Mitochondrial ion channels as oncological targets · 2014. 11. 10. · REVIEW Mitochondrial ion...

REVIEW

Mitochondrial ion channels as oncological targetsL Leanza1, M Zoratti2, E Gulbins3 and I Szabo1

Mitochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channelfunctions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function,such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species productionand mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential tocompromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivoevidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, theouter membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus onmitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance.Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promisingtargets for cancer treatment.

Oncogene advance online publication, 27 January 2014; doi:10.1038/onc.2013.578

Keywords: mitochondria; ion channels; direct targeting; pharmacology

MITOCHONDRIA; ION HOMEOSTASIS AND APOPTOSIS;IMPACTS ON SIGNALING AND CANCERThe mitochondrial inner membrane (IMM) surrounding the matrixpermits the formation of an electrochemical proton gradient thatdrives aerobic adenosine triphosphate (ATP) synthesis, and theouter membrane (OMM) delimits an intermembrane space (IMS)between the OMM and IMM. Proteins with fundamental roles incell death, such as cytochrome c and SMAC/Diablo, can bereleased from the IMS upon receipt of a proapoptotic signal andcan lead to commitment of the cell to apoptosis. The releasedserine protease HtrA2/Omi, apoptosis-inducing factor and endo-nuclease G also contribute to the executive phase of apoptosis.1

Much effort has been devoted to identifying drugs capable ofinducing cancer cell death by stimulating OMM rupture/permeabilization and release of these proteins.2 Mitochondrialion channels are therapeutically relevant to cancer because theycan influence both OMM and IMM permeability. Members of theB-cell lymphoma 2 (Bcl-2) family and MAC (mitochondrialapoptosis-induced channel) are directly involved in OMMpermeabilization, but they do not function as bona fide ionchannels. Excellent reviews are available on their role in thisprocess;3,4 therefore, the present review focuses on themitochondrial voltage-dependent anion channel (VDAC, alsoknown as porin) among the OMM channels. Selective inductionof IMM permeabilization in cancer cells is also a potentialtherapeutic strategy because mitochondrial energy productionbecomes compromised when the IMM becomes freely permeableto solutes, which leads to cell death.

As a consequence of changes in ion flux across the IMM,alterations of mitochondrial function occur that produce severeeffects on the overall fitness, including ATP production, of theseorganelles. Most available data on ion channels or ion fluxpathways that modulate cancer cells can be ultimately reconciled

with the idea that the perturbation of ion homeostasis withinmitochondria leads to cancer cell death via reactive oxygenspecies (ROS) production, mitochondrial calcium overload andmetabolism changes, but other mechanisms of action might alsoexist. Indeed, mitochondria are crucial for maintaining intracellularCa2þ homeostasis and produce ROS, which are two importantfactors for cancer cell survival and proliferation. A detailedoverview of the above processes is beyond the scope of thisreview; thus, we briefly describe the general mechanisms bywhich ion channels may affect ROS production, mitochondrialcalcium homeostasis and metabolism.

The matrix-negative difference in electrical potential across theIMM (DCm, ranging between � 150 and � 180 mV) is maintainedby the proton pumps of the respiratory chain (RC). Consequently,cations (Kþ and Ca2þ ) flow from the IMS (where the concentra-tion of ions is comparable to cytosolic concentrations because theOMM is permeable to these ions) to the matrix when a permeationpathway opens, as was demonstrated several years ago byexperiments with ionophores such as valinomycin. The electrical-equivalent anion outflow is considered much less relevantbecause the anion pool in the mitochondrial matrix is limitedcompared with the cytosolic Kþ pool. To compensate for chargemovement (unless permeating counterions are also present,which is feasible in experimental settings but unusual underphysiological conditions), the RC must increase the rate of protontransfer from the matrix to the IMS. To increase RC activityaccording to the chemiosmotic model, the transmembraneelectrochemical proton gradient (D~mH, composed of mainlyDCm) must decrease. Thus, passive charge flow and D~mH (DCm)are coupled, and the opening of Kþ or Ca2þ channels in the IMMwill lead to depolarization. Clearly, this model also functions inreverse; closing cation channels is expected (and observed) tolead to an increase of a D~mH (DCm) (that is, hyperpolarization).

