Home | Molecular Cancer Therapeutics - Mitochondrial ......Small Molecule Therapeutics Mitochondrial Targeting of Metformin Enhances Its Activity against Pancreatic Cancer StepanaBoukalova1,JanStursa2,LukasWerner2,3,ZuzanaEzrova1,JiriCerny1,Ayenachew

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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on July 4, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 7, 2016; DOI: 10.1158/1535-7163.MCT-15-1021

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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.

Mol Cancer Ther; 15(12) December 2016 Molecular Cancer Therapeutics2876




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

www.aacrjournals.org Mol Cancer Ther; 15(12) December 2016 2877




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,

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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.

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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

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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.






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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.






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

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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.

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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.






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-


Boukalova et al.




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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2016;15:2875-2886. Published OnlineFirst October 7, 2016.Mol Cancer Ther Stepana Boukalova, Jan Stursa, Lukas Werner, et al. Pancreatic CancerMitochondrial Targeting of Metformin Enhances Its Activity against

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Home | Molecular Cancer Therapeutics - Mitochondrial ......Small Molecule Therapeutics Mitochondrial Targeting of Metformin Enhances Its Activity against Pancreatic Cancer StepanaBoukalova1,JanStursa2,LukasWerner2,3,ZuzanaEzrova1,JiriCerny1,Ayenachew