Gamma linolenic acid regulates gap junction communication in endothelial cells and their interaction with tumour cells

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(4), 307-316

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G a m m a linolenic acid r e g u l a t e s gap j u n c t i o n c o m m u n i c a t i o n in e n d o t h e l i a l cells and their i n t e r a c t i o n w i t h t u m o u r cells W. G. Jiang, 1 R. P. Bryce, 2 R. E. M a n s e l ~ 1University Department of Surgery, University of Wales College of Medicine, Cardiff, UK 2Scotia Pharmaceuticals Ltd, Stirling, Scotland, UK

Summary Tumour-endothelial cell adhesion forms a key role in the establishment of distant metastases. This study examined the effect of gamma linolenic acid (GLA), an anti-cancer polyunsaturated fatty acid (PUFA), on both the gap junction communication of human vascular endothelial cells and tumour cell-endothelial interactions. By using scrape loading of Lucifer yellow dye, we showed that GLA at non-toxic levels increased Lucifer yellow transfer, indicating improved gap junction communication. The fatty acid also corrected the communication that was reduced by the mitogenic and motogenic factor HGF/SF. GLA inhibited the tyrosine phosphorylation of connexin-43, a protein that formed gap junction in this cell. When human tumour cells were added to quiescent or HGF/SF-activated endothelial cells, the presence of GLA reduced adhesion of tumour cells to the endothelium. It is concluded that GLA reduces tumour-endothelium adhesion, partly by improved gap junction communications of the endothelium.

INTRODUCTION Establishment of distant metastases is a multi-step process which comprises a number of separate but highly dependent steps. ~,2The adherence of turnout cells to the vascular endothelial lining and their subsequent migration through it is central to this process of metastatic development. Turnout cells in the circulation that survive immune attack must first 'dock' to the endothelium using carbohydrate-carbohydrate interactions and, after transition, firmly 'lock' to the endothelium before migration and invasion of the endothelium layer can occur. The latter process requires certain adhesion molecules including selectins, ICAMs, VCAlVl and integrins. Gap junctions are aggregations of intercellular channels that provide a direct pathway for the diffusion of small molecules (1-2000 Da) between adjacent cells so allowing a number of cells to respond following stimulation/insult to single cell or a small group of cells. Gap Received 5 June 1996 Accepted 18 June 1996 Correspondence to: Dr W. G. Jiang, Tel: 44 1222 744712; Fax: 44 1222 761623; E-mail:jiangw @cf.ac.uk

junctions are formed by the circular arrangement of six protein molecules termed connexins, resulting in a 'cylindrical pore' within the cell membrane. Reduced gap junction communication has been implicated in pathological processes such as carcinogenesis and certain steps in the metastasic cascade. 3-5 It has been shown that invasion and metastasis promoting factors may decrease gap junction communication and thus result in increased cell motility and dissociation? -9 Clinical studies indicate that connexin expression is associated with tumour progression, including invasion and metastasis, x° Following tumour cell adhesion to the endothelium, the endothelial cells may fail to communicate information via gap junctions and this may result in cellular responses that further facilitate tumour cell attachment and invasion of the endothelial cells. In this study we examined the effect of gamma linolenic acid (GLA) on gap junction communication in human vascular endothelial cells. GLA is an n-6 polyunsaturated fatty acid, which has been shown to have anti-proliferation and cytotoxic effects on a range of cancer cells in vitro, including breast, prostate, pancreatic cancer and hepatoma cells. 11-17Inhibition of tumour cell growth by some cytokines is also dependent on the 307

