Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c
Trang 1Inhibition of the MEK/ERK signaling pathway by the novel
and endothelial cell proliferation
Gianfranco Pintus1,2, Bruna Tadolini1,2, Anna Maria Posadino1,2, Bastiano Sanna1,2, Marcella Debidda1,2, Federico Bennardini2,3, Gianni Sava4and Carlo Ventura1,2
1
Department of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research,2Division of Cell Biology, National Institute of Biostructures and Biosystems, and3Department of Drug Sciences, University of Sassari, Italy;4Callerio Foundation, Institutes for Biological Research, Trieste, Italy
Imidazolium trans-imidazoledimethyl
sulfoxide-tetrachlo-roruthenate (NAMI-A) is a novel ruthenium-containing
experimental antimetastatic agent Compelling evidence
ascribes a pivotal role to endothelial cells in the orchestration
of tumor angiogenesis and metastatic growth, suggesting
antiangiogenic therapy as an attractive approach for
anticancer treatment In this context, activation of the
mitogen-activated protein kinase (MAPK)/extracellular
signal-regulated kinase (ERK) signaling pathway has been
found fundamental in transducing extracellular stimuli that
modulate a number of cellular process including cell
prolif-eration, migration and invasion Here we show that
expo-sure of the transformed endothelial cell line ECV304 to
NAMI-A significantly inhibited DNA synthesis, as well as
the expression of the proliferating cell nuclear antigene
(PCNA) These responses were associated with a marked
down-regulation of ERK phosphorylation in
serum-cultured cells In addition, NAMI-A markedly reduced
serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity NAMI-A was also able to inhibit the phos-phorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation
Keywords: ruthenium compound; signal transduction; gene expression; cell proliferation; cancer
Uncontrolled cell proliferation, as well as neoplastic growth,
consistently associate with the functional abrogation of
different intracellular checkpoint pathways Among these,
the mitogen-activated protein kinase (MAPK)/extracellular
signal-regulated kinase (ERK) pathway has been proposed
to play a crucial role in the modulation of cellular process
such as proliferation, differentiation and development [1]
Interestingly, a MAPK-related pathway has also been
reported to be involved in neoplastic transformation [2], and
an increase in MAPK expression and activity reported in
carcinoma cells suggest that its overexpression may be of critical relevance in the maintenance of tumor cell growth [3] The finding that the ERK signaling pathway is involved
in tumor cell migration and invasion [4,5], as well as in tubular formation induced by insulin in human endothelial cells [6], suggests a crucial role for the MAPK/ERK pathway in the modulation of extracellular stimuli leading
to angiogenesis and metastatic growth Within this context, the c-myc protooncogene emerged as a major conductor among the different genes involved in both primary and metastatic tumor growth This protooncogene is intimately implicated in the control of cell proliferation and its overexpression is detected in many tumor cell types [7] Amplified c-myc expression has also been reported to correlate with metastatic growth [8,9], and an involvement
of the Ras/Raf signaling pathway in the modulation of its expression has been proposed [10] These findings, and the discovery that transfection of constitutively active MAPK/ ERK kinase confers tumorigenic and metastatic potentials
to NIH3T3 cells [11], suggest that pharmacological mani-pulation of this signal transduction pathway may represent
a promising strategy for handling of human diseases including cancer and metastasis
New blood vessel formation is a prerequisite step for metastasis dissemination, a phenomenon that mainly occurs
Correspondence to G Pintus, Department of Biomedical Sciences,
Division of Biochemistry, Laboratory of Cardiovascular Research,
University of Sassari, Viale San Pietro 43/B, 07100 Sassari.
Fax: + 39 079228120, E-mail: gpintus@uniss.it
Abbreviations: NAMI-A, imidazolium trans-imidazoledimethyl
sulfoxide-tetrachlororuthenate; MAPK, mitogen-activated protein
kinase; ERK, extracellular signals-regulated kinase; MEK, MAPK/
ERK kinase; GPCR, G-protein coupled receptor; RTK, receptor
tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; PCNA, proliferating cell nuclear antigen; PMA,
phorbol 12-myristate 13-acetate.
