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Biochemical and Biophysical Research Communications 303 (2003) 572–575
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Homocysteine is a potent inhibitor of human tumor cell gelatinases
Teresa Chavarrıa, Salvador Rodrıguez-Nieto, Francisca S
anchez-Jimenez,
*
Ana R. Quesada, and Miguel A. Medina
Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of M
alaga, E-29071 M
alaga, Spain
Received 10 February 2003
Abstract
Extracellular matrix-degrading gelatinases are mainly involved in tumor invasion and metastasis. Previous experimental data
from our group and others suggested that homocysteine could have a potential modulatory role on the proteolytic balance at the
extracellular matrix. Therefore, we studied the effects of homocysteine on extracelluar matrix-degrading proteases using model
human tumor cell lines and a combination of in vitro fluorogenic assay and zymographic techniques. Homocysteine is shown to be
the thiol compound with the most potent inhibitory activity on matrix metalloproteinase 9. Zymographies reveal that matrix
metalloproteinase 2 is, at least, as sensitive to inhibition by homocysteine as matrix metalloproteinase 9 is. This study opens new
ways to the potential pharmacological use of thiol compounds.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Homocysteine; Matrix metalloproteinases; Gelatinases; HT-1080 cells; U2-OS cells
Tumor progression from a benign in situ neoplasia to
a malignant tumor involves the acquisition of the capability for invasion and metastasis, enabling cancer
cells to escape the primary tumor mass and to colonize
new terrain in the body. In fact, these distant settlements
of tumor cells are the cause of most of human cancer
deaths [1].
Invasion and metastasis are two very complex processes with genetic and biochemical basis not fully understood yet. However, they are closely allied processes
at the mechanistic level, since both use similar operational strategies, involving changes in their adhesion to
their microenvironment and activation of their capabilities to remodel extracellular matrix [2] through a shift
of the proteolytic balance towards proteolysis [3]. An
essential step is the breakdown and removal of extracellular matrix, mediated by several proteases, the most
important of which appear to be matrix metalloproteinases (MMP) and serine proteases. In fact the MMPs,
72 and 92 kDa type IV collagenases or gelatinases
(MMP-2 and MMP-9, respectively) are mainly involved
in tumor invasion and metastasis [4,5].
*
Corresponding author. Fax: +34-95-2132000.
E-mail address: [email protected] (M.A. Medina).
Homocysteine is a sulfur-containing, non-proteinogenic amino acid biosynthesized from methionine which
takes a key place in common in between folate cycle and
activated methyl cycle [6]. An imbalance of homocysteine metabolism has been associated with several disorders and diseases [6–9]. Recently, homocysteine has
been shown to induce the expression and synthesis of the
tissue inhibitor of metalloproteinases-1 (TIMP-1) in a
variety of cells [10]. In addition, exposure of cultured
endothelial cells to homocysteine reduces cell proliferation, increases the amount of telomere length lost per
population doubling, and increases the expression of cell
surface molecules linked to vascular diseases such as
intracellular adhesion molecule 1 and plasminogen activator inhibitor 1 [11–14]. Very recently, homocysteine
has been shown to inhibit angiogenesis in vitro and in
vivo [15,16]. These findings suggest that homocysteine
may alter extracellular matrix homeostasis on diverse
tissular backgrounds.
The purpose of the present study was to examine the
effects of homocysteine on extracellular matrix-degrading proteases using human tumor cell lines. Herein, we
demonstrate that, in fact, homocysteine strongly inhibits
human tumor cell gelatinases (matrix metalloproteinases
2 and 9).
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0006-291X(03)00382-6
T. Chavarrıa et al. / Biochemical and Biophysical Research Communications 303 (2003) 572–575
Materials and methods
Cell culture. Cells were maintained in DulbeccoÕs modified EagleÕs
medium (DMEM) containing glucose (4.5 g/L) and glutamine (2 mM),
supplemented with the antibiotics penicillin (50 IU/mL), streptomycin
(50 lg/mL), and amphotericin (1.25 lg/mL) and with 10% fetal bovine
serum.
Fluorogenic assay of MMP-9 activity. Purification of HT-1080
tumor cell MMP-9 protein was carried out as described elsewhere
[17]. Prior to their use for the in vitro assay of activity, samples of
purified MMP-9 were completely preactivated by treatment with
1 mM 4-aminophenylmercuric acetate (APMA) for 12 h at 37 °C. We
used a fluorogenic assay by using a peptide substrate (supplied by
Bachem) highly specific for this gelatinase, namely, 2,4-dinitrophenylPro-b-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-(N-Me-Abz)-NH2 . This
substrate is cleaved by MMP-9, releasing 2,4-dinitrophenol, the
quenching group of the molecule, and N-methyl-anthranylic acid (NMe-Abz), the fluorogenic group. Previous tritation of the activity of
our purified samples of MMP-9 made us to choose 5% (v/v) MMP-9
suspension in assay buffer (50 mM Tris–HCl, pH 7.5, 10 mM CaCl2 ,
0.1 M NaCl, and 0.1% (w/v) Brij-35) as the optimal final concentration of enzyme for the activity assay. Activity assays were carried out
in wells of 96-well plates, with 5% (v/v) purified MMP-9 solution,
5 lM peptide substrate, and different concentrations of thiols in a
final volume of 100 lL assay buffer. Activity was followed for 30 min
at 37 °C in a fluorescence plate reader FL600FA (Bio-Tek, Winooski,
VT) at 360/460 nm excitation/emission set, a gain of 100 and bottom
lecture mode. Samples with 20 mM EDTA added were used as
positive controls of inhibition.
Conditioned media and zymography for the gelatinolytic activity of
MMP-2 and MMP-9. Conditioned media were prepared as previously
described [18,19]. The gelatinolytic activity of MMP-2 and MMP-9
delivered to the conditioned media by the different tumor cells was
detected by using gelatin zymography, as follows. Samples were subjected to non-reducing SDS/PAGE with gelatin (1 mg/mL) added to
the 10% resolving gel. After electrophoresis, gels were washed twice
with 50 mM Tris–HCl, pH 7.4, supplemented with 2% Triton X-100,
and twice with 50 mM Tris–HCl, pH 7.4. Each wash was for 10 min
and with continuous shaking. After the washes, the gels were incubated
at 37 °C for 12–16 h immersed in a substrate buffer (50 mM Tris–HCl,
pH 7.4, supplemented with 1% Triton X-100, 5 mM CaCl2 , and 0.02%
Na3 N). Afterwards, the gels were stained with Coomassie blue R-250
and the bands of gelatinase activity could be detected as non-stained
bands in a dark, stained background. Alternatively, to detect direct
effect of homocysteine and other thiols on the in situ gelatinolytic
activity of samples, different concentrations of homocysteine and other
thiols were added to the substrate buffer during the incubation of gels
containing control conditioned media. Quantitative analysis was
carried out by using the NIH Image 1.6 program.
573
teine could be involved in the modulation of angiogenesis and tumor invasion [6]. We have tested such a
hypothesis and, in fact, we have previously shown that
homocysteine exerts anti-angiogenic effects on cultured
endothelial cells [16] and anti-invasive effects on tumor
cells [19]. Since tumor invasion and metastasis require
remodeling of extracellular matrix, and gelatinases
(MMP-2 and 9) are mainly involved in this remodeling
[2,3], we investigated whether homocysteine can inhibit
both activities. We also compared the effects of homocysteine with those of other thiols, since the pharmacological potential of thiols as inhibitors of gelatinases
has been previously shown [25,26].
Fig. 1 shows that while cysteine, glutathione, Nacetyl-cysteine and 2-mercaptoethanol completely inhibited MMP-9 activity only at concentrations in the mM
range, homocysteine behaved as a much more potent
inhibitor, abolishing MMP-9 activity at a concentration
Results and discussion
An essential step in tumor invasion and metastasis
(but also in angiogenesis) is a proteolytic degradation of
extracellular matrix [2,3,20–22]. In fact, a positive correlation among tumor invasiveness and extracellular
matrix protease levels has been shown [4,23].
Homocysteine is one of the main circulating thiols in
humans, with plasma levels higher than those of glutathione (4 lM) in healthy individuals and even higher
than those of cysteine (200 lM) in cases of severe
hyperhomocysteinemia [7,24]. Based on recent findings
by other groups, we recently postulated that homocys-
Fig. 1. Inhibitory effect of thiols on in vitro MMP-9 activity. The
fluorimetric assay for MMP-9 activity described in Materials and
methods was carried out with 5% (v/v) purified MMP-9 (APMA-preactivated) and different concentrations of homocysteine (A) or other
thiols (B). Data are means SD of, at least, three different activity
assays (only two assays for 2-mercaptoethanol).
