Three vasoactive peptides, endothelin-1, adrenomedullin and urotensin-II, in human tumour cell lines of different origin : expression and effects on proliferation
Kazuhiro TAKAHASHI*, Kazuhito TOTSUNE+, Tomomi KITAMURO*,
Masahiko SONE*, Osamu MURAKAMI+ and Shigeki SHIBAHARA*
*Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-I Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan, and +Department of Medicine, Tohoku University School of Medicine, 2-I Seiryo-machi, Aoba- ku, Sendai, Miyagi 980-8575, Japan
ABSTRACT
Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adreno- medullin and urotensin-II, are expressed in various kinds of tumour cells. In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines : T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC- RCW (renal carcinoma). Reverse transcriptase-PCR showed expression of endothelin-I mRNA in seven out of the eight cell lines, the exception being BeWo cells. ETA receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells. ETB receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and Hela cells. Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells. Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24 h decreased cell numbers to approx. 84% and 90% of control respectively. Treatment with the ETA receptor antagonist BQ-610 (I AM) significantly decreased cell number to about 90% of control, whereas the ETB receptor antagonist BQ-788 had no significant effect. On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1 µM) had no significant effects on cell number. These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours. The effect of endothelin-1 on tumour growth appears to be mediated by the ETA receptor.
INTRODUCTION
Endothelin-1 (ET-1) is produced and secreted by various kinds of tumours, and may act as an autocrine or paracrine growth stimulator for the tumour cells [1,2]. Adrenomedullin (AM), a potent vasodilator peptide originally discovered in phaeochromocytoma, is also produced and secreted by various non-endocrine tumours as well as endocrine tumours [3,4]. AM has a
growth-stimulating effect on cultured tumour cells [3]. Urotensin-II (U-II) is a somatostatin-like cyclic peptide, originally isolated from fish spinal cords [5]. Human U- II has a more potent vasoconstrictor action than ET-1 [6], while it exerts vasodilator effects on some arteries [7]. Because U-II is expressed in various human tissues [8] and stimulates the cell growth of vascular smooth muscle cells [9], it is interesting to study possible proliferative actions of U-II on tumour cells.
Abbreviations: AM, adrenomedullin; APUD, ‘Amine and Precursor Uptake Decarboxylation’; ET-1, endothelin-1; IR-, immunoreactive; RT-PCR, reverse transcriptase-PCR; U-II, urotensin-II.
Correspondence: Dr Kazuhiro Takahashi (e-mail ktaka-md@mail.cc.tohoku.ac.jp).
We have previously reported expression of these three peptides in various human tumour cell lines [2,4,10-15]. In the present study, we first confirmed the expression of ET-1 and ET receptors in eight human tumour cell lines. The effects of antibodies against ET-1 and AM, ET antagonists and exogenously added peptides on cell growth were then studied in T98G glioblastoma cells.
MATERIALS AND METHODS
Materials
ET-1, human AM and human U-II were obtained from the Peptide Institute (Osaka, Japan). The antiserum against ET-1 (BP6) was a gift from Professor S. R. Bloom and Dr M. A. Ghatei (Hammersmith Hospital, London, U.K.) [16]. The antiserum against human AM (no. 102) was raised in a rabbit in our laboratory [17]. The ET receptor antagonists BQ-610 and BQ-788 were obtained from Peninsula Laboratories, Inc. (Belmont, CA, U.S.A.).
Cell culture
T98G glioblastoma cells, IMR-32 neuroblastoma cells, NB69 neuroblastoma cells, BeWo choriocarcinoma cells, SW-13 adrenocortical carcinoma cells, DLD-1 colonic carcinoma cells, HeLa cervical carcinoma cells and VMRC-RCW renal carcinoma cells were cultivated as previously reported [14,18]. The culture media were harvested for peptide extraction and the cells were har- vested for RNA extraction.
RIA for ET
The culture media were extracted using Sep-Pak C18 cartridges (Waters, Milford, MA, U.S.A.), as previously reported [17]. Immunoreactive (IR)-ET in the media was measured by a specific RIA, as reported previously [16]. The assay showed 60% and 70% cross-reaction with ET-2 and ET-3 respectively.
Reverse transcriptase-PCR (RT-PCR)
Total RNA was extracted from cultured cells by the guanidine isothiocyanate/caesium chloride method. RT- PCR analysis of ET-1, the ETA receptor and the ETB receptor was performed as previously reported [11]. The sense primer for ET-1 was 5’-ATGGATTATTTGCT- CATGATTTT-3’, and the antisense primer was 5’- CTTGGGATCATGAAAAGATGATTT-3’.
Cell growth assay
The effects of an anti-ET-1 antibody (BP6) [16], an anti- AM antibody (no. 102) [17], ET receptor antagonists (BQ-610 and BQ-788) and exogenously added synthetic peptides on cell proliferation were studied in T98G glioblastoma cells using the Cell Counting Kit-8 (Dojindo Kagaku Co.), as previously reported [19]. T98G glioblastoma cells were cultivated in minimum essential
medium supplemented with 5% (v/v) fetal bovine serum at 37 ℃ under 5% CO2. The cells were seeded at a density of 0.3 x 10-4 cells/0.3 ml per well. Under these conditions, T98G cells reach confluence in about 7 days. After seeding, T98G cells were cultured for 24 h, and then the medium was replaced by fresh medium with 5 % (v/v) fetal bovine serum containing anti-ET-1 antibody (dilution 1:10000), anti-AM antibody (dilution 1: 10000), non-immune rabbit serum (dilution 1:10000), an ET antagonist (0.1 or 1 µM BQ-610, 0.1 or 1 µM BQ- 788), ET-1 (0.1 µM), human AM (0.1 µM) or human U-II (0.1 µM). The cells were cultured for a further 24 h, and then the number of cells was assessed by the Cell Counting Kit-8.
