Endogenously produced nitric oxide mitigates sensitivity
of melanoma cells to cisplatin
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Citation
Godoy, L. C., C. T. M. Anderson, R. Chowdhury, L. J. Trudel,
and G. N. Wogan. Endogenously Produced Nitric Oxide
Mitigates Sensitivity of Melanoma Cells to Cisplatin.
Proceedings of the National Academy of Sciences 109, no. 50
(December 11, 2012): 20373-20378.
As Published
http://dx.doi.org/10.1073/pnas.1218938109
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Wed May 25 19:21:39 EDT 2016
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http://hdl.handle.net/1721.1/79378
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Detailed Terms
Endogenously produced nitric oxide mitigates
sensitivity of melanoma cells to cisplatin
Luiz C. Godoya, Chase T. M. Andersona, Rajdeep Chowdhurya, Laura J. Trudela, and Gerald N. Wogana,b,1
Departments of aBiological Engineering and bChemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by Gerald N. Wogan, November 1, 2012 (sent for review May 17, 2011)
chemoresistance
| cancer | caspase-3
M
elanoma is a particularly aggressive and chemotherapy-resistant cancer (1, 2). Current strategies for improving treatment efficacy are based on critical signaling pathways that control
melanoma progression to develop targeted therapies that will
promote long-lasting remission; however, current therapeutic approaches show limited efficacy and limited cure rates (2). Cisplatin,
a DNA-damaging compound that triggers apoptotic cell death, is
commonly used in the treatment of malignant melanoma. Although cisplatin is highly effective in the treatment of many types
of cancer, melanoma is relatively resistant to its effects (3, 4).
Although a combination of mechanisms may underlie resistance to
this drug (4, 5) it is believed that low sensitivity can develop during
treatment or constitute an intrinsic property of tumor cells. In
most scenarios, these mechanisms involve inhibition of cisplatininduced apoptosis (1, 4) and stimulation of survival signals (6).
Nitric oxide (NO) is a bioactive molecule generated by NO
synthases (NOSs) in mammalian cells. By interacting with numerous diverse biomolecules, NO and its derivatives participate
in multiple cellular responses, from neurotransmission to regulation of vascular tone to immune responses and tumorigenesis
(7). In melanoma and other cancers, NOS activity is associated
with tumor growth (7–10), malignant transformation (9, 11),
angiogenesis (11), and resistance to apoptosis (6, 12). Of note,
www.pnas.org/cgi/doi/10.1073/pnas.1218938109
an important process regulated by NO is apoptosis (13), which
is dependent on S-nitrosation (14, 15), the covalent binding of
NO to protein cysteine residues that leads to functional alterations. This posttranslational modification accounts for guanylyl
cyclase-independent signaling properties of low levels of NO,
and has been involved in the regulation of an increasing number
of biological processes (13, 16–18). Imbalanced S-nitrosation is
linked to the etiology of several diseases (17), and it has been
suggested that cancer cells may exploit this biomolecular modification to escape cell death (18).
A strong correlation has been shown between the prevalence
of tumor cells expressing inducible NOS (iNOS; i.e., NOS2) and
shortened survival of patients with advanced melanoma (6, 9, 12,
19, 20). Constitutive expression of iNOS has been detected in
most metastatic melanomas and melanoma cell lines (9, 19, 20),
and it has been suggested that NO contributes to tumor survival
(12, 21). In addition, inhibition of NO production increases
sensitivity to cisplatin in vitro (22) and acts synergistically with
cisplatin to reduce tumor development in experimental melanoma (23). However, molecular mechanisms through which endogenous NO protects tumors have not been defined. Here we
show that NO produced endogenously by melanoma cells is an
important regulator of their biology and decreases their sensitivity to cisplatin. Moreover, our results indicate that increased
protein S-nitrosation is associated with the phenotype of cells
able to escape drug-induced apoptosis.
Results
Based on clinical and experimental evidence correlating NO with
poor response to cisplatin (15, 21–23), we initially confirmed that
endogenously generated NO promotes resistance to cisplatininduced apoptosis in three human melanoma cell lines. First, we
demonstrated constitutive expression of iNOS by immunoblot
(Fig. 1A). Expression of other NOS isoforms was inconsistent
or not detected. Although NO was not secreted at detectable
amounts into culture medium by melanoma cells, analysis of
whole-cell extracts by reduction/chemiluminescence using an NO
analyzer showed that NO was present at low nanomolar levels
(Fig. 1 B and C). Complementary studies with the fluorescent
dye 4,5-diaminofluorescein diacetate (DAF-2DA) confirmed the
presence of NO and its derivatives in intact, live cells (Fig. S1A),
as well as decreased intracellular NO upon incubation with the
iNOS inhibitor 1400W (Fig. S1B).
