-
Small Molecule Therapeutics
Mitochondrial Targeting of Metformin EnhancesIts Activity
against Pancreatic CancerStepanaBoukalova1, Jan Stursa2,
LukasWerner2,3, Zuzana Ezrova1, Jiri Cerny1,
AyenachewBezawork-Geleta4, Alena Pecinova5, Lanfeng Dong4, Zdenek
Drahota5, and Jiri Neuzil1,4
Abstract
Pancreatic cancer is oneof the hardest-to-treat types
ofneoplasticdiseases. Metformin, a widely prescribed drug against
type 2diabetes mellitus, is being trialed as an agent against
pancreaticcancer, although its efficacy is low. With the idea of
deliveringmetformin to its molecular target, the mitochondrial
complex I(CI), we tagged the agent with the mitochondrial vector,
triphe-nylphosphonium group. Mitochondrially targeted
metformin(MitoMet) was found to kill a panel of pancreatic cancer
cells three
to four orders of magnitude more efficiently than found for
theparental compound. Respiration assessment documented CI as
themolecular target for MitoMet, which was corroborated by
molec-ular modeling. MitoMet also efficiently suppressed
pancreatictumors in three mouse models. We propose that the novel
mito-chondrially targeted agent is clinically highly intriguing,
and it has apotential to greatly improve the bleak prospects of
patients withpancreatic cancer. Mol Cancer Ther; 15(12); 2875–86.
�2016 AACR.
IntroductionCancer is one of the most serious pathologies in
industrialized
countries with rather grim prognosis (1). Emerging
evidenceindicates that cancer is primarily a metabolic disease
arising inresponse to disturbance in cell energy homeostasis (2,
3). Manyproto-oncogenes and tumor suppressors have been shown
toregulate cell metabolism (4). A link between metabolic
disordersand cancer is supported also by epidemiological evidence
indi-cating that pathologies like type 2 diabetes mellitus (T2DM)
areassociated with increased risk of different types of
malignancies,pancreatic cancer being a prime example (5).
Retrospective aswellas epidemiological and clinical studies
indicate that therapy withmetformin, the first-line drug of choice
for treating T2DM, isassociated with decreased incidence of cancer
and increasedsurvival rate of patients with different types of
tumors (6–9).
Metformin is considered to be a promising drug in regard
totreatment and prevention of pancreatic cancer, one of the
mostfatal humanpathologies (10–12). The agent, which has been
usedfor therapy of diabetes since 1950s (13), is recognized as a
safedrug. Recent in vitro studies have demonstrated that
metforminacts directly on tumor cells to suppress their
proliferation(14–16), targeting also pancreatic cancer stem cells
(17). Theclinical impact of these observations is currently
unclear, as the
antiproliferative effects of metformin become apparent only
atsupra-pharmacological concentrations of the drug (18).
At the molecular level, metformin primarily targets
mitochon-dria, inhibiting complex I of the respiratory chain (19,
20).Alternations inmitochondrial function are believed to be
respon-sible for anticancer effects ofmetformin, as it restricts
the ability oftumor cells to cope with energetic stress (21). This
concept issupported by emerging literature showing that
mitochondrialfunction is tightly linked to cancer (22).
Recent reports document that mitochondrial respiration
isimportant for tumor initiation, progression, and
metastasis(23–25). This led us to coin the hypothesis that
targeting mito-chondrial complexes may be an efficient way to treat
cancer(26, 27). With this in mind, we designed, synthesized, and
testedfor their anticancer efficacy severalmitochondrially targeted
drugsacting via mitochondria that were tagged with
triphenylpho-sphonium (TPPþ; refs. 28, 29). This delocalized
cationic groupanchors small molecules with pro-oxidant function at
the inter-face of the mitochondrial inner membrane (MIM) and
matrix(30), allowing the drugs to accumulate at their primary site
ofaction. We now applied TPPþ tagging to metformin in order
tomaximize its activity, and report that this approach
enhancestoxicity of the parental drug towards pancreatic cancer
cells bythree to four orders of magnitude, making
mitochondriallytargeted metformin (MitoMet), an exceptionally
promisinganti-cancer agent.
Materials and MethodsFor information regarding the number of
cells seeded in each
experiment, see Supplementary Table S1.
Chemicals and reagentsAll chemicals were purchased from
Sigma-Aldrich if not stated
otherwise. Stock solutions of metformin (Enzo Life Sciences)
andthe TPPþ-modified compounds were prepared by their dissolvingin
water. For animal experiments, the compounds were dissolvedin
PBS.
1Institute of Biotechnology, Czech Academy of Sciences,
Vestec,Czech Republic. 2Institute of Chemical Technology in Prague,
CzechRepublic. 3Biomedical Research Centre, University Hospital
HradecKralove, Czech Republic. 4School of Medical Science, Griffith
Univer-sity, Southport, Qld, Australia. 5Institute of Physiology,
Czech Acad-emy of Sciences, Prague, Czech Republic.
Note: Supplementary data for this article are available at
Molecular CancerTherapeutics Online
(http://mct.aacrjournals.org/).
CorrespondingAuthors: Jiri Neuzil, Griffith University, Parkland
Avenue, South-port, Qld 4222, Australia. Phone: 07 55529109; Fax:
07 55528444; E-mail:[email protected]; and Stepana
Boukalova, [email protected]
doi: 10.1158/1535-7163.MCT-15-1021
�2016 American Association for Cancer Research.
MolecularCancerTherapeutics
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Synthesis of TPPþ-tagged metforminThe synthesis and
physico-chemical properties of MitoMet
(compound 9), norMitoMet (compound 7), C6 MitoMet (com-pound10),
andC6norMitoMet (compound8; Fig. 1) is describedin detail in the
SupplementaryMethods. 11C-TPPwas prepared asdescribed earlier
(31).
