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Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ETA antagonists
  1. H Alia,
  2. M Loizidoua,
  3. M Dashwoodb,
  4. F Savagea,
  5. C Shearda,
  6. I Taylora
  1. aDepartment of Surgery, Royal Free and University College Medical School, London, UK, bDepartments of Molecular Pathology and Clinical Biochemistry, and Surgery, Royal Free and University College Medical School, London, UK
  1. Professor I Taylor, Department of Surgery, Royal Free and University College Medical School, Charles Bell House, 67–73 Riding House Street, W1P 7LD, London UK.m.loizidou{at}ucl.ac.uk

Abstract

BACKGROUND The vasoactive peptide endothelin 1 (ET-1) acts via two receptors, endothelin receptors A (ETA) and B (ETB). ET-1 is overexpressed by human cancers in vivo and in vitro and may be mitogenic for cancer cells.

METHOD To elucidate if ET-1 is a growth regulator the following were investigated in human colorectal cancer cell lines (LIM1215 and HT29): ET-1 production by ELISA; ET receptor expression using radioligand autoradiographic techniques; and responsiveness to ET-1, and to ETA and ETB antagonism by growth measurements.

RESULTS ET-1 was produced by LIM1215 and HT29 cells (21.3 and 41.7 fmol/ml/106 cells (24 hours); 22.6 and 71.7 fmol/ml/106 cells (48 hours), respectively). ETA and ETB receptors were expressed by both cell lines. Addition of ET-1 resulted in a dose dependent increase in cell numbers which was significant at 10−8–10−9 M for LIM1215, with the greatest increase at 10−8 M (32.7% and 28.4% increase above controls at 48 hours and 72 hours; p<0.05) and at 10−8–10−9 M for HT29, with the greatest increase at 10−9 M (13.4% and 15.7% increase above controls at 48 hours and 72 hours; p<0.05). ETAantagonists BQ123 and BQ610, but not the ETB antagonist BQ788, inhibited ET-1 induced proliferation of both LIM1215 and HT29 (p<0.05).

CONCLUSION ET-1 can stimulate the proliferation of colorectal cancer cell lines via the ETA, but not the ETB, receptor.

  • endothelin-1
  • endothelin receptor
  • colorectal cancer
  • colorectal cancer cell lines

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The endothelins are a family of 21 amino acid peptides. There are three members in the endothelin family: endothelin 1, 2, and 3 (ET-1, ET-2, and ET-3). These peptides act via the G protein coupled to receptor subtypes A (ETA) and B (ETB); ET-1 and ET-2 bind to ETA with a higher affinity than ET-3 and all isopeptides display similar affinities for ETB.1-3 ET-1 was first isolated from porcine aortic endothelial cells and has potent vasoactive activity.4 However, ET-1 is now recognised as having multiple pathophysiological activities, including mitogenesis.5-7 Increased ET-1 levels have been detected in plasma and tissue samples from patients with solid malignant tumours, including liver, lung, prostate, and breast cancers.8-12 ET-1 is also produced in vitro by several cancer cell lines, including colonic, pancreatic, ovarian, breast, stomach, and prostate.13-16 Shichiri and colleagues have proposed that ET-1 acts as an autocrine growth factor.17Specifically in ovarian cancer cells, ET-1 has been shown to give an autocrine mitogenic stimulus via the ETAreceptor.18

In colorectal cancer, raised levels of ET-1 have been detected in the plasma of patients with and without metastasis to the liver. Furthermore, ET-1 has been demonstrated immunohistochemically in tumour epithelial cells, stroma, and the blood vessels of both primary colorectal cancer and liver metastases.19-21Autoradiographic studies have detected the presence of binding sites (putative receptors) for ET-1 in colorectal cancer specimens and normal tissue, including the nerve supply of the colon.22-24Although colorectal cancer cells have been shown to secrete ET-1 in vitro, it is not known if this peptide acts as a mitogen for these cells.