1Department of Biology, University of Padova, Padova, Italy; 2CNR Institute of Neuroscience and Department of Biomedical Sciences, University of Padova, Padova, Italy and3Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany. Correspondence: Professor I Szabo, Department of Biology, University of Padova, Viale GColombo 3, Padova PD, 35138, Italy.E-mail: [email protected] or [email protected] 15 August 2013; revised 4 December 2013; accepted 5 December 2013

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Such alterations may in turn have consequences on the rate ofsuperoxide formation.5,6 Hyperpolarization lowers the efficacy ofcytochrome c oxidase,7 induces the reduction of RC complexesand intermediates and thereby increases the probability of a one-electron transfer to oxygen at respiratory complex I. Complex-III-dependent ROS formation can also be significant at highrespiration rates (depolarized conditions) because of a highconcentration of semiquinone at center ‘O’ (SQo or QP) of thebc1 complex.8 Thus, enhanced superoxide (O2

� ) productionoccurs in both the matrix and IMS.7,9–11 In addition, recentdata have highlighted that complex II also generates superoxideor hydrogen peroxide molecules at the flavin site IIF whenelectrons are supplied by either succinate or the reducedubiquinone pool.12 Irrespective of the site of production, whichmight depend on the substrates being oxidized,13 the superoxideanion is the precursor of most ROS. ROS can diffuse to differentsubcellular compartments. Increased ROS production is importantfor the maintenance and evolution of the cancerous phenotype.14

The effects of ROS vary according to the stage of carcinogenesis.In the tumor initiation phase, ROS may cause DNA damage andmutagenesis, whereas in established cancer they may act as signalmediators through the Akt pathway and stimulate proliferation,conferring a growth advantage.15 ROS may also inhibit PyruvateKinase M2, the embryonic form produced by cancer cells, therebydiverting reducing equivalents to the pentose phosphatepathway.16

Conversely, ROS release can induce cell death by damagingproteins, lipids and DNA and thus act as anticancer agents.17–19 Inaddition, the activation of pro-apoptotic kinases, such as apoptosissignal-regulating kinase 1 (ASK1) and death-associated proteinkinases (DAPK; 5 members),20,21 by ROS is of fundamentalimportance.15,17–21 Oxidative stress can also activate the pro-apoptotic Bax and permeability transition pore (MPTP) (seebelow),22 whose prolonged opening leads to the loss of themitochondrial D~mH, mitochondrial swelling, cristae remodelingand pro-apoptotic factor release.23 Bax and other Bcl-2 familyproteins regulate OMM permeabilization through pore formationand possible regulation of mitochondrial fission and fusion.24

In light of this dichotomy, both antioxidant and pro-oxidantapproaches have been proposed to antagonize cancer.25–28

Antioxidants are expected to be useful for cancer prevention,whereas pro-oxidants are desirable to potentially and selectivelyinduce cancer cell death. Chronic metabolic oxidative stress, likelyoccurring in most cancer cell types, can indeed be exploitedbecause these cells exhibit an increased sensitivity to ROS-inducedapoptosis.26 Therefore, therapies that specifically target the RC,either directly or indirectly, to further elevate ROS productionshould selectively kill cancer cells. Because pharmacologicalmodulation of mitochondrial ion channel function is expected tolead to changes in membrane potential, targeting these channelsto increase ROS levels above a critical threshold might be one wayto induce cancer cell apoptosis, as detailed below.

Furthermore, altered ion fluxes might lead, via hyperpolariza-tion, to an increased driving force for Ca2þ entry into the matrix,whereas cation (Kþ or Mg2þ ) channel opening-induced depolar-ization decreases Ca2þ uptake. Mitochondria have the capacity toaccumulate Ca2þ , using the mitochondrial calcium uniporter as ahighly selective ion channel,29 to a much higher concentrationthan found in the cytoplasm. Thus, mitochondria shape cytosoliccalcium signals.30 The mitochondrial matrix possesses a Ca2þ

buffering system and calcium exit pathways (the Naþ /Ca2þ

exchanger and transient opening of the permeability transitionpore) that normally prevents excessive [Ca2þ ] buildup (see, forexample, Drago et al.31). However, after apoptotic stimuli,sustained Ca2þ release from the endoplasmic reticulum (‘ERstress’) may lead to high mitochondrial matrix Ca2þ levels thatcauses prolonged opening of the permeability transition poreand subsequent loss of mitochondrial D~mH. Thus, Ca2þ overload

disrupts energy production, releases proapoptotic proteinsinto the cytoplasm and enables apoptosis and/or necrosis.31–33

In addition, mitochondrial Ca2þ accumulation also leadsto enhanced ROS production through multiple potentialmechanisms.34 Although the exact mechanistic role ofmitochondrial Ca2þ homeostasis in tumorigenesis (beyond ROSproduction and modulation of apoptosis) is not well understood,mitochondrial Ca2þ is an important signal transducer that caninitiate pathways mediating cell death. Therefore, drugs increasingcalcium uptake may efficiently kill cancer cells; however, how toselectively target cancer cells instead of healthy cells by inducingmitochondrial calcium accumulation remains unclear.