Cell cultureA panel of human pancreatic cancer cell lines and
nonmalig-
nant control cell lines was used. PANC-1, MiaPaCa-2,
BxPC-3,AsPC-1 cells, BJ skin fibroblasts, MRC-5 lung fibroblasts,
MCF-10A breast epithelial cells, and EA.hy926 endothelial
hybridomacells were purchased from ATCC within last 6 years. PaTu
8902cells were obtained from DSMZ in 2011. HFP1 skin
fibroblastswere a kind gift from K. Smetana (Institute of Anatomy,
CharlesUniversity, Prague, Czech Republic) (32). The last
authenticationof the cell lines was performed in 2016 using STR
profiling. Cellswere routinely cultured in DMEM (PANC-1, PaTu 8902,
BJ, MRC-5, HFP1, and EA.hy926; Lonza) or RPMI (BxPC-3 and
AsPC-1cells; Lonza) supplemented with 10% FBS (Life
Technologies),nonessential amino acids (Life Technologies),
L-glutamine, andantibiotics, at 37�Cand5%CO2.MiaPaCa-2 cellswere
cultured inGibco DMEM (Life Technologies) with the same
supplementsas for the other cell lines. DMEM containing 5% horse
serum, 20ng/mL epidermal growth factor (Life Technologies), 0.5
mg/mLhydrocortisone, 100 ng/mL cholera toxin, 10 mg/mL insulin,
andantibiotics was used for MCF10A culture.
Crystal violet assayThe effect of tested compounds on cell
proliferation was
assessed by the crystal violet assay. Cell were exposed to
theagents for 48 hours, unless stated otherwise, and fixed with
4%
paraformaldehyde in PBS for 20 minutes at 37�C. Cells werethan
washed with PBS, stained with crystal violet (0.05% inwater) for 1
hour. After 3 washing cycles, the crystal violet dyewas extracted
with 1% SDS and absorbance was determined at595 nm.
Impedance based assay/real-time cell analysisCells were seeded
in the e-plate 96 (ACEA Biosciences) in 100
mL media per well and were transferred into xCelligence
real-timecell analysis (RTCA) SP station (ACEA Biosciences) located
inhumidified 37�C chamber with 5%CO2. Tested compoundswereapplied
24 hours post-plating. Impedance was measured indefined intervals
for 100 hours. The data were evaluated usingthe RTCA software.
Western blot analysisTreated cells and nontreated controls were
harvested and lysed
in RIPA buffer supplemented with protease and
phosphataseinhibitors. Protein lysates were separated by SDS-PAGE
andtransferred to a nitrocellulose membrane (Bio-Rad
Laboratories).After probing with specific antibodies, proteins were
detectedusing SuperSignal West Femto Maximum Sensitivity Substrate
orPierce ECL WB Substrate (Thermo Scientific). Primary
antibodiesfor cleaved caspase-3 (#9664), AMP-activated protein
kinase a(AMPK; #2532), phospho-AMPKa (p-AMPK; Thr172;
#2531),acetyl-CoA carboxylase (ACC; #3662), p-ACC (Ser79;
#11818),raptor (#2280), p-raptor (Ser792; #2083), mammalian target
ofrapamycin (mTOR; #2972), p-mTOR (#2971), and b-actin(#5125) were
purchased from Cell Signaling. PANC-1 cells usedfor AMPK pathway
analysis were cultivated in media withdecreased glucose
concentration (1 g/L). Western blot (WB)
Figure 1.
Structures of the compounds used in this study.
Boukalova et al.
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signals were quantified using Quantity One analysis
software(Bio-Rad).
Cell death assessmentPANC-1 or BJ cells were seeded in 12-well
plates and allowed to
attach overnight. After 48-hour incubation with
norMitoMet,metformin or vehicle, both adherent and floating cells
werecollected, washed with PBS, resuspended in 100 mL of
annexinbinding buffer and incubated with 0.3 mL fluorescein
isothiocy-anate (FITC)-labeled annexin V (Apronex) for 30 minutes.
Pro-pidium iodide (PI) was added to identify cells with
disruptedplasma membrane. Annexin V-positive fraction was
determinedbyflow cytometry (FACSCalibur or LSRFortessa;
BDBiosciences).
Generation of stable NDI1 transgenic lineNDI1 and control pWPI
vectors containing GFP were trans-
fected into HEK293T cells using lipofectamine 3000
(Invitrogen)together with psPAX2 and pMD2.G packaging vectors. The
result-ing lentiviruses were used for transduction of parental PaTu
8902cells. Fluorescence-activated cell sorting for GFP-positive
cellsusing FACS Aria Fusion (BD Biosciences) was performed to
selectfor NDI1-expressing and control cells.
High-resolution respirometryRespiration of intact cells andCI,
complex II (CII) or glycerol-3-
phosphate dehydrogenase (G3PDH)-specific respiration in
per-meabilized cells was assessed using Oxygraph-2k
(Oroboros).Procedure details are described in the Supplementary
Methods.
Glycerol-3-phosphate dehydrogenase-mediated respiration inbrown
adipose tissue mitochondria
Newborn 10-day-old rats of Wistar strain were used to
obtaininterscapular brown adipose tissue. The tissue was
homogenizedinmedia containing 320mmol/L sucrose, 10mmol/L Tris-HCl,
1mmol/L EDTA, and 0.5 mg/mL BSA with pH adjusted to
7.4.Mitochondria were isolated by differential centrifugation
andresuspended in homogenization media with no BSA added.Isolated
mitochondria were stored at �80�C. Frozen-thawedmitochondria
suspended in the K medium (80 mmol/L KCl,10 mmol/L Tris-HCl, 5
mmol/L K-phosphate, 3 mmol/L MgCl2,1 mmol/L EDTA, pH 7.4) were
utilized for high-resolutionrespirometry using Oxygraph-2k. The
inhibitory effect of metfor-min and norMitoMet on GPDH-mediated
respiration stimulatedby 10 mmol/L glycerol 3-phosphate (G3P) was
determined bystepwise addition of tested compounds directly into
the chamberof the Oxygraph-2k instrument.
Seahorse XF metabolic flux analysisExtracellular acidification
rates (ECAR) and oxygen consump-
tion rates (OCR), respective measures of glycolytic flux
andmitochondrial respiration, were assessed for a panel of
pancreaticcancer cell lines using the Seahorse XF-24 analyzer
(SeahorseBiosciences). Cells were plated in the Seahorse XF24 cell
culturemicroplates in standard culture media. After 24 hours, the
medi-um was replaced with Seahorse XF base medium supplementedwith
0.2% BSA and 10mmol/L glucose, and themicroplates wereplaced in
non-CO2 incubator for 30 to 60 minutes. The assayprotocol consisted
of four consecutive injection steps in which 1mmol/L oligomycin,
0.5 mmol/L FCCP, 1 mmol/L FCCP, and thecombination of 100mmol/L
2-deoxyglucose, 1 mmol/L rotenone,and 1 mg/mL antimycin A were
added. Maximal respiration wasdetermined as the maximal OCR
stimulated by FCCP. The ele-
vated rate of glycolysis after oligomycin addition is referred
to asglycolytic capacity. After terminating the measurement, cells
werelysed in the RIPA buffer and the protein content was
determinedusing the Pierce BCA Protein Assay Kit (Thermo
Scientific). Datawere normalized to the amount of protein present
in each well ofthe microplate.
Detection of reactive oxygen species generation andmitochondrial
membrane potential (Dcm,i)
Cells were seeded in 12-well plates, left to attach
overnight,and treated as indicated. 15 minutes before collecting
the cells,5 mmol/L 20,70-dichlorofluorescin diacetate (DCF-DA) and
50nmol/L tetramethylrhodamine methyl ester (TMRM), probes
formonitoring ROS production and Dym,i, respectively, were
added.Harvested cells were resuspended in PBS containing 50
nmol/LTMRM and analyzed by flow cytometry (FACS Calibur). The
levelof TMRM fluorescence in cells with Dym,i dissipated by
CCCPpretreatment was used as a baseline for Dym,i
measurements.MitoSOX Red dye (Life Technologies) applied at 1.25
mmol/Lconcentration for 15 minutes was used to detect
mitochondrialsuperoxide production by flow cytometry.
Computer modelingThe recently deposited crystal structure of
yeast CI from Yarrowia
lipolytica (PDB ID 4wz7; ref. 33) was used for modeling.
Thegeometry of a set of possible tautomeric forms of MitoMet
wasoptimized using theDFT-Dmethod (34)with TPSS functional andTZVP
basis set (35). The effect of water solvation was treatedimplicitly
using COSMO (36) with e ¼ 78.4. All optimizationswere performed in
the TurboMole suite of programs. Optimizedgeometry of two most
stable forms of MitoMet was used for thedocking study. The Python
Molecular Viewer (PMV 1.5.6 rc3) wasused to set the docking
parameters. MitoMet was then allowed tosample docking poses in a
box (90 � 90 � 90 grid points, 1.0 Åspacing) covering the lower
part of the peripheral arm (Qmodule)and the transmembrane PP module
of the membrane arm. Resultsof five separate docking runs for
eachMitoMet formwere collectedemploying AutoDock Vina version
1.1.2. The program3V (37)wasused to identify internal cavities
connecting the iron-sulfur clusterswith the ubiquinone (UbQ)
binding cavity in the crystal structure.
Animal experimentsImmunocompromised, athymic female Balb c/nu-nu
mice
(Charles River Laboratories) were subcutaneously injected with2
� 106 PANC-1 or 5 � 106 PaTu 8902 cells per animal. Thegrafted
PANC-1 cells formed slow-growing tumors after 2 weeklag phase. When
the PANC-1 tumors reached about 5 mm3,mice were divided into
norMitoMet, metformin, and controlgroups receiving treatment (125
mmol/kg of norMitoMet or1500 mmol/kg of metformin) or the vehicle
by oral gavage 3times perweek (Mo/We/Fri). PaTu8902 cells formed
fast-growingtumors several days after tumor cell implantation.
Treatment wasstarted when tumors reached about 80 mm3. To overcome
thepossible low bioavailability of the agents after oral delivery,
micewere treated intraperitoneally using 4.4 mmol/kg of
norMitoMet(maximal tolerated dose), 1,500mmol/kg ofmetformin or
vehicleadministrated daily. Tumor growth was monitored using
theVevo770 ultrasound imaging device equipped with the RMV708or
RMV704 probe (VisualSonics). All mice were cared for andmaintained
in accordance with the Animal Welfare Act of theCzech Republic.
MitoMet and Pancreatic Cancer
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Statistical analysisStatistical analysis was performed using
GraphPad Prism 6
software. Statistical significance was determined by
one-wayANOVA followed by Tukey's or Dunnett's multiple
comparisontests. The results from xenograft experiments were
statisticallyevaluated by ANOVA followed by Sidak's multiple
comparisontest. Statistical significance is reported as follows:
ns, P > 0.05; �, P� 0.05; ��, P� 0.01, ���, P� 0.001; and ����,
P� 0.0001. Data areexpressed as mean � SEM.
ResultsMitochondrially targeted analogs of metformin
aremuchmoretoxic to PANC-1 cells than metformin
We prepared a panel of mitochondrially targeted analogs
ofmetformin (Fig. 1). This includes MitoMet comprising a metfor-min
core tagged with the TPPþ group via a 10-C spacer (com-pound 9) as
well as norMitoMet, lacking a methyl group on the
nitrogen adjacent to the 10-C spacer (compound 7). To see
theimportance of the length of the spacer, we prepared
correspond-ing compounds featuring a 6-C spacer (compound 8 and
10,respectively). These agents were tested for their toxicity
toward thehuman pancreatic cancer cell line PANC-1. Figure 2A and
Bdocuments a surprising finding that MitoMet was some three tofour
orders of magnitude more efficient than the parental met-formin.
For example, the IC50 value for norMitoMet, which wasabout 20-fold
more efficient than MitoMet, was 0.9 mmol/L,whereas it was 14
mmol/L for metformin. The corresponding6-C analogs of MitoMet and
norMitoMet were at least 100-foldless efficient than their 10-C
counterparts. Figure 2C and D showsthat 11C-TPP, that is, a
compound with straight undecyl chaintagged with TPPþ, was more
efficient in suppressing viability ofPANC-1 cells than was
norMitoMet. However, 11C-TPP wassimilarly toxic toward a panel of
nonmalignant cell lines astoward PANC-1 cells, whereas norMitoMet
was much less toxic,
A EC
G
10-1 100 101 1020
50
100
norMitoMet (μmol/L)
Via
bilit
y( %
)
PANC-1MiaPaCa-2PaTu 8902BxPc-3AsPC-1
0
10
20
30
4080
120
IC5
0(μ
mol
/L)
24 h48 h
** ***
**
*** *** **
H
B D F
10-1100 101 102 103 104 1050
50
100
Concentration (μmol/L)
Via
bilit
y(%
)
norMitoMetMitoMet
C6 MitoMetC6 norMitoMet
Metformin
0.1 1 10 1000
50
100
Concentration (μmol/L)
Via
bilit
y(%
)
PANC-1 11C-TPPPANC-1 nMMBJ 11C-TPPBJ nMM
0
50
100
Concentration (μmol/L)
Via
bilit
y(%
)
nMM 4.5 g/l glcnMM 1 g/l glcMet 4.5 g/l glcMet 1 g/l glc
10-1100101102103104105
IC50
(μm
ol/L
)
0.1
1
10
100
IC50
(μm
ol/L
)
norMitoMet11C-TPP
*
*******
***
10-1
100
101103104105
IC50
(μm
ol/L
)
1 g/l glc4.5 g/l glc
***
Figure 2.
Mitochondrial targeting substantiallyincreases the
antiproliferative activity ofmetformin. A, PANC-1 cells
wereincubated with increasingconcentrations of metformin
andmitochondrially targeted biguanidinesfor 48 hours, and the
effect on cellviability was determined by the crystalviolet assay.
Inhibition dose–responsecurves were plotted for calculation ofIC50
values (B). C and D, Effect of 48-hour treatment with norMitoMet
(nMM)or 11C-TPP on viability of PANC-1 cellsand a panel of
nonmalignant cells—BJskin fibroblasts, MCF10A breastepithelial
cells, MRC-5 lung fibroblasts,HFP1 skin fibroblasts, and
EA.hy926endothelial-like cells. EA.hy926 cellswere used in
confluent state as anin vitromodel of the endothelium. E andF,
PANC-1 cells were incubated withincreasing concentrations of
norMitoMetormetformin (Met) for 48hours inDMEMcontaining either 4.5
or 1 g/L glucose(glc). The cytotoxic effects werecompared using the
crystal violet assay.G, Different pancreatic cancer cell lineswere
tested for their sensitivity tonorMitoMet (48-hour treatment)
usingthe crystal violet assay.H,Comparison ofnorMitoMet IC50 values
for pancreaticcancer cells and noncancer BJfibroblasts.
Boukalova et al.
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indicating selectivity of the mitochondrially targeted agent.
Tox-icity of norMitoMet was not dependent on glucose level,
whichwas found for metformin (Fig. 2E and F), consistent with
theliterature (38).
norMitoMet is toxic to and causes apoptosis in pancreaticcancer
cell lines
In the next set of experiments, we explored the effect of
themostefficient of the newly synthesized agents norMitoMet, using
apanel of pancreatic cancer cells. The agent suppressed viability
inall of them with a considerable difference for the
individuallines. Figure 2G and H and Supplementary Table S2
documenta similar strong effect of the agent after 48-hour
treatment onPANC-1 and MiaPaCa-2 cells, with the IC50 values of 0.9
and 0.8mmol/L, respectively. PaTu 8902 cells were slightly more
resistant
(IC50 ¼ 2.3 mmol/L), whereas BxPC-3 and AsPC-1 were
mostresistant, with IC50 comparable to noncancer BJ fibroblasts
(20.0and 17.1 mmol/L, respectively, compared to 13.2 mmol/L for
BJcells).
Using the xCelligence apparatus, we assessed the effect
ofnorMitoMet on proliferation of pancreatic cancer cells. Figure3A
shows plots for proliferation of PANC-1 cells in the presence
ofvarious concentrations of norMitoMet, and Fig. 3B shows
thederived normalized slopes. These data indicate that the effect
ofnorMitoMet on cell proliferation develops gradually in terms
ofdays. After 48 to 72 hours, the cell growth ceased and the
numberof cells started to decline as documented by the negative
values ofthe growth curve slope. The xCelligence assay was used
forcalculation of IC50 values for inhibition of proliferation of
indi-vidual pancreatic cancer cell lines and noncancer BJ
fibroblasts. As
32168421–0.5
0.0
0.5
1.0
1.5
norMitoMET (μmol/L)
Nor
mal
ized
slop
e
24 h48 h72 h
0.5–10–20 0 2010 4030 50 7060 80Time (h)
012345678
Cel
l ind
ex
ControlnMM 0.5 μmol/LnMM 2 μmol/LnMM 5 μmol/L
A B
C
0
20
40
60
IC5
0(μ
mol
/L) h24 h48
h72
***
*** **
**** *** ******* **** ****
1684210.50
5
10
15
20
25
Concentration (μmol/L)
Ann
exin
posi
tive
(%)
norMitoMETMetformin
0
*******
****
*
D
E
0
20
40
60
Cle
aved
cas
p-3/
actin
ratio **
****
Control
nMM 10 μmol/L
Annexin V-FITC
Pro
pidi
um io
dide
14
9.8
3.8
5.5
Contr1 μmol/L24 h
1 μmol/L48 h
5 μmol/L24h
5 mmol/L24 h
5 μmol/L48 h
5 mmol/L48 h Stau
norMitoMET Metformin
Cleavedcaspase-3
β-Actin
20 kD
15 kD
37 kD
p-AMPK
AMPK
p-ACC
ACC
p-Raptor
Raptor
p-mTOR
mTOR
β-Actin
Contr
Contr
ol
Stau
rospo
rin
1 μmol/L 5 mmol/L5 μmol/LnMM Met
AICAR
F
Figure 3.
Time-dependent effects of norMitoMeton proliferation of a panel
of pancreaticcancer cell lines.A,Real-time analysis
ofnorMitoMet-induced effects on PANC-1cell line growth using the
RTCAxCelligence system. The arrow marksthe time of norMitoMet
addition.B, Time- and concentration-dependenteffects of norMitoMet
treatment on theslopeof PANC-1 growth curve.Negativevalues of the
growth curve slopeindicate that the number of attachedcells
decreased during themeasurement time period. C,Comparison of IC50
values fornorMitoMet in different cancer cell linesdetermined from
the growth curves atdifferent time points. AsPC-1 cell linewas
excluded from this assay as it wasfound to be unsuitable for
impedance-based measurements. D, Effect ofnorMitoMet and metformin
applied for48 hours on cell death in PANC-1 cells asdetermined by
the annexin V assay.E, Western blot analysis of cleavedcaspase-3 in
PANC-1 cells treated withnorMitoMet (1 and 5 mmol/L) ormetformin (5
mmol/L) for 24 and 48hours. Cells incubated with 0.5
mmol/Lstaurosporine for 24 hourswere used asa positive control. F,
PANC-1 cells wereexposed to norMitoMet and metforminat the
concentrations shown for 24hours or to 2 mmol/L
5-aminoimidazole-4-carboxamideribonucleotide (AICAR) for 2 hours as
apositive control, and Western blots forproteins as indicated
performed.
MitoMet and Pancreatic Cancer
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F
A E
B
C
D
10–210–1100 101 102 103 1040
50
100
Concentration (μmol/L)
CIR
espi
ratio
n(%
)MetforminnorMitoMet
IC50=4.9 μmol/L
IC50=2.1 mmol/L
10–1 100 101 102 1030
50
100
norMitoMet (μmol/L)R
espi
ratio
n(%
)
Glutamate + MalateSuccinate
100 101 102 103 104 1050
20
40
60
80
100
Concentration (μmol/L)
GP
DH
Res
pira
tion
(%)
norMitoMetMetformin
0
2
4
6
8
10
IC5
0(μ
mol
/L)
**
**** ********
0
50
100
norMitoMet (μmol/L)
Max
imal
resp
iratio
n[p
mol
/(s*1
06ce
lls)]
Glutamate + MalateSuccinate
****
****
*****
*
0
20
40
60
80
Res
pira
tion
[pm
ol/(s
*106
)]
****
I
G
0
0.5
1
1.5
0
2
4
6
8
Rot
enon
eIC
50
(μm
ol/L
) norMitoM
etIC5
0 (μmol/L)
*** **
H
010203040506070
100
* **
*
%of
Con
trol
GFPNDI1 GFP
norMitoMet 4 μmol/L
GFPNDI1 GFP
Boukalova et al.
Mol Cancer Ther; 15(12) December 2016 Molecular Cancer
Therapeutics2880
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presented in Fig. 3C and Supplementary Table S2, the level
ofsuppression was time-dependent and the cell lines were
suscep-tible similarly as shown for viability (cf. Fig. 2G and H
andSupplementary Table S2) with PANC-1 cells slightly more
sus-ceptible in this case than MiaPaCa-2 cells. The decreased
prolif-eration rate induced by norMitoMet was associated with
cell-cyclearrest as documentedby increasedG0 fraction anddecreased
S andG2 fractions (Supplementary Fig. S1). To see whether toxicity
ofnorMitoMet results also in activation of apoptosis, we
testedPANC-1 cells for binding of annexin V and for cleavage
ofcaspase-3. Figure 3D and E document that norMitoMet
triggersapoptosis at levels as low as 1 mmol/L, whereas metformin
wasinactive even at 5 mmol/L and at 48 hours of treatment. In
BJfibroblasts, norMitoMet did not induce any apoptosis even
whenused in 10 times higher concentration than in PANC-1
cells(Supplementary Fig. S2).
Potential mechanisms of metformin's antitumorigenic
effectinclude activation of AMPK signaling (11, 12). To compare
theeffect of norMitoMet and metformin on the AMPK pathway,
weanalyzed the phosphorylation status of AMPK and its down-stream
targets ACC, raptor, and mTOR. Figure 3F and Supple-mentary Fig. S3
document that norMitoMet activates the AMPKpathway when used in 3
to 4 order of magnitude lower concen-tration compared to metformin.
As metformin-induced AMPKactivation is believed to be related to
reduced cellular energycharge resulting from the inhibition of
respiration (11), wecompared the effect of metformin and norMitoMet
on the ATPcontent in PANC-1 cells. Intracellular ATP levels were
decreasedby the compounds used in the same concentration range
thatactivates AMPK (Supplementary Fig. S4).
norMitoMet acts by targeting mitochondrial complex IWe next
assessed the effect of norMitoMet on mitochondrial
respiration. Mitochondrial complex I (CI)-dependent
respirationwas suppressed in PANC-1 cells by norMitoMet with IC50
of 4.9mmol/L, whereas it was some 3 orders of magnitude higher
formetformin (Fig. 4A). Figure 4B documents that the IC50 values
forinhibition of respiration via CI are similar (some 2.4–8
mmol/L)for all pancreatic cancer cell lines tested. The sensitivity
of the cellsto norMitoMet-induced inhibition of CI respiration does
notcorrelate with their susceptibility to the toxic effects of the
agent(cf. Fig. 2H). The IC50 values derived from dose responses
toacutely applied norMitoMET are most probably underestimated,as
this compound is characterized by time-dependent activity.
InPANC-1, norMitoMET is able to fully inhibit CI-mediated
respi-
ration but also routine respiration of intact cells in a dose as
low as2 mmol/L and 24-hour incubation time (SupplementaryFig. S5).
Figure 4C and D show that CII is a very weak target fornorMitoMet,
as the CII-dependent respiration was suppressedonly at levels of
the agent >100 mmol/L. Because it has beenreported that
G3PDH-dependent respiration is a major target forthe effect of
metformin in liver cells (39), we tested the effect ofnorMitoMet
andmetformin onG3PDH-dependent respiration inbrownadipose
tissuemitochondria, where a considerable portionof respiration is
driven by G3P. Figure 4E documents that nor-MitoMet suppressed
G3PDH respiration considerably at levels>100 mmol/L and
metformin at levels >10 mmol/L. This findingfor norMitoMet is
comparable with its effect on CII-dependentrespiration. Comparing
the contribution of the three differenttypes of respiration (CI-,
CII-, and G3PDH-dependent) revealedthat PANC-1 cells respire
similarly viaCI andCII, whereasG3PDHcontributes only by �10% to
total respiration (Fig. 4F).
To further document CI as a molecular target of MitoMet,
westably overexpressed the yeast NADH dehydrogenase NDI1 inPaTu
8902 cells and compared the effect of norMitoMet onrespiratory rate
in NDI1-expressing and control vector-trans-fected cells. NDI1
expression increased the routine respiratoryrate and the
respiration supported by NADH-linked substrates,whereas it resulted
in slight decrease in succinate-stimulatedoxygen consumption
(Supplementary Fig. S6). In NDI1-expres-sing cells, norMitoMet was
not able to fully inhibit the NADH-linked respiration contrary to
control cells (Fig. 4G). Moreover,the sensitivity to
antiproliferative effects of norMitoMet androtenone were suppressed
in NDI1-transfected cells, as docu-mented by elevated IC50 values
for these compounds (Fig. 4H).Thus, NDI1 expression allows recovery
of mitochondrial elec-tron-transport activity in norMitoMet-treated
cells and, at thesame time, reduces the impact of norMitoMet
treatment oncellular viability.
To localize the possible binding site for MitoMet in CI,
weperformed molecular modeling using the recently publishedcrystal
structure of Yarrowia lipolytica CI resolved at 3.6 Å
(33).Wehaveoptimized the geometry offive cationic formsofMitoMetin
order to identify the most probable protonation state. At theDFT-D
level we have identified two nearly isoenergetic, moststable
structures, whereas the remaining structures represent min-ima less
stable by tens of kcal/mol. The two structures are axiallychiral
formsof one protonation state. The interconversionof thesetwo forms
is connected with a barrier of �7 kcal/mol. We havethus used both
these forms for the subsequent docking study. As
Figure 4.norMitoMet inhibits CI respiration as revealed by
high-resolution respirometry. A,Dose–response effect of norMitoMet
andmetformin on CI-mediated respiration ofpermeabilized PANC-1
cells. Glutamate together with malate were used as substrates. B,
IC50 values for norMitoMet-induced inhibition of CI respiration
fordifferent pancreatic cancer cell lines. C, Comparison of
norMitoMet effects on CI- and CII-mediated respiration in PANC-1
cells utilizing glutamate plusmalate and succinate, respectively.
D, PANC-1 cells were pretreated for 24 hours with increasing
concentrations of norMitoMet and subjected to
high-resolutionrespirometry measurements. CI and CII respiratory
capacity determined in CCCP-uncoupled state was assessed. E,
Frozen-thawed mitochondria isolatedfrom brown adipose tissue of
newborn rats were used to compare the effects of norMitoMet and
metformin on GPDH respiration using the Oxygraph-2k.Glycerol
3-phosphate (10 mmol/L) was used as a substrate. F, Comparison of
routine respiratory rates in nonpermeabilized PANC-1 cells with the
rates ofpermeabilized cells respiring on combination of glutamate
and malate, succinate, and glycerol 3-phosphate, respectively. G,
PaTu 8902 cells were stablytransfected with the NDI1-coding vector
or control vector (GFP). The cells were then pretreated with 4
mmol/L norMitoMet for 24 hours. Routine respiration
innonpermeabilized cells and glutamate andmalate-stimulated
respiration in permeabilized cells in basal andCCCP-uncoupled
statewere compared.H, IC50 values forrotenone and norMitoMet (48
hours exposure) in PaTu 8902 cells transfected with NDI1 or the
control vector were determined by the crystal violet assay.I, Left:
the crystal structure of complex I (with indicated N, Q, PD, and PP
modules). The broken red line indicates movement of electrons from
the catalyticcenter of CI to their acceptor, UbQ; right: the
structure of CI is shown using its lateral view with the boxed area
containing the UbQ binding cavity (graysurface) into which MitoMet
can bind. The boxed area is enlarged, indicating predicted most
probable positions of two MitoMet forms inside the cavity, with
thepotential effect on electron flow.
MitoMet and Pancreatic Cancer
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summarized in Fig. 4I, we have identified similar high
affinity(sub-micromolar) binding mode for both MitoMet forms
insidethe UbQ-binding pocket. The poses share the same binding
cavityas well as orientation of the metforminmoiety with the
predictedposition of UbQ (33, 40). Supported by experimental
observa-tions, this suggests thatMitoMETmay affect UbQ
interactionwithCI, that is, its function within CI.
Pancreatic cancer cell lines exert different OCR
andextracellular acidification
To learnmore about the bioenergetics in the studied
pancreaticcancer cell lines, we utilized the Seahorse instrument to
assesstheir OCR and ECAR. Figure 5A presents the OCR and ECARcurves
for PANC-1 and BxPC-3 cells, whereas Fig. 5B shows OCRand ECAR
values for all five studied lines, documenting both thebasal
andmaximal respiration, as well as glycolysis and
glycolyticcapacity. The results revealed an inverse correlation
betweenrespiration and glycolysis for the tested lines. We next
calculatedthe ratios between maximal OCR and ECAR values, which
isplotted in Fig. 5C. This shows that the highest ratio was found
forPANC-1 cells that are highly susceptible to norMitoMet,
whereasthe least susceptible BxPC-3 and AsPC-1 cells showed the
lowestOCR/ECAR ratio, suggesting that the level of toxicity of
norMi-toMet to pancreatic cancer cells is driven by the respiratory
andglycolytic state of the cells. In other words, the higher
respiratoryand the lower glycolytic activity of the cells, the more
susceptiblethey are to the mitochondrially targeted analog of
metformin.
norMitoMet dissipates Dcm,i, causes ROS generation,
andsuppresses growth of pancreatic tumors
Because agents targetingmitochondrial complexes are expectedto
alter the mitochondrial function, we tested norMitoMet for
itseffect on Dym,i and ROS generation. Figure 6A documents that
5mmol/L norMitoMet caused strong dissipation of Dym,i. Similarlyit
caused considerable generation of ROS as assessed using
theprobeDCF-DA (Fig. 6B). Because
interferencewithmitochondrialcomplexes is assumed to cause
generation of superoxide withinmitochondria, we also used
theMitoSOX probe. Figure 6C revealsthat already at 1 mmol/L and at
6 hours, norMitoMet causedsignificant increase in mitochondrial
superoxide. The increase inROS production at least partially
mediates the norMitoMet-induced apoptosis in PANC-1 cells, as
documented by thedecreased level of apoptosis in cells treated with
the anti-oxidantNAC (Fig. 6D and E).
Finally, we tested the effect of norMitoMet on the growth
ofexperimental pancreatic cancer. For this, xenografts were
preparedby subcutaneous implantation of PANC-1 and PaTu 8902 cells
innude mice. Figure 6F and G reveals about 50% inhibition oftumor
progression in norMitoMet-treated mice. In PANC-1-derived tumors,
norMitoMet applied orally displayed similartumor suppression effect
as metformin used in 10-fold higherdose. In the very aggressive
PaTu8902-derived tumors,metformindosed via intraperitoneal routewas
not able to suppress the tumorprogression, whereas 2 orders of
magnitude lower dose of nor-MitoMet significantly reduced the
growth of tumors. NorMitoMet
A B
C
****
**** ****
**** **** ***
**** **** ns ns
MiaPaCa-2
PaTu 8902
BxPC-3
AsPC-1
0 50 1000
10
20
30
OC
R(p
mol
/min
/μg
prot
ein)
PANC-1BxPC-3
0 50 1000
5
10
Time (min)
EC
AR(m
pH/m
in/μ
gpr
otei
n)
0
10
20
30
OC
R(p
mol
/min
/μg
prot
ein)
BasalrespirationMaximalrespiration
0
1
2
3
Omy FCCP FCCPRot + Ama
+2-DG
Max
imal
OC
R/E
CA
R
0
5
10
ECA
R(m
pH/m
in/μ
gpr
otei
n)
GlycolysisGlycolyticcapacity
Figure 5.
