Download Title Page. Redox Signaling and Bioenergetics Influence Lung

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Title Page.
Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the
Isoflavone, ME-344.
Yefim Manevich, Leticia Reyes, Carolyn D. Britten, Danyelle M. Townsend, Kenneth D. Tew
Departments of Cell and Molecular Pharmacology and Experimental Therapeutics (Y.M., L.R.,
University of South Carolina, Charleston, SC.
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K.T.); Medicine (C.B.); and Drug Discovery and Biomedical Sciences (D.T.) of the Medical
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Running Title Page.
A) Running title: Redox active isoflavone in lung cancer.
B) Corresponding author: Kenneth D. Tew
Address: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, 173
Ashley Ave., BSB358, Charleston, SC 29425
Phone: 843 792 2514.
Fax: 843 792 2475
C) Text pages: 14
Tables: 0
Figures: 8
References: 34
Abstract words: 203
Introduction: 381
Discussion: 1321
D) Abbreviations: arbitrary units (au); dimethylsulfoxide (DMSO); extracellular acidification rates
(ECAR); human lung embryonic fibroblasts (IHLEF); inner mitochondrial membrane (IMM);
nicotinamide nucleotide transhydrogenase (NNT); oxygen consumption rates (OCR); reactive
oxygen species (ROS); p-triflouromethoxyphenylhydrazone (FCCP)
E) Section: Chemotherapy, Antibiotics and Gene Therapy.
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E-mail: [email protected]
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Abstract.
ME-344 is a second-generation derivative natural product isoflavone presently under
clinical development. ME-344 effects were compared in lung cancer cell lines either intrinsically
sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts
(IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines,
causing redox stress, decreased mitochondrial ATP production and subsequent disruption of
mitochondrial function. In a dose dependent manner the drug caused instantaneous and
significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by
ME-344, with specific effects on the respiratory chain (resistance correlated with higher
glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased
extracellular acidification rates (ECAR) in drug-resistant cells (significantly less in drug-sensitive
cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug sensitive cells,
ME-344 dose-dependently increased intracellular generation of ROS and caused oxidation of
total (mainly GSH) and protein thiols and concomitant immediate increases in NAD(P)H levels.
We conclude that ME-344 causes complex, redox-specific and mitochondria-targeted effects in
lung cancer cells which differ in extent from normal cells, correlate with drug sensitivity and
provide indications of a beneficial in vitro therapeutic index.
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pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells, (quantitatively
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Introduction.
Natural product isoflavones have been studied as agents useful in cancer prevention (de Souza
et al., 2010; Leclercq and Jacquot, 2014). However, one isoflavone, phenoxodiol has cytotoxic
antitumor activities more characteristic of a therapeutic agent and consequently, consideration
has been given as to how this agent and some of its derivatives might induce cell death (Sapi et
al., 2004; Aguero et al., 2005; Silasi et al., 2009). Mechanistically, pro-apoptotic effects may
result from interference with mitochondrial functions (Kamsteeg et al., 2003), interference with
(Morre and Morre, 2003; Herst et al., 2007). Downstream of such events, phenoxodiol
derivatives can activate caspase-dependent and independent apoptotic pathways, perhaps
through AKT signaling events (Wang et al., 2011; Pant et al., 2014). Related isoflavones alter
mitochondrial membrane potential, deplete ATP, reduce steady state levels of cytochrome c
oxidase (complex IV of electron transport chain) and might eventually inhibit mTOR activity
(Alvero et al., 2011). One commonality of these effects is interference with mitochondrial energy
production. Related to phenoxodiol, ME-344 (Fig. 1) is a second-generation derivative of
triphendiol currently under development as a clinical therapeutic drug candidate in both small
cell lung and ovarian cancers. ME-344 is the lead compound selected from a number of
analogues, where structure activity relationship analyses revealed that the ring hydroxyl groups
provide characteristics prerequisite for cytotoxicity (Silasi et al., 2009). Initial in vitro cytotoxicity
screens demonstrated that only 20 of 240 human tumor cell lines (mostly solid tumor origins)
were intrinsically (naturally) resistant to the effects of ME-344. Within the context of the present
study, this provided the opportunity to compare the effects of ME-344 in these cell lines and
compare with an immortalized cell line of lung fibroblast origin. Such an approach obviates the
complexity of using drug selection conditions to derive acquired resistant clones and facilitates
the identification of traits that might be important to the mechanism of action of ME-344. In turn,
such information should have relevance to how the drug is clinically applied. Our results indicate
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plasma membrane electron transport and/or cell surface thiol: disulfide exchange reactions
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that ME-344 impacts redox homeostasis and may act in mitochondria through interference with
pathways that regulate switching from oxidative phosphorylation to glycolysis. Our present data
implicate certain proteins and pathways that may be linked with the drug’s mechanism of action.
Methods.
SHP-77–small cell lung carcinoma (epithelial cells, sensitive); H-460 large cell lung cancer
(epithelial cells, sensitive) cells were purchased from NCI (Frederick, MD) and cultured
according to supplier recommendations. Primary immortalized human lung embryonic
fibroblasts (MRC-5); SW 900 – Grade IV, squamous cell lung carcinoma (epithelial cells,
resistant), NCI-H596 – adenosquamous lung carcinoma (epithelial cells, resistant) were
purchased
from
ATCC
(Manassas,
VA)
and
cultured
according
to
the
supplier
recommendations. A panel of 240 cell lines was initially screened by MEI Inc. and the present
lines chosen to represent natural (intrinsic) sensitivity or resistance to ME-344.
Seahorse Bioscience™ experiments
For standard and glycolytic mitochondrial stress test experiments, 96-well plates (XF96,
Seahorse Bioscience, USA) with attached confluent cells were used in the Seahorse
Bioscience™ apparatus in MUSC COBRE Metabolomics Core. Standard protocols for OCR and
ECAR recording were used. Incubation time and ME-344 concentration dependence of OCR
and ECAR were analyzed. The results were normalized for cell number in each well and
presented as mean ±SE, for n=8.
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Tissue culture.
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Cell viabilities and fluorometric detection of caspase-3 protease.
We used a standard Caspase-3/CPP32 Fluorometric Assay Kit from BioVision Inc. (Milpitas, CA
USA) to study the effects of ME-344 induction of apoptosis in lung cancer cells and
immortalized lung fibroblasts. Cells were grown in standard Petri dishes (60 mm) to ~80%
confluence and attached cells (control (DMSO) or ME-344 treated) were washed with PBS (3
times), lyzed, combined in reaction buffer and fluorescent substrate DEVD-AFC and incubated
at 37oC for 1.5h. Finally, samples were placed in a quartz cuvette (5x5x40 mm) and the intensity
nm) recorded using a standard kinetic mode (resolution 0.1 sec) of the QM-4 fluorometer for
200 sec. The resultant emissions were averaged and plotted as MEAN±SD, n=400. We
recorded kinetics of caspase-3 activation during incubation of cells with ME-344 for 0, 2, 4 and 6
hours. Each time point was measured in triplicate and final results were presented as
MEAN±SD.
Viability after incubation with ME-344 for 0, 2, 4, and 6 hours at 37oC were assessed. After
incubation, cells were washed with PBS and detached from Petri dishes with trypsin, optimized
for each cell type. After washing cells with fresh media, their counts/viabilities were determined
using a Cellometer™ Auto T4 (Nexcelom Bioscience LLC, Lawrence, MA) and trypan blue
exclusion. All experiments were in triplicate and results presented as MEAN±SD.
Real time kinetic detection of intracellular ROS and NAD(P)H.
For ROS/NAD(P)H fluorescent analyses, cells were grown as confluent monolayers adherent to
Aclar© plastic slides (14x25 mm). These slides were placed in a quartz cuvette (10x10x40 mm)
with PBS (with 100µM of CaCl2, 37oC) at a 45o angle to excitation light and the dynamics of
NAD(P)H emission (under constant stirring) were detected (Ex 340, Em 460 nm) before and
after ME-344 (or other effectors) in real-time kinetics with a resolution of 0.1 sec using a QM-4
fluorometer (PTI, NJ; (Manevich et al., 2002). In other experiments adhered cells were labeled
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of the DEVD-AFC cleavage product (AFC: 7-amino-4-trifluoromethyl coumarin, Ex/Em=400/505
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with specific dyes (ROS measurements) and similarly analyzed in the QM-4 fluorometer.
Intracellular O2*- was monitored with CellTracker™ Deep Red fluorescent dye (Ex/Em, 630/650
nm, Life technology, CA); intracellular H2O2 generation with H2DCF-DA dye (Sigma); *OH
radical with 3-CCA-DA dye (Manevich et al., 1997). Live cells attached to Aclar® slides were
incubated in complete media with 5µM of the appropriate dye for 45 min at 370C. Slides with
labeled cells were washed with 3 times with PBS to eliminate excess dye before placing in a
quartz cuvette for data recording.
Incubation medium from cell cultures in 6-well plastic plates was replaced with 5% metaphosphoric acid (MPA) at 4ºC. After 3 min, the plates were scraped and extracts centrifuged at
15,000xg for 5 min at 4ºC. The supernatants were then neutralized with 0.1M Tris-acetate
buffer. Adenine nucleotides in the extracts were separated using a Model 1525 Binary Breeze
HPLC pump equipped with a Model 717 Plus Autosampler and detected using a Model 2487
UV-Vis Detector (all from Waters, Milford, MA). Experiments were performed using a C18, 5µm,
4.6x150-mm reverse phase column (SunFire™, Waters), isocratic elution (1ml/min) with 100mM
Na-phosphate buffer (pH 5.5) and UV detection at 260 nm. The MPA lysates (25µl) were
injected into the column and ATP, ADP and AMP were detected at approximate retention times
of 4.1, 4.9 and 11.5 min, respectively (Maldonado et al., 2013). Authenticity of detection was
confirmed by spiking actual samples with known amounts of ATP, ADP and AMP as standards.
Appropriate peak areas were quantified using Empower 2 software (Waters) and quantification
of individual compounds achieved with use of particular calibration curves with purified ATP,
ADP and AMP standards.
HPLC analysis of GSSG.
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HPLC analyses of ATP, ADP and AMP.
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Attached cells were incubated with ice-cold 5% (final concentration) MPA. After 3-5 min, cells
were scraped, and the extracts centrifuged at 15,000g for 5 min at 4 °C. The supernatants were
collected and used for analysis. GSSG in the extracts was detected using an HPLC method
similar to that described for ATP (ADP, AMP) analyses. The supernatants from cell lysates
treated with MPA (25µl) were injected into the column, and the GSSG peak was detected
(absorbance at 260 nm) at a retention time of ~2.85 min. Authenticity of detection was
confirmed by spiking actual samples with known amounts of GSSG as standards. Peak areas
were quantified using Empower 2 software (Waters) and quantification of GSSG was achieved
Fluorescent analysis of low molecular weight and protein reduced thiols.
Detection and quantification of sulfhydryls is based on their specific reaction with the maleimide
probe TG-1 (Calbiochem), which becomes fluorescent (Bowers et al., 2012). Cell lysates were
passed through a SEC micro-column (Bio-Spin 6; cut off ~6 kDa, Bio-Rad) to separate proteins
from lower molecular weight thiols. Both SEC processed and intact samples were diluted in a
quartz cuvette 1:100 (by volume) with 20mM PB (pH7.4) at 370C under constant stirring in a
sample holder of the QM-4 fluorometer. After addition of TG-1 (DMSO solution, final
concentration 5μM), the fluorescent detection (Ex 379 nm, Em 513nm) of labeled sulfhydryls
was performed using a standard kinetic mode of the QM-4 to reach saturation. Where pertinent,
background fluorescence’s of cell lysates, effects of DMSO and background fluorescence of
TG-1 in 20mM PB (pH 7.4 at 370C) were subtracted from the final results. The final emission
values were averaged for 50s (500 time points) using Sigma Plot 10.0 software (Systat
Software, Inc., San Jose, CA, USA) and presented as the mean ±SD.
Fluorescent analysis of cell surface thiols.
Either lung cancer or immortalized primary (MRC-5) cells attached to Aclar© plastic slides
(~85% confluent) were treated with ME-344 for 30 min in complete media. After washing these
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using calibration curves with purified GSSG.
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slides in PBS (with 100uM of CaCl2) they were placed in a quartz cuvette with PBS (with 100uM
CaCl2) at 4oC (to prevent TG-1 endocytosis) similar to that for NAD(P)H detection. Both before
and after addition of 5µM TG-1, emissions were detected in real-time kinetics until saturation.
Background fluorescence for the cells and TG-1 were each subtracted from the final results.
Samples were also corrected for DMSO and the final TG-1 fluorescence was normalized for cell
number and presented as mean ±SD for 3 independent experiments. After those
measurements, cells were detached from the slides and their counts/viabilities estimated using
Cellometer™ Auto T4 (Nexcelom Bioscience LLC, Lawrence, MA) and trypan blue exclusion.
treatment with ME-344 (30 min) and TG-1.
Mitochondrial membrane depolarization measurements in real-time kinetics.
Cells on the Aclar© plastic slides (~85% confluent) were incubated with 5.0µM of JC-9
fluorescent dye (Life technology, CA) in complete media for 45 min at 370C. Labeled cells were
washed 3 times with PBS containing 150µM CaCl2 and placed in a quartz cuvette similar to the
above measurements for NAD(P)H dynamics. JC-9 fluorescence was measured as a ratio of
(485/529nm monomeric dye fluorescence) to (535/590nm J-aggregate fluorescence) in realtime kinetics before and after addition of indicated amounts of those agents shown (ME-344 and
glucose).
Statistical analysis.
We used SigmaStat 3.5 (Systat Software, Inc., San Jose, CA, USA) for statistical analysis of
data. One-way ANOVA was used for statistical relevance of data comparison.
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Cell viabilities under our experimental conditions were very close (98-99%) before and after
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Results.
Recent work has implicated inhibition of mitochondrial complex I as a possible contributory
mechanism for this class of drugs (Lim et al., 2015). We now provide further interrogation
through SeaHorse technology and mitochondrial-stress testing measurements of the effects of
ME-344 (Fig. 1) on oxygen consumption and extracellular acidification rates (OCR and ECAR)
in drug-sensitive (SHP-77 and H460) and resistant (H596 and SW900) lung cancer and IHLEF
(MRC-5) cell lines.
ME-344 caused a substantial and essentially instantaneous inhibition of OCR (similar in extent
to oligomycin) in the lung cancer cell lines, correlating with the natural sensitivity of cells to the
drug. Application of an uncoupler p-triflouromethoxyphenylhydrazone (FCCP) caused a fast
recovery in the drug-resistant lung cancer cells, but was substantially retarded in the sensitive
counterpart (Fig. 2, panel A). Subsequent addition of antimycin A and rotenone completely
inhibited respiration, with the effect more pronounced in resistant cells. Mitochondrial stress
testing of the same cells revealed a substantial increase of ECAR in resistant cells after ME-344
(Fig. 2, panel B), an effect that was barely detectable in sensitive cells. The impact of other
standard stressors on ECAR was similar for both sensitive and resistant cell lines (Fig. 2, panel
B).
A further interesting difference was found between the resistant and sensitive cells. While
comparing the effects of oligomycin on cells treated with either DMSO or ME-344, we measured
OCR inhibition and normalized it for the OCR value before oligomycin addition. Pretreatment of
cells with ME-344 inhibited the effects of subsequent oligomycin, but only in the sensitive cell
lines. In the resistant cells, ME-344 was shown to potentiate the effects of oligomycin. (Fig. 2,
panel C). The effects of ME-344 on OCR were concentration-dependent and reached saturation
at ~57.4µM (ED50~11.5 µM, Fig. 2, panel D).
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Mitochondrial stress in lung cancer cells.
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Glycolytic mitochondrial stress test.
We next studied the effect of ME-344 on glycolysis in the lung cancer cells. Pretreatment of
sensitive lung cancer cells resulted in an increase of ECAR (Fig. 3, panel A) after addition of
glucose. Interestingly the effects of oligomycin treatment were suppressed in sensitive lung
cancer cells following pretreatment with ME-344 (Fig. 3, panel B).
(Fig.3, panel B) compared to resistant (averaged H596 and SW900) lung cancer cell lines (Fig.
3, panel B). While ME-344 decreased the glycolytic response in IHLEF MRC-5 cells (Fig. 3,
panel B), in resistant cells, there was a greater decrease (Fig. 3, panel B). Subsequent
treatments with oligomycin caused a pronounced inhibitory response in both sensitive and the
primary MRC-5 cells and an increased response in resistant cells (Fig. 3, panels B). The effects
of ME-344 on glycolysis were concentration dependent (Fig. 3, panel C) in the sensitive cells
and reached saturation at ~57.4 µM (ED50~11.5 µM) a value similar to that for OCR. Our data
also suggest that ME-344 had dose dependent effects upon glycolytic stress that were different
between the immortalized primary cells and drug resistant cell lines (ED50~11.5 µM, Fig. 3,
panel D).
Bioenergetic and redox effects in cancer and immortalized primary cells.
Following ME-344, ATP levels were decreased ~12% in sensitive cells, with essentially no
depletion in resistant cells (Fig. 4, panel A). ME-344 also mildly altered redox stress in sensitive
compared to resistant cells (as detected by GSSG levels; Fig. 4, panel B). Initial ATP to ADP
hydrolysis was two-fold lower in primary cells (Fig.4, panel C) when compared to sensitive cells
(Fig. 4 panel D). ATP to ADP hydrolysis and redox stress (GSSG) were practically unaffected by
ME-344 in primary cells (Fig. 4, panel C) but the former was dose-dependently facilitated in
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Initial ECAR values under conditions of glucose deprivation were lower in IHLEF (MRC-5) cells
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sensitive lung cancer cells (Fig. 4, panel D). These data again imply a degree of specificity for
ME-344 effects in sensitive lung cancer cells.
Cytotoxicity and apoptosis.
Cytotoxicity of ME-344 was assessed through its effects on apoptosis and viability. The drug
rapidly (two hours) induced apoptosis in the sensitive cell lines (H460 and SHP-77; Fig.5, panel
A) through an activation of caspase-3 in a time dependent manner. In contrast, there was
minimal (if any) induction of apoptosis in the IHLEF (MRC-5) and resistant cells. Fig.5, panel B
IHLEF (MRC-5) these effects were minimal. Fig. 5, panel C shows the dose effects of ME-344
on caspase-3 activation in all cell types. The ED50 of ME-344-mediated caspase-3 activation
occurred at 2.13µM. Fig. 5, panel D shows viability of sensitive, resistant lung cancer and IHLEF
after 24h incubation with 11.48µM of ME-344. Approximately 90% of the sensitive cells were
killed, but only minimal cytotoxicity was apparent in resistant and IHLEF cells.
ME-344 effects on intracellular oxidant stress.
ME-344-mediated inhibition of OCR may contribute to oxidative stress through impairment of
the mitochondrial electron transfer chain. ME-344 induced pronounced, concentrationdependent generation of H2O2 and *OH in sensitive cells (SHP-77 and H460; ED50 ~11.5 µM,
Fig. 6, panels A - C). This effect was not observed in resistant cells and was minimal in IHLEF
(Fig. 6, panels B and D), implying some degree of specificity of ME-344 towards the sensitive
lung cancer cells. ME-344 treatments did not lead to superoxide anion radical generation in
either lung cancer cells or IHLEF (Fig. 7, panel A).
ME-344 effects on general redox stress.
NAD(P)H is a universal intracellular source of electrons for redox reactions. Both NADH (mainly
mitochondrial) and NADPH (mainly cytosolic) have indistinguishable biofluorescences. Dynamic
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shows that progressive death of sensitive cells occurred after ME-344, but in resistant cells and
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changes in NAD(P)H fluorescence frequently accompany active redox signaling and have been
used previously for evaluation of drugs that impact intermediate metabolism (Biaglow et al.,
1998). Our present data indicate that under conditions of glucose deprivation, ME-344 treatment
of sensitive cells (H460) caused a rapid increase and subsequent slow decrease of NAD(P)H to
below normal resting levels (Fig. 7, panel B, red trace). As detected by fluorescence
measurements, subsequent addition of glucose to the same cells resulted in a rapid increase in
the generation and cycling of NAD(P)H (Fig. 7, panel B, red trace). A similar dose of glucose
given before ME-344 did not alter NAD(P)H, but further addition of ME-344 induced a similar
Such effects did not occur in resistant (H596, Fig.7 panel B, black trace) or IHLEF (not shown).
Since proton fluxes control mitochondrial functions, we measured the dynamics of inner
mitochondrial membrane potential (IMM) changes (ΔΨmit) in sensitive (H460), resistant (SW900)
cells and IHLEF using a ratiometric fluorescent dye JC-9 (Fig. 7, panels C and D). Our data
showed rapid hyper-polarization of IMM immediately following ME-344 addition (under glucose
deprivation conditions), which was significant (~12%) and sustained in sensitive (H460) cells
(Fig.7, panel C). In IHLEF and resistant cells the effects were smaller (~5%), and hyperpolarization was followed by rapid depolarization of IMM, which was slower and of smaller
intensity in resistant cells (Fig. 7, panel D). In the cancer cells the addition of glucose after ME344 caused a fast, sustained depolarization of IMM almost up to resting levels (Fig. 7, panel C).
Similar treatments of IHLEF caused slower depolarization of IMM, exceeding resting levels by
~6% and further hyperpolarization of IMM in resistant cells (Fig.7, panel D). These results again
suggest that ME-344 has some degree of specificity for effecting mitochondrial proton fluxes in
sensitive cells.
ME-344 effects on intra- and extra-cellular redox states.
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rapid increase and subsequent cycling of NAD(P)H fluorescence (Fig. 7, panel B, green trace).
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The dynamics of ME-344-mediated induction of oxidant stress (H2O2 and *OH, Fig. 6, panel A
and C) were further investigated using fluorescent detection of intracellular thiol oxidation. The
data show a progressive increase of redox stress (corresponding with decreases in reduced
thiols) in lung cancer cells with saturation approximately one hour after drug treatment (Fig. 8).
Maximal decreases in reduced thiols were seen in sensitive cells (Fig. 8, panel A). Interestingly,
ME-344 induced redox stress was reversible in IHLEF (Fig. 8, panel B) inferring a plausible
therapeutic advantage. ME-344 triggered pronounced oxidation of cell surface thiols with similar
kinetics and magnitude (~40-50% in one hour) in both sensitive cells and IHLEF (Fig. 8, panels
Discussion.
Because of the hydroxyl constituents on its phenol rings (Silasi et al., 2009), ME-344 has some
properties that distinguish it from most of this family of chemicals. Of particular importance, our
present results demonstrate that at low micromolar concentrations, cytotoxic effects are not
induced in either immortalized normal lung fibroblasts or in lung cancer cell lines that have
intrinsic resistance to the drug. Since ME-344 is in early stage clinical trials, there would be
benefit in defining mechanism(s) of action. In the present study, we describe a series of drug
effects, which may be associated with a beneficial therapeutic index and can be linked with the
unusual cytotoxic properties of the drug. Previous work has identified inhibition of energy
production downstream of isoflavone effects, so we focused our attention on redox signaling
and its consequent impact on bioenergetics in lung cancer cell lines sensitive and resistant to
the drug. The cell lines were chosen from a preliminary screen of over 200 where those
identified as resistant have not been previously exposed to drug selection.
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C and D).
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Energy production through the mitochondrial respiratory chain requires an intensive flux of
protons from the matrix into the inner-membrane space. Inhibition of respiration through
impairment of electron flow diminishes this proton flux and causes IMM depolarization (Alberts,
2002). ME-344 can inhibit complex I of the respiratory chain and cause dissipation of inner
mitochondrial membrane potential (Lim et al., 2015). Our OCR inhibition data indicate that
sensitive lung cancer cells follow a similar response pattern. Instead of depolarization, ME-344
causes hyper-polarization of the inner mitochondrial membrane serving to minimize the
inhibitor of the F0 component (proton pump) of ATP synthase, which regulates proton efflux into
the matrix from the inter-membrane space (Shchepina et al., 2002). In the sensitive cells, ME344 may be targeting the oligomycin-sensitive F0 component of ATP synthase and some degree
specificity is implied, since ATP levels and redox signaling were affected only in these sensitive
cells. On the basis of dose, the efficacy of ME-344 is less than that of oligomycin (compare
11.5µM to 1.0µM), but the specificity of this effect may be beneficial for the eventual therapeutic
index of ME-344. Also in sensitive lung cancer cells ME-344 dose dependently and effectively
(~50% at 23.0µM, 30 min incubation) facilitated ATP hydrolysis. This did not occur in IHLEF
cells perhaps again indicating some degree of selectivity for transformed cells. Taken together,
these data are consistent with one interpretation to suggest that ME-344 targets the inner
mitochondrial membrane proton channel (F0 component of ATP synthase), causes reduced
levels of ATP production, alters proton fluxes and initiates inner membrane hyperpolarization.
This would provide a rational explanation for the unusually rapid and immediate effects of ME344 on bioenergetics and redox signaling in these cells. In this regard, there are existing
examples of other isoflavones that target ATP synthase (Zheng and Ramirez, 2000; Chinnam et
al., 2010).
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subsequent impact of oligomycin on OCR and ECAR. In this context, oligomycin is a known
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Under hypoxic conditions glycolysis is a complementary pathway for cancer cells to generate
ATP. We showed that resting ECAR in immortalized primary cells was ~2-fold lower than in
cancer cells. With glucose deprivation, ME-344 had little effect on ECAR in any of the cells.
Glycolytic rates in sensitive lung cancer cells were ~54.0 µpH/min but because of the Warburg
effect (Vander Heiden et al., 2009) were expectedly lower in IHLEF cells (~12.0 µpH/min with
non-glycolytic acidification at ~4.0 µpH/min). In resistant lung cancer cells ME-344 did not
evince any glycolytic response. While this could be interpreted to suggest a direct impact of ME344 on glycolysis, at this stage, such a conclusion would be premature. In addition, the results
synthase (following oligomycin addition) only in the sensitive cells and IHLEF.
Two other effects of ME-344 in sensitive cancer cells are described. Drug treatment was linked
with increases in intracellular hydroxyl radicals (*OH) and NAD(P)H. Generation of *OH is
surprising, because traditionally isoflavones are not viewed as pro-oxidants. Biological formation
of *OH radicals can occur through Fenton chemistry, where transition metals react with H2O2 or
other organic peroxides (Winterbourn, 1995). In our present study we show that ME-344
increases H2O2 generation in cytosol and such a process would be consistent with interference
with the mitochondrial respiratory chain and subsequent leakage of electrons (Halliwell et al.,
1992) causing cytosolic redox stress. Again, this would be consistent with our data showing
progressive thiol oxidation. Moreover, this process has been previously shown to induce heme
oxygenase (HO-1; (Choi and Alam, 1996)). In addition, Park et al showed that certain
isoflavones can induce HO-1 in primary astrocytes (Park et al., 2011) and this can be
accompanied by its translocation from the ER to mitochondria (Bindu et al., 2011; Bansal et al.,
2014). The first intermediate step of HO-1 catalysis produces Fe2+, an effective component of
the Fenton reaction. It is plausible that this sequence of events could be involved in the
mechanism by which ME-344 induces cytotoxicity. The precise chain of events by which ME344 impacts HO-1 induction and/or translocation to mitochondria requires further investigation,
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for estimating glycolysis (Fig. 3, panel B) indicate that the drug may selectively inhibit ATP
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but it is perhaps pertinent that HO-1 has been suggested as a possible anticancer target (Choi
and Alam, 1996).
The rapid drug-induced increases in NAD(P)H could also result from direct or indirect effects on
the F0 component of ATP-synthase. In turn, this could be linked with IMM hyper-polarization and
compensatory proton fluxes, which could influence nicotinamide nucleotide transhydrogenase
(NNT), an enzyme localized in the inner mitochondrial membrane catalyzing NADH-mediated
reduction of NADP+ to NADPH (Mather et al., 2004; Gameiro et al., 2013). NNT-controlled
(Gameiro et al, 2013). In concert with some of the other results, the immediacy of the effects of
ME-344 on NAD(P)H in the sensitive lung cancer cells could also imply that the drug has a
direct or indirect impact on this enzyme, although further experimentation in this area will be
required.
Earlier genetic characterizations of the lung cancer cell lines used here shed a little light on
targeting redox pathways and their plausible influence drug response data. For example, SHP77 cells express the cell surface markers that are reminiscent of HLA-DR macrophages and
hematopoietic cells and these may influence cell attachment (Ruff et al., 1986). These are not
found in H460 (NSCLC) cells and may help to explain cell surface thiol differences (Davidson et
al., 2004) and perhaps influence ME-344 sensitivities (likely only the magnitude of such
responses). In the resistant cells, SW900 (Wesley et al., 2004) and H596 cells (Nguyen et al.,
2015) are each characterized by the Notch1 trans-membrane receptor, linked with the malignant
phenotype and absent from the sensitive cell lines. In theory, this difference might also influence
drug response. In general, however, the quantitative aspects of the differences in response to
ME-344 are unlikely to be a function of simple monogenic differences between the cell lines.
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catalysis is dynamic, efficient and does seem to parallel the instantaneous effects of ME-344
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In summary, our study shows that ME-344 is an unusual isoflavone with mitochondria-centered
activities engendering pronounced and specific inhibition of bioenergetics and redox signaling
pathways in lung cancer cells that are sensitive to the drug. At equivalent concentrations these
effects do not occur in naturally resistant or immortalized primary cells. Two major mitochondrial
enzymes: ATP-synthase and NNT are potentially direct or indirect targets for ME-344.
Hypothetically, translocation of HO-1 into mitochondria as well as its activity could also be
influenced by ME-344 and further studies in this area are warranted. The clinical use of ME-344
either as a single agent or in drug combinations may be contingent upon the cancer cell’s
dependent upon glycolysis would be more responsive to the drug. In addition, the drug has
lower toxicities in immortalized primary cells indicating that at least in vitro, an advantageous
therapeutic index may be attainable.
Acknowledgements.
The authors also wish to thank Dr. C. Beeson, Ms. G. Beeson and Mr. B. Hoover of the
Metabolomics Core of the COBRE for their help and advice in conducting SeaHorse
experiments.
Authorship contributions.
Participated in research design: Manevich, Britten, Townsend, Tew
Conducted experiments: Manevich, Reyes, Townsend.
Performed data analysis: Manevich, Townsend, Tew.
Wrote or contributed to the writing of the manuscript: Manevich, Tew.
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balance between oxidative phosphorylation and glycolysis. As a prediction, patient tumors more
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Footnotes
This work was supported by grants from the National Institutes of Health [Grants CA08660,
CA117259, and NCRR P20RR024485 - COBRE in Oxidants, Redox Balance and Stress
Signaling] and support from the South Carolina Centers of Excellence program and was
conducted in a facility constructed with the support from the National Institutes of Health, [Grant
RR015455] from the Extramural Research Facilities Program of the National Center for
Research Resources. Two of us CDB and KDT hold endowed Chairs from the South Carolina
SmartState program. Supported in part by the Drug Metabolism and Clinical Pharmacology
shared Resource, Hollings Cancer Center, Medical University of South Carolina. Further
financial support was through an unrestricted grant from MEI Pharma Inc.
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Legends for Figures.
Fig. 1. Structure of ME-344.
Fig. 2. Standard mitochondrial stress test traces for lung cancer cells. OCR (panel A) and
ECAR (panel B) changes upon addition of: ME-344 (4.0µg/ml), O (oligomycin, 0.5µM), F
(FCCP, 1.0µM), and A+R (antimycin A, 1.0µM + rotenone, 1.0µM) to lung cancer H460
(sensitive) and H596 (resistant) cells in standard SeaHorse® 96-well microplates. The traces are
representative of 6 independent experiments (results were essentially the same for SHP-77
Panel C: ME-344 effects on ATP synthase inhibition by oligomycin. Presented as a difference
(%) between OCR with or without ME-344 before and after oligomycin. Data are normalized for
values before oligomycin addition and represent mean ±SD for 3 independent experiments, &,
ε
and # represent statistical significance p≤0.05. Panel D: ME-344 dose effects on OCR changes
(% of initial OCR w/o effector). The data were averaged for either sensitive or resistant cells and
presented as mean ±SE for 6 independent experiments, p≤0.009.
Fig.3. Effects of ME-344 on glycolysis in lung cancer and IHLEF cells. Glycolysis stress
tests were presented as ECAR changes upon addition of: ME-344 (11.4μM), Glu (glucose,
10.0mM), O (oligomycin, 1.0µM), and 2-DG (2-deoxy-glucose, 10.0mM) using SHP-77 lung
cancer cells (sensitive, panel A). Trace is representative of 6 independent experiments. The
averaged differences between ME-344 (11.48 µM) and DMSO (same volume)-treated sensitive
(H460 and SHP-77), IHLEF MRC-5, and resistant (H596 and SW900) cells are presented in
panel B. The differences are statistically significant only where sensitive cells are compared with
either resistant or IHLEF cells (p≤0.001). Data were normalized for cell numbers. Panel C: ME344 dose effects on glycolysis (% of initial ECAR changes after glucose addition, averaged for
sensitive (H460, SHP-77) cells). Panel D: ME-344 dose effects on glycolysis (% of initial ECAR
changes after addition of glucose) averaged for IHLEF (MRC-5) and resistant (H596, SW900)
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(sensitive) and SW900 (resistant) lung cancer cells). Data were normalized for cell numbers.
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cells. All data are presented as mean ±SD for 6 independent experiments, and are statistically
different (p≤ 0.001).
Fig.4. Effects of ME-344 on bioenergetics and redox stress in IHLEF and lung cancer
cells. HPLC analyses of ATP (panel A) and GSSG (panel B) in intact (DMSO, black bars) and
ME-344 treated (11.48 µM), white bars) sensitive (SENS) or resistant (RES) cell lysates. Data
were averaged for sensitive (H460, SHP-77) or resistant (H596, SW900) cells and presented as
mean ±SD for 3 independent experiments. HPLC analyses of ME-344 dose effects on ATP/ADP
(MRC-5, panel C) or sensitive cells (H460, panel D) are presented as mean ±SE for 3
independent experiments. ATP/ADP changes in sensitive (H460) cells were statistically different
(p≤0.05).
Fig. 5. Me-344 effects on caspase-3 activation and cell viability. The kinetics of ME-344
(11.4μM) inhibition of caspase-3 activity (normalized for DMSO) in: H596 and SW-900
(resistant; empty squares and circles), H460 and SHP-77 (sensitive; filled squares and circles)
and MRC-5 (IHLEF, filled stars) are shown in Panel A. The temporal viability of sensitive (filled
squares and circles), resistant (empty squares and circles) cells and of IHLEF (MRC-5, filled
stars) after ME-344 (11.4μM) is presented in panel B. The dose effects of ME-344 on caspase-3
activity in all cell types are presented in panel C. The viabilities of sensitive (H460), resistant
(H596) cells and IHLEF (MRC-5) after 24h incubation with ME-344 (5.74 and 11.48 µM) are
presented in panel D. Data are mean ±SD for 3 independent experiments.
Fig.6. ME-344 treatments linked with hydrogen peroxide and *OH generation. ME-344
(11.4μM) effects on intracellular H2O2 (*OH) generation in H460 sensitive (black traces) and
H596 resistant (gray traces) cells in real time kinetics are presented in panels A and C. The
traces are representative of three independent experiments and similar for SHP-77 (sensitive)
and SW900 (resistant) cells. The averaged dose effects of ME-344 on intracellular generation of
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ratios (black bars) and GSSG (normalized for its value before treatment, gray bars) in IHLEF
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averaged H2O2 (*OH) in sensitive (H460 and SHP-77, filled circles), resistant (H596 and SW900,
empty circles) cells and IHLEF (MRC-5, filled stars) are presented in panels B and D as mean
±SD for 3 independent experiments, where the effects are statistically different (p≤ 0.001). The
lines represent sigmoid fit (3 parameters) using Sigma Plot software. The arrows indicate points
where effector was added.
Fig.7. ME-344 effects on the dynamics of superoxide radical generation, NAD(P)H redox
and inner mitochondrial membrane potentials (Ψin). Panel A: ME-344 (11.4μM) addition
dye in real-time kinetics) in H460 (sensitive; black trace) and H596 (resistant; gray trace) cells.
Panel B: ME-344 (11.4μM) effects on NAD(P)H emission either before (red trace) or after (green
trace) glucose (Glu, 2.0mM) as indicated by arrows in sensitive (H460) cells or in IHLEF (MRC5, black trace). Traces are representative of three independent experiments and similar for
SHP-77 (sensitive, not shown) cells. Panels C and D: effects of ME-344 (11.4μM) and glucose
(2.0mM, arrows) on the IMM potentials of sensitive (H460, solid line) cells, IHLEF (MRC-5,
dotted and dashed line) and resistant (SW900, dashed line) cells; detected with JC-9 ratiometric
fluorescent dye in real-time kinetics. Traces are representative of 3 independent experiments
and were similar for SHP-77 sensitive and H596 resistant cells.
Fig. 8. ME-344 effects on dynamics of intra- and extra-cellular thiol redox status in lung
cancer and IHLEF cells. Averaged reduced thiol (sulfhydryls) dynamics in lysates of sensitive
(H460 and SHP-77, panel A), and IHLEF (MRC-5, panel B) before (DMSO control [CTR], filled
circles and stars) and after incubation with ME-344 (22.8μM, empty circles and stars), analyzed
with sulfhydryl-specific fluorescent dye TG-1. Emissions were normalized for protein
concentrations and presented as mean ±SE for 3 independent experiments. Sensitive (H460
and SHP-77 cells, panel C) and IHLEF (MRC-5, panel D) were treated with ME-344 (22.8μM)
for the indicated times and cell surface thiol (sulfhydryls) were detected with TG-1. Emissions
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does not affect intracellular superoxide radical generation (detected with Deep Red fluorescent
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were normalized for cell numbers, averaged and presented as mean ±SE for 3 independent
experiments.
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