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ORIGINAL PAPER
The nitric oxide donor sodium nitroprusside requires the 18 kDaTranslocator Protein to induce cell death
Luba Shargorodsky • Leo Veenman •
Beatriz Caballero • Yelena Pe’er • Svetlana Leschiner •
Julia Bode • Moshe Gavish
Published online: 28 April 2012
� Springer Science+Business Media, LLC 2012
Abstract Various studies have shown that several lethal
agents induce cell death via the mitochondrial 18 kDa
Translocator Protein (TSPO). In this study we tested the
possibility that nitric oxide (NO) is the signaling compo-
nent inducing the TSPO to initiate cell death process. Cell
viability assays included Trypan blue uptake, propidium
iodide uptake, lactate dehydrogenase release, and DNA
fragmentation. These assays showed that application of the
specific TSPO ligand PK 11195 reduced these parameters
for the lethal effects of the NO donor sodium nitroprusside
(SNP) by 41, 27, 40, and 42 %, respectively. TSPO
silencing by siRNA also reduced the measured lethal
effects of SNP by 50 % for all of these four assays. With
2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetra-
zolium-5-carboxyanilide (XTT) changes in metabolic
activity were detected. PK 11195 and TSPO knockdown
fully prevented the reductions in XTT signal otherwise
induced by SNP. Collapse of the mitochondrial membrane
potential was studied with the aid of JC-1 (5,50,6,60-tetra-
chloro-1,10,3,30-tetraethyl-benzimidazolylcarbocyanine
chloride). PK 11195 and TSPO knockdown reduced,
respectively by 36 and 100 %, the incidence of collapse of
the mitochondrial membrane potential otherwise induced
by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to
detect mitochondrial reactive oxygen species generation
due to SNP. PK 11195 and TSPO knockdown reduced this
effect of SNP by 65 and 100 %, respectively. SNP did not
affect TSPO protein expression and binding characteristics,
and also did not cause TSPO S-nitrosylation. However,
b-actin and various other proteins (not further defined)
were S-nitrosylated. In conclusion, TSPO is required for
the lethal and metabolic effects of the NO donor SNP, but
TSPO itself is not S-nitrosylated.
Keywords 18 kDa mitochondrial Translocator Protein
(TSPO) � Nitric oxide (NO) � Protein S-nitrosylation �Apoptosis � Reactive oxygen species (ROS)
Abbreviations
DWm Mitochondrial membrane potential
ANOVA Analysis of variance
ANT Adenine nucleotide transporter
Bmax Maximal binding capacity
BSA Bovine serum albumin
BST Biotin-switch technique
CAPON A cytosolic adaptor and scaffold protein
associated with nitric oxide synthase
CBR Central-type benzodiazepine receptor
CCCP Carbonyl cyanide
3-chlorophenylhydrazone
CysNO S-Nitrosocysteine
Dexras1 A member of the Ras family of small
G-proteins
DMEM Dulbecco’s Modification of Eagle’s
Medium
EDT Ethylenediaminetetraacetic acid
eNOS Endothelial NO synthase
Leo Veenman and Luba Shargorodsky contributed equally.
L. Shargorodsky � L. Veenman � B. Caballero � Y. Pe’er �S. Leschiner � M. Gavish (&)
Department of Molecular Pharmacology, Faculty of Medicine,
Rappaport Family Institute for Research in the Medical Sciences,
Technion-Israel Institute of Technology, Ephron Street,
Bat-Galim, P.O.B. 9649, 31096 Haifa, Israel
e-mail: [email protected]
J. Bode
Abteilung Padiatrie I, Zentrum Kinderheilkunde und
Jugendmedizin, Universitatsmedizin Gottingen, Gottingen,
Germany
123
Apoptosis (2012) 17:647–665
DOI 10.1007/s10495-012-0725-2
Page 2
ErPC3 Erucylphosphohomocholine
FACS Fluorescence assisted cell counting
FBS Fetal bovine serum
GAPDH Glyceraldehyde-3-phosphate
dehydrogenase
GSNO S-Nitroso-L-glutathione
HEN buffer 100 mM Hepes, 1 mM EDTA, 0.1 mM
neocuproine
HENS buffer HEN containing 1 % SDS
HEPES 4-(2-Hydroxyethyl)-1-
piperazineethanesulfonic acid
HRP Horse radish peroxidase
HS Horse serum
iNOS Inducible NO synthase
JC-1 5,50,6,60-Tetrachloro-1,10,3,30-tetraethyl-
benzimidazolylcarbocyanine chloride
Kd Equilibrium dissociation constant
Ki Binding affinity of the inhibitor
(in displacement assays)
LDH Lactate dehydrogenase
MEM-EAGLE Minimum Essential Medium Eagle
MMTS Methylmethane thiosulfonate
NAO 10-N-Nonyl-Acridine Orange
nNOS Neuronal NO synthase
NO Nitric oxide
NOS Nitric oxide synthase
PAP7 PBR associated protein 7
PBS Phosphate buffered saline
PC12 A cell line derived from a
pheochromocytoma of the rat adrenal
medulla
PI Propidium iodide
pk 10 A protein of 10 kDa MW
PK 11195 1-(2-Chlorophenyl-N-methyl-1-
methylpropyl)-3-isoquinoline-carbox-
amide
PRAX-1 PBR associated protein 1
Protein-SNO S-Nitrosylated proteins
RNS Reactive nitrogen species
Ro5 4864 7-Chloro-5-(4-chlorophenyl)-1,3-
dihydro-1-methyl-2H-1,4-benzodiazepin-
2-one
ROS Reactive oxygen species
RT-PCR Real time polymerase chain reaction
Scr Sham control scrambled siRNA cells
SD Standard deviation
SDS Sodium dodecyl sulfate
SDS-PAGE SDS-polyacrylamide gel
siRNA cells U118MG TSPO knockdown cells
SNO S-Nitrosothiol
SNO-RAC SNO precipitation using resin-assisted
capture
SNP Sodium nitroprusside
Tris Tris(hydroxymethyl)aminomethane
U118MG Human glioblastoma cell line
TSPO Mitochondrial 18 kDa Translocator
Protein
VDAC Voltage dependent anion channel
XTT 2,3-Bis[2-methoxy-4-nitro-5-
sulphophenyl]-2H-tetrazolium-5-
carboxyanilide
Introduction
The 18 kDa mitochondrial Translocator Protein (TSPO),
formerly known as peripheral-type benzodiazepine recep-
tor (PBR), can be found in peripheral tissues and in glial
cells in the brain [1–6]. The primary intracellular location
of TSPO is the outer mitochondrial membrane, while it can
also be associated with the cell nucleus and outer cell
membrane [7–11]. TSPO have been implicated in: apop-
tosis, steroidogenesis, oxidative stress, mitochondrial res-
piration, immune response, inflammation, glial activation,
ischemia, regulation of the mitochondrial membrane
potential (DWm), cell proliferation, cell differentiation,
tumorigenicity, and mental and neuropathological disor-
ders (for reviews, see [2–5, 11–15]). TSPO knockdown and
TSPO ligand studies have shown that the TSPO can induce
cell death via reactive oxygen species (ROS) generation,
cardiolipin oxidation, collapse of the DWm, and mito-
chondrial cytochrome c release [16–18]. Alternatively, it
has been suggested that TSPO may play a role in the
protection of cells from ROS damage [19, 20]. It was found
that various agents can induce cell death via activation of
the TSPO [16–18]. However, none of these agents in these
studies interacted directly with the TSPO [16–18]. Thus,
the question remained how these diverse agents can mod-
ulate TSPO function, i.e. which is the intermediate intra-
cellular factor that acts directly on the TSPO. One
candidate we considered was nitric oxide (NO), potentially
causing S-nitrosylation of the TSPO.
Like TSPO, NO is known to be involved in cell death
induction [21–26]. In healthy brain, NO is produced mainly
by neuronal NO synthase (nNOS) found in a small subset
of neurons, and endothelial NO synthase (eNOS) found in
endothelial cells [27]. In diseased brain, processes related
to damage such as hypoxia and pathogens activate induc-
ible NOS (iNOS) in microglia and astrocytes [28, 29]. It is
well known that NO can affect protein function via S-nit-
rosylation (by covalent attachment of NO to cysteine thi-
ols) [30, 31]. Aberrant S-nitrosylation is implicated in
tumorigenicity and neurodegeneration [32–34]. NO can
cause cell death by interference with various cellular
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mechanisms: inhibition of the mitochondrial respiratory
chain; collapse of the DWm; inhibition of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH); and generation of
ROS and reactive nitrogen species (RNS). Such processes
deplete the cell of ATP, leading to cell necrosis [35].
