Peroxiredoxin 3 Is a Redox-Dependent Target of
Thiostrepton in Malignant Mesothelioma Cells
Kheng Newick1,5, Brian Cunniff1, Kelsey Preston1, Paul Held2, Jack Arbiser3, Harvey Pass4,
Brooke Mossman1,5, Arti Shukla1,5, Nicholas Heintz1,5*
1Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, United States of America, 2BioTek Instruments, Winooski, Vermont, United
States of America, 3Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia, United States of America, 4Department of Cardiothoracic
Surgery, New York University Langone Medical Center, New York, New York, United States of America, 5Vermont Cancer Center, University of Vermont, Burlington,
Vermont, United States of America
Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for
cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and
strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed
FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The
mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited
expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental
activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC),
indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase
2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic
mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic
mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by
NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels
by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its
cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant
network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2
network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-
dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.
Citation: Newick K, Cunniff B, Preston K, Held P, Arbiser J, et al. (2012) Peroxiredoxin 3 Is a Redox-Dependent Target of Thiostrepton in Malignant Mesothelioma
Cells. PLoS ONE 7(6): e39404. doi:10.1371/journal.pone.0039404
Editor: Sumitra Deb, Virginia Commonwealth University, United States of America
Received March 14, 2012; Accepted May 24, 2012; Published June 25, 2012
Copyright: ? 2012 Newick et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The John Sterling Family Memorial grant from the Mesothelioma Applied Research Foundation, the Lake Champlain Cancer Research Organization, the
Vermont Cancer Center, the Vermont Ladies Auxiliary of the Veterans of Foreign Wars, and a training grant in Environmental Pathology from the NIEHS (T32
ES007122-29). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The MARF, LCCRO and
VFW grants supporting this work do not have federal grant numbers, only internal numbers associated with UVM accounting. The website for the Mesothelioma
Applied Research Foundation is curemeso.org. There is no website for the Lake Champlain Cancer Research Organization, a charitable organization that funds
cancer research at the Vermont Cancer Center, which has the web address vermontcancer.org. The Vermont Ladies Auxilary of the VFW website is www.
ladiesauxvfw.org. The website for the NIEHS in www.niehs.nih.gov.
Competing Interests: Paul Held is a former postdoctoral fellow of Nicholas Heintz who is employed by BioTek Instruments. He provided expertise on measuring
cellular oxidant levels; no other author has any affiliation with BioTek Instruments, and none have products in development, patent applications, or consulting
arrangements with BioTek. In the past students from the Heintz laboratory have participated with Dr. Held in the development of commercial assays for BioTek
plate readers from other companies that are posted as application notes on BioTek Instruments’ website. This does not alter the authors’ adherence to all the
PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Malignant mesothelioma (MM) is a type of cancer originating
from the mesothelial lining of the pleural and peritoneal cavities
. It is a deadly malignancy primarily associated with exposure to
asbestos, with an annual incidence of 2000–3000 cases in the
United States . Due to long latency periods, the risk of
developing MM increases with age , and the incidence of MM
is expected to rise in regions where asbestos use has been banned,
as well as in countries where protection from occupational
exposures is presently lacking [2,3]. Pleural malignant mesothe-
lioma is the most common type of mesothelioma , and it
primarily affects men, with a men-to-women ratio of 5:1. Effective
therapy for MM is lacking, with average survival estimated at less
than 2 years .
We are interested in developing new approaches to treating
MM, and have begun investigation of FOX family proteins in this
disease entity. The FOX (for forkhead box) family encompasses
over 100 proteins that play important roles in development, cell
proliferation, cell survival, metabolism, stress responses and aging
(reviewed in [4,5]). The forkhead superfamily of transcription
factors is characterized by a common DNA binding domain first
identified in the Drosophila melanogaster forkhead gene product
[4,5,6]. Several members of the FOX family of transcriptional
regulators, including FOXO3a and FOXM1, have emerged as
important therapeutic targets in human malignancies .
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The FOX family member FOXM1 regulates the expression of
genes involved in cell survival and cell cycle progression, including
S phase entry [4,7] and transition through mitosis [8,9,10].
Alternative splicing results in three protein isoforms: FOXM1A,
which acts as a transcriptional repressor , and FOXM1B and
FOXM1C, which are transcriptional activators . FOXM1 is
not expressed in non-cycling cells and is induced in response to
growth factor stimulation via the E2F pathway . FOXM1 has
an N-terminal auto-inhibitory domain, and N-terminal deletion
mutants of FOXM1C are constitutively active, whereas activation
of the full length protein requires growth factor signaling .
FOXM1 enters the nucleus during G2 in an ERK-dependent
manner , and is degraded during exit from mitosis by APC/
Cdh1, an event required for regulated entry into the next S-phase
[9,10]. Depletion of FOXM1 in mouse models of cancer markedly
impedes tumor progression (reviewed in [4,6]), indicating FOXM1
is an important factor in tumor progression. The oncogenic splice
isoforms FOXM1B and/or FOXM1C are over-expressed in all
carcinomas examined to date , but not in quiescent tissues,
suggesting FOXM1 may represent a therapeutic target in many
human solid tumor types.
Chronic oxidative stress has long been recognized as a pheno-
typic feature of many cancers [14,15,16], and certain tumors
appear to rely on the enhanced production of reactive oxygen
species (ROS) for cell proliferation. FOXM1 has emerged recently
as an important cell cycle regulator that sits at the interface
between oxidative stress, aging, and cancer [4,6,17]. Expression of
FOXM1 is inhibited by antioxidants and induced by hydrogen
peroxide (H2O2), albeit through unknown mechanisms .
FOXM1 counteracts oncogenic Ras-induced oxidative stress
through the up-regulation of antioxidant enzymes that include
mitochondrial manganese superoxide dismutase (MnSOD), cata-
lase, and peroxiredoxin 3 (PRX3) . A screen for small
molecules that inhibit the transcriptional activity of FOXM1
identified siomycin A , a thiazole antibiotic very similar to
thiostrepton (TS), one of a large family of multicyclic peptide
antibiotics produced by diverse bacteria . TS induces cell cycle
arrest and selectively kills breast cancer cells through down-
regulation of FOXM1 protein and RNA expression , and acts
synergistically with other agents to promote tumor cell apoptosis
. TS has modest anti-tumor activity in a xenoplant model of
breast cancer in mice .
