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Tumor Biology |
Human Tumors Immunobiology Unit, Department of Experimental Oncology [O. S., M. C., I. B., A. A.], and Division of Pathology [G. T.], Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m) and down-regulation of Bcl-2 protein
level after iNOS inhibition. AMG treatment triggered release of
caspase-1, enzymatic activation of caspase-3, and degradation of
poly(ADP-ribose) polymerase, one of the main caspase-3 substrates.
Melanoma cell apoptosis induced by iNOS inhibition was completely
blocked by peptide inhibitors of caspase-1 and caspase-3 (Ac-DEVD-CHO
and AC-YVAD-CHO) or by an exogenous NO donor, sodium nitroprusside, or
by addition of serum. Finally, comparison of control and AMG-treated
melanoma cells by pathway-specific gene array analysis indicated that
inhibition of NO synthesis led, before induction of apoptosis, to
up-regulation of mRNA levels of genes involved in the apoptosis
pathway such as Bax, caspase-1, caspase-3, caspase-6,
gadd45ß, mdm2, and TRAIL. Taken
together, these results indicate that melanoma cell survival is
regulated by endogenous NO resulting from iNOS activity. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Even tumor cell susceptibility to immune response can be modulated by
escape mechanisms that block susceptibility to apoptosis induced by
triggering cell surface death receptors as FAS/CD95 and
TRAIL3
-R. In fact, over-expression of cellular FLICE inhibitory protein
(11)
in human melanoma can prevent both CD95-mediated
(12)
and TRAIL-mediated apoptosis (13)
, and
murine models (14)
implicate these resistance mechanisms
in tumor escape from immunity in vivo. Furthermore, melanoma
cells can resist TNF-
-induced apoptosis by a mechanism involving the
homologue of Slimb proteins, which can up-regulate nuclear factor-
B
activity by targeting its inhibitor IkB for degradation
(15)
.
Negative regulation of apoptosis in human melanoma cells is mediated by
additional processes depending on loss of gene expression, associated
with tumor progression or on activation of genes not expressed in
normal cells. For example, the expression of the c-Kit
proto-oncogene is lost during tumor progression, thus abolishing tumor
cell susceptibility to apoptosis induced by stem cell factor, the c-Kit
ligand (16)
. In addition, melanomas, but not normal
melanocytes, express
vß3 integrin
(17)
, a receptor the interaction of which with collagen
promotes tumor cell survival by increasing the expression of the
antiapoptotic gene Bcl-2 (18)
. Furthermore,
melanoma, but not normal melanocytes, express the antiapoptotic gene
survivin (19)
, and overexpression of
tyrosinase-related protein-2 can protect melanoma cells from
UVB-induced apoptosis (20)
.
Taken together, these data suggest that development of resistance to
induction of apoptosis may be considered a hallmark of tumor
progression in human melanoma and highlights the need for the
elucidation of new molecular pathways of susceptibility to the
activation of programmed cell death in this tumor. In search for such
alternative routes, we investigated the potential role of endogenous NO
in survival and apoptosis of human melanoma cells. Available evidence
indicates that endogenous or exogenous NO can exert an antiapoptotic
function in different normal cells in response to proapoptotic stimuli
as TNF-, actinomycin D, CD95-mediated signaling, removal of growth
factors, or UV irradiation (21, 22, 23, 24, 25)
. However, in contrast
with normal cells, no evidence is available on a possible function of
endogenous NO as survival factor in human melanoma. On the contrary,
several reports have provided evidence indicating that NO promotes
tumor cell apoptosis in neoplastic cells of different histological
origin (26, 27, 28, 29)
. In addition, murine models have shown
that proliferation, tumorigenicity, and metastasis formation of
melanoma cells can be inhibited when NO production is induced
(30
, 31)
.
In contrast with these findings, in this study we provide evidence indicating that constitutive production of endogenous NO, by inducible NOS, has an antiapoptotic function promoting survival of human melanoma cells but not of normal melanocytes. These data suggest that new strategies aimed at targeting iNOS expression/function in melanoma cells may provide a new tool to inhibit the survival of neoplastic cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mAbs and Immunofluorescence Analysis.
