Content uploaded by Soraya L. Valles
Author content
All content in this area was uploaded by Soraya L. Valles on May 02, 2015
Content may be subject to copyright.
R E S E A R C H Open Access
Stress hormones promote growth of B16-F10
melanoma metastases: an interleukin 6- and
glutathione-dependent mechanism
Soraya L Valles
1
, María Benlloch
2
, María L Rodriguez
1
, Salvador Mena
1
, José A Pellicer
1
, Miguel Asensi
1
,
Elena Obrador
1
and José M Estrela
1*
Abstract
Background: Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its
interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is
regulated by cancer cell-independent mechanisms.
Methods: Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into
syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic foci using high-
performance cell sorting. Stress hormones and IL-6 levels were measured by ELISA, and CRH expression in the brain
by in situ hybridization. DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6 was measured using specific
transcription factor assay kits. IL-6 expression was measured by RT-PCR, and silencing was achieved by transfection
of anti-IL-6 small interfering RNA. GSH was determined by HPLC. Cell death analysis was distinguished using
fluorescence microscopy, TUNEL labeling, and flow cytometry techniques. Statistical analyses were performed using
Student’s t test.
Results: Plasma levels of stress-related hormones (adrenocorticotropin hormone, corticosterone, and noradrenaline)
increased, following a circadian pattern and as compared to non-tumor controls, in mice bearing B16-F10 lung or
liver metastases. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion
of IL-6 in B16-F10 cells in vitro. Corticosterone- and noradrenaline-induced transcriptional up-regulation of IL-6 gene
involves changes in the DNA binding activity of nuclear factor-κB, cAMP response element-binding protein,
activator protein-1, and nuclear factor for IL-6. In vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA,
treatment with a glucocorticoid receptor blocker (RU-486) or with a β-adrenoceptor blocker (propranolol), increased
hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth. Corticosterone, but not NORA, also
induced apoptotic cell death in metastatic cells with low GSH content.
Conclusions: Our results describe an interorgan system where stress-related hormones, IL-6, and GSH coordinately
regulate metastases growth.
Keywords: Metastases, Glutathione, Interleukin 6, Stress hormones, Apoptosis
* Correspondence: jose.m.estrela@uv.es
1
Department of Physiology, University of Valencia, 15 Av. Blasco Ibañez,
46010, Valencia, Spain
Full list of author information is available at the end of the article
© 2013 Valles et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Valles et al. Journal of Translational Medicine 2013, 11:72
http://www.translational-medicine.com/content/11/1/72
Background
Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is in-
volved in cell protection against free radicals, and in
many cellular functions being particularly relevant
in cancer cells by regulating carcinogenic mechanisms;
sensitivity against xenobiotics, ionizing radiation and
some cytokines; DNA synthesis; cell proliferation; the
protection against tumor microenvironment-related ag-
gression, apoptosis evasion, colonizing ability, and
multidrug and radiation resistance [1-3].
Recently, using the highly metastatic B16 melanoma
F10 (B16-F10) cell line (a classical model with very high
metastatic potential), we reported that interleukin 6
(IL-6) (mainly of tumor origin) facilitates GSH release
from hepatocytes and its interorgan transport through
the blood circulation to metastatic growing foci [4].
γ-Glutamyl transpeptidase (GGT) cleaves extracellular
GSH, releasing γ-glutamyl amino acids and cysteinyl
glycine, which is further cleaved by membrane-bound
dipeptidases into cysteine and glycine [1,5]. Free γ-
glutamyl-amino acids, cysteine, and glycine entering the
cell serve as GSH precursors [6]. In agreement with these
facts, we found that tumor GGT activity and the
intertissue flow of GSH, where the liver plays a key role,
regulate GSH content of B16 melanoma cells and thereby
their metastatic growth [7,8]. Nearly half of the GSH re-
leased by rat hepatocytes is transported across the sinus-
oidal membrane into the blood plasma for delivery to
other tissues [9].
The role of IL-6 in cancer metastases is complex and
mayinvolvea)autocrineandparacrinemechanismsofIL-6
activity; b) direct growth stimulatory activity through
activation of several signaling mechanisms; c) attraction of
circulating immune and cancer cells to specific organs (e.g.
lungs, brain, or liver) where IL-6 can be overexpressed;
d) stimulation of neoangiogenesis and vascular remodeling;
and e) promotion of inflammatory reactions and immune
scape, thus contributing to an immune microenvironment
that is favorable to tumor progression (e.g. [10-13]). In
addition, anticancer treatments, such as the chemothera-
peutic agents doxorubicin or paclitaxel and radiation ther-
apy, can also facilitate IL-6 release by tumor cells [14].
Therefore, taking into account the pro-cancer roles of IL-6,
it is not surprising that elevated serum levels of IL-6 and
sIL-6R have been associated to chemoresistance with poor
clinical outcome in different human cancers [15]. Many
cancer cells, including e.g. prostate, breast, and colon
cancer or melanoma, may produce large amounts of IL-6
and express the IL-6R/gp80 and gp130 receptor subunits,
which allow them to respond to IL-6 stimulation even in
an autocrine manner [13]. However, whether this capacity
to overproduce IL-6 is constitutive in metastatic cells and/
or regulated by cancer cell-independent mechanisms
is unknown.
The nervous, endocrine, and immune systems interact
trying to maintain physiological homeostasis under condi-
tions that induce systemic cytokine production [16]. Stress
has been suggested as a promoter of tumor growth
and angiogenesis in different in vivo models [17]. The
hypothalamus-pituitary-adrenal (HPA) axis, a main coord-
inator of the stress response, can be stimulated by cytokines
(e.g.IL-1,IL-6,orαTNF) during the course of different im-
mune, inflammatory, and neoplastic processes [18]. IL-6 is
an essential corticotropin-releasing hormone (CRH)-inde-
pendent stimulator of the pituitary-adrenal axis [19]. Acti-
vation of the HPA axis causes an increased secretion of
adrenocorticotropin hormone (ACTH), which stimulates
synthesis and release of glucocorticoids from the adrenal
glands [20]. Glucocorticoids have been used widely in con-
junction with other treatments for patients with cancer be-
cause(inadditiontootherpotential benefits) they have
proapoptotic properties in different cancer cell types; never-
theless glucocorticoids may also induce a resistant pheno-
type (still undefined) and, thereby, facilitate fast growth
and metastases of different solid tumors [21]. Stress-re-
lated pathophysiological concentrations of cortisol have
been shown to increase IL-6 production by human squa-
mous cell carcinoma cells [22]. Besides, cancer associated-
chronic stress can be associated in turn with a sympathetic
system-induced increase in catecholamines production
[23]. Moreover noradrenaline (NORA), at stress-related
concentrations, has been shown to up-regulate VEGF,
IL-, and IL-6 expression in different human melanoma cell
lines [24].
Therefore it is plausible that glucocorticoids and/or cat-
echolamines may influence IL-6 production by growing
metastatic cells. The main objective of the present contri-
bution was to explore the possibility that the IL-6/GSH
interorgan cycle [4], as a metastases growth promoting ac-
tivity, could be regulated by stress-related hormones under
in vivo conditions.
Materials and methods
Culture of B16-F10 melanoma cells
Murine B16-F10 melanoma cells (from the ATCC,
Rockville, MD) were cultured in serum-free Dulbecco's
modified Eagle's medium (DMEM; Gibco, Grand Island,
NY),pH7.4,supplementedwith10mMHEPES,40mM
NaHCO
3
, 100 U/ml penicillin and 100 μg/ml streptomycin
[8]. Cells were harvested by incubation for 5 min with
0.05% (w/v) trypsin (Sigma, St. Louis, MO) in PBS (10 mM
sodium phosphate, 4 mM KCl, 137 mM NaCl), pH 7.4,
containing 0.3 mM EDTA, followed by the addition of 10%
calf serum to inactivate the trypsin. Cell numbers were de-
termined using a Coulter Counter (Coulter Electronic Inc.,
Miami, FL). Cell integrity was assessed by trypan blue ex-
clusion and leakage of lactate dehydrogenase activity [8].
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 2 of 14
http://www.translational-medicine.com/content/11/1/72
Animals
Syngenic male C57BL/6J mice (12 weeks old) from
Charles River Laboratories (Barcelona, Spain) were fed ad
libitum on a standard diet (Letica, Barcelona, Spain). Mice
were kept on a 12-h light/12-h dark cycle with the room
temperature maintained at 22°C. Procedures involving ani-
mals were in compliance with international laws and pol-
icies (EEC Directive 86/609, OJ L 358. 1, December 12,
1987; and NIH Guide for the Care and Use of Laboratory
Animals, NIH Publ. No. 85-23, 1985). Experimental re-
search on mice was performed with the approval of the
ethics committee on animal research of the University of
Valencia (Spain).