However, NO can also induce apoptosis, for example via:
(i) induction of p53; (ii) endoplasmic reticulum dysfunc-
tion; (iii) mitochondrial cytochrome c release; and (iv)
activation of MAP kinase pathways [35]. NO has also been
suggested to be involved in tumorigenicity and neurode-
generation [35–37]. NOS may be linked to the TSPO via
scaffolding proteins, including CAPON in association with
PBR associated protein 7 (PAP7, also known as acyl-CoA
binding domain containing 3, ACBD3) and Dexras1 [38,
39]. CAPON is a cytosolic adaptor and scaffold protein
associated with NOS. Dexras1 is a member of the Ras
family of small G-proteins. Thus, we assumed that NO may
act via the TSPO to induce cell death.
With the present study we sought to determine whether
the TSPO indeed is involved in the induction of cell death
due to application of NO donors, in particular, whether NO
donors may directly affect TSPO.
Methods
Materials
S-Nitroso-L-glutathione (GSNO) and S-Nitrosylated Pro-
tein Detection Assay Kits were from Cayman Chemical
Company (Ann Arbor, MI). Various cell culture materials,
Trypan blue, cell proliferation kits (XTT based), and EZ-
ECL Chemiluminescence Detection Kits were from Beit
Haemek—Biological Industries (Israel). Dulbecco’s phos-
phate buffered saline (PBS) with CaCl2 and MgCl2, bovine
serum albumin (BSA), sodium nitroprusside dehydrate
(SNP), clonazepam, 7-chloro-5-(4-chlorophenyl)-1,3-dihy-
dro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5 4864),
1-(2-chlorophenyl-N-methyl-1-methylpropyl)-3-isoquino-
line-carbox-amide (PK 11195), 10-N-Nonyl-Acridine
Orange (NAO), propidium iodide (PI), carbonyl cyanide
3-chlorophenylhydrazone (CCCP), anti-b-actin antibody
(mouse anti-human), anti-VDAC antibody (mouse anti-
human), sodium ascorbate, sodium nitrite, L-cysteine,
methylmethane thiosulfonate (MMTS) and Ponceau
staining solution were from Sigma-Aldrich (Rehovot,
Israel). Cytotoxicity Detection Kit (LDH kit), Cell Death
Detection ELISAPLUS Kit (Cell Death Kit), Protease
inhibitor (Complete) were from Roche (Mannheim, Ger-
many). Bradford solution and Coomassie blue, were from
Bio-Rad (Munich, Germany). [3H]PK 11195 was from
New England Nuclear (Boston, MA). CytoScint was
purchased from MP Biomedicals (Irvine, CA). Whatman
GF/C filters were obtained from Tamar (Mevaseret Zion,
Israel). Nitrocellulose membranes were from Hybond
ECL, Amersham Biosciences (Buckinghamshire, Eng-
land). Dried milk powder was from Carnation (Glendale,
CA). Anti-human TSPO anti-serum from rabbit was pre-
pared in our laboratory [17]. Anti-VDAC and anti-b-actin
antibodies were from Sigma, Rehovot, Israel. The sec-
ondary antibodies (anti-rabbit and anti-mouse—IgG
linked to horseradish peroxidase) were from Healthcare,
Amersham (Buckinghamshire, England). Western-
BrightTM-ECL HRP western blotting detection kit was
purchased from Advansta (Menlo Park, CA). Kodak
BioMax MR Film was obtained from Scientific Imaging
Film (Chalon-sur-Saone, France). The fluorescent dye
5,50,6,60-tetrachloro-1,10,3,30tetraethylbenzimidazolylcarb
ocyanine iodide (JC-1) was from Calbiochem (Merck,
Darmstadt, Germany). Thiopropyl Sepharose 6B was
purchased from GE Healthcare (Amersham, Bucking-
hamshire, England).
Cell cultures and experimental conditions
The human glioblastoma cell line, U118MG, and the rat
pheochromocytoma cell line, PC12, were cultured at 37 �C
in an atmosphere of 5 % CO2 and 90 % relative humidity.
These tumor cells (U118MG) and neuronal like cells (PC12)
were chosen, because cell death is an important mechanism
in tumorigenicity and neuronal death due to disease and
injury. PC12 cells were incubated in DMEM supplemented
with 8 % fetal bovine serum (FBS), 8 % heat-inactivated
horse serum (HS), 1 % glutamine, and 0.1 % penicillin
(100,000 units/ml) ? streptomycin (100 mg/ml) solution.
U118MG cells were cultured in MEM-EAGLE supple-
mented with 10 % FBS, 2 % glutamine and 0.05 % genta-
mycin (50 mg/ml). U118MG TSPO knockdown cells
(siRNA cells), and their sham control scrambled siRNA
cells (Scr) prepared in our laboratory, were cultured in
MEM-EAGLE supplemented with 10 % FCS, 2 % gluta-
mine and 0.05 % hygromycin (30 lg/ml) [18]. Western blot
and real time polymerase chain reaction (RT-PCR) were
applied routinely to determine the persistence of the TSPO
knockdown, see below.
For 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetra-
zolium-5-carboxyanilide (XTT inner salt) and lactate
dehydrogenase (LDH) assays, the cells were grown in 96
well-plates for 24 h (2 9 104 cells seeded per well). For
S-nitrosylation assays by resin-assisted capture (SNO-RAC)
cells were grown in 10 cm Petri dishes (seeded 1 9 106 cells
per dish) for 96 h reaching 70–80 % confluence. For all other
experiments 1.25 9 105 cells were seeded in 6 well plates
and grown for 72 h until reaching 60–70 % confluence. For
RT-PCR analysis as verification for TSPO knockdown,
1 9 104 cells were seeded in 75 cm2 cell culture flasks
Apoptosis (2012) 17:647–665 649
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(Sarstedt, Nuembrecht, Germany) in triplicates and cul-
tured for 2 days (until they reached 90 % confluence).
Then, floating and trypsinized adherent cells were col-
lected, as described further below. For each assay applying
NO donors, culture medium of the cells was replaced with
new medium with reduced serum levels (i.e. 0.5 % FBS for
U118MG; 0.5 % FBS and 0.5 % HS for PC12), and the
required cell culture conditions were applied, including
incubation with the appropriate compounds (Table 1). To
assay the effects of the NO donor SNP on cell viability, the
cells were incubated in the dark for 24 h in the appropriate
medium and then collected for the different analyses. In
addition, to determine S-nitrosylation of proteins by SNP
application, cells were incubated with NO donors for
5 min, 10 min, 15 min, 30 min, 60 min, and 24 h, as
required. The incubations included the NO donors SNP and
S-nitrosoglutathione (GSNO) at different concentrations
(500, 750, and 1000 lM), as described in previous studies
[40–43]. For a positive control of S-nitrosylation of TSPO,
also the NO donor S-Nitrosocysteine (CysNO) was applied
(1 mM for 10 min), as described previously [44]. The
vehicle for SNP, GSNO, and CysNO was PBS (Biological
Industries) (1 % of the total volume of medium). In addi-
tion, the cells received the TSPO specific ligands PK 11195
(25 lM), with and without NO donors. All the ligands were
applied 1 h before the NO donor and then also together
with the NO donor. For the negative control of the ligand
applications, we used the vehicle (medium ? 1 % ethanol,
i.e. vehicle control). Positive controls were applied as
required for the various assays. The concentration of PK
11195 applied to study TSPO related effects were as
described previously [17]. SNP, GSNO, and CysNO were
applied according to methods described previously [44–
47]. After NO donor and TSPO ligand application we
assayed cell death, including apoptosis, mitochondrial
activity, mitochondrial ROS generation, DWm, TSPO
expression and binding characteristics, and assays for
protein S-nitrosylation as described in detail below.
Collection of material
Material was collected for various complementary methods
used to assay cell death as a consequence of NO donor
application. For LDH assays, assaying cell death/viability
[48], 96 well plates were centrifuged (2509g, 10 min) and
cell free supernatant was collected for further analysis. For
XTT assays, assaying metabolic activity/proliferation/and/
or cell viability [49–51], cells were left in their original 96
well plates. For application of the S-Nitrosylated Protein
Detection Assay Kit to assay S-nitrosylation of proteins in
general, cells were collected with the wash buffer supplied
by the kit of Cayman Chemical Company. To determine
specific S-nitrosylation of TSPO, VDAC and b-actin, as
well as S-nitrosylation of proteins in general, cells were
rinsed with cold PBS, and then incubated for 30 min at
4 �C with shaking in lysis buffer (PBS, 0.1 % SDS, 1 %
triton X-100 plus protease inhibitors), and then the cells
were collected by scraping to optimize protein yield.
For all other assays, including RT-PCR, western blot,
binding analysis, and all of the flow cytometry assays [PI,
10-N-Nonyl-Acridine Orange (NAO) and 5,50,6,60-tetra-
chloro-1,10,3,30-tetraethyl-benzimidazolylcarbocyanine
chloride (JC-1)], culture medium was collected and the
cells were trypsinized (3 min, at 37 �C). After trypsiniza-
tion and collection, the cell suspensions were centrifuged
(2009g, 10 min at 4 �C), then the cell pellets were washed
in 1 ml of PBS, and centrifuged again. For RT-PCR cell
were pelleted by centrifugation 10009g, 7 min, 20 �C. The
final pellets were immediately processed for the assays in
question. For TSPO binding assays, the pellets were snap-
frozen in liquid nitrogen and stored at -70 �C until further
use. For western blot analysis of TSPO protein levels and
for assays of protein S-nitrosylation, cell pellets were lysed
and the lysate stored at -70 �C until further use.
