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Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells.
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PMID:  22237009     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
12(S)-Lipoxygenase (LOX) and its product 12(S)-hydroxyeicosatetraenic (HETE) acid have been implicated in angiogenesis and tumour invasion in several tumour types while their role in colorectal cancer progression has not yet been studied. We have analysed 12(S)-LOX expression in colorectal tumours and found gene expression up-regulated in colorectal cancer specimens for which the pathology report described involvement of inflammation. Using cell line models exposed to 12(S)-HETE or over-expressing 12(S)-LOX malignant cell growth as well as tumour cell migration was found to be stimulated. Specifically, Caco2 and SW480 cells over-expressing 12(S)-LOX formed fewer colonies from sparse cultures, but migrated better in filter-migration assays. SW480 LOX cells also had higher anchorage-independent growth capacity and a higher tendency to metastasise in vivo. Knock-down or inhibition of 12(S)-LOX inhibited cell migration and anchorage-independent growth in both 12(S)-LOX transfectants and SW620 cells that express high endogenous levels of 12(S)-LOX. On the cell surface E-cadherin and integrin-β1 expression were down-regulated in a 12(S)-LOX-dependent manner disturbing cell-cell interactions. The results demonstrate that 12(S)-LOX expression in inflammatory areas of colorectal tumours has the capacity to induce an invasive phenotype in colorectal cancer cells and could be targeted for therapy.
Authors:
T Klampfl; E Bogner; W Bednar; L Mager; D Massudom; I Kalny; C Heinzle; W Berger; S Stättner; J Karner; M Klimpfinger; G Fürstenberger; P Krieg; B Marian
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-12-29
Journal Detail:
Title:  Experimental cell research     Volume:  318     ISSN:  1090-2422     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  2012 Apr 
Date Detail:
Created Date:  2012-03-05     Completed Date:  2012-04-27     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  768-78     Citation Subset:  IM    
Copyright Information:
Copyright © 2011 Elsevier Inc. All rights reserved.
Affiliation:
Department of Internal Medicine 1, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090 Vienna Austria.
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MeSH Terms
Descriptor/Qualifier:
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / metabolism
Arachidonate 12-Lipoxygenase / genetics*,  metabolism
Caco-2 Cells
Cell Movement / genetics*
Colorectal Neoplasms / enzymology*,  genetics*,  metabolism
Humans
Phenotype
Tumor Cells, Cultured
Up-Regulation*
Chemical
Reg. No./Substance:
59985-28-3/12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; EC 1.13.11.31/Arachidonate 12-Lipoxygenase
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Full Text
Journal Information
Journal ID (nlm-ta): Exp Cell Res
Journal ID (iso-abbrev): Exp. Cell Res
ISSN: 0014-4827
ISSN: 1090-2422
Publisher: Academic Press
Article Information
© 2012 Elsevier Inc.
License:
Received Day: 11 Month: 11 Year: 2011
Revision Received Day: 15 Month: 12 Year: 2011
Accepted Day: 21 Month: 12 Year: 2011
pmc-release publication date: Day: 01 Month: 4 Year: 2012
Print publication date: Day: 01 Month: 4 Year: 2012
Volume: 318 Issue: 6
First Page: 768 Last Page: 778
ID: 3314953
PubMed Id: 22237009
Publisher Id: YEXCR8904
DOI: 10.1016/j.yexcr.2011.12.017

Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells
T. Klampfla1
E. Bognera1
W. Bednara1
L. Magera
D. Massudoma
I. Kalnya
C. Heinzlea
W. Bergera
S. Stättnerb
J. Karnerb
M. Klimpfingerb
G. Fürstenbergerc
P. Kriegc
B. Mariana Email: brigitte.marian@meduniwien.ac.at
aDepartment of Internal Medicine 1, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090 Vienna Austria
bSocial Medical Centre South, Vienna, Austria
cGerman Cancer Research Centre, Heidelberg, Germany
Corresponding author at: Institute of Cancer Research, Department of Medicine 1, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria. Fax: + 43 1 4277 9651. brigitte.marian@meduniwien.ac.at
1T.K, E.B and WB. contributed to this manuscript equally.

