[CANCER RESEARCH 62, 6467– 6469, November 15, 2002]
Advances in Brief
The Neurotransmitter ␥-Aminobutyric Acid Is an Inhibitory Regulator for the
Migration of SW 480 Colon Carcinoma Cells1
Jan Joseph, Bernd Niggemann, Kurt S. Zaenker, and Frank Entschladen2
Institute for Immunology, Witten/Herdecke University, 58448 Witten, Germany
Abstract
Materials and Methods
␥-Aminobutyric acid (GABA) is the inhibitory neurotransmitter in the
brain, also playing a role in diseases like epilepsy. We now show that this
inhibitory neurotransmitter can also reduce migratory activity in SW 480
colon carcinoma cells. GABA reduced the norepinephrine-induced migratory activity of these cells within a three-dimensional collagen matrix to
spontaneous migration levels, as was analyzed by time-lapse videomicroscopy. This inhibitory effect of GABA was mediated by the serpentine
receptor GABAB and was intracellularly transduced by a decrease of
the cyclic AMP concentration. Cancer cell migration is thus regulated
by neurobiological signals, opening new possibilities for pharmacological
agonists in cancer therapy.
Cell Culture. The colon carcinoma cell line SW 480 (American Type
Culture Collection, Manassas, VA) was maintained at standard conditions as
described previously (2).
Cell Migration Assay. The cell migration assay was performed as described
in detail previously (9). In short, cell locomotion within three-dimensional collagen lattices was recorded by time-lapse videomicroscopy overnight. For the
analysis of migratory activity, 30 cells of each sample were randomly selected, and
two-dimensional projections of paths were calculated by computer-assisted cell
tracking in 20-min step intervals.
Measurement of Cellular cAMP. For the measurement of changes in the
cellular cAMP, 6 ⫻ 104 cells were incubated for 20 min at 37°C with either
medium alone or with 10 ␮M norepinephrine, 100 ␮M GABA, or the combination of both neurotransmitters. For positive control, the cells were incubated
with 500 ng/ml cholera toxin or 500 ng/ml pertussis toxin (both Sigma,
Deisenhofen, Germany) under the same conditions. After incubation, cells
were lysed, and the cAMP level was measured using a cAMP enzyme-linked
immunoassay system (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom) as described by the manufacturer.
Flow-Cytometrical Measurement of Cytosolic Calcium. For the investigation of changes in cytosolic calcium by treatment with norepinephrine or
GABA, the SW 480 colon carcinoma cells were loaded with fluo-3/a.m. as
previously described with minor modifications (10), and the calcium-induced
fluo-3/a.m. fluorescence was measured immediately after addition of norepinephrine or GABA alone or in combination.
Introduction
Evidence is growing that the migration of tumor cells is not solely
a consequence of genetic alterations but is regulated by a multitude of
epigenetic factors. Chemokines, neurotransmitters, and other structurally nonrelated ligands of serpentine receptors are known as important
initiators of migratory activity (1). We have previously reported on the
initiation of colon carcinoma cell migration by the neurotransmitter
norepinephrine (2). In a further development of our studies on the
influence of neurotransmitters on the migration of cancer cell migration, we investigated the effect of the inhibitory neurotransmitter
GABA3 on the norepinephrine-induced migration of SW 480 colon
carcinoma cells. GABA is the major inhibitory neurotransmitter of the
central nervous system, where it has been shown to play a role in
pathological conditions like epilepsy (3). GABA can, however, also
be found in neural and nonneural tissues outside the brain (4, 5),
including the gastrointestinal tract (6). Principally, GABA acts on two
classes of cellular receptors: (a) the ionotropic GABAA and GABAC
receptors are oligomeric chloride channels (6, 7); and (b) the metabotropic GABAB receptor is a member of the serpentine or seven-helices
receptor family and is therefore related to chemokine receptors and
catecholaminergic receptors, which both have been shown to be
involved in the regulation of leukocyte and tumor cell migration (1,
8). Herein, we describe the signal transduction of the antidromic
effects of norepinephrine and GABA on the regulation of tumor cell
migration.
Received 8/21/02; accepted 10/4/02.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Bruno und Helene Joester Foundation, Cologne,
Germany, and the Fritz Bender Foundation, Munich, Germany.