General protein measurement
Protein levels, as required for binding assays and western
blot, were measured according to Bradford [52], using BSA
as a standard.
TSPO binding
To detect changes in TSPO binding characteristics after
application of the NO donor SNP, binding assays with
[3H]PK 11195 were performed. The cell pellets stored at
-70 �C were thawed and homogenized in 1 ml of PBS
using a Kinematika Polytron (setting 6) for 10 s. Binding
of [3H]PK 11195 to membranes of the various cell lines
was conducted as previously described [16–18]. The
Table 1 Scheme for NO donor and TSPO ligand application
Group Vehicle control NO donor TSPO ligand NO donor ? TSPO ligand
Treatment Medium ? 1 % Ethanol Medium ? 1 %
Ethanol ? SNP or GSNO
Medium ? 1 %
Ethanol ? PK 11195
Medium ? 1 % Ethanol ? SNP or
GSNO ? PK 11195
NO donors: SNP sodium nitroprusside, GSNO S-nitrosoglutathione
650 Apoptosis (2012) 17:647–665
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reaction mixture contained 400 ll of the homogenized
membranes in question (40 lg protein) and 25 ll of [3H]PK
11195 solution (0.18–6 nM final concentration) in the
absence (total binding) or presence (non-specific binding) of
75 ll unlabeled PK 11195 (10 lM final concentration). After
incubation for 90 min at 4 �C, the samples were vacuum
filtered through Whatman GF/C filters, washed three times
with 4 ml of 50 mM potassium phosphate buffer (pH 7.4).
Then the filters were placed in vials containing 4 ml of
CytoScint (MP Biomedicals). Radioactivity was counted
after 12 h with the 1600 CA Tri-Carb liquid scintillation
analyzer. The binding density (Bmax) and equilibrium dis-
sociation constant (Kd) were determined by Scatchard anal-
ysis of saturation curves of [3H]PK 11195 binding.
Displacement assays were applied to study whether
application of SNP could interfere directly with TSPO
binding to its ligand [3H]PK 11195. For these displacement
studies, rat kidney membranes homogenates were used as
previously described [53]. The reaction mixture contained
400 ll of homogenized kidney membranes (5 mg of
homogenate/ml) and 25 ll of [3H]PK 11195 (6 nM final
concentration) in the absence (total binding) or presence of
various concentrations (from 10-9 to 10-4 M) of SNP.
After incubation for 60 min at 4 �C, samples were filtered
under vacuum over Whatman GF/B filters and washed
three times with 5 ml of 50 mM Tris–HCl buffer, pH 7.4.
The filters were incubated in CytoScintTM and, after 12 h,
radioactivity was counted with the liquid scintillation
analyzer. Ki values were calculated by the equation
Ki = IC50/(1 ? C/Kd), where C = [3H] PK 11195 con-
centration, IC50 = concentration causing 50 % inhibition
of [3H]PK 11195 binding and Kd = 2 nM (from Scatchard
analysis of [3H]PK 11195 binding to kidney membranes).
Western blot
This analysis was done in order to assess relative protein
levels of TSPO after application of the NO donors, in
particular SNP, as well as to verify the stability of the
TSPO knockdown. Furthermore, western blot was applied
to determine the relative amount of S-nitrosylated proteins,
including S-nitrosylated TSPO after application of SNP
and CysNO. Collected samples with equal amount of
protein (10 lg protein/lane) were prepared in 29 sample
buffer (0.125 M 2-amino-2-(hydroxymethyl) propane-1,3-
diol (Tris)–HCl, pH 6.8, glycerol (20% v/v), SDS (sodium
dodecyl sulfate) (4% w/v), 0.14 M b-mercaptoethanol, and
bromophenol blue (0.005% w/v). The SDS–polyacryl-
amide gels were run and analyzed as described previously
[18, 54]. Rabbit anti-human TSPO antiserum 1:1,000 pre-
pared in our laboratory was used [17]. Mouse anti-human
VDAC antibody 1:5,000 was also used, and mouse anti-
human b-actin antibody 1:5,000 was used as a loading
reference. The primary antibodies were labeled with
IgG secondary antibody linked to horseradish peroxidase
(anti-rabbit and also anti-mouse IgG 1:5,000). Binding
of antibodies to their antigens was detected with the
WesternBrightTM-ECL western blotting detection kit (Advansta,
Menlo Park, CA). Labeling was captured on X-Omat blue XB-1
Kodak scientific Imaging Film. The results were analyzed using
densitometry.
RT-PCR
Obtained pellets were used for RNA extraction (RNeasy�
Mini Kit, Qiagen, Germany). The concentrations of RNAs
were determined by NanoDropTM measurement (Nano-
Drop Technologies, Wilmington, DE). Reverse transcrip-
tion of RNA was performed with iSript cDNA Synthesis
Kit (BioRad, Hercules, CA), according to the instructions
of the manufacturer. RT-PCR was performed using 10 ng
target cDNA and 3 pmol of each primer in SYBR-Green-
Mix (SYBR Green, Applied Biosystems, Foster City, CA).
The reaction was carried out at a final volume of 10 ll in
384 well optical plates. For each sample triplicates were
performed and water was taken for negative control. 18S
was chosen as internal standard as genes whose expression
should not be changed by treatment of the cells. The
reaction was carried out at the ABI PRISM 7900HT Fast
Real Time PCR System (Applied Biosystems, Life Tech-
nologies Corporation, Carlsbad, CA) with 40 cycles of 15 s
at 95 �C and one minute at 60 �C followed by the disso-
ciation measurement.
Housekeeper sequence for RT-PCR primer:
18S Forward: 50-AACTTTCGATGGTAGTCGCCG-30
18S Reverse 50-GGATGTGGTAGCCGTTTCTCAG-30
TSPO sequence for RT-PCR primer:
TSPO_Forward: 50-TTGGAGGAAGGCCTATTCACT-30
TSPO_Reverse: 50-GCCGCATTCACCTTGAAGA-30
Data analysis was performed using SDS2.4 software.
Viability assays
Viability assays were performed on normal U118MG cells
(wild type), PC12 cells (wild type), U118MG TSPO
knockdown cells (siRNA) and their background control,
i.e. U118MG Scr cells to determine the effects of the NO
donors and the TSPO ligand PK 11195. Trypan blue and PI
uptake defined dead cells were analyzed as described pre-
viously [18]. LDH release into the cell culture medium was
determined using a Cytotoxicity Detection Kit (Roche) as
described by the manufacturer. Evaluation of cytotoxicity
was calculated according to the formula suggested by the
Apoptosis (2012) 17:647–665 651
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manufacturer (Roche): % LDH released = [(A - B)/(C -
B)] 9 100; where A = LDH activity in the culture medium
of cells treated with NO donor; B = LDH activity of cul-
ture medium from untreated cells; and C = LDH activity
after total cell lysis with 1 % Triton.
Apoptosis
It is known that one of the TSPO functions is regulation of
apoptotic rates [12, 14, 16–18, 55–57]. Thus, we wanted to
determine whether our treatments with NO donors affect
this TSPO function. Cell morphology, including rounding
and blebbing analyzed microscopically was indicative of
the occurrence of apoptosis, as described previously [17,
18]. DNA fragmentation indicative of apoptotic cell death
was assayed with the Cell Death Detection ELISAPLUS
(Cell Death Kit) from Roche Applied Science (Mannheim,
Germany), as described previously [17, 18].
Mitochondrial and metabolic activity assays
Several parameters of metabolic activity and mitochondrial
activity/dysfunction following the various treatments were
assayed. The XTT assay is based on reduction of XTT
(normally yellow in PBS) by mitochondrial dehydrogen-
ases of viable cells, while extracellular reduction of XTT
may involve NADH coenzyme Q-dependent ferricyanide
reductases and a ubiquinone-dependent plasma membrane
electron transport chain [58]. We measured reduction of
XTT by means of the Cell proliferation-XTT based assay
kit (Biological Industries), as described by the manufac-
turer. To assay incidence of collapse of the DWm, JC-1 was
used as described previously [17, 18, 57].
Oxidative stress
Oxidative stress localized to mitochondria was assessed by
fluorescence measurement of NAO. NAO is able to bind
with high affinity to non-oxidized cardiolipin in a 2:1 ratio.
In the case of oxidized cardiolipin, NAO has been reported
to bind cardiolipin with a decreased affinity reflected by
lower levels of green fluorescence [13, 18, 59]. The NAO-
based assay gives clear results regarding the role of TSPO
in ROS generation [14]. Other assays attempting to relate
TSPO to oxidative stress at best only presented equivocal
results in our hands [14].