Introduction

Lipoxygenases (LOX) and their eicosanoid products are involved in many physiological and pathophysiological processes like growth, differentiation, vascularisation, inflammation and arteriosclerosis [1,2]. Specifically, 12- and 15-LOX enzymes have been described as regulators of inflammation and immune-response [3]. Leukocyte, reticulocyte and epidermal forms of the enzyme are well characterised as to their product pattern and physiological function [3–5]. By contrast, the platelet-derived 12(S)-LOX (ALOX12) is still insufficiently understood. It produces almost exclusively 12(S)-hydroxyeicosatetraenic acid (HETE) [6,7] that has been shown to act as a growth and/or survival factor in gastric [8], pancreatic [9], melanoma and prostate tumour cells [10,11] and to induce a metastatic phenotype in prostate cancer [12]. In addition 12(S)-HETE may also affect other tissue components to enhance neovascularisation and tumour progression [10,12–15]. For this reason 12(S)-LOX is regarded as a protumorigenic gene [16]. Protumorigenic roles of LOX-enzymes are also suggested by the observation that inhibitors of LOX isoenzymes like quercetin, nordihydroguaretic acid or baicalein can protect from carcinogenesis in both the skin and the colon [17–19]. Such inhibitors are not specific for individual LOX enzymes, however, so that a more specific approach is needed to demonstrate the actual role of 12(S)-LOX in tumour development.

In colorectal cancer protumorigenic impact has been unequivocally demonstrated for cyclooxygenase 2 (COX-2) and its prostaglandin products [20] as well as for 5-LOX and its product leukotrien B4 [21,22]. Concerning 12(S)-LOX a genetic polymorphism that produces a more active enzyme is related to a higher cancer risk [23] suggesting that there is a protumorigenic impact of 12(S)-LOX on CRC. The pathophysiological impact of the enzyme on CRC has not yet been analysed in any detail, however. This study therefore, undertakes to determine the expression of 12(S)-LOX in colorectal carcinomas as compared to normal mucosa as well as the cell biological consequences of 12(S)-HETE and 12(S)-LOX expression in colorectal tumour cell lines.


Materials and methods
Tissue specimen

Tissue specimens were obtained from colorectal carcinomas and normal tumour-free mucosa at the resection margin from patients suffering from colorectal cancer. The study has been approved by the local ethics committee and the patients had given their informed consent. The tissue specimens were collected immediately after surgery and frozen in liquid N2 until extraction.

Cell lines and transfection

SW480, SW620, and Caco2 colon carcinoma cells were obtained from the American Type Culture Collection. The cell lines were kept under standard tissue culture conditions using Minimal Essential Medium (MEM) containing 10% fetal calf serum (FCS).

12(S)-LOX over-expressing SW480 and Caco2 cells were produced by electroporation as described before [24]. Populations stably over-expressing 12(S)-LOX were cultivated in the presence of 1.6 and 0.2 mg/ml geneticin (G418) respectively.

Eicosanoid mediators, inhibitors and cell treatment

12(S)-HETE (0.1 mg/ml in ethanol) was purchased from Cayman chemicals (Ann Arbor, MI). Arachidonic acid (AA) and baicalein were obtained from Sigma (St. Louis, MO). Cells were plated at a density of 5 × 104 in 24-well plates and left to attach for 24 h before exposure to the compounds diluted into HEPES-buffered MEM containing 1 mg/ml bovine serum albumin (BSA). Experiments on 12(S)-LOX transfectants and their respective controls were done in the presence of 10 μM AA to provide sufficient substrate for eicosanoid synthesis.

Growth parameters

Viable cell number was determined by neutral red uptake as reported previously [24].

For clonogenicity assays cells were plated at a density of 100 cells/well (SW480) or 200 cells/well (Caco2, SW620) into six-well plates in growth medium. Unattached cells were removed 24 h later and the cultures then left to grow for 7 days. The number of colonies was assessed after staining with crystal violet.