Statistics
Data are shown as means+S.E.M. unless otherwise stated. The statistical analysis was performed by one- way ANOVA followed by Fisher’s PLSD multiple- comparison test.
RESULTS
RT-PCR showed expression of ET-1 mRNA in seven out of the eight cell lines, the exception being Be Wo cells (Figure 1). ETA receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in other cells. ETB receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but weakly in T98G and Hela cells. Consistent with the RT-PCR results, IR-ET was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 cells ( < 0.6 fmol . 24 h-1. 105 cells-1) or BeWo cells (< 1.4 fmol . 24 h-1. 105 cells-1), either in the present study or in our previous studies [10,11,14,15]. We have previously reported the secretion of IR-ET by T98G cells (17.6+0.6 fmol . 24 h-1. 105 cells-1; n = 5) [11], SW-13 cells (11.0±0.8
T98G
IMR-32
BeWo
SW-13
DLD-1
VMRC-RCW
NB69
HeLa
ET-1
582 bp
ETAR
299 bp
ETBR
428 bp
A
100
*
% cell number
80
60
40
20
0
control ET Ab AM Ab
B
100
*
% cell number
80
60
40
20
0
control
10-7 10-6 10-7 10-6 M
BQ610
BQ788
C
100
% cell number
80
60
40
20
0
control ET-1 AM U-II
fmol . 24 h-1.105 cells-1) [10], DLD-1 cells (0.86± 0.05 fmol . 24 h-1. 105 cells-1) [15] and VMRC-RCW cells (0.75 ±0.03 fmol .24 h-1.105 cells-1) [14]. IR-ET was also detected in the culture medium of NB69 cells (1.00 ±0.07 fmol .24 h-1.105 cells-1) and HeLa cells (2.00±0.09 fmol .24 h-1.105 cells-1).
Because T98G cells secrete both ET-1 and AM, and express both ETA and ETB receptors, we studied the effects of the three peptides, ET-1, AM and U-II, on the proliferation of T98G cells. Treatment of T98G cells
with an anti-ET-1 antibody or an anti-AM antibody for 24 h decreased the cell number to approx. 84 % and 90 % of control respectively (Figure 2A). Treatment with the ETA receptor antagonist BQ-610 (1 µM) significantly decreased the cell number to approx. 90% of control (P < 0.05 compared with control), whereas the ETB receptor antagonist BQ-788 had no significant effect (P> 0.1; Figure 2B). Exogenously added ET-1, AM and U-II, each at a concentration of 0.1 µM, had no significant effects on the number of T98G cells (Figure 2C).
DISCUSSION
More than 30 years ago, A. G. E. Pearse showed that peptide-secreting cells have common morphological features, and proposed his famous ’ Amine and Precursor Uptake Decarboxylation (APUD) theory’ [20]. There has been accumulating evidence, however, that ET-1 and AM are produced and secreted from various non-APUD tumour cells, such as glioblastomas and colonic car- cinomas. Further, we have recently reported expression of U-II in various human tumour cell lines [13,14]. The pathophysiological roles of these peptides produced by non-APUD tumours, however, have not been clarified. Because ET-1, AM and U-II modulate the proliferation of various types of cells [1-4,9,19,21], it is plausible that these peptides act as autocrine or paracrine growth stimulators in these tumours.
T98G gliobastoma cells secrete both IR-ET and IR- AM abundantly, and express both ETA and ETB receptor mRNAs. We therefore studied the effects of an anti-ET antibody and ET receptor antagonists on the growth of T98G cells. The results showed that endogenously secreted ET-1 stimulated the growth of tumour cells, and this effect was mediated by the ETA receptor. We cannot exclude the possibility that the ETB receptor mediates the action of ETs on growth in other types of normal or tumour cells, because ETB receptor mRNA was only weakly expressed in T98G cells. Exogenously added ET- 1, AM or U-II had no significant effects on the number of T98G cells. This may be due to the fact that endogenously secreted ET-1 and/or AM may be sufficient to maintain the growth of T98G cells under the conditions used. Further studies are required to clarify whether U-II has effects on the growth of tumour cells.
The present study has raised the possibility that ETA receptor antagonists may be useful in the treatment of certain types of malignant tumours. Experimental studies using animal models and clinical studies in human have shown that ET receptor antagonists are useful in the treatment of some cardiovascular diseases, such as con- gestive heart failure, diabetic nephropathy and pulmon- ary hypertension [22-24]. It is expected that ET receptor antagonists will be used clinically for the treatment of some cardiovascular diseases in future. If the therapy
using ET receptor antagonists could reduce the risk of cancer death in patients with cardiovascular diseases, such a therapy should be the most favourable choice among several possible therapies for cardiovascular dis- eases.
ACKNOWLEDGMENTS
This work was supported in part by grants-in-aid for Scientific Research (B) and (C), and on Priority Areas (A), from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from Renal Anaemia Research (2000 and 2001). We are grateful to Professor S. R. Bloom and Dr M. A. Ghatei (Hammersmith Hos- pital, London, U.K.) for their gift of the anti-ET-1 serum (BP6).
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