Protein S-nitrosylation
The ability of SNP in our paradigm to S-nitrosylate pro-
teins in general was investigated with the S-Nitrosylated
Protein Detection Assay Kit (Cayman) according to the
instructions of the manufacturer. Using this method, free
SH groups are first blocked with buffer A containing
blocking reagent, as supplied by the kit) and any S-NO
bonds left present in the sample are then cleaved with
Buffer B containing reducing and labeling reagents, sup-
plied by the kit. Biotinylation of the newly formed SH
groups provides the basis for visualization by streptavidin-
based colorimetric detection (applying S-nitrosylation
detection reagent I, including HRP, provided with the kit)
after running SDS-PAGE gels and transfer of the proteins
to membranes. For the detection of S-nitrosylated proteins,
the membranes were incubated overnight at 4 �C with
S-nitrosylation detection reagent I, 1:75. HRP signal
indicative of S-nitrosylation was detected with the EZ-
ECL-detection reagent while using the X-Omat blue XB-1
Kodak scientific Imaging Film.
S-nitrosylation of specific proteins was assayed by bio-
tin switch assay for protein S-nitrosothiols (SNOs), and
precipitation using resin-assisted capture (SNO-RAC) [44,
47]. Briefly, control wild type U118MG cells as well as
cells treated with 1 mM SNP (5, 10 and 15 min) were lysed
as described above. Then, 1 mg of lysed protein was
diluted in 1.25 ml final volume of HEN buffer (100 mM
Hepes, 1 mM EDTA, 0.1 mM neocuproine) containing
2.5 % SDS and 0.1 % MMTS. (Protein thiols are blocked
with MMTS). This ‘‘blocking’’ reaction was performed at
50 �C for 30 min with frequent agitation. After blocking,
proteins were precipitated with three volumes of cold
acetone and incubated at -20 �C for 20 min. The mixture
was then centrifuged at 20009g for 5 min, washed three
times with 70 % acetone and re-suspended in HENS buffer
(HEN containing 1 % SDS). This material was added to
50 ll of thiol-reactive resin slurry (Thiopropyl Sepharose
6B) in the presence of 20 mM sodium ascorbate (ascorbate
converts SNOs to free thiols). Following rotation in the
dark overnight, the resin was washed 4 9 with 1 ml HENS
buffer, and then 2 9 with 1 ml HENS/10 buffer (HENS
diluted 1:10). Nascent thiols are covalently trapped with
the resin-bound 2- or 4-pyridyl disulfides. Captured pro-
teins were eluted with 40 ll HENS/10 containing 100 mM
2-mercaptoethanol for 20 min at room temperature, and
30 ll each eluant mixed with 10 ll of 29 sample buffer to
be used for SDS-PAGE. Positive and negative controls
were also included in the SDS-PAGE. For positive con-
trols, cells were treated with 1 mM of the robust S-nitro-
sylation promoter CysNO (instead of SNP) for 10 min at
37 �C and 5 % CO2 as described previously [44, 47]. Non-
ascorbate samples were prepared in parallel with the SNO-
RAC assays to serve as negative controls for each sample
to avoid false positives. Specific anti-bodies were applied
to the proteins transferred to the membranes to detect
S-nitrosylated forms of TSPO, VDAC, and b-actin, as
described in the section western blot. In addition to the
immunoblots of the membranes, we also applied
652 Apoptosis (2012) 17:647–665
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Coomassie blue staining to the SDS-PAGE gels with sol-
ubilized proteins collected before pull down of S-nitrosy-
lated proteins (starting material), as well as after pull down
of S-nitrosylated proteins (Protein-SNO). Coomassie blue
staining of gels with S-nitrosylated proteins collected after
pull down was done to determine the range of molecular
weights of S-nitrosylated proteins by SNP treatment, in
comparison to the controls.
Statistical analysis
Sufficiently large experimental and control groups (n C 4)
were used for each assay. Results are expressed as
means ± SD. One-way analysis of variance (ANOVA)
was performed. Bartlett’s test for homogeneity of variance
was done. When appropriate, Kruskal–Wallis non-para-
metric ANOVA, was performed. For parametric post hoc
analysis Tukey–Kramer was applied. The non-parametric
Mann–Whitney or Dunn’s multiple analysis comparison
were used as post hoc tests, depending on the sample sizes
(for n \ 5 Mann–Whitney was used exclusively). When
comparing only between two groups, depending on the
significant difference between the SDs, Mann–Whitney or
Student’s t test were performed as appropriate. p \ 0.05
was considered statistically significant. The program used
for statistical analysis was Instat version-2 (GraphPad
Software, San Diego, CA).
Results
The two different NO donors, GSNO (500 lM) and SNP
(1,000 lM) strongly enhanced DNA fragmentation levels
measured with the Cell Death Kit indicative of apoptosis in
the two cell lines, U118MG (human) and PC12 (rat)
(Fig. 1a, b). In contrast to SNP, applying 1,000 lM of
GSNO to PC12 cells caused full cell detachment and
rounding of the cells, while apoptotic levels appeared lower
than observed with 500 lM (data not shown). Thus, as the
effects of SNP were well controllable, reliable, and well
distinguishable both from negative control as well as
positive control (Fig. 1c–i), SNP was the NO donor of
choice of the present study. Cell viability of U118MG and
PC12 was also assayed with the LDH kit after application
of SNP (500 lM and 1,000 lM) (Fig. 1c, d). In particular,
applying 1,000 lM SNP on PC12 and U118MG cells
resulted in a twofold and fivefold increase in cell death,
respectively, compared to vehicle control. Compared to the
positive control (1 % Triton treatment causing 100 % cell
death), applying 1,000 lM of SNP to PC12 and U118MG
cells achieved 49 and 53 % cell death, respectively
(Fig. 1c, d). 500 lM SNP was not toxic at all in PC12
cells, but still caused a fourfold increase in cell death in
U118MG cells, compared to vehicle control, i.e. attaining
about 42 % cell death compared to the positive control
(Fig. 1c, d). Assaying cell viability with Trypan blue dye
showed that SNP at 1,000 lM caused 26 and 46 % cell
death of the total cell population in PC12 and in U118MG
cells, respectively (Fig. 1e, f). The vehicle treated controls
only displayed 4 and 5 % cell death, respectively. Mea-
suring cell viability applying PI uptake with the aid of
FACS, also showed that U118MG cells are sensitive to
SNP. For this PI assay, U118MG cells were treated with
three different concentrations of SNP (500, 750, and
1000 lM) (Fig. 1g–i). Representative effects of SNP
(1,000 lM) treatment on cell death and its control detected
with PI in U118MG cells are presented in Fig. 1g, h. In
these examples, in the control only 6 % of the cells present
PI inclusion (Fig. 1g), while in the SNP treated cells 44 %
of the cells present PI inclusion (Fig. 1h). In Fig. 1i is
shown that average cell death levels were 8 % in vehicle
treated cell cultures as determined with the PI analysis.
SNP causes cell death in a dose-dependent manner
(Fig. 1i). As with the other assays, the PI inclusion showed
that 1,000 lM of SNP caused robust cell death of the total
cell population of U118MG (average of 46 %). Thus,
comparing these effects of SNP and GSNO makes clear
that U118MG cells exposed to 1,000 lM of SNP provide
the most clear cut results amenable to further assays. As
mentioned above, 1,000 lM of GSNO did not provide well
interpretable results. We also took into consideration that
the U118MG cell line was more sensitive to SNP than the
PC12 cell line (Fig. 1c, b). Therefore, we continued
working with the U118MG cells, applying the most
effective concentration of SNP (1,000 lM). We chose this
dose of SNP resulting in a level of cell death (46 %) that
would allow for up-regulation as well as down-regulation
of cell death.
Figure 2a shows that the application of 1,000 lM of the
NO donor SNP to U118MG cells causes a significant
decrease in signal of XTT reduction (by 71 %) indicative
of compromised metabolic activity (and/or a reduction in
cell number). This is in accord with the viability assays
showing reduced cell viability after SNP exposure (46 %
cell death, Fig. 1f). Furthermore, JC-1 assays with FACS
indicate significantly enhanced incidence of collapse of the
DWm by SNP, i.e. in U118MG cells exposed to 1,000 lM
of SNP maintenance of the DWm was only 30 % of that
observed in vehicle control (Fig. 2b).
As hypothesized in the ‘‘Introduction’’, we found that
the specific TSPO ligand PK 11195 attenuates cell death
induced by the NO donor SNP and this reduction in cell
death by PK 11195 is significant (n = 4, p \ 0.05), as
observed by Trypan blue uptake (Fig. 3a). In particular,
U118MG cells treated with 1,000 lM of SNP showed
46 % cell death, but when 25 lM of PK 11195 was added
Apoptosis (2012) 17:647–665 653
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to SNP treated cells, the cell death rate went down to 27 %.
In the vehicle control groups the cell death levels were 5 %
of the total population. Similar to the effects observed with
Trypan blue, PI uptake indicated that PK 11195 added to
the SNP-treated cells significantly reduced cell death rate
from 44 to 32 % (Fig. 3b). In the vehicle control cell death
levels were 6 % as determined with PI inclusion.
As expected, micrographs of the cells (Fig. 4) demon-
strated morphological signs of apoptosis induced by SNP,
including rounding and blebbing of cells (Fig. 4b), while in
vehicle treated cells the number of apoptotic cells was low
(Fig. 4a). With addition of PK 11195 to SNP exposed cells
there was a decrease in the number of the apoptotic cells in
comparison to those exposed to SNP without PK 11195
(Fig. 4c). In accord with the microscopic observations,
DNA fragmentation measured by the Cell Death Kit
demonstrated that addition of PK 11195 to SNP exposure,
in comparison to SNP alone, reduced DNA fragmentation
levels by 42 % (Fig. 5a). Also in this case, application of
PK 11195 significantly counteracted the decrease in
Fig. 1 Cell death induced by the NO donors GSNO and SNP in PC12
and U118MG cells. In (a) and (b) the y-axes present optical density
(OD) marking DNA fragmentation as an indicator for apoptosis levels
caused by GSNO (500 lM) and SNP (1,000 lM) applied for 24 h to
PC12 cells (a) and U118MG cells (b). In (c) and (d) is shown that
SNP (at 500 and 1,000 lM for 24 h) induces cell death in PC12 and
U118MG cell lines as measured by LDH release. The positive control
is cells treated with medium containing 1 % Triton which caused total
cell lysis. The y-axes in (c) and (d) mark the OD levels of the LDH
signal. In (e) and (f) Trypan blue uptake is used to indicate induction
of cell death by the NO donor SNP in PC12 cells (e) and in U118MG
cells (f). The y-axes in (e) and (f) mark the percentage of Trypan blue
positive cells from total population of cells. In (g), (h), and (i), PI
uptake indicates SNP induced cell death in U118MG cells. g,
h Representative FACS results of PI uptake: g vehicle treated cells;
h cells treated with SNP (1,000 lM for 24 h). M1 in (g) and
(h) represents the live cells and M2 represents the dead cells. i Cell
death induction of U118MG cells by different concentrations of the
NO donor SNP is shown, as determined by PI inclusion. The y-axis
marks the percentage of dead cells having taken up PI. The x-axis
marks SNP concentrations (0, 500, 750, and 1000 lM for 24 h).
*p \ 0.05, **p \ 0.01, and ***p \ 0.001 versus vehicle control.
(‘‘0’’ and ‘‘control’’ is vehicle control as indicated in the panels).##p \ 0.01 and ###p \ 0.001 versus SNP 500 lM (n = 6 for all
groups)
654 Apoptosis (2012) 17:647–665
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metabolic activity due to SNP in U118MG cells as mea-
sured with XTT, virtually restoring the normal level of
metabolic activity (Fig. 5b). SNP by itself caused a 73 %
reduction in metabolic activity. Assays of the collapse of
the DWm with the aid of JC-1 showed that the incidence of
DWm collapse with SNP alone was 72 %, while SNP in
combination with PK 11195 showed only 46 % incidence
of DWm collapse (Fig. 5c).
The role of TSPO in the lethal effects of SNP was fur-
ther checked by using TSPO knockdown. Viability assays
were performed on TSPO knockdown cells, i.e. TSPO
siRNA U118MG cells (siRNA) and on sham control cells,
i.e. scrambled control U118MG cells (Scr). siRNA cells
have only about 30 % of the TSPO protein levels observed
in Scr cells [18]. In accord with this previous report
applying immunoblot, the present study applying RT-PCR
assays showed that gene expression of the TSPO was
reduced to 20 % of the levels detected in Scr control cells
(n = 6, p \ 0.01, for each assay), as described also else-
where [60]. Our present results show that TSPO knock-
down counteracts cell lethality induced by 1,000 lM SNP
(Fig. 6). The Trypan blue assays showed that Scr cells
treated with 1,000 lM of SNP presented a more than
twofold increase in cell death compared to Scr vehicle
control, whereas cell death of siRNA cells was not sig-
nificantly increased (Fig. 6a). Similar results were found
with application of 750 lM of SNP [61]. Furthermore,
while the LDH assay showed a more than twofold increase
in cell death of SNP-treated Scr cells, compared to the Scr
control cells, only negligible effects of SNP were observed
in TSPO knockdown cells (Fig. 6b). The results of PI
uptake also showed that TSPO knockdown protects against
cell death induced by SNP. In particular, TSPO knockdown
caused a twofold reduction in cell death otherwise induced
by 1,000 lM of SNP, compared to sham control (Fig. 6c).
The results in Fig. 6d show that 1,000 lM of SNP caused a
twofold increase in DNA fragmentation levels in Scr cells,
compared to vehicle treated control, while in TSPO
knockdown cells (siRNA), no such effect could be dis-
cerned whatsoever (Fig. 6d).
Application of the NO donor SNP also had effects on
various aspects of mitochondrial function under TSPO
control assayed in this study (Fig. 7). Scr cells treated with
SNP show a significant reduction of 80 % in their meta-
bolic activity compared to their vehicle control, as detected
with the XTT assay (Fig. 7a). In contrast, TSPO knock-
down siRNA cells did not show any difference between
vehicle control and SNP treated cells in this respect
(Fig. 7a). Applying the JC-1 assay, Scr cells exposed to
SNP showed a significant reduction, of more than 50 %,
regarding the incidence of DWm collapse compared to their
vehicle control, while siRNA cells showed no significant
difference between SNP treatment and vehicle control,
Fig. 2 SNP affects mitochondrial function in U118MG cells. a SNP
(1,000 lM) causes a decrease in mitochondrial activity in U118MG
cells, as determined with XTT assays (n = 8). In (a) the y-axis
indicates OD levels as a measure for relative mitochondrial activity.
b SNP (1,000 lM) causes collapse of the DWm as measured by JC-1
fluorescence (n = 6). In (b) the y-axis indicates the drop in red/green
fluorescence ratio of SNP treated cells normalized to control (set at 1),
which is indicative for the incidence of collapse of the DWm.
***p \ 0.001 for SNP treatment versus vehicle control (control)
Fig. 3 PK 11195 reduces lethal effects of SNP. U118MG cells were
treated with 1,000 lM of SNP with or without 25 lM of PK 11195
(PK) for 24 h, or with vehicle (control). a Lethal effects of SNP and
protective effects of PK 11195 as measured by Trypan blue uptake.
The y-axis indicates % of cells that were Trypan blue positive per
sample (n = 4 for all groups in a). b The lethal effects of SNP and
protective effects of PK 11195 as measured by PI uptake. The y-axis
indicates the % of cells that were PI positive per sample (n = 6 for all
groups in b). *p \ 0.05 for SNP treatment versus vehicle control;#p \ 0.05 for SNP in combination with PK 11195 (SNP ? PK)
versus SNP alone (n = 6 for all groups)
Apoptosis (2012) 17:647–665 655
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regarding modulation of the DWm (Fig. 7b). SNP-treated
Scr cells showed a significant (70 %) increase in mito-
chondrial ROS generation (as assayed by NAO), as com-
pared to the Scr control cells, while TSPO knockdown
siRNA cells receiving the same treatment were not affected
(Fig. 7c). Representative examples of NAO assays are
shown in Fig. 7d. The data on metabolic activity, modu-
lation of the DWm, and mitochondrial ROS generation as a
consequence of SNP exposure (Fig. 7) are in accord with
the cell viability assays employing Trypan blue, PI, LDH,
and DNA fragmentation assays (Fig. 6).
In order to check whether the NO donor SNP, at a
concentration of 1,000 lM, had an effect on TSPO
expression, we performed binding analyses including
maximal binding capacity (Bmax) and the equilibrium dis-
sociation constant (Kd) of [3H]PK 11195 as well as western
blot analysis in normal U118MG cells. Binding analysis
showed that Bmax and Kd for SNP-treated cells did not
differ significantly from the control (Table 2). Also with
western blot we could not detect differences in TSPO
expression in U118MG cells exposed to 1,000 lM of SNP
for 24 h compared to vehicle treated control cells (Fig. 8).
Neither did PK 11195 exposure, or the combination of PK
11195 with SNP, affect TSPO protein expression (Fig. 8).
In addition, displacement experiments were performed on
rat kidney homogenates in order to evaluate potential
binding of SNP or NO to the TSPO. This assay showed that
SNP was not able to displace the TSPO ligand [3H] PK
11195 (Ki [ 10,000 nM). These data indicate that SNP
application does not affect TSPO binding characteristics or
expression in cell culture, and that SNP or NO generated by
SNP does not bind to TSPO.