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presence of poly-unsaturated fatty acids (PUFAs). 14 These anti-cancer effects are seen particularly with gamma linolenic acid (GLA), dihomo-gamma linolenic acid (DGLA), and eicosapentaenoic acid (EPA). We have recently reported another property of GLA, exerted on human colon cancer cells, namely the inhibition of motility and in vitro invasive properties. 17 We further showed that this fatty acid is capable of inducing E-cadherin expression in a range of human cancer cells (colon, mammary, liver, lung cancer and malignant melanoma cell), 17,18and may also reduce tumour-matrix interaction by the inhibition of tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. 19 Some PUFAs may inhibit certain cell adhesion molecules such as Eselectin and VCAM-I which may be required in tumourendothelium interactions. 2° Furthermore, by its ability to inhibit HGF/SF-induced motility and invasion, 17 we hypothesised that GLA may have an effect on tumourendothelial interactions and therefore conducted this study. We report here that GLA exerted inhibitory effects on endothelial-tumour cell interactions (colon and breast cancer cells), in particular those induced by HGF/SF. These results may be attributed to improved gap junction communication induced by GLA. MATERIAL AND METHODS

Human vascular endothelial cells (ECV304), human breast cancer cells MDA MB 231, human colon cancer cells HT115 were from ECACC (European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) and were maintained in M199 and DMEM with 10% FCS, respectively. Linoleic acid (LA), gamma linolenic acid (GLA), oleic acid (OA), eicosapentaenoic acid (EPA), alphalinolenic acid (ALA) and arachidonic acid (AA) were purchased from Sigma (Poole, Dorset, UK). Fatty acids were initially dissolved in ethanol, stored in liquid nitrogen, and diluted in culture medium immediately before use (ethanol final concentration < 0.01%). HGF/SF was a gift from Dr T Nakamura (Osaka, Japan). 1, l'-dioctadecyl3,3,3',3'-tetramethylindocarbocyanine parchlorate (DiI) was from Molecular Probes Inc., Eugene, Oregon, USA. Cytodex-2 carrier beads were obtained from Pharmacia, FITC-labelled goat anti-mouse IgG ab2 was obtained from Sigma, rabbit anti-connexin 43 from Zymed Laboratories, Inc., San Francisco USA, rabbit anti-mouse IgG from DAKO Ltd. Lucifer yellow CH was from Sigma (Poole, Dorset, E@and). Protein A/G agarose and monoclonal anti-phosphotyrosine antibody were from Santa-Cruz Biotechnologies (Santa Cruz, California, USA). Peroxidase conjugated secondary antibodies were from Bio-Rad Laboratories Ltd (Bio-Rad House, Maylands Avenue, Hertfordshire, England).

Scrape loading of Lucifer yellow

This was based on a method reported by E1-Fouly et al.21 Briefly, confluent ECV304 cells were washed and treated with medium alone, medium containing fatty acids, cytokine or their combination. After washing off the reagents, cells were resuspended in HEPES-buffered medium with 0.5 mg/ml Lucifer yellow. The monolayer was then quickly scraped with a sterile surgical blade and incubated for 2 min at 37°C prior to washing. Cells were immediately examined under a fluorescence Hoffmann microscope (Leica Cambridge Ltd, Cambridge, England, UK). Positive cells with fluorescent dye were counted under a higher power field.

Tumour-endothelial cell interaction

This was based on a method we recently described using the fluorescent label, DiI, a dye that can be incorporated into cellular membrane and nontransferable between cells (Hiscox and Jiang, manuscript in submission). Briefly, tumour cells were loaded with DiI (1 ~g/ml) at 37°C for 25 rain and then washed. They were then added to a confluent ECV304 monolayer for varying time periods. At the end of this incubation, unbound ceils were washed off and bound cells and ECV304 were lysed with 0.1% SDS for 60 minutes. Lysate fluorescence (arising from adherent tumour cells) was measured on a multichannel fluorescence plate reader (Denly, Sussex, UK) at excitation )~ = 540 nm and emission )~ = 590 nm. The following regimes were adopted in the experiments: GLA or HGF/SF pre-treated ECV304 or tumour cells; GLA or HGF/SF present in the adhesion system, GLA and HGF/SF present in combination. Adhesion is presented here as the adhesion index: (fluorescent unit of treated cellblank)/(control cell-blank).