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T. Chavarrıa et al. / Biochemical and Biophysical Research Communications 303 (2003) 572–575
as low as 75 lM. At 0.1 mM, the potent non-physiological reductant agent 2-mercaptoethanol only inhibited
MMP-9 activity by 60 5%, whereas the physiologically
relevant thiols, cysteine and glutathione produced no
significant inhibition of MMP-9 activity (12 13% and
22 12%, respectively). These data show that homocysteine seems to be the most potent thiol inhibitor of
tumor gelatinases, probably due to its higher chemical
reactivity as compared with other naturally occurring
thiols [27]. It must be stressed that complete inhibition of
MMP-9 activity is achieved at homocysteine concentrations almost 300-fold lower than those required to
achieve this effect by using other thiols.
Homocysteine indeed inhibits gelatinases by directly
blocking enzymatic activity. This block could be caused
by a competitive affinity of homocysteine for the zinc
ion necessary for the enzymatic activity of gelatinases
[28]. An activation of pro-MMP-2 by low concentrations of homocysteine has been demonstrated previously
[29]. This activation is claimed to be in accordance with
the ‘‘cysteine-switch’’ mechanism [30], occurring without
further autoproteolysis of the enzyme. These previous
data are not in contradiction with our own data, since
our in vitro assays were done on APMA-preactivated
MMP-9 purified enzyme. In fact, the same previous
study shows that APMA-activated pro-MMP-2 can be
inhibited by homocysteine [29].
Both HT-1080 and human osteosarcoma U2-OS cells
produce and release both gelatinases, but HT-1080 releases more MMP-9 than MMP-2 and U2-OS releases
more MMP-2 than MMP-9 [18]. To show whether this
inhibitory effect of homocysteine is also exerted on
Fig. 2. Effects of thiols and NADH added to the substrate buffer of the
gelatin zymography assay. Conditioned media from control, untreated
U2-OS cells were used for gelatin zymography. After electrophoresis,
5 mM of one of the tested thiols or NADH was added to substrate
buffer.
Table 1
Homocysteine inhibits the secretion of gelatinases by HT-1080 and
U2-OS cells
Cell line
MMP-9 activity (%)
MMP-2 activity (%)
HT-1080
U2-OS
47 3
62 1
38 3
27 1
Experiments were carried out as described in Materials and
methods in the absence or presence of 5 mM homocysteine. Data are
given as percentages as compared to control values and they are
means SD from two different, independent gelatin zymography
assays.
MMP-2, conditioned media from U2-OS cells were used
for gelatinolytic assays where the thiol was added to
substrate buffer. In this assay, 0.1 mM homocysteine did
not produce any significant effect either on the MMP-2
and the MMP-9 proteolytic bands (results not shown).
Fig. 2 shows that 5 mM homocysteine, glutathione or
cysteine added to the substrate buffer completely abolished the in situ reconstitution of gelatinase activity in
zymographic assays. Another thiol compound, namely,
2-mercaptoethanol only partially inhibited the gelatinases and the reduced compound NADH had no effect.
Conditioned media from both tumor cell lines exposed for 24 h to 5 mM homocysteine contained much
lower MMP-2 and MMP-9 levels than control, untreated cells, as determined by gelatin zymography (Fig.
3 and Table 1). Although the homocysteine concentration here used in cell culture experiments is rather high
as compared with the concentrations in plasma under
physiological and pathological conditions, this is unavoidable because cells in culture upregulate the metabolism of homocysteine considerably. It has been
recently shown that such high extracellular concentrations of homocysteine are required to observe cellular
responses to homocysteine treatment [12]. In fact, similar high concentrations of homocysteine have been used
in recent relevant studies involving cultured endothelial
cells [11–16]. However, the known limitations of cell
cultures make us to be cautious regarding the actual
physiological relevance of our data. Further studies on
in vivo models would be necessary to elucidate this issue.
Modulation of extracellular and intracellular thiols is
being investigated as a promising strategy in cancer
prevention [31]. Other mechanisms related to its redox
status are likely to contribute to these effects. Further
studies in this direction are warranted. In any case, our
results are biochemically relevant and of potential
pharmacological interest.
Acknowledgments
Fig. 3. Effects of homocysteine on the secretion of gelatinases by HT1080 and U2-OS cells. Conditioned media from control and 5 mM
homocysteine-treated cells were used for gelatin zymography as
described in Materials and methods.
This work was partially supported by Grants PTR1995-0619 (CICYT, MCYT, Spain), OTRI-PAI-02-06 and funds from group CVI267 (Andalusian Government), and a Grant from Fundaci
on Ram
on
T. Chavarrıa et al. / Biochemical and Biophysical Research Communications 303 (2003) 572–575
Areces (Spain). Thanks are due to Miss Auxiliadora L
opez Jimenez for
her excellent technical help.
[17]
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