeocin. The orientation of the insert in the pcDNA3.1 vector was confirmed by sequencing on an automated sequencer (Applied Biosystems 377), using fluorescent methodology. Vector constructs i.e. pcDNA3/15-LO-1S-sense; pcDNA3/15-LO-1AS-antisense and pcDNA3.1 used for transfection studies, to generate stable PC-3 cell lines, were purified with an Endofree plasmid kit (Qiagen, Valencia, CA).

Parental PC-3 cells were individually transfected with 10 µg of plasmid containing 15-LO-1 inserts in sense (pcDNA3/15-LO-1S-sense) and antisense (pcDNA3/15-LO-1AS-antisense) orientation and vector alone without insert (pcDNA3.1; for mock transfection) using FuGENETM 6 transfection reagent, respectively. Two days later, the transfected cells were split 1:15. Selection was then initiated with 50 µg/ml Zeocin (determined by the kill-curve assay) in the PC-3 cells to select cells that express resistance to this marker. Individual resistant clones were isolated 3 weeks later and expanded into cell lines. Transfected cells were maintained in the RPMI medium containing 10% FBS and 50 µg/ml Zeocin. After selection, resistant clones were chosen from the total population by limiting dilution to a single-cell/well. 15-LO-1 mRNA expression was stable along the passages. To screen and characterize cell lines for 15-LO-1 protein and RNA levels, western and RT–PCR were performed. One clone of each type i.e. PC3-15LOS, PC3-15LOAS and PC3-Zeo was selected and expanded into individual flasks. 15-LO-1 enzyme activity was determined in these cell lines as described below.

RT–PCR analysis
Briefly, RT–PCR of 15-LO-1 mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in PC3-15LOAS cells was performed using total RNA extracted by standard methodology (18). Total RNA (1 µg) is reverse transcribed using gene specific primers for 15-LO-1 and GAPDH as previously described. PCR products were separated by 2% agarose gel electrophoresis. For semi-quantification, DNA was stained with ethidium bromide and analyzed densitometrically.

Western blot analysis
Antibodies

The isotypes and specificities of monoclonal and polyclonal antibodies (Abs) are as follows: polyclonal CheY antibody-IgG1 specific for 15-LO-1 (obtained from Dr E.Sigal, CA), monoclonal antibody-IgG1 specific for MIB-1 (Ki67; Immunotech, FL) and antibody-IgG1 specific for factor VIII (Novocastra, CA).

SDS–PAGE and western blotting

Detection of 15-LO-1 by western blotting was performed similarly as previously described (18). Briefly, separated proteins on SDS gels were transferred onto individual PVDF membranes by electroblotting. Ponceau S staining of the blots was conducted to ensure equivalent loading. The nitrocellulose membranes were incubated with their respective antibodies (1:10 000 dilution) for 1 h at room temperature. Following incubation with either goat anti-rabbit (1:8000 dilution) or donkey anti-rabbit (1:2000 dilution) IgG peroxidase second antibody, proteins were visualized by using the Luminol/Enhancer (ECL) solutions as described by the manufacturer. Samples were compared with 15-LO-1 standards and ß-actin.

Cell morphology
PC-3 cells (1x106) growing in complete RPMI medium for 3 days were observed for cellular morphology using a Zeiss LSM510 laser-scanning microscope using differential interference contrast (DIC) at 20x magnification. Scanned images were documented.

Determination of [3H]thymidine incorporation
PC-3 cell lines were grown on 6-well culture plates for 2 days. Cells were then starved in depletion buffer (RPMI containing 20 mmol/l HEPES pH 7.4, 0.2% BSA, 0.4% FBS) for 24 h, and then continuously cultured in the depletion medium that contained 1 µCi/ml 3H isotope (DuPont-New England Nuclear, Boston, MA). After 20 h of incubation, the medium was aspirated and cells were rapidly washed twice with 1 ml cold PBS solution and once with 1 ml 10% trichloroacetic acid (TCA) and incubated in 1 ml fresh 10% TCA at 4°C for 30 min.