To assess impacts of endogenous NO on the physiology of
melanoma cells, growth was evaluated in cells cultured in presence of the broad-spectrum NOS inhibitor N-monomethyl-Larginine (NMA), which resulted in reduced growth in all cases
(Fig. 1D). Sensitivity to cisplatin was also affected by endogenous
NO, as shown by enhanced killing in cells treated with NMA
(Fig. 1E). Similar results were seen in A375 cells treated with 2(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
Author contributions: L.C.G., R.C., L.J.T., and G.N.W. designed research; L.C.G., C.T.A., R.C.,
and L.J.T. performed research; L.C.G. contributed new reagents/analytic tools; L.C.G., C.T.A.,
R.C., and G.N.W. analyzed data; and L.C.G. and G.N.W. wrote the paper.
The authors declare no conflict of interest.
1
To whom correspondence should be addressed. E-mail: wogan@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1218938109/-/DCSupplemental.
PNAS | December 11, 2012 | vol. 109 | no. 50 | 20373–20378
APPLIED BIOLOGICAL
SCIENCES
Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the
inducible isoform of NO synthase (iNOS) and elevated levels of
nitrotyrosine, a product derived from NO. Although several lines
of evidence suggest that NO promotes tumor growth and
increases resistance to chemotherapy, it is unclear how it shapes
these outcomes. Here we demonstrate that modulation of NOmediated S-nitrosation of cellular proteins is strongly associated
with the pattern of response to the anticancer agent cisplatin in
human melanoma cells in vitro. Cells were shown to express iNOS
constitutively, and to generate sustained nanomolar levels of NO
intracellularly. Inhibition of NO synthesis or scavenging of NO
enhanced cisplatin-induced apoptotic cell death. Additionally,
pharmacologic agents disrupting S-nitrosation markedly increased
cisplatin toxicity, whereas treatments favoring stabilization of
S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of
the proapoptotic enzyme caspase-3 was higher in cells treated
with a combination of cisplatin and chemicals that decreased NO/
SNOs, whereas lower activity resulted from cisplatin combined
with stabilization of SNOs. Constitutive protein S-nitrosation in
cells was detected by analysis with biotin switch and reduction/
chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin
treatment, resulting in augmented S-nitrosation of caspase-3 and
prolyl-hydroxylase-2, the enzyme responsible for targeting the
prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are
inhibited by S-nitrosation, our data thus indicate that modulation
of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the
efficacy of chemotherapy.
B
A
C
nM NO2
SK Mel SK Mel
A375 100 28
1000
28
150
0
Nitrite (nM)
M
el
10
5
SK
SK
A3
7
600
400
LB
NO signal (mV)
800
M
el
iNOS
200
120
90
60
30
100
0
A375
SK Mel 28 SK Mel 100
Time
A375
1.01
*
0.8
0.6
0.4
_
1.2
1
0.8*
0.6
0.4+
SK Mel 28
_
+
E
SK Mel 100
*
_
G
Relative Survival
1.2
*
0.8
0.6
0.4
PTIO
iPTIO
Cisplatin
_
_
_
+
_
_
_
+
_
_
_
+
+
+
+
_
SK Mel 100
*
0.6
**
_
_
**
_ _ +
_
+ +
_
+
+ +
_ _
+
_
+ +
80
80
*
60
60
40
40
20
20
00
Control
PTIO
Cisplatin PTIO+
Control PT IOCisplatin
PT IO+Cisplati
Cisplatin
+
(PTIO; which scavenges NO-derived species) or with the iNOS
inhibitor 1400W (Fig. S2) and subsequently challenged with
cisplatin (Fig. 1F). Cells treated with an inactive form of PTIO
before challenge with cisplatin displayed the same extent of cell
death as those treated with cisplatin alone, confirming that NO
was involved (Fig. 1F). Further, annexin V staining showed that
scavenging of NO led to increased apoptosis and significantly
enhanced the toxicity of cisplatin (Fig. 1G).