Transfection of red fluorescent protein
The pDsRed-2 vector (Clontech Laboratories Inc., Palo
Alto, CA) was used to engineer B16-F10 melanoma clones
stably expressing red fluorescent protein (RFP). Cultured
B16-F10 cells were transfected as previously described [4].
High-Performance Cell Sorting (DAKO, Copenhagen,
Denmark) was used to select geneticin-resistant B16-F10
clones expressing the RFP (B16-F10-RFP) and showing
high fluorescence emission. These cells were seeded in 96
wells plates, and their growth was followed by immune-
fluorescence microscopy to select clones showing stable
fluorescence emission.
Experimental metastases
Hepatic or lung metastases were produced by i.v. injec-
tion (portal vein or tail vein, respectively) into anesthe-
tized mice (Nembutal, 50 mg/kg i.p.) of 10
5
viable
B16-F10-RFP suspended in 0.2 ml DMEM. Mice were
cervically dislocated 10 days after tumor cell inoculation.
Livers and lungs were fixed with 4% formaldehyde in
PBS (pH 7.4) for 24 hours at 4°C and then parafin-
embedded. Metastases volume (mean % of organ volume
occupied by metastases) was determined as earlier
described [25].
Isolation of B16-F10 melanoma cells from metastatic foci
Isolation of B16-F10 melanoma cells from metastatic foci
was performed as previously described [4]. Briefly, tissues
containing tumor cells were obtained by surgical means.
Cell dispersion was carried out in minced tissue by
trypsinization and collagenase digestion. Cells were washed
three times in PBS and resuspended in 1 ml of ice-cold
PBS, filtered through a 44-μm pore mesh and analyzed
using a MoFlo High-Performance Cell Sorter (DAKO).
Fluorescent B16-F10-RFP cells were separately gated for
cell sorting and collected into individual tissue culture
chambered slides (Nalge Nunc International Corp.,
Naperville, IL). Then the sorted tumor cells were harvested
andplatedin25-cm
2
polystyrene flasks (Falcon Labware).
Measurement of adrenocorticotropin hormone,
corticosterone, and norepinephrine levels
Plasma levels of ACTH (Calbiotech, Inc., Spring Valley,
CA), corticosterone (Kamiyama Biomedical Co., Seattle,
WA), and NORA (IBL, Hamburg, Germany) were quanti-
fied by ELISA according to the instructions of the
suppliers.
CRH expression in the brain (in situ hybridization)
Sections of 10 μm of the paraventricular nucleus (PVN)
were cut according to a mouse brain atlas (Allen Insttute
for brain science, http://www.brain-map.org) on a cryo-
stat, mounted on polysine microscope slides (Menzel-
Gläzer, Braunschweig, Germany), and stored at -80°C for
24 h. Then sections were fixed in 4% paraformalde-
hyde, further permeabilized by proteinase K treat-
ment, acetylated twice with 0.25% acetic anhydride in
0.1 M triethanolamine, and dehydrated in a graded
ethanol series.
Hybridization, carried out as described before [26], was
performed using specific 48-mer,
35
S-labeled oligonucleo-
tide probes for murine CRH mRNA (5
0
-GGC CCG CGG
CGC TCC AGA GAC GGA TCC CCT GCT CAG CAG
GGC CCT GCA-3
0
) [27]. Hybridized slices were exposed
to BioMax MR film (Kodak, Rochester, NY). The mRNA
expression of CRH in the PVN was quantified as gray
density minus background in digitized images using
the NIH ImageJ 1.6 program (http://rsb.info.nih.gov/ij).
Bilateral measures were taken from two to four PVN sec-
tions for each mouse, which were pooled to provide indi-
vidual means per mouse. For tissue background, the
optical density of a nonhybridized region outside the PVN
was measured.
Measurement of IL-6 levels
Blood samples were centrifuged at 14,000 rpm for 10
min at 4°C to separate the serum. Concentration of
IL-6 in the serum was determined using commercially
available mouse cytokine ELISA kits from Innovative
Research (Novi, MI).
DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6
Nuclear extracts were prepared with a nuclear extraction
kit (Millipore, Billerica, MA). The DNA binding activity
of nuclear factor-κB (NF-κB, p65/p50) and activator
protein-1 (AP-1, c-Jun/c-Fos) in nuclear extract was de-
termined by the NF-κB or AP-1 EZ-TFA transcription
factor assay kits (Millipore) according to the manufac-
turer’s protocols; whereas the DNA binding activity of
cAMP response element-binding protein (CREB) and
nuclear factor for IL-6 expression (NF-IL-6) were deter-
mined by specific ELISA-based TransAm
™
(Active Motif
North America, Carlsbad, CA) assay kits following man-
ufacturer’s procedures.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 3 of 14
http://www.translational-medicine.com/content/11/1/72
Transfection of anti-IL-6 small interfering RNA
IL-6 silencing was performed as previously described in
detail [4]. The PSilencer 3.1-H1 linear vector from
Ambion Inc. (Austin, TX) was used to obtain long term
gene silencing. The siRNA molecules targeting IL-6
mRNA were purchased from Ambion. The RNA duplex
against IL-6 had the sequence 5
0
-GGA CAU GAC AAC
UCA UCU CTT-3
0
(sense) and 5
0
-GAG AUG AGU
UGU CAU GUC CTG-3
0
(antisense). The negative con-
trol vector that expresses a hairpin siRNA with limited
homology to any known sequences in mice was provided
by the vector kit (Ambion). Stably transfected clones were
selected in medium containing 0.5 mg/ml Geneticin
(Invitrogen). Established clones were grown in medium
supplemented with 10% FCS and 0.5 mg/ml Geneticin. Si-
lencing was confirmed by immunoblotting.
GSH determination
GSH was determined, following procedures previously de-
scribed [28], by liquid chromatography-mass spectrometry
using a Quattro micro triple-quadrupole mass spectrometer
(Micromass, Manchester, UK) equipped with a Shimadzu
LC-10ADVP pump and SCL-10AVP controller system with
an SIL-10ADVP autoinjector (Shimadzu Corporation,
Kyoto, Japan). Cell processing was performed according to
published methodology, where rapid N-ethylmaleimide de-
rivatization was used to prevent GSH auto-oxidation [29].
Cell death and cell cycle analysis
Apoptotic and necrotic cell death were distinguished by
using fluorescence microscopy. For this purpose, isolated
cells were incubated with Hoescht 33342 (10 mM; which
stains all nuclei) and propidium iodide (10 mM; which
stains nuclei of cells with a disrupted plasma membrane),
for 3 min, and analyzed using a Diaphot 300 fluorescence
microscope (Nikon, Tokyo, Japan) with excitation at 360
nm. Nuclei of viable, necrotic, and apoptotic cells were ob-
served as blue round nuclei, pink round nuclei, and
fragmented blue or pink nuclei, respectively. About 1,000
cells were counted each time. DNA strand breaks in apop-
totic cells were assayed by using a direct TUNEL labeling
assay (Boehringer, Mannheim, Germany) and fluorescence
microscopy following manufacturer’s methodology. Quanti-
tative determination of mitochondrial membrane potential,
measurement of H
2
O
2
, flow cytometry determination of O
2
.-
generation, and measurements of cytochrome c release and
caspase 3 activity, were performed as previously described
[30]. Cell-cycle phase distribution was determined by ana-
lytical DNA flow cytometry as previously described [25].
Compartmentation of B16-F10 cells
Cultured cells were harvested (see above), washed twice in
DMEM, and resuspended in ice-cold Krebs-Henseleit bi-
carbonate medium (pH 7.4). Rapid separation of cytosolic
and mitochondrial compartments, and calculation of
mitochondrial volume, were performed as previously de-
scribed [31].
Cellular electroporation
Transient plasma membrane permeabilization was obtained
using an electroporation unit for eukaryotic cells (BioRad,
Hercules, CA). The field strength applied to each sample
was of 1.0 kV/cm with a time constant of 50 ms.
RT-PCR and detection of mRNA
Total RNA was isolated using the TRIzol kit from
Invitrogen and following the manufacturer’s instructions.
cDNA was obtained using a random hexamer primer and a
MultiScribe Reverse Transcriptase kit, as described by the
manufacturer (TaqMan RT Reagents, Applied Biosystems,
Foster City, CA). APCR master mix and AmpliTaq Gold
DNA polymerase (Applied Biosystems) were then added
containing the specific primers (Sigma-Genosys) previously
reported [4] for IL-6 and glyceraldehyde-3P-dehydrogenase
(GAPDH). Real-time quantitation of the mRNA relative to
GAPDH was performed with a SYBR Green I assay, and a
iCycler detection system (Biorad, Hercules, CA). Target
cDNA was amplified as follows: 10 min at 95°C, then 40 cy-
cles of amplification (denaturation at 95°C for 30 sec and
annealing and extension at 60°C for 1 min per cycle). The
increase in fluorescence was measured in real time during
the extension step. The threshold cycle (C
T
)wasdeter-
mined, and then the relative gene expression was expressed
as: fold change= 2
–Δ(ΔCT)
,whereΔC
T
=C
T
target –C
T
GAPDH, and Δ(ΔC
T
)=ΔC
T
treated - ΔC
T
control.