Anchorage-independent growth was determined from 5000 cells/well in 0.25% agar prepared in RPMI medium containing 20% FCS and incubated for 2–3 weeks before counting the number of colonies microscopically.

Cell migration assay

Cell migration was analysed by filter migration assay as described before [25]. Specifically, 0.5 × 105 cells/cm² were seeded into 8-μm-pore-size filters (Becton Dickinson-Falcon, Franklin Lakes, NJ) and migration periods of 24 h (SW480, SW620) or 48 h (Caco2) were permitted.

Tumour growth in vivo

SW480-LOX and SW480-co cells were harvested, washed with PBS, and suspended in Ringer's solution. 1 × 106 cells in 50 μl Ringer's solution were subcutaneously injected into the rear flanks of immunodeficient SCID/Balb/c recipient mice (female, aged 4 weeks, Harlan Winkelmann, Borchen, Germany). Tumour formation was monitored periodically by palpation, and the tumour size was determined using a Vernier caliper. Tumour volume was calculated using the formula (smaller diameter2 × larger diameter)/2. All experiments were performed in triplicate and carried out according to the Austrian and FELASA guidelines for animal care and protection. Tissue sections of experimental tumours were analysed by immunohistochemistry using antibodies directed against cytokeratin 20.

Mouse lungs were prepared for immunohistochemistry and stained with a monoclonal antibody recognising Ki67 (Novacastra, Leica Microsystems, Wetzlar, Germany). Serial sections were scored for Ki67-positive cells/field of vision and the following scores were assigned: < 1 cell/field = 0, 1–5 cells/field = 1; 5–10 cells/field = 2; 10–20 cells/field = 3; > 20 cells/field = 4.

Knock-down of 12(S)-LOX expression

To knock down 12(S)-LOX expression 400 pmol (Caco2 transfectants), 10 nmol (SW480 transfectants) or 30 nmol (SW620 cells) of an antisense phosphothioate oligonucleotide directed against 12(S)-LOX with the sequence 5′-CTCAGGAGGGTGTAAACA-3′ [26] was introduced by lipofection. Lipofectamin (Invitrogen Life Technologies, Carlsbad, CA) was used for Caco2 cells and siLentFect (BioRad, Hercules, CA) for SW480 and SW620 cells. Controls were transfected with a scrambled oligonucleotide, the sequence of which was 5′-AAGATT GCGCGACGATGA-3′ [26]. To determine the efficiency of 12(S)-LOX down-regulation the production of 12(S)-HETE was determined 48 and 96 h after transfection.

Reverse transcription-PCR analysis of gene expression

Total RNA was isolated from subconfluent cultures or frozen colon tissue specimens using Trifast (PeqLab, Erlangen, G) and chloroform extraction. 5 μg aliquots were used to prepare cDNA using RevertAid MMuLV reverse transcriptase (Fermentas, Burlington, Ontario) and random hexamer primers (GE Healthcare, Piscataway, NJ, USA).

The expression of 12(S)-LOX mRNA, E-cadherin, integrin β1 and GAPDH was then determined by real time PCR on an ABI PRISM 7000 system (Applied Biosystems, Foster City, CA, USA) using Taqman Gene expression assays listed in Table 2 of supplemental materials. Quantification of gene expression was calculated by the ΔΔCt method using GAPDH as the internal control gene. Selected experiments were also done by standard RT-PCR using the primers and conditions listed in Table 1 of supplemental materials.

E-cadherin and integrin β1 on the cell surface

Cells were harvested by limited trypsinisation and washed thoroughly. 106 cells were then re-suspended in 100 μl PBS. For detection of E-cadherin cells were incubated with 10 μg/ml mouse anti-human E-Cadherin (HECD-1; Alexis, San Diego) for 2 h on ice followed by a 1-h incubation with 20 μl of a phycoerythrin-labelled second antibody (Rat anti mouse—PE, Becton Dickinson). Detection of integrin β1 was done using a PE-labelled antibody recognising total integrin β1 (clone MAR4, Becton Dickinson). Control samples were incubated with control IgG. Analysis was performed using a FACScalibur (Becton Dickinson).