Cell motility

The cell dissociation assay was essentially the same as that described by Rosen et al. a2 Cells were cultured with Cytodex-2 beads (5 mg/ml) for 24 h. The beads were then harvested and washed in culture medium. A small sample of this suspension was used to quantify total cell number attached to the beads: cells were dissociated from the carrier beads with hydrochloric acid, the nuclei stained with crystal violet, and then counted with a haemocytometer. The cell number in the original suspension was then adjusted to 5 x 105 cells/ml. Miquots of this suspension of beads with attached cells were placed into 96 well multi-plates (Nunc, Denmark) and fatty acids were then added to the cells. After 24 h culture, the plates were emptied and washed with BSS buffer to remove all the carrier beads. Cells which had detached from the

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(12%). Following electrophoresis, proteins were blotted onto nitro-cellulose sheets and blocked in 10% skimmed milk for 60 minutes before probing with anti-connexin 43 (1:50 dilution) and peroxidase-conjugated secondary antibody (1:2000). Protein bands were visualised with an enhanced chemiluminescence (ECL) system (Amersham, UK).

beads and which were bound to the bottom of the wells were fixed in buffered formalin and counted after staining with 0.5% crystal violet. Immunoprecipitation, SDS-PAGE and Western blotting

Cells were pelleted and lysed in HCMF buffer containing 0.5% SDS, 0.5% Triton, 2 m M CaC12, 100 gg/ml PMSF, 1 ~tg/ml leupeptin, 1 gg/ml aprotinin and 10 mM sodium orthovanadate for 30 minutes. Protein concentrations were measured using fluorescamine and quantified by using a multi-fluoroscanner (Denly, Sussex, UK). For immunoprecipitation, equal amounts of protein were incubated with an anti-phosphotyrosine antibody for 60 min before the addition of protein A/G agarose. Following extensive washing, the beads were then added to sample buffer, boiled and subject to electrophoresis. Equal amounts of protein from each cell sample (controls and treated) were resolved on a poly-acrylamide gel

Cell growth assay

To exclude the possibility of cytoxicity exerted by fatty acids, DNA quantity was determined with Hoescht 33528 assay. 17 Cells were treated with 0.01% SDS for 60 rain at the end of culture and then Hoechst 33258 was added (final concentration = 1.0gg/ml). Calf thymus DNA (Sigma) was used as an internal standard. Fluorescence was measured with a Mukifluoroscanner (Denly, UK), at excitation 36Onto and emission 460 nm.

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D Fig. 1 Lucifer yellow transfer with ECV304 cell. A: control, B: with HGF/SF 20 ng/ml, C: with GLA 50 pM, D: the combination of GLA and HGF/SF. The dye transfer was markedly increased when cells were treated with GLA (shown is 40 minutes treatment). HGF/SF otherwise reduced the expression but this was reversed by the inclusion of GLA (D).

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RESULTS

of positive cells (C) compared with control (A). In order to determine whether this effect could be repeated on cells treated with an inhibitory agent, cells were also treated with HGF/SF, a lcnown inhibitor of gap junction communication. 23 We found that HGF/SF reduced Lucifer yellow transfer (Fig. 1B), an effect reversed by GLA (Fig. 1D). GLA at concentrations higher than 10 bLM pro-

The effect of GLA on Lucifer yellow transfer

We firstly determined the effect of GLA on the transfer of Lucifer yellow in ECV304 cells. Endothelial cells, after pre-treatment with gamma linolenic acid increased dye transfer. Figure 1 shows that GLA increased the number 30

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GLA regulates gap junction communication in endothelial cells

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was also concentration dependent (Fig. 6A). In contrast to GLA, other fatty acids showed variable effects (Fig. 4B). The effect of GLA was further tested on endothelial cells pretreated with HGF/SF. As expected, tumour cells showed an increased adhesion to HGF/SF-activated ECV304 cells (Fig. 5C,6B) with a peak effect at approximately 20 ng/ml (Fig. 6B). Inclusion of GLA in the assay system avoided the stimulation of adhesion exerted by HGF/SF alone (Fig. 5D,6B). While both cell types (Fig. 7A-HTll5 and 7B-MDA MB 231) showed similar responses, the effect of GLA and HGF/SF appeared as early as 5 min and reached a maximum in approximately 40 min (Fig. 7).