(Received 25 June 2002, revised 3 September 2002,
accepted 11 October 2002)
Trang 2through tumor-induced angiogenesis [12] Therefore,
tar-geting endothelial cells with antiangiogenic drugs may
represent a useful implementation in the current
antineo-plastic approaches In this regard, newly synthesized
molecules are frequently proposed for being added to the
antitumoral or antimetastatic arsenal NAMI-A
(imidazo-lium trans-imidazoledimethyl
sulfoxide-tetrachlororuthe-nate) is a new ruthenium-based compound active against
lung metastasis in vivo [13] and tumor cell invasion in vitro
[14] So far, the molecular mechanisms by which this novel
ruthenium complex exerts its antimetastatic activity are
largely unknown
In this study, we attempted at elucidating the molecular
target(s) and the possible mechanism(s) involved in
NAMI-A action, by assessing its effects on cell proliferation,
ERK1/2 activation and c-myc gene expression in ECV304, a
spontaneously transformed human endothelial cell line
M A T E R I A L S A N D M E T H O D S
Cell culture
ECV304 is a spontaneously transformed, immortal
endo-thelial cell line established from the vein of an apparently
normal human umbilical cord This line displays high
proliferation rates along with the capability to induce tumor
in nude mice and has been proposed as a suitable model for
providing novel insights into the mechanisms governing
angiogenesis under both physiological and pathological
conditions [6,15] ECV304 were provided by the European
Collection of Animal Cell Cultures (Salisbury, UK) Cells
were grown in medium M199 supplemented with 10% fetal
bovine serum (Life Technologies, Paisley, UK),
100 UÆmL)1 penicillin, and 100 lgÆmL)1 streptomycin
(Sigma, St Louis, MO, USA) Cells were maintained in a
standard culture incubator with humidified air containing
5% CO2at 37C In selected experiments, cells were
serum-starved by incubation in a serum-free medium M199
containing antibiotics for 24 or 48 h before use
Determination of DNA synthesis
ECV304 cells were stimulated to growth in 24-well plates
(Falcon, Oxnard, CA, USA) for the indicated times in the
presence of medium M199, containing 10% fetal bovine
serum in the absence or presence of 100 lM NAMI-A
During the last 24 h, cells from each experimental group
were added with 1 lCiÆmL)1[methyl-3H]thymidine (specific
activity 5 CiÆmmol)1, Amersham Pharmacia Biotech,
Buck-inghamshire, UK) At the end of each experiment, the
medium was removed and the cell monolayer in each well
was washed twice with phosphate buffered saline (NaCl/Pi)
(120 mM NaCl, 2.5 mM KCl, 8.5 mM NaH2PO4, 1.5 mM
KH2PO4) pH 7.3, exposed to 5% trichloroacetic acid
(500 lL) for 5 min and then fixed in methanol (500 lL)
Finally, the cells were digested by the addition of 25M
formic acid (500 lL) Each formic acid digest was
trans-ferred with one rinse of NaCl/Pi(1 mL) to a scintillation vial
containing 3.5 mL of INSTA-GEL scintillation fluid
(Pack-ard instrument Co., Meriden, CT, USA), and radioactivity
was determined by liquid scintillation counting using a
Wallac 1215 RackBeta liquid scintillation counter (LKB
Instrument Inc., Gaithersburg, MD, USA) [16]
Immunoblot analysis Serum-starved or growing ECV304 cells were treated as described in the figure legends Immunoblotting analysis was performed as previously described [17] At the end of each experimental point, the medium was removed and cells were detached with 0.1% trypsin plus 0.02% EDTA in NaCl/Pi, pH 7.3, and pelleted by centrifugation at 1000 g for 5 min The pellet was washed with NaCl/Pi, centrifuged
as above and then resuspended in 100 lL of a chilled lysis buffer (50 mMHepes, pH 7.5, 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mMsodium vanadate,
50 mMsodium fluoride, 20 mM2-glycerophosphate, 0.1 lM okadaic acid, 1 mM phenylmethanesulfonyl fluoride,
20 lgÆmL)1 aprotinin, 50 lgÆmL)1 leupeptin, and 10 lM pepstatin) The samples were sonicated for 10 s (Branson, sonifer B-12, setting 3) and incubated at 4C for 15 min Lysates were then centrifuged at 10 000 g for 15 min (4C) and analyzed for the protein content Each sample was added with Laemmli sample buffer and boiled for 4 min Equal amounts of sample protein (5–10 lg per lane) and prestained molecular mass markers (Santa Cruz Biotech-nology, Inc., Santa Cruz, CA, USA) were fractionated by SDS/PAGE with a 12% acrylamide gel Proteins were transferred to nitrocellulose in 25 mM Tris/HCl, 192 mM glycine, and 10% methanol at 4C for 12–16 h at a constant current of 50 mA or for 2 h at 300 mA with similar results Nitrocellulose membranes were incubated in
20 mM Tris/HCl, pH 7.6, 137 mMNaCl, 0.2% Tween 20 with 5% nonfat dried milk for 1 h, washed three-times in Tween 20 (3, 3, 5 min) and incubated for 1 h with primary antibody in Tween 20 containing 1% milk Incubation was performed at room temperature for nonphospho-antibodies and overnight at 4C for phospho-specific antibodies Proteins of interest were detected using specific antibodies against PCNA, c-myc (Santa Cruz Biotechnology), MEK1/
2, ERK-1/2, phospho-MEK1/2 and phospho-ERK1/2 (New England Biolabs, Inc Beverly, MA, USA) The following dilutions were used for individual antibodies against different proteins: MEK1/2 and ERK1/2 (1 : 1600), phospho-ERK1/2 and phospho-MEK1/2 (1 : 1000), PCNA and c-myc (1 : 1000) After further washing in Tween 20, membranes were incubated for 1 h with horse-radish peroxidase-linked anti-IgG secondary Ig diluted