Western blot

Whole cell lysates were obtained by homogenising cultures in RIPA buffer (50 mM Tris/HCl pH 7.4, 500 mM NaCl, 1% NP-40, 0.5% Na-DOC, 0.1% SDS, 0.05% NaN3) supplemented with complete protease inhibitor mix (1:50; Roche Diagnostics, Mannheim, Germany). Integrin β1 protein levels were determined by western blotting from 50 μg of protein per lane using primary antibodies directed against integrin β1 (1:1000; 4706, Cell Signalling, Boston, MA) and β-actin (1:5000; AC-15, Sigma, St. Louis, MO, USA). Secondary antibodies were conjugated to horseradish peroxidase and detection achieved by chemiluminescence.

12(S)-HETE ELISA

5 × 104 cells/ well were plated into 24 well plates and 10 μM AA (Sigma) was added 24 h later. 12(S)-HETE production was determined from culture supernatants using an indirect ELISA assay purchased from R&D (Minneapolis, MN) according to the manufacturer's instructions. 100 μl of the supernatant were added to the plates together with an alkaline phosphatase-labelled HETE as a tracer and polyclonal anti-12(S)-HETE antibody. After 2 h of incubation at room temperature wells were washed extensively and bound tracer was detected photometrically.


Results
Expression of 12(S)-LOX in colorectal tumours

Expression of 12(S)-LOX was determined by quantitative RT-PCR from 50 colorectal tumour specimens. For 16 of these tumours the pathology report described ulcerated areas and/or inflammatory cells recruited into the tumour tissue. Based on the role of 12-LOX enzymes in inflammation regulation we chose to analyse these tumours separately from the remaining 34 tissue specimens for which no signs of inflammation was reported. Quantification was obtained relative to the house keeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) and normalised to the respective normal, tumour-free tissue at the resection margin. The mean relative expression for the 16 tumour specimens was 2.29 ± 0.52 (range 0.004–7.24; higher than normal mucosa at p = 0.0121) with 12(S)-LOX mRNA up-regulated in 10/16 specimens and down-regulated in only 2/16. No up-regulation and even down-regulation was observed in tumour-specimens for which little or no inflammatory component was described in the pathology report (mean relative LOX-expression 0.381 ± 0.380; range 0.00001–1.14). Calculation relative to ß-actin and microglobulin as alternative house keeping genes did not alter the results (data not shown).

Biological effects of 12(S)-HETE on colorectal cancer cells

To investigate the biological impact of 12(S)-LOX expression and the 12(S)-HETE product on colorectal cancer cells cell line models were chosen due to their endogenous 12(S)-LOX expression levels (see supplemental materials). To assess the biological effects of the 12(S)-LOX product 12(S)-HETE in colorectal tumour cells the mediator was diluted into the medium of SW620, SW480 and Caco2 cultures and cell number was determined 24 h later. Viability was increased by 12(S)-HETE in the highly differentiated, slowly growing Caco2 cells about 25% above the control. The eicosanoid did not enhance viability in the rapidly growing SW480 and SW620 cells (Fig. 1a). However, growth of SW480 cells in soft agar was stimulated by 12(S)-HETE in a concentration dependent manner (p < 0.001 by one-way ANOVA) reaching 1.6-fold at a concentration of 100 nM. SW620 cells that were obtained from a lymph node metastasis served as positive control for agar growth (Fig. 1b). Caco2 cells did not produce sufficient agar colonies to be quantified (data not shown). By contrast, clonogenicity of SW480 cells was inhibited by 12(S)-HETE (Fig. 1c; p < 0.05 by one-way ANOVA). Effects of 12(S)-HETE on the migration of SW480 or Caco2 cells were not detected (data not shown).