The biotin-switch technique (BST) (Figs. 9, 10) was used
to assay if S-nitrosylation of TSPO by SNP is implicated in
the activation of its pro-apoptotic functions. For this pur-
pose, we performed immunodetection by applying our
TSPO specific antibody to western blots of S-nitrosylated
proteins after treatments with SNP and application of the
positive control (the robust S-nitrosylation promoter, CysNO
[44, 47] (Fig. 9a). First of all, total TSPO levels (starting
material) were not affected by CysNO and SNP (Figs. 8,
9b). Remarkably, S-nitrosylation of TSPO by SNP was not
detected (Fig. 9a). However, the positive control (CysNO)
did show S-nitrosylation of TSPO (Fig. 9a). Omission of
ascorbate from the reaction mixture demonstrated the lack of
false positivity (Fig. 9a). We also did not detect S-nitrosy-
lation by SNP of VDAC, which is closely associated with
the TSPO (Fig. 9c, d). However, VDAC was S-nitrosylated
by the positive control (CysNO) (Fig. 9c). To our knowl-
edge there are no other reports specifically dealing with
potential S-nitrosylation of VDAC, although it possesses
several cysteine residues [62].
In Fig. 10a we show the general patterns of molecular
weights of S-nitrosylated proteins after application of SNP
(1 mM) for different time periods (5, 10, and 15 min), and
also after application of the NO donor of the positive control
(1 mM of CysNO for 10 min), by Coomassie blue staining
of S-nitrosylated proteins run on a 12 % polyacrylamide gel,
in comparison to vehicle control, as described in ‘‘Meth-
ods’’. Various proteins of different molecular weights were
S-nitrosylated by SNP, as compared with vehicle control
(Fig. 10a), while total protein levels (starting material) were
not affected by the different treatments (Fig. 10b). Further-
more, we found that the cytoskeletal protein b-actin was one
of the proteins that could be S-nitrosylated by SNP
(Fig. 10c). In particular, treatment with SNP resulted in
45 % more S-nitrosylated b-actin than observed in the
vehicle control (p \ 0.01, n = 6) (Fig. 10c). S-nitrosylation
of b-actin by the positive control treatment (CysNO) was 2.8
fold more than detected in the vehicle control (p \ 0.001
versus vehicle control cells) (Fig. 10c). Total protein level of
b-actin (starting material) was not affected by SNP or in the
A B C
Fig. 4 Micrographs of effects of SNP treatment of U118MG cells.
The arrows indicate rounding and blebbing of cells. a Vehicle treated
cells; b cells treated with 1,000 lM of SNP for 24 h; c cells treated
with 1,000 lM of SNP in combination with 25 lM of PK 11195.
Compared to vehicle control treated cells (a), the NO donor SNP
causes abundant rounding and blebbing of cells and reduces the cell
density (b), which is attenuated by the TSPO ligand PK 11195 (c)
656 Apoptosis (2012) 17:647–665
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positive control (CysNO) (Fig. 10d). Also for the non-TSPO
proteins, omission of ascorbate from the reaction mixture
demonstrated the lack of false positivity for S-nitrosylation
(Fig. 10a). Noteworthy, we also used the S-Nitrosylated
Protein Detection Assay Kit (Cayman), as described in
‘‘Methods’’, and with this technique we also found S-nitro-
sylation of various proteins after 24 h of treatment with
SNP.
Discussion
Cell death, including apoptosis, induced by various agents
such as CoCl2, erucylphosphohomocholine (ErPC3), etc.,
Fig. 5 PK 11195 (PK) counteracts induction of cell death related
processes otherwise induced by SNP in U118MG cells. a PK 11195
reduces measured DNA fragmentation compared to treatment with
SNP alone. The y-axis indicates DNA fragmentation levels (repre-
sented in OD) (n = 6 for all groups in a). b PK 11195 attenuates
decrease in measured mitochondrial activity otherwise caused by
SNP, as measured by XTT assays. The y-axis indicates mitochondrial
activity as determined with XTT assay (represented in OD) (n = 8 for
all groups in b). c PK 11195 reduces DWm collapse induced by SNP
as measured by JC-1 fluorescence. The y-axis indicates normalized
red/green fluorescence ratio of JC-1, whose reduction is indicative of
DWm collapse (n = 6 for all groups in c). Control is vehicle control
as indicated in each panel. #p \ 0.05 versus SNP; **p \ 0.01 versus
control. ***p \ 0.001 versus vehicle control
Fig. 6 TSPO knockdown counteracts cell lethality induced by SNP
in U118MG cells. a Trypan blue uptake shows that SNP does not
induce cell death in TSPO knockdown cells (siRNA), but does induce
cell death in the control cells (Scr) cells. In (a) the y-axis indicates the
percentage of Trypan blue positive cells from total population of
cells. b LDH release also indicates that SNP causes cell death in Scr
cells, but not in siRNA cells. c PI uptake also shows that TSPO
knockdown (siRNA) protects against cell death induced by SNP. In
(c) the y-axis indicates the relative levels of dead cells having taken
up PI. The results are normalized to vehicle control, which was set as
100 % in both Scr and siRNA. d TSPO knockdown prevents DNA
fragmentation induced by SNP. The y-axis indicates DNA fragmen-
tation levels (OD) as an indicator for apoptosis. In (a), (b), and
(d) control is vehicle control, *p \ 0.05 versus Scr control.
***p \ 0.001 versus Scr (n = 6 for all groups)
Apoptosis (2012) 17:647–665 657
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includes activation of the TSPO leading to ROS generation
at mitochondrial levels, oxidation of cardiolipins, collapse
of the Dwm, caspase activation, and induction of DNA
fragmentation as components of the mitochondrial apop-
tosis cascade in human glioblastoma U118MG cells, as
described previously [13, 16–18, 63]. These are processes
that are also induced by NO [64–67]. Furthermore, nNOS
may be linked to the TSPO via the scaffolding protein
CAPON, which binds to the G protein Dexras1, which in
turn binds to PAP7 [38, 39]. As this variety of lethal agents
(CoCl2, ErPC3, etc.) require the presence of TSPO to
induce cell death, we assumed that NO may be a common
mediator for all these lethal agents targeting the TSPO.
With the present study we showed that application of two
different NO donors, GSNO and SNP, induced apoptosis
both in U118MG human glioblastoma cells and in PC12 rat
pheochromocytoma cells. Because of the consistency of
the observed effects, we applied 1,000 lM of SNP to the
U118MG cell line for the most part of this study. The
concentrations and time periods of the NO donor applica-
tions used in the present study are as described previously
by others (see ‘‘Methods’’), and we found them to be
effective also in our pilot studies (e.g. Fig. 1). With this
approach we set out to determine potential interactions
between TSPO and NO in relation to cell death induced by
SNP. For this purpose, we applied TSPO knockdown and
their appropriate controls, and also assayed the effects of
the TSPO ligand PK 11195 on U118MG cells.
As the TSPO is able to activate the mitochondrial
apoptosis cascade, we decided to assay various aspects of
mitochondrial functions as well as indicators of cell death
after application of SNP. With the aid of the XTT kit, we
measured metabolic activity, which is also indicative to
cell proliferation and viability according to the manufac-
turer of the kit (Biological Industries). This showed that
SNP caused a reduction of 73 % in XTT signal. As the cell
death levels (46 % of the total population), measured with
Trypan blue and PI uptake, do not fully account for the
73 % reduction in XTT signal, we assume that SNP
application also affects metabolic processes in living cells,
in addition to cell death induction in our cell cultures. We
found here that SNP causes collapse of the DWm, which is
known to initiate cell death processes [35, 68–70]. TSPO
also is able to regulate the DWm [12, 13, 17, 18, 57]. With
the present study, we found that the TSPO specific ligand
PK 11195 was able to significantly reduce the cell death
levels effects of SNP on U118MG cells. The concentration
of PK 11195 (25 lM) used is the same concentration that
Fig. 7 Effects of SNP on mitochondrial functions is prevented by
TSPO knockdown in U118MG cells. a XTT assays show that in
siRNA cells SNP does not affect mitochondrial activity, as it does in
Scr cells. The y-axis indicates OD levels of XTT product, as an
indicator for relative mitochondrial activity (n = 8 for all groups).