We hypothesized that protein modification by S-nitrosation
was a potential mechanism through which NO exerted the aforementioned effects. Results supporting that hypothesis showed that
pretreatment of cells with dinitrochlorobenzene (DNCB), which
favors the stabilization of S-nitrosothiols (SNOs) by inhibiting
the activity of thioredoxin reductase (24–26), increased survival
of A375 cells challenged with cisplatin (Fig. 2A). Conversely,
transient treatment of cells with a nontoxic concentration of the
reducing agent DTT to disrupt SNOs markedly increased cisplatin toxicity in a dose-dependent manner (Fig. 2B), corroborating the findings obtained with DNCB and suggesting S-nitrosation
as a regulator of chemosensitivity. Pretreatment with DTT also
increased sensitivity to cisplatin, rendering cells responsive to
concentrations that did not cause cell death in the absence of the
reducing agent (Fig. 2C).
We subsequently investigated cell signaling targets potentially
affected by NO during these responses. Immunoblot analysis
showed that activation of Akt by phosphorylation, a survival signal,
was not affected by an NMA-mediated decrease in NO concentration during the response to cisplatin in A375 or SK Mel 28 cells
(Fig. 3A). Similarly, expression of p53 and its downstream regulator MDM2, both of which are involved in responses to DNA
damage, was not significantly affected by decreased NO production. Phosphorylation activation of NFκB p65 was changed only
in SK Mel 28 cells when NMA was combined with cisplatin. On
the contrary, ERK1/2 was clearly activated in both cell lines, as
indicated by increased phosphorylation when cells were exposed
to a combination of NMA and cisplatin (Fig. 3A), a treatment
that resulted in enhanced cell death (Fig. 1E).
20374 | www.pnas.org/cgi/doi/10.1073/pnas.1218938109
SK Mel 28
0.8
NMA
Cisplatin
F
_
A375
1
0.4
+
NMA
1.0
1.2
Relative Survival
Relative Growth
1.2
Apoptotic cells (%)
D
Fig. 1. Endogenous NO mitigates cisplatin-induced apoptosis. (A) Constitutive expression of iNOS in the human melanoma cells A375, SK Mel 100, and SK Mel 28, as analyzed by
immunoblot. (B and C) Melanoma cells produce NO. Intracellular nitrite concentration was determined in whole-cell
homogenates as an indicator of NO production by using an
NO analyzer. Nitrite in the samples is reduced to NO in the
purge vessel, and NO is detected by the NO analyzer. A representative plot of the chemiluminescent signal generated
by the reaction of samples-derived NO with ozone within the
NO analyzer is shown in B. Nitrite standards (100–1,000 nM)
are also depicted. LB, lysis buffer. Intracellular concentrations
of NO are shown in C, as calculated based on standard curves
generated with nitrite. (D) NO sustains melanoma cell growth.
Cells were grown in presence of the NOS inhibitor NMA
(5 mM) for 12 d and counted under light microscopy with
a hemocytometer (*P < 0.05). (E–G) NO decrease enhances
cisplatin toxicity. (E) Cells grown in 96-well microplates in
presence of NMA for 72 h were treated with 12.5 μM (A375
and SK Mel100) or 40 μM (SK Mel28) cisplatin for 24 h. Viability was estimated with the WST-1 reagent (*P < 0.05 vs.
cisplatin). (F) A375 cells were grown for 24 h in microplates
with 75 μM PTIO to scavenge NO-derived species, followed by
addition of 12.5 μM cisplatin. Cell viability was quantified
with the WST-1 reagent 24 h later. To verify that the effect of
PTIO was caused specifically by the scavenging of NO species,
a control with inactive PTIO (iPTIO) was performed, in which
PTIO was presaturated with NO before cell treatment (*P <
0.01). (G) A375 cells grown for 24 h in presence of 75 μM PTIO
were challenged with cisplatin. The level of apoptosis was
evaluated by flow cytometry after annexin V-FITC staining
24 h later (*P < 0.05). Values shown are means ± SD.
As ERK1/2 activation is essential for induction of apoptosis
by cisplatin (4, 27, 28), expression of a number of pro- and antiapoptotic proteins was evaluated under the aforementioned conditions (Fig. 3A). As shown in Fig. 3B, NOS inhibition with NMA
during challenge with cisplatin in A375 cells reduced the levels of
the key antiapoptotic protein Bcl-2, whereas the opposite effect
was observed for proapoptotic proteins Bax and, more strikingly,
PUMA. These results show that NO was a critical component in
the regulation of cisplatin-induced cell death in this model.
Because the apoptotic effector enzyme caspase-3 is a known
target for inhibition by S-nitrosation (29), we investigated whether
NO effects on cisplatin-induced cell death were attributable to
changes in its activity resulting from modulation of S-nitrosation.
Indeed, A375 cells grown in presence of NMA to reduce NO
production and challenged with cisplatin displayed 20% higher
caspase-3 activity than cells treated with cisplatin alone (Fig. 4A).
Similarly, disruption of SNOs by a nontoxic dose of DTT enhanced cisplatin-induced caspase-3 activity early after addition of
cisplatin (Fig. 4B). On the contrary, stabilization of S-nitrosation
with DNCB inhibited 50% of caspase-3 activity in response to
cisplatin (Fig. 4C). These data suggest that S-nitrosation is a possible mechanism underlying the effects of NO, partially mitigating cisplatin toxicity by inhibiting caspase-3 activity and interfering with apoptosis.
In view of results indicating that NO fosters chemoresistance,
we investigated the hypothesis that increased production of NO
induced by cisplatin contributed to enhanced survival. Analysis
of A375 cells surviving a 24-h treatment with cisplatin revealed
a significant increase in NO content (Fig. 5 A and B), supporting
this interpretation; concurrent treatment with the NOS inhibitors
NMA and L-N6-(1-Iminoethyl)lysine hydrochloride (L-NIL) completely eliminated detectable levels of NO in cell homogenates.
To verify whether the increment in NO production induced by
cisplatin had the potential to modulate cell signaling via a corresponding increase in protein S-nitrosation, we next determined
levels of S-nitrosation in proteins known to be targets of this
modification by using the biotin switch technique. Analysis of
proteins isolated from cell extracts showed a twofold increase in
Godoy et al.
Relative Survival
A
apoptosis (caspase-3) and survival (PHD2), both known targets
for inhibition by this modification (29, 30), confirms an association between S-nitrosation and increased drug-resistance.
1.2
1.2
**
1.0
1.0
*
0.8
0.8
Discussion
Low levels of endogenous or exogenous NO enhance progression
of cancer in vitro and in vivo (7, 13, 18, 21, 23, 31). Strong clinical
and experimental evidence shows a correlation between production of NO by tumor cells and reduced survival of patients
with advanced melanoma (11, 19, 32), as well as poor response to
chemotherapy and radiation therapy (17, 22, 23, 33, 34). The
matrix of regulatory mechanisms underlying these interactions
is undoubtedly multifactorial and complex. Although NO can
promote angiogenesis (11) and tumor-protective mutations (9,
13), the focus of the present study was to investigate the role of
low levels of endogenously produced NO in signaling pathways
affected by treatment with cisplatin. Our findings confirm earlier
reports of constitutive synthesis of NO by human metastatic
melanoma cells and extend them by showing that NO production
results in muted responses to cisplatin in vitro, with S-nitrosation
representing a potentially important biochemical way through
which it exerts these effects.
The role of NO in tumorigenesis and tumor progression has
been an object of intense investigation, stimulated by the expression of NOS in many types of cancer cells, its correlation
with reduced patient survival, and its potential as a therapeutic
target (11, 13, 19). NOS expression by tumor cells has been
DNCB
DNCB
DNCB
DNCB++ Cisplatin
Cisplatin
0.6
0.6
0.0
0
0.5
0.5
1.0
1.0
1.5
1.5
DNCB (μM)
DNCB (μM)
B
1.2
Relative Survival
1
**
0.8
*
0.6
0.4
0.2
0
0.625 mM DTT 1.25 mM DTT Cisplatin -
C
+
+
-
+
-
+
+
+
+
A
A375
_
_
_
NMA
Cisplatin
+
_
_
_
+
+
+
SK Mel 28
_
+
_
+
+
+
Phospho NFκ
κB p65
***
1. 0
NFκB p65
Cisplatin
Cisplatin
*
0.8
*
0.6
Phospho ERK1/2
DTT+Cisplatin
DTT + Cisplatin
ERK1/2
Phospho Akt
0.4
Akt
0.2
p53
0.0
MDM2
10
10 0
Cisplatin (μM)
Fig. 2. S-nitrosation as a possible player in the response to cisplatin. (A)
S-nitrosation favors resistance to cisplatin. A375 cells grown in microplates
were treated for 24 h with increasing concentrations of DNCB, which favors
stabilization of SNOs through inhibition of thioredoxin reductase (24), followed by the challenge with 12.5 μM cisplatin. Cell death was analyzed
24 h later (*P < 0.05, **P < 0.01). (B) Breakdown of SNOs increases cisplatin
toxicity. Cells grown as described earlier were transiently treated for 1 h with
the indicated concentrations of DTT to destabilize protein SNOs, followed
by addition of 12.5 μM cisplatin. Cell viability was quantified 24 h later (*P <
0.05, **P < 0.01). (C) Disruption of SNOs increases sensitivity to cisplatin.
A375 cells grown in microplates were treated as in B with 1 mM DTT and
subsequently challenged with increasing doses of cisplatin. Viability was estimated 24 h later (*P < 0.05, ***P < 0.001). In all three experiments, cell survival was determined with the WST-1 reagent. Values shown are means ± SD.
S-nitrosation of caspase-3 in response to cisplatin in A375 and
SK Mel28 cells (Fig. 5C). In addition, prolyl-hydroxylase 2 (PHD2),
the enzyme that targets the prosurvival transcription factor hypoxia-inducible factor-1α (HIF-1α) for degradation, displayed a
fivefold increase in S-nitrosation upon cisplatin challenge (Fig.
5D), reflected in a corresponding accumulation of HIF-1α (Fig.
5E). Therefore, increased S-nitrosation of enzymes involved in
Godoy et al.
β-actin
B
Bcl-2
1.2
Relative density
1
Bax
2
**
1. 5
6
1
4
0 .3
0 .5
2
0
0
0
0 .9
0 .6
NMA
Cisplatin
*
_
_
+
_
_
+
+ +
_
_
+
_
_
+
+ +
PUMA
8
**
_
_
+
_
_
+
+ +
Fig. 3. NO modulates cell signaling in response to cisplatin. (A) ERK1/2 is
regulated by endogenous NO. A375 and SK Mel 28 cells were grown in
presence of 5 mM NMA for 72 h followed by addition of cisplatin (12.5 μM
and 40 μM, respectively). Cell extracts were prepared and analyzed by immunoblot 24 h later. Total Akt, NF-κB, and ERK1/2 were detected on membranes stripped and reprobed after detection of the respective phosphorylated forms. (B) NO modulates apoptotic proteins. A375 cells grown as
described in A were submitted to immunoblot analysis of pro- and antiapoptotic proteins. Bar charts represent the densitometry analysis of protein
bands (averaged from three identical experiments). β-Actin is shown as a
loading control (*P < 0.05 and **P < 0.01 vs. cisplatin alone). Values shown
are means ± SD.
PNAS | December 11, 2012 | vol. 109 | no. 50 | 20375
APPLIED BIOLOGICAL
SCIENCES
Relative Survival
1. 2
Relative caspase activity
A
2.5
*
2 .20
1.5
1.10
0.5
0 .00
_
_
NMA
Cisplatin
_
+
_
+
Relative caspase activity
B
+
+
*
1. 2
1.2
*
1. 1
1.1
11
0.9
0.9
Cisplatin
Cisplatin
DTT + Cisplatin
0.8
0.8
0
2
4
C
Relative caspase activity
Hours with cisplatin
*
10
10
88
66
44
22
00
DNCB
Cisplatin
_
_
_
+
+
+
Fig. 4. NO-mediated inhibition of caspase-3 during cisplatin-induced apoptosis may be a result of S-nitrosation. (A) NO decrease enhances caspase-3
activity. A375 cells were grown in presence of 5 mM NMA for 72 h before
treatment with 12.5 μM cisplatin for 24 h and measurement of caspase-3
activity in cell lysates with the substrate DEVD-pNA (*P < 0.05). (B) Disruption of SNOs increases caspase-3 activity. Cells were transiently treated for
1 h with 1 mM DTT to decrease protein S-nitrosation, followed by addition of
cisplatin and caspase-3 activity measurements (*P < 0.05 vs. respective activity
with cisplatin alone). (C) Increased SNOs reduce activation of caspase-3. Cells
were treated for 2 h with 1 μM DNCB to promote increased protein S-nitrosation. Subsequently, 12.5 μM cisplatin was added and caspase-3 activity was
estimated 24 h later (*P < 0.01). Values shown are means ± SD.
described in breast cancer, colon cancer, lung cancer, and melanoma, among many other cancers (15, 18, 21, 34, 35). High
20376 | www.pnas.org/cgi/doi/10.1073/pnas.1218938109
levels of cytokine- or viral vector-driven expression and activity
of NOS have been shown to lead to tumor cell killing, usually
requiring the participation of proinflammatory factors, such as
TNF-α, γ-IFN, and IL-1 (7, 21). At high concentrations, NO
produced by stromal, immune, or cancer cells themselves causes
nitrosative stress, which leads to tumor death by DNA and protein damage (9). In contrast, noninduced, i.e., constitutive, expression of iNOS results in production of low, nontoxic levels of
NO (15), which favors growth and progression of tumor cells, as
our present results and other reports (13, 16, 23) demonstrate.