TCA-insoluble material was washed twice with 95% ethanol and fixed cellular material was solubilized in 0.1 N NaOH at 24°C for 2 h. Samples were divided into six wells. Three wells were used for incorporation measurements and three wells were used for cell counting. The 3H-isotope incorporation was determined by liquid scintillation spectrometry. Cells were counted with a Coulter counter. The data were normalized as counts per minute (c.p.m.) per 106 cells and finally expressed as the fold over mock condition.

Cell proliferation (MTT) and inhibition assay
Cell proliferation was measured in PC-3 cells using the Boehringer Mannheim Cell Proliferation kit (MTT) according to manufacturer's instructions. Briefly, after cell growth for 72 h, the MTT labeling reagent was added and the OD at 490 nm determined 24 h later. The corrected absorbance (control blanks) was used to determine increase in cell proliferation.

For inhibition assays, various concentrations (0.1, 0.5 and 1 µM in 0.1% ethanol in PBS) of PD146176 (28) (experimental) versus 0.1% ethanol in PBS only (control) were added on day zero and MTT assays performed on cells grown for 72 h. Cells grown in parallel were also harvested and survival estimated from those that excluded 0.2% trypan blue (29). Cell numbers, however, did not fall below those seeded at day 1 indicating that PD146176 exerted no cytotoxicity effects.

Growth in soft agar
PC-3 cells were suspended in 0.3% agar with complete RPMI medium, plated at a density of 1x104 cells per 85-mm dish, previously coated with 0.5% agar and maintained at 37°C. On day 20, plates were stained with the 1 ml vital stain 2-(p-isodophenyl)-3-(p-nitrophenyl)-5-phenyltertazolium chloride hydrate (250 mg/l in PBS) (Sigma, St Louis, MO) for 24 h as previously described (30). Colonies were counted and divided into small (<10 cells) and large (>50 cells) colonies. Colonies >150 µm in diameter were scored using a microscope at x10 magnification.

Animal experiments
Animal experiments were performed according to the guidelines of the Committee on Experimental Animals of Emory University. Athymic male BALB/C nude (nu/nu) mice (6–8 weeks old), congeneically inbred, were obtained from Charles River (Wilmington, MA). They were housed in single sterile animal cages under laminar flow hoods in a temperature controlled room with a 12 h light/dark schedule and fed autoclaved chow and water ad libitum. All mice were maintained in the pathogen-free Biogen animal facility (BSL-II facility) for at least 2 weeks before each experiment. Genetically engineered PC-3 derivatives PC3-15LOS, PC3-15LOAS, PC3- Zeo (mock-transfected) and parental PC-3 cells were individually injected subcutaneously (s.c.) (2x106 cells in 100 µl PBS—counted in a microcytometer chamber) for these studies. The shortest and longest diameter of the tumors was measured with callipers and tumor volume (cm3) was calculated using the following standard formula: (the shortest diameter)2 x (the longest diameter)x0.5.

There were no significant differences in body weight among the groups throughout the experiment. Tumor volumes were measured twice a week for 6 weeks. Animals were killed after 6 weeks, the tumors harvested, cut into two equal parts and placed in liquid nitrogen or in a –80°C freezer. All tumors were subjected to western analysis, 15-LO-1 enzyme activity assay and immunohistochemical analysis. Simultaneously, organs such as spleen, liver and lung were analyzed immunohistochemically [hematoxylin and eosin (H&E) staining] for metastasis.

Immunohistochemical analysis of tumor samples from athymic mice
Sections of formalin-fixed, paraffin-embedded tissue (5 µ) were tested for the presence of 15-LO-1 [1:1600], MIB-1 [1:50] and factor VIII [1:20], using an avidin–biotin complex technique and steam heat-induced antigen retrieval as previously described (18).

Image cytometric of 15-LO-1, factor VIII and MIB-1 quantitation
Quantitation of 15-LO-1, factor VIII and MIB-1 was performed as described previously (18). For angiogenesis study, sections were graded for the extent of neovascular formation (NVES). Grading: 0, no vessels; 1, scattered vessels; 2, minimal vessels per tissue section; and 3, maximal vessels per tissue section. Results were calculated as a mean of angiogenesis score. Weidner et al. (31,32) have studied angiogenesis in a large and diverse array of other tumors, including breast, melanomas, gliomas, lung, bladder and prostate cancers using factor VIII (von Willibrand factor), CD-31 or CD-34, respectively.