Over-expression of 12(S)-LOX in colorectal tumour cells

To achieve sustained 12(S)-HETE production 12(S)-LOX over-expressing cells were constructed from both Caco2 and SW480 cells. The SW480-LOX cultures have been characterised previously [24]. Stable Caco-LOX transfectants expressed 14-fold higher levels of 12(S)-LOX compared to vector-transfected cells and secreted about 5 nM 12(S)-HETE into their medium upon AA addition (supplemental materials Figure 1s). That amount of mediator was sufficient to induce increased viability: in the presence of AA to support 12(S)-HETE production both over-expressing cell lines achieved a growth and/or survival advantage over the respective controls (supplemental materials Figure 2s). The growth promoting effect was even more obvious in soft agar media. Colony number obtained from SW480-LOX was increased 1.7-fold above the controls and almost reached the number of colonies derived from untransfected SW620, which is a more malignant cell clone producing higher amounts of 12(S)-HETE (Fig. 2a). The growth of Caco-LOX cells was not sufficient to be analysed in soft agar assays. By contrast, colony formation was inhibited by 33% in Caco2 transfectants and by 24% in SW480-LOX cells which was equal to colony formation in SW620 cultures (Fig. 2b). Cell migration was stimulated in 12(S)-LOX transfectants as compared to controls so that Caco-LOX cells migrated like SW480 controls and SW480-LOX cells displayed activity similar to SW620 cells (Fig. 2c).

To assess the cells' malignant properties in vivo they were injected s.c. into the rear flank of SCID mice and tumour growth monitored for 7 weeks. Both LOX- and control (co)-transfectants formed tumours of epithelioid, cytokeratin 20 expressing cells (Fig. 3a) and local tumour growth did not differ between both groups (Fig. 3b). Metastatic potential was determined from the presence of Ki67-positive tumour cells in the lungs of tumour bearing mice. Single metastatic cells were identified in the vicinity of major vessels (Fig. 3c) and scored from serial sections as described in Materials and methods. The resulting mean score in SW480-LOX groups was about double the score in SW480-co mice (Fig. 3d; p = 0.0281).

Inhibition of 12(S)-HETE production

For inhibition of 12(S)-LOX-activity the flavonoid baicalein was used that reduced 12(S)-HETE production with an IC50 of 0.7 μM and decreased cell viability with an IC50 in the range of 6–8 μM [24]. In addition the inhibitor blocked cell migration in untransfected SW480 and SW620 cells as well as SW480-co and SW480-LOX transfectants (Fig. 4a). Baicalein effects on anchorage-independent growth were determined in SW620 cells and were also inhibited (Fig. 4b). Unexpectedly, clonogenicity was also inhibited by 0.7 μM as well as 6 μM baicalein (Fig. 4c) which may be due to effects of baicalein on other cellular targets that also cause loss of viability (e.g. growth signalling).

To obtain a more specific inhibition 12(S)-LOX expression was knocked down by anti-sense nucleotides as described by Tang et al. [26]. In SW620 cells this caused an 80% reduction of 12(S)-LOX mRNA and 12(S)-HETE production that was maintained for 4 days (supplemental materials Figure 3s a, b). Knock down in 12(S)-LOX over-expressing SW480 and Caco2 cells was less efficient but did reach a ≥ 50% reduction after 2 transfections both on the RNA- and the HETE-production level (supplemental materials Figure 3s c, d, e). This procedure caused an increase in clonogenicity (Fig. 5a) as well as a reduction in migration (Fig. 5b) and in anchorage-independent growth (Fig. 5c).

Expression of genes related to cell interactions

For an initial analysis of gene expression Caco2 cells transiently over-expressing 12(S)-LOX were used because of their high HETE production upon AA addition (about 20 nM; see supplemental materials Figure 2s b). RNA was isolated 24 h after supplementation of the culture medium with 20 μM AA and 96 metastasis-related genes were assessed using a focused micro-array. 12 of these genes produced signals robust enough for analysis and were significantly down-regulated compared to control (supplemental materials Table 3). Prominent among the down-regulated genes were the cell surface molecules integrin β1 (ITGB1), integrin α5 (ITGA5), and E-cadherin (CDH1). The effect on expression of these three genes was verified by standard RT-PCR. While integrin α5 did not appear down regulated in the PCR reaction, the inhibitory effect could be verified for E-cadherin and integrin β1 (Fig. 6a).