Measurement of OCR and ECARusing the Seahorse XF analyzer.A,OCR
and ECAR values for PANC-1and BxPC-3 cells in the presence of10
mmol/L glucose. During thecourse of the experiment, the cellswere
exposed to oligomycin (omy),FCCP, and the combination ofrotenone
(Rot), antimycin A (Ama),and 2-deoxyglucose (2-DG)at the time
points indicated.B, Comparison of OCR and ECARvalues for a panel of
pancreaticcancer cell lines in basal state(basal respiration,
glycolysis), afteroligomycin addition (glycolyticcapacity), and in
FCCP-induceduncoupled state (maximalrespiration). C, The ratio
ofFCCP-stimulated OCR tooligomycin-stimulated ECARvalues.
Statistical evaluation of thedata is provided on the right.
Boukalova et al.
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CBA
0
50
100
nMM Exposure (h)
TMR
Mflu
ores
cenc
e(%
)0.5 μmol/L5 μmol/L
2418126
*** **
********
**** ****
24181260100
200
300
400
nMM Exposure (h)D
CF
fluor
esce
nce
(%)
0.5 μmol/L5 μmol/L
* ******
**
****
********
1684210.5100
150
200
250
300
norMitoMET (μmol/L)
Mito
SO
Xflu
ores
cenc
e(%
) 6 h18 h
24 h
* **
*****
****
******
****
****
****12 h
******
********
D0
Control nMM
1 mm
PANC-1
25201510500
10
20
30
Time (days)
Control
MetforminnorMitoMET
***
******
*
GF
D0
D7D7
D14D14
D21D21
nMMControl
2 mmD0 D0
D7 D7
D14 D14
PaTu 8902
1510500
500
1,000
1,500
Days
Tum
orvo
lum
e(m
m3 )
Tum
orvo
lum
e(m
m3 )
ControlnorMitoMetmetformin
*
***
NAC 12 mmol/L
norMitoMET (µmol/L)
1 51 5 10 10+ + +
β-Actin
Cleaved caspase-3
+ 20 kD
15 kD
37 kD
0
5
10
15
Ann
exin
posi
tive
(%) **
*
nMM (μmol/L) 1 51 10105+NAC 5 mmol/L + +
nMM (μmol/L) 10510
50
100
NAC
- indu
ced
inhi
bitio
n(%
)
*****
NAC 12 mmol/L
D E
Figure 6.
The effects of norMitoMet on mitochondrial potential, ROS
production, and tumor growth in pancreatic cancer models. A, Flow
cytometry analysis of mitochondrialmembrane potential in PANC-1
cells after incubation with norMitoMet for indicated time
intervals. The graph indicates the change in TMRM
fluorescencerelative to control conditions (no treatment).
norMitoMet-induced ROS production in PANC-1 cells determined by DCF
(B) and MitoSOX fluorescence (C).D, Representative image showing
Western blot analysis of cleaved caspase-3 in PANC-1 cells treated
with norMitoMet for 48 hours in the presence or absenceof 12 mmol/L
NAC. NAC-induced suppression of cleaved caspase-3 level is
quantified in the lower plot. E, Annexin V-staining was used to
evaluate theeffect of NAC treatment on norMitoMet-induced apoptosis
in PANC-1 cells. NACwas applied at the same time as norMitoMet in
bothD and E. Progression of tumors inmice injected with (F) PANC-1
or (G) PaTu 8902 cells quantified using ultrasound imaging system.
Representative ultrasound images of PANC-1 xenografttumors of
control and norMitoMet-treated Balb c/nu-nu mice at different time
points are shown on the right in panels F and G. n ¼ 5 and 6 for
each experimentalgroup for PANC-1 and PaTu 8902-derived tumors,
respectively.
MitoMet and Pancreatic Cancer
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was found to inhibit with a similar extent
alsoMiaPaCa-2-derivedtumors (Supplementary Fig. S7A). We did not
observe any effectof treatment on body weight of the animals or
their behavioralpattern at any dosing (Supplementary Fig. S7B).
DiscussionDespite a considerable progress in molecular medicine
with
focus on neoplastic diseases, pancreatic cancer is still on the
rise.Apart from patients where resection is an option, there is
nocurrent cure for this pathology, with the prescribed
therapeuticregimen only modestly increasing the 5-year survival of
patientsand with some 80% relapse of the disease (41, 42). About
90% ofpancreatic cancer patients are positive for oncogenic K-Ras
(41).Interestingly, a recent report showed that ablation of K-Ras
inpancreatic cancer causes demise ofmajority of themalignant
cells,with a small population surviving, featuring high level of
mito-chondrial respiration and characteristics of cancer stem-like
cells,capable of tumor initiation (23). This indicates that tumor
ini-tiation/progression may be driven by or be dependent on
mito-chondrial function. This is consistent with growing body
ofreports that tumors are aberrant tissues with deregulated
metab-olism and that metabolic reprogramming is critical for
tumorinitiation, progression and metastasis, critically involving
mito-chondria (2–4, 23–25), and that metabolism can be a target
foranticancer therapy (43).
An intriguing anticancer target, yet to be fully exploited,
aremitochondria, in particular mitochondrial respiratory
complexes(26, 27). Mitochondrial CII has been recently reported as
a noveltarget for the anticancer agent a-tocopheryl succinate
(a-TOS;refs. 44–46). CI has also been suggested as a target, for
example inthe context of breast cancer (47), and, interestingly,
formetformin(19, 20), a drug of choice for T2DM, which has also
been impliedas a potential agent against pancreatic cancer (11, 12,
48). Inpancreatic cancer, metformin is believed to act via
regulation oftumor cell metabolism (49).
Because metformin is very inefficient against cancer
cells,including pancreatic cancer cells, suppressing their
prolifera-tion/inducing apoptosis only at high concentrations (20,
50),we decided to adapt a novel approach that is based on
sendingthe agent where it matters, that is, to the interface of the
MIMand the matrix, the site of mitochondrial complexes, by meansof
tagging it with the delocalized cationic TPPþ group.
Veryunexpectedly, this increased the efficacy of the
mitochondriallytargeted agent compared to that of metformin by 3 to
4 ordersof magnitude. Of the analogs we synthetized,
norMitoMet,featuring a 10-C linker between the metformin and
TPPþ
moieties and lacking one methyl on the metformin structure,was
the most efficient one.
Usinghigh-resolution respirometry, we found that
norMitoMetpreferentially suppressed respiration via CI, with the
IC50 valuessome 100-fold lower than those for CII. It inhibited
with similarefficacy as found for CII-dependent respiration also
respirationdependent on G3PDH, a major target of metformin in liver
cells(39). Because G3P-driven respiration contributed to overall
mito-chondrial respiration only marginally, we ruled it out as a
majortarget for MitoMet. Expression of the yeast NADH
dehydrogenaseNDI1, which is known to be able to bypass CI and
reverse theeffects of metformin in cancer cells (20), was able to
partiallyrescue the effect of MitoMet on respiration and cellular
viability,further corroborating CI as a molecular target of the
agent.