Expression of results and statistical analyses
Data are presented as the means + S.D. for the indicated
number of different experiments. Statistical analyses were
performed using Student’s t test, and P values < 0.05 were
considered significant.
Results
Stress hormones in metastatic tumor-bearing mice
Stress-relative responses in rodents under stressful condi-
tions can be evaluated by measuring plasma levels of cor-
ticosterone and NORA (main circulating glucocorticoid
and catecholamine, respectively) [32,33]. As shown in
Figure 1. A corticosterone levels in plasma peak at 12 h,
right before the begin of the dark active phase in mice.
However corticosterone levels were significantly higher in
B16-F10 (lung metastases)-bearing mice than in control
non-tumor-bearing mice (Figure 1A). In agreement with
this finding plasmatic ACTH levels were also higher in
metastases-bearing mice than in controls, and also
followed a circadian pattern (ACTH was higher before
corticosterone levels peaked, and lower during the dark
active phase) (Figure 1B). Besides NORA levels in plasma
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 4 of 14
http://www.translational-medicine.com/content/11/1/72
were also higher in metastases-bearing mice than in con-
trols (Figure 1C). Similar results were found in mice bear-
ing B16-F10 metastases growing in the liver (not shown),
thus suggesting a general mechanism not dependent on
the site of metastases growth.
Corticosterone and noradrenaline stimulate IL-6
expression and secretion in metastatic cells
Our next step was to investigate if corticosterone and/or
NORA, at pathophysiologically relevant concentrations,
could influence IL-6 expression and/or secretion in meta-
static cells. For this purpose B16-F10 cells were cultured in
thepresenceofcorticosteroneand/orNORA,whichwere
incubated at mean peak plasmatic values (approx. 350 ng
corticosterone/ml and/or 5.5 ng NORA/ml in mice bearing
B16-F10 metastases versus 150 ng corticosterone/ml and/
or 3.0 ng NORA/ml in control non-tumor-bearing mice)
for a period of 6 h (see Figure 1). As a double control, B16-
F10-RFP cells isolated from lung metastases were also
assayed. A shown in Table 1, both corticosterone and
NORA significantly increase IL-6 expression and secretion
in B16-F10 cells. Although, when both hormones were
added together, IL-6 expression and secretion values were
not significantly different from those found using cortico-
sterone alone (Table 1). Moreover, B16-F10-RFP cells
(isolated from lung metastatic growing foci) showed higher
IL-6 expression and secretion as compared to control B16-
F10 cells, which is not surprising since these cells have been
exposed to higher corticosterone and NORA levels under
in vivo conditions. In agreement with this idea, in vitro ex-
posure to B16-F10-RFP cells to corticosterone and/or
NORA did not up-regulate IL-6expressionand/orsecre-
tion as compared to B16-F10-RFP controls (Table 1).
Human and murine melanoma cells express high-affinity
glucocorticoid receptors (GCRs) [34]; and the presence of
adrenoceptors (ARs) has been also detected in different
melanoma cells [24]. Moreover, the presence of GCRs [35]
and βARs [36] in B16 melanoma cells has been reported.
Thus, we investigated if corticosterone- and NORA- in-
duced up-regulation of metastatic cell IL-6 production is a
mechanism specifically bound to GCRs and/or ARs. For
thispurposeweusedmifepristone(RU-486)toblock
GCRs [37] and propranolol to block βARs[38].Addition
of RU-486 (50 μM) or propranolol (50 μM) to cultured
B16-F10 cells (2 h before hormones addition) completely
abolished the corticosterone- or NORA-induced increase
in IL-6 secretion displayed in Table 1 (data not shown).
The ARs-mediated effect was specific to βARs since the
α-adrenergic antagonist prazosin (5 μM) had no effect on
the ability of corticosterone and/or NORA to induce IL-6
expression (not shown). Therefore corticosterone and
NORA-induced up-regulation of IL-6 in metastatic cells
indeed appears to involve interaction of these hormones
with their specific receptors.
Transcriptional regulation of the IL-6 gene by
corticosterone and noradrenaline
Previous work by several groups showed that NF-κB, NF-
IL-6, AP-1, CREB, interferon-regulatory factor-1, and spe-
cificity protein 1 can interact with the IL-6 promoter to
Figure 1 Corticosterone, ACTH, and noradrenaline levels in
plasma of non-tumor- and B16-F10-bearing (lung metastases)
mice. Corticosterone, ACTH, and noradrenaline levels were measured
as indicated under Materials and methods. Blood was collected from
the tail vein during the 24-h period. Data are mean values + S.D.
(error bars)of7–8 different animals. *P< 0.05 comparing B16-F10
(7 days after tumor inoculation)-bearing mice versus non-tumor
-bearing mice; +P< 0.05 comparing 15 h versus 6 h.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 5 of 14
http://www.translational-medicine.com/content/11/1/72
initiate mRNA synthesis [13]. GCRs coordinate with NF-
κB to regulate expression of different pro-inflammatory
cytokines, including IL-6 [39]. Whereas β-adrenergic
stimulation, via the cAMP-protein kinase A signaling
pathway, leads to activation of AP-1 [40]. Therefore, we
investigated the effect of these hormones on activation of
NF-κB, CREB, AP-1, and NF-IL-6, which correspond to
the four major transcriptional regulatory sites present in
the IL-6 promoter region of the IL-6 gene [13]. As shown
in Figure 2, corticosterone increases DNA binding activity
of NF-κB (p65 and p50) in cultured B16-F10 cells
(Figure 2A), whereas NORA increases DNA binding activ-
ity of phosphorylated CREB (P-CREB, which in addition
can mediate β-adrenergic stimulation of c-Fos via protein
kinase A [41] (Figure 2C) and the AP-1 complex (c-Jun
and c-Fos) (Figure 2B). DNA binding activity of NF-IL-6
was significantly increased by corticosterone but not by
NORA (Figure 2D), which is interesting because single
binding sites for NF-IL6 and NF-kappa B are present in
the promoter of the IL 6 gene [42]. In fact NF-IL6 and
NF-kappa B synergistically activate transcription of IL-6
and other cytokines [42]. Therefore interaction of cortico-
sterone and NORA with their receptors is linked, via
intracellular signaling cascades, with the molecular mech-
anism promoting IL-6 expression.
Cell cycle distribution (approximate percentage of cells
in G0/G1, S, and G2/M phases, n = 5) was 48.0 ± 5.2,
29.4 ± 3.4, and 23.6 ± 2.4 in controls growing exponen-
tially (24 h after seeding, as the controls in Figure 2);
and 66.0± 4.2, 18.2 ± 1.8, and 15.8 ± 2.6 in cells close to
quiescence (66 h after seeding). However results
reported in Figure 2 (Effect of corticosterone and NORA
on activation of NF-κB, CREB, AP-1, and NF-IL-6) for
cells cultured × 24 were not significantly different from
those found in cells cultured × 66 h. Thus indicating
that, in the standard cultured conditions used in our ex-
periments, GCRs- and ARs-dependent signaling mecha-
nisms (and likely expression of these receptors) are
not affected by the cell cycle distribution along the
culture time.
Tumor-derived IL-6 facilitates activation of the pituitary-
adrenal axis
The major neuroendocrine response mediating stress adap-
tation is activation of the HPA axis, with stimulation of
CRH and vasopressin, leading to pituitary ACTH secretion
and increases in glucocorticoid secretion from the adrenal
cortex [43]. Tumor derived cytokines have been suggested
to activate the HPA axis [44,45]. Interestingly IL-6 in par-
ticular appears essential for activation of the HPA axis
during immunological challenge in the absence of hypo-
thalamic input from CRH [19]. Moreover it has been
reported that suppressor of cytokine signaling-3, stimulated
by IL-6 and cAMP, is involved in the negative regulation of
CRF gene expression [46]. Thus we investigated if, in mice
bearing B16-F10 melanoma metastases, increased circulat-
ing IL-6 did affect CRH production. As shown in Figure 3,
CRH expression in the hypothalamic PVN was significantly
lower in metastatic B16-F10-bearing mice than in non-
tumor-bearing controls, whereas serum IL-6 increased.
Moreover, CRH expression was similar in mice bearing
lung or liver metastases (Figure 3). Thus suggesting again a
systemic mechanism which does not depend on the site of
metastatic growth. To investigate if tumor-derived IL-6 is
directly linked to changes in CRH expression under in vivo
conditions, we inoculated intravenously control B16-F10
cells and B16-F10 cells transfected with siRNA specific for
IL-6 (B16-F10/IL-6-siRNA). As shown in Table 2, down
regulation of tumor IL-6 expression in mice bearing B16-
F10/IL-6-siRNA metastases associated with a decrease in
circulating IL-6 levels. Besides CRH expression increased in
B16-F10/IL-6-siRNA-bearing metastases as compared to
B16-F10 controls, whereas ACTH levels were similar in
both cases (Table 2). Thus indicating, as previously sug-
gested [19], that IL-6 may act in metastatic tumor-bearing
hosts as a CRH-independent pituitary stimulator.
Table 1 Effect of corticosterone and NORA on IL-6 expression and secretion by B16-F10 and B16-F10-RFP cells in vitro
Tumor cells Additions Expression (-fold change) Secretion (pg/10
6
cells x 24 h)
B16-F10 None 1.0 ± 0.1 833 ± 170
Corticosterone 2.5 ± 0.4
*
1654 ± 266
*
NORA 1.7 ± 0.2
*
1257 ± 214
*
Corticosterone+NORA 2.4 ± 0.3
*
1712 ± 247
*
B16-F10-RFP None 3.0 ± 0.3
*+
1966 ± 331
*+
Corticosterone 3.3 ± 0.4
*+
2184 ± 287
*+
NORA 3.0 ± 0.3
*+
1763 ± 294
*+
Corticosterone+NORA 3.6 ± 0.5
*+
2477 ± 315
*+
IL-6 expression and release to the extracellular medium were measured (48 and 72 h after seeding, respectively) as indicated under Materials and methods.
Corticosterone and/or NORA were incubated (at the concentrations indicated in the text) 24 h after seeding and were present in the incubation medium for 6 h,
then the medium was renewed. Data are mean values + S.D. (n= 5-6) calculated for the indicated period. *P< 0.01 comparing both cell types and all conditions
versus control B16-F10 cells; +P< 0.01 comparing all additions under B16-F10-FRP cells versus their equivalents under B16-F10 cells.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 6 of 14
http://www.translational-medicine.com/content/11/1/72
It is also plausible that CRH expression could be
influenced by IL-6-dependent factors (missed in B16-
F10 with IL-6 expression silencing). Nevertheless, as
shown in Table 2, tumor IL-6 silencing increases (x 2-
fold) hypothalamic CRH expression in B16-F10 tumor-
bearing mice. Besides tumor IL-6 silencing associates
with a decrease in serum IL-6 (approx. 70% less in mice
bearing lung or liver metastases, Table 3). Thus, without
ruling out the possibility mentioned above, down-
regulation of tumor IL-6 appears a main factor influen-
cing hypothalamic CRH expression. A fact anticipated
by the work of Bethin et al. [19] and confirmed by
our studies.
Treatment with glucocorticoid receptor or β
adrenoceptor blockers inhibits metastatic growth
Whether a decrease in circulating IL-6 levels was linked to
metastatic activity, and whether this mechanism was regu-
lated by corticosterone and/or NORA, was our next step
forward. To address these key questions we compared
physiological saline-treated B16-F10 metastases (lung or
liver)-bearing controls with metastases-bearing mice
treated with the GCR blocker (RU-486) or the βAR
blocker (propranolol), and with mice inoculated with B16-
F10/IL-6-siRNA cells. As shown in Table 3, tumor IL-6
expression and circulating IL-6 levels decreased in all
groups as compared to controls, whereas hepatic GSH
levels increased. However, plasma levels of corticosterone
Figure 2 NF-κB, CREB, AP-1, and NF-IL-6 DNA binding activity
in nuclear extracts of B16-F10 melanoma cells treated with
corticosterone or NORA. Corticosterone (C) and NORA were
incubated (at the concentrations indicated in Table 1) 18 h after
seeding and were present in the incubation medium for 6 h.
Nuclear extracts were then obtained as described under Materials
and methods. Results are means + S.D. (error bars) of 4-5
independent experiments. The significance test refers to the
comparison between each experimental condition versus controls
(serum treated) (*P< 0.01).
Figure 3 CRH expression and serum IL-6 levels in non-tumor-
and B16-F10 (metastases)-bearing mice. Measurements were
performed in non-tumor-bearing and in tumor-bearing mice
(7 days after inoculation). mRNA expression of CRH in the
hypothalamic PVN was evaluated as described under Materials and
methods. CRH expression data (optical density arbitrary units, AU),
measured at 0 h (circadian time, see Figure 1), are expressed as
mean values + S.D. (error bars) of 6–7 different animals. For IL-6
levels determination blood was collected from the tail vein every 6
h during the 24-h period of the indicated day, and data are mean
values of the peak serum cytokine concentrations + S.D. (error bars)
(pg/ml) measured in 7–8 different animals. *P< 0.01 comparing
B16-F10-bearing mice versus non-tumor-bearing mice.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 7 of 14
http://www.translational-medicine.com/content/11/1/72
and NORA were significantly similar in all groups
(Table 3). The decrease in circulating IL-6 levels found in
RU-486- or propranolol-treated mice, or in mice inocu-
lated with B16-F10/IL-6-siRNA cells, associated with
lower metastases growth either in lung or liver (Table 3).
However, metastases volume in mice inoculated with B16-
F10/IL-6-siRNA cells was smaller (P< 0.05) than that
found in RU-486- or propranolol-treated mice (Table 3).
Thus suggesting that, perhaps, pathophysiological levels of
corticosterone and/or NORA have some anti-cancer ef-
fects which are absent if GCRs and/or βARs are blocked.
Glucocorticoids (such as dexamethasone) are widely
used in cancer therapy and may have cell type-specific
pro- or antiapoptotic effects, although when applied at
high therapeutic doses their anti-tumor effects prevail
[34]. Nevertheless there are very limited data regarding
possible direct effects of stress hormones, at in vivo patho-
physiological levels, on cancer cell proliferation [49]. Some
early findings in leukemia research suggested a possible
link between GSH and glucocorticoids effects in cancer
cells [50]. Maung et al. [51] found in their study of newly
diagnosed leukemia patients a positive correlation between
GSH levels and prednisolone resistance. Later Anderer
et al. [52] reported the implication of polymorphisms in
the GSH-S-transferase genes for glucocorticoid sensitivity
in childhood acute leukemia. Besides, it has been shown
that GSH levels in metastatic cells can regulate growth
and death mechanisms [2,3]. However, whether GSH
levels in metastatic cells influence stress hormones effects
is unknown. Thus, in the next step, we investigated if
corticosterone and/or NORA regulate growth and/or
death mechanisms in B16-F10 cells, and if these effects
are GSH dependent.
Corticosterone induces cell death in metastatic cells with
low GSH content
We evaluated the effect of corticosterone and NORA
(at pathophysiological concentrations) on cell growth and
viability using B16-F10 cell subsets with different GSH
content. B16-F10 cells cultured to low density (12 h after
seeding) show a high GSH content (40 + 5 nmol/10
6
cells,
n=7); whereas these cells, when incubated in the presence
of 1 mM BSO (L-buthionine (SR)-sulphoximine, the non-
toxic and selective GSH synthesis inhibitor [1]), show a
low GSH content (14 + 3 nmol/10
6
cells, n=7) under the
same culture conditions. As shown in Table 4, cortico-
sterone (but not NORA) decreased growth and viability of
B16-F10 cells with low GSH content but not of those with
high GSH content.
Corticosterone-induced cell death in B16-F10 cells with
low GSH content was further analyzed. As shown in
Figure 4, as a consequence of exposure to corticosterone,
most dying cells displayed apoptotic features and only a
small percentage was identified as necrotic. The percentage
of apoptotic cells obtained by using Hoescht 33342 and
propidium iodide or the TUNEL technique (see under
Materials and methods) was similar (not shown). Molecular
activation of apoptosis in B16-F10 cells with low GSH
content was confirmed as shown in Table 5, where
Table 3 In vivo effect of RU-486, propranolol, and siRNA-induced down regulation of tumor IL-6 expression, on
circulating levels of IL-6, corticosterone and NORA, hepatic GSH and metastases growth
Treatment
Physiological saline RU-486 Propranolol IL-6-siRNA
Metastases Lung Liver Lung Liver Lung Liver Lung Liver
Tumor IL-6 expression (-fold change) 1.0 ± 0.1 1.0 ± 0.2 0.4 ± 0.1
**
0.3 ± 0.15
**
0.6 ± 0.2
**
0.7 ± 0.15
*
0.15 ± 0.05
**
0.2 ± 0.1
**
Serum IL-6 (pg/ml) 355 ± 81 432 ± 69 123 ± 36
**
154 ± 49
**
206 ± 77
*
255 ± 60
**
107 ± 40
**
124 ± 33
**
Liver GSH (μmol/g of tissue) 4.5 ± 05 3.8 ± 0.4 6.8 ± 0.6
**
5.9 ± 0.5
**
6.2 ± 0.5
**
5.5 ± 0.5
**
7.3 ± 0.6
**
7.1 ± 0.5
**
Plasma NORA (ng/ml) 406 ± 65 447 ± 72 337 ± 59 412 ± 77 426 ± 68 394 ± 59 433 ± 67 360 ± 45
*
Plasma NORA (ng/ml) 7.9 ± 1.5 8.7 ± 1.4 7.3 ± 1.2 8.1 ± 1.6 7.5 ± 1.1 8.5 ± 1.7 8.2 ± 1.3 9.2 ± 1.6
Metastases volume (%) 6.4 ± 09 8.5 ± 1.2 4.0 ± 0.6
**
5.0 ± 0.7
**
4.9 ± 0.5
**
5.8 ± 0.9
**
3.0 ± 0.7
**
3.7 ± 0.8
**
RU-486 (20 mg/kg body wt.) [47] or propranolol (10 mg/kg body wt.) [48] were administered i.p. with daily frequency, starting 24 h after tumor cell inoculation. All
measurements (see under Material and methods) were performed 7 days after tumor cell inoculation. Plasma levels of corticosterone and NORA were measured at 12 h
(circadian time, see Figure 1). Metastases volume is indicated as the mean percentage of organ volume occupied by metastases. The data show mean values + S.D. for
9–10 different experiments. **P< 0.01, *P< 0.05 comparing each condition versus physiological saline-treated controls.
Table 2 Effect of siRNA-induced tumor IL-6 silencing on
hypothalamic CRH expression and circulating levels of
IL-6 and ACTH in B16-F10 tumor-bearing mice
Lung metastases
B16-F10 B16-F10/IL-6-siRNA
Tumor IL-6 expression (-fold change) 1.0 ± 0.2 0.15 ± 0.05
*
Serum IL-6 (pg/ml) 347 ± 75 132 ± 34
*
Hypothalamic CRH expression (AU) 40 ± 12 85 ± 16
*
Plasma ACTH (pg/ml) 240 ± 50 255 ± 63
Gene transfections were performed as explained under Materials and
methods. CRH expression data are expressed as optical density arbitrary units
(AU). All measurements were performed 7 days after tumor cell inoculation.
The data show mean values ± S.D. for 5–6 different experiments. *P< 0.01
comparing B16-F10/IL-6-siRNA-bearing mice versus control
B16-F10-bearing mice.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 8 of 14
http://www.translational-medicine.com/content/11/1/72
corticosterone-induced reactive oxygen species (ROS) gen-
eration (being mitochondria their principal source in cells)
associates with mitochondrial GSH (mtGSH) and ATP de-
pletion, a decrease in mitochondrial membrane potential,
and an increase in cytosolic cytochrome c level and caspase
3 activity.
Depletion of mtGSH (which cannot be synthesized by
mitochondria and must be transported from the cytosol)
may facilitate mitochondrial membrane permeabilization,
permeability transition pore opening, and the release of
apoptosis-inducing molecular signals [30]. Indeed when
GSH levels were increased, after loading the cells with
GSH ester (which readily enters the cell and delivers free
GSH, [30]), mtGSH increased and the mitochondria
dysfunction-dependent/corticosterone-induced molecular
activation of cell death was prevented (Table 4). To prove
that mtGSH is directly involved (and because there are
no specific inhibitor of the GSH transport into mitochon-
dria) we used cell electroporation in the presence of
10 mM L-glutamate (a competitive inhibitor of the
mtGSH transport). In the presence of BSO, GSH ester,
and L-glutamate, cytosolic GSH levels in B16-F10 cells in-
creased up to 42 + 6 nmol/10
6
cells (n=6) (see control
values in Table 4); whereas when corticosterone was
added mtGSH remained low (2.0 + 0.4 nmol/10
6
cells)
(n=6) (see control values in Table 5) and the molecular
activation of apoptosis (as in Table 5) was not prevented
(data not shown). Thus indicating that cellular GSH,
and mtGSH in particular, regulate the mechanism of
corticosterone-induced metastatic cell death.
Discussion
Neuroendocrine mediators, IL-6, and metastases growth
In the present work we have observed that stress-re-
lated hormones (corticosterone and NORA) promote
Table 4 Effect of corticosterone and NORA on growth and viability of B16-F10 cell subsets with different GSH contents
Culture time
12 h 48 h
B16-
F10
cells
Additions GSH
(nmol/
10
6
cells)
Cell number (10
6
cells) GSH
(nmol/
10
6
cells)
Cell number (10
6
cells)
Viable Dead Viable Dead
High GSH content
No addition 40 ± 5 2.01 ± 0.26 0.02 ± 0.01 46± 7 5.24 ± 0.36
*
0.15 ± 0.04
*
+ Corticosterone 5.06 ± 0.17 0.18 ± 0.03
+NORA 5.57 ± 0.43 0.14 ± 0.03
+Corticosterone+NORA 4.82 ± 0.30 0.20 ± 0.05
Low GSH content
No addition 14 ± 3
+
1.93 ± 0.15 0.05 ± 0.01
+
8 ± 2 2.24 ± 0.45
+
0.15 ± 0.03
*
+ Corticosterone 0.86 ± 0.15
+
0.76 ± 0.14
+
+NORA 2.07 ± 0.26
+
0.13 ± 0.05
+Corticosterone+NORA 0.75 ± 0.18
+
0.80 ± 0.17
+
Cells were cultured as described under Materials and methods. To allow maintenance of a high intracellular GSH content GSH ester (1 mM) was added 12 h after
seeding. To maintain a low intracellular GSH content BSO (200 μM) was added 2 h after seeding. Corticosterone and/or NORA were incubated (at the
concentrations indicated in Table 1) 18 h after seeding and were present in the incubation medium for 6 h, then the medium was renewed and GSH ester and
BSO were added again. Data are mean values + S.D. (n= 7) calculated for the indicated period. *P< 0.01 comparing 42h of culture time versus 12 h; +P< 0.01
comparing low versus high GSH content.
Figure 4 Type of death induced by corticosterone in B16-F10
melanoma cells with low GSH content. The experimental
procedure was that indicated in Table 4. Cell death was analyzed
using fluorescence microscopy as described under Materials and
methods. Cell death analysis was performed every 2 h during the
24 h period after corticosterone removal from the cultured medium.
Non-viable cells includes all cells marked as necrotic or apoptotic
along the experiment. Data are mean values + S.D. (error bars)
(n= 5-6) calculated for the indicated period. *P< 0.01 comparing
cells treated with corticosterone versus control B16-F10 cells.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 9 of 14
http://www.translational-medicine.com/content/11/1/72
overexpression of IL-6 in metastatic B16-F10 cells
(Table 1). Plasmatic levels of these hormones (including
pituitary ACTH) follow a physiological circadian pattern,
but were higher in metastases-bearing mice (Figure 1). To
date the majority of neuroendocrinological research deal-
ing with stress and accelerated tumor growth has focused
on suppressed immune response to malignant tissue [53].
However recent molecular and animal studies have begun
to identify specific signaling pathways suggesting an im-
pact of neuroendocrine mediators on tumor growth and
metastasis [49]. Nevertheless, as shown by different stud-
ies (mainly in vitro), the effects of stress-related hormones
on tumor cell proliferation can be either stimulatory or in-
hibitory [53]. This apparent paradox may depend, as it has
been suggested, on (yet undefined) differences between
cancer cells [53].
Corticosterone, GSH, and metastatic cell viability
Here we show that GSH levels in metastatic cells, which
are regulated by the IL-6/GSH interorgan cycling activity
(where the liver provides GSH to metastases) [4], directly
influence the effect of corticosterone on tumor cell viabil-
ity (Table 4). Pathophysiologically relevant levels of cor-
ticosterone or NORA increase IL-6 secretion by B16-F10
cells (Table 1), which subsequently will promote hepatic
GSH release [4]. Although these in vitro studies clearly
support corticosterone- and NORA-induced increased ex-
pression and secretion of IL-6 by B16-F10 cells, it is
unclear if the same mechanisms are operating in the com-
plexity of pathophysiological conditions in tumor-bearing
mice. Nevertheless preliminary results obtained in our lab
show that in B16 cells isolated from lung or liver meta-
static foci (where metastatic cell GCR has been knocked
down, previously to their inoculation, using specific
shRNA), IL-6 expression and secretion is decreased
(Estrela et al., unpublished results).
Additionally, potential cross-talk mechanisms, such as
up-regulation of GCRs by IL-6 [54] or synergistic activation
of IL-6 response element by IL-6 and glucocorticoids [55],
could further potentiate this signaling mechanism. How-
ever, only corticosterone decreased tumor cell viability
(Table 4) by activating mitochondria-dependent apoptosis
(Figure 4 and Table 5). GSH (one of the key endogenous ef-
fectors involved in regulating activation of cell death path-
ways [56]), if maintained at high levels, was capable of
preventing the corticosterone-induced apoptosis (Table 5).
In particular mtGSH oxidation facilitates opening of
the mitochondrial permeability transition pore complex
and, in consequence, can be a causal factor in the
mitochondrion-based mechanism that leads to cell death
[3]. Thus corticosterone-induced increase in ROS gener-
ation must contribute to mtGSH depletion (Table 5). In this
scenario several studies in thymocytes have implicated ROS
in glucocorticoid-induced apoptosis signaling, and more re-
cently it has been shown that hydrogen peroxide signaling
is required for glucocorticoid-induced apoptosis in lymph-
oma cells [57]. Nevertheless the critical molecular targets
or sensors of hydrogen peroxide during glucocorticoid-
induced apoptosis signaling remain to be elucidated.
It is noteworthy to mention that activation of neutral
sphingomyelinase, which is induced by hydrogen peroxide,
is required for glucocorticoid-induced apoptosis in thymo-
cytes [57].
IL-6, GSH, and the molecular mechanisms promoting
tumor growth
Malignant melanoma cells, as well as practically all cancer
cells, can release different growth factors and cytokines,
which (in addition of their autocrine and paracrine effects)
are potential systemic signals [58]. IL-6 serves as a major
regulatory cytokine in the human body [59]. Solid tumor
cells may secrete high levels of IL-6 (as shown here, a
process stimulated by stress hormones, Table 1), which in
turn promotes fundamental processes in cancer growth
and metastasis including angiogenesis, proliferation, at-
tachment, and invasion [13,49]. Previously we reported, in
Table 5 ROS generation and the molecular activation of apoptosis upon corticosterone administration to B16-F10 cells
with low GSH content
Parameters Controls + Corticosterone +Corticosterone
+GSH ester
H
2
O
2
(nmol/10
6
cells) 0.45 ± 0.09 1.74 ± 0.35* 0.73± 0.20*
+
O
2
.-
(ΔFL1) 2.10 ± 0.39 4.33 ± 0.41* 2.65 ± 0.27
+
mtGSH (nmol/10
6
cells) 3.2 ± 0.5 1.6 ± 0.4* 6.4 ± 1.0*
+
MMP (TPM accumulation ration ratio, %) 95 ± 4 52 ± 9 90 ± 7
+
mtATP (mM) 1.07 ± 0.24 0.66 ± 0.23 0.95 ± 0.18
Cytosolic cytochrome C (% of control) 100 ± 11 165 ± 27 112 ± 15
+
Caspase 3 (pmol/10
6
cells x min) 1.36 ± 0.21 3.15 ± 0.77 1.79 ± 0.26
+
Corticosterone was added as indicated in the caption to Table 1. GSH ester (1 mM) was added 6 h before corticosterone addition. BSO was added to all cells as
indicated in the caption to Table 4. Measurements were performed 12 h after corticosterone removal. Data are means + S.D. of 5 or 6 different experiments.
*P< 0.01 comparing all conditions versus untreated controls; +P< 0.01 comparing cells treated with corticosterone + GSH ester versus those treated with
corticosterone alone.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 10 of 14
http://www.translational-medicine.com/content/11/1/72
metastatic B16-F10 melanoma-bearing mice, that tumor
IL-6 silencing causes a significant decrease in circulating
IL-6 and in hepatic GSH efflux, and consequently an in-
crease in liver GSH content [4]. Thus suggesting that
tumor-derived IL-6 release is the main factor inducing
GSH release form the liver. Nevertheless, it is plausible
that in the liver, a major producer of IL-6, hepatic IL-6
may play a prevalent role particularly at early stages of
metastatic invasion. A mechanism that may be further po-
tentiated by tumor-derived factors [10,13]. Furthermore
IL-6 may also provide tumor cells with mechanisms to es-
cape cell death induced by stress and cytotoxic drugs, such
as increased expression of several survival proteins, i.e.
Bcl-2, Bcl-xL, Mcl-1, survivin, and XIAP [49]. The mech-
anism by which the transcription of specific eukaryotic
genes is redox regulated is complex, however, it has
been proposed that redox-sensitive transcription factors
containing reactive thiols in their DNA binding regions
(including e.g. NF-κB, AP-1, HIF-1, p53, or FoxO) play an
essential role in this process [60]. Redox-sensitive cysteine
residues sense and transduce changes in cellular redox sta-
tus caused by the generation of ROS, reactive electrophilic
species, reactive nitrogen species, and the presence of oxi-
dized thiols [61]. Oxidation of such cysteines is converted
into signals that control cell regulatory pathways and in-
duction of gene expression [61]. Transcription factors in-
cluding p53, NF-κB, and FoxO family can directly regulate
the expression of different Bcl-2 family members [62].
Therefore it would be possible that GSH levels, by directly
regulating the activity of redox-sensitive transcription fac-
tors and/or by decreasing ROS, may affect expression of
proteins involved in regulating apoptosis. Furthermore,
IL-6, as stressed in the introduction section, facilitates the
interorgan transport of GSH to metastatic growing cells,
thus favoring their growth and resistance [4]. In addition,
as shown in the present report, metastatic cells with high
GSH content are more resistant to corticosterone-induced
apoptosis (Table 4).
Physiological neuroendocrine systems and metastases
growth
Within the tumor microenvironment ARs and GCRs in
cancer, stromal cells, and tumor associated macrophages
are activated by agonists from circulating blood; but,
additionally, by catecholamines from sympathetic nerve
fibers [40]. Additionally, it is also plausible that specific
tissue/organ (such as liver or lung)-derived factors (still
undefined) may contribute to GCR and AR expression
by metastatic cells.
Moreover the brain can monitor immune status and
sense peripheral cancer-related inflammation through two
main pathways: neural and humoral. The neural mechan-
ism would rely upon direct activation of vagus nerve
Figure 5 The stress hormones/hypothalamic-pituitary-adrenal axis-dependent regulation of the IL-6/GSH interorgan cycling activity: a
systemic mechanism promoting metastases growth. IL-6 (mainly of tumor origin) potentiates the release of pituitary ACTH. Stress hormones
released by the suprarenal glands upregulate IL-6 expression and secretion by metastatic cells, which in turns increases GSH release from the
liver. Tumor GGT degrades plasma GSH, providing extra Cys for metastic cell GSH synthesis. Other (unknown) tumor-derived molecular signals,
acting as GSH release activators in other cancers, the role of tissue specific microenvironments, or the possible influence of tumor innervation in
metastatic cell behavior, are open questions.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 11 of 14
http://www.translational-medicine.com/content/11/1/72
afferent sensory fibers by cytokines, or indirectly through
chemoreceptive cells located in vagal paraganglia [40].
Moreover neurotransmitters are known to regulate the mi-
gratory activity of tumor cells, and secondly, nerve fibers
are used as routes for perineural invasion [63]. Therefore it
is plausible that metastatic cell populations, as suggested
by the present results, use physiological neuroendocrine
mechanisms to promote growth of highly aggressive (high
GSH content) cell subsets.
Clinical applications
α- and β-ARs protein expression is associated with poor
clinical outcome in breast cancer [64], thus suggesting a
possible role for targeted therapy using ARs antagonists.
In fact three recent population studies have translated
laboratory investigations into a clinical setting and con-
cur in presenting evidence that suggest a role for
β-blockers in reducing metastases, tumor recurrence
and specific mortality in breast cancer [65]. Besides
RU-486, a GR antagonist, is used for treatment of several
cancers, such as breast, ovarian, prostate, and glaucoma,
and has been shown to sensitize renal carcinoma cells to
TRAIL-induced apoptosis through up-regulation of DR5
and down-regulation of c-FLIP(L) and Bcl-2 [66]. More-
over, the first approved anti-IL-6 agent, tocilizumab, actu-
ally acts against the IL-6 receptor; whereas other anti-IL-6
compounds, including e.g. elsilimomab (a mouse mono-
clonal anti-IL-6 antibody) or CNTO 328 (an anti-IL-6
chimeric monoclonal antibody), are now following clinical
trials (see http://clinicaltrials.gov). In addition to this we
have reported a feasible strategy to deplete cytosolic and
mitochondrial GSH in metastatic cells, under in vivo con-
ditions, which includes a L-glutamine-enriched diet and
an anti-Bcl-2 antisense therapy [3].
An obvious question is whether these effects are just
present in the B16-F10 metastatic model. We have prelim-
inary data in other experimental models, including non-
metastatic human A375 melanoma and HT-29 colorectal
cancer (xenografted into nu/nu nude mice) (tumor vol-
umes >400 mm
3
) and metastatic Lewis lung carcinoma
(10 days after i.v. inoculation into C57BL/6 mice, tail vein),
where increased circulating levels of ACTH, cortico-
sterone and NORA, associate with a decrease in hepatic
GSH levels (in all cases as compared to non-tumor-bear-
ing controls) (not shown). Thus suggesting that findings
in the B16-F10 model are common to, at least, some other
metastatic and non-metastatic models.
Conclusions
The present results suggest the existence of an HPA-metas-
tases circuit linked to the previously reported metastases-
liver IL-6/GSH cycle. Figure 5 schematically summarizes
these interorgan relationships.
Abbreviations
IL: Interleukin; GSH: Glutathione; GGT: γ-glutamyl transpeptidase; B16-F10: B16
melanoma F10 subline; HPA: Hypothalamus-pituitary-adrenal axis;
CRH: Corticotropin-releasing hormone; ACTH: Adrenocorticotropin hormone;
NORA: Noradrenaline; RFP: Red fluorescence protein; PVN: Paraventricular
nucleus; NF-κB: Nuclear factor-κB; AP-1: Activator protein-1; CREB: CAMP
response element-binding protein; NF-IL-6: Nuclear factor for IL-6;
GAPDH: Glyceraldehyde-3P-dehydrogenase; GCRs: Glucocorticoid receptors;
ARs: Adrenoceptors; BSO: L-buthionine (SR)-sulphoximine; ROS: Reactive
oxygen species; mtGSH: Mitochondrial GSH.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
SLV, MLR, SM, JAP, MA, EO, and JME: Performed experiments. JME: Planned
the studies and wrote the manuscript. All authors have read and approved
the final manuscript.
Acknowledgements
This research was supported by grant (SAF2009-07729 and IPT-010000-2010-
21) from the Ministerio de Economía y Competitividad (http://www.idi.
mineco.gob.es), Spain.
Author details
1
Department of Physiology, University of Valencia, 15 Av. Blasco Ibañez,
46010, Valencia, Spain.
2
Faculty of Medicine, San Vicente Martir Catholic
University, 2 Calle Quevedo, 46001, Valencia, Spain.
Received: 22 November 2012 Accepted: 12 March 2013
Published: 22 March 2013
References
1. Meister A: Selective modification of glutathione metabolism. Science 1983,
220:472–477.
2. Estrela JM, Ortega A, Obrador E: Glutathione in cancer biology and
therapy. Crit Rev Clin Lab Sci 2006, 43:143–181.
3. Ortega A, Mena S, Estrela JM: Glutathione in cancer cell death. Cancers
2001, 3:1285–1310.
4. Obrador E, Benlloch M, Pellicer JA, Asensi M, Estrela JM: Intertissue flow of
glutathione (GSH) as a tumor growth-promoting mechanism: interleukin
6 induces GSH release from hepatocytes in metastatic B16 melanoma-
bearing mice. J Biol Chem 2011, 286:15716–15727.
5. Zhang H, Forman HJ, Choi J: Gamma-glutamyl transpeptidase in
glutathione biosynthesis. Methods Enzymol 2005, 401:468–483.
6. Meister A: Glutathione deficiency produced by inhibition of its synthesis,
and its reversal; applications in research and therapy. Pharmacol Ther
1991, 51:155–194.
7. Hanigan MH: Expression of gamma-glutamyl transpeptidase provides
tumor cells with a selective growth advantage at physiologic
concentrations of cyst(e)ine. Carcinogenesis 1995, 16:181–185.
8. Obrador E, Carretero J, Ortega A, Medina I, Rodilla V, Pellicer JA, Estrela JM:
gamma-Glutamyl transpeptidase overexpression increases metastatic
growth of B16 melanoma cells in the mouse liver. Hepatology 2002,
35:74–81.
9. Ballatori N, Rebbeor JF: Roles of MRP2 and oatp1 in hepatocellular export
of reduced glutathione. Semin Liver Dis 1998, 18:377–387.
10. Hodg DR, Hurt EM, Farrar WL: The role of IL-6 and STAT3 in inflammation
and cancer. Eur J Cancer 2005, 41:2502–2512.
11. Barton BE: Interleukin-6 and new strategies for the treatment of cancer,
hyperproliferative diseases and paraneoplastic syndromes. Expert Opin
Ther Targets 2005, 9:737–752.
12. Rose-John S, Waetzig GH, Scheller J, Grötzinger J, Seegert D: The IL-6/sIL-6R
complex as a novel target for therapeutic approaches. Expert Opin Ther
Targets 2007, 11:613–624.
13. Ara T, Declerck YA: Interleukin-6 in bone metastasis and cancer
progression. Eur J Cancer 2010, 46:1223–1231.
14. Emmenegger U, Kerbel RS: Cancer Chemotherapy counteracted.
Nature 2010, 468:637–638.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 12 of 14
http://www.translational-medicine.com/content/11/1/72
15. Wang Y, Niu XL, Qu Y, Wu J, Zhu YQ, Sun WJ, Li LZ: Autocrine production
of interleukin-6 confers cisplatin and paclitaxel resistance in ovarian
cancer cells. Cancer Lett 2010, 295:110–123.
16. Sternberg EM: Neural-immune interactions in health and disease. J Clin
Invest 1997, 100:2641–2647.
17. Reiche EM, Nunes SO, Morimoto HK: Stress, depression, the immune
system, and cancer. Lancet Oncol 2004, 5:617–625.
18. Besedovsky HO, Del Rey A, Klusman I, Furukawa H, Monge Arditi G,
Kabiersch A: Cytokines as modulators of the hypothalamus-pituitary
-adrenal axis. J Steroid Biochem Mol Biol 1991, 40:613–618.
19. Bethin KE, Vogt SK, Muglia LJ: Interleukin-6 is an essential, corticotropin-
releasing hormone-independent stimulator of the adrenal axis during
immune system activation. Proc Natl Acad Sci USA 2000, 97:9317–9322.
20. Fauci AS: Mechanisms of the immunosuppressive and anti-inflammatory
effects of glucocorticosteroids. J Immunopharmacol 1978, 1:1–25.
21. Herr I, Pfitzenmaier J: Glucocorticoid use in prostate cancer and other
solid tumours: implications for effectiveness of cytotoxic treatment and
metastases. Lancet Oncol 2006, 7:425–430.
22. Bernabé DG, Tamae AC, Biasoli ÉR, Oliveira SH: Stress hormones increase
cell proliferation and regulates interleukin-6 secretion in human oral
squamous cell carcinoma cells. Brain Behav Immun 2011, 25:574–583.
23. Antoni MH, Lutgendorf SK, Cole SW, Dhabhar FS, Sephton SE, McDonald PG,
Stefanek M, Sood AK: The influence of bio-behavioural factors on tumour
biology: pathways and mechanisms. Nat Rev Cancer 2006, 6:240–248.
24. Yang EV, Kim SJ, Donovan EL, Chen M, Gross AC, Webster Marketon JI,
Barsky SH, Glaser R: Norepinephrine upregulates VEGF, IL-8, and IL-6
expression in human melanoma tumor cell lines: implications for
stress-related enhancement of tumor progression. Brain Behav Immun
2009, 23:267–275.
25. Carretero J, Obrador E, Anasagasti MJ, Martin JJ, Vidal-Vanaclocha F, Estrela
JM: Growth-associated changes in glutathione content correlate with
liver metastatic activity of B16 melanoma cells. Clin Exp Metastasis 1999,
17:567–574.
26. Lachize S, Apostolakis EM, van der Laan S, Tijssen AM, Xu J, de Kloet ER,
Meijer OC: Steroid receptor coactivator-1 is necessary for regulation of
corticotropin-releasing hormone by chronic stress and glucocorticoids.
Proc Natl Acad Sci USA 2009, 106:8038–8042.
27. Veenema AH, Reber SO, Selch S, Obermeier F, Neumann ID: Early life stress
enhances the vulnerability to chronic psychosocial stress and
experimental colitis in adult mice. Endocrinology 2008, 149:2727–2736.
28. New LS, Chan EC: Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18)
column chemistries for the UPLC-MS-MS analysis of glutathione,
glutathione disulfide, and ophthalmic acid in mouse liver and human
plasma. Chromatogr Sci 2008, 46:209–214.
29. Asensi M, Sastre J, Pallardo FV, Garcia De La Asuncion J, Estrela JM, Vina JA:
High-performance liquid chromatography method for measurement of
oxidized glutathione in biological samples. Anal Biochem 1994,
217:323–328.
30. Benlloch M, Mena S, Ferrer P, Obrador E, Asensi M, Pellicer JA, Carretero J,
Ortega A, Estrela JM: Bcl-2 and Mn-SOD antisense oligodeoxynucleotides
and a glutamine-enriched diet facilitate elimination of highly resistant
B16 melanoma cells by tumor necrosis factor-alpha and chemotherapy.
J Biol Chem 2006, 281:69–79.
31. Ortega AL, Carretero J, Obrador E, Gambini J, Asensi M, Rodilla V, Estrela JM:
Tumor cytotoxicity by endothelial cells. Impairment of the mitochondrial
system for glutathione uptake in mouse B16 melanoma cells that
survive after in vitro interaction with the hepatic sinusoidal
endothelium. J Biol Chem 2003, 278:13888–13897.
32. Sakakibara H, Koyanagi A, Suzuki T, Suzuki A, Ling L, Shimoi K: Effects of
animal care procedures on plasma corticosterone levels in
group-housed mice during the nocturnal active phase. Exp Anim 2010,
59:637–642.
33. Lucot JB, Jackson N, Bernatova I, Morris M: Measurement of plasma
catecholamines in small samples from mice. J Pharmacol Toxicol Methods
2005, 52:274–277.
34. Dobos J, Kenessey I, Tímár J, Ladányi A: Glucocorticoid receptor expression
and antiproliferative effect of dexamethasone on human melanoma
cells. Pathol Oncol Res 2011, 17:729–734.
35. Ristic-Fira A, Vujcic M, Krstic-Demonacos M, Kanazir D: Identification and
characterization of glucocorticoid receptors in B16 mouse melanoma
cells. Endocr Regul 1999, 33:109–115.
36. Tsuji M, Kuno T, Tanaka C, Ichihashi M, Mishima Y: Beta-adrenergic
receptors of B16 melanoma cell. Arch Dermatol Res 1983, 275:415–416.
37. Im A, Appleman LJ: Mifepristone: pharmacology and clinical impact in
reproductive medicine, endocrinology and oncology. Expert Opin
Pharmacother 2010, 11:481–488.
38. Schuller HM: Beta-adrenergic signaling, a novel target for cancer
therapy? Oncotarget 2010, 1:466–469.
39. Smoak KA, Cidlowski JA: Mechanisms of glucocorticoid receptor signaling
during inflammation. Mech Aging Dev 2004, 125:697–706.
40. Cole SW, Sood AK: Molecular pathways: beta-adrenergic signaling in
cancer. Clin Cancer Res 2012, 18:1201–1206.
41. Boutillier AL, Barthel F, Roberts JL, Loeffler JP: Beta-adrenergic stimulation
of cFOS via protein kinase A is mediated by cAMP regulatory element
binding protein (CREB)-dependent and tissue-specific CREB-independent
mechanisms in corticotrope cells. J Biol Chem 1992, 267:23520–23526.
42. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T,
Akira S: Transcription factors NF-IL6 and NF-kappa B synergistically
activate transcription of the inflammatory cytokines, interleukin 6 and
interleukin 8. Proc Natl Acad Sci USA 1993, 90:10193–10197.
43. McEwen BS: Physiology and neurobiology of stress and adaptation:
central role of the brain. Physiol Rev 2007, 87:873–904.
44. Turnbull A, Prehar S, Kennedy A, Little R, Hopkins S: Interleukin-6 is an
afferent signal to the hypothalamo-pituitary-adrenal axis during local
inflammation in mice. Endocrinol 2003, 144:1894–1906.
45. Lee JH, Yoo SB, Kim NY, Cha MJ, Jahng JW: Interleukin-6 and the
hypothalamic-pituitary-adrenal activation in a tumor bearing mouse.
Int J Neurosci 2008, 118:355–364.
46. Kageyama K, Tamasawa N, Suda T: Signal transduction in the
hypothalamic corticotropin-releasing factor system and its clinical
implications. Stress 2011, 14:357–367.
47. Li YF, He RR, Tsoi B, Li XD, Li WX, Abe K, Kurihara H: Anti-stress effects of
carnosine on restraint-evoked immunocompromise in mice through
spleen lymphocyte number maintenance. PLoS One 2012, 7:e33190.
48. Sarabdjitsingh RA, Kofink D, Karst H, de Kloet ER, Joëls M: Stress-induced
enhancement of mouse amygdalar synaptic plasticity depends on
glucocorticoid and ß-adrenergic activity. PLoS One 2012, 7:e42143.
49. Moreno-Smith M, Lutgendorf SK, Sood AK: Impact of stress on cancer
metastasis. Future Oncol 2010, 6:1863–1881.
50. Tissing WJ, Meijerink JP, den Boer ML, Pieters R: Molecular determinants of
glucocorticoid sensitivity and resistance in acute lymphoblastic
leukemia. Leukemia 2003, 17:17–25.
51. Maung ZT, Hogarth L, Reid MM, Proctor SJ, Hamilton PJ, Hall AG: Raised
intracellular glutathione levels correlate with in vitro resistance to
cytotoxic drugs in leukaemic cells from patients with acute
lymphoblastic leukemia. Leukemia 1994, 8:1487–1491.
52. Anderer G, Schrappe M, Brechlin AM, Lauten M, Muti P, Welte K, Stanulla M:
Polymorphisms within glutathione S-transferase genes and initial
response to glucocorticoids in childhood acute lymphoblastic leukaemia.
Pharmacogenetics 2000, 10:715–726.
53. Thaker PH, Sood AK: Neuroendocrine influences on cancer biology.
Semin Cancer Biol 2008, 18:164–170.
54. Snyers L, De Wit L, Content J: Glucocorticoid up-regulation of high-affinity
interleukin 6 receptors on human epithelial cells. Proc Natl Acad Sci USA
1990, 7:2838–2842.
55. Takeda T, Kurachi H, Yamamoto T, Nishio Y, Nakatsuji Y, Morishige K-I,
Miyake A, Murata Y: Crosstalk between the interleukin-6 (IL-6)-JAK-STAT
and the glucocorticoid-nuclear receptor pathway: synergistic activation
of IL-6 response element by IL-6 and glucocorticoid. J Endocrinol 1998,
159:323–330.
56. Franco R, Cidlowski JA: Glutathione efflux and cell death. Antioxid Redox
Signal 2012, 17:1676–1693.
57. Tome ME, Jaramillo MC, Briehl MM: Hydrogen peroxide signaling is
required for glucocorticoid-induced apoptosis in lymphoma cells.
Free Radic Biol Med 2011, 51:2048–2059.
58. Lázár-Molnár E, Hegyesi H, Tóth S, Falus A: Autocrine and paracrine
regulation by cytokines and growth factors in melanoma. Cytokine 2000,
12:547–554.
59. Sansone P, Bromberg J: Targeting the interleukin-6/Jak/stat pathway in
human malignancies. J Clin Oncol 2012, 30:1005–1014.
60. Arrigo AP: Gene expression and the thiol redox state. Free Radic Biol Med
1999, 27:936–944.
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 13 of 14
http://www.translational-medicine.com/content/11/1/72
61. Antelmann H, Helmann JD: Thiol-based redox switches and gene
regulation. Antioxid Redox Signal 2011, 14:1049–1063.
62. Leibowitz B, Yu J: Mitochondrial signaling in cell death via the
Bcl-2 family. Cancer Biol Ther 2010, 9:417–422.
63. Mancino M, Ametller E, Gascón P, Almendro V: The neuronal influence on
tumor progression. Biochim Biophys Acta 1816, 2011:105–118.
64. Powe DG, Voss MJ, Habashy HO, Zänker KS, Green AR, Ellis IO, Entschladen
F: Alpha- and beta-adrenergic receptor (AR) protein expression is
associated with poor clinical outcome in breast cancer: an
immunohistochemical study. Breast Cancer Res Treat 2011, 130:457–463.
65. Powe DG, Entschladen F: Targeted therapies: Using β-blockers to inhibit
breast cancer progression. Nat Rev Clin Oncol 2011, 8:511–512.
66. Min KJ, Jang J, Lee JT, Choi KS, Kwon TK: Glucocorticoid receptor
antagonist sensitizes TRAIL-induced apoptosis in renal carcinoma cells
through up-regulation of DR5 and down-regulation of c-FLIP(L) and
Bcl-2. J Mol Med (Berl) 2012, 90:309–319.
doi:10.1186/1479-5876-11-72
Cite this article as: Valles et al.:Stress hormones promote growth of
B16-F10 melanoma metastases: an interleukin 6- and glutathione-
dependent mechanism. Journal of Translational Medicine 2013 11:72.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Valles et al. Journal of Translational Medicine 2013, 11:72 Page 14 of 14
http://www.translational-medicine.com/content/11/1/72