2
To whom requests for reprints should be addressed, at Institute for Immunology,
Witten/Herdecke University, Stockumer Str. 10, 58448 Witten, Germany. E-mail:
[email protected].
3
The abbreviations used are: GABA, ␥-aminobutyric acid; cAMP, cyclic AMP; PLC,
phospholipase C; PKA/C, protein kinase A/C; PTK, protein tyrosine kinase.
Results and Discussion
The Inhibition of Norepinephrine-induced Migration of Colon
Carcinoma Cells by GABA Requires the Engagement of GABAB
Receptors. Norepinephrine induces migration of SW 480 colon carcinoma cells (2). We now report that this induced tumor cell migration
is completely inhibited by GABA (Fig. 1A). Norepinephrine induces
migration from 42 ⫾ 13% spontaneously locomoting cells to
63 ⫾ 3%, whereas GABA alone had no effect (46 ⫾ 4% locomoting
cells) but abolished norepinehrine-induced migration (37 ⫾ 3%). We
investigated the receptor for the regulatory function of GABA by
specific agonists (Fig. 1, B and C). We used baclofen as a specific
agonist for GABAB receptors and isoguvacine for GABAA receptors.
Baclofen but not isoguvacine inhibited the norepinephrine-induced
migration (Fig. 1, B and C). After treatment with baclofen, the
norepinephrine-induced migration was reduced from 52 ⫾ 5% down
to 33 ⫾ 4% locomoting cells, whereas treatment with isoguvacine had
no effect (44 ⫾ 3% locomoting cells with norepinephrine versus
45 ⫾ 2% locomoting cells with norepinephrine and isoguvancine).
Thus, the inhibitory function of GABA on the migration of these
tumor cells is mediated by GABAB receptors.
The Inhibitory Effect of GABA Is Mediated by the Regulation
of Cellular cAMP. It is well known from heart muscle cells that
binding of norepinphrine leads to activation of stimulating G proteins
(11). These G proteins regulate the activity of the adenylyl cyclase,
which generates the second messenger cAMP (12). In turn, cAMP
binds to multiple effector molecules, which are involved in the regulation of the cytosolic calcium concentration. A key effector mole-
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GABA INHIBITS SW 480 TUMOR CELL MIGRATION
(Fig. 2A). The addition of GABA to norepinephrine-treated cells
significantly reduced the norepinephrine-induced increase of cAMP.
Therefore, an increase of locomotor activity is coupled to an increase
of cellular cAMP, whereas a reduction of cellular cAMP decreases
migratory activity.
Cells treated with dibutyryl-cAMP in combination with GABA and
Fig. 1. Effects of GABA on the migration of SW 480 colon carcinoma cells. The
migration of the cells was induced by 10 ␮M norepinephrine (Nor). GABA (A), the
GABAB-specific agonist baclofen (B), or the GABAA-specific agonist isoguvacine (C)
were added at a concentration of 100 ␮M. The graphs show mean values of three
independent experiments (90 cells were analyzed/sample).
Fig. 2. Role of cAMP in the regulation of SW 480 colon carcinoma cell migration. In
A, the concentration of intracellular cAMP after treatment with 10 ␮M norepinephrine or
100 ␮M GABA alone or in combination was analyzed by an enzyme-linked immunoassay.
Statistical significance of the cAMP reduction by GABA in norepinephrine-treated cells
was calculated using Student’s t test (P ⫽ 0.02; indicated by an asterisk). Cholera (CTX)
and pertussis (PTX) toxins were used as positive controls. The diagram shows values ⫾ SD of three independent experiments. In B, cells were treated with either 1 ␮M
dibutyryl-cAMP (db-cAMP) alone or in combination with norepinephrine and GABA.
The graph shows mean values of three independent experiments (90 cells were analyzed/
sample).
cule is the protein kinase A, which phosphorylates phospholamban.
Phosphorylation of phospholamban leads to its release from sarcoplasmatic/endoplasmatic reticulum calcium ATPase, which sequestrates cytosolic calcium into intracellular stores (13).