1 : 5000 (Bio-Rad, Hercules, CA, USA) and immunoreac-tive proteins were detected by ECL as described by the supplier (Amersham Pharmacia Biotech, Buckinghamshire, UK) The intensities of autoradiographic bands were measured with a laser densitometer (ImageQuant Compu-ting Densitometer 300/325, Molecular Dynamics, Sunny-valle, CA, USA) data are representative of three or more independent experiments
Assessment of MAPK/ERK1/2 activity Serum-starved ECV304 cells were treated as described in the figure legends At the end of each experiment, cells were washed in ice-cold NaCl/Piand removed from the flask by gentle scraping in lysis buffer (50 mM Hepes, pH 7.5,
150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxy-cholate, 1 mM sodium vanadate, 50 mM sodium fluoride,
20 mM 2-glycerophosphate, 0.1 lM okadaic acid, 1 mM phenylmethanesulfonyl fluoride, 20 lgÆmL)1 aprotinin,
Trang 350 lgÆmL)1leupeptin, and 10 lMpepstatin) After 15 min
on ice, insoluble material was removed by sedimentation for
20 min at 100 000 g, and ERK1/2 was recovered by
immunoprecipitation with anti-(ERK1/2) Ig [17] Briefly,
each cell extract (400 lg) was mixed with 10 lL of
anti-(ERK1/2) Ig for 1 h, and then 30 lL of 50% protein A–
Sepharose in lysis buffer was added for an additional 1 h
MAPK/ERK activity was estimated as previously described
[18], using the serine/threonine kinase SPA assay kit
(Amersham Pharmacia Biotech) The immune complex
was recovered by sedimentation for 5 min in a
micro-centrifuge, washed three times with 0.5 mL NaCl/Pi
containing 1% Nonidet P-40 and 2 mMsodium vanadate
and then dissolved in the reaction mixture (25 mMHepes,
p H 7.5, 10 mMMgCl2, 20 mM2-glycerophosphate, 0.5 mM
sodium vanadate, 0.5 mM EDTA, 10 mM dithiothreitol,
10 lgÆmL)1 leupeptin, 6 lM pepstatin), containing
[c-33P]ATP (specific activity 2500 CiÆmmol)1, Amersham
Pharmacia Biotech) Samples were then incubated for 1 h at
37C, pelleted by centrifugation at 14 000 g and the
resulting pellet was transferred to a scintillation vial The
radioactivity was determined by liquid scintillation
counting
Determination of c-myc gene expression by RT-PCR
analysis
Serum-starved or growing ECV304 cells cultured in T25
culture flasks (Falcon, Oxnard, CA, USA), were treated as
described in figure legends At the indicated time points,
total RNA was extracted, reverse transcribed and amplified
according to a previously described procedure [19] Total
RNA (1 lg) from ECV304 cells was reverse-transcribed for
45 min at 37C The reaction was performed in a solution
of 25 lL containing, 50 mMTris/HCl pH 8.3, 75 mMKCl,
3 mMMgCl2, 10 mMdithiothreitol, 0.2 mMof each dNTP,
0.1 lg of oligo dT, 200 units of M-MLV reverse
transcrip-tase (Life Technologies) The reaction mixture was then
heated at 95C for 5 min to inactivate the enzyme PCR
amplification was performed in 25 lL of a reaction mixture
containing, 5 lL of the reverse transcribed cDNA, 20 mM
Tris/HCl pH 8.3, 50 mMKCl, 1.5 mMMgCl2, 2.5 U of Taq
polymerase (Life Technologies, Paisley, UK), 0.2 mM of
each dNTP, and 50 pmol of each sense and antisense primer
that had previously dissolved in Tris/EDTA solutions (Tris
10 mM p H 8.0, EDTA, 1 mM pH 8.0) The number of
amplification cycles was determined experimentally for each
primer pairs by using 0.5 lCi of [a-32P]dCTP (specific
activity 3000 CiÆmmol)1, Amersham Pharmacia Biotech)
and establishing the point at which exponential
accumula-tion plateaus Using 30 PCR cycles, the products of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and c-myc amplification were all within the linear phase of
the reaction Indeed, similar conditions have been
previ-ously reported for semiquantitative analysis of gene
expres-sion [19] The position of PCR fragments was evaluated by
comp arison with a DNA molecular mass marker (Gibco
BRL) GAPDH mRNA was used for each sample as an
internal control for mRNA integrity and equal loading The
levels of radioactivity incorporated into c-myc product were
normalized by comparison with the levels of radioactivity
incorporated into the GAPDH product from the same
sample Specific primers directed against human sequences
for c-myc and GAPDH and PCR conditions were as previously described [20]
Analysis of c-myc gene transcription by nuclear runoff assay
To prepare ECV304 nuclei, cells were washed with ice-cold NaCl/Piand lysed with 0.5% Nonidet P-40 solution [10 mM Tris HCl, 10 mMNaCl, 3 mM MgCl2, and 0.5% NP-400 (v/v), pH 7.4] Nuclei were isolated by centrifugation and resuspended in a 40% glycerol buffer [50 mM Tris HCl, 40% (v/v] glycerol, 5 mM MgCl2, and 0.1 mM EDTA,
pH 8.3] In vitro nascent transcription was performed as described in detail elsewhere [21], with a minor modification Briefly, 90 lL of nuclei suspension were added with 100 lL