b JC-1 fluorescence indicates that TSPO knockdown attenuates DWm
collapse, as it is normally induced by SNP in Scr cells. The y-axis
indicates red/green fluorescence ratio marking JC-1 polymerization
normalized to control, which reduction is indicative for the incidence
of collapse of the DWm (n = 6 for all groups). c NAO fluorescence
indicates that TSPO knockdown prevents cardiolipin oxidation
induced by SNP, as it normally occurs in Scr cells with SNP
exposure. The y-axis indicates normalized NAO signal for oxidized
cardiolipins (M1 in d), which is indicative to mitochondrial ROS
generation. Vehicle treated control groups for both siRNA and Scr
cells were set at ‘‘1’’ (n = 9 for all groups). d Representative FACS
results of NAO signal vehicle treated Scr cells (left hand figure); Scr
cells treated with SNP (1,000 lM) (right hand figure); M1 in each
histogram indicates the population of cells showing low intensity
NAO labeling, representing the % of cells displaying oxidized
cardiolipins. In (d) the x-axes indicate fluorescence intensity of NAO
signal, and the y-axes indicate the number of cells related to the
measured fluorescence intensity. ***p \ 0.001 versus Scr control;
**p \ 0.01 versus Scr control; *p \ 0.05 versus Scr control
b
658 Apoptosis (2012) 17:647–665
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showed protection against lethal effects of ErPC3 and
CoCl2 in these cells in previous studies [13, 17, 18]. In
contrast to the isoquinoline, PK 11195, which has high
affinity for the TSPO in all species studied so far, the
benzodiazepine Ro5 4864 shows only a low affinity for the
TSPO in the human brain and glioma (as well as kidney
and colon) [71, 72]. In particular, while [3H]PK 11195
binds with high affinity (Kd of about 2 nM) to human
cerebral cortex, kidney, and colon membranes, the specific
binding of [3H]Ro5 4864 is barely detectable (nonspecific
binding about 90 % of the total binding) [71]. Furthermore,
unlabeled PK 11195 is two orders of magnitude more
potent than unlabeled Ro5 4864 in displacing [3H]PK
11195 specific binding from human cerebral cortex and
kidney membranes [71]. Thus, we assume that the high
affinity of the isoquinoline PK 11195 for TSPO in human
cells is responsible for PK 11195’s ability to prevent the
lethal effects of the NO donor SNP in U118MG cells. In
another study, applying benzodiazepines, we found that the
TSPO ligand Ro5 4864 did not significantly modulate
effects of SNP in the human U118MG cells [61]. Treat-
ment with the benzodiazepine clonazepam, which is a CBR
specific ligand, also did not show any significant effect in
this regard [61]. Clonazepam is well known to not bind to
TSPO.
Apart from preventing cell death induced by SNP, PK
11195 also reduced metabolic activity, and collapse of the
DWm otherwise induced by SNP. Collapse of the DWm,
leading to cytochrome c release and the activation of cas-
pase-3 and caspase-9, is one of the initiating steps for the
mitochondrial apoptosis cascade [64, 68, 73]. Other studies
Table 2 Effects of the NO donor SNP on TSPO binding characteristics in U118MG cells
U118MG cells SNP treatment Vehicle treatment
Bmax 2146 ± 675 (fmol/mg protein) 2040 ± 424 (fmol/mg protein)
Kd 4.4 ± 2.0 nM 3.1 ± 1.2 nM
TSPO binding characteristics (Bmax and Kd) were determined with binding assays applying [3H]PK 11195 (n = 9 for all assays)
Control SNP + PK
β-Actin (42 KDa)
TSPO (18 KDa)
SNPPK
A
B
Fig. 8 Western blot of 18 kDa TSPO protein expression in U118MG
cells after exposure for 24 h to 1 mM of SNP (SNP), 25 lM of PK
11195 (PK), and the combination of 1 mM of SNP and 25 lM of PK
11195 (SNP ? PK). a A representative western blot of 18 kDa TSPO
protein expression. b-Actin was used as a loading control. b A bar
graph presenting the relative 18 kDa TSPO levels. No significant
differences were observed between the different types of exposure.
Control is application of vehicle
Fig. 9 Lack of TSPO and VDAC S-nitrosylation due to SNP (1 mM)
treatment of U118MG cells. Omission of ascorbate is applied as a
negative control (-), as described in ‘‘Methods’’. (?) Underneath
blots indicates the regular procedure with addition of ascorbate. As
positive control, cells are exposed to 1 mM of CysNO. a Represen-
tative western blot showing that SNP is not able to S-nitrosylate
18 kDa TSPO, while the positive control (CysNO) does S-nitrosylate
18 kDa TSPO. b Representative western blot showing that neither
SNP, nor the positive control CysNO, do affect normal 18 kDa TSPO
levels (starting material). c Representative western blot showing that
SNP is not able to S-nitrosylate VDAC, while the positive control
(CysNO) does S-nitrosylate VDAC. d Representative western blot
showing that neither SNP, nor the positive control CysNO, do affect
normal VDAC levels (starting material)
Apoptosis (2012) 17:647–665 659
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applying TSPO knockdown and TSPO ligands have shown
that the occurrence of these events of the mitochondrial
apoptosis cascade can be modulated by the TSPO [12, 13,
16–18, 74, 75]. For the present study, we also used TSPO
siRNA knockdown U118MG cells in comparison to their
sham controls (Scr), prepared previously in our laboratory
[18]. Comparable to the effects of PK 11195, our various
assays to test cell death, and cell death related processes,
showed that TSPO knockdown also counteracts cell
lethality induced by SNP. Thus both approaches for mod-
ulation of TSPO activity, i.e. application of the TSPO
ligand PK 11195 as well as TSPO knockdown by genetic
manipulation, implicate that TSPO is involved in the lethal
and metabolic effects of the NO donor SNP.
Interestingly, we found that applying 25 lM of PK
11195 to TSPO knockdown cells did not have any effect on
cell lethality, collapse of the DWm, metabolic activity, or
mitochondrial ROS generation, still occurring to TSPO
Fig. 10 Protein S-nitrosylation due to SNP (1 mM) treatment of
U118MG cells. Omission of ascorbate is applied as a negative control
(-), as described in ‘‘Methods’’. (?) Underneath blots indicates the
regular procedure with addition of ascorbate. As positive control,
cells are exposed to 1 mM of CysNO. a Coomassie stained gel
showing that CysNO (1 mM) exposure for 10 min (as positive
control) and SNP exposure for different time periods (5, 10 and
15 min) do S-nitrosylate proteins (Protein-SNO) of U118MG cells.
b Coomassie stained gel showing that SNP does not affect general
protein levels (starting material) in U118MG cells. Also the positive
control (CysNO) does not affect general protein levels (starting
material). In (a) and (b) the MW markers of the Precision Plus
ProteinTM Standards, KaleidoscopeTM (Bio-Rad) are presented on the
left hand sides of the gels. c Representative western blot showing that
SNP can cause S-nitrosylation of b-actin, displayed at the bottom ofthe figure. The bar chart presents the relative optical density of bands
labeled for S-nitrosylated b-actin showing that SNP, as well as the
positive control (CysNO), can cause S-nitrosylation of b-actin. The
y-axis indicates in arbitrary units the optical density of Actin-SNO
labeled bands. d Representative western blot indicating that SNP and
CysNO do not affect normal b-actin levels (Starting material).
**p \ 0.01 versus vehicle control. Vehicle = vehicle control. ‘‘-’’
Underneath blots indicates omission of ascorbate (negative control).
‘‘?’’ Underneath blots indicates inclusion of ascorbate (regular
procedure). TSPO-SNO, b-actin-SNO, and Protein-SNO are S-nitro-
sylated TSPO, b-actin, and total protein, respectively. 1 mM of SNP,
and 1 mM of CysNO as positive control, was applied for each assay
660 Apoptosis (2012) 17:647–665
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knockdown cells exposed to SNP. This makes sense as PK
11195 is a TSPO specific ligand and our siRNA U118MG
TSPO knockdown cells display reduced levels of TSPO
expression ([18]; present study). This point is not irrelevant
as PK 11195 is used at a concentration high above its
dissociation constant (Kd) for the TSPO (25 lM vs.
±2.5 nM). An interesting question is why concentrations
of PK 11195, well above its Kd for TSPO, are needed to
induce TSPO specific effects. We assume that such high
concentrations of PK 11195 may be needed to compete
successfully with endogenous TSPO ligands potentially
having higher affinity for the TSPO than PK 11195. In a
generalized postulation, we suggest that TSPO specific
ligands, such as PK 11195, at concentrations up to 50 lM
can act as antagonists. Here we mean with antagonist the
capability to attenuate TSPO’s pro-apoptotic function. At
higher concentrations still, however, PK 11195 can induce
cell death, and we suggest that the latter is due to non-
TSPO specific effects. This postulation is presented by
various experimental studies by us targeting this question,
as well as by an extensive overview of experimental studies
by others [12–18]. We believe that endogenous activators
of the TSPO are able to induce TSPO’s pro-apoptotic
function, which appears to be blocked by TSPO knock-
down and synthetic TSPO ligands such as PK 11195.
Further studies are required to obtain better insights into
this possibility.