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duced the effect (Fig. 2A), which appeared shortly after treatment (Fig. 3A). HGF/SF at concentrations higher than 1 ng/ml reduced the communication (Fig. 2B). This effect of HGF/SF was observed to be transient as cells showed signs of recovery after 3 h (Fig. 3B). The inhibitory effect of HGF/SF was prevented when GLA was included with the cytokine (Fig. 2B and 3B). This enhancement of the dye transfer appeared to be GLA specific. Of the six fatty acids tested, GLA showed marked effects whilst linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), oleic acid (OA) and alpha linolenic acid (ALA) showed a less profound effect (Fig. 4A). GLA affected tumour endothelial interaction

To further determine the effect of modified communication exerted by GLA, HGF/SF, or their combinations, we tested the interaction of tumour cells with the endothelial cells. When ECV304 endothelial cells were treated with GLA, a marked reduction of adhesion of both tumour cell lines to ECV304 was seen (Fig. 5&B), even when GLA was removed during this period. The effect © Pearson Professional Ltd 1997

We further examined the function (tyrosine phosphorylation) of connexin 43 which forms gap junctions in the endothelial cells. Immunoprecipitation and Western blotting revealed that HGF/SF induced tyrosine phosphorylation of connexin 43 in endothelial cells (Fig. 8A), an effect which was inhibited by the inclusion of gamma linolenic acid (Fig. 8B). Effect of HGF/SF on ECV304 dissociation

HGF/SF induced an increase in £CV304 dissociation, a response similar to that seen with epithelial cells and this was markedly reduced in the presence of GLA (Fig. 9). This reflected an increased motile nature of the cells stimulated by HGF/SF. The cell growth assay indicated that the concentrations of GLA used in this study were not toxic to the cells (data not shown). DISCUSSION

In this study we report that GLA inhibited tumourendothelium interactions, an effect partly attributable to improved gap junction communication within the endothelium. Endothelium presents a barrier against metastatic tumour cells, preventing their escape from the circulation and therefore the establishment of a new tumour site. Upon tumour cell adhesion to the endothelial, increased motility and retraction occur which will expose the basement membrane. 24-27 This is followed by tumour adhesion and destruction of the basement membrane and migration. 2s-3° Measures facilitating the tumour-endothelium interaction may increase the chances of a successful formation of metastasis, while those that inhibit the interaction may reduce the risk of such a development. GLA, which inhibits this process may therefore reduce the likelihood of tumour extravasation, HGF/SF may, in con-

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Fig. 5 Visualization of tumour endothelial adhesion using Dil. A: control, B: GLA (50 gM), C: HGF/SF (20 ng/ml); D: GLA+HGF/SF. ECV304 cells were pretreated with GLA for 40 min and then Dil-labelled tumour cell (MDA MB 231 ) were added. This was incubated for a further 40 min prior to washing. At the end of the experiment, cells are washed and resuspended in HEPES-buffer DMEM, and photographed on an Hoffman fluorescence microscope (Leica, MPS32). Tumour cells, which were stained with red colour (where arrows indicated in A), adhered to endothelial cells (where arrow heads indicated in A). The adhesion was increased by HGF/SF (C). The interactions were reduced when GLA was present (B and D) when compared with corresponding controls (A and C respectively).