Tissue incubations and HPLC analysis
Five to 10 mg amount of either PC-3 cells grown in vitro or PC-3 cell derived tumor tissues from nude mice were individually analyzed for 15-LO-1 enzyme activity similar to a method previously described (18). Straight phase analyses were performed. Authentic standards of 12(S)-HETE, 15(S)-HETE, 13(S)-HODE, 9(S)-HODE and PGB1 were obtained from Cayman (Ann Arbor, MI) and used. Radiolabeled [14C]linoleic acid (40–60 mCi/mmol) was from DuPont-New England Nuclear. All solvents were HPLC grade and were from Baker (Phillipsburg, NJ).

Vascular endothelial growth factor (VEGF) analysis by ELISA
PC-3 cells (1.5x106) were grown in complete RPMI medium (containing 10% FBS) in triplicate for 2 days until they were 80–90% confluent. The growth medium was removed, cells briefly washed with RPMI medium without FBS and equal volumes of fresh RPMI medium containing 2% FBS was added and cells were allowed to grow further for 24 h. The medium was harvested and tested for VEGF using a Quantakine Human VEGF ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Results were expressed as picograms per milliliter (pg/ml) of growth medium. This experiment was performed in triplicate twice. The final values of VEGF concentration shown are subtracted values from control (i.e. RPMI medium containing 2% FBS).

Statistical analysis
All experimental data are analyzed statistically by Kendall's tau and Fisher's exact tests.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Characterization of 15-LO-1, sense and antisense PC-3 cells
We have chosen the PC-3 cell line as our model (33) for this study because of the very low level of 15-LO-1 expression, the cells are androgen-independent and PC-3 cell line itself produces aggressive undifferentiated tumors in nude mouse models. In order to study the biological function of 15-LO-1, we generated stable transfectants of PC-3 cells that over-express 15-LO-1. We obtained >200 ({approx}70 colonies/cell line) resistant colonies, and by RT–PCR and western analysis, we chose colonies individually (data not shown) expressing 15-LO-1 in the sense (15-LOS) and antisense (15-LOAS) orientations. PC-3 parental, PC3-Zeo (mock-transfected), PC3-15LOS and PC3-15LOAS cells (Figure 1AGo, lanes 4 and 5) were tested for 15-LO-1 expression by RT–PCR as shown in Figure 1AGo. 15-LO-1 mRNA was easily detected in the PC3-15LOS and PC3-15LOAS cells. Furthermore, 15-LO-1 protein expression analyzed by western blot analysis, as shown in Figure 1BGo, is highly expressed only in PC3-15LOS (Figure 1BGo, lane 5), whereas 15-LO-1 protein is undetectable in PC3-15LOAS, PC-3 parental and PC3-Zeo cells, respectively. However, we have shown previously that 15-LO-1 is detected in parental PC-3 cells by immunoprecipitation (18).



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  		Fig. 1. (A) RT–PCR analysis of 15-LO-1 transcript. Lane 1, PCR control with 15-LO-1 cDNA; lane 2, parental PC-3 cells; lane 3, cells transfected with empty vector (PC3-Zeo); lane 4, PC3-15LOAS cells; lane 5, PC3-15LOS cells. The band intensities were compared with GAPDH. (B) Western blot analysis of 15-LO-1 protein expressed in PC-3 cell lines (indicated by arrows). Total RNA and protein was isolated from these cell lines. RT–PCR of RNA and western analysis of protein was performed as described in Materials and methods. Lane 1, standard 15-LO-1 protein; lane 2, parental PC-3 cells; lane 3, cells transfected with empty vector (PC3-Zeo); lane 4, PC3-15LOAS cells; lane 5, PC3-15LOS cells. The band intensities were compared with ß-actin protein.

 
15-LO-1 enzymatic activity
The ability of PC-3 cells to form 13-hydroxyoctadecadienoic acid (13-HODE) from exogenous linoleic acid (LA), the preferred substrate of 15-LO-1, was used to confirm that the expressed 15-LO-1 in PC3-15LOS cells has enzymatic activity. Exogenous LA was metabolized and converted to several metabolites by lysates prepared from PC3-15LOS cells with the major metabolite, 13-HODE, eluting as a peak at 66–68 min retention time. The metabolite co-eluted with an authentic standard of 13-HODE (Figure 2Go). There is a >10-fold increase in 15-LO-1 enzyme activity in PC3-15LOS cells compared with PC3-15LOAS, parental PC-3 and PC3-Zeo cells. The formation of 13-HODE was inhibited by the addition of NDGA, a lipoxygenase inhibitor, but not by the addition of the prostaglandin H synthase inhibitor, indomethacin (data not shown).



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  		Fig. 2. Enzyme (15-LO-1) activity profile of linoleic acid metabolite products from PC-3 cells was measured as described in Materials and methods. Arrows indicate 13-HODE and prostaglandin (PG) peaks.

 
Effect of 15-LO-1 overexpression on cell growth
As shown in Figure 3Go, a 2–3-fold increase in growth of PC-3 (parental), PC3-Zeo (control) and 15-LO-1AS was observed when supplemented with 13-(S)-HODE (34 µM) in growth medium. 13-HODE did not alter the growth of PC3-15LOS cells. Addition of linoleic acid (LA), which is present in the serum, did not alter the growth rate of any of the cells. Overall, PC3-15LOS grew the fastest, and adding 13-(S)-HODE only slightly increased the growth. This suggests that sufficient 13-(S)-HODE was formed by the 15-LO-1 present in these PC3-15LOS cells to maintain cell growth at maximum proliferation levels.



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  		Fig. 3. 13-(S)-HODE and linoleic acid (LA) effects on PC-3 cell proliferation rates after 72 h. 13-(S)-HODE is added at a concentration of 34 µM and LA at 50 µM and MTT assay performed. The data shown are means ± SE of six determinations. *Significant difference at P < 0.01 versus control.

 
Addition of PD146176 (obtained from Parke-Davis, MI), a specific inhibitor of 15-LO-1 (28), inhibited the growth of PC-3 cell lines in 72 h in a concentration-dependent manner with an optimum concentration of 1 µM (Figure 4Go). The inhibition data also points to an important observation. PD146176 did not cause significant inhibition in growth of PC3-15LOAS cells, which do not express 15-LO-1 enzyme. This result suggests that the reduction in cell growth by PD146176 was dependent on 15-LO-1 overexpression and enzyme activity. The data also support the hypothesis that 15-LO-1 alters the growth characteristic of PC-3 cells



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  		Fig. 4. Inhibition of PC-3 cells by the 15-LO-1 specific inhibitor, PD146176. Cells were exposed to increasing concentrations of PD146176 (0, 0.1, 0.5 and 1 µM in 0.1% ethanol in PBS) and cell death is determined using the trypan blue assay. Values represent the mean percentage of cell viability from three independent experiments (in duplicate).

 
To further explore the effect of 15-LO-1 overexpression on cell growth, PC-3 clones were incubated with [3H]thymidine to evaluate DNA synthesis. Figure 5Go, illustrates that the overexpression of 15-LO-1 increases thymidine incorporation by ~4-fold (3.9 ± 0.3-fold, n = 3 in triplicate, P < 0.01) compared with other cell lines. We propose that PC-3 cells overexpressing 15-LO-1 retain characteristics of PC-3 cells and have been conferred with a more aggressive growth rate compared with other PC-3 cells.



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  		Fig. 5. Effect of 15-LO-1 overexpression on PC-3 cell growth. All PC-3 cell lines were grown on 6-well plates with 10% FBS for 2 days. Cells were depleted with RPMI containing 20 mmol/l HEPES pH 7.4, 0.2% BSA and 0.4% FBS. After depletion for 24 h, cells were continuously cultured in the depletion medium containing 1 µCi/ml [3H]thymidine for a further 20 h. Cells were washed and treated for determination of 3H isotope incorporation as described in Materials and methods. Each point is an average (mean ± SEM) from three separate samples. *P < 0.01 versus mock; n = 4.