of 2· reaction buffer (10 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 0.3M KCl, 5 mM dithiothreitol, 1 mM each of ATP, GTP, and CTP), and 5 lL of [a-32P]UTP (3000 CiÆmmol)1), followed by incubation at room tem-perature for 15 min DNA was digested by incubating the transcription mixture for 5 min at room temperature in the presence of 1 lL of 20 000 UÆmL)1 RNase-free DNase Nuclear RNA was isolated by using the Trizol reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK), followed by purification on RNAMATRIXTM(BIO 101, inc Vista, CA, USA) Radiolabeled nuclear RNA was then subjected to a solution hybridization RNase protection assay, as previously described [21] Briefly, a 354-base pair fragment amplified from the human genomic c-myc gene [20] was inserted into pCRII-TOPO (Invitrogen Ltd, Paisley, UK) Equal counts of 32P-labeled RNA ( 5 · 106c.p.m.) were hybridized for 12 h at 55C in the presence of an unlabeled antisense c-myc RNA probe generated by transcription of the plasmid linearized with BamHI Samples were then incubated with a combination
of RNase A and T1 and exposed to proteinase K The protected fragments were recovered after phenol chloro-form extraction and electrophoretically separated in a polyacrylamide nondenaturing gel Autoradiographic exposure was performed for 48 h on Kodak X-Omat film with an intensifying screen 32P-labeled nuclear RNA was also hybridized with unlabeled antisense GAPDH mRNA synthesized from a SacI-linearized pCRII-TOPO vector, containing a 788-base pair fragment amplified from the human genomic GAPDH gene [20] GAPDH mRNA was utilized as a constant mRNA for control
Statistical analysis The statistical analysis of the data was performed using the unpaired Student’s t-test, assuming a P < 0.05 as the limit
of significance All values are given as means ± SE of at least three independent experiments
R E S U L T S
NAMI-A inhibits ECV304 proliferation and PCNA expression
To test whether NAMI-A may affect cell proliferation, we first examined the rate of DNA synthesis by following [3H]thymidine incorporation in ECV304 cells Figure 1A shows that 100 lM NAMI-A significantly inhibited DNA
Trang 4synthesis in cells cultured under standard conditions This
effect was evident after 24 h of treatment and was still
consistent in cells exposed to the ruthenium compound for
48 and 72 h, indicating the capability of NAMI-A to
markedly affect cell proliferation In these time course
experiments we utilized 100 lMNAMI-A since it proved to
be the most effective concentration in counteracting DNA
synthesis over a 1–200 lMrange (Fig 1B)
Further analysis of the inhibitory response elicited by
NAMI-A on ECV304 growth was accomplished by
inves-tigating the effect of NAMI-A on proliferating cell nuclear
antigen (PCNA), a cell growth-related protein Growing
cells were exposed to the drug for the indicated times and
PCNA expression was evaluated by Western blotting
analysis using specific anti-PCNA Ig The current
experi-mental data show that cell exposure to NAMI-A
time-dependently inhibited PCNA protein expression, as
compared to untreated cells This effect was detectable after
24 h of cell treatment and reached the maximal amplitude at
72 h (Fig 1C)
NAMI-A inhibits both ERK1/2 activation and activity The MAPK/ERK cascade, including ERK1/2, normally promotes cell proliferation, as indicated by the strong correlation between ERK1/2 activation and both DNA synthesis and PCNA expression [22,23] Here, we assessed both ERK1/2 activation and activity in either serum- or phorbol 12-myristate 13-acetate (PMA)- stimulated ECV304 cells that had been exposed to NAMI-A Cells were treated with 100 lMNAMI-A for the times indicated and the activation of ERK1/2 was assessed by immunoblot analysis with antibodies that recognize the activated phos-phorylated form of this kinase Drug treatment was able to down regulate serum-induced ERK1/2 phosphorylation, compared to untreated cells Such an inhibitory effect was already detectable after 0.5 h incubation, increased upto
6 h, then decreased at 48 h (Fig 2A) In ECV304 cells, ERK1/2 phosphorylation was also enhanced by exposure of serum-free cells to 100 nMPMA (Fig 2B) NAMI-A or the selective MEK/ERK1/2 inhibitor PD98059 completely prevented PMA-generated ERK1/2 phosphorylation in serum-free cells, indicating the capability of both drugs to selectively inhibit the PMA-dependent signaling leading to the phosphorylation of ERK1/2 isoenzymes (Fig 2B)
To verify the involvement of a PKC-dependent pathway
in both serum- and PMA-generated ERK1/2 phosphoryla-tion, the selective PKC inhibitor GF109203X was used in separate experiments As reported in the Fig 3A and B, the exposure of ECV304 cells to GF109203X was able to partially suppress serum-induced ERK phosphorylation while completely inhibiting that elicited by the PMA In addition, NAMI-A inhibited MEK1/2 phosphorylation with a magnitude similar to that of ERK1/2 inhibition (Fig 3C,D), suggesting that NAMI-A-mediated inhibition
of ERK1/2 phosphorylation may be lying upstream of ERK1/2
Using a serine/threonine kinase SPA assay kit, the capability of NAMI-A to affect the ERK1/2 phosphoryla-tion activity was also assessed Consistent with immunoblot analyses, a 15-min exposure to 100 lM NAMI-A signifi-cantly but not completely inhibited serum-stimulated kinase activity, which was still detected after 1 h of drug treatment