The aims of our study were to investigate: firstly, the production of ET-1 and expression of ETA and ETB receptors by two colorectal cancer cell lines; secondly, the ability of ET-1 to stimulate the proliferation of these cell lines; and finally, the potential of ET receptor antagonism as an inhibitor of growth.

Materials and methods

The human colorectal cancer cell lines HT29 (ECACC, Salisbury, Wiltshire, UK) and LIM1215 (kindly donated by Dr M O'Hare, Ludwig Institute for Cancer Research, London, UK) were used. The cells were routinely cultured at 37°C with 5% CO2 in 95% air in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS), l-glutamine 2 mM, penicillin 100 IU/ml and streptomycin 100 μg/ml (all from Imperial Laboratories, Basingstoke, Hants, UK).

ET-1 PRODUCTION

Cells were plated to obtain 85–90% confluence by 72 hours. They were seeded and left to grow in 10% FCS containing medium for the first 24 hours. This was replaced by serum free medium and supernatants were collected after 24 and 48 hours and stored at −70°C until assayed. At each time point, the number of cells were counted on a haemocytometer in order to relate production of ET-1 to cell numbers. ET-1 in the media was measured using an ET-1 kit for an ELISA (Nycomed Amersham, Little Chalfont, Buckinghamshire, UK) and the absorbance read at 550 nm on a plate reader (MRX Denley, Billinghurst, West Sussex, UK). Baseline readings of ET-1 were provided by assaying serum free DMEM. The assay has a sensitivity of 1–32 fmol per well for a 96 well plate and specificity of 98% for ET-1; cross reactivity with ET-2 is <2%, and with ET-3 <0.01%.

ENDOTHELIN RECEPTOR EXPRESSION

Cultured cells were trypsinised and cytospins were prepared using 106 cells per spot. The slides were incubated with 150 pM125I ET-1 (specific activity 2000 Ci/mM) and non-specific binding was determined by coincubation of alternate slides with 500 nM unlabelled ET-1 at room temperature. After incubation, cells were dried and autoradiographs generated by apposing to Hyperfilm 3H (Nycomed Amersham) for up to eight days. Film was processed according to the manufacturer's instructions and cells were stained with haematoxylin and eosin for histology. Cellular binding was achieved by post fixing incubated cells with paraformaldehyde vapour (80°C for two hours), coating them with molten nuclear emission (Ilford K2, Mobberly, Cheshire, UK), and exposing them for eight days, after which emulsion was processed and cells stained with Hand E.25Endothelin receptor subtypes were identified using 150 pM125I PD151242 for ETA or 125I BQ3020 for ETB (specific activity 2000 Ci/mM for both (Nycomed Amersham)).

GROWTH UNDER THE INFLUENCE OF ET-1 AND ENDOTHELIN RECEPTOR ANTAGONISTS

Twenty thousand cells per well were plated into 24 well plates and grown for 24 hours in fully supplemented medium. The cells were washed and incubated in serum free medium containing 10−7–10−12 M ET-1/well for 48 or 72 hours. At each time point the cells were fixed in 10% formaldehyde and cell number measured using the methylene blue assay.26 The optimum concentrations of ET-1 for stimulation of growth from this experiment were used to investigate receptor antagonism.

ETA receptor antagonists BQ123 or BQ610 (100 nM), or ETB receptor antagonist BQ788 (100 nM) were added to each well with or without ET-1 at 10−8 M (LIM1215) or 10−9 M (HT29). The plates were fixed with 10% formaldehyde at 48 and 72 hours; cell number was measured using the methylene blue assay.

Results

PRODUCTION OF ET-1 IN HUMAN COLORECTAL CANCER CELLS

The production of ET-1 by the colorectal cell lines LIM1215 and HT29 was measured in media conditioned for 48 or 72 hours. Values were corrected for a baseline reading of 0.6 fmol/ml (medium only). For LIM1215 there was only a small increase with time from 21.3 fmol/ml/106 cells at 24 hours to 22.5 fmol/ml/106 cells at 48 hours. In contrast, HT29 secreted a higher concentration of ET-1 at 24 hours (41.7 fmol/ml/106cells) which further increased to 71.7 fmol/ml/106 cells at 48 hours.

ENDOTHELIN RECEPTOR EXPRESSION IN HT29 AND LIM1215 COLORECTAL CANCER CELLS

Binding for ET-1, ETA antagonist (PD151242), and ETB agonist (BQ3020) in both cell lines was demonstrated by autoradiography. Total binding was clearly in excess of non-specific binding for ET-1 and its receptor subtypes for both LIM1215 and HT29 colorectal cancer cells, suggesting the presence of both receptors in these cell types (fig 1).

Figure 1

Binding for endothelin receptors (ETA and ETB) on LIM1215 (left) and HT29 (right) cell cytospins was demonstrated by autoradiography. Slides were incubated with ETA antagonist (125I PD151242) or ETB agonist (125I BQ3020) for total binding. Non-specific binding was determined by incubation with excess unlabelled ligand. Total binding was clearly in excess of non-specific binding, suggesting the presence of both receptors in these cell types.

EFFECT OF EXOGENOUS ADDITION OF ET-1 AND RESPONSE TO ETA AND ETB ANTAGONISTS

Addition of ET-1 to both LIM1215 and HT29 resulted in a dose dependent increase in cell number (figs 2, 3). For LIM1215 this was maximal at 10−8 M with a rise of 32.7% and 28.4% above controls at 48 and 72 hours, respectively. In contrast, the maximal number of cells for HT29 occurred with 10−9 M ET-1 when there was an increase of 13.4% and 15.7% above control numbers at 48 and 72 hours, respectively.

Figure 2

Effect of addition of endothelin 1 (ET-1) on LIM1215 cell growth. Cells were grown for 48 or 72 hours in the presence of increasing concentrations of ET-1 (10−12 M to 10−7 M; shown on the x axis as 12 to 7). Cell growth was measured using the methylene blue assay and read as absorbance (equivalent to cell numbers) at 650 nm. Results are shown as mean (SD). Statistically significant growth is shown as *p<0.05 or **p<0.01 (Student's paired t test). C, control.

Figure 3

Effect of addition of endothelin 1 (ET-1) on HT29 cell growth. Cells were grown for 48 or 72 hours in the presence of increasing concentrations of ET-1 (10−12 M to 10−7 M, shown on the x axis as 12 to 7). Cell growth was measured using the methylene blue assay and read as absorbance (equivalent to cell numbers) at 650 nm. Results are shown as mean (SD). Statistically significant cell growth is shown as *p<0.05 or **p<0.01 (Student's paired t test). C, control.

Competitive ETA antagonists BQ123 and BQ610 significantly inhibited ET-1 induced proliferation of both LIM1215 and HT29 at 48 and 72 hours (figs 4, 5). ETB antagonists had no effect on ET-1 stimulated proliferation.

Figure 4

Effect of endothelin 1 (ET-1) and/or endothelin receptor antagonists on LIM1215 cell growth. Cells were grown for 48 or 72 hours in the presence of 10−8 M ET-1 and/or ETA receptor antagonist BQ123 or BQ610, or ETBreceptor antagonist BQ788, all at 100 nM. Cell growth was measured using the methylene blue assay and read as absorbance (equivalent to cell numbers) at 650 nm. Results are shown as mean (SD). Cell numbers were significantly higher in the ET-1 group compared with control cells (*p<0.05, Student's t test); cell numbers from groups treated with both ET-1 and ETA antagonists were similar to control levels, and were significantly lower than in the ET-1 group (†p<0.05, Student's t test).

Figure 5

Effect of endothelin 1 (ET-1) and/or endothelin receptor antagonists on HT29 cell growth. Cells were grown for 48 or 72 hours in the presence of 10−9 M ET-1 and/or ETA receptor antagonist BQ123 or BQ610, or ETBreceptor antagonist BQ788, all at 100 nM. Cell growth was measured using the methylene blue assay and read as absorbance (equivalent to cell numbers) at 650 nm. Results are shown as mean (SD). Cell numbers were significantly higher in the ET-1 group compared with control cells (*p<0.05, Student's t-test); cell numbers from groups treated with both ET-1 and ETA antagonists were similar to control levels, and were significantly lower than in the ET-1 group (†p<0.05, Student's t test).