Expression of iNOS and of Bcl-2 was determined by intracytoplasmic
immunofluorescence in saponin-permeabilized cells as described
(33)
. A FITC-labeled antibody directed to human iNOS
(FITC-macNOS, clone 6; Transduction Laboratories, Lexington, KY) was
used. For detection of intracellular Bcl-2, permeabilized cells were
stained with an anti-Bcl-2 mAb (anti-Bcl-2, clone 7; Transduction
Laboratories), followed by incubation with fluorescein-conjugated
F(ab')2 fragments of a goat-antimouse IgG + IgM (H+L) antibody (Jackson ImmunoResearch, West Grove, PA).
Negative controls for iNOS staining consisted of cells stained only
with an FITC-labeled IgG2a control isotype (Becton Dickinson,
Sunnyvale, CA). Cells stained with a mAb to vimentin (Roche
Diagnostics, Monza, Italy) were used as a positive control for cell
permeabilization. After staining and washing, the samples were analyzed
for expression by a FACScalibur cytofluorimeter (Becton Dickinson).
Immunohistochemical Analysis of Melanoma Lesions.
Immunohistochemical analysis was performed on routinely formalin- or
Bouin’s-fixed and paraffin-embedded specimens as described previously
(34)
. To optimize immunodetection of MIB-1 and Bcl-2,
antigen unmasking was performed (34)
. Staining with
anti-iNOS and anti-eNOS was conducted using polyclonal rabbit
antibodies (Oxis International, Portland, OR) in PBS-1% BSA, 0.1%
sodium azide. A mouse monoclonal anti-Bcl-2 antibody (Dako Corp.) and
anti-Ki-67 mAb (MIB-1; Immunotech, Marseille, France) were used.
Apoptosis-induced DNA fragmentation in melanoma lesions or in melanoma
cell lines was estimated with the TdT in situ Apoptosis
Detection Kit-HRP/DAB (Genzyme, Cambridge, MA), according to the
manufacturer’s specifications. Melanoma cell lines, cultured in
serum-free medium and treated with the specific iNOS inhibitor
aminoguanidine hemisulfate salt (AMG; Sigma, St. Louis, MO), a
structural analogue of the iNOS substrate
(L-arginine; Ref. 35
), and
subsequently formalin-fixed and paraffin-embedded were used as positive
controls for the apoptosis detection kit.
Determination of Nitrite Concentration in Melanoma and Melanocyte
Supernatants.
Five x 105 melanoma cells or
normal melanocytes were cultured for 24 h in DMEM (Celbio, Milan,
Italy) without serum. Inhibition of iNOS enzymatic activity was
performed by culturing cells in the presence of 0.1–4 mM
AMG. Supernatants were then collected, and nitrite levels were tested
using a nonenzymatic NO assay (Byoxytech nitric oxide non-enzymatic
assay; Oxis International, Portland, OR). The threshold of
nitrite concentration by this assay is 1 µM.
Detection of Apoptosis and Caspase Activity.
Subconfluent monolayers of melanoma cells or of normal melanocytes were
incubated for 48 h with/without serum and in the presence or
absence of AMG (0.1–2 mM). In some experiments, melanoma
cells were cultured for 48 h in the presence of 100
µM N-(3-(aminomethyl)benzyl)acetamidine
(1400W; Alexis Biochemicals, San Diego, CA), a different and specific
iNOS inhibitor (36)
. As positive control of apoptosis,
melanoma cells were treated with 50 µM
etoposide (Bristol-Myers Squibb, Sermoneta, Italy) or 50
µM cisplatin (Bristol-Myers Squibb).
Quantification of apoptotic cells was performed by the Annexin V-FITC
kit (Bender MedSystems, Vienna, Austria). Apoptosis-induced DNA
fragmentation was also estimated by the TUNEL assay. Inhibition of
AMG-induced apoptosis was evaluated by the Annexin V-FITC kit in the
presence of 2 or 5 µM of caspase-3 inhibitor
Ac-DEVD-CHO (Calbiochem, La Jolla, CA) and 0.5
µM of caspase-1 inhibitor AC-YVAD-CHO (Alexis,
San Diego, Ca). Caspase-1 levels were tested in tumor supernatants by
an ELISA test (Bender MedSystems). Caspase-3 enzymatic activity was
measured in cell extracts by using CASPASE-3 Cellular Activity Assay
Kit PLUS-AK-703 (QuantiZyme Assay System; Biomol Research Laboratories,
Plymouth Meeting, PA).
3
[H]Thymidine Incorporation and Cell Cycle
Analysis.
Melanoma cells (104 cells/well) were seeded in
flat-bottomed, 96-well plates (Costar, Corning, NY) and incubated for
72 h in RPMI 1640 with (0.1%) or without FCS, in the absence or
presence of the iNOS inhibitor AMG (0.1–2 mM).
[3
H]Thymidine (DuPont NEN Life Science Products, Boston,
MA) incorporation was assessed during the last 18 h of culture as
described (33)
. The assay was performed in six replicate
wells, and data were expressed as mean [3
H] content
(cpm). Cell cycle analysis was carried out on cells treated first with
paraformaldehyde and ethanol and then with PI (Sigma). Twenty thousand
events were acquired on a FACScalibur instrument, and analysis of cell
cycle phases was performed with the aid of the Modfit software (Becton
Dickinson).
Mitochondrial Membrane Potential Assay.
The mitochondrial membrane potential (m)
was quantitated by flow cytometric analysis of cells stained with
DiOC6(3)
(Calbiochem/France Biochem, Meudon, France). DiOC6(3)
is a dye
that incorporates into mitochondria in strict dependence of the
m (37)
. Melanoma cells
stimulated for 24 h in presence or in absence of AMG (4
mM), with or without SNP (50 µM), were
incubated in 20 nM DiOC6(3)
for 15 min at 37°C. Cells
were then washed in PBS and analyzed on a FACScalibur flow cytometer
(Becton Dickinson).
Western Blot Analysis for PARP Degradation.
Melanoma cells in serum-free RPMI 1640 were treated with AMG (from 0.1
to 8 mM) for 48 h. Cells were then lysed with a buffer
containing 2 M Tris-HCl (pH 8.0), 0.5 M EDTA
(pH 8.0), NP40, and protease inhibitors (10 µg/ml aprotinin, 1
mM phenylmethylsulfonyl fluoride, 20 µM
pepstatin, and 40 µM leupeptin). Protein samples (10
µg) were electrophoresed under reducing conditions on 8%
polyacrylamide minigels. The proteins were then electroblotted to a
polyvinylidene difluoride membrane (Hybond-P; Amersham Life Sciences,
Buckinghamshire, United Kingdom), and the membrane was blocked with 10
mM Tris and 150 mM NaCl (TBS) plus 0.1% Tween
containing 5% nonfat milk. The membrane was rinsed with TBS/Tween
0.1% and then incubated for 2 h at room temperature in the
presence of a 1:1000 dilution of a rabbit antibody specific for the p85
fragment of PARP (Promega Corp., Madison, WI). After washing, the
membrane was incubated for 45 min at room temperature with a 1:200
dilution of a biotinylated-antirabbit IgG (Amersham). The blot was then
washed with TBS/Tween 0.1% and incubated for 30 min with
streptavidin-horseradish peroxidase (1:400; Amersham). Development was
performed by the chemiluminescence method (ECL) following the
manufacturer’s protocol (Amersham). Lysates of Jurkat cell line
treated for 6 h with the anti-FAS CH11 mAb (Immunotech, Marseille,
France) were used as positive and negative controls, respectively, for
Western blot detection of PARP cleavage.
Gene Expression Levels by Pathway-specific Array Analysis.
Expression of 23 genes involved in the apoptosis pathways and of two
housekeeping genes (ß-actin and
GAPDH) was compared in melanoma cells treated or not with
AMG (4 mM) for 24 h in serum-free medium by
the Human-Apoptosis-2 GE-Array kit (SuperArray, Inc., Bethesda, MD). To
this end, total RNA was extracted with the use of RNeasy Mini kit
(Qiagen, Inc., Hilden, Germany). cDNA probes were synthesized from
total RNA samples using SuperArray’s proprietary GEAprimer mix as
reverse transcriptase primers. The cDNA probes were then hybridized to
gene-specific cDNA fragments spotted on the GEArray nylon membranes.
The relative expression levels of the various genes, as detected by
analysis with a 425 Phosphorimager (Molecular Dynamics, Sunnyvale, CA),
were estimated by comparing signal intensity to
ß-actin and GAPDH spots and then
quantitated by densitometry of autoradiograms, after background
subtraction, using the ImageQuant software (Molecular Dynamics).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, all melanoma cell lines and normal melanocytes expressed iNOS.
Expression of iNOS in vivo was confirmed by
immunohistochemistry in tissue sections of metastases corresponding to
the metastatic cell lines shown in Table 1
(data not shown). Although
intratumor heterogeneity was common for all markers, expression of iNOS
was found in most neoplastic cells (Fig. 1)
. Neoplastic cells in these lesions stained also with a control
antibody to eNOS. In all lesions (10 were analyzed), a common pattern
emerged; iNOS positivity was associated with variable expression of
proliferation markers (MIB-1), of antiapoptotic genes
(Bcl-2), and with complete, or almost complete, lack of
apoptosis in neoplastic cells as evaluated by the TUNEL assay (see Fig. 1
for representative results).
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. However, in the presence of 0.5–4
mM AMG, a specific iNOS inhibitor (33)
,
supernatant levels of nitrite from two melanoma cell lines, and from
normal melanocytes were significantly reduced after 24–48 h (Fig. 2)
. Similar experiments, performed in the presence of L-NAME,
a nonselective iNOS inhibitor, failed to reduce nitrite levels in
melanoma cells supernatants (Fig. 2)
, in agreement with the results
indicating that L-NAME itself can be a source of
nonenzymatically produced NO (39)
.
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A, panel 1), a marked increase in the proportion of cells
staining with Annexin V alone (early apoptosis) was observed in the
presence of 0.1 and 2 mM AMG (Fig. 3
A,
panels 2 and 3). Furthermore, at the highest AMG dose
(Fig. 3
A, panel 3), an increase in cells staining with both
Annexin V and PI (late apoptosis/secondary necrosis) was detected. The
induction of melanoma cell apoptosis by iNOS inhibition was confirmed
by culturing cells for 48 h in the presence of 100
µM of
N-(3-(aminomethyl)benzyl)acetamidine, a different and
iNOS-specific inhibitor (data not shown). Interestingly, apoptosis
induced by low or high AMG dose was completely prevented by the
presence of FCS (Fig. 3
A, panels 6 and 7) or by
addition of an exogenous NO donor, SNP (Fig. 3
A, panel 4).
By contrast, apoptosis of melanoma cells induced by 50
µM etoposide or cisplatin was not inhibited by
SNP (data not shown).
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B, panel 1). This was attributable to the
removal of growth factors, but the proportion of apoptotic melanocytes
(seen mainly in the Annexin V-positive/PI-positive, upper right
quadrant) was slightly reduced in the presence of AMG (Fig. 3
B, panel 2) and not increased, as seen with melanoma cells.
Further evidence for an antiapoptotic role of endogenous NO was
obtained by looking at DNA fragmentation in AMG-treated tumor cells by
the TUNEL method. As seen in Fig. 4
, in comparison with cells kept in medium alone (panel 1),
most AMG-treated cells became TUNEL positive at 48 h (panel
2), and the effect of AMG was almost completely inhibited by the
presence of the exogenous NO donor SNP (panel 3).
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). As seen when looking at apoptosis, the effect of AMG on
3
[H]thymidine incorporation by melanoma cells
was counteracted in most instances by the presence of serum (Table 2)
,
although a significant reduction in
3
[H]thymidine incorporation was evident in some
cell lines at the highest AMG doses, even in the presence of serum
(compare response of melanoma cell lines 6, 13, and 14 in the presence
of serum at 2 mM AMG versus control).
Furthermore, the inhibitory effect of highest dose of AMG was
completely abrogated by an exogenous NO donor (SNP; Table 2
). However,
the effect of AMG on 3
[H]thymidine
incorporation by melanoma cells reflected only increased cell death by
apoptosis and not a reduced proliferation of neoplastic cells, as shown
by cell cycle analysis, indicating that AMG did not affect proportion
of cells in G0-G1, S, or
G2-M phases, either in the presence or absence of
serum (data not shown).
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and B, in two melanoma cell lines, AMG induced
a marked down-modulation of Bcl-2 protein, an effect that was
completely prevented in the presence of the exogenous NO donor SNP.
Reduced Bcl-2 levels were also associated with evidence of altered
mitochondrial membrane potential (m), as
determined by the reduction in uptake of the potential sensitive dye
DiOC6(3)
(37)
. As shown in Fig. 5
C, AMG-treated
cells showed a reduced DiOC6(3)
staining, an effect that was partly
reversed by SNP.
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. This release was inhibited by the addition of 50
µM SNP (Fig. 6A)
. In melanoma cell
extracts, AMG induced a significant increase in caspase-3/CPP32
enzymatic activity, as determined by evaluation of degradation of the
DEVD-pNA substrate (Fig. 6B)
. An exogenous NO donor, SNP,
significantly inhibited AMG-induced caspase-3 activation (Fig. 6B)
. The enzymatic activation of caspase-3, after NO
synthesis inhibition, was also documented by additional experiments
aimed at determining the proteolytic cleavage of one of its main
natural substrates, PARP. As shown in Fig. 6
C, Lanes 5 and
6, inhibition of endogenous NO synthesis by AMG led to PARP
cleavage, as documented by the appearance, on Western blots, of an
Mr 85,000 cleavage product in cell
lysates from melanoma cell lines. This cleavage product, absent from
melanoma cell lines, not treated with AMG (Fig. 6
C, Lane 3),
was induced at low levels at the lowest (0.1 mM)
AMG concentration (Fig. 6
C, Lane 4) and reached the highest
level at AMG 2 mM (Fig. 6
C, Lane 5).
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, compare the lower right quadrants of C and
D with B). A complete block of apoptosis was
observed in the presence of 5 µM of caspase-3
inhibitor Ac-DEVD-CHO and 0.5 µM of caspase-1
inhibitor AC-YVAD-CHO (Fig. 7
, compare D and B).
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. Expression of several other genes, including Bad, Bcl-2, Bcl-w,
Bcl-x, c-myc, Fas, gadd45, nuclear factor-B,
DR5, p21Waf1, and
caspase-10, was not significantly affected by AMG treatment
(Fig. 8)
. Furthermore, several other genes (e.g., FAS-L,
caspase-5, caspase-7, and p53) were not expressed in
untreated cells, and AMG treatment did not induce them (data not
shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m reduction,
and caspase activation). These data suggest that induction of cell
death by iNOS inhibition was strictly dependent on reduced NO level,
and that constitutively produced endogenous NO is an antiapoptotic
factor in human melanoma. Inhibition of iNOS led, before induction of apoptosis, to changes in expression levels of several genes. The increased mRNA expression of caspase-1, caspase-3, and caspase-6 appears consistent with the role of at least two of these proteases (caspase-1 and caspase-3) in the apoptosis induced by iNOS inhibition. Furthermore, modest but significant increases were observed for expression levels of Bax and gadd45ß. The proapoptotic function of Bax (40) and the role of gadd45 proteins in activating JNK/stress-activated protein kinase-dependent cell death through binding of MTK1/MEK4 (the upstream regulator of JNK/stress-activated protein kinase; Ref. 43 ) suggest that endogenous NO levels can impact on several pathways involved in regulation of programmed cell death in melanoma cells. However, the most impressive relative change in gene expression level was observed for mdm2, a finding that appears particularly intriguing in the light of the known functions of this gene. In fact mdm2, one of the targets of the transcriptional activator function of p53, is predominantly involved in a negative feed back loop inhibiting p53 activity (44) . However, p53 was not expressed nor induced by NO inhibition in several melanoma cell lines where up-regulation of mdm2 was observed. This suggests that NO levels can impact on a p53-independent pathway affecting mdm2 expression levels. In agreement with our results, a recent report has indicated that mdm2 has a proapoptotic role in p53-deficient medullary thyroid carcinoma cells and that this effect is associated with down-regulation of Bcl-2 (45) .
iNOS expression and release of NO in supernatants was detected in both melanoma cells and melanocytes. In agreement with these data, NOS activity has been documented previously in both melanoma and melanocytes, but increased NO levels have been described by Joshi et al. (46) in the transformed cells in comparison with the normal ones, suggesting to these authors a possible role of NO in promoting metastasis formation by maintaining a vasodilator tone in blood vessels around the tumor. However, in contrast with melanoma cells, AMG treatment of melanocytes did not promote apoptosis but rather reduced programmed cell death of these cells resulting from growth factor withdrawal (47) . These data suggest that endogenous NO exerts an antiapoptotic role in transformed but not in normal cells of the melanocyte lineage.
Analysis of the mechanisms of apoptosis induced in melanoma cells
indicated that inhibition of NO synthesis, by block of iNOS activity,
activated a mitochondrial pathway of apoptosis. In fact, treatment of
cells with AMG reduced the mitochondrial transmembrane potential
(m), as evaluated with the potential sensitive dye DiOC6(3)
, and
promoted a down-regulation of Bcl-2 protein levels. In agreement with
in vitro data, immunohistochemical analysis of tissue
sections revealed that expression of iNOS was associated with Bcl-2
positivity and with complete lack of TUNEL-positive neoplastic cells.
These results should be interpreted in the light of the current
evidence on the role of mitochondria and Bcl-2 in programmed cell death
(40)
. Results initially obtained in lymphocytes indicated
that reduction in mitochondrial membrane potential is an early step in
apoptosis (37)
. This alteration is associated with
translocation of apoptogenic cytochrome c,
apoptosis-inducing factor, procaspase-2, and procaspase-9 to the
cytoplasm (48, 49, 50)
. Interestingly, recent results indicate
that the conserved NH2-terminal homology domain
(BH4) of antiapoptotic members of the Bcl-2 family, including Bcl-2
itself, can negatively regulate these early steps of apoptosis
(51)
. In fact, the BH4 domain inhibits the activity of the
voltage-dependent anion channel of mitochondria, thereby preventing
m loss and cytochrome c release (51)
, and
thus inhibiting apoptosis. Furthermore, it has been shown that
activated caspase-3 can cleave Bcl-2, thus promoting cytochrome
c release from mitochondria (52)
. The overall
picture emerging from these data suggests that caspase-3 activation,
detected after NO inhibition in melanoma cells, may contribute to the
observed down-regulation of Bcl-2. Bcl-2 reduction, in turn, may remove
an inhibitory mechanism that normally prevents the early changes (such
as loss of m) associated with the mitochondrial pathway of
apoptosis.
However, alternative mechanisms can be considered. For example, antigen-induced programmed cell death in B cells, in vitro, can be prevented by exogenous NO through a mechanism that inhibits the drop in Bcl-2 levels, thus implying a link between Bcl-2 and NO signaling (23) . More recent results indicate that protection of keratinocytes and endothelial cells from UV-induced apoptosis is mediated through endogenous NO-mediated increase in Bcl-2 expression (53) . These data suggest that the levels of endogenous and/or exogenous NO may contribute to the expression of Bcl-2, independently from caspase-mediated Bcl-2 degradation. These mechanisms may contribute to the effect of down-regulation of Bcl-2 in melanoma cells, described in this study, upon inhibition of endogenous NO synthesis.
It is also to be pointed out, as shown in leukemia cells, that
mitochondrial permeability transition, and even cytochrome c
release, are not sufficient events for induction of apoptosis in the
absence of caspase activation (54)
. In agreement with
these data, evidence was obtained in this study indicating that at
least two key executioners of apoptosis, caspases-1 and caspase-3, were
involved in melanoma cell death induced by block of iNOS activity. This
was suggested by experiments of caspase-1 release and of enzymatic
activation of caspase-3. More importantly, experiments with caspase-1
and caspase-3 inhibitors (Ac-DEVD-CHO and Ac-YVAD-CHO) indicated that
both caspases mediated apoptosis activated by iNOS inhibition. These
data suggest that, in melanoma cells, endogenous NO may normally
prevent caspase activation. This possible function of NO is in
agreement with data obtained in hepatocytes and endothelial cells,
where it has been shown that apoptosis induced by removal of growth
factors, or by TNF-, can be prevented by exogenous or endogenous NO,
and that in these instances, NO acts through inhibition of caspase-1,
caspase-3, and caspase-8 activation (21, 22
, 55)
. A
function of NO as caspase inhibitor is consistent with the discovery of
S-nitrosylation of cysteine residues as a regulatory
mechanism to control caspase activation (56
, 57)
. Indeed,
recent results have indicated that NO can inhibit by
S-nitrosylation, the activation of at least seven caspases
(58
, 59)
and of other enzymes involved in apoptotic
pathways as JNK (60)
.
Finally, melanoma cells in vivo were found to express both a constitutive (eNOS) and the inducible (iNOS) forms of the enzymes that synthesize NO. Furthermore, although not investigated in this study, the third form of NOS (neuronal NOS) has been shown to be frequently expressed in metastatic melanoma cells (61) . However, inhibition of iNOS alone by AMG resulted in a profound reduction in NO release and promoted cellular apoptosis. These results suggest that efficient induction of melanoma cell apoptosis does not require a complete block of NO production and that the constitutive NOS may contribute to only a minor fraction of the total NO output of melanoma cells. Thus, targeting of iNOS expression/function may be sufficient to achieve inhibition of neoplastic cell survival.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by a fellowship
from Fondazione Italiana per la Ricerca sul Cancro, Milan (to O. S.)
and by Grants from Associazione Italiana per la Ricerca sul Cancro,
Milan. 
2 To whom requests for reprints should be
addressed, at Human Tumors Immunobiology Unit, Department of
Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei
Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390817; Fax:
39-02-2390630; E-mail: Anichini{at}istitutotumori.mi.it
3 The abbreviations used are: TRAIL, tumor
necrosis factor-related apoptosis-inducing ligand; TNF, tumor necrosis
factor; iNOS, inducible nitric oxide synthase; eNOS, endothelial NOS;
TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end
labeling; AMG, aminoguanidine; PARP, poly(ADP-ribose) polymerase; SNP,
sodium nitroprusside; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide;
L-NAME,
NG-nitro-L-arginine methyl
ester; mAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; PI, propidium iodide; ICE, interleukin-1ß converting
enzyme; JNK, c-Jun-NH2-terminal kinase.
Received 5/31/00. Accepted 10/23/00.
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J. A. Sosman and I. Puzanov Molecular targets in melanoma from angiogenesis to apoptosis. Clin. Cancer Res., April 1, 2006; 12(7): 2376s - 2383s. [Abstract] [Full Text] [PDF]
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 P. A. Ascierto, S. Scala, G. Castello, A. Daponte, E. Simeone, A. Ottaiano, G. Beneduce, V. De Rosa, F. Izzo, M. T. Melucci, et al. Pegylated Arginine Deiminase Treatment of Patients With Metastatic Melanoma: Results From Phase I and II Studies J. Clin. Oncol., October 20, 2005; 23(30): 7660 - 7668. [Abstract] [Full Text] [PDF]
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M. Zanon, A. Piris, I. Bersani, C. Vegetti, A. Molla, A. Scarito, and A. Anichini Apoptosis Protease Activator Protein-1 Expression Is Dispensable for Response of Human Melanoma Cells to Distinct Proapoptotic Agents Cancer Res., October 15, 2004; 64(20): 7386 - 7394. [Abstract] [Full Text] [PDF]
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K. Kawakami, M. Kawakami, and R. K. Puri Nitric Oxide Accelerates Interleukin-13 Cytotoxin-Mediated Regression in Head and Neck Cancer Animal Model Clin. Cancer Res., August 1, 2004; 10(15): 5264 - 5270. [Abstract] [Full Text] [PDF]
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C.-H. Tang and E. A. Grimm Depletion of Endogenous Nitric Oxide Enhances Cisplatin-induced Apoptosis in a p53-dependent Manner in Melanoma Cell Lines J. Biol. Chem., January 2, 2004; 279(1): 288 - 298. [Abstract] [Full Text] [PDF]
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S. Ekmekcioglu, J. A. Ellerhorst, J. B. Mumm, M. Zheng, L. Broemeling, V. G. Prieto, A. L. Stewart, A. M. Mhashilkar, S. Chada, and E. A. Grimm Negative Association of Melanoma Differentiation-associated Gene (mda-7) and Inducible Nitric Oxide Synthase (iNOS) in Human Melanoma: MDA-7 Regulates iNOS Expression in Melanoma Cells Mol. Cancer Ther., January 1, 2003; 2(1): 9 - 17. [Abstract] [Full Text] [PDF]
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R. M. Medeiros, A. Morais, A. Vasconcelos, S. Costa, D. Pinto, J. Oliveira, P. Ferreira, and C. Lopes Outcome in Prostate Cancer: Association with Endothelial Nitric Oxide Synthase Glu-Asp298 Polymorphism at Exon 7 Clin. Cancer Res., November 1, 2002; 8(11): 3433 - 3437. [Abstract] [Full Text] [PDF]
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M. Ikeguchi, T. Ueta, Y. Yamane, Y. Hirooka, and N. Kaibara Inducible Nitric Oxide Synthase and Survivin Messenger RNA Expression in Hepatocellular Carcinoma Clin. Cancer Res., October 1, 2002; 8(10): 3131 - 3136. [Abstract] [Full Text] [PDF]
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N. J. Torok, H. Higuchi, S. Bronk, and G. J. Gores Nitric Oxide Inhibits Apoptosis Downstream of Cytochrome c Release by Nitrosylating Caspase 9 Cancer Res., March 1, 2002; 62(6): 1648 - 1653. [Abstract] [Full Text] [PDF]
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G. Pietra, R. Mortarini, G. Parmiani, and A. Anichini Phases of Apoptosis of Melanoma Cells, but not of Normal Melanocytes, Differently Affect Maturation of Myeloid Dendritic Cells Cancer Res., November 1, 2001; 61(22): 8218 - 8226. [Abstract] [Full Text] [PDF]
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 S.-i. Fujii, K. Shimizu, T. Shimizu, and M. T. Lotze Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ Blood, October 1, 2001; 98(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies Blood, October 1, 2001; 98(7): 2220 - 2228. [Abstract] [Full Text] [PDF]
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) and treated with 4 mM AMG (
) for 24 h at
37°C. cDNA probes were synthesized from total RNA samples using
SuperArray’s proprietary GEAprimer mix as reverse transcriptase
primers. The relative expression levels of the various genes was
estimated by comparing signal intensity to GAPDH spots and then
quantitated by densitometry of autoradiograms, after background
subtraction. Expression levels of bax, caspase-1, caspase-3,
gadd45ß, mdm2, TRAIL, and
caspase-6 genes were significantly higher in AMG-treated
cells than in untreated samples. *, P < 0.05; **, P < 0.01;
***, P < 0.001, two-way ANOVA
followed by Bonferroni test.