Mitochondrial calcium uptake is important for oxidativephosphorylation regulation35 and energy metabolism controlbecause it enhances the rate of NADH production bymodulating the enzymes of the Szent-Gyorgyi–Krebs cycle andfatty acid oxidation pathways.36,37 In general, strategies thatincrease energy and metabolite production in energy-demandingcancer cells promote a high glycolytic flux rate and thusallow enhanced tumor cell growth.38,39 Some IMM channels,by regulating DC, matrix volume, matrix acidification andROS production, would be expected to impact the functionalityof mitochondrial RC complexes. Indeed, enhanced substrateoxidation with matrix volume expansion has beendemonstrated,40 and pharmacological activation of calcium-induced potassium channels by NS11021, which affectsmitochondrial matrix volume, leads to enhanced respiratorycontrol.41 Interestingly, the respiration rate and oxidativephosphorylation efficiency were found to be increased inAugust rats that are characterized by an increased rate ofATP-dependent mitochondrial potassium transport as comparedwith Wistar rats that have lower ATP-dependent potassiumchannel activity.42 A recent paper reported the physicalinteraction and functional coupling of the mitochondrial large-conductance calcium-activated potassium channel (mtBKCa) andcomplex IV of the RC.43 Whether this physical interaction alsooccurs in other channels and plays a direct role in the regulationof mitochondrial energy fitness by ion channels is an importantand unresolved question. Finally, the changes in matrixvolume resulting from channel opening are expected to lead tostructural cristae reorganization that has recently been shownto affect respiratory supercomplex organization and respirationefficiency.44

Along with IMM channels, a role for the OMM channel VDAC inmetabolism regulation has become clear; the glycolytic enzymehexokinase (HK), when bound to VDAC1, regulates metabolitetrafficking (including ATP)45,46 through the OMM channel andprovides cancer cells with metabolic advantages. Furthermore,VDAC1-bound HK is less sensitive to inhibition by its product,glucose-6-phosphate,47 and avoids product inhibition that furtherboosts a high glycolytic rate. Thus, IMM and OMM channels areexpected to affect mitochondrial metabolism and RC functionthrough multiple mechanisms. Therefore, their modulation maylead to cancer cell metabolism alteration, ultimately promotingcell death, as discussed below. In summary, OMM and IMMchannel modulation may affect cancer cell fate through differentsignaling mechanisms.

THE IN VITRO AND IN VIVO TARGETING OF MITOCHONDRIALION CHANNELS IN CANCERThe mitochondrial channels characterized over the past twodecades include the outer membrane-localized VDAC and, in theIMM, potassium channels such as mtKATP, mtBKCa, mtIKCa, mtKv1.3,mtTASK-3, the nonselective permeability transition pore MPTP,chloride channels, the magnesium-permeable Mrs-2, the calciumuniporter, uncoupling proteins and proteins that have beenshown to function as ion channels in other membranes but not

Mitochondrial ion channelsL Leanza et al

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Oncogene (2014) 1 – 13 & 2014 Macmillan Publishers Limited

yet directly observed in mitochondrial membranes.48–51 Here, wediscuss the in vitro and in vivo evidence for ion channels involvedin cancer cell death obtained through either pharmacological orgenetic means (Figure 1). Data from animal models clearlydemonstrate the importance of mitochondrial channels and theirpotential as therapeutic cancer targets. The possible roles ofindividual channels in cell death in relation to the processesdiscussed above are summarized in Figure 2 and are described ingreater detail below. Pharmacological targeting of these channels(summarized in Table 1) and possible pitfalls are also criticallydiscussed.