Discussion

The effect of ET-1 on colorectal cancer cell growth was investigated in vitro. The two colorectal cancer cell lines used in this study secreted ET-1, as has been demonstrated for other cancers. Levels of ET-1 measured in the medium were similar to those previously reported for HT29 of 23 fmol/ml/106 cells.15

ET-1 acts via two receptors, ETA and ETB, which were found to be present on LIM1215 and HT29 by autoradiography. This technique, by utilising radiolabelled agonists and antagonists which display high affinity for receptors, not only demonstrated the presence of the receptors but also indicated that they occurred in a functional state.27 Ovarian cancer cell lines have also been shown to produce ET-1 and express ETA and ETBreceptors.18 28 29

The mitogenic potential of exogenous ET-1 on these cell lines was assessed. This peptide was found to significantly increase cell number for both cell lines with a greater increase for LIM1215 compared with HT29. The difference may be related to the greater endogenous production of ET-1 by HT29: if the mitogenic effect occurs via one or both of the receptors and these receptors are partially occupied by endogenous ET-1, then addition of further ET-1 will have less of an effect than in a cell line where the receptors are available for binding. The concentrations of ET-1 (10−9 M and 10−8 M) which resulted in an increase in cell growth are 10 000 times higher than circulating plasma levels of ET-1 in patients with colorectal cancer. However, locally (in tissues), much higher levels may occur.30 For other cell lines, for example Swiss 3T3 fibroblasts, vascular smooth muscle, and ovarian cancer cell lines, a mitogenic effect of ET-1 was produced at 10−10M, only 10-fold lower than that required to produce an effect on LIM1215 and HT29.5-7 18

The effect of ET-1 on LIM1215 and HT29 was mediated via the ETA, and not the ETB receptor, as demonstrated by the ability of BQ123 and BQ610 to prevent an increase in cell number on addition of ET-1. ETA has also been demonstrated to be the receptor through which the mitogenic effect of ET-1 is mediated for ovarian cancer cell lines18 and for a number of other cell lines.5 6 9 30 In contrast, BQ788, the ETBantagonist, had no effect on cell number.

In the only in vitro cancer model in which ET-1 mitogenic signalling has been studied, ovarian cancer cells were used. Binding of ET-1 to the ETA G protein coupled receptor resulted in activation of phospholipase C activity and Ca2+/PKC signalling, which are the classical effectors of G protein signalling. Furthermore, other intracellular targets activated included: tyrosine kinases (for example, focal adhesion kinase p125FAK), p42 mitogen activated protein kinase (MAPkinase), and immediate early response genes (for example, fos).28 This suggests that ET-1 not only uses phospholipase C/PKC pathways but cross talks with tyrosine kinase cascades. These intracellular steps have been implicated in mitogenic signalling via ETA in a variety of cell types, including fibroblasts and vascular smooth muscle cells.30-34

In conclusion, ET-1 can stimulate net cell growth of LIM1215 and HT29 colorectal cancer cell lines via the ETA receptor. Whether this effect is mediated via a mitogenic stimulus as in ovarian cancer cells and other non-cancer cells, or an anti-apoptotic signal, or a combination of the two, has not yet been demonstrated in this model. However, the findings of Asham and colleagues19 and Shankar and colleagues20 that ET-1 is produced by colorectal cancers combined with the data from this study are consistent with the notion that ET-1 may act as a mitogen in colorectal cancer and that there may be some therapeutic potential in the use of ETA antagonists.

References

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Footnotes

  • Abbreviations used in this paper:
    ET
    endothelin
    ETA
    endothelin receptor A
    ETB
    endothelin receptor B
    DMEM
    Dulbecco's modified Eagle medium
    FCS
    fetal calf serum
    PBS
    phosphate buffered saline

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