A priori, we assumed that NO, as generated for
example by SNP, acts directly on the TSPO. However, we
found that SNP did not affect TSPO’s expression or
binding characteristics, neither did SNP interfere with
[3H]PK 11195 binding to TSPO. Thus, SNP neither did
appear to damage the TSPO nor to interact directly via a
ligand binding mechanism with the TSPO. Nonetheless,
applying TSPO knockdown and the TSPO ligand PK
11195, our study shows that SNP application causes sev-
eral effects that are dependent on the presence of the
TSPO. Thus, we assumed that SNP may cause TSPO
S-nitrosylation. It is well known that S-nitrosylation can
affect protein function [31, 76–78]. In addition, co-local-
ization of NOS enzymes with target proteins, including
direct interactions, may be an important determinant of
S-nitrosylation [31]. As NOS may be closely associated
with the TSPO [38, 39], we assumed that NO generated by
NOS may affect TSPO function. Furthermore, human
TSPO possesses two cysteine residues, one at the N-ter-
minus and the other one at the C-terminus, therefore we
considered it likely that the TSPO can be nitrosylated [79,
80]. Such a nitrosylation of the TSPO would affect
TSPO’s interactions with molecules at both sides of the
outer mitochondrial membrane, i.e. it would affect TSPO
function. With the present study we found that the NO
donor SNP causes S-nitrosylation of various proteins,
including b-actin. Thus, our study suggests that S-nitro-
sylation of various proteins due to SNP exposure may
contribute to the induction of cell death by SNP. However,
S-nitrosylation of TSPO by SNP did not appear to occur.
Nonetheless, application of CysNO as a positive control
for detection of S-nitrosylation of TSPO showed that
TSPO in U118MG cells can be nitrosylated. In any case,
TSPO appears to be relatively resistant against S-nitrosy-
lation due to application of SNP. Thus, it appears that
S-nitrosylation of the TSPO is not required to allow TSPO
to initiate cell death processes. Noteworthy, the other cells
of our study, PC12 cells, are from rat, where TSPO does
not possess cysteine residues and thus cannot be S-nitro-
sylated [81]. Nevertheless, the effects of SNP on rat PC12
cells are not much different from what we observed in the
human U118MG cells (Fig. 1). This indicates that indeed
S-nitrosylation of the TSPO is not required for SNP to
exert its effects observed in the present study. It appears
that S-nitrosylation of proteins other than TSPO may lead
to activation of the TSPO, although at present it is not
clear how. Interestingly, Dexras1, which presents a link
between NOS and TSPO, can be nitrosylated [82]. Our
experiments, assaying protein S-nitrosylation, demon-
strated in our paradigm that protein S-nitrosylation takes
place within a short time after NO donor application, and
subsides after that. Thus, we assume that SNP in our
model affects the cell machinery soon after application,
for example by nitrosylating various proteins. This then
over an extended period of time leads to cell death
including apoptosis. Apart from inducing S-nitrosylation
of various proteins, the NO donor SNP can also be cyto-
toxic by other means, for example due to cyanide pro-
duction, as a consequence of SNP degradation [83]. This,
however, requires light [84]. Our experiments were per-
formed in the dark, excluding the possibility of cell death
induction due to degradation products of SNP. Interest-
ingly, in the light, our application of SNP did not induce
cell death (unpublished results). We assumed that degra-
dation of SNP in the light prevented NO production by
SNP and thereby pre-empted cell death induction due to
SNP application. In any case, our experiments performed
in the light do not support the possibility that cyanide
products due to SNP application present the major cause
of cell death detected under conditions of darkness.
Nonetheless, SNP was not able to S-nitrosylate TSPO in
our model. We consider the possibility that NO generated
by NOS may be more effective for S-nitrosylation of
TSPO than can be achieved with SNP. Interestingly, NOS
can be found in the vicinity of the TSPO [38, 39]. How-
ever, we have found that glutamate applied at toxic levels
([25 mM) to U118MG cells induces S-nitrosylation of
b-actin, but not of TSPO (unpublished results). Furthermore,
applying TSPO knockdown to these cells prevents the
Apoptosis (2012) 17:647–665 661
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toxic effects of glutamate (unpublished results). It is well
known that glutamate can activate NOS to generate NO
leading to S-nitrosylation of various proteins [85]. One of
the proteins that can be S-nitrosylated in this way is
Dexras1, which is closely associated with the TSPO [38,
39, 82]. Also by applying NH4Cl as a toxic agent we could
induce cell death and S-nitrosylation of various proteins,
but no nitrosylation of the TSPO (unpublished results). It
is known that NH4Cl can cause collapse of the DWm via
NOS activation [86]. Thus, SNP, glutamate, and NH4Cl
can cause cell death via S-nitrosylation of proteins, and
this induction of cell death can be prevented by deacti-
vation of the TSPO. Other proteins than Dexras1 that are
known to interact with the TSPO are: VDAC, ANT,
PRAX-1, PAP7, and pk 10 [4, 12, 13]. VDAC, however,
which is also closely associated with the TSPO, was not
S-nitrosylated by our application of SNP. To our knowl-
edge, S-nitrosylation of VDAC is not considered a major
function modifier for this protein. We are also not aware
of studies addressing S-nitrosylation of ANT, PRAX-1,
PAP7, and pk 10. The question remains by which mech-
anism the NO donor SNP (and other agents such as
glutamate, NH4Cl, CoCl2, and ErPC3) modulate mito-
chondrial function and cell death, i.e. the question remains
which agent(s) interact immediately with the TSPO to
activate its cell death function. Apart from potential
S-nitrosylation of proteins closely associated with TSPO,
it may be that steroids regulate TSPO functions, as various
steroids are known to affect TSPO expression in vivo [11,
12]. Recent data by us suggest that dexamethasone may
reduce TSPO expression in T98G cells, in correlation with
reduction in cell death levels otherwise induced by various
agents, such as SNP, CoCl2, and ErPC3 (unpublished
results). Note that SNP, CoCl2, and ErPC3 do require the
TSPO to induce cell death [17, 18, and the present study].
Thus, it cannot be excluded that locally produced steroids
may affect TSPO expression in relation to cell death
induction. Compounds we have also considered are por-
phyrins, which are putative endogenous TSPO ligands
[87], however, it appears that interactions between por-
phyrins and TSPO primarily serve to regulate porphyrin
levels rather than that porphyrins regulate TSPO’s cell
death functions [88–92].
Conclusion
In the present study we show that the activation of the
TSPO due to application of NO donors, in particular SNP,
leads to cell death including collapse of the DWm, ROS
generation at mitochondrial levels, cardiolipin oxidation,
DNA fragmentation, and rounding and blebbing of cells
(Fig. 11). The results of this study indicate conclusively
that the TSPO is involved in cell death caused by the NO
donor SNP, as well as SNP effects on mitochondrial
functions. Our results suggest that the cell death inducing
effects of the NO donor SNP is due to S-nitrosylation of
various proteins other than the TSPO (Fig. 11). Other
cytotoxic effect of SNP, however, cannot be excluded. A
question remaining is how in the end TSPO is activated by
SNP (or other lethal agents) to induce cell death; for
example: is an endogenous TSPO ligand released, or do
proteins typically in conjugation with the TSPO regulate its
function. It for example may be that S-nitrosylation of
Dexras1, a protein associated with TSPO, may contribute
to the effects of SNP operated by way of TSPO activation.
Other proteins closely associated with the TSPO are
VDAC, ANT, pk 10, PAP7, and PRAX-1 (see reviews, [4,
12]). Alternatively, regulation of TSPO gene expression,
including post-transcriptional processes, may be the driv-
ing force to induce its capability to initiate cell death
processes. In any case, the presence of the TSPO is spe-
cifically required to achieve at least part of the lethal and
metabolic effects of exposure to the NO donor SNP.
Fig. 11 The present study shows that the NO donor SNP is able to
S-nitrosylate proteins, inducing TSPO to activate the mitochondrial
apoptosis cascade, including mitochondrial ROS generation, cardio-
lipin oxidation, and DWm collapse. The DWm collapse may also
contribute to cell death other than apoptosis. While activation of the
TSPO apparently does not require TSPO’s S-nitrosylation, TSPO still
is required for cell death to occur in this experimental model. Which
exactly are the intermediate agents between general protein S-nitro-
sylation and activation of the TSPO in this experimental model is
unknown. Steroids and/or proteins closely associated with TSPO, and/
or regulation of gene expression of the TSPO can be suggested
662 Apoptosis (2012) 17:647–665
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Acknowledgments Moran Benhar and the co-workers of his labo-
ratory are thankfully acknowledged for their help with the S-nitro-
sylation assays. The expert assistance of Ilana Spanier and Esther
Messer is acknowledged. Research support was provided by: Nie-
dersachsen-Israel Project (MG, LV, JB), Technion-John Hopkins
Joint Research Program (S-HS, MG, LV), Johnson & Johnson (MG,
LV), L. Aronberg Research Fund in Neurology (MG, LV), Sch-
warzbach Medical Research Fund (MG, LV). Dr. Beatriz Caballero
thankfully acknowledges her post-doctoral fellowship (Programa
Cların) from FICYT (Gobierno del Principado de Asturias), Spain.
Dr. Leo Veenman acknowledges his support by a joint grant from the
Center for Absorption in Science of the Ministry of Immigrant
Absorption and the Committee for Planning and Budgeting of the
Council for Higher Education under the framework of the KAMEA
Program.
Conflict of interest The authors declare that they don’t have con-
flict of interest.
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