The outer membrane-localized VDACThe major protein of the OMM, VDAC or porin, is required forcancer cell survival and participates in apoptotic signaling. A largebody of literature examines the role of VDAC in the regulation ofapoptosis.48,52 VDAC is being studied as a cancer-specific targetbecause tumor cells have increased glycolysis and VDACexpression.53 The role of VDAC1 and the other isoforms, VDAC2and VDAC3, in cell death is complex,54–56 but importantly, in vivoevidence shows that in cancer cells, the association of HK withVDAC1 protects against mitochondrial-mediated apoptosis.Therefore, disruption of the HK-VDAC1 complex represents anattractive therapeutic cancer target. Overexpression ofhexokinase-1 and 2 (HK2) and their association with VDAC areimportant characteristics of glycolytic cancers.57 The expression ofVDACs has also been found to be elevated in cancerous cellscompared with normal cells and may be altered withchemotherapy. High VDAC levels are an unfavorable prognosticfactor;53 moreover, VDAC downregulation by RNA interferencereduces cancer growth.58 This evidence may seem at odds withthe finding that VDAC overexpression induces apoptosis,59–61 butit illustrates how the functional meaning of a biological parameterdepends on context. VDAC upregulation in cancer goes hand inhand with HK2 upregulation and can be considered a componentof glycolytic upregulation. HK2 binding to VDAC, which allows for

ATP transport out of mitochondria, leads to a cancer cell metabolicadvantage (termed the Warburg effect), and it antagonizes celldeath through the inhibition of Bax-induced cytochrome crelease62,63 and/or inhibition of the mitochondrial permeabilitytransition (MPT).64 HK dissociation from VDAC seems to favor cell

matrix IMS

Kv1.3

IKCa

UCPs

MPTP

IMS

cytosol

MCU

VDA

C

MA

C

ASIC1a

K+K+

K+K+

Ca2+Ca2+

matrix IMS

solutes,ions

solutes,ions

Na+?Na+

H+?H+?

K+K+ TASK-3

Figure 1. Outer and inner mitochondrial membrane ion channels with documented roles in cell death regulation. For details on MAC, wesuggest consulting the recent review by the Kinnally group.4 With regard to other channels, detailed descriptions are provided in the text. Forthe mitochondrial UCP2, and ASIC1a channels, the nature of the ions transported under physiological conditions remains unknown (questionmarks). Except for ASIC1a and SK2, the involvement of all other channels in cancer cell death has been documented either by pharmacologicalor genetic means.

Pro-deathKinases

MPTPCyt c

detachment/release

CELL DEATH

Δψm

ROSMito & cytosolic

Ca2+ homeostasis

Glycolyticmetabolism

HK

UCP2

??

Kv1.3 IKCa MCU VDAC

Figure 2. Mitochondrial ion channels and their main proposed rolesin cell death regulation in cancer cells. Ion channels involved in theregulation of mitochondrial membrane potential, ROS production,calcium homeostasis and metabolism are indicated. Mitochondrialchannels with well documented role in cell death in cancer cells areshown. See text for details and other possible mechanisms.


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& 2014 Macmillan Publishers Limited Oncogene (2014) 1 – 13

Table 1. Pharmacological modulation of mitochondrial ion channels and use in cancer cells

Channel Cancer cell studies Agents Development stage Observations

VDAC Adenocarcinoma88

Non-small-cell lungcancer53

Glioblastoma57

HELA cervical cancer cells58

Melanoma65

Prostate cancer cell line65

Breast cancer cell line65

Lymphoblastic leukemia65

Multiple myeloma66

El-4 lymphoma65

Furanonaphthoquinone(FNQs)88

Preclinical development FNQ acts on VDAC and causesalteration of mitochondrialmembrane potential, NADH-dependent ROS production andcytochrome c release (in ref. 88)

Clotrimazole74 Clinical trial for Sickle cell disease(SCD) (see ref. 52 in ref. 74)

Commonly used as fungicide74

Inhibits KCa3.1 calcium-dependentpotassium channel73

Erastin86 In vitro studies86

PRLX 93936, erastin homolog inclinical trial for multiple myeloma(NCT01695590)

RAS/RAF/MEK-dependent action86

Oxidative cell death (not apoptosis)was induced via VDAC2 or VDAC3 (inref. 88)

Methyl jasmonates65 Preclinical development65,66

Topical application in cancerousskin lesions in humans67

Plant hormones induce MOMP inisolated mitochondria.Disruption of the HK–VDACinteraction;189 activation of BAX andCaspase-3 via ROS production66,68

Indirect activation of MPTP3-Bromopyruvate77 Clinical trial in hepatocellular

carcinoma79A pyruvate mimeticActs as an inhibitor of glycolysis78

but also as a covalently alkylatingagentPyruvylates certain proteins atcysteine residues79

MPTP Melanoma117

ProstateBreast cancerLymphoblastic leukemia112

Chronic lymphocyticleukemia113

Acute promyelocyticleukemia105

Multiple myeloma112,113

CSA133 Clinical use for different diseases CSA inhibits MPTP98,99

4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide(GSAO)105,106

Development forphase I clinical trial105

Novel anti-neovascular agent thatinteracts with redox active,mitochondrial protein dithiols inendothelial cells105

Mastoparan-likesequences107–109

Preclinical development107 (inclinical trial but not for cancer)

Cell penetrant, mitochondriotoxicand apoptogenic107

Betulinic acid2,115,190 Phase I/II clinical trialsNCT00346502

Steroid-like structure.Induces MOMP in isolatedmitochondria2,189,190

Gold complex AUL12111 Preclinical development Inhibits complex IIncreases ROS levels leading toactivation of GSK3a/b, favoring MPTPopening111

CD437115 Preclinical developmentRetinoid analog NRX195183 is inclinical trial (NCT00675870)

Synthetic retinoid (triterpenoid) ableto promote MOMP independently ofnuclear receptors189

Berberine117,118 In vitro studies117,118 Induces mitochondrial fragmentationand depolarization, oxidative stressand decreased ATP levels128

Honokiol119,120 In vitro studies on different cancercell lines and in clinical trial formicrobial infection, anxiety,oxidative stress and plateletaggregation119

Induces MPTP-dependent celldeath119,120

a-Bisabolol121 In vitro studies on glioma celllines;121

Preclinical, ex–in vivo studies onhuman acute leukemia191

Induces intrinsic apoptotic pathwaythrough the loss of mitochondrialinner transmembrane potential andrelease of cytochrome c121

Shikonin122 In vitro studies122

Clinical trial on human lungcancer192

Induces necroptosis122


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death through the disruption of aerobic glycolysis and the cell’senergy balance by altering the interaction of Bcl-2 family proteinswith mitochondria and by regulation of ROS production andfacilitation of VDAC oligomer formation.48,55 A class of novelapoptosis-inducing anticancer drugs, which currently comprisessmall molecules such as clotrimazole, bifonazole (both fungicides,in the tens-of-micromolar range) and methyl jasmonate (MJ), actby disrupting the HK-VDAC1 complex (Table 1).

Among these small molecules, MJ has been demonstrated to beactive in preclinical models of melanoma, prostate and breastcancer, lymphoblastic leukemia and multiple myeloma,65,66 alsoindependently of p53 status and has been tested in at least onepilot human study.67 However, the major obstacle for adoptingjasmonates as anticancer agents is their relatively high dosageneeded to exert their action. It should be also noted that differentmechanisms of action from the mechanism described above havealso been envisioned. MJ activates Caspase-3 through ROSproduction,68 and another study showed that it alsodownregulates the expression of proliferating cell nuclearantigen.69 Furthermore, MJ can bind to members of the Aldo-keto reductase family 1,70 which are potential tumor biomarkersinvolved in steroid metabolism.71,72 The fact that MJ does notaffect nontransformed, intact cells is compatible with mostsuggested mechanisms. Whether MJ can also induce cell deathin the absence of the proapoptotic Bax/Bak, which occurs in

chemoresistant cancer cells, remains unknown. Clotrimazole couldhave multiple effects because it also acts as a membrane-permeable inhibitor of the KCa3.1 calcium-dependent potassiumchannel that has also been localized to the IMM73 and seems to beinvolved in apoptosis regulation.74 In addition, peptidescorresponding to the amino-terminus of both HK-I75 and HK-II62

and a cell-permeable HK-II-based peptide76 may be potentiallyuseful as pharmacological tools.

The HK-VDAC complex (more specifically, HK) can also betargeted by the alkylating agent 3-bromopyruvate (3BP) thathas been reported efficacious in in vitro and in vivo studies (forexample, see reviews in refs 77–80) and possesses strongpotential as an anticancer agent in humans.79,81 In animalmodels, 3BP showed high efficacy against advanced-stagemalignant tumors by inhibiting both glycolysis andmitochondrial energy generation, possibly by interfering withthe HK-VDAC1 complex.80 Thus, 3BP has been proposed to targetmetabolism and block energy supplies. In addition, GAPDH,82

components of the RC,83 ER and lysosomes are targets of thiscompound. Its remarkable specificity for cancer cells may bepartially attributed to the presence of glycolytic pathway (HK andGAPDH) members among these targets; however, most of 3BPspecificity likely stems from facilitated entry into glycolytic cancercells via the lactate transporter.84 The transcription factor ATF2,which elicits oncogenic and tumor-suppressor activities in

Table 1. (Continued )

Channel Cancer cell studies Agents Development stage Observations

Kv1.3 T-cell leukemia cell line133

Chronic B lymphocyticleukemia (CLL)134

Osteosarcoma cell lineMelanoma133

Colon rectum cancer193

Breast cancer193

Prostate cancer193

Psora-4133,134 Preclinical development.Methoxypsoralen is in clinical trialfor cutaneous T-cell lymphomaNCT00056056

Blocks inward Kþ flux inducing ROS,mitochondrial depolarization,cytochrome c release and celldeath133

PAP-1133,134 Preclinical development Blocks inward Kþ flux inducing ROS,MT depolarization, cyt c release andcell death133

Clofazimine133 Preclinical development, ex–in vivostudies on chronic lymphocyticleukemia134 Used clinically to treatleprosy and autoimmunediseases137

Induces mtROS production andtriggers cell death133

IKCa Colon cancer cells73

Melanoma74

Breast cancer193

Prostate cancer193

Pancreas cancer193

Clotrimazole73 Clinical trial for Sickle cell disease(see ref. 52 in ref. 78)

Systemic application is not possiblebecause of hepatotoxicity induced bynonspecific effects on cytochromeP45074

TRAM-3473,74,77,78,194 Clotrimazole analog, lackingcytochrome P450 inhibitory effects,able to enhance TRAIL-inducedapoptosis via the mitochondrialpathway74

UCP2 Breast cancer168

leukemia, ovarian, bladder,esophagus, testicular,colorectal, kidney,pancreatic, lung andprostate tumors162,163

Genipin168 In vitro and in vivo studies168 Abolishes UCP2-mediated protonleak168

TASK-3 Ovarian cancer158 Zinc andmethanandamide

Nonspecific inhibitors ofplasmamembrane TASK-3

Abbreviations: ATP, adenosine triphosphate; CSA, cyclosporin A; GSK3a/b, glycogen synthase kinase-3a/b; MOMP, mitochondrial outer membranepermeabilization; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; TASK-3, TWIK-related acid-sensitive Kþ channel-3; TRAIL,tumor necrosis factor-related apoptosis-inducing ligand; UCP2, uncoupling protein-2; VDAC, voltage-dependent anion channel. This table summarizes thepharmacological tools used to elucidate the role of the indicated ion channels in several cancer cell types, with either in vitro or in vivo studies. SK2, MCU andASIC1a are not mentioned in this table because pharmacological evidence for their involvement in cancer cell death regulation is not available. See text forfurther details.


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melanoma and nonmalignant skin cancer, respectively, has beenrecently identified as a potent disruptor of the HK1–VDAC1complex. When localized to mitochondria in a protein kinase C-E-dependent manner, ATF2 increases mitochondrial permeabilityand promotes apoptosis.85

The heterocycle erastin has been found to selectively inducethe death of cells with mutations in the oncogenes HRas, KRasand BRaf in a VDAC2/3-dependent manner. Apparently, thiscompound induces oxidative stress involving VDAC throughan unknown mechanism.86 An erastin homolog, PRLX 93936,is currently being evaluated in a clinical trial in multiplemyeloma patients (http://www.cancer.gov/clinicaltrials). Finally, theanticancer agent furanonaphthoquinone, isolated from Avicenniaplants, induces caspase-dependent apoptosis via ROS productionby acting on VDAC. VDAC has also been proposed to mediate exitof superoxide anions from the intermembrane space, and itsclosure caused internal oxidative stress and sensitized mito-chondria to Ca2þ /ROS-induced MPT.87 Moreover, the anticanceractivity of furanonaphthoquinone and ROS production wereenhanced by VDAC1 overexpression.88

VDAC has also been proposed to directly participate in OMMpermeabilization and mediate cytochrome c release, either byoligomer formation48 or by the formation of a large porecomprising VDAC and Bax/Bak (Tsujimoto et al.89 but seeMartinez-Caballero et al.90). Knockdown of VDAC1 preventedcisplatin-induced conformational activation of Bax91 or selenite-induced cytochrome c release.92 However, Bax-inducedcytochrome c release from mitochondria isolated from wild-typeor VDAC1-, VDAC3- and VDAC1/VDAC3-null cells was reported tobe identical.93 In any case, the binding of anti-apoptotic Bcl-2 andBcl-xL to VDAC1 (with resulting porin activity inhibition)94

produced anti-apoptotic actions.95 A recent study reported thatconstructs consisting of cell-penetrating Antp fused to VDAC1-derived sequences, which prevented the interaction of VDAC1 andHK, Bcl-2 and Bcl-xL, were effective in the selective eradication of Bcells from patients with chronic lymphocytic leukemia.96

In summary, a systematic search for compounds that act onVDAC to antagonize cancer remains to be performed, but thosecompounds already identified are promising.

Inner membrane-localized permeability transition poreA number of cytotoxic agents and cellular stressors trigger theloss of IMM permeability. This process is most often caused byMPT that is considered to be a final common pathway of variousforms of cell death.97,98 In fact, MPT results in a ‘bioenergeticcatastrophe’; the transmembrane electrochemical proton gradientdissipates, ATP synthesis ceases and respiratory substrates are lostfrom the mitochondrial matrix. MPT is caused by the opening of alarge, Ca2þ -activated and oxidative stress-sensitive pore (MPTP)that creates an IMM permeable to ions and solutes up to 1500 Damolecular weight and leads to matrix swelling. This pore coincideswith the mitochondrial megachannel that has been studied bypatch clamp and characterized by conductances up to 1.5 nS.99

MPTP has recently been proposed to be formed by dimers of theFoF1 ATP synthase and Cyclophilin D.98,100 MPTP/mitochondrialmegachannel opening is considered to account for a substantialportion of the tissue damage caused by ischemia/reperfusion andoxidative stress.

In cancer cells, signaling pathways are activated that rendermitochondria more resistant to MPT induction.101–103 Becausechemotherapeutic agents cause oxidative stress, they mayactivate signals that induce cell death through MPTP opening.104

MPT inducers have potential oncological applications, butpotential side effects involving the nervous system or cardiactissues can be expected. A consistent number of compounds(often used at relatively high concentrations) have been shown toinduce oxidative stress and/or disruption of Ca2þ homeostasis

along with MPT. Some of the MPTP-targeting molecules, such as4-(N-(S-glutathionylacetyl) amino) phenylarsenoxide, are currentlybeing evaluated in clinical trials as promising drugs againstrefractory tumors.105,106 Mitochondria-penetrating peptides, suchas mastoparan-like sequences, peptides of the innate immunesystem and molecules developed by the Kelley group,107–109 alsoinduce MPT and could become candidates for future clinical trials.

MPTP can also be indirectly activated (or inhibited) throughmodulatory signaling pathways. For example, in cancer modelscontaining constitutively active extracellular signal-regulatedkinase, this kinase acts through the glycogen synthase kinase-3b/Cyclophilin D axis to repress cell death by MPT inducers suchas arachidonic acid or BH3-mimetic EM20-25.110 Induction ofoxidative stress by the gold complex AUL12 can lead to activationof glycogen synthase kinase-3a/b that favors MPTP opening.111 Alarge portion of MPTP-opening inducers (direct or indirect) forwhich in vivo antitumor activities have been reported2 are naturalcompounds, including jasmonates,112–114 betulinic acid, thesynthetic retinoid CD437,115,116 berberine,117,118 honokiol,119,120

a-bisabolol121 and shikonin122 (Table 1). Among these com-pounds, betulinic acid is currently in a phase I /II clinical trial fordysplastic nevi (http://www.cancer.gov/clinicaltrials). A retinoidanalog, NRX 195183, is also in a phase II trial for acutepromyelocytic leukemia (http://www.cancer.gov/clinicaltrials).

A specific, powerful MPTP inhibitor is also available: cyclosporinA, a cyclic endecapeptide.123–125 Cyclosporin A inhibits the MPTPthrough binding to matrix cyclophilin, a peptidyl-prolyl cis–transisomerase, and acts as an immunosuppressant by inhibitingcalcineurin. Further evidence for the link between MPTP andcancer is illustrated by the observation that patients treated withcyclosporin A to prevent rejection of organ transplants have ahigh incidence of cancer.126

The IMM potassium channel Kv1.3As mentioned above, IMM-localized potassium channels arepredicted to participate in the regulation of mitochondrialmembrane potential, volume and ROS production. Our researchgroups have identified a potassium channel, mtKv1.3, in the IMMof several cell types and have shown with mitochondria from cellsexpressing or lacking this channel that mtKv1.3 activity indeed hasan impact on DCm and on ROS production.127,128 Similar to someother IMM channels,50 mtKv1.3 is the mitochondrial counterpart ofthe plasma membrane-localized Shaker family potassium channel,Kv1.3. Its crucial role in apoptosis became evident because theexpression of a mitochondria-targeted Kv1.3 construct wassufficient to induce cell death upon apoptotic stimuli in CTLL-2T lymphocytes that lack Kv channels and are otherwise resistant toapoptosis. A physical interaction between Bax and mtKv1.3 hasbeen demonstrated in apoptotic cells, and Bax has been shown toinhibit channel activity at nM concentrations.129,130 Incubation ofKv1.3-positive isolated mitochondria with Bax or specific mtKv1.3inhibitors triggered apoptotic events, including membranepotential changes (hyperpolarization followed by depolarizationbecause of MPTP opening), ROS production and cytochrome crelease; however, Kv1.3-deficient mitochondria were resistant tothese inhibitors. The highly conserved Bax lysine residue 128 wasshown to protrude into the intermembrane space.131 This residueis responsible for the inhibitory effect of Bax on Kv1.3 activitybecause it mimics a crucial lysine residue in Kv1.3-blockingpeptide toxins. Indeed, mutant BaxK128E did not exert its effectson Kv1.3 and mitochondria, and it did not mediate apoptosis inBax/Bak double-knockout mouse embryonic fibroblasts, indicatingthat the toxin-like action of Bax on Kv1.3 triggers the abovedescribed mitochondrial events.130

Three membrane-permeable inhibitors of Kv1.3, Psora-4, PAP-1and clofazimine, are able to induce cell death at mM concentra-tions by directly targeting mtKv1.3 in different cancer cell lines, in


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http://www.cancer.gov/clinicaltrials



contrast to the membrane-impermeable high-affinity Kv1.3inhibitors ShK and margatoxin.132,133 Genetic deficiency or smallinterfering RNA-mediated downregulation of Kv1.3 abrogated theeffects of the membrane-permeable drugs that also killed cells inthe absence of Bax and Bak, in agreement with the abovedescribed model (Figure 3). In vivo, intraperitoneal injection ofclofazimine in an orthotopic melanoma B16F10 mouse modelreduced tumor size by 90%, without causing obvious sideeffects.133 Recently, promising results were obtained withprimary human cancer cells from patients with chroniclymphocytic leukemia, where drugs significantly and exclusivelydecreased the survival of pathologic B cells (which express higherlevels of Kv1.3 compared with B cells from normal subjects) byinducing the crossing of a critical ROS threshold.134 Thus, theselective apoptosis-inducing action of these drugs on tumor cellsseems to be related to a synergistic effect of an altered cancer cellredox state and higher Kv1.3 expression.134,135 Interestingly,As2O3, a clinically active antileukemic agent, has been reportedto inhibit mitochondrial respiratory function, increase ROSgeneration and enhance the activity of other O2

.� -producingagents against cultured and primary patient leukemia cells.136

Because clofazimine is already used clinically to treat someautoimmune diseases and leprosy137 and shows an excellentsafety profile, targeting mtKv1.3 is a feasible cancer therapy for atleast some cancer types. Interestingly, altered Kv1.3 expression isobserved in several different cancer cell lines and tumors,138 and acorrelation between Kv1.3 expression and cell sensitivity toclofazimine has been reported.139 In addition, the ability ofthese drugs to induce cell death in the absence of Bax and Bak,similar to other agents,140–142 could be useful becausedownregulation of these pro-apoptotic proteins represents acommon tumor cell resistance mechanism.143–146

Calcium-dependent potassium channelsThe activity of an intermediate conductance potassium channel(IKCa, KCa3.1) has been recorded from the inner mitochondrial

membranes of human cancer cells.73,147. IKCa can be selectivelyinhibited by low concentrations of clotrimazole and TRAM-34. Thislatter drug has been reported to synergistically increase thesensitivity of melanoma cells to the death receptor ligand TRAIL(tumor necrosis factor-related apoptosis-inducing ligand) via itsaction on mtIKCa.74 However, the effects of TRAM-34 onmitochondrial bioenergetics have not yet been investigated indetail, but TRAM-34 has been reported to induce hyper-polarization of the mitochondrial membrane (which is expectedif an influx of positively charged ions/molecules is inhibited),confirming that functional IKCa is expressed in the IMM.Interestingly, TRAM-34 application induced Bax translocation tothe mitochondria, representing an early step of apoptosis. BothTRAIL and TRAM-34 are characterized by a relatively good safetyprofile, suggesting that co-administration of these two drugsmight be exploited to treat melanoma.

In addition to the intermediate conductance channels, thepresence of small-conductance calcium-activated potassium (SK2/KCa2.2) channels in the IMM has been recently reported.148,149 Itspharmacological activation in a neuronal cell line exposed to toxicglutamate levels attenuated the loss of the mitochondrialtransmembrane potential, blocked mitochondrial fission,prevented the release of proapoptotic mitochondrial proteinsand reduced cell death.149 SK2 channel opening preventedmitochondrial calcium overload