 
Cell morphology and anchorage independent growth of PC-3 cells
Cell morphology of PC-3 cell lines was examined under the microscope. PC3-15LOS cells were markedly larger in size (Figure 6CGo) compared with other cell lines (Figure 6A, B and DGo). PC3-15LOAS cells show rounding up (blebs as shown by arrows in Figure 6DGo) after 72 h, whereas none were observed in either parental PC-3, PC3-15LOS or mock-transfected PC3-Zeo cells. Anchorage independent growth is considered as an in vitro test for tumorigenesis. Therefore, the PC-3 cells were also examined for anchorage independent growth in a semisoft agar medium. All PC-3 cell lines exhibited anchorage independent growth in soft agar. As shown in Table IGo, the PC3-15LOS cells were able to form more colonies >150 µm (83.0 ± 12.0 colonies per 104 cells plated) in soft agar compared with other PC-3 cell lines used. Moreover, there is a significant difference (P < 0.01) in the ability to form colonies in agar between PC3-Zeo (mock-transfected) (20.0 ± 2.0 colonies/104 cells plated) compared with PC3-15LOS cells. However, the ability of PC3-15LOAS cells to form colonies in agar was reduced (15.0 ± 3.0 colonies/104 cells plated). These data suggest a correlation between 15-LO-1 expression in PC-3 cells and an increased survival/growth advantage in anchorage-independent conditions.



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  		Fig. 6. Morphology of PC-3 cell lines. Cells (1x106) were cultured on a tissue culture flask and incubated in complete RPMI medium at 37°C for 72 h. After washing with PBS, the cells were observed at 20x magnification using a laser scanning microscope as described in Materials and methods. (A) Parental PC-3; (B) PC3-Zeo (mock-transfected); (C) PC3-15LOS; and (D) PC3-15LOAS cells. Bar represents 50 µm.

 

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  		Table I. Number of PC-3 cell colonies per 104 cells in soft agar
 
Tumorigenesis in nude mouse model
Since 15-LO-1 expression enhanced proliferation of PC-3 cells and their ability to grow on soft agar, we investigated whether 15-LO-1 overexpression would alter the ability of PC-3 cells to form tumors in nude mice. PC3-15LOS, PC3-15LOAS, PC-3 parental and PC3-Zeo cells were injected subcutaneously [5x106 cells (in PBS)/animal] into 10 individual athymic mice. Control mice were injected with sterile PBS only. The mice were observed for 6 weeks to monitor tumor growth. One mouse from each set of 10 mice injected with PC3-15LOS and parental PC-3 died in the third week. However, the frequency of tumor formation was increased and the average tumor size at 42 days was larger in PC3-15LOS (8/10) [Figures 7 and 8GoGo] compared with PC3-15LOAS (1/10) mice. Control mice did not show any tumor growth. Also, there was no significant difference in the weight of mice among groups. After 6 weeks, the mice were killed, the tumors harvested, cut into two equal parts and immediately placed either in liquid nitrogen or in a –80°C freezer. Organs such as spleen, liver, lungs and vertebra were removed and analyzed for metastasis by H&E staining.



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  		Fig. 7. Representative athymic mice showing tumor growth by PC-3 cells. Mice were individually injected subcutaneously with PC-3 cells (2x106 in 100 µl PBS) and monitored for tumor growth for 6 weeks. (A) Two mice injected with PC3-15LOAS, one showing a small tumor; (B) the upper mouse represents mice injected with parental PC3 cells and the lower represents those injected with PC3-Zeo cells; and (C) mice injected with PC3-15LOS both show large tumors. Arrows indicate location of tumors.

 


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  		Fig. 8. Average tumor volumes of tumors in nude mice. Mice injected with PC-3 cells were monitored for tumor growth. Tumor volumes (cm3) were measured twice a week for 6 weeks.