In contrast, the exposure of ECV304 cells to 100 lM NAMI-A completely prevented the phorbol ester-induced ERK1/2 activity at all times assessed (Fig 4A) The effect elicited by NAMI-A on both serum- and PMA-induced ERK1/2 activity was dose-dependent and 100 lM was the most effective concentration in down regulating ERK1/2 activity (Fig 4B,C)
NAMI-A-induced ERK1/2 inhibition down regulates c-myc gene expression
To investigate whether NAMI-A-induced inhibition of ERK1/2 signaling could affect the expression of a cell growth-related gene, we assessed c-myc gene expression in growing cells treated either with NAMI-A or the MAPK/ ERK inhibitor PD98059 The addition of 100 lMNAMI-A
to the incubation medium significantly inhibited serum-elicited c-myc gene expression as compared with untreated cells This effect was already evident 1 h after NAMI-A exposure and reached a maximum at 3 h (Fig 5A) A down regulation of c-myc mRNA expression was also observed in
Fig 1 NAMI-A inhibits DNA synthesis and PCNA expression in
ECV304 cells (A) Growing cells were treated for the indicated times
with medium M199, containing 10% fetal bovine serum in the absence
(j) or p resence (s) of 100 l M NAMI-A *, significantly different from
fetal bovine serum (B) Growing cells were treated for 72 h with
medium M199, containing 10% fetal bovine serum in the absence
(0 l M ) or p resence (s) of the indicated concentration of NAMI-A *,
Significantly different from fetal bovine serum (C) Growing cells were
treated for the indicated times with medium M199, containing 10%
fetal bovine serum in the absence (j) or presence (s) of 100 l M
NAMI-A Equal amounts of protein from each sample were subjected
to SDS/PAGE and analyzed by immunoblotting The upper and lower
part of panel C show, respectively, the PCNA immunoreactivity and
the quantitative immunodensity expressed as percentage of control
(time 0) *, significantly different from fetal bovine serum.
Trang 5serum-cultured ECV304 cells exposed to PD98059
(Fig 5B) Interestingly, both the inhibition of MEK/ERK
pathway and the exposure of ECV304 cells to NAMI-A
resulted in down regulation of c-myc gene expression with
superimposable magnitudes and kinetics profiles Figure 5C
shows the dose–response effect of NAMI-A on ECV304
c-myc mRNA expression As reported for DNA synthesis
and ERK1/2 activity, 100 lMNAMI-A resulted to be the
concentration most effective in inhibiting c-myc gene
expression (Fig 5C) Furthermore, NAMI-A addition to
PD98059-treated cells failed to produce any additive
inhibition of the residual c-myc gene expression induced
by serum, suggesting the possibility that both NAMI-A and
PD98059 might have exerted their effect by following the same signal transduction pathway (Fig 5C)
We next investigated whether NAMI-A may also induce c-myc down-regulation in serum-free cells exposed to PMA,
an experimental condition, which has been previously shown to elicit a NAMI-A inhibitable increase in ERK1/2 activity Figure 6A shows that in serum-free cells, 100 nM PMA increased c-myc gene expression Such an effect was evident at 1 h, peaked after 3 h of treatment, thereafter progressively declined and returned to the control value at
12 h PMA-induced increase in c-myc gene expression
Fig 2 NAMI-A and PD98059 down regulate serum-induced ERK1/2
phosphorylation (A) Growing cells were stimulated for the indicated
times with medium M199, containing 10% fetal bovine serum in the
absence (j) or presence (s) of 100 l M NAMI-A * Significantly
different from fetal bovine serum (B) Serum-starved ECV304 cells
were stimulated for the indicated times with medium M199 containing
100 n M PMA (j), or pretreated for 1 h with either 50 l M PD98059
or 100 l M NAMI-A and then stimulated with medium M199
con-taining 100 n M PMA in the presence of either 50 l M PD98059 (h) or
100 l M NAMI-A (s) *, Significantly different from PMA; §
signifi-cantly different from time 0 The upper part of each panel shows
representative autoradiograms corresponding to ERK1/2 and
phos-pho ERK1/2 immunoreactivity while the lower part of each panel
reports the quantitative analysis of phospho ERK1/2 immunodensity
expressed as percentage of control (individual densities from ERK1
and 2 bands were added upto generate one single ERK1/2 value).
Fig 3 NAMI-A inhibits MEK1/2 activation and GF109203X down regulates ERK1/2 phosphorylation (A,B) Serum-starved ECV304 cells were pretreated for 1 h with 5 l M GF109203X (+) and then stimu-lated for 1 h with medium M199 containing 10% fetal bovine serum (A) or 100 n M PMA (B) in the presence (+) or absence (–) of 5 l M
GF109203X (C,D) Serum-starved ECV304 cells were pretreated for
1 h with 100 l M NAMI-A (+) and then stimulated for 1 h with medium M199 containing 10% fetal bovine serum (C) or 100 n M
PMA (D) in the presence (+) or absence (–) of 100 l M NAMI-A * Significantly different from fetal bovine serum; §, significantly different from PMA The upper part of each panel shows representative auto-radiograms corresponding to the immunoreactivity of ERK1/2 and phospho ERK1/2 (A,B), and MEK1/2 and phospho MEK1/2 (C,D) The lower part of each panel reports the quantitative analysis of phospho ERK1/2 (A,B), and phospho MEK1/2 (C,D) immunodensity expressed as percentage of control (individual densities from ERK1 and 2 bands were added upto generate one single ERK1/2 value).
Trang 6occurred in a dose-dependent fashion, reaching the maximal
amplitude at 100 nM over a concentration range of
10–200 nMPMA (Fig 6B) Both NAMI-A and PD98059
dose-dependently counteracted and ultimately abolished the
increase in c-myc mRNA expression elicited by PMA
(Fig 6C,D), suggesting a close relationshipbetween the
inhibitory effect of NAMI-A on ERK1/2 activation and
NAMI-A- induced down regulation of c-myc gene
expres-sion
NAMI-A inhibits c-myc gene transcription and protein
expression
To establish whether the decrease in c-myc mRNA
expres-sion elicited by NAMI-A might have occurred at the
transcriptional level, we assessed the rate of transcription of
the c-myc gene by using an in vitro nuclear run-off
transcription assay A remarkable decrease in c-myc gene
transcription was observed in nuclei isolated from serum-stimulated ECV304 cells that had been exposed for the times indicated to 100 lM NAMI-A, as compared with nuclei from untreated control cells (Fig 7A) The transcriptional decrease induced by NAMI-A exhibited a time-course that overlaid that observed in RT-PCR experiments, being evident at 1 h and reaching a maximum after 3 h (Fig 7A)
In addition, the down-regulatory effect of NAMI-A on gene transcription was paralleled by a decrease in c-myc protein expression, as confirmed by immunoblotting experiments (Fig 7B)
Fig 5 NAMI-A and PD98059 down regulate serum-induced c-myc gene expression with superimposable magnitudes and kinetic profiles (A) Growing cells were stimulated with medium M199 containing 10% (v/v) fetal bovine serum in the absence (j) or presence (s) of 100 l M
NAMI-A (B) Growing cells were stimulated with medium M199 containing 10% fetal bovine serum in the absence (j) or presence (half shaded square) of 50 l M PD98059 (C) Growing cells were stimulated for 3 h with medium M199 containing 10% fetal bovine serum in the absence (0 l M ) or p resence (d) of the indicated concentration of NAMI-A Cells were also treated with 100 l M NAMI-A + 50 l M
PD98059 for 3 h The upper part of Panel A shows representative ethidium bromide stained gels of the reaction products obtained using
5 lL of the RT products after 30 cycles of PCR amplification The lower part of panel A, and panels B, C report the expression of c-myc mRNA levels detected by[ 32 P]dCTP-PCR Individual results were normalized to GAPDH mRNA detected in each samp le and exp ressed
as a ratio to GAPDH *, Significantly different from fetal bovine serum.
Fig 4 NAMI-A partially inhibits serum-induced ERK 1/2 activity but
completely counteracts the activity induced by PMA (A) Serum-starved
ECV304 cells were left untreated (U) or pretreated for 1 h with 100 l M
NAMI-A (N) Cells were then stimulated for the indicated times with
medium M199 containing either 10% fetal bovine serum (open bars)
or 100 n M PMA (hatched bars) in the absence (–) or presence (+) of
100 l M NAMI-A ERK1/2 activities are expressed as c.p.m 32 P
incorporated per mg protein per hour *, Significantly different from
fetal bovine serum; §, significantly different from PMA; #, significantly
different from time 0 (B,C) Serum-starved ECV304 cells were left
untreated (U) or pretreated for 1 h with the indicated NAMI-A
con-centration (NAMI-A) Cells were then stimulated for 1 h with medium
M199 containing either 100 n M PMA (B) or 10% fetal bovine serum
(C), in the presence of the indicated concentrations of NAMI-A.
ERK1/2 activities are expressed as percentage of control *
Signifi-cantly different from U.