We could not detect NO production by melanoma cells using the
Saville–Griess assay, but were able to detect and quantify it by
using an NO analyzer and DAF-2DA, both of which are sensitive
to nanomolar concentrations of NO and its metabolites. Under
these circumstances, the signaling qualities of NO are of increased
importance, potentially modulating protein activity within signaling pathways by S-nitrosation, which may, for example, contribute to the relative resistance of melanoma cells to cisplatininduced apoptosis, as our data indicate.
Lack of information specifically addressing the molecular basis
for tumor-protective properties of NO prompted us to investigate the involvement of its signaling properties in the response of
melanoma cells to cisplatin. A key feature of chemoresistance
is the ability of cancer cells to resist drug-induced apoptosis (1, 4,
7, 18). Of note, S-nitrosation is known to regulate apoptosis in
cancer cells, whereas these effects of NO have not been observed
in normal melanocytes (12, 13, 16). This reinforces the proposition that tumor cells may exploit S-nitrosation–based mechanisms to resist cell killing (18). Our results show that suppression
of NO production during treatment with cisplatin led to increased activation of ERK1/2 and enhanced cell death compared
with cisplatin alone. Even though ERK1/2 activation has been
linked to survival signaling, a number of reports have shown that
this kinase complex is critical for induction of apoptosis by platinum agents (4, 27, 28), which induce anomalous, sustained
hyperactivation of ERK1/2 (28, 36). In fact, we observed that
conditions leading to augmented ERK1/2 activation and increased
apoptosis also decreased expression of the antiapoptotic protein
Bcl-2 and up-regulated expression of the proapoptotic factors
PUMA and Bax, which function in association in the initiation
of apoptosis (37). These data support the interpretation that cell
fate after chemotherapy depends on alteration of the balance
between anti- and prosurvival components (4, 12), and show that
NO plays a key role in the process. Collectively, our findings clearly
show an association between NO-induced S-nitrosation and altered cytotoxicity of cisplatin in melanoma cells. In themselves,
however, they do not provide definitive evidence that S-nitrosation
is responsible for effects on signaling pathways such as that shown
in Fig. 3; this will require further investigation.
In addition to the altered level of proteins involved in the
response to cisplatin, we found that, under the same condition,
caspase-3 and PHD2 displayed increased S-nitrosation, a protein
modification known to inhibit their functions (29, 30, 34). Caspases are the ultimate effectors of apoptosis, and their activity
is attenuated in cells resistant to cisplatin (4). NO interferes with
caspase activity by S-nitrosation of the active site (29), and also
by impairing the cleavage of proenzymes, which is required for
their activation (16, 24). On the contrary, HIF-1α, whose degradation is governed by PHD2, favors several aspects of tumorigenesis, including resistance to chemotherapy and radiation
therapy (38, 39). NO promotes stabilization of HIF-1α directly,
via S-nitrosation of its oxygen-dependent domain (33), or indirectly, by inhibiting PHD2 (30, 34). Our data show that cisplatin
induces accumulation of HIF-1α, which could be partially attributable to the observed increased S-nitrosation inhibition
of PHD2. Our findings of inducible increases in S-nitrosation of
caspase-3 and PHD2 are previously unrecognized features of
the complex mechanisms of drug resistance. Additional research
is needed to confirm the significance of S-nitrosation of these
proteins in the context of numerous other potential regulatory
mechanisms (1–5, 11, 40).
Godoy et al.