In addition to the G protein-mediated signaling, Luttrell et al. (14)
described an activation of PTKs via ␤-arrestin after ␤2-adrenoceptor
engagement. We have shown that a PTK-dependent activation of the
PLC␥ is a crucial regulator for the norepinephrine-induced migration
of colon carcinoma cells (2). Two second messengers are generated by
the PLC␥: (a) inositol-1,4,5-trisphosphate, which opens intracellular
calcium channels; and (b) diacylglycerol, which activates the PKC␣.
In this context, it is important to stress that an activation of the PKC
with the diacylglycerol analogue phorbol-12-myristate-13-acetate is a
sufficient start signal for the induction of very high locomotor activity
in colon carcinoma cells (15).
In our experiments on the regulatory signal transduction of the
norepinephrine- and GABA-mediated effects on cell migration, we
focused on the G protein-mediated regulation of cellular cAMP.
Norepinephrine induced an increase of cellular cAMP by 45.2%,
whereas GABA reduced the cellular cAMP concentration by 48.5%
Fig. 3. Flow-cytometrical measurement of changes of the intracellular calcium concentration after treatment with norepinephrine and GABA. Cells were loaded with the
calcium-dye fluo-3/a.m. and subjected to flow cytometry immediately after addition of the
neurotransmitters. The diagram shows values ⫾ SD of three independent experiments.
The mean fluorescence intensity of the control was adjusted to a value close to 700.
Treatment of the cells with 5 ␮g/ml ionomycin served as a positive control and led to an
increase to a mean fluorescence intensity of 1971 ⫾ 302 (data not shown). Statistically
significant changes versus control are indicated by an asterisk (Student’s t test; P ⬍ 0.05).
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GABA INHIBITS SW 480 TUMOR CELL MIGRATION
scribed PLC␥ activation catalyzing the production of inositol-1,4,5trisphosphate (2), which leads to the fast release of calcium from the
endoplasmatic reticulum (arrow A in Fig. 4). The second effect of
norepinephrine, the activation of the ATP-dependent sequestration of
calcium by increased levels of cAMP, is not sufficient to override the
ATP-independent calcium release from the endoplasmatic reticulum
resulting in the observed increase of cytosolic calcium (arrow B in
Fig. 4). Addition of GABA to norepinephrine-treated cells did not
change cytosolic calcium as compared with norepinephrine alone
(Fig. 3). GABA reduces the cAMP concentration (Fig. 2A) and causes
thereby a reduced calcium sequestration. This interruption of the
previously described calcium cycling (10) results in the reduction of
locomotor activity (arrow C in Fig. 4).
In conclusion, the induced migration of SW 480 colon carcinoma
cells is inhibited by the neurotransmitter GABA. Our results lend
themselves to the investigation of the possible use of GABAB agonists
for the chemoprevention of metastasis development. Accordingly,
Kawabata et al. (16) provided evidence for a preventive role of GABA
in the frequency of azoxymethane-induced colonic adenocarcinoma in
rats. Using this novel knowledge of neurobiological influences and the
concerned intracellular pathways of tumor cell migration, it might be
possible to develop strategies to delay or prevent metastasis formation
either by blocking the involved receptors or by interfering with the
intracellular signal transduction.
References
Fig. 4. Model of the signal transduction pathways regulating the norepinphrine-induced
locomotion and the inhibition of migration by GABA. This model is an extension of the
pathway published in Ref. 2 (u). The norepinephrine-induced pathways, which are
marked by the arrows A and B, lead to a release of calcium from the endoplasmatic
reticulum and its sequestration, thereby promoting a calcium cycling within the cell. The
GABA-induced pathway, which is marked by the arrow C, interrupts this cycling by
inhibiting the calcium sequestration. AC, adenylyl cyclase; PLB, phospholamban.
norepinephrine developed a migratory activity of 55 ⫾ 18% locomoting cells, which was similar to norepinephrine alone (55 ⫾ 15%
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Research.
The Neurotransmitter γ-Aminobutyric Acid Is an Inhibitory
Regulator for the Migration of SW 480 Colon Carcinoma Cells
Jan Joseph, Bernd Niggemann, Kurt S. Zaenker, et al.
Cancer Res 2002;62:6467-6469.
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