 
Histological and immunohistochemical findings
Histopathological examinations (by H&E staining) from a fraction of whole tumor tissues revealed no apparent differences in morphology of the tumor tissues among all groups of mice studied. Tumor cells from all xenografts had clear cytoplasm and pale, round nuclei and did not contain mucus. Also there were no significant differences in the histological score of necrosis among the groups. Metastasis was undetectable in spleen, liver, lungs and vertebral bone (data not shown). Immunohistochemistry for 15-LO-1, Ki-67 (proliferation marker) and factor VIII (angiogenesis marker) in tumors, quantified as previously described by us (18), indicate levels as PC3-15LOS > PC3 = PC3-Zeo > PC3-15LOAS, respectively (Figure 9Go). Interestingly, there is an increase in angiogenesis in tumors formed from 15-LO-1-expressing PC-3 cells (Figure 9Go; panel 2B) as compared with those formed from parental PC-3 cells (data not shown). The level of angiogenesis was identical to PC3-Zeo (Figure 9Go; panel 1B) and significantly reduced in tumors formed by PC3-15LOAS cells (Figure 9Go; panel 3B), as demonstrated by factor VIII immunostaining. These data strongly suggests that 15-LO-1 overexpression may cause an increase in angiogenesis.



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  		Fig. 9. Immunostaining (brown) of tumors from nude mice (representative) with polyclonal antibodiies for 15-LO-1, factor VIII (as an angiogenesis marker) and MIB-1 (as a proliferation marker for Ki67) (100x). Panel 1, PC3-Zeo; panel 2, PC3-15LOS; panel 3, PC3-15LOAS. (A) 15-LO-1; (B) factor VIII (angiogenesis) and (C) MIB-I (proliferation).

 
Vascular endothelial growth factor (VEGF) secretion by PC-3 cells
To obtain additional evidence to support the hypothesis for an increase in angiogenesis in the tumors, the angiogenic factor VEGF was measured by ELISA. As shown in Table IIGo, the level of VEGF secreted into the media by PC3-15LOS cells was 3–4-fold (1129 ± 44 pg/ml) higher compared with parental PC-3 (363 ± 22 pg/ml) and PC3-Zeo cells (380 ± 25 pg/ml). However, PC3-15LO-AS cells secreted similar VEGF (330 ± 32 pg/ml) levels compared with parental PC-3 and P3-Zeo cells but less compared with PC3-15LOS cells (Table IIGo). These results suggest that 15-LO-1 overexpression causes an increase in VEGF levels in PC-3 cells; also supported by observations in the tumor immunohistochemistry analyses. This observation also provides evidence that the expression of 15-LO-1 in prostate cancer increases tumor growth, possibly via an increase in angiogenesis.


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  		Table II. Vascular endothelial growth factor (VEGF) analysis by ELISA
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The presence of 15-LO-1 at high levels in PCa epithelium and the correlation between expression and Gleason grade suggests 15-LO-1 might be a key enzyme that contributes to the development of the malignant phenotype in PCa (18). To identify the possible role of 15-LO-1 in PCa cancer, we have developed human prostate epithelial cell lines (using parental PC-3 cells) that overexpress 15-LO-1 or express 15-LO-1 in the antisense orientation. We confirmed by RT–PCR and western analysis that 15-LO-1 is expressed in PC3-15LOS cell lines and confirmed by HPLC analysis a 10-fold greater 15-LO-1 activity in PC3-15LOS cells compared with PC3-15LOAS, parental PC3 and PC3-Zeo cells. The expression of 15-LO-1 appears to confer on the cells a clear growth advantage. The addition of the metabolic product of 15-LO-1, 13-(S)-HODE, to the growth medium caused a 2–3-fold increase in the growth of PC-3 (parental), PC3-Zeo (control) and PC3-15LOAS cells, but incubation with the substrate of 15-LO-1, LA, did not alter growth. This result is in agreement with the low 15-LO-1 activity of the cells and suggests 13-HODE is responsible for the biological response. PC3-15LOS cells, which overexpress 15-LO-1, exhibited a high growth rate that was reduced by the addition of a specific 15-LO-1 inhibitor. PC3-15LOS cells demonstrated an increased ability to grow in agar compared with other PC-3 cells suggesting an increase in tumorigenicity of these cells. This was further supported by tumor formation in the nude mouse model. PC3-15LOS cells yield a greater number of tumors and a larger tumor volume compared with parental PC-3, PC3-Zeo (controls) and PC3-15LOAS cells. The expression of 15-LO-1 did not appear to alter metastases since none were detectable in the lungs, liver, lymph nodes and bones (vertebra) of nude mice.