B
*
1000
control
LB
NMA
L-NIL
NO (nM)
nM
NO
NO signal (mV)
1200
800
600
400
200
0
_
Time
+
Cisplatin
Relative density
C
A375
SK Mel 28
22
22
11
11
00
00
Casp-3-SNO
_
Cisplatin
_
+
D
+
E
Relative density
12
9
HIF-1α
6
3
_
0
+
Cisplatin
Asc
_
+
Control
_
PHD2-SNO
+
Cisplatin
Fig. 5. Protein S-nitrosation modulation in response to cisplatin. (A and B)
NO content increases in response to cisplatin. A375 cells were treated with
12.5 μM cisplatin for 24 h or with the NOS inhibitor NMA (5 mM) or the
selective iNOS inhibitor L-NIL (1 mM) for 72 h. The NO content was measured
in a NO analyzer by using homogenates prepared from surviving cells. A
representative plot of the chemiluminescent signal proportional to the NO
content in the samples is depicted in A; intracellular NO concentration is
shown in B. LB, lysis buffer (*P < 0.05). (C and D) Increased S-nitrosation of
specific targets during response to cisplatin. Cells treated with cisplatin for
24 h were submitted to the biotin switch assay for labeling of SNOs with
biotin. The biotinylated (i.e., S-nitrosated) protein fraction was isolated with
avidin-conjugated agarose beads and submitted to immunoblot analysis for
identification of the S-nitrosated targets caspase-3 (C) and PHD-2 (D). For
caspase-3, the proenzyme band is shown. Bar charts represent the densitometry analysis of protein bands. Increased signal in presence of ascorbate,
as demonstrated in D, verifies the specific detection of protein S-nitrosation,
as ascorbate releases NO from SNOs, allowing for biotin to bind the nascent
thiol groups (24). (D) Cisplatin-induced accumulation of HIF-1α. A375 cells
were treated or not with cisplatin for 24 h and submitted to immunoblot
analysis for HIF-1α. Values shown are means ± SD.
We showed that, in response to cisplatin, melanoma cells increased NO levels and up-regulated S-nitrosation of specific
proteins in a fashion that promoted survival signals (HIF-1α
accumulation via S-nitrosation inhibition of PHD2) and inhibited apoptosis (through S-nitrosation inhibition of caspase-3 activity). S-nitrosation and denitrosation of cellular proteins are
regulated through an enzymatic complex, in which thioredoxins,
thioredoxin reductase, thioredoxin-interacting protein, and Snitrosoglutathione reductase orchestrate the cellular pool of SNOs
and, therefore, protein function (24, 41). In addition, the fact
that S-nitrosation of a given protein depends on its subcellular
localization, rather than solely on the amount of available NO,
further emphasizes that S-nitrosation is a finely controlled mechGodoy et al.
anism (17, 29, 41), an imbalance of which is linked to genesis of
several pathologic states (13, 16–18, 41). We conjecture that,
during the response to cisplatin, a process is activated in which
NO concentration increases and SNO levels are modulated by
the aforementioned enzymes (together with other mechanisms
yet to be identified), enabling cancer cells to mitigate apoptosis.
To characterize the regulation of cisplatin-induced apoptosis
by S-nitrosation, we used DTT and DNCB to promote disruption
and stabilization of intracellular SNOs, respectively. By using a
similar approach, other investigators employed DTT to show the
involvement of SNOs in the protective role of NO against cell
death in neurons (42). DTT has also been instrumental in the
demonstration of S-nitrosation of proteins such as caspases (43),
Bcl-2 (18), and PHD2 (30). DNCB, which irreversibly inhibits
thioredoxin reductase (25, 26), has been shown to increase the
amount of S-nitrosylated caspase-3 (24) and PHD2 (30). Of note,
because these compounds do not affect exclusively SNOs (i.e.,
DTT) or thioredoxin reductase (i.e., DNCB), other concurrent
effects of these chemicals cannot be ruled out. However, interpretation of our results as evidence of S-nitrosation–mediated
phenomena is supported by the responses to NOS inhibitors
(NMA, L-NIL, 1400W) and the NO scavenger PTIO as well.
Although it has been shown that PTIO may promote oxidation
of NO and even formation of the nitrosating agent N2O3 under
certain conditions (44), those used in our investigation do not
support such chemistry.
Our finding that decreased NO concentrations (and possibly
disruption of SNOs) substantially increase sensitivity of cells to
cisplatin is particularly noteworthy. Agents of the platinum family
are highly toxic and induce serious side effects in renal, neural,
myeloid, and auditory tissues (5), considerably limiting their usefulness and emphasizing the need for means to optimize their
efficacy. Several investigators have suggested therapeutic protocols combining NO-decreasing strategies with cisplatin or other
anticancer drugs (21, 23, 30, 31, 33, 34). Protocols already in
clinical use, which include N-acetylcysteine, rosiglitazone, iNOS
antisense oligomers, and physical activity, reportedly decreased
S-nitrosation in other pathologic conditions (17). Therefore, further exploration of combination of cisplatin with NO-decreasing
(denitrosating?) agents may be a promising strategy to potentiate
its desirable effects while diminishing side effects by enabling the
use of lower doses.
Materials and Methods
Chemicals and Antibodies. Cisplatin [cis-diamminedichloroplatinum(II)], DNCB,
and DTT were purchased from Sigma-Aldrich. NMA and PTIO were from
EMD-Calbiochem. Antibodies were as follows: iNOS, caspase-3, PUMA, and
Bcl-2 from Santa Cruz; p53 and β-actin from EMD-Calbiochem; HIF-1α from
BD Transduction Laboratories, MDM2 from Millipore, and PHD2 from AbCam.
All other antibodies were from Cell Signaling Technologies.
Cell Culture. Human melanoma cell lines A375, SK Mel 28, and SK Mel 100
(provided by Elizabeth A. Grimm, MD Anderson Cancer Center, Houston, TX)
were grown at 37 °C and 5% (vol/vol) CO2 in DMEM (A375) or RPMI (SK Mel
cells) supplemented with 10% (vol/vol) FBS (Atlanta Biologicals), 0.2 mM
L-glutamine, 10 U/mL penicillin, 10 μg/mL streptomycin, and 1 mM sodium
pyruvate (Lonza).
Cell Proliferation and Cytotoxicity. WST-1 reagent (Roche) was used to analyze
cell growth and viability. Ten microliters of reagent was added per 100 μL of
culture medium, and the absorbance was read at 450 nm after incubation
for 30 min at 37 °C.
Quantitative Analysis of NO. Cells were grown in medium containing 12.5 μM
cisplatin for 24 h. For measurement of NO content in homogenates, cells
were washed in PBS solution at 4 °C and lysed in PBS solution containing 1%
Nonidet P-40, 2 mM N-ethylmaleimide, 0.2 mM diethylenetriaminepentaacetic dianhydride, and protease inhibitors. After three instant freeze/thaw
cycles (−80/37 °C), lysates were passed through a 29-gauge needle to reduce
viscosity and spun at 2,000 × g for 10 min at 4 °C. Protein concentration was
measured in the soluble phase, and aliquots containing 2 mg of protein
were brought up to 100 μL with lysis buffer and kept on ice for analysis
PNAS | December 11, 2012 | vol. 109 | no. 50 | 20377
APPLIED BIOLOGICAL
SCIENCES
cisplatin
A
by using a Sievers NO analyzer (280i; GE Analytical Instruments). The liquidsample protocol used (45) measures nitrite and various nitroso species after
cleavage by a reaction mixture (45 mM potassium iodide and 10 mM iodine
in acetic glacial acid at 60 °C) in the purge vessel of the NO analyzer and
subsequent determination of the NO released into the gas phase by its
chemiluminescent reaction with ozone, which is quantified by a photomultiplier. For nitrite-only measurements, iodine was omitted from the
mixture and the procedure was carried out at room temperature. NO concentrations were calculated based on standard curves generated with sodium nitrite. The background signal from lysis buffer alone was subtracted
from the values obtained for cell homogenates.
S-Nitrosation Analysis. S-nitrosation was detected by the biotin switch technique as described elsewhere (43), with modifications. Unless otherwise
stated, all reagents were from Sigma. After treatments, cells were rinsed
with PBS solution containing 1 mM EDTA and 0.1 mM neocuproine, followed
by lysis in 25 mM Hepes, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 1% Nonidet
P-40, and protease inhibitors. From each sample, 0.5 mg of total protein was
used for the assay. The volume of samples was adjusted to 650 μL with HEN
buffer (100 mM Hepes, pH 8, 1mM EDTA, 0.1 mM neocuproine) and SDS
2.5% (wt/vol; final concentration). Free thiols were blocked with methyl
methanethiosulfonate (0.15% final concentration) and incubation for
30 min at 50 °C with frequent, vigorous vortexing. Excess methyl methanethiosulfonate was removed by acetone precipitation and gentle rinse of
protein pellets with chilled 70% (vol/vol) acetone, followed by resuspension
in HEN buffer containing 1% SDS. Biotinylation of SNOs was performed
with a final concentration of 50 μM [N-(6-(Biotinamido)hexyl)-3′-(2′-pyridyldithio)-propionamide] (EZ-Link Biotin-HPDP, in DMSO; Thermo Scientific)
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20378 | www.pnas.org/cgi/doi/10.1073/pnas.1218938109
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Statistical Analysis. All results are from experiments performed at least three
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treatments were analyzed by one-way ANOVA and complemented by the
Student–Newman–Keuls multiple comparisons test.
NO Detection and Analyses of Apoptosis. Details concerning NO detection
with DAF-2DA and analyses of apoptosis are provided in SI Materials
and Methods.
ACKNOWLEDGMENTS. This work was supported by National Cancer Institute Program Project Grant 5 P01 CA26731 and National Institute of Environmental Health Sciences Grant ES02109.
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