While TS inhibits protein synthesis through binding to
ribosomes in prokaryotes , its mechanism of action in
mammalian cells is not well understood. TS has been reported
to bind directly to FOXM1  and to inhibit proteosome activity
leading to induction of the cyclin-dependent kinase inhibitor p21
[24,25,26]. In melanoma cells, but not primary melanocytes, TS
induces oxidative stress and impairs the activity of the proteosome,
eliciting proteotoxic stress, upregulation of heat shock and stress
response gene expression, and apoptosis . Here we have
investigated the mechanism of action of TS in malignant
mesothelioma (MM) cells, and report that TS acts via redox-
dependent mechanism that includes covalent adduction of PRX3,
a mitochondrial peroxidase linked to suppression of apoptosis
[28,29]. In cancer cells elevated expression of the mitochondrial
thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-PRX3
antioxidant network is an adaptive response to increased
mitochondrial oxidative stress. Since inhibiting expression of
PRX3 sensitizes cells to apoptosis [28,29], this mitochondrial
antioxidant enzyme has emerged as a therapeutic target in cancer
. Here we show that disabling PRX3 by TS correlates with
FOXM1 expression, and cell death. The cationic triphenylmeth-
production, inhibition of
ane gentian violet (GV), which inhibits expression of TRX2 ,
the mitochondrial oxidoreductase required for reduction of PRX3
during its catalytic cycle, markedly enhances both modification of
PRX3 and the cytotoxic activity of TS. These studies provide
a combinatorial approach for inhibiting FOXM1 expression and
impairing tumor cell viability that may prove particularly useful in
MM and other tumor types characterized by aberrant mitochon-
drial oxidant production.
MM Tumors and Cells Express FOXM1
To determine if FOXM1 is a relevant therapeutic target in
MM, we examined FOXM1 expression in tissue arrays of human
MM tumor specimens with a polyclonal FOXM1 antibody at
multiple dilutions (1:1000, 1:2000 and 1:3000), as in another study
. At dilutions of 1:3000, there was no staining in control tissues
such as liver (Fig. 1A). Since the significance of cytoplasmic
staining of FOXM1 is not well understood, specimens were scored
for nuclear staining on a scale of 0–3. Specimens stained at the
1:3000 dilution showed predominantly nuclear staining and were
scored by two observers: 0= no positive nuclei, 1=,5% positive
nuclei, 2=.5% and ,50% positive nuclei, and 3=.50%
positive nuclei. Regardless of subtype, the majority of MM tumors
expressed nuclear FOXM1 in 50% or more of the cells (Fig. 1B).
In general, the epithelioid compartments of mesothelioma tumor
specimens displayed nuclear or cytoplasmic staining, with little
staining of connective tissue elements. Quantification of FOXM1
mRNA expression in MM tumor specimens and matched control
tissues from four individuals showed that FOXM1 mRNA is
elevated in human MM (Fig. 1C), confirming the results obtained
To define an experimental model, we then investigated the
expression of FOXM1 in several human MM cell lines, using the
hTERT immortalized human mesothelial cell line LP9 as
a control. While HM, H2373 and several other human MM cell
lines form tumors when injected into the peritoneal cavity of Fox
Chase SCID mice, LP9 does not produce tumors in this
background (B. Mossman and A. Shukla, unpublished data).
When assessed by immunoblotting, the protein isoforms of
FOXM1 differed between LP9 and MM cell lines (Fig. 2A), with
LP9 expressing a species with reduced electrophoretic mobility
that may represent splice isoform FOXM1A. MM cells lines
express modestly higher levels of FOXM1 mRNA (Fig. 2B), and
qRT-PCR for specific FOXM1 mRNA splice variants A, B and C
showed that HM cells express the oncogenic FOXM1B and
FOXM1C isoforms relative to splice isoform A at higher levels
than LP9 cells (Fig. 2C), a result confirmed for the HP-1 and
H2373 MM cell lines (data not shown). Incubation of cells in low
serum conditions resulted in loss of FOXM1 expression in LP9
cells, but not in three MM cell lines (Fig. 2D), indicating that
expression of FOXM1 in LP9 cells requires mitogens, but in MM
cells is mitogen-independent.
FOXM1 protein isoforms are predicted to range between 75–
85 kD, but isoforms that migrate with apparent molecular weights
as large as 120 kD were detected by immunoblotting (Fig. 2A, D),
as in other studies [32,33]. To ensure that the antibodies used in
these studies detect FOXM1, small interfering RNA (siRNA) was
used to selectively extinguish FOXM1 expression, resulting in loss
of all immunoreactive bands detected by the FOXM1 antibody
(Fig. 2E). Due to the complexity of post-translational modifications
to FOXM1 [32,33], the precise relationship between the protein
isoforms observed by gel electrophoresis and the various mRNA
species is not known.
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TS Inhibits the Expression of FOXM1 in MM Cells by
a Redox-dependent Mechanism
The dose-dependent effects of TS on FOXM1 mRNA and
protein expression were examined in LP9 and MM cells, and as
reported by others , TS inhibited expression of FOXM1 by
down-regulating levels of FOXM1 mRNA (not shown) and
protein (Fig. 3A, lanes 2–5). However, we observed cell-type
specific effects of TS in human MM cells. TS has been reported to
act as a proteosome inhibitor [24,26], but in MM and LP9 cells
the proteosome inhibitor MG132 stabilized FOXM1 in the
presence of TS (Fig. 3B, lanes 1–8). Treatment of LP9 cells and
a panel of mesothelioma cell lines (e.g. H2373, MO, HP-1 and
HM) with 10 mM MG-132 overnight did not extinguish FOXM1
expression (Fig. 3C, lanes 3–12), whereas MG-132 inhibited
expression of FOXM1 in Met5A cells, a mesothelial cell line
transformed by SV40 (Fig. 3C, lanes 1–2). Additionally, in contrast
to the results reported for breast cancer cells , examination of
mitogen-activated protein kinase (MAPK) signaling pathways
showed TS markedly increased phospho-ERK1/2 levels in
a dose-dependent fashion in HM cells (Fig. 3D). Given the
sensitivity of ERK1/2 to ROS , we examined the effect of pre-
incubating cells with N-acetyl-L-cysteine (NAC), which increases
cellular glutathione levels , on the activity of TS. HM cells
were treated overnight with 1 mM NAC, the medium was
removed, and cells then were exposed to TS for 16 hr in standard
growth medium as before. Expression of FOXM1, cell morphol-
ogy and the phosphorylation state of the MAPK p38, JNK and
ERK1/2 were examined as endpoints. Pre-incubation of cells with
NAC completely blocked the effects of TS on expression of
FOXM1 (Fig. 3A, lanes 6–10), activation of ERK1/2 (Fig. 3D,
lane 6), and cell morphology (Fig. 3E), indicating that inhibition of
FOXM1 expression by TS in MM cells is redox-dependent. TS
had no effect on the phosphorylation state of JNK or p38 MAPK
kinase (not shown). While prolonged activation of ERK1/2 by
ROS or asbestos is incompatible with cell cycle progression and
cell viability , blocking ERK1/2 signaling with U0126 did not
rescue cells from TS-induced cytotoxicity nor did it restore
FOXM1 expression (data not shown). These results indicated
activation of ERK1/2 by TS is redox-dependent, but activation of
the ERK signaling pathway is not an obligate step in the inhibition
of FOXM1 expression in MM cells.
Production of Mitochondrial Oxidants in MM Cells
FOXM1 expression responds to the redox status of cells, as low
levels of hydrogen peroxide increase FOXM1 expression while the
antioxidant TEMPOL inhibits it . Based on these observa-
tions, and the sensitivity of the effects of TS to NAC, we examined
cellular oxidant production and anti-oxidant defenses as potential
Figure 1. Human MMs express FOXM1. A) Paraffin-embedded sections from human MM tissue specimens were examined for FOXM1 expression
using immunohistochemistry; shown are representative results for the indicated tumor types at a 1:3000 dilution of primary antibody, which
accentuates nuclear localization of FOXM1. No signal for FOXM1 was observed in normal human liver. B) Nuclear expression of FOXM1 was scored for
the indicated tumor types, using 0= no positive nuclei, 1=,5% positive nuclei, 2=.5% and ,50% positive nuclei, and 3=.50% positive nuclei.
Significant levels of nuclear FOXM1 expression were observed in all MM tumor types. There was no significant difference in nuclear FOXM1
expression between mesothelioma tumor types. C) Total RNA was extracted from normal mesothelial tissue (N) or mesothelioma (T) from four
patients and examined for FOXM1 mRNA expression relative to HPRT using qRT-PCR. Using the Student’s t-test, relative expression (RQ) for FOXM1
transcript in tumor tissue was increased as compared to normal mesothelium (p =0.0017).
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targets of TS. Studies with hydro-Cy3, a fluorescent probe for
superoxide , showed under standard growth conditions HM
cells produce more cellular superoxide than LP9 cells, both in low
serum conditions and when stimulated with serum (Fig. 4A). Co-
staining with nitroblue tetrazolium (NBT), which is reduced by
superoxide and detects superoxide produced by NADPH oxidases
, and MitoTracker Deep Red indicated that the majority of
cellular superoxide was derived from mitochondria (Fig. 4B).
MitoSOX Red and flow cytometry was used to compare
mitochondrial oxidant levels in MM cells to LP9 cells. MitoSOX
Red, a fluorescent probe targeted to mitochondria that is
responsive to superoxide and other oxidants, is considered
a suitable probe for general mitochondrial oxidative stress .
LP9, HM and H2373 cells in log phase growth were loaded with
MitoSOX Red for 30 min, trypsinized and immediately analyzed
by flow cytometry, which showed MM cells produce significantly
more mitochondrial oxidants than do LP9 cells (Fig. 4C). Co-
staining with a DNA dye showed that increased levels of
mitochondrial oxidant production occur at all phases of the MM
cell cycle (data not shown). Generally all MM cell lines examined
to date appear to constitutively produce 2–3 times more
mitochondrial oxidants than do LP9 cells (Fig. 4A and C), and
ratiometric imaging with a redox-sensitive green fluorescent
protein (roGFP) targeted to mitochondria confirmed this general
phenotypic property in a representative panel of MM cell lines
When compared to LP9 cells, MM cells appear to have
adapted to enhanced levels of mitochondrial oxidant production
Figure 2. Expression of FOXM1 in LP9 mesothelial cells and MM cells lines. A) FOXM1 expression was examined by immunoblotting of cell
extracts; note that LP9 expresses a larger isoform of FOXM1 than the MM cell lines. B) Total RNA was extracted from the indicated cell lines in log
phase growth, and FOXM1 mRNA expression relative to HPRT (RQ) was determined by qRT-PCR. C) Isoform-specific RT-PCR was used to examine
expression of FOXM1A, FOXM1B, and FOXM1C mRNA in the indicated cell lines. HM cells expressed increased levels of the oncogenic isoforms B and
C compared to LP9 controls while levels of isoform A were relatively similar. D) LP9 mesothelial cells and HM, HP-1 and H2373 MM cells were
incubated in medium containing 10% FBS (H) or medium containing 0.25% FBS (L) for 48 hours, and cell extracts were examined for FOXM1
expression by immunoblotting. Note that MM cells continue to express FOXM1 in the absence of mitogenic stimulation. E) HM cells were transfected
with control siRNA (ct) and siRNA directed toward all FOXM1 splice variants (si), and 24 hr later cell extracts were examined for expression of FOXM1
by immunoblotting. Specific knockdown of FOXM1 mRNA resulted in the loss of all immunoreactive FOXM1 bands, validating the antibodies used in
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by up-regulating expression of thioredoxin reductase 2 (TR2) and
peroxiredoxin 3 (PRX3), components of a major mitochondrial
anti-oxidant network (Fig. 4E). PRX3 is responsible for 90% of
the peroxidase activity in mitochondria [28,29], and both TR2
and PRX3 are known to be over-expressed in human MM
tumors [40,41,42]. Total cellular thioredoxin reductase (TR)
activity also was significantly increased in MM cells as compared
to LP9 cells (Fig. 4F). Together these results indicated that the
mitochondria of MM cells consistently produce significantly
higher levels of mitochondrial oxidants, and that MM cells adapt
to this pro-oxidant state by up-regulating the TR2/TRX2/
PRX3 antioxidant network.
PRX3 is a Redox-dependent Target of TS
When examining the dose-dependent effects of TS on the
cellular TR network, we observed that treatment of MM or LP9
cells with TS at concentrations at or above 2.5 mM altered the
electrophoretic mobility of PRX3, producing modified forms of
PRX3 with apparent molecular weights ranging from 44–50 kD
(Fig. 5A), approximately the molecular weight of disulfide-bonded
PRX3 dimers. However, these immunoreactive species were
maintained when heated to 95uC for more than 10 min in SDS
sample buffer with dithiothreitol (DTT), indicating the modified
species of PRX3 are resistant to denaturation by detergents or
reduction by DTT. Based on experiments with the bacterial
protein TipAS, in which cysteine residues are directly adducted by
reactive dehydroalanine moieties in TS , recombinant human
PRX3 (rPRX3) was incubated with TS in vitro. These experi-
ments showed TS induces a modified form of rPRX3 in vitro with
similar electrophoretic mobility as was observed for PRX3 in cell
extracts (Fig. 5B). Reduction of rPRX3 with sodium cyanobor-
ohydride increased modification of rPRX3 by TS (Fig. 5B, lane 2),
while NAC inhibited modification of PRX3 by TS (Fig. 5B, lane
5), supporting the possibility that TS directly adducts cysteine
residues in PRX3. It does not appear that TS quantitatively
adducts all cysteine residues in PRX3, for the thiol-reactive agent
N-ethyl-maleimide (NEM) altered the mobility of the TS:rPRX3
complex (Fig. 5B, lane 3). The dehydroalanine residues in TS have
been shown to bind 3–4 free cysteines , and addition of NAC
along with TS to PRX3 reduced by sodium cyanoborohydride
resulted in multiple species of PRX3 with altered mobility. Under
no condition was rPRX3 quantitatively modified by TS in vitro.
Like other 2-Cys peroxiredoxins, PRX3 functions as an obligate
homodimer, and disulfide-bonded dimers are generated during the
resolving step of the catalytic cycle [28,44]. Using denaturing but
not reducing gel electrophoresis conditions that preserve disulfide
bonds, the relative distribution of PRX3 monomers was compared
to PRX3-S-S-PRX3 dimers, which can only be generated during
the PRX3 catalytic cycle. In the presence of increasing concentra-
tions of TS, monomer PRX3 was detected until concentrations of
Figure 3. TS inhibits FOXM1 expression through a redox-dependent mechanism. A) HM cells were treated with the indicated
concentration of TS for 18 hr (lanes 1–5), or were pre-incubated with 1 mM NAC for 16 hr prior to exposure to TS (lanes 6–10). Cell extracts were
examined for FOXM1 expression by immunoblotting as before. B) The indicated cell lines were incubated with 2.5 mM TS, or 2.5 mM TS and 1 mM of
the proteosome inhibitor MG132, for 18 hr and FOXM1 expression was examined by immunoblotting of cell extracts. C) The indicated cell lines were
incubated for 16 hr with or without 10 mM MG132 as indicated and cell lysates were examined for FOXM1 expression by immunoblotting. D) HM cells
were incubated with the indicated concentration of TS for 18 hr and levels of phospho-ERK1/2 (pERK1/2) and total ERK1/2 were assessed by
immunoblotting. To test sensitivity of induction of phospho-ERK1/2 by TS to NAC, cells were pre-incubated with 1 mM NAC overnight and then
exposed to 5.0 mM TS as before (lane 6). HM cells express significantly higher levels of ERK2 versus ERK1. E) HM cells were treated with 10 mM TS, with
or without pre-incubation with 1 mM NAC, for 8 hr and examined by phase-contrast microscopy. Cell rounding, membrane retraction and other early
morphological changes induced by TS were attenuated by pre-incubating HM cells with NAC.
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Figure 4. Enhanced mitochondrial superoxide production by MM cells. A) Equivalent numbers of LP9 and HM cells were deprived of serum
for 72 hr, then incubated in medium with either low serum (0.25% FBS) or high serum (10% FBS) and loaded with the fluorescent reporter hydro-Cy3.
After 30 min relative fluorescence was measured in triplicate; data are expressed as relative fluorescence units (RFU) +/2 the standard error of the
mean. B) LP9 mesothelial cells (row b) and HM MM cells (row c) were incubated with nitroblue tetrazolium (NBT) and MitoTracker Deep Red and
imaged by confocal microscopy. Colocalization of the NBT and MitoTracker Deep Red signals indicated that the majority of superoxide in LP9 and HM
cells is derived from mitochondria. Row a represents images of LP9 cells without staining with NBT. C) H2373 and HM MM cells and LP9 controls were
loaded with MitoSOX Red for 30 min and analyzed by flow cytometry for mitochondrial superoxide production. D) The indicated cell lines were
transfected with an expression vector for mitochondrial roGFP, a genetically-encoded reporter that is responsive to mitochondrial redox status. The
relative redox status of the different cell lines was examined by ratiometric live cell imaging as described previously [67,68] Higher 400:495 ratios are
indicative of an increased oxidation state in cellular mitochondria. E) Total RNA was prepared from LP9, HM and H2373 cells and relative levels of
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TS at or above 2.5 mM (Fig. 5C), when all PRX3 was observed to
migrate at the apparent molecular weight of PRX3 dimers. These
results indicate complete loss of reduced PRX3 monomers, either
through direct modification by TS or disulfide bond formation
between homodimeric subunits during the PRX3 catalytic cycle,
and are indicative of induction of severe mitochondrial oxidative
Gentian Violet Potentiates the Formation of Modified
The cationic triphenylmethane gentian violet (GV) has antitu-
mor activity that is redox-dependent [30,45]. Recently GV was
shown to impair mitochondrial antioxidant capacity by inhibiting
the expression of TRX2, but not TR2 . In mitochondria
TRX2 is the oxidoreductase that reduces disulfide-bonded PRX3
dimers, and thereby is required for regenerating reduced PRX3
during the 2-Cys peroxiredoxin catalytic cycle . In HM cells,
GV inhibited the expression of TRX2 in a dose-dependent
manner, with significant inhibition at doses as low as 0.5 mM
(Fig. 6A). GV had no effect on the expression of TR2, but like TS,
GV inhibited the expression of FOXM1 (Fig. 6B).
When combined with TS, GV resulted in marked increases in
the levels of modified PRX3 (Fig. 6C, lanes 5–6). When HM or
other MM cells were pre-incubated with NAC, no modified form
of PRX3 was observed (Fig. 6C, lanes 11–12, and data not shown).
L-buthionine-S,R-sulfoximine (BSO), an inhibitor of cellular
glutathione synthesis , had no significant effect on modifica-
tion of PRX3 by TS (lanes 8–9). Analysis of mitochondrial redox
expression of PRX3 and TR2 mRNA in comparison to HPRT were determined by qRT-PCR. Data are plotted as fold-increase in the MM cell lines as
compared to LP9. F) Cell extracts were prepared from LP9, HM and H2373 cells and assayed for total cellular thioredoxin reductase (TR) activity. Data
are expressed as arbitrary units (AU) per 25 mg cell extract.
Figure 5. The electrophoretic mobility of PRX3 is modified by TS. A) HM cells were treated with the indicated concentration of TS for 18 hr,
and cell extracts were prepared, heated in SDS sample buffer at 95uC for 10 min, resolved by gel electrophoresis, and PRX3 expression was assessed
by immunoblotting. Treatment of HM cells with increasing doses of TS caused the formation of a modified species of PRX3 that migrated with an
apparent molecular weight of about 45–50 kD. B) Recombinant human PRX3 (rPRX3) was incubated with 10 mM TS in solution in vitro using
conditions described by Chiu and colleagues , and samples were resolved by gel electrophoresis and examined by immunoblotting. Pre-
treatment of rPRX3 with sodium cyanoborohydride (NaCNB) accentuated the reactivity of TS (lane 2) whereas pre-incubation with NAC inhibited it
(lane 5). Addition of TS and N-ethyl-maleimide (NEM) or NAC to rPRX3 treated with NaCNB resulted in multiple species of immunoreactive PRX3 (lanes
3 and 4), whereas treatment with NACNB alone (lane 7) had no effect. C) HM cells were treated with the indicated concentration of TS for 18 hr, and
cell extracts were prepared in the absence of reducing agents as described previously . Samples (20 mg protein/lane) were resolved under
denaturing but not reducing conditions, and the relative distribution of PRX3 monomers to dimers was assessed by immunoblotting.
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status using the redox-responsive fluorescent dye MitoSOX Red
and flow cytometry showed TS induces increased levels of
mitochondrial oxidants at 2.5 mM or above (Fig. 6D), in excellent
agreement with the concentrations of TS that promote modifica-
tion of PRX3. At concentrations that inhibit expression of TRX2
or above (e.g. .0.5 mM), GV increased mitochondrial oxidant
levels, and when added together, TS and GV caused production of
even higher levels of mitochondrial oxidants (Fig. 6D). GV alone
Figure 6. GV accentuates modification of PRX3 by TS. A) HM cells were treated with the indicated concentration of GV for 18 hr, cell extracts
were prepared, and expression of TRX2 and TR2 was assessed by immunoblotting. Actin was used as a loading control. B) HM cells were treated with
the indicated concentration of GV for 16 hr, cell extracts were prepared and examined for FOXM1 expression by immunoblotting. Actin was used as
a loading control. C) HM cells were treated with the indicated concentration of TS, TS plus GV, or TS after pre-incubation with either BSO or NAC. Cell
extracts were prepared and assessed for PRX3 expression by immunoblotting as before. Actin was used as a loading control. D) HM cells were treated
with the indicated concentration of TS, GV or TS plus GV for 18 hr, loaded with MitoSOX Red for 40 min, and examined by flow cytometry. Data are
expressed as the log of the relative fluorescent signal (FL2) versus the number of cells. NT indicates no treatment. E) HM cells were treated with TS, GV
or TS plus GV at the indicated concentrations, and modification of PRX3 was examined by immunoblotting as before. Note that under these
conditions the electrophoretic mobility of the entire cellular pool of PRX3 was modified in cells treated with TS plus GV.
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did not alter the electrophoretic mobility of PRX3 (Fig. 6E, lanes
4–5), but at markedly cytotoxic doses of GV and TS the
electrophoretic mobility of the entire cellular pool of PRX3 was
modified (Fig. 6E, lanes 6–7). Under these conditions all
immunoreactive species of PRX3 migrated in excess of 44–
50 kD under denaturing and reducing conditions.
Based on the ability of GV to enhance modification of PRX3,
we tested the effect of GV on cell viability in response to exposure
to TS. HM cells were plated in 96 well dishes and exposed to
1.0 mM TS alone, approximately half the ID50(2.3+/20.3 mM),
or 1.0 mM TS and increasing concentrations of GV. Using a four
parameter linear regression model, the combination of 1 mM TS
plus GV was 3.3-fold more potent than GV alone (Fig. 7A), and in
the reverse format 0.05 mM GV plus TS was 175-fold more potent
than TS alone (Fig. 7B). Phenylethyl isothiocyanate (PEITC),
a redox-active agent which affects PRX3 oxidation state , also
enhanced the cytotoxic activity of TS (not shown). We conclude
from these studies that TS disables the TR2-TRX2-PRX3
network by covalently adducting PRX3, and that GV enhances
adduction of PRX3 by TS by inhibiting expression of TRX2. The
mechanism by which disruption of PRX3 activity and increased
levels of mitochondrial oxidants inhibit FOXM1 expression is not
The role of ROS in cell physiology is complex, with the source,
species, rate of production, steady state concentration and
subcellular location contributing to its effects on specific targets
that control cell fate decisions . As the primary source of
cellular ROS, mitochondria play a central role in redox signaling
. Mitochondria from tumor cells commonly display altered
morphology, changes in membrane potential, and perturbations in
energy metabolism, consequences of neoplastic transformation
and uncontrolled cell proliferation [48,49,50]. Enhanced con-
sumption of glucose during aerobic glycolysis is associated with
increased mitochondrial superoxide production . While it is
possible that superoxide may signal directly, it more likely is
rapidly dismutated by MnSOD to H2O2, which is capable of
diffusing to the cytoplasm and modulating signaling cascades that
regulate mitogenesis, cell migration, drug resistance and other
processes of malignancy (reviewed in ). There are a large
number of studies that have targeted perturbations in ROS
metabolism as a therapeutic strategy in cancer, and induction of
intolerable levels of oxidative stress generally appears to be a more
effective strategy than blocking oxidant production [53,54].
Based on studies that show FOXM1 responds to cellular redox
status, albeit through unknown mechanisms , we explored the
effect of cellular redox status on the activity TS in a cell culture
model of human MM. MM shares several features with ‘‘reactive
oxygen-driven tumors’’ , and the studies here show MM cells
in culture constitutively produce 2–3-fold more mitochondrial
oxidants than non-transformed LP9 mesothelial cells. We expect
that responses to TS will differ between tumor cell types based on
specific perturbations in oxidant metabolism and the array of
redox-responsive targets expressed in different cell types. For
example, in breast cancer cells TS inhibits activation of ERK1/2
, whereas in MM cells TS activates ERK1/2, and activation is
sensitive to the antioxidant NAC (Fig. 3C). MM cells apparently
adapt to a pro-oxidant mitochondrial state by up-regulating
expression of TR2 and PRX3, both of which are over-expressed in
human mesothelioma [40,41,42].
Our results indicate TS disables the TR2/TRX2/PRX3
mitochondrial anti-oxidant pathway by covalently adducting
cysteine residues in PRX3, similar to the adduction of a cysteine
residue by TS in the bacterial protein TipAS . TS covalently
binds TipAS with a 1:1 stoichiometry, and the peptide harboring
the modified cysteine peptide is increased in MW by 1664 Da, in
excellent agreement with the MW of TS . The covalent
modification of a cysteine residue in TipAS by TS is mediated by
dehydroalanine moieties and is resistant to reducing and de-
naturing agents , as is the modified form of human PRX3
induced by TS. As for TipAS, PRX3 modification by TS is
sensitive to competition by free thiols. TS has three dehydroala-
nine residues capable of reacting with thiols , and PRX3
dimers have six cysteine residues (three per monomer), and one
plausible interpretation of our results is that TS covalently cross
links PRX3 homodimers through cysteine residues (or some other
amino acid). The peroxidatic cysteine residues in peroxiredoxins
[28,29,41,44,47,52], suggesting the cysteine residues intimately
involved in peroxidase activity may be preferred targets.
Inhibition of TRX2 expression by GV markedly enhanced
modification of PRX3 by TS (Fig. 6). Since TRX2 is the
oxidoreductase required for reduction of PRX3 disulfide-bonded
dimers, and only a small fraction of rPRX3 was modified by TS in
vitro, this suggests that catalytic intermediates with specific
oxidation states that accumulate in the PRX3 reaction cycle in
the absence of TRX2 may be preferred substrates for TS. Each
head-to-tail PRX3 dimer has two reaction centers composed of the
peroxidatic cysteine in one subunit in apposition to the resolving
cysteine in the other [28,29]; these two catalytic centers are
oriented in opposite directions in the PRX3 dimer and may
function independently. In PRX3 dimers containing one disulfide
bond, TS may interact with thiols in the other catalytic site, or
with Cys65, which has no known role in catalysis. Since we did not
observe a species of PRX3 with an increase in apparent molecular
weight of about 2 kD, we expect that the complex structure of TS
promotes specific binding to surfaces of the PRX3 dimer, thereby
orienting two or more dehydroalanine moieties in TS in close
proximity to reactive thiols in each subunit, leading to cross-linking
and enzymatic inactivation of PRX3.
In higher concentrations of TS, PRX3 also becomes hyperox-
idized (not shown), but it is not known if this intermediate is
a preferential target of TS. Indeed, the modified forms of PRX3
do not migrate as a single species, and mass spectrometry and
mutagenesis of PRX3 will be required to identify the amino acid
residues adducted by TS in vitro, in cells, and in response to
treatment with GV. Moreover, we expect that TS reacts with
other redox-dependent targets in MM cells, and global analysis of
protein modification by TS will be required to identify additional
proteins that may play a role in the anti-tumor activity of TS. Our
present results indicate, however, than PRX3 is more reactive with
TS than either PRX1 or PRX2 (not shown), although preferential
accumulation of TS in mitochondria might account for differences
PRX3 is consistently up-regulated in prostate cancers , is
over-expressed in greater than 90% of hepatocellular carcinomas
, and increases in abundance during malignant progression of
cervical cancer . Inhibition of PRX3 expression in breast
cancer cells induces cell cycle arrest and impairs cell proliferation
. Since elevated expression of PRX3 is linked to resistance to
apoptosis, inhibiting the peroxidase activity of PRX3 has been
proposed as a therapeutic target in cancer . Here we have
detected a link between mitochondrial oxidant metabolism and
FOXM1 expression, and our present evidence indicates that TS
acts on this axis through covalent adduction of PRX3, leading to
increases in mitochondrial oxidant production and/or general
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mitochondrial oxidative stress. Of particular interest is the
mechanism by which high levels of oxidants impair FOXM1
expression, and one intriguing possibility is that this phenotypic
response results from redox-dependent activation of the anti-
proliferative transcription factor FOXO3a, which is required to
elicit responses to many chemotherapeutic agents [5,59,60]. Since
FOXO3a and FOXM1 regulate many of the same genes, it is
possible that a redox-dependent interplay between the activation
of these transcription factors regulates responses to TS. Suppres-
sion of FOXM1 expression does not appear to be linked to
activation of ERK1/2 by TS, for inhibition of ERK activity with
U0126 in the presence of by TS did not restore FOXM1
expression (not shown).
There are several important implications to these findings. A
number of approaches have emerged for exploiting FOXM1 as
a therapeutic target in other malignancies [61,62,63,64]. FOXM1
is an attractive target since it is not expressed in quiescent cells ,
and is required for cell cycle progression and viability in many
tumor cell types. TS alone has modest anti-tumor activity ,
and the observation that low doses of GV markedly potentiate
modification of PRX3 by TS provides a combinatorial approach
for inactivating the mitochondrial TR2/TRX2/PRX3 anti-
oxidant network and inducing cytotoxicity (Fig. 8). Disabling this
antioxidant network also may increase sensitivity to conventional
drugs that induce apoptosis. Further work will be required to
optimize the ratio of TS to GV that provides selectivity for tumor
versus normal cells, and to test these approaches in models that
more closely approximate tumor environments in vivo, which no
doubt differ significantly from cell culture conditions.
Our approach may be particularly appropriate for intractable
tumor types characterized by high levels of mitochondrial oxidant
production. For example, melanoma cell lines have been grouped
into two categories based on the expression of anti-apoptotic and
anti-oxidant genes, including PRDX3 . TS induces apoptosis in
Figure 7. GV potentiates the cytotoxicity of TS in MM cells. A) HM cells were plated in duplicate in 96 well plates, and treated with either
increasing concentrations of GV or TS alone, or 1.0 mM TS with increasing concentrations of GV. After 4 days cells were stained with crystal violet and
total cellular mass was assessed by absorbance of methanol-soluble dye at 540 nM. Using the four parameter non-linear regression model in Gen5
software (BioTek Instruments), 1 mM TS with increasing [GV] was estimated to be approximately 3.3-fold more potent than increasing concentrations
of GV alone. B) HM cells were plated in 96 well dishes and treated with the indicated concentrations of TS and GV, or with 0.05 mM GV with increasing
concentrations of TS. After 4 days total cell mass was quantified by staining with crystal violet as above. A constant concentration of 0.05 mM GV with
increasing [TS] was estimated to be approximately 175 times more potent than increasing concentrations of TS alone.
Peroxiredoxin 3 Is a Target of Thiostrepton
PLoS ONE | www.plosone.org10June 2012 | Volume 7 | Issue 6 | e39404
melanoma cells in a redox-dependent manner, for NAC rescues
melanoma cells from TS toxicity , but the role of PRX3 in cell
death in this context is not known. We suggest documenting the
expression levels of PRX3 may prove useful in identifying
malignancies susceptible to combinations of TS and GV. Finally,
GV is safe in humans , and TS has no overt toxicity in animal
models , suggesting that once optimal ratios of the two
compounds that exploit the differences in mitochondrial oxidant
metabolism between normal mesothelial cells and MM tumor cells
are refined, translation to clinical applications might proceed
Materials and Methods
Cell Lines and Cell Culture
Four pleural human MM cell lines were used in these studies.
HMESO1 (HM) was obtained from J. Testa (Fox Chase Cancer
Center, Philadelphia, PA); this cell line was originally isolated by
Reale and colleagues . HP-1 and H2373 were established from
human MMs after surgical resection , and were verified to be
mesothelial using a calretinin antibody. Morphologically, HM and
HP-1 are epithelioid, while H2373 is fibrosarcomatoid. LP9,
a human mesothelial cell line immortalized with hTERT, was
obtained from J. Rheinwald (Dana Farber Cancer Institute,
Boston, MA). Cell lines were validated by STR DNA fingerprint-
ing using the Promega CELL ID System (Promega, Madison, WI).
The STR profiles are of human origin, and did not match known
DNA fingerprints in the Cell Line Integrated Molecular Authen-
tication database (http://bioinformatics.istge.it/clima/), but will
serve as a reference for future work. In several experiments, the
MM cell lines HEC, PET, ROB and PRO established by C.
Verschraegen (Vermont Cancer Center) were also examined. Cells
were maintained in DMEM-F12 with hydrocortisone, insulin,
transferrin, and selenite with 10% fetal bovine serum (FBS,
GIBCO) as previously described . For low serum conditions,
cells were incubated for 48–72 hours in complete medium
containing 0.25% FBS.
Cell lysates were prepared as previously described . Protein
concentrations were determined using Bradford assays (Bio-Rad,
Hercules, CA). Lysates (20–25 mg protein/well) were resolved by
SDS-PAGE and prepared for immunoblotting as previously
described . Blots were incubated at 4uC overnight with rabbit
anti-FOXM1 K19 polyclonal antibody (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA) at a 1:500 dilution in blocking buffer, and
after washing protein bands were visualized with the Western
Lightning chemiluminescent detection system (Perkin Elmer,
Waltham, MA) using secondary antibodies coupled to horse
radish peroxidase. Blots were re-probed with a mouse anti-actin
antibody (Millipore, Billerica, MA) to verify equal protein loading.
FOXM1 Isoform-specific PCR
The detection of specific FOXM1A, B and C mRNA splice
variants via RT-PCR was performed as previously described .
FOXM1 siRNA-mediated Knockdown
Cells were grown to 70% confluence, and medium was changed
to serum-free OPTI-MEM before preparing siRNA stocks.
Mixtures of FOXM1 siRNA (GGACCACUUUCCCUACUUU)
 and scramble control (GCUCCUUUCGUCUCACAUAUU)
were prepared in OPTI-MEM as described by the manufacturer
(Dharmacon, Lafayette, CO). Cells were transfected with siRNA
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in OPTI-
MEM; after 4–6 hours, FBS was added to a final concentration of
10%. Media was changed to DMEM-F12 complete media 24 hrs
Figure 8. Cooperative effects of GV and TS on the TR2/TRX2/PRX3 antioxidant network. Superoxide from the electron transport and
chain and other sources is converted by MnSOD to H2O2, which is then metabolized by PRX3 to water. Disruption of PRX3 activity by covalent
adduction of cysteine residues by TS increases mitochondrial oxidant levels, which repress FOXM1 expression through unknown mechanisms.
Inhibition of TRX2 expression by GV potentiates the activity of TS, increasing its cytotoxicity. Combinatorial approaches to inactivating the TR2/TRX2/
PRX3 antioxidant network provide an approach for disabling an important adaptive response in MM, and may enhance the sensitivity of MM to
conventional chemotherapeutic drugs.
Peroxiredoxin 3 Is a Target of Thiostrepton
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after initial transfection. Cell extracts were prepared for immuno-
blotting as before .
Measurement of ROS Production
Hydro-Cy3 , a fluorescent probe specific for intracellular
superoxide and hydroxyl radicals, was a gift from N. Murthy
(Georgia Institute of Technology, Atlanta, GA). Cells were plated
in black clear-bottom 96-well microplates (Corning, Lowell, MA),
incubated in starvation medium for 48 hrs prior to stimulation
with complete medium containing 0.25% or 10% FBS for 45
minutes at 37uC. Cells were then washed in phenol red- and
serum-free DMEM-F12 medium, loaded with 5 mM hydro-Cy3
prepared in phenol red- and serum-free DMEM-F12, and read at
535ex/560emevery minute for 30 minutes using a BioTek Synergy
H4 Hybrid microplate reader (BioTek Instruments, Winooski,
Thioredoxin Reductase Activity Assay
Cells were scraped from culture dishes in 50 mM Tris/Cl,
pH 7.5, with 1 mM EDTA, sonicated, and centrifuged at
14,000 rpm for 10 min. Assays contained 20 mg lysate protein,
50 mM Tris/Cl, pH 7.5, 1 mM EDTA, 8 mM NADPH, 6 mg/
ml insulin with or without purified recombinant thioredoxin.
Reaction mixtures (80 ml) were incubated at 37uC, and after 1 hr
reactions were terminated by adding 920 ml of 8 M guanidine-
HCl containing 1 mM DTNB and read at 412 nm. Activity is
expressed as A412 per mg protein after subtraction of reaction
controls without substrate.
RNA Preparation and Gene Expression
Trizol total RNA extraction and subsequent DNase treatment
were performed as described by the manufacturer (Qiagen,
Valencia, CA). Patient tumor tissues were homogenized using
a Ultraturex homogenizer. cDNA was prepared using the High
Capacity cDNA Reverse Transcription kit (Applied Biosystems,
Foster City, CA). Assays for RNA expression were done in
triplicate using Assay On Demand (Applied Biosystems) for
Changes in relative mRNA expression levels in MM cell lines
were compared to those in LP9 cells, and in tumor specimens to
matched normal mesothelium. qPCR was performed on an
Applied Biosystems (ABI) Prism 7900HT Sequence Detection
System using the SDS v2.2 software.
Patient Materials and Immunostaining of Tissue Arrays
Tissue microarrays were obtained through the Mesothelioma
Virtual Tissue Bank; they included duplicate paraffin-embedded
sections for 46 tumors, (28 epithelial/epithelioid, 9 biphasic, 3
sarcomatoid, 4 papillary and 2 desmoplastic specimens). The
histological diagnosis was not specified for 13 specimens, and these
were not included in our analysis. Arrays were stained by
immunohistochemistry (IHC) as described previously , using
polyclonal FOXM1 antibody C-20 (Santa Cruz Biotechnology) at
dilutions of 1:1000, 1:2000 and 1:3000. Specimens stained with
the 1:3000 dilution showed predominantly nuclear staining and
were scored by two independent observers: 0= no positive nuclei,
1=,5% positive nuclei, 2=.5% and ,50% positive nuclei, and
3=.50% positive nuclei.
The cationic triphenylmethane gentian violet (GV) was a kind
gift from J. Arbiser (Emory University, Atlanta, GA). Thiostrepton
was from EMD Biochemicals.
Cell Growth Assays
Cells were plated in 96-well plates at a density of 1500 cells per
well. The following day, cells were treated with test compounds in
complete medium with 10% FBS. After 4 days cells were washed
with PBS, fixed in 3.7% para-formaldehyde (PFA) and stained for
30 min with 0.1% crystal violet in water. To quantify crystal violet
staining, the dye was dissolved in 100% methanol absorbance was
read at 540 nm. Signals were normalized by subtracting the
background signal from wells treated in the same fashion, but with
no cells. To determine the relative potency (REP) of combinations
of drugs to individual compounds the data were plotted using a 4-
parameter non-linear regression model with the Gen5 software,
using responses to the parent compound as the reference curve
(BioTek Instruments, Winooski, VT).
MitoSOX Red Flow Cytometry
After indicated treatments cells were loaded with 5 mM
MitoSOX Red in tissue culture medium for 30 minutes. Cells
were washed with Hanks buffered salt solution with calcium and
magnesium (HBSS), collected by brief trypsinization, centrifuged
and washed twice in HBSS, and re-suspended in 1% bovine serum
albumin (BSA) in HBSS and analyzed by flow cytometry. To
measure oxidized MitoSOX Red, cells were excited at 488 nm
and emission was collected in the FL2 channel. No dye and
menadione-treated cells were used as controls for each experiment
(data not shown).
Measurement of Mitochondrial Redox Status by Mito-
Cells were plated in 35 mm glass bottom imaging dishes
(MatTek, Ashland, MA) and transiently transfected with mito-
chondrial targeted pEGFP-R12 (roGFP2) using Lipofectamine
2000 (Invitrogen). The following day media was changed to CO2-
independent media (Invitrogen) supplemented with all other
components of complete MM media and imaged on a Nikon
Ti-E inverted microscope with a 100X 1.49 NA objective in
a heated environmental chamber. To determine the oxidation
state of the probe, fluorescence images were collected with an
Andor iXon X3 EMCCD camera (Andor Technology, Belfast,
UK) after excitation at 400 nm and 495 nm using a diode
illuminator (Lumencor, Beaverton, OR); emission was collected at
525 nm for both excitation wavelengths. Individual cells were
imaged every minute for 30 minutes and the ratio of emission from
400 (oxidized) and 495 (reduced) was measured to determine the
amount of oxidized probe in each cell line tested. Quantification of
signals was determined using the NIS-Elements software (Nikon
Instruments, Melville, NY) and is graphically depicted as the mean
of the 400/495 ratio.
Cells were plated on glass coverslips in 35 mm tissue culture
dishes in complete media. 2.0 mg/ml of nitroblue tetrazolium
(Sigma) was added to each plate in HBSS for 40 min. A final
concentration of 250 nM of Mitotracker Deep Red (Invitrogen)
was added directly to each plate for the final 20 min of NBT
staining. Cells were subsequently washed and fixed in 3.7% PFA
and mounted on glass slides in mounting media. Images were
collected on a Zeiss LSM 510 META confocal laser scanning
imaging system using a Plan-Neofluar 40X objective. NBT images
were visualized using differential interference contrast (DIC) while
MitoTracker Deep Red was visualized through excitation at
633 nM and emission collection at 650 nm.
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Incubation of Recombinant PRX3 with TS
Recombinant human PRX3 (Prospec Biochemicals, East
Brunswick, NJ) was treated with TS as described by Chui and
colleagues . Briefly, 2 mg of rPRX3 protein in 20 mM Tris-
HCl at pH 8 and 10% glycerol was reduced with sodium
cyanoborohydride (0.5 ml of 1 mM freshly made stock/20 ml
reaction mix). After incubating for 5 min at room temperature,
excess sodium cyanoborohydride was removed by adding 1 ml
acetone. After the addition of TS to a final concentration of 5 mM,
the reaction was incubated at room temperature for another
15 min. Selected samples were treated with NAC (final concen-
tration of 1 mM) or alkylated with N-ethyl-maleimide for 15
minutes at room temperature. Samples were then heated in SDS
sample buffer and subjected to SDS-PAGE and immunoblotting
blotting as described above.
For in vitro experiments, at least two independent replicates were
performed (n=2 to 4 samples/group/experiment). Statistical
significance was evaluated by analysis of variance (ANOVA) using
the Student Neuman-Keul’s procedure for adjustment of multiple
pairwise comparisons between treatment groups. Values of
p,0.05 were considered statistically significant. One asterisk
indicates a p value ,0.05, two asterisks p,0.01, three asterisks
p,0.001, and four asterisks p,0.0001.
We thank Michael Becich and the National Virtual Mesothelioma Bank for
MM tissue microarrays, Niren Murthy for hydro-Cy3, Nicole Bishop of the
University of Vermont Microscopy Imaging Center for immunohisto-
chemical analyses of FOXM1 expression, Ben Fleishman for photography
of tissue specimens, and the University of Vermont Advanced Genome
Technology Core for technical assistance. The humanized redox-re-
sponsive GFP targeted to mitochondria (Mito-roGFP) was a generous gift
provided by J. Andre Melendez, whose laboratory generated this construct
based on work originally reported by S. Jim Remington [67,68].
Conceived and designed the experiments: KN BC PH NH. Performed the
experiments: KN BC KP PH. Analyzed the data: KN BC PH NH.
Contributed reagents/materials/analysis tools: JA HP BM AS. Wrote the
paper: KN BC NH.
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