How 15-LO-1 expression alters the growth characteristic of PC-3 cells in not known. 13-(S)-HODE, formed by 15-LO-1, up-regulates the epidermal growth factor receptor (EGFR) signaling pathway, which enhances growth, but can also serve as a ligand for peroxisome proliferation receptor (PPAR{gamma}) (11,24,34). In addition, our results also suggest that the expression of 15-LO-1 up-regulated angiogenesis, which might play a key role in the enhanced tumorigenesis. In PC-3-derived tumor tissues immunostained for 15-LO-1, angiogenesis (factor VIII) and proliferation (MIB-1), we observed that tumors with high levels of 15-LO-1 expression display greater angiogenesis [microvessel number (NVES)] compared with other tumors. What is particularly intriguing is that the PC3-15LOAS-derived tumor tissue (evaluated from the one mouse out of 10 that developed a small tumor) displayed a drastic reduction in angiogenesis. Overall tumors revealed: PC3-15LOS > PC3 = PC3-Zeo > PC3-15LOAS when analyzed for levels of 15-LO-1, MIB-1 and factor VIII, respectively. Enhanced angiogenesis is support by a higher level of expression of angiogenic factor VEGF in human PC-3 cells overexpressing 15-LO-1. Therefore, overexpression of 15-LO-1 in PC-3 cells might provide favorable conditions for angiogenesis and malignant growth of tumors in nude mice.

One potential mechanism could be that the 13-(S)-HODE signaling cascade acts as a potent and powerful survival factor for endothelial cells of newly formed immature blood vessels. It is clear that in vivo growth of tumors is dependent upon angiogenesis (35) and that the formation of new blood vessels is critical to this process (36). The PC-3 cell line that overexpresses 15-LO-1 also secretes high levels of vascular endothelial growth factor (VEGF) as compared with parental PC-3 cell lines. Further, the cell line expressing antisense 15-LO-1 showed the lowest level of VEGF production. VEGF expression patterns in this study were also correlated with the ability of the PC-3 epithelial cell lines to form tumors in nude mice and to initiate angiogenesis. Although it is unknown at present whether 13-(S)-HODE is present in endothelial cells or not, it is indeed intriguing to speculate that 13-(S)-HODE secreted by proliferating prostate epithelial cells could favor angiogenesis in endothelial cells. Thus we also hypothesize that high concentrations of 13-(S)-HODE could be an apoptosis survival factor for endothelial cells. Hence, it is theorized that PCa epithelial cells overexpressing 15-LO-1, being androgen-independent and unresponsive to anti-androgen therapy, might be responsive to 15-LO-1 inhibitors. This in turn would result in rapid apoptosis of the endothelial cells comprising immature tumor vessels causing a secondary, but much more massive, wave of apoptotic cell death in tumor cells surrounding the regressive/dying vessels. This secondary cell death process could lead to the regression of tumor mass. Although this hypothesis has yet to be proven, it is interesting to note that 15-LO-1 expression levels do correlate with more aggressive tumors.

Our observations in this report using PC-3 cell lines suggest that overexpression or inappropriate expression of 15-LO-1 in prostate epithelial cells cause proliferation and aggressive tumors. This study underscores the importance of 15-LO-1 overexpression in PCa and warrants further investigation.


   Notes
 
4 To whom correspondence should be addressed Email: kelavkar{at}emory.edu Back


   Acknowledgments
 
This work was supported in part by American Cancer Society–Winship Cancer Center seed grant (to U.P.K). We thank the histopathology laboratory for tissue sectioning, Diane Lawson and Debbie Sexton for immunohistochemistry (Emory University, GA) and Mark Geller for HPLC analysis (NIEHS, NC).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 27, 2001; revised July 5, 2001; accepted July 12, 2001.


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