To explore the possible binding site for MitoMet in CI,
weperformed molecular modeling of its interaction with the
respi-ratory complex using its recently published crystal structure
(33).Our previousmodeling of interaction ofmitochondrially
targetedvitaminE succinate (MitoVES; refs. 28, 29)documents
associationof the TPPmoiety of the agentwith the interface between
theMIMand matrix, whereas the succinyl moiety interacts with the
prox-imal UbQ-site of CII (51). On the contrary, the wholemolecule
ofMitoMet resides inside CI, within its UbQ site. While
shorteningof the spacer of MitoVES from 11-C to 5-C results in
losing theanticancer activity of the agent due to the notion that
the freecarboxyl group of the succinyl moiety cannot reach the
proximalUbQ site, this is unlikely the reason for MitoMet. Rather,
the factthat C6 MitoMet is less efficient than MitoMet with 10-C
spacercan be explained likely due to higher hydrophobicity of the
latter.
Importantly, we found that MitoMet suppressed pancreaticcancer
in two mouse models by some 50% using 10- to 20-foldlower dose than
found formetformin by us and as reported in theliterature for the
latter (20, 50). The anticancer efficacy of the agentdidnot show
the3 to4 log gain in efficacy compared tometforminfound for
toxicity toward cultured pancreatic cancer cells. Thismay be due to
the mode of administration of the drug and itsresulting slower
uptake following gavage. Indeed, norMitoMetadministrated
intraperitoneally suppressed the growth of veryaggressive tumors
derived from PaTu 8902 cell line, which werenot affected by
300-fold higher doses of metformin. We arecurrently developing
pro-drugs based on MitoMet that wouldresult in better uptake of the
drug and its delivery to the pancreaticcancer tissue.
Targeting of CI by MitoMet is linked to deregulation of
themitochondrial function, as proposed for classfivemitocans
actingby targeting the electron transport chain (26, 27).
Themechanismby which suppression of CI-dependent respiration by
MitoMet isrelayed to cell-cycle arrest and induction of apoptosis
in pancre-atic cancer cells is currently unclear, although a
parallel can bedrawnwith the proposed effect ofmetformin. This
agent has beensuggested to act by bioenergetics deregulation, such
as modulat-ing the AMPK/mTOR pathway, an important regulator of
cell-cycle progression (11, 12, 52), which we show to be affected
alsoby MitoMet. It cannot be excluded that there are
additionalmechanisms by which MitoMet acts. For example elevated
ROSproduction induced by MitoMet may have beneficial effects,
ascancer cells are more vulnerable to oxidative stress than
normalcells (26). Indeed, we have demonstrated that
MitoMet-inducedapoptosis in pancreatic cancer cells is
ROS-dependent, as it wassuppressed by pretreatmentwithNAC. An
innovative approach tounderstanding the effect of agents like
MitoMet stems from therecent train of thought that respiration is a
prerequisite for tumorinitiation and progression, as well as for
the metastatic disease(2–4, 23–25). That anticancer agents,
including metformin,deregulate cancer cell metabolism has been
proposed (49). Avery attractive option is that suppression of
respiration observedfor MitoMet and pancreatic cancer cells is
linked to generation ofessential metabolites that are substrates
for important biosyn-thetic pathways, such as the de novopyrimidine
synthesis as shownrecently (53, 54). Related to the perception of
the importance ofrespiration for tumor growth, we found that
MitoMet was moretoxic toward pancreatic cancer cell lines
thatweremore dependenton respiration and less on glycolysis.
In conclusion,wehavedesigned, synthesized and tested
anovelanticancer agent based on the most frequently prescribed
anti-
Mol Cancer Ther; 15(12) December 2016 Molecular Cancer
Therapeutics2884
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T2DM drug, metformin. We report here on an unprecedentedfinding
that mitochondrially targeted metformin (MitoMet) ismore toxic
toward pancreatic cancer cells by some 3 to 4 orders ofmagnitude
compared to the parental compound. MitoMet is,therefore, a very
promising anticancer drug against a pathologythat is at present
largely beyond treatment. The attractiveness ofMitoMet stems from
the fact that it is based on an approved andwidely used drug,
facilitating its potential translation into a drugof choice against
pancreatic cancer.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: S. Boukalova, L.
Werner, L. Dong, J. NeuzilDevelopment of methodology: S. Boukalova,
L. Werner, L. Dong, Z. Drahota,J. NeuzilAcquisition of data
(provided animals, acquired and managed patients,provided
facilities, etc.): S. Boukalova, Z. Ezrova, J. Cerny, A.
Bezawork-Geleta,A. Pecinova, L. Dong, Z. DrahotaAnalysis and
interpretation of data (e.g., statistical analysis,
biostatistics,computational analysis): S. Boukalova, J. Stursa, L.
Werner, J. Cerny, A. Beza-work-Geleta, J. NeuzilWriting, review,
and/or revision of the manuscript: S. Boukalova, J. Stursa,L.
Werner, A. Bezawork-Geleta, L. Dong, J. Neuzil
Administrative, technical, or material support (i.e., reporting
or organizingdata, constructing databases): L. WernerStudy
supervision: S. BoukalovaOther (Design of the synthesis; synthesis
of mitochondrially targeted met-formin derivatives; purification,
analysis and identification of preparedmetformin derivatives.): J.
Stursa
AcknowledgmentsThe NDI1-containing pWPI vectors and the empty
counterparts were a
generous gift from Professor Navdeep S. Chandel.
Grant SupportThis work was supported in part by Australian
Research Council Discov-
ery grant, Czech Science Foundation grant (GA15-02203S), and
CzechMinistry of Health grant (AZV 16-31604.A) to J. Neuzil.
Further supportwas provided by BIOCEV CZ.1.05/1.1.00/02.0109 and
Mitenal CZ.2.16/3.1.00/21531 from the ERDF, RVO: 86652036 and the
Ministry of Educa-tion, Youth and Sports of the Czech Republic
(LO1220) at the CZ-OPEN-SCREEN: National infrastructure for
chemical biology.
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicatethis fact.
Received January 5, 2016; revised August 30, 2016; accepted
September 20,2016; published OnlineFirst October 7, 2016.
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Pancreatic CancerMitochondrial Targeting of Metformin Enhances Its
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