E-cadherin expression was easily detectable on both the RNA and protein level in Caco2 cells but not in SW480 and SW620 cultures (Figs. 6b, d). E-cadherin protein on the cell surface of SW480 and SW620 was about 10% the amount seen on Caco2 cells (Fig. 6d). Over-expression of 12(S)-LOX in Caco2 cells induced a 40% reduction of E-cadherin at the cell surface that was reversible upon knock down of 12(S)-LOX expression (Fig. 6d).

Similarly, integrin β1 which is part of the fibronectin and collagen I receptors was expressed in the cell lines in different amounts (Caco2 > SW480 > SW620) at both the RNA and the protein levels (Figs. 6c, e). Over-expression of 12(S)-LOX in both Caco2 and SW480 cells reduced integrin β1 at the cell surface by 20% (Fig. 6e) and total integrin β1 by 30% (Fig. 6f). By contrast, inhibition of 12(S)-LOX by either knock down (SW620, Fig. 6e) or exposure to baicalein (both SW480-LOX and SW620) increased integrin protein (Fig. 6g).


Discussion

12(S)-LOX (ALOX12) introduces a hydroxyl-group at position 12 of arachidonic acid to produce 12(S)-HETE [2,27], a mediator involved in tumour progression and metastasis in melanoma [28,29], prostate cancer [30], pancreatic cancer [31] as well as in angiogenesis in several tumour types reviewed in [32]. In prostate cancer 12(S)-LOX-over expression was correlated with advanced disease [13,33]. Our study now shows up-regulation of 12(S)-LOX in colorectal tumours for which an inflammatory component was described in the pathology report but not in tumours with little signs of inflammation. The study did not use specimens from IBD or IBD-related cancer. A correlation with staging or grading parameters could not be observed. This is in agreement with a recent survey of eicosanoid levels in colorectal tumours that did not report a general increase in 12(S)-HETE [34] and may indicate that both enzyme and product may be derived from or produced as a reaction to the inflammatory tumour microenvironment.

In colorectal cancer cell lines 12(S)-LOX expression was generally low except for SW620 cells that were obtained from a lymph node metastasis. This cell line produced about twice the amount of 12(S)-HETE when compared with SW480 cells that originated from the respective primary tumour and the well differentiated Caco2 cells. Other cell lines derived from metastases (e.g. T84) did not produce more LOX or HETE. For this two explanations are possible: (1) 12(S)-LOX expression is not tumour cell autonomous, but arises from the tumour microenvironment or (2) 12(S)-LOX expression is transiently up-regulated in response to an inflammatory micro-environment.

Independent of their source the results obtained with our in vitro models suggests a role of the enzyme and its product in tumour progression and metastasis. 12(S)-HETE increased cell viability in standard cultures of the well differentiated, slowly growing cell line Caco2. In cultures of SW480 and SW620 cells that already have a much higher growth potential the mediator had no additional impact on growth under standard conditions, but it strongly supported anchorage-independent growth of SW480 cells. Effective concentrations in both cases were 10 and 100 nM. Similarly, over-expression of 12(S)-LOX in Caco2 or SW480 cells conferred a growth/survival advantage in standard cultures and increased growth of SW480-LOX over-expressing cells in soft agar media. Over-expressing cell lines secreted about 5–10 nM 12(S)-HETE into their media, which on the low end of the concentration range for which biological effects of exogenous 12(S)-HETE have been observed in our cells. As endogenous HETE has a limited half-life when exposed to oxygen, it may well be less effective than HETE continuously produced in an autocrine manner.

Both our results from HETE-exposed cells and from 12(S)-LOX over-expressers confirm reports assigning survival activity to 12(S)-HETE in other tumour types [26,35,36]. In addition, 12(S)-LOX expression reduced clonogenicity which under our conditions—removal of non-attached cells after 24 h—largely reflects decreased cell attachment. Concomitantly cell migration in Caco2 and SW480 cells was stimulated effectively increasing their capability to invade. The combined results from the functional assays (clonogenicity, anchorage-independent growth, cell migration) define a malignant phenotype increasing in the sequence Caco2 < SW480 < SW620. Over-expression of 12(S)-LOX reduced clonogenicity but stimulated anchorage-independent growth and cell migration. In summary these biological effects shift the tumour cell characteristics towards higher degrees of malignancy. An impact on the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) that also indicate tumour progression was not observed in our analysis (data not shown).

In vivo SW480-LOX cells did not form larger tumours than the respective controls, which seems to indicate that the growth and survival impact of 12(S)-HETE is not sustained in vivo. With regard to this observation it has to be taken into account that SW480 cells already have a very high potential for local tumour growth so that it may not be possible to increase it further—similar to the absence of a 12(S)-HETE growth effect in standard cultures. LOX-over-expressing SW480 cells did, however, have a stronger tendency to metastasise in line with their increased growth capacity in soft agar and enhanced migratory potential. Again this indicates a more aggressive phenotype. A reverse shift was achieved by inhibition of LOX-activity by baicalein or by knock-down of 12(S)-LOX expression in SW620 cells as well as Caco2-LOX and SW480-LOX transfectants demonstrating LOX-dependency of the effects.

Pro-metastatic effects of 12(S) HETE have also been described for breast cancer cells where the mediator comes from 15-LOX-1 (ALOX15) and enhanced intravasation into lymph vessels [15]. The HETE induces migration of the lymph endothelial cells in an NFκB-dependent manner in these cells [37]. In prostate cancer 12(S)-LOX-derived 12(S)-HETE increased anchorage-independent growth [33], similar to our observations. Attachment and spreading on the matrix proteins fibronectin and collagen I was increased in the 12-LOX over-expressing prostate cells [33]. By contrast, 12(S)-LOX over-expressing colon cancer cells displayed decreased colony formation using an assay format reflecting both cell attachment and growth potential. As viability assays indicate a positive effect of 12(S)-HETE on growth and survival, we do conclude that in contrast to prostate cancer cells colon carcinoma cells decrease their interaction with the culture substrate in response to the mediator. This is also supported by the down-regulation of integrin β1 protein which is part of the receptors for both fibronectin and collagen I [38] by 12(S)-LOX over-expression in both Caco2 and SW480 cells. Integrin β1 has been shown to inhibit cell migration [39] and is lost in advanced colorectal tumours [40]. Its down-regulation was reversed by both baicalein and 12(S)-LOX knock-down indicating that 12(S)-HETE is instrumental in this process.

In Caco2 cells not only integrin β1 was subject to down-regulation by 12(S)-LOX, but E-cadherin was similarly reduced diminishing cell-cell contacts in a 12(S)-LOX-dependent manner. Like many other malignant cells the more undifferentiated cell lines SW480 and SW620 did no longer express detectable amounts of E-cadherin, as it is lost with tumour progression and epithelial mesenchymal transition [41].

In summary, our results show the induction of a migratory, metastatic phenotype in 12(S)-LOX transfected colorectal tumour cells. This is a pre-requisite for metastasis while the cells move towards the metastatic site.


Conflict of interest statement

None of the authors have any conflict of interest to declare.


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Appendix A  Supplementary data

Supplementary materials.


Click here for additional data file (mmc1.pdf)


Acknowledgments

This work was supported by a grant from the Austrian Science Fund (P16328).


Article Categories:
  • Research Article

Keywords: Abbreviations AA, arachidonic acid, HETE, hydroxyeicosatetraenic acid, LOX, lipoxygenase..
Keywords: Keywords Lipoxygenase, 12(S)-HETE, Cell migration, Malignant phenotype, E-cadherin, Integrin.

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