trast, increase the risk. In fact, data from some animal studies 31,32and preliminary h u m a n studies showing possible signs of anti-metastatic properties exerted by GLA,33 m a y be partially attributed to the effects reported here. The pro-invasion and pro-metastatic effect of HGF/SF

may therefore also be attributed to its promoting effect on tumour-endothelium interaction reported here? 4-36 Although GLA has been shown to selectively increase the expression of E-cadherin in certain tumour cells, the t u m o u r cells we chose to use in this study are E-cadherin

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negative 17,18 and tumour-endothelial cell interactions in this situation do not require E-cadherin. Although reduced expression of selectin and VCAM which will be required in this reaction, has been reported to be induced by certain PUFAs, 2° the inhibition on the interaction of t u m o u r and endothelium reported here is unlikely to be via this regulation, due largely to the immediate effect © Pearson Professional Ltd 1997

after treated (as short as 5 min). We have demonstrated here another mechanism involved in the GLA effect on tumour-endothelium interaction i.e. improvement of gap junction communication in endothelial cells. Gap junction communication is reduced in malignant cells and this has been postulated as playing a role in carcinogenesis. 3-9,37,38 This change can been seen together with

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damaged cell adhesion properties. 39 Clinical studies indicate that level of connexin expression in tumours is associated with tumour progression, invasion and metastasis.l° It has been shown that reduced gap junction communication is associated with increased cell motilityY This is indeed the case with HGF/SF and ECV304 endothelial cells. HGF/SF markedly reduced the communication and increased cell-cell dissociation/motility. Tyrosine phosphorylation of connexins (CX43) which makes up the gap junction has been shown to correlate with reduced communication, indicating that in some way the phosphorylation may in this case reduce functional gap junctions. 4°-44 Agents that can up-regulate the expression of connexin and also its function include carotenoids and they may reduce carcinogenesis? 5-47 Other factors that regulate the channels include cyclic nucleotides, diacylglycerol, hapatanol and halothane. Modification of tyrosine phosphorylation of connexins is correlated with blockade of gap junction communication (induced by phorbol esters and kepone) and cell tumorigenicityY '4s Our data here confirm this relationship, in which HGF/SF induced tyrosine phosphorylation of connexin 43 and a simultaneous reduction of gap junction communication. These effects were reversed by GLA. Vitamin D3 and retinoic acid are also capable of inducing gap junction communicationY This study shows that GLA at concentrations lower than 100BM enhances the communication, but at higher concentrations is less effective. A similar concentration-related effect has been seen previously with vitamin D.49 In summary, this study shows that GLA exerts an inhibitory effect on the adhesion of E-cadherin-negative breast and colon cancer cells to endothelial cells. We argue that this effect can be attributed to an improved gap-junction communication.

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ACKNOWLEDGEMENTS

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We would like to thank the Welsh Scheme for the Development of Social and Health Research for supporting this work, Dr T Nakamura for providing human recombinant HGF/SF.

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REFERENCES II

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HGF (ng/ml) Fig. 9 Effect of HGF/SF, GLA or their combination on ECV302 cell dissociation. Cytodex-2 was used as cell carrier. Shown is number of cells dissociated from the carrier beads. HGF/SF stimulated dissociation, which was minimised in the presence of GLA.

1. Hart I. R., Goode N. T., Wilson R. E. Molecular aspects of the metastatic cascade. Biochim Biophy Acta 1989; 989: 65-84. 2. Jiang W. G., Puntis M. C. A., Hallett M. B. Molecular and cellular basis of cancer invasion and metastasis: implication for treatment. BrJ Surg 1994; 81:1576-1590. 3. HolderJ. W., Elmore E., Barrett J. C. Gap junction function and cancer. Cancer Res 1993; 53: 3475-3485. 4. HotzwagenblattA., ShallowayD. Gap junctional communication and neoplastic transformation. Crit Rev Oncagenesis 1993; 4: 541-558.

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5. Budunova I. V., Carbajal S., Viaje A., Slaga T.J. Connexin expression in epidermal-cell lines from sencar mouse skin tumors. Mol Carcinogenesis 1996; 15: 190-201. 6. Grossman H. B., Liebert M., Lee I. W., Lee S. W. Decreased connexin expression and intercellular communication in h u m a n bladder-cancer cells. Cancer Res 1994; 54: 3062-3065. 7. Yamasaki H., Mesnil M., Omori Y., Mironov N., Krutovskikh V. Intercellular communication and carcinogenesis. Mutagenesis 1995; 333: 181-188. 8. Uchida Y., Matsuda K., Sasahara K., Kawabata H., Nishioka M. Immunohistochemistry of gap-junctions in normal and diseased gastric-mucosa of humans. Gastroenterology 1995; 109: 1492-1496. 9. Mehta P. P., Lokeshwar B. L., Schiller P. C., Bendix M. V., Ostenson R. C., Howard G. A., Roos B. A. Gap-junctional communication in normal and neoplastic prostate epithelial cells and its regulation by cAMP. Molecular Carcinogenesis 1996; 15: 18-32. 10. Kamibayashi Y., Oyamada Y., Mori M., Oyamada M. Aberram expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in-vivo. Carcinogenesis, 1995; 16: 1287-1297. 11. Begin M. E., Ells G., Das U. N., Horrobin D. F. Differential killing of h u m a n carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Nag Cancer Inst 1986; 77: 1053-1062. 12. Tiwari R. K., Mukhopadhyay B., Telang N. T., Osborne M. P. Modulation of gene expression by selected fatty acids in h u m a n breast cancer cells. AnticancerRes 1991; 11: 1383-1388. 13. Rose D. P., Connolly J. M., Meschter C. L. Effect of dietary fat on h u m a n breast cancer growth and lung metastasis in nude mice. JNatl Cancerlnst 1991; 83: 1491-1495. 14. Newman M.J. Inhibition of carcinoma and melanoma cell growth by type 1 transforming growth factor beta is dependent on the presence of polyunsaturated fatty acids. Proc Natl Acad Sci USA 1990; 87: 5543-5547. 15. Takeda S., Horrobin D. F., Manku M., Sire P. G., Ells G., Simmons V. Lipid peroxidation in h u m a n breast cancer cells in response to gamma-iinolenic acid and iron. Anticancer Res 1992; 12: 329-333. 16. Horrobin D. F. Essential fatty acids, lipid peroxidation, and cancer, in 'omega-6 essential fatty acids'. Horrobin D. F., ed. New York: Wiley-Liss, 1990: 351-378. 17. Jiang W. G., Hiscox S., Hallett M. B., Scott C., Horrobin D. F., Puntis M. C. A. Inhibition of hepatocyte growth factor/scatrer factor induced motility and invasion of h u m a n cancer cells by gamma linolenic acid. BrJ Cancer 1995; 71: 744-752. 18. Jiang W. G., Hiscox S., Hallett M. B., Horrobin D. F., Mansel R. E., Puntis M. C. A. Regulation of the expression of E-cadherin in h u m a n colon cancer cells by gamma linolenic acid. Cancer Res 1995; 55: 5043-5048. 19. Jiang W. G., Hiscox S., Puntis M. C. A. et al. Gamma linolenic acid (GLA) inhibits tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin and tumour cell-matrix interaction. IntJ Oncology, 1996; 8: 583-587. 20. Collie-Duguid E. S. R and Wahle K. W.J. Inhibitory effect of fish oil n-3 polyunsaturated fatty acids on the expression of endothelial cell adhesion molecules. Biochem Biophys Res Commun 1996; 220: 969-974. 21. Ei-Fouly M. H., Trosko J. E., Chang C. C. Scrape-loading and dye transfer. A rapid simple technique to study gap junctional intercellular communication. Exp Cell Res 1987; 168: 422-430. 22. Rosen E. M., Meromsky L, Setter E., Vinter D. W., Goldberg I. D. Quantitation of cytokine-stimulated migration of endothelium

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