Trang 7D I S C U S S I O N
The process of angiogenesis involves the formation of new
blood vessels from established vasculature and is essential
for progressive tumor growth and metastasis The finding
that such a pathological condition mainly depends on
endothelial cell proliferation, migration and invasion [12]
has sparked an interest in identifying cellular models of
angiogenesis as well as molecular mechanisms amenable to
endothelial cell proliferation In this context, ECV304 a
spontaneously transformed endothelial cell line, has been
recently proposed as a suitable model for the investigation
of the signaling mechanisms governing angiogenesis under
both normal and pathological conditions [6,15] A recent
investigation reports that ERK1/2 is constitutively active in
ECV304 cells and that such a condition plays a key role in the alteration of the growth behavior of these cells [24] Constitutively active components of the ERK pathway have been shown to be associated with neoplastic growth [3] and
an increase in ERK-related signaling has been found to represent a common outcome for a number of molecular events leading to angiogenesis [6] and metastatic growth [25,26] These observations prompt the hypothesis that pharmacological manipulation of the ERK pathway may prove rewarding in counteracting such pathological process
A number of extracellular stimuli, including serum, have been reported to activate ERK via Ras/Raf/MEK/signaling [27,28] This activation is mediated, at least in part, by G-protein coupled receptors (GPCRs), stimulation of membrane phospholipid hydrolysis, and activation of the diacylglycerol sensitive isoforms of protein kinase C (PKC) [28] Thus, phorbol esters such PMA also powerfully activated the ERK cascade either by c-raf-mediated phos-phorylation [29] or in a Ras-dependent manner [30] The
Fig 7 NAMI-A inhibits c-myc gene transcription and protein expres-sion (A) Nuclear run-off transcription assay Growing cells were sti-mulated for the indicated times with medium M199 containing 10% (v/v) fetal bovine serum in the absence (–) or presence (+) of 100 l M
NAMI-A and then processed as described in materials and methods Representative autoradiograms of the ribonuclease protection analysis
of c-myc mRNA are shown in panel A Autoradiographic exposure was for 2 days on Kodak X-Omat film with an intensifying screen Row a, transcription of the c-myc gene Row b, GAPDH mRNA On the right are indicated the position of 350- or 800-bp radiolabeled DNA markers, showing that the single protected fragments migrated with a molecular size comparable to c-myc (bp354) or GAPDH (bp788) mRNA (B) Growing cells were treated for the indicated times with medium M199, containing 10% (v/v) fetal bovine serum in the absence (–) or presence (+) of 100 l M NAMI-A Equal amounts of protein from each sample were subjected to SDS/PAGE and analyzed
by immunoblotting Representative autoradiograms of the immuno-blotting analysis correspond to c-myc immunoreactivity are shown in the panel B.
Fig 6 NAMI-A and PD98059 completely inhibits PMA-induced c-myc
gene expression (A) Serum-starved ECV304 cells were stimulated for
the indicated times with medium M199 containing 100 n M PMA.
*, Significantly different from time 0 (B) Serum-starved ECV304 cells
were stimulated with the indicated concentrations of PMA for 3 h.
*, Significantly different from 0 n M PMA (C,D) Serum-starved
ECV304 cells were left untreated (U) or pretreated for 1 h with the
indicated concentration of PD98059 (PD) or NAMI-A (N) Cell were
then stimulated for 3 h in medium M199 containing 100 n M PMA, in
the abesence (U), or presence of the indicated concentration of either
PD98059 (PD) or NAMI-A (N) *, significantly different from PMA.
The upper part of panels A and B, shows representative ethidium
bromide stained gels of the reaction products obtained using 5 lL of
the RT products after 30 cycles of PCR amplification The lower part
of panels A, B and panels C, D report the expression of c-myc mRNA
levels detected by [32P]dCTP-PCR (30 cycles) Individual results were
normalized to GAPDH mRNA detected in each sample and expressed
as a ratio to GAPDH.
Trang 8present results showing the ability of NAMI-A to
com-pletely abolish ERK1/2 activation elicited by PMA in
serum-free cells, clearly indicate the specificity of the drug in
inhibiting phorbol ester-generated signals leading to
ERK1/2 phosphorylation Whereas, the finding that
expo-sure of serum-cultured cells to NAMI-A markedly,
although not completely, inhibited ERK1/2
phosphoryla-tion indicates that the wide-scale profiling of intracellular
signals leading to ERK1/2 activation may have not been
entirely encompassed by the drug-treatment Such a
hypo-thesis is further confirmed by the observation that,
differ-ently from the effect of serum, the stimulatory effect of
PMA on ERK1/2 activity could also be totally abolished by
NAMI-A Within this context, the present observation that
the level of phospho-ERK1/2, as well as the peak increase in
ERK1/2 activity, was significantly lower in the presence of
PMA than in serum-stimulated cells suggests, as previously
reported [31,32], that multiple signaling mechanisms
occurred in ERK1/2 activation induced by serum The
finding that PKC inhibition only partially down regulated
serum-generated ERK1/2 phosphorylation, but abolished
PMA-primed activation, indicates that serum-induced
ERK1/2 phosphorylation may both result from the
activa-tion of PKC-dependent and -independent pathways, while
that evoked by PMA mainly proceeded through PKC/Ras/
Raf/MEK-dependent signals In addition, the finding that
PMA-induced MEK1/2 activation was completely
preven-ted by the drug treatment strongly suggests that
NAMI-A-mediated inhibition of ERK1/2 phosphorylation may be
lying upstream of ERK1/2
Recent observations indicate that activation of Raf by
PMA may trigger the same signaling pathway as oncogenic
Raf, or Raf activation by Ras in combination with tyrosine
phosphorylation [30] Moreover, it is now established that
PKC activation may both result from GPCR- and receptor
tyrosine kinase (RTK)-mediated signaling and that the
activation of ERK by GPCRs closely parallels that
employed by RTK [33] On the whole, these observations
suggest that the inhibitory effect elicited by NAMI-A on
ERK signaling might have occurred by affecting a
serum-generated PKC-dependent Ras/Raf/MEK signaling
upstream to ERK1/2 Such a hypothesis is supported by
the finding that, similar to NAMI-A, exposure of
PMA-stimulated cells to either PD98059 or GF109203 completely
counteracted ERK1/2 activation, indicating that
PMA-generated signals leading to ERK1/2 phosphorylation are
under a tight control of the PKC/Ras/Raf/MEK pathway
The present results showing the capability of NAMI-A to
drastically reduce c-myc mRNA levels indicate c-myc gene
expression as a part of the molecular patterning affected by
this new compound The present observation that the
selective MEK/ERK inhibitor PD98059 significantly
reduced c-myc mRNA level in serum-cultured cells indicates
that c-myc gene expression may be at least in part mediated
by the activation of this signal transduction pathway In the
current study, a number of interrelated observations suggest
that the inhibitory effect elicited by NAMI-A and PD98059
on c-myc mRNA expression might have occurred through a
common intracellular pathway First, both ERK1/2
activa-tion and c-myc gene expression were markedly inhibited
following the exposure of serum-feeded cells to NAMI-A
Second, both the MEK/ERK inhibitor PD98059 and
NAMI-A inhibited c-myc gene expression with a similar
time-course and by superimposable magnitudes Third, NAMI-A failed to further decrease c-myc mRNA expres-sion in PD98059-treated, serum-cultured cells Since both PD98059 and NAMI-A could not completely inhibit c-myc gene expression in serum-cultured cells, it is possible that protooncogene expression may also have been induced by serum through signal(s) unrelated to ERK1/2
The finding that, in serum-free cells PD98059 completely suppressed PMA-induced c-myc mRNA expression, strongly indicates a crucial role of the Ras/Raf/MEK/ ERK pathway in modulating phorbol ester-activated mechanism(s) leading to c-myc gene expression Consistent with the present results is the finding that the c-myc gene promoter contains conserved binding sites for the Ets family [34] and that both phorbol ester and Ras are able to induce expression from Ap-1/Ets-driven promoters via the upstream MEK/ERK effector Raf [35] The finding that NAMI-A completely inhibited PMA-induced ERK1/2 activation and activity and totally counteracted phorbol ester-primed c-myc gene expression heavily suggests that the inhibitory effect of NAMI-A on c-myc gene expression may have occurred by suppressing ERK1/2 activation and activity elicited by PMA-generated signals
Unlike the ras gene family, mutations within the coding sequence appear not to be an important feature in converting c-myc from a protooncogene to oncogene Rather, abnormally high transcription of c-myc, at an inappropriate stage of the cell cycle or during differenti-ation, leads to oncogenic transformation [36] In this regard, our results showing the ability of NAMI-A to down regulate c-myc gene transcription as well as protein expres-sion may be of particular relevance Interestingly, human melanoma cells derived from metastatic lymph node displayed cisplatin resistance, higher tumorigenic ability and increased c-Myc expression, as compared with cell lines derived from the primary tumor The finding that the treatment with c-myc antisense oligodeoxynucleotides abrogated cisplatin resistance and induced apoptosis in a metastatic lymph node cell line suggests that pharmacolo-gical manipulation of c-myc gene expression may have an important role in metastasis control [37] Within this context, NAMI-A has displayed a better therapeutic effect
on the growth of mouse lung metastases, when compared to the reference drugs, including cisplatin [38]
The exact molecular sequelae of events underlying the observed effects of NAMI-A on ERK1/2 activation as well
as c-myc gene expression remain to be elucidated and are the subject for future investigations Nevertheless, the cellular targets and signaling mechanisms uncovered in the present study are associated with the modulation of critical growth regulatory decisions in different cell types Moreover, growing evidence supports the view that unplugging the currently dissected pathways from regulatory control may associate with abnormalities in the architectural plans of cell growth and differentiation, ultimately ensuing in the appearance of a malignant phenotype [5,11,25,26] These findings, along with the recent observation that NAMI-A inhibited tumor cell invasion of matrigel [14], strongly suggest that the down regulation of ERK1/2 currently elicited by NAMI-A may be a part of the molecular mechanism by which this drug exerts its antimetastatic activity in vivo Such a view may be further inferred by the following observations The NAMI-induced inhibition of
Trang 9ERK1/2 activation and activity was attained in a
trans-formed endothelial cell line and it is now evident that the
endothelial cell plays a crucial role in angiogenesis [39] and
that it might affect the growth and differentiation of
neighboring cells [40] The endothelial cell is also the target
cell for tumor neovascularization, a process that closely
associates with tumor growth and metastasis [12], and in
this context, NAMI-A has been shown to inhibit
angiogen-esis [41] and to induce apoptosis in endothelial cells [42] In
conclusion, the inhibitory effects of NAMI-A on ERK1/2
activation and c-myc gene expression observed here may
represent a prominent feature in the control of pathological
processes of the blood vessel wall and in the modulation of
angiogenic stimuli leading to neoplastic growth
A C K N O W L E D G E M E N T S
This study represents work done within the frame of the COST D20/
0005/01 and supported by grants from Ministero della Sanita` (Attivita`
di Ricerca Finalizzata – 2000), Ministero dellIstruzione,
dell’Univer-sita` e della Ricerca (Fondo Integrativo Speciale per la Ricerca – 2001;
Programmi di Ricerca Cofinanziati – 2001)’ and University of Sassari
Young Researchers Grant We thank Ms Annalisa Cossu for her
technical assistance.
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