Content uploaded by Jan Blecha
Author content
All content in this area was uploaded by Jan Blecha on Aug 10, 2018
Content may be subject to copyright.
Author’s Accepted Manuscript
Antioxidant defense in quiescent cells determines
selectivity of electron transport chain inhibition-
induced cell death
Jan Blecha, Silvia Magalhaes Novais, Katerina
Rohlenova, Eliska Novotna, Sandra Lettlova,
Sabine Schmitt, Hans Zischka, Jiri Neuzil, Jakub
Rohlena
PII: S0891-5849(17)30714-1
DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2017.07.033
Reference: FRB13410
To appear in: Free Radical Biology and Medicine
Received date: 30 May 2017
Revised date: 24 July 2017
Accepted date: 30 July 2017
Cite this article as: Jan Blecha, Silvia Magalhaes Novais, Katerina Rohlenova,
Eliska Novotna, Sandra Lettlova, Sabine Schmitt, Hans Zischka, Jiri Neuzil and
Jakub Rohlena, Antioxidant defense in quiescent cells determines selectivity of
electron transport chain inhibition-induced cell death, Free Radical Biology and
Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2017.07.033
This is a PDF file of an unedited manuscript that has been accepted for
publication. As a service to our customers we are providing this early version of
the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting galley proof before it is published in its final citable form.
Please note that during the production process errors may be discovered which
could affect the content, and all legal disclaimers that apply to the journal pertain.
www.elsevier.com
Blecha et al.: Selectivity of electron transport chain inhibition
1
Antioxidant defense in quiescent cells determines selectivity of electron
transport chain inhibition-induced cell death
Jan Blechaa,b, Silvia Magalhaes Novaisa, Katerina Rohlenovaa, Eliska Novotnaa, Sandra
Lettlovaa, Sabine Schmittc, Hans Zischkac,d, Jiri Neuzila,e,*, Jakub Rohlenaa,*
a Institute of Biotechnology, Czech Academy of Science, BIOCEV, Vestec, Prague-West,
Czech Republic
b Faculty of Science, Charles University, Prague, Czech Republic
c Institute of Toxicology and Environmental Hygiene, Technical University Munich, Munich,
Germany
d Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German
Research Center for Environmental Health, D-85764 Neuherberg, Germany
e School of Medical Science, Griffith University, Southport, Qld, Australia
*Correspondence: Jakub Rohlena, Molecular Therapy Group, Institute of Biotechnology,
Academy of Sciences of the Czech Republic, BIOCEV, Prumyslova 595, 25250 Vestec,
Prague-West, Czech Republic; email: jakub.rohlena@ibt.cas.cz
or Jiri Neuzil, Mitochondria, Apoptosis and Cancer Research Group, School of Medical
Science and Menzies Health Institute Queensland, Griffith University, Southport, Qld 4222,
Australia; e-mail: j.neuzil@griffith.edu.au
ABSTRACT
Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer
therapy. It is particularly effective in tumors with high ETC activity where ETC-derived
reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted
compounds are tolerated on the organismal level remains unclear. As most somatic cells are
in non-proliferative state, the features associated with the ETC in quiescence could account
Blecha et al.: Selectivity of electron transport chain inhibition
2
for some of the specificity observed. Here we report that quiescent cells, despite increased
utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death
induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by
the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense,
principally by the superoxide dismutase 2 – thioredoxin axis. In contrast, under conditions of
glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with
intracellular ATP depletion but not with ROS. This is related to the inability of quiescent
cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose
uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible
for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused
healthy tissues, where antioxidant defense imparts specificity. However, in conditions of
limited glucose, e.g.in poorly perfused tumors, ETC disruption causes rapid depletion of
cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we
propose that antioxidant defense in quiescent cells is aided by local glucose limitations to
ensure selectivity of ETC inhibition-induced cell death.
ABBREVIATIONS
BNE, blue native electrophoresis; CAT, catalase; CCCP, carbonyl cyanide 3-
chlorophenylhydrazone; DCF, 2’,7’-dichlorofluorescin diacetate; DHE, dihydroethidium;
DHR123 dihydrorhodamine 123; ETC , electron transport chain; FCCP, carbonyl
cyanide p-(trifluoromethoxyl)-phenyl-hydrozone; GPX1, glutathione peroxidase-1; GSR,
glutathione reductase; hrCNE, high-resolution clear native electrophoresis; NAC, N-acetyl
cysteine; PEITC, phenethyl-isothiocyanate; PRX3, peroxiredoxin-3; ROS, reactive oxygen
species; SCs, supercomplexes; SOD1, superoxide dismutase 1; SOD2, manganese superoxide
dismutase; TRX2, thioredoxin-2; TXNRD2 thioredoxin reductase 2; WB, western blot
Key words: Electron transport chain, supercomplexes, antioxidant defense, SOD2,
thioredoxin
Blecha et al.: Selectivity of electron transport chain inhibition
3
INTRODUCTION
Mitochondria play a key role in multiple cellular processes, including energy production,
metabolism and cell death. Functional mitochondria are essential in tumorigenesis [1-3], and
targeting of the mitochondrial electron transport chain (ETC) emerges as an effective strategy
to suppress and eliminate cancer cells [4-8]. ETC consists of four macromolecular protein
complexes (referred to as CI, CII, CIII and CIV) that associate further into supercomplexes
(SCs) [9, 10], and work together to maintain mitochondrial inner membrane potential, used
by the mitochondrial ATP synthase (a.k.a complex V, CV) to produce ATP. Because of high
electron traffic and presence of redox centers, the ETC is a significant source of reactive
oxygen species (ROS) [11, 12]. To counter ETC-derived ROS, mitochondria are equipped
with several antioxidant mechanisms, such as manganese superoxide dismutase (SOD2), as
well as the glutathione and thioredoxin systems [13-15]. Inhibition of the ETC interferes with
mitochondrial ATP synthesis, but also substantially increases ETC-derived ROS production,
leading to cell death [4]. The susceptibility to cell death upon ETC inhibition will therefore
be governed by the degree of cell’s reliance on mitochondrial ATP production, and by the
balance between the propensity to form and detoxify ETC-derived ROS.
Recent findings indicate that ETC targeting is particularly effective in cancer cells and
subtypes with increased dependence on mitochondrial respiration and higher SC assembly [5,
16-18]. At the same time, pharmacologically relevant ETC inhibitors, such as metformin or
the recently developed mitochondrial-targeted agents such as MitoTam and MitoVES, are
well tolerated in animals, suggesting selectivity for cancer cells [5, 19]. However, despite its
obvious translational relevance, the molecular reasons for this selectivity are unclear.
Blecha et al.: Selectivity of electron transport chain inhibition
4
While cancer cells are hard-wired for proliferation, the majority of normal somatic cells are
in a non-proliferative, post-mitotic state. Features associated with quiescence and non-
proliferative state could therefore partially account for the specificity we observed
previously: quiescent cells were resistant to agents that induce ROS from the ETC [20, 21],
but molecular reasons remained uncharacterized. Hence, specific configuration of the ETC in
quiescent cells, different handling of ROS, or changes in the dependence on mitochondrial
ATP production could all significantly contribute to the therapeutic window associated with
the ETC inhibition in cancer. Understanding the paradigms that govern sensitivity to cell
death in quiescent cells under various circumstances is therefore of high importance for the
development of efficient and non-toxic ETC-directed cancer therapy.
In the current study we attempted to resolve the selectivity issue by characterizing the ETC
and the ability to form and detoxify ROS upon ETC inhibition in proliferating and quiescent,
non-transformed cells. Using pharmacological and genetic manipulations, we show that the
capacity to detoxify ROS is increased in quiescent cells, significantly contributing to the
resistance of these cells to ETC inhibition, despite their increased reliance on the ETC.
Furthermore, under conditions of nutrient limitations expected to occur in poorly vascularized
areas of tumors, ETC inhibition brings an additional benefit by interfering with mitochondrial
ATP production, further improving specificity.
Blecha et al.: Selectivity of electron transport chain inhibition
5
MATERIAL AND METHODS
Reagents
All reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), unless stated
otherwise.
Cell culture
The cells used in the study were obtained from ATCC and were authenticated. The Ea.hy926
cells were cultured in high glucose (4.5 g/l) (Lonza, Basel, Switzerland) or low glucose (1
g/l) (Lonza) DMEM media supplemented with 10% fetal bovine serum (ThermoFisher,
Waltham, MA, USA), hypoxanthine aminopterin thymidine (HAT) supplement 2%
(ThermoFisher) and 1% antibiotics (penicilin G 100U/ml and streptomycin 100 μg/ml) and,
for low glucose media only, L-glutamine 0,03% in a 5% CO2, 37°C incubator. MCF10A cells
were cultured in DMEM:F12 (Lonza) media supplemented with 5% horse serum (Sigma-
Aldrich, H1138), human insulin 0.1% (I9278), 20 ng/mL epidermal growth factor
(ThermoFisher), hydrocortisone 0.5 mg/ml, cholera toxin 100 ng/mL. For the experiments the
media was changed for low glucose or high glucose DMEM supplemented as DMEM:F12
above. All cultures were negative for mycoplasma as determined by the MycoAlert kit
(Lonza). For experiments requiring pharmacological treatments, equal numbers of cells (5-7
x105) were exposed to the studied compounds. For quiescent cultures, the cells were seeded
into 24-well plates at 80% density, and cultured for 4 days post-confluence. For proliferating
cultures, cells were seeded into 6-well plates at 2x105 cells per well two days before the
experiment. 1 ml of medium per well were used for both proliferating and quiescent cultures.
Lentiviral production and RNA interference
Blecha et al.: Selectivity of electron transport chain inhibition
6
pLKO1 plasmids containing shRNA sequences against the targeted transcripts were obtained
from Sigma-Aldrich. The specific shRNA used were as follows: SOD2 - TRCN0000320739,
GPX1TRCN0000046231, TXNRD2 - TRCN0000433391, GSR -TRCN0000046425.
Lentiviral particles were produced in Hek293-FT cells using second generation psPAX and
pMD.2G plasmids and calcium phosphate or Lipofectamine 3000 (ThermoFisher)
transfections. Virus-containing medium was collected after 48 h, centrifuged at 3,000 x g for
15 min, and in some cases the viruses were concentrated by overnight incubation with PEG-it
(SBI-System Biosciences, Palo Alto, CA, USA). The supernatants were aliquoted and stored
at -80ºC. To induce silencing, the lentivirus-containing supernatants were added to cells
grown in normal culture medium overnight. The transduced cells were selected by
puromycin, and the level of silencing was assessed by western blotting (WB).
Respiration assessment
Cellular respiration was assessed by Seahorse XF24 analyzer (Seahorse Biosciences, Santa
Clara, CA, USA). For quiescent cultures the cells were seeded at 80% density in the Seahorse
XF 24-well microplates and cultured 4 days post-confluence. Proliferating cells were seeded
at 20,000 per well two days before the experiment. Medium was switched before the
experiment for the base medium (D5030) of pH 7.4, containing 0.2% BSA and 10 mM
glucose. Oxygen consumption rate (OCR) was measured in the basal state and after
sequential addition of 10 µM oligomycin, 1 and 2 µM carbonyl cyanide p-
(trifluoromethoxyl)-phenyl-hydrazone (FCCP) and combined 10 µM rotenone plus 25 µM
antimycin. Data were normalized to DNA content measured by the Quant-iT™ PicoGreen®
dsDNA reagent (ThermoFisher). To determine the efficacy of ETC inhibition by the tested
compounds, the respiration of proliferating endothelial Ea.hy926 cells under both low
glucose (1 g/l) and high glucose (4.5 g/l) conditions was monitored by the Oroboros
Blecha et al.: Selectivity of electron transport chain inhibition
7
Oxygraph 2-K instrument (Oroboros Instruments, Innsbruck, Austria) as described [22],
using 2x106 of non-permeabilized cells per chamber. Basal routine respiration was
established after adding succinate (10 mM) and ADP (3 mM), after that pharmacological
agents were added as shown below.
SDS-PAGE and Western blot analysis
SDS-PAGE and WB analysis were performed using standard methods as detailed elsewhere
[1]. Antibodies used were: anti-peroxiredoxin III (SantaCruz, Dallas, TX, USA, sc-23973),
anti-thioredoxin 2 (Abcam, Cambridge, United Kingdom, ab185544), anti-SOD1 (SantaCruz,
sc-11407), anti-catalase (Abcam, ab1877), anti-GPX-1 (Abcam, ab108427), anti-SOD2
(Acris, Herford, Germany, AP03024-PU-N), anti-GSR (Proteintech, Manchester, United
Kingdom, 18257-1-AP), anti-TXNRD2 (Abcam, ab180493). The anti-β-Actin HRP
conjugate (CellSignaling, Danvers, MA, USA, #5125) was used for visualization.
Blue native electrophoresis (BNE) and high-resolution clear native electrophoresis
(hrCNE)
Mitochondria were isolated using a Balch-style homogenizer as detailed elsewhere [4, 23].
The published protocols were modified by using hand-driven 1 ml disposable syringes
(Becton Dickinson, Franklin Lakes, NJ, USA). BNE followed by immunoblotting and hrCNE
followed by in-gel activity assays was performed using digitonine-solubilised mitochondria
as described [24]. Primary antibodies used were as follows. CI - anti-NDUFB8 (Abcam,
ab110242), CII - anti-SDHA (Abcam, ab14715), CIII - anti-UQCRFS1 (Abcam, ab14746),
CIV - anti-MTCO2 (Abcam, ab110258), CV - anti-ATP5A (Abcam, ab110273). Samples
used for native gels were also run on SDS-PAGE/WB to verify equal mitochondrial loading
using anti-mtHSP70 antibody (ThermoFisher, MA3-028).
Blecha et al.: Selectivity of electron transport chain inhibition
8
Mitochondrial NAD(P)H measurements
Mitochondrial NADH was measured by two-photon microscopy using Zeiss LSM880 NLO
inverted microscope, equipped with Chameleon Ultra II and Compact OPO MP lasers
(Coherent, Santa Clara, CA, USA) using non-descanned BiG-2 GaAsP detector, 63x/1.4
Plan-Apochromat oil immersion objective, 740 nm laser line at 8 mW power (6 mW in
scanning mode during image acquisition which includes blanking), and emission range 390-
480 nm. The cells were grown in glass-bottom microscopy plates in normal culture medium,
pre-treated with 1 nM TMRM for 30 min and examined either untreated, exposed to 5 µM
CCCP, or to 1.25 µM rotenone. Mitochondrial NAD(P)H was quantified via the Fiji software
by segmenting cells using thresholding and averaging signal over mitochondrial ROIs. 3-4
independent experiments were performed, with more than 150 cells per condition imaged in
each experiment.
Detection of cell death and ROS
For the detection of cell death, cells were seeded before the experiment as described above. If
required, the cells were pre-incubated with acetylcysteine 1 mM (NAC) or auranofin 0.75 µM
(Enzo Life Sciences, Farmingdale, NY, USA) for 1 or 2 h, respectively. Incubation time with
the tested compounds was 22 h, after which cell death was assessed by flow cytometry using
Annexin V/propidium iodide (PI) staining as described [4]. Cells positive for Annexin V, PI,
or Annexin V/PI were considered dead. The tested agents were as follows: H2O2, phenethyl
isothiocyanate (PEITC), rotenone, piericidin A (Enzo), myxothiazol, oligomycin A, and
CCCP. MitoVES and MitoTam were synthetized in-house [5, 6]. For ROS measurements,
cell cultured as described were exposed to the tested compounds for one hour. For the last 20
min, the cells were co-incubated with 5 μM 2′,7′-dichlordihydrofluorescein diacetate (DCF),
Blecha et al.: Selectivity of electron transport chain inhibition
9
5 μM dihydroethidium (DHE) or 10 μM dihydrorhodamine 123 (DHR123). The level of
fluorescence was assessed by flow cytometry as described [4].
Assessment of glucose uptake, lactate production, and glucose content in culture media
Glucose uptake was evaluated after 15 min incubation with 50 μM 2-nitrobenzodeoxyglucose
(2-NBDG; ThermoFisher) by flow cytometry as described [5]. In some experiments, cells
were pre-treated for 6 hours by 0.5 or 5 µM MitoVES or 7.5 µM myxothiazol. Lactate
concentration in the culture medium was assessed after 19 h of cell cultivation using lactate
detection kit (TrinityBiotech, Bray, Ireland), following manufacturer’s instructions with all
volumes scaled down 10 times. Glucose content in the cultured media was determined after 6,
16, 24 and 38 h of cultivation under normal experimental conditions (proliferating cells in 6-
well format, quiescent cells in 24-well format, both in 1 ml of media) using low and high
glucose media and Amplex™ Red Glucose/Glucose oxidase assay kit (ThermoFisher)
following manufacturer’s instructions. Data were normalized to glucose content in fresh
media.
Evaluation of intracellular ATP
Intracellular ATP levels were assessed by the CellTiter-Glo kit (Promega, Madison, WI,
USA) following manufacturer’s instructions in 96 well format. Data were normalized to the
cell number measured by crystal violet in a parallel 96-well plate, and are shown as relative
to non-treated controls.
Measurements of surface GLUT1 exposure
Cells were exposed to the indicated agents and collected by trypsinization, incubated for 30
min in 100 ul PBS with 1:200 Anti-GLUT1 antibody (Abcam, ab15309), sedimented by
Blecha et al.: Selectivity of electron transport chain inhibition
10
centrifugation and resuspended in PBS with 1:100 secondary Cy3-stained antibody (Sigma-
Aldridge). Both incubations were performed at 37°C, 5%CO2 incubator. Fluorescence
intensity was determined by flow cytometry.
In-gel assessment of antioxidant protein activity
In-gel activity of antioxidant proteins was determined as described [25], with minor changes.
Samples were prepared on the Misonix Sonicator 3000 instrument (QSonica, Newtown, CT,
USA) at 30% power using microtip ~3 W, 3x15 s with 25 s pause performed on ice. The
incubation time in the SOD2 activity assay was reduced to 7 min.
NADPH/NADP and GSH/GSSG measurements
Cells were seeded in a 96 well plate and ratios of GSH/GSSG and NADPH/NADP+ were
determined using luminescence assays (GSH/GSSG-Glo™, Promega and NADP/NADPH-
Glo™, Promega) according to manufacturer’s instructions.
Cell cycle evaluation and visualization of adherence junctions
For cell cycle assay, cells were harvested by trypsin, stained by Hoechst 33342 (10 μg/ml) for
35 min at 37°C, 5% CO2 in normal culture media. After that pyronin Y was added to the final
concentration of 0.5 μg/ml for 15 min. 2x105 of stained cells were examined by flow
cytometry at a flow rate of 400 cells/s. Hoechst-low/pyronin Y-low cells were assigned to G0
phase of the cell cycle. For detection of adherence junctions, cells grown on coverslips were
permeabilized and blocked with 5% fetal bovine serum, 0.075% Tween-20, 0.075% Triton-
X100, 100 mM glycine (Serva, Heidelberg, Germany) in PBS for 1 h, incubated with anti-VE
cadherin (Santa Cruz, sc-9989) or anti-E-cadherin (Santa Cruz, sc-21791) primary antibodies
(1:100) for 1 h at room temperature, followed by 1 h incubation with anti-mouse Alexa Fluor
Blecha et al.: Selectivity of electron transport chain inhibition
11
647-conjugated antibody (Thermo Fisher, 1:1000). Samples were embedded in DAPI-
containing mowiol and imaged by the Leica TCS SP8 confocal microscope (Leica
Microsystems, Wetzlar, Germany). Images were deconvolved in Huygens Professional
software and processed in Fiji.
Statistical analysis
Data were analyzed using the GraphPad Prism 5.04 software, and are presented as mean
values ± S.E.M. of at least three independent experiments, unless indicated otherwise.
Statistical significance was determined using unpaired Student's t-test or ANOVA when
suitable; p < 0.05 was considered statistically significant.
RESULTS
Electron transport chain activity is increased in quiescent cells.
Increased ETC assembly in cancer cells has been linked to the susceptibility to ETC-
dependent cell death [5]. To understand if this relationship is maintained in non-transformed
quiescent cells, we first examined the ETC utilization and assembly in proliferating and
quiescent endothelial-derived Ea.hy926 and breast epithelia-derived MCF10A cells. To
investigate the effect of nutrient availability (which can be scarce for example in poorly-
perfused areas of tumors), the cells were grown in high (4.5 g/l) and low (1 g/l) glucose
media that are expected to represent situations of ample and limited glucose supply. Both
tested cell types undergo contact growth inhibition upon reaching confluence, as evidenced
by the increase of the G0 cell cycle subpopulation (Fig S1 A,B) and assembly of adherence
junctions (Fig. S1 C,D), and represent therefore a plausible experimental model of quiescence
induced by contact inhibition. Respiration measurements revealed that oxygen consumption
Blecha et al.: Selectivity of electron transport chain inhibition
12
was increased in quiescent cells in both cell types (Fig. 1A, B), suggesting that ETC activity
is elevated when cells become quiescent. On the other hand, lactate production and glucose
uptake were reduced in quiescent cells (Fig. 1C,D), indicating lower rate of glycolysis, in
agreement with previously-reported data [26-28]. We then performed a more detailed
analysis of the ETC in Ea.hy926 cells. Blue native electrophoresis (BNE) of mitochondria
isolated from proliferating and quiescent cells revealed more SCs in quiescent cells (Fig. 1E),
and in-gel activity measurements using high resolution clear native electrophoresis (hrCNE)
demonstrated that these SCs were enzymatically active (Fig. 1F). This suggests that the ETC
in quiescent cells is primed for increased performance, consistent with the proposed role of
respiratory SC [29, 30]. Finally, in situ examination by two photon microscopy documented
that mitochondrial NADH was significantly elevated upon addition of the CI inhibitor
rotenone in quiescent but not proliferating cells (Fig. 2 A,B). This points to increased
mitochondrial NADH production in quiescent cells, which is at baseline avidly consumed by
the ETC, but is substantially elevated when CI activity is suppressed by rotenone. In
summary, these results demonstrate that ETC gains prominence in quiescence. Consequently,
ETC blockade should have more profound consequences in quiescent cells than in
proliferating (i.e. cancer) cells.
Quiescent cells are protected from cell death induced by the ETC blockade when
glucose is not limiting.
While in cancer the dependence on oxidative phosphorylation and higher ETC SC assembly
correlate with sensitivity to ETC inhibition, in non-transformed cells this relationship has not
been rigorously tested. Importantly, ETC blockade may result in both the suppression of
Blecha et al.: Selectivity of electron transport chain inhibition
13
mitochondrial ATP generation due to an impaired proton-motive force, but also in the
stimulation of ROS production, due to accumulating electrons at the ETC complexes.
However, the relative importance of elevated ROS or ATP depletion in cell death induction
may vary depending on the cellular state (proliferating or quiescent), and on the nutritional
status (availability of glucose). We therefore exposed proliferating and quiescent Ea.hy926
and MCF10A cells to three distinct classes of agents. The first group consisted of compounds
that generate ROS without acute inhibition of the ETC, hydrogen peroxide and phenethyl-
isothiocyanate (PEITC) [31], (Fig. S2 A,B), expected to induce cell death solely in an ROS-
dependent manner. The second group comprised agents that block the ETC (Fig. S2 C-F), the
CI inhibitors rotenone, pierecidin A and MitoTam [5], the CII inhibitor MitoVES [4, 6], and
the CIII inhibitor myxothiazol. These compounds may either induce ROS via the ETC, but
may also suppress mitochondrial ATP synthesis. The third group contained agents that
specifically block mitochondrial ATP generation, the ATP synthase inhibitor oligomycin, and
mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Fig. S2 G,H).
When compared to quiescent cells, proliferating cells under high glucose conditions were
more susceptible to ROS inducers (Fig. 3 A,G) ETC inhibitors (Fig. 3 B,H), as well as to
oligomycin and CCCP (Fig 3 C,I). Under low glucose, this sensitivity pattern was maintained
only for pure ROS inducers (Fig. 3 D,J). Importantly, however, upon treatment with ETC
inhibitors (Fig. 3 E,K), oligomycin and CCCP (Fig. 3 F,L), quiescent cells became highly
susceptible to cell death induction. This suggests that the sensitivity of quiescent cells to ETC
and ATP synthase inhibitors, but not to pure ROS inducers, is significantly altered based on
the availability of glucose. Moreover, under conditions of ample glucose supply, quiescent
cells are much less sensitive to cell death induction by ROS inducers and inhibitors of the
ETC.
Blecha et al.: Selectivity of electron transport chain inhibition
14
ETC inhibition in quiescent cells leads to ATP depletion when ambient glucose is below
the physiological range.
To properly interpret the above results, we first assessed glucose levels at various time points
in the cell-conditioned media. This revealed that in low glucose media, the glucose
concentration is less than 20% of the initial 5.5 mM concentration (corresponding to less than
1 mM) at the time of the treatment (16 h after the media change), while in high glucose media
it was about half of the initial 25 mM concentration (10-15 mM) (Fig. 4 A). Thus, in low
glucose cultures the glucose concentration is significantly below physiological levels of well
perfused tissues (about 5 mM) [32], but in high glucose media such conditions of glucose
limitation are not reached. ATP production by glycolysis requires glucose, therefore
generation of ATP by oxidative phosphorylation may gain prominence when glucose is
limiting. For this reason, we assessed the consequences of ETC blockade on intracellular
ATP levels. To this end we exposed proliferating and quiescent cells to ETC inhibitors and
disruptors of mitochondrial ATP production described above, and assessed total cellular ATP
by a luminescence-based assay. While this treatment did not affect ATP levels in
proliferating or quiescent cells in high glucose media (Fig 4 B,C), it resulted in a severe
depletion of ATP in quiescent cells cultured in low glucose media (Fig. 4 D,E). This ATP
depletion correlated with enhanced cell death (cf. Fig. 3 E,F,K,L), suggesting that the
shortage of intracellular ATP is the defining factor for cell death in quiescent cells when
glucose is below the normal physiological range, but not when glucose is in ample supply. In
support of this notion, when quiescent cells were exposed to ETC inhibitors in media
containing the physiological glucose level (5.5 mM), depletion of intracellular ATP was not
observed (Fig. 4 F).
Blecha et al.: Selectivity of electron transport chain inhibition
15
Quiescent cells cannot compensate for the loss of mitochondrial ATP synthesis when
glucose is limiting.
ATP depletion in quiescent cells after inhibition of the ETC at sub-physiological glucose
levels suggests that quiescent cells may be unable to compensate for the defect in
mitochondrial ATP production when glucose is a limiting factor. As the observed ATP
depletion could result from the failure of quiescent cells to switch to glycolysis, we assessed
changes in the rate of glucose uptake upon ETC inhibition in Ea.hy926 cells to characterize
the possible compensatory regulation. Indeed, we found that upon ETC blockade the
proliferating cells increased their glucose uptake much more efficiently than quiescent cells
(Fig. 4 G). Glucose uptake is mediated by glucose transporters [33], therefore we next
assessed the level of GLUT1 using flow cytometry after immunostaining of non-
permeabilized cells. This analysis revealed that whereas ETC suppression significantly
increased the level of GLUT1 in proliferating cells, little or no increase was observed in
quiescent cells (Fig. 4 H). Hence, the reduced flexibility of glucose uptake in quiescent cells
renders these cells vulnerable to ETC blockade in glucose concentrations significantly below
the physiological range.
Generation of ROS upon ETC inhibition corresponds to cell death when glucose is not
limiting.
Having established that factors other than ATP depletion determine sensitivity to cell death in
quiescent cells at physiological glucose concentrations, we next assessed the relevance of
increased ROS as another major consequence of ETC blockade [4, 11, 12]. We used flow
Blecha et al.: Selectivity of electron transport chain inhibition
16
cytometry and 2’,7’-dichlorofluorescin diacetate (DCF) to assess the amount of ROS
produced upon the administration of tested compounds to proliferating and quiescent
Ea.hy926 and MCF10A cells and compared these results to cell death levels presented in Fig.
3. For ROS inducers hydrogen peroxide and PEITC, the magnitude of ROS generation
correlated with the cells’ sensitivity to cell death both in high (Fig. 5 A,G) and low (Fig. 5
D,J) glucose conditions. In contrast, for ETC inhibitors oligomycin and CCCP the correlation
between ROS generation and cell death was found only under high glucose media (Fig. 5
B,C,H,I), and this was confirmed by additional ROS probes dihydrorhodamine (DHR123)
and dihydroethidium (DHE) (Fig. S3). However, no clear correlation between cell death and
ROS was apparent under low glucose conditions (Fig. 5 E,F,K,L). These results support the
notion that the level of formed ROS determines the level of cell death induced by ETC
inhibition when glucose is not limiting. In addition, lower ROS in quiescent cells, despite
their more active ETC, suggests that these cells can neutralize ROS more efficiently.
Antioxidant defense is upregulated in quiescent cells.
To further elaborate why quiescent cells produce less ROS upon ETC inhibition and why
they are protected from cell death despite their more active ETC, we investigated the status of
relevant antioxidant defense components. In mitochondria, this includes SOD2 that detoxifies
superoxide, and glutathione and thioredoxin systems that inactivate hydrogen peroxide [14,
15]. In Ea.hy926 cells, the expression of mitochondrial antioxidant defense proteins SOD2
and glutathione peroxidase-1 (GPX1) was elevated in quiescent cells, while peroxiredoxin-3
(PRX3), thioredoxin-2 (TRX2) were not significantly changed, irrespective of glucose
concentration (Fig. 6 A). Cytoplasmic catalase (CAT) and superoxide dismutase 1 (SOD1)
Blecha et al.: Selectivity of electron transport chain inhibition
17
were slightly increased in proliferating cells (Fig. 6 A). This was corroborated by in-gel
activity assays for superoxide dismutase, glutathione peroxidase and catalase (Fig. 6 C,
quantified in Fig. 6 E), showing that the enzymatic activity of SOD2 and glutathione
peroxidase was significantly increased in quiescent cells. In MCF10A cells, the protein
expression of antioxidant defense components was similar in proliferating and quiescent cells
both in high and low glucose (Fig. 6 B), but the in-gel activity assays revealed that the
enzymatic activity of GPX and SOD2, but not that of SOD1 or catalase, is increased in
quiescent cells (Fig. 6 D, quantified in Fig. 6 E). This was again independent of glucose
concentration. Furthermore, the ratio of reduced to oxidized glutathione was reduced and
NADPH/NADP ratios were increased in quiescent cells (Fig. 6 E,F). Collectively, this
suggests that the mitochondrial antioxidant defense is more active in quiescent cells and these
cells are well equipped to deal with the oxidative insult induced by ETC inhibition.
Antioxidant defense is functionally relevant in protection of quiescent cells from ETC
blockade-induced cell death.
To evaluate the functional relevance of the elevated antioxidant defense for the protection of
quiescent cells from ETC-induced cell death under the condition where glucose is not
limiting, we used lentiviral delivery of shRNA to suppress its individual protein components.
We performed these experiments in Ea.hy926 cells, as the downregulation of antioxidant
defense components was only poorly tolerated by MCF10A cells. SOD2 controls
mitochondrial superoxide, which is the major ROS species produced when ETC is blocked
[11]. Downregulation of SOD2 by a specific shRNA resulted in a significantly increased cell
death upon treatment of quiescent cells with ETC inhibitors, while little or no effect of SOD2
Blecha et al.: Selectivity of electron transport chain inhibition
18
knockdown was observed in proliferating cells (Fig. 7 A). This was accompanied by
increased ROS production in quiescent cells, as assessed both by dihydroethidium (DHE), a
fluorescent probe that preferentially detects superoxide, and by DCF, a more general
oxidative stress indicator (Fig. 7 B,C). We then knocked down GPX1, which inactivates
hydrogen peroxide in mitochondria in a glutathione-dependent manner. GPX1 knock-down
had much less effect on cell death upon application of ETC inhibitors in quiescent cells than
SOD2 knockdown (Fig. 7 D), but resulted in an increased ROS signal (Fig. 7 E). This
indicates that in GPX1 knock-down cells, ROS is uncoupled from cell death. Similar to
GPX1, knock-down of glutathione reductase (GSR), a glutathione recycling enzyme, did not
significantly increase cell death in quiescent cells (Fig. 7 F), and the effect on ROS was
moderate (Fig. 7 G). Finally, interference with thioredoxin reductase 2 (TXNRD2), a
mitochondrial thioredoxin recycling enzyme [34], significantly increased cell death (Fig. 7
H), and also increased ROS generation (Fig. 7 I). In line with the shRNA data, thioredoxin
reductase inhibitor auranofin-dependent cell death was increased in quiescent cells treated
with ETC inhibitors more than in proliferating cells (Fig. 7 J).
To complement the above results, we also took an inverse approach and boosted the available
antioxidant capacity by administration of the antioxidant N-acetyl cysteine (NAC). While
little or no effect of this treatment was observed in quiescent cells, a significant reduction in
cell death was detected in proliferating cells (Fig. 7 K). Importantly, in low glucose media the
knock downs of antioxidant enzymes described above did not affect cell death in quiescent
cells (Fig. S4 A-D), in line with the notion that ROS are not the causal agents of cell death in
that situation. Collectively, these results demonstrate that antioxidant defense is functionally
relevant in protecting quiescent cells from cell death, and determines to a large degree their
sensitivity to ETC inhibition in a situation when cells are not affected by nutrient limitation.
Blecha et al.: Selectivity of electron transport chain inhibition
19
DISCUSSION
Targeting of the ETC holds promise for cancer therapy and has demonstrated its efficacy and
low toxicity in multiple pre-clinical cancer models [5, 6, 8]. Considerable effort has been
invested in uncovering the rules that govern susceptibility of cancer cells to ETC inhibition-
induced cell death and a direct correlation has been found between oxygen consumption,
assembly of ETC SCs and sensitivity to ETC disruption [5]. On the other hand, the question
why these ETC disrupting agents are well tolerated on the organismal level and in non-
cancerous cells has received much less attention, even though it is of high importance for
efficient and save cancer therapy. For this reason, the key finding of the present study is that
the elevated antioxidant defense in non-proliferating cells ensures resistance to ETC
inhibition in a situation when glucose is not limiting.
Most somatic cells in an organisms are in a quiescent, post-mitotic state, and features
associated with quiescence may play an important role in resistance to ETC inhibition, as
suggested by our previous work [20, 21]. In the current study we found that quiescent cells
feature increased respiration, elevated SC assembly and mitochondria that produce more
NADH, consistent with fully active TCA cycle [26]. Apparently, the ETC gains prominence
in quiescence, and quiescent non-transformed cells should be susceptible to cell death
induced by ETC inhibition, resulting either from increased ROS generation or mitochondrial
ATP loss (discussed below). Elevated NADH is expected to stimulate ROS generation from
the ETC [11]. Indeed, NADH accumulation is efficiently induced in mitochondria of
quiescent cells upon ETC blockade.
Blecha et al.: Selectivity of electron transport chain inhibition
20
Notwithstanding the above reasoning, inhibition of ETC under ample glucose supply leads to
reduced ROS production and reduced cell death in quiescent cells when compared to
proliferating cells, suggesting that ROS detoxification is increased in quiescent cells as
proposed previously [35-37]. Data presented herein point to ROS, and not to ATP depletion,
being the dominant factor in cell death induction upon ETC inhibition in quiescent cells when
glucose is not limiting, and imply that ROS detoxification, not the ETC status or increased
ETC-mediated ATP production, determine sensitivity to cell death in quiescent cells. This is
in direct contrast to cancer cells, where correlation between ETC activity/assembly and the
efficacy of ETC inhibitors to induce cell death has been established in multiple studies [4, 5,
17, 18, 38, 39]. Furthermore, it is in line with reports showing that redox modulation may be
an effective anti-cancer strategy [31, 40].
Pharmacological and genetic manipulations of SOD2 and TXNRD2, the mitochondrial
thioredoxin reductase [34], revealed that the SOD2 - thioredoxin axis plays a principal role in
efficient protection of quiescent cells from ETC blockade-induced ROS and cell death. In
contrast, interference with the glutathione system (GPX1 and GSR) led to some elevation of
ROS upon ETC blockade in quiescent cells without increase in cell death, suggesting that this
system plays a less important role in quiescent cells. This is consistent with the notion that
superoxide is the primary species of ROS-induced damage, possibly leading to direct
oxidation of various mitochondrial components [41-43]. The thioredoxin system may counter
this oxidative damage, consistent with its role in the regulation of cell death [14, 44-46]. In
agreement, deletion of SOD2, TRX2 or TXNRD2 in mice is incompatible with life [47-49],
while genetic inactivation of GSR or GPX1 is not associated with lethality [50, 51].
The sensitivities of proliferating vs. quiescent cells to ETC inhibition change dramatically in
the conditions of glucose limitation. Here, a substantial decrease of cellular ATP upon ETC
Blecha et al.: Selectivity of electron transport chain inhibition
21
blockade in quiescent cells was observed, which correlates with increased cell death.
Downregulation of antioxidant defense components did not increase cell death any further,
suggesting that cell death in this situation is largely ROS-independent, instead relating to
acute bioenergetic stress. In contrast to quiescent cells, proliferating cells strongly upregulate
glucose uptake upon ETC suppression by increasing the level of glucose transporters, which
likely compensates for the loss of ATP production in mitochondria. This effect may be
particularly important for scavenging glucose when its availability is low, and may explain
why quiescent cells are more sensitive to ETC inhibition when glucose is below its normal
physiological range. A similar mechanism has been reported in cancer cells, where the failure
to upregulate glucose transporters and glucose uptake sensitized to ETC inhibition only when
glucose availability was limiting [52].
To interpret the results of this study, it is important to realize that the high and low glucose
media represent situations of ample and limited glucose supply. In low glucose media, the
glucose concentrations found in our system within the time frame of the experiment are much
lower than those that are regularly encountered in vivo in well perfused tissues [32, 53]. In
fact, when ETC inhibitors were applied in fresh low (1 g/l) glucose media corresponding to
the physiological 5 mM glucose concentration, no ATP depletion was observed. This means
that in normal healthy tissues where perfusion is intact, glucose does not present a limiting
factor, and it is expected that effective inactivation of ETC blockade-induced ROS by
antioxidant defense will play the primary role in governing sensitivity. On the other hand, in
poorly perfused regions, such as hypoxic areas of tumors where the availability of nutrients is
low, the application of ETC inhibitors may significantly accelerate cell death in a ROS-
independent manner. This is consistent with reports of low glucose availability in tumors [53-
55], and with previous findings showing that ETC inhibition was highly effective in non-
Blecha et al.: Selectivity of electron transport chain inhibition
22
perfused hypoxic regions [56]. We therefore propose that the mechanism for the observed
selectivity of ETC inhibition in cancer is two-fold. First, elevated antioxidant defense limits
ROS production upon ETC inhibition in somatic quiescent cells that do not suffer from
glucose limitation, the large majority of cells in an organism. Second, in poorly perfused
tumors where glucose is limiting, the application of ETC inhibitors will lead to bioenergetic
catastrophe, amplifying the ROS-related effects, and broadening effectivity to cancer cells
that might have a ‘quiescent’-like phenotype [17].
The mechanism discussed above is not exhaustive, particularly because not all somatic cells
in the body are quiescent. In addition, factors specific to each ETC inhibitor, such as its
pharmacological properties, blood-brain barrier penetrance, specific accumulation in tumor
tissues and off-target effects will also be important, and so will be the rate of ROS production
induced by the inhibitor for the given level of ETC suppression. Consistent with this, we
propose that ETC inhibitors that induce maximal ROS will kill proliferating cancer cells most
efficiently. Yet, this study introduces a general concept that is relevant to all ETC-targeted
agents that produce ROS, and describes the molecular mechanisms with the potential to
contribute to the specificity observed [5, 19]. To conclude, ETC targeting represent a new,
promising approach for treatment of multiple cancer types, including neoplasias that are
difficult-to-treat, and resistant populations of cancer cells within tumors [5, 16, 18, 38]. As
such, this study is relevant for future development and optimization of very interesting, novel
anti-cancer agents that target the ETC and present potential broad-spectrum therapeutics for a
range of malignancies.
CONCLUSION
Blecha et al.: Selectivity of electron transport chain inhibition
23
In summary, we propose that specificity of ETC inhibition in cancer is in part conferred by
the increased antioxidant defense in non-proliferating non-transformed cells. ROS production
from the ETC is the dominant factor that determines susceptibility to cell death induced by
ETC disruption in a situation when glucose is not limiting. Hence, ETC inhibitors that induce
maximal ROS will kill proliferating cancer cells most efficiently while maintaining
specificity. On the other hand, in a situation of glucose limitation characteristic for tumors,
ATP depletion upon ETC disruption gains prominence, and non-proliferating cells become
highly susceptible to cell death because they are unable to upregulate glucose uptake. This
will supplement the ROS-related effects and optimize impact towards cancer, including
cancer cells with a dormant-like phenotype.
FUNDING SOURCES
This work was supported by Czech Science Foundation [grants no. 16-22823S, 17-20904S,
16-12719S], Czech Health Research Council [grant no. 16-31604A], Grant Agency of
Charles University [grant no. GAUK 98215], ERDF and MEYS CR [CZ.1.05/1.1.00/02.0109
BIOCEV, LQ1604 NPU II], institutional support from Czech Academy of Sciences [RVO:
86652036]. Imaging Methods Core Facility at BIOCEV is supported by large infrastructure
Czech-BioImaging project provided by MEYS [LM2015062]. Funders had no involvement in
study design, the collection, analysis or interpretation of data.
ACKNOWLEDGEMENTS
Blecha et al.: Selectivity of electron transport chain inhibition
24
We would like to thank the Imaging Methods Core Facility at BIOCEV, Faculty of Sciences,
Charles University, for their support with obtaining imaging and flow cytometry data
presented in this paper.
AUTHOR’S DISCLOSURE STATEMENT
J.N. is an inventor of a patent ‘Tamoxifen analogues for treatment of neoplastic diseases,
especially with high Her2 protein level’. The authors declare no additional competing
financial interests.
REFERENCES
[1] A.S. Tan, J.W. Baty, L.F. Dong, A. Bezawork-Geleta, B. Endaya, J. Goodwin, M.
Bajzikova, J. Kovarova, M. Peterka, B. Yan, E.A. Pesdar, M. Sobol, A. Filimonenko, S.
Stuart, M. Vondrusova, K. Kluckova, K. Sachaphibulkij, J. Rohlena, P. Hozak, J. Truksa, D.
Eccles, L.M. Haupt, L.R. Griffiths, J. Neuzil, M.V. Berridge, Mitochondrial genome
acquisition restores respiratory function and tumorigenic potential of cancer cells without
mitochondrial DNA, Cell Metab. 21(1) (2015) 81-94.
[2] F. Weinberg, R. Hamanaka, W.W. Wheaton, S. Weinberg, J. Joseph, M. Lopez, B.
Kalyanaraman, G.M. Mutlu, G.R. Budinger, N.S. Chandel, Mitochondrial metabolism and
ROS generation are essential for Kras-mediated tumorigenicity, Proc. Natl. Acad. Sci. U. S.
A. 107(19) (2010) 8788-93.
[3] L.F. Dong, J. Kovarova, M. Bajzikova, A. Bezawork-Geleta, D. Svec, B. Endaya, K.
Sachaphibulkij, A.R. Coelho, N. Sebkova, A. Ruzickova, A.S. Tan, K. Kluckova, K.
Judasova, K. Zamecnikova, Z. Rychtarcikova, V. Gopalan, L. Andera, M. Sobol, B. Yan, B.
Pattnaik, N. Bhatraju, J. Truksa, P. Stopka, P. Hozak, A.K. Lam, R. Sedlacek, P.J. Oliveira,
M. Kubista, A. Agrawal, K. Dvorakova-Hortova, J. Rohlena, M.V. Berridge, J. Neuzil,
Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial
DNA-deficient cancer cells, Elife 6 (2017).
[4] K. Kluckova, M. Sticha, J. Cerny, T. Mracek, L. Dong, Z. Drahota, E. Gottlieb, J. Neuzil,
J. Rohlena, Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II
in cell death initiation, Cell Death Dis. 6 (2015) e1749.
Blecha et al.: Selectivity of electron transport chain inhibition
25
[5] K. Rohlenova, K. Sachaphibulkij, J. Stursa, A. Bezawork-Geleta, J. Blecha, B. Endaya, L.
Werner, J. Cerny, R. Zobalova, J. Goodwin, T. Spacek, E. Alizadeh Pesdar, B. Yan, M.N.
Nguyen, M. Vondrusova, M. Sobol, P. Jezek, P. Hozak, J. Truksa, J. Rohlena, L.F. Dong, J.
Neuzil, Selective disruption of respiratory supercomplexes as a new strategy to suppress
Her2high breast cancer, Antioxid. Redox. Signal. 26(2) (2017) 84-103.
[6] L.F. Dong, V.J. Jameson, D. Tilly, J. Cerny, E. Mahdavian, A. Marin-Hernandez, L.
Hernandez-Esquivel, S. Rodriguez-Enriquez, J. Stursa, P.K. Witting, B. Stantic, J. Rohlena, J.
Truksa, K. Kluckova, J.C. Dyason, M. Ledvina, B.A. Salvatore, R. Moreno-Sanchez, M.J.
Coster, S.J. Ralph, R.A. Smith, J. Neuzil, Mitochondrial targeting of vitamin E succinate
enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II, J. Biol.
Chem. 286(5) (2011) 3717-28.
[7] J. Rohlena, L.F. Dong, S.J. Ralph, J. Neuzil, Anticancer drugs targeting the mitochondrial
electron transport chain, Antioxid. Redox. Signal. 15(12) (2011) 2951-74.
[8] G. Cheng, J. Zielonka, O. Ouari, M. Lopez, D. McAllister, K. Boyle, C.S. Barrios, J.J.
Weber, B.D. Johnson, M. Hardy, M.B. Dwinell, B. Kalyanaraman, Mitochondria-targeted
analogues of metformin exhibit enhanced antiproliferative and radiosensitizing effects in
pancreatic cancer cells, Cancer Res. 76(13) (2016) 3904-15.
[9] H. Schagger, K. Pfeiffer, Supercomplexes in the respiratory chains of yeast and
mammalian mitochondria, EMBO J. 19(8) (2000) 1777-83.
[10] R. Acin-Perez, P. Fernandez-Silva, M.L. Peleato, A. Perez-Martos, J.A. Enriquez,
Respiratory active mitochondrial supercomplexes, Mol. Cell 32(4) (2008) 529-39.
[11] M.P. Murphy, How mitochondria produce reactive oxygen species, Biochem. J. 417(1)
(2009) 1-13.
[12] M.D. Brand, Mitochondrial generation of superoxide and hydrogen peroxide as the
source of mitochondrial redox signaling, Free Radic. Biol. Med. 100 (2016) 14-31.
[13] T. Rabilloud, M. Heller, M.P. Rigobello, A. Bindoli, R. Aebersold, J. Lunardi, The
mitochondrial antioxidant defence system and its response to oxidative stress, Proteomics
1(9) (2001) 1105-10.
[14] A.G. Cox, C.C. Winterbourn, M.B. Hampton, Mitochondrial peroxiredoxin involvement
in antioxidant defence and redox signalling, Biochem. J. 425(2) (2009) 313-25.
[15] M. Mari, A. Morales, A. Colell, C. Garcia-Ruiz, J.C. Fernandez-Checa, Mitochondrial
glutathione, a key survival antioxidant, Antioxid. Redox. Signal. 11(11) (2009) 2685-700.
[16] R. Haq, J. Shoag, P. Andreu-Perez, S. Yokoyama, H. Edelman, G.C. Rowe, D.T.
Frederick, A.D. Hurley, A. Nellore, A.L. Kung, J.A. Wargo, J.S. Song, D.E. Fisher, Z. Arany,
Blecha et al.: Selectivity of electron transport chain inhibition
26
H.R. Widlund, Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF,
Cancer Cell 23(3) (2013) 302-15.
[17] A. Viale, P. Pettazzoni, C.A. Lyssiotis, H. Ying, N. Sanchez, M. Marchesini, A. Carugo,
T. Green, S. Seth, V. Giuliani, M. Kost-Alimova, F. Muller, S. Colla, L. Nezi, G. Genovese,
A.K. Deem, A. Kapoor, W. Yao, E. Brunetto, Y. Kang, M. Yuan, J.M. Asara, Y.A. Wang,
T.P. Heffernan, A.C. Kimmelman, H. Wang, J.B. Fleming, L.C. Cantley, R.A. DePinho, G.F.
Draetta, Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial
function, Nature 514(7524) (2014) 628-32.
[18] A. Roesch, A. Vultur, I. Bogeski, H. Wang, K.M. Zimmermann, D. Speicher, C. Korbel,
M.W. Laschke, P.A. Gimotty, S.E. Philipp, E. Krause, S. Patzold, J. Villanueva, C. Krepler,
M. Fukunaga-Kalabis, M. Hoth, B.C. Bastian, T. Vogt, M. Herlyn, Overcoming intrinsic
multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-
cycling JARID1B(high) cells, Cancer Cell 23(6) (2013) 811-25.
[19] L.F. Dong, V.J. Jameson, D. Tilly, L. Prochazka, J. Rohlena, K. Valis, J. Truksa, R.
Zobalova, E. Mahdavian, K. Kluckova, M. Stantic, J. Stursa, R. Freeman, P.K. Witting, E.
Norberg, J. Goodwin, B.A. Salvatore, J. Novotna, J. Turanek, M. Ledvina, P. Hozak, B.
Zhivotovsky, M.J. Coster, S.J. Ralph, R.A. Smith, J. Neuzil, Mitochondrial targeting of
alpha-tocopheryl succinate enhances its pro-apoptotic efficacy: a new paradigm for effective
cancer therapy, Free Radic. Biol. Med. 50(11) (2011) 1546-55.
[20] J. Rohlena, L.F. Dong, K. Kluckova, R. Zobalova, J. Goodwin, D. Tilly, J. Stursa, A.
Pecinova, A. Philimonenko, P. Hozak, J. Banerjee, M. Ledvina, C.K. Sen, J. Houstek, M.J.
Coster, J. Neuzil, Mitochondrially targeted alpha-tocopheryl succinate is antiangiogenic:
potential benefit against tumor angiogenesis but caution against wound healing, Antioxid.
Redox. Signal. 15(12) (2011) 2923-35.
[21] L.F. Dong, E. Swettenham, J. Eliasson, X.F. Wang, M. Gold, Y. Medunic, M. Stantic, P.
Low, L. Prochazka, P.K. Witting, J. Turanek, E.T. Akporiaye, S.J. Ralph, J. Neuzil, Vitamin
E analogues inhibit angiogenesis by selective induction of apoptosis in proliferating
endothelial cells: the role of oxidative stress, Cancer Res. 67(24) (2007) 11906-13.
[22] J. Truksa, L.F. Dong, J. Rohlena, J. Stursa, M. Vondrusova, J. Goodwin, M. Nguyen, K.
Kluckova, Z. Rychtarcikova, S. Lettlova, J. Spacilova, M. Stapelberg, M. Zoratti, J. Neuzil,
Mitochondrially targeted vitamin E succinate modulates expression of mitochondrial DNA
transcripts and mitochondrial biogenesis, Antioxid. Redox. Signal. 22(11) (2015) 883-900.
[23] S. Schmitt, F. Saathoff, L. Meissner, E.M. Schropp, J. Lichtmannegger, S. Schulz, C.
Eberhagen, S. Borchard, M. Aichler, J. Adamski, N. Plesnila, S. Rothenfusser, G. Kroemer,
H. Zischka, A semi-automated method for isolating functionally intact mitochondria from
cultured cells and tissue biopsies, Anal. Biochem. 443(1) (2013) 66-74.
Blecha et al.: Selectivity of electron transport chain inhibition
27
[24] M. Vondrusova, A. Bezawork-Geleta, K. Sachaphibulkij, J. Truksa, J. Neuzil, The effect
of mitochondrially targeted anticancer agents on mitochondrial (super)complexes, Methods
Mol. Biol. 1265 (2015) 195-208.
[25] C.J. Weydert, J.J. Cullen, Measurement of superoxide dismutase, catalase and
glutathione peroxidase in cultured cells and tissue, Nat. Protoc. 5(1) (2010) 51-66.
[26] J.M. Lemons, X.J. Feng, B.D. Bennett, A. Legesse-Miller, E.L. Johnson, I. Raitman,
E.A. Pollina, H.A. Rabitz, J.D. Rabinowitz, H.A. Coller, Quiescent fibroblasts exhibit high
metabolic activity, PLoS Biol. 8(10) (2010) e1000514.
[27] S. Schoors, K. De Bock, A.R. Cantelmo, M. Georgiadou, B. Ghesquiere, S.
Cauwenberghs, A. Kuchnio, B.W. Wong, A. Quaegebeur, J. Goveia, F. Bifari, X. Wang, R.
Blanco, B. Tembuyser, I. Cornelissen, A. Bouche, S. Vinckier, S. Diaz-Moralli, H. Gerhardt,
S. Telang, M. Cascante, J. Chesney, M. Dewerchin, P. Carmeliet, Partial and transient
reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis, Cell Metab.
19(1) (2014) 37-48.
[28] I. Diamond, A. Legg, J.A. Schneider, E. Rozengurt, Glycolysis in quiescent cultures of
3T3 cells. Stimulation by serum, epidermal growth factor, and insulin in intact cells and
persistence of the stimulation after cell homogenization, J. Biol. Chem. 253(3) (1978) 866-
71.
[29] E. Maranzana, G. Barbero, A.I. Falasca, G. Lenaz, M.L. Genova, Mitochondrial
respiratory supercomplex association limits production of reactive oxygen species from
complex I, Antioxid. Redox. Signal. 19(13) (2013) 1469-80.
[30] J.A. Letts, K. Fiedorczuk, L.A. Sazanov, The architecture of respiratory supercomplexes,
Nature 537(7622) (2016) 644-648.
[31] D. Trachootham, Y. Zhou, H. Zhang, Y. Demizu, Z. Chen, H. Pelicano, P.J. Chiao, G.
Achanta, R.B. Arlinghaus, J. Liu, P. Huang, Selective killing of oncogenically transformed
cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate, Cancer Cell
10(3) (2006) 241-52.
[32] M.Z. Shrayyef, J.E. Gerich, Normal glucose homeostasis, in: L. Poretsky (Ed.),
Principles of diabetes mellitus, Springer US, Boston, MA, 2010, pp. 19-35.
[33] M. Mueckler, B. Thorens, The SLC2 (GLUT) family of membrane transporters, Mol.
Aspects Med. 34(2-3) (2013) 121-38.
[34] S.R. Lee, J.R. Kim, K.S. Kwon, H.W. Yoon, R.L. Levine, A. Ginsburg, S.G. Rhee,
Molecular cloning and characterization of a mitochondrial selenocysteine-containing
thioredoxin reductase from rat liver, J. Biol. Chem. 274(8) (1999) 4722-34.
Blecha et al.: Selectivity of electron transport chain inhibition
28
[35] G.J. Kops, T.B. Dansen, P.E. Polderman, I. Saarloos, K.W. Wirtz, P.J. Coffer, T.T.
Huang, J.L. Bos, R.H. Medema, B.M. Burgering, Forkhead transcription factor FOXO3a
protects quiescent cells from oxidative stress, Nature 419(6904) (2002) 316-21.
[36] E.H. Sarsour, S. Venkataraman, A.L. Kalen, L.W. Oberley, P.C. Goswami, Manganese
superoxide dismutase activity regulates transitions between quiescent and proliferative
growth, Aging Cell 7(3) (2008) 405-17.
[37] J. Naderi, M. Hung, S. Pandey, Oxidative stress-induced apoptosis in dividing
fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of
quiescent cells to oxidative stress, Apoptosis 8(1) (2003) 91-100.
[38] B. Yan, M. Stantic, R. Zobalova, A. Bezawork-Geleta, M. Stapelberg, J. Stursa, K.
Prokopova, L. Dong, J. Neuzil, Mitochondrially targeted vitamin E succinate efficiently kills
breast tumour-initiating cells in a complex II-dependent manner, BMC Cancer 15 (2015) 401.
[39] S. Boukalova, J. Stursa, L. Werner, Z. Ezrova, J. Cerny, A. Bezawork-Geleta, A.
Pecinova, L. Dong, Z. Drahota, J. Neuzil, Mitochondrial targeting of metformin enhances its
activity against pancreatic cancer, Mol. Cancer Ther. 15(12) (2016) 2875-2886.
[40] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated
mechanisms: a radical therapeutic approach?, Nat. Rev. Drug Discov. 8(7) (2009) 579-91.
[41] Z. Dong, S. Shanmughapriya, D. Tomar, N. Siddiqui, S. Lynch, N. Nemani, S.L. Breves,
X. Zhang, A. Tripathi, P. Palaniappan, M.F. Riitano, A.M. Worth, A. Seelam, E. Carvalho, R.
Subbiah, F. Jana, J. Soboloff, Y. Peng, J.Y. Cheung, S.K. Joseph, J. Caplan, S. Rajan, P.B.
Stathopulos, M. Madesh, Mitochondrial Ca2+ uniporter is a mitochondrial luminal redox
sensor that augments MCU channel activity, Mol. Cell 65(6) (2017) 1014-1028 e7.
[42] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N.
Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, Vlasova, II, Q. Zhao, M. Zou, P. Di, D.A.
Svistunenko, I.V. Kurnikov, G.G. Borisenko, Cytochrome c acts as a cardiolipin oxygenase
required for release of proapoptotic factors, Nat. Chem. Biol. 1(4) (2005) 223-32.
[43] M. Ott, J.D. Robertson, V. Gogvadze, B. Zhivotovsky, S. Orrenius, Cytochrome c
release from mitochondria proceeds by a two-step process, Proc. Natl. Acad. Sci. U. S. A.
99(3) (2002) 1259-63.
[44] K.K. Brown, S.E. Eriksson, E.S. Arner, M.B. Hampton, Mitochondrial peroxiredoxin 3
is rapidly oxidized in cells treated with isothiocyanates, Free Radic. Biol. Med. 45(4) (2008)
494-502.
[45] A.G. Cox, K.K. Brown, E.S. Arner, M.B. Hampton, The thioredoxin reductase inhibitor
auranofin triggers apoptosis through a Bax/Bak-dependent process that involves
peroxiredoxin 3 oxidation, Biochem. Pharmacol. 76(9) (2008) 1097-109.
Blecha et al.: Selectivity of electron transport chain inhibition
29
[46] Y. Chen, J. Cai, D.P. Jones, Mitochondrial thioredoxin in regulation of oxidant-induced
cell death, FEBS Lett. 580(28-29) (2006) 6596-602.
[47] M. Conrad, C. Jakupoglu, S.G. Moreno, S. Lippl, A. Banjac, M. Schneider, H. Beck,
A.K. Hatzopoulos, U. Just, F. Sinowatz, W. Schmahl, K.R. Chien, W. Wurst, G.W.
Bornkamm, M. Brielmeier, Essential role for mitochondrial thioredoxin reductase in
hematopoiesis, heart development, and heart function, Mol. Cell. Biol. 24(21) (2004) 9414-
23.
[48] L. Nonn, R.R. Williams, R.P. Erickson, G. Powis, The absence of mitochondrial
thioredoxin 2 causes massive apoptosis, exencephaly, and early embryonic lethality in
homozygous mice, Mol. Cell. Biol. 23(3) (2003) 916-22.
[49] Y. Li, T.T. Huang, E.J. Carlson, S. Melov, P.C. Ursell, J.L. Olson, L.J. Noble, M.P.
Yoshimura, C. Berger, P.H. Chan, D.C. Wallace, C.J. Epstein, Dilated cardiomyopathy and
neonatal lethality in mutant mice lacking manganese superoxide dismutase, Nat. Genet. 11(4)
(1995) 376-81.
[50] L.K. Rogers, T. Tamura, B.J. Rogers, S.E. Welty, T.N. Hansen, C.V. Smith, Analyses of
glutathione reductase hypomorphic mice indicate a genetic knockout, Toxicol. Sci. 82(2)
(2004) 367-73.
[51] Y.S. Ho, J.L. Magnenat, R.T. Bronson, J. Cao, M. Gargano, M. Sugawara, C.D. Funk,
Mice deficient in cellular glutathione peroxidase develop normally and show no increased
sensitivity to hyperoxia, J. Biol. Chem. 272(26) (1997) 16644-51.
[52] K. Birsoy, R. Possemato, F.K. Lorbeer, E.C. Bayraktar, P. Thiru, B. Yucel, T. Wang,
W.W. Chen, C.B. Clish, D.M. Sabatini, Metabolic determinants of cancer cell sensitivity to
glucose limitation and biguanides, Nature 508(7494) (2014) 108-12.
[53] T.B. Rodrigues, E.M. Serrao, B.W. Kennedy, D.E. Hu, M.I. Kettunen, K.M. Brindle,
Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose,
Nat. Med. 20(1) (2014) 93-7.
[54] A. Hirayama, K. Kami, M. Sugimoto, M. Sugawara, N. Toki, H. Onozuka, T. Kinoshita,
N. Saito, A. Ochiai, M. Tomita, H. Esumi, T. Soga, Quantitative metabolome profiling of
colon and stomach cancer microenvironment by capillary electrophoresis time-of-flight mass
spectrometry, Cancer Res. 69(11) (2009) 4918-25.
[55] J. Goveia, A. Pircher, L.C. Conradi, J. Kalucka, V. Lagani, M. Dewerchin, G. Eelen, R.J.
DeBerardinis, I.D. Wilson, P. Carmeliet, Meta-analysis of clinical metabolic profiling studies
in cancer: challenges and opportunities, EMBO Mol. Med. 8(10) (2016) 1134-1142.
[56] X. Zhang, M. Fryknas, E. Hernlund, W. Fayad, A. De Milito, M.H. Olofsson, V.
Gogvadze, L. Dang, S. Pahlman, L.A. Schughart, L. Rickardson, P. D'Arcy, J. Gullbo, P.
Nygren, R. Larsson, S. Linder, Induction of mitochondrial dysfunction as a strategy for
Blecha et al.: Selectivity of electron transport chain inhibition
30
targeting tumour cells in metabolically compromised microenvironments, Nat. Commun. 5
(2014) 3295.
Blecha et al.: Selectivity of electron transport chain inhibition
31
Fig. 1. Electron transport chain activity and assembly is enhanced in quiescent cells.
(A, B) Oxygen consumption rate (OCR, pMoles/min) profiles in (A) Ea.hy926 (B) and
MCF10A cells in both low glucose (1 g/l) and high glucose (4.5 g/l) was measured using
Seahorse Bioscience XF24 extracellular flux analyzer. Data correlated to DNA content are
present as means ± S.E.M. from at least three independent experiments. (C) Excreted lactate
was measured in the culture medium after 19 hours cultivation. Data represent means ±
S.E.M. of 5 independent experiments, and are correlated to cell number. The symbol *
indicates values significantly different between proliferating and quiescent cells. (D) Glucose
consumption of Ea.hy926 and MCF10A cells was measured upon 24 h pre-incubation in low
glucose media (1 g/l glucose) by flow cytometry using 2-NBDG. Data are correlated to
quiescent Ea.hy926 cells in high glucose condition. Data represent means ± S.E.M., n≥4. The
symbol * indicates values significantly different between proliferating and quiescent cells.
(E) Isolated mitochondrial fraction were probed for the presence of SCs by WB following
BNE with the following antibodies: CI, NDUFB8; CII, SDHA; CIII, MTCO2; CIV,
UQCRFS1; and CV, ATP5A; mtHSP70 was used as loading control. A representative
experiment is shown. (F) In-gel assessment of enzymatic activity of respiratory SCs was
performed in isolated mitochondrial fraction following hrCNE as follows: CI - NADH:NTB
reductase, CII - succinate:NTB reductase, CIII - DAB oxidase, CIV- DAB:cytochrome c
reductase, CV- ATP hydrolase. A representative experiment is shown.
Fig. 2. In-situ measurements of mitochondrial NAD(P)H indicate increased NADH
utilization by the ETC in quiescent cells. (A) Mitochondrial NAD(P)H signal was
measured by two-photon microscopy in control, CCCP (5μM) or rotenone (1.25 μM)-treated
Blecha et al.: Selectivity of electron transport chain inhibition
32
cells. Intensity is color-coded as shown; note the increased NAD(P)H signal in mitochondria
of rotenone-treated quiescent cells. Regions marked by yellow rectangles are magnified for
clarity. Scale bars indicate 10 μm. Representative images are shown. (B) Average values of
NAD(P)H signal quantified in Fiji as described in ‘Materials and methods’. Data represent
the means ± S.E.M. of 4 independent experiments. The symbol * indicates values
significantly different between control and rotenone-treated cells.
Fig. 3. Glucose availability affects sensitivity of quiescent cells to cell death induced by
inhibitors of the ETC and ATP synthesis, and not by oxidant agents. (A-F) Ea.hy926 and
(G-H) MCF10A cells cultivated in high glucose (4.5 g/l glucose, A-C, G-I) or low glucose (1
g/l glucose, D-F, J-L) media, respectively, were treated with H2O2 (800 mM), PEITC (10
µM), rotenone (Rot, 2 µM), piericidin (Pier, 80 µM high glucose, 0.05 µM low glucose),
MitoTam (mTAM, 4 µM high glucose and 2 µM low glucose), MitoVES (mVES, 10 µM),
myxothiazol (Myxo, 15 µM high glucose and 0,1 µM low glucose), CCCP (75 µM high
glucose and 10 µM low glucose), oligomycin (Omy, 25 µM high glucose and 0.5 µM low
glucose) for 22 h. The cells were stained with Annexin V-FITC/PI, and evaluated by flow
cytometry as described in ‘Materials and methods’. Data represent the means ± S.E.M., n≥4.
The symbol * indicates values significantly different between proliferating and quiescent
cells.
Fig. 4. Cellular ATP is depleted in quiescent cells upon ETC inhibition in sub-
physiological glucose levels. (A) Glucose concentration in cell-conditioned media was
determined after 6, 16, 24 and 38 h of cultivation using amplex red-based assay. Data are
Blecha et al.: Selectivity of electron transport chain inhibition
33
correlated to fresh media and presented as means ± S.E.M., n=3. (B, D) Ea.hy926 and (C, E)
MCF10A cells in (B, C) high and (D, E) low glucose media were treated for 0.5 h with
rotenone (2 µM), piericidin (5 µM), mVES (10 µM), myxothiazol (15 µM), oligomycin (25
µM), CCCP (75 µM) and ATP level was assessed using luminescent assay. Results were
corrected to cell number measured by crystal violet in a parallel plate. Data represent means ±
S.E.M. of 3 independent experiments, the symbol * indicates significant differences to
untreated control. (F) Quiescent Ea.hy926 cells in fresh low glucose (5.5 mM) media or low
glucose media cell-conditioned for 18 hours were exposed to the indicated agents as
described above, and the ATP content was determined. Data represent means ± S.E.M., n=3.
The symbol * indicates significant differences to untreated control. (G) Glucose uptake was
assessed using 2-NBDG staining and flow cytometry after 6 h treatment with MitoVES (0.5
µM low glucose or 5 µM high glucose) and myxothiazol (7.5 µM). Data shown represent
means ± S.E.M. of at least 3 independent experiments, and are expressed relative to the
untreated control. The symbol * indicates values significantly different between proliferating
and quiescent cells. (H) Surface GLUT1 was determined by immunostaining of non-
permeabilized cells using primary anti-GLUT1 IgG and secondary Cy3 labelled anti-Rabbit
IgG and flow cytometry. Cells were pretreated for 2 h by MitoVES (5 µM) and myxothiazol
(7.5 µM). Data shown represent means ± S.E.M. of 4-7 independent experiments, and are
expressed relative to the untreated control. The symbol * indicates values significantly
different from untreated control.
Fig. 5. ROS production upon treatment by oxidant agents and oxidative
phosphorylation inhibitors is reduced in quiescent cells. (A-F) Ea.hy926 cells cultivated
Blecha et al.: Selectivity of electron transport chain inhibition
34
in high glucose (4.5 g/l glucose, A-C) or in low glucose (1 g/l glucose, D-F) media were
treated with H2O2 (800 μM), PEITC (3 µM), rotenone (rot, 20 µM), piericidin (pier, 50 µM
high glucose, 10 µM low glucose), MitoTAM (mTAM, 7.5 µM) MitoVES (mVES, 10 µM),
myxothiazol (myxo, 15 µM), CCCP (75 µM), and oligomycin (omy, 2.5 µM) for 1 h,
followed by staining with the DCF fluorescent probe. (G-L) MCF10A cells cultivated in high
glucose (4.5 g/l glucose, G-I) or in low glucose (1 g/l glucose, J-L) media were treated with
H2O2 (800 μM), PEITC (10 µM), rotenone (2 µM), piericidin (50 µM), mVES (10 µM),
myxothiazol (25 µM), CCCP (75 µM), oligomycin (2.5 µM) for 1 h and then stained using
the DCF fluorescent probe. Data are expressed relative to the non-treated controls and
represent the mean ± S.E.M. of more than 4 independent experiments. The symbol * indicates
values significantly different in proliferating and quiescent cells.
Fig. 6. Activity of antioxidant defense is increased in quiescent cells. Whole cell lysates
from (A) Ea.hy926 and (B) MCF10A cultured in high and low glucose media were assessed
by WB for expression of the indicated antioxidant proteins. Data represent 3 independent
experiments. (C-E) In-gel activities of indicated antioxidant enzymes were assessed in (C)
Ea.hy926 and (D) MCF10A cells in high and low glucose media. (E) Data were quantified by
densitometry, and correlated to quiescent cells in high glucose media. Data shown are means
± S.E.M. of at least 4 independent experiments. (F, G) GSH/GSSG ratio (F) and
NADPH/NADP+ ratio (G) was determined by luminescence assay in whole cell extracts.
Data shown are means ± S.E.M., n=4. The symbol * indicates significantly different values
between proliferating and quiescent cells.
Blecha et al.: Selectivity of electron transport chain inhibition
35
Fig. 7. Antioxidant defense protects quiescent cells from cell death when glucose is not
limiting. (A-C) Ea.hy926 cells were silenced for SOD2 and assessed for (A) cell death by the
Annexin V-FITC/ PI method, (B) ROS production by DHE and (C) DCF. (D,E) GPX1-
silenced cells were assessed for (D) cell death and (E) ROS production by DCF. (F,G) GSR
silenced cells were assessed (F) for cell death and (G) ROS production by DCF. (H,I)
TXNRD2-silenced cells were assessed for (H) cell death and (I) ROS production by DCF.
Data represent means ± S.E.M., n≥4., are given relative to cells expressing non-silencing
shRNA treated in the identical manner, and measured as described in the legends to figures 3
and 5. The symbol * indicates values significantly different between cells expressing specific
shRNA and non-silencing shRNA. (J) Cell were pre-treated by auranofin (0.75 µM) for 2 h,
followed by MitoVES (mVES, 10 µM) and myxothiazol (myxo, 15 µM) treatment. After 22
h, cell death was assessed using Annexin V-FITC/ PI staining. Data are shown as means ±
S.E.M. of at least 5 independent experiments. (K) Cells were pre-treated with NAC (1 mM)
for 1 h, followed by rotenone (5 µM), MitoVES (mVES, 10 µM) and myxothiazol (myxo, 15
µM) treatment. After 22 h, cell death was assessed using Annexin V-FITC/PI staining. Data
shown are means ± S.E.M., n≥5. The symbol * indicates values significantly different with or
without (J) auranofin and (K) NAC pre-treatment.
1 Fig. S1. Confluent cells undergo cell cycle arrest and form adherence
junctions. (A, B) Analysis of cell cycle distribution of (A) Ea.hy926 and
(B) MCF10A cells was performed by double staining for Hoechst 33342 and
Pyronin Y. Flow cytometry was used to assign cells to G0/G1/S/G2M phase
of the cell cycle based on the level of Hoechst 33342 and Pyronin Y staining.
The symbol * indicates, compared to proliferating cells, significantly
increased G0 phase, the symbol # significantly decreased G1 phase, the
Blecha et al.: Selectivity of electron transport chain inhibition
36
symbol † significantly decreased G2/M phase, the symbol ‡ significantly
decreased S phase. Data are shown as means ± S.E.M. of at least 5
independent experiments. (C, D) Adherence junctions in (C) Ea.hy926 and
(D) MCF10A cells were detected by immunostaning for VE-cadherin and E-
cadherin, respectively, and analyzed by confocal microscopy. Representative
images are shown from 3 independent experiments.
Fig. S2. The effect of investigated agents on cellular respiration. Oxygen consumption by
proliferating Ea.hy926 cultured in both low glucose (1 g/l) and high glucose (4.5 g/l) media
was monitored by the Oroboros Oxygraph instrument. Basal respiration was established after
adding succinate (10 mM) and ADP (3 mM), and was followed by titration with
pharmacological agents indicated in A-J.
Fig S3. Confirmation of ROS production upon ETC inhibition in proliferating cells.
Proliferating and quiescent Ea.hy926 cells in high glucose media were exposed to MitoVES
(10 μM) and myxothioazol (15 μM) for 1 h, and ROS formation was assessed using (A)
DHR123 or (B) DHE by flow cytometry. The symbol * indicates values significantly
increased in proliferating compared to quiescent cells, n≥3.
Fig. S4. Antioxidant defense does not protect quiescent cells from cell death when
glucose is limiting. Ea.hy926 cells cultured in low glucose media were silenced for SOD2
(A), GPX1 (B), GSR (C) and TXNRD2 (D). The cells were treated with indicated
compounds as describe in figure 3 for 22 h and cell death was assessed by the Annexin V/PI
method. Data shown are means ± S.E.M. of 4 independent experiments, and are shown
relative to non-silencing control.
Blecha et al.: Selectivity of electron transport chain inhibition
37
HIGHLIGHTS
A mechanism is proposed for selectivity of ETC disruption in cancer
Susceptibility to ETC inhibitors is linked to cell proliferation
Quiescent, but not proliferating cells, are protected when glucose is not limiting
Protection is conferred by antioxidant defense that eliminates ETC-derived ROS
In limiting glucose conditions (as in a tumor) protection is no longer effective
Blecha et al.: Selectivity of electron transport chain inhibition
38
Blecha et al.: Selectivity of electron transport chain inhibition
39
Blecha et al.: Selectivity of electron transport chain inhibition
40
Blecha et al.: Selectivity of electron transport chain inhibition
41
Blecha et al.: Selectivity of electron transport chain inhibition
42
Blecha et al.: Selectivity of electron transport chain inhibition
43
Blecha et al.: Selectivity of electron transport chain inhibition
44
Blecha et al.: Selectivity of electron transport chain inhibition
45
