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Stress hormones promote growth of B16-F10 melanoma metastases: An interleukin 6- and glutathione-dependent mechanism

Authors:
  • School of Medicine, Univesity of Valencia

Abstract and Figures

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.
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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
Students 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].
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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-
turers 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-
ufacturers procedures.
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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 manufacturers 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 manufacturers 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 Students 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
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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)of78 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
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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.
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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 67 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 78 different animals. *P< 0.01 comparing
B16-F10-bearing mice versus non-tumor-bearing mice.
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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
910 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 56 different experiments. *P< 0.01
comparing B16-F10/IL-6-siRNA-bearing mice versus control
B16-F10-bearing mice.
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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.
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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.
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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.
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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.
Authorscontributions
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
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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.
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... Therefore, based on this background, it is plausible that glucocorticoids and catecholamines may influence melanoma growth and IL-6 production in its metastatic cells. Further work in the B16-F10 melanoma model showed that plasma levels of ACTH, corticosterone and noradrenaline increase in mice bearing B16-F10 lung or liver metastases, as compared to non-tumor-bearing controls [98]. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in the B16-F10 cells, which involves changes in the DNA binding activity of NF-κB, cAMP response elementbinding protein, AP-1, and nuclear factor for IL-6 [98]. ...
... Further work in the B16-F10 melanoma model showed that plasma levels of ACTH, corticosterone and noradrenaline increase in mice bearing B16-F10 lung or liver metastases, as compared to non-tumor-bearing controls [98]. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in the B16-F10 cells, which involves changes in the DNA binding activity of NF-κB, cAMP response elementbinding protein, AP-1, and nuclear factor for IL-6 [98]. Moreover, in vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA, treatment with the GR blocker RU-486 or with propranolol (a β-adrenoceptor blocker), increased hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth [98]. ...
... Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in the B16-F10 cells, which involves changes in the DNA binding activity of NF-κB, cAMP response elementbinding protein, AP-1, and nuclear factor for IL-6 [98]. Moreover, in vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA, treatment with the GR blocker RU-486 or with propranolol (a β-adrenoceptor blocker), increased hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth [98]. In addition, IL-6 may also promote mechanisms to avoid the stress-and/or cytotoxic drug-induced metastatic cell death (e.g., increased expression of several survival proteins, such as Bcl-2, Bcl-xL, Mcl-1, survivin, and XIAP) [98,99]. ...
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Circulating glucocorticoids increase during stress. Chronic stress, characterized by a sustained increase in serum levels of cortisol, has been associated in different cases with an increased risk of cancer and a worse prognosis. Glucocorticoids can promote gluconeogenesis, mobilization of amino acids, fat breakdown, and impair the body’s immune response. Therefore, conditions that may favor cancer growth and the acquisition of radio- and chemo-resistance. We found that glucocorticoid receptor knockdown diminishes the antioxidant protection of murine B16-F10 (highly metastatic) melanoma cells, thus leading to a drastic decrease in their survival during interaction with the vascular endothelium. The BRAFV600E mutation is the most commonly observed in melanoma patients. Recent studies revealed that VMF/PLX40-32 (vemurafenib, a selective inhibitor of mutant BRAFV600E) increases mitochondrial respiration and reactive oxygen species (ROS) production in BRAFV600E human melanoma cell lines. Early-stage cancer cells lacking Nrf2 generate high ROS levels and exhibit a senescence-like growth arrest. Thus, it is likely that a glucocorticoid receptor antagonist (RU486) could increase the efficacy of BRAF-related therapy in BRAFV600E-mutated melanoma. In fact, during early progression of skin melanoma metastases, RU486 and VMF induced metastases regression. However, treatment at an advanced stage of growth found resistance to RU486 and VMF. This resistance was mechanistically linked to overexpression of proteins of the Bcl-2 family (Bcl-xL and Mcl-1 in different human models). Moreover, melanoma resistance was decreased if AKT and NF-κB signaling pathways were blocked. These findings highlight mechanisms by which metastatic melanoma cells adapt to survive and could help in the development of most effective therapeutic strategies.
... Other research has also emphasized the connection between stress-induced neuroendocrine factors, proinflammatory cytokines, and the metastatic potential of melanoma. Using a B16F10 melanoma metastasis mouse model, it was shown that increased levels of stress-related hormones are associated with the promotion of lung and liver metastases [47]. In in vitro studies using the B16F10 melanoma cell line, high levels of corticosterone and NE induced an increased expression and secretion of IL-6. ...
... This cytokine has an important role in liver metastatic invasion. IL-6 can also generate an increased expression of proteins such as B-cell lymphoma (Bcl)-2, Bcl-xl, myeloid leukemia cell (Mcl)-1, survivin, and X-linked inhibitor of apoptosis protein (XIAP), providing cancerous cells with an important survival mechanism [47]. Corticosterone and NE may generate transcriptional upregulation of the IL-6 gene with changes in DNA binding activity of nuclear factor-kB (NF-κB), activator protein-1 (AP-1), and nuclear factor for IL-6 [47]. ...
... IL-6 can also generate an increased expression of proteins such as B-cell lymphoma (Bcl)-2, Bcl-xl, myeloid leukemia cell (Mcl)-1, survivin, and X-linked inhibitor of apoptosis protein (XIAP), providing cancerous cells with an important survival mechanism [47]. Corticosterone and NE may generate transcriptional upregulation of the IL-6 gene with changes in DNA binding activity of nuclear factor-kB (NF-κB), activator protein-1 (AP-1), and nuclear factor for IL-6 [47]. ...
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Melanoma is one of the most aggressive skin cancers with a sharp rise in incidence in the last decades, especially in young people. Recognized as a significant public health issue, melanoma is studied with increasing interest as new discoveries in molecular signaling and receptor modulation unlock innovative treatment options. Stress exposure is recognized as an important component in the immune-inflammatory interplay that can alter the progression of melanoma by regulating the release of neuroendocrine factors. Various neurotransmitters, such as catecholamines, glutamate, serotonin, or cannabinoids have also been assessed in experimental studies for their involvement in the biology of melanoma. Alpha-MSH and other neurohormones, as well as neuropeptides including substance P, CGRP, enkephalin, beta-endorphin, and even cellular and molecular agents (mast cells and nitric oxide, respectively), have all been implicated as potential factors in the development, growth, invasion, and dissemination of melanoma in a variety of in vitro and in vivo studies. In this review, we provide an overview of current evidence regarding the intricate effects of neuroendocrine factors in melanoma, including data reported in recent clinical trials, exploring the mechanisms involved, signaling pathways, and the recorded range of effects.
... It is also possible to use RNAi in therapy, involving B16-F10 cells, intradermally -after administration of preparations in this manner, the level of GAPDH (3-phosphotridal aldehyde dehydrogenase) has been reduced, the overexpression of which is often recorded in neoplastic cells and the c-myc gene has been silenced by inducing the apoptosis of B16-F10 melanoma cells [38]. Thanks to siRNA it has been also possible to inhibit the growth of melanoma in this cell line by silencing IL-6 -cytokines that are pro-inflammatory and proangiogenic, as well as coordinating cancer growth with stress hormones and glutathione [39]. In the present paper, significant inhibition of tumour growth was also observed, as well as a decrease in the dynamics of the growth of the mass and volume of tumours after subcutaneous administration of transfectants. ...
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Introduction: Gene therapy is an innovative form of treatment of genetic diseases, in which psiRNA molecules silencing specific genes are applied. Aim: The study evaluated the anti-tumour effect of psiRNA silencing preparations of the vascular endothelial growth factor (VEGF) and Sry-related HMG-Box gene 10 (SOX10) on melanoma (B16-F10) by inhibiting angiogenesis. Material and methods: The preparations based on plasmid vectors psiRNA silencing the gene SOX10 and VEGF that form complexes with cationic lipid (psiRNA/carrier) have been developed. psiRNA preparations were tested on the mouse melanoma cell line B16-F10, both in vitro and in vivo. The silencing activity of transfected melanoma cells with the obtained psiRNA preparations was examined using the qPCR and Western blot methods. The anti-tumour activity of psiRNA preparations on melanoma tumour cells was then evaluated in a mouse in vivo model. Results: In vitro studies have shown that the B16-F10 cells efficiently transfect non-viral preparations - psiRNA: Lyovec (74-89%). Worth mentioning is the fact that silencing SOX10 in B16-F10 melanoma cells increases the expression of the COL18A1 gene (compared to the preparation inhibiting only VEGF), which codes the endostatin to stop angiogenesis. In vivo results show that the level of haemoglobin in tumours of mice treated with psiRNA formulations was over 6 times lower than controls and tumour mass was 60-80% lower. Conclusions: The novel study proves that simultaneous inhibition of SOX10 and VEGF enhances the antiangiogenic action and thus contributes to a significant halt of disease development. In addition, these data expand knowledge about SOX10 regulation and functions.
... Stress and cancer both come as a guest simultaneously, so the role of stress modulating factors in cancer development has been well documented (Krizanova et al., 2016). An early study in different carcinoma proves that various tumours like melanoma (Valles et al., 2013), breast cancer (Vandewalle et al., 1990), pituitary cancer (Reisine et al., 1983), and pancreatic cancer (Reisine et al., 1983;Zhang et al., 2011) do express functional adrenergic receptors. The expression of receptors on these tumours helps them to survive and proliferate. ...
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... Previous studies found that catecholamines by binding to ARs facilitated tumor development [47]. Activation of either α-AR [48] or β-AR [49] triggers the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/c-Jun signaling pathway and mediates the behaviors of malignancies [50,51]. ...
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... Furthermore, several reports suggest that GCs stimulate expression of anti-apoptotic genes and hence antagonize the ability of cytotoxic drugs to successfully induce cell death [31]. Moreover, in vivo studies on stress-induced GCs suggest a positive relationship between GCs and melanoma progression [32,33]. ...
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Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy.
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Aims: Chronic exposure to environmental toxicants, such as paraquat, has been suggested as a risk factor for Parkinson's disease (PD). Although dopaminergic cell death in PD is associated with oxidative damage, the molecular mechanisms involved remain elusive. Glutaredoxins (GRXs) utilize the reducing power of glutathione to modulate redox-dependent signaling pathways by protein glutathionylation. We aimed to determine the role of GRX1 and protein glutathionylation in dopaminergic cell death. Results: In dopaminergic cells, toxicity induced by paraquat or 6-hydroxydopamine (6-OHDA) was inhibited by GRX1 overexpression, while its knock-down sensitized cells to paraquat-induced cell death. Dopaminergic cell death was paralleled by protein deglutathionylation, and this was reversed by GRX1. Mass spectrometry analysis of immunoprecipitated glutathionylated proteins identified the actin binding flightless-1 homolog protein (FLI-I) and the RalBP1-associated Eps domain-containing protein 2 (REPS2/POB1) as targets of glutathionylation in dopaminergic cells. Paraquat induced the degradation of FLI-I and REPS2 proteins, which corresponded with the activation of caspase 3 and cell death progression. GRX1 overexpression reduced both the degradation and deglutathionylation of FLI-I and REPS2, while stable overexpression of REPS2 reduced paraquat toxicity. A decrease in glutathionylated proteins and REPS2 levels was also observed in the substantia nigra of mice treated with paraquat. Innovation: We have identified novel protein targets of glutathionylation in dopaminergic cells and demonstrated the protective role of GRX1-mediated protein glutathionylation against paraquat-induced toxicity. Conclusions: These results demonstrate a protective role for GRX1 and increased protein glutathionylation in dopaminergic cell death induced by paraquat, and identify a novel protective role for REPS2.
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The intracellular redox status is a tightly regulated parameter which provides the cell with an optimal ability to counteract the highly oxidizing extracellular environment. Intracellular redox homeostasis is regulated by thiol-containing molecules, such as glutathione and thioredoxin. Essential cellular functions, such as gene expression, are influenced by the balance between pro- and antioxidant conditions. The mechanism by which the transcription of specific eukaryotic genes is redox regulated is complex, however, recent findings suggest that redox-sensitive transcription factors play an essential role in this process. This review is focused on the recent knowledge concerning some eukaryotic transcription factors, whose activation and DNA binding is controlled by the thiol redox status of the cell.
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Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.
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Glutathione (GSH) is the most abundant nonprotein thiol in cells and has multiple biological functions. Glutathione biosynthesis by way of the γ‐glutamyl cycle is important for maintaining GSH homeostasis and normal redox status. As the only enzyme of the cycle located on the outer surface of plasma membrane, γ‐glutamyl transpeptidase (GGT) plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate‐limiting substrate, for intracellular de novo synthesis of GSH. GGT also initiates the metabolism of glutathione S‐conjugates to mercapturic acids by transferring the γ‐glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine. GGT is expressed in a tissue‐, developmental phase‐, and cell‐specific manner that may be related to its complex gene structure. In rodents, there is a single GGT gene, and several promoters that generate different mRNA subtypes and regulate its expression. In contrast, several GGT genes have been found in humans. During oxidative stress, GGT gene expression is increased, and this is believed to constitute an adaptation to stress. Interestingly, only certain mRNA subtypes are increased, suggesting a specific mode of regulation of GGT gene expression by oxidants. Here, protocols to measure GGT activity, relative levels of total and specific GGT mRNA subtypes, and GSH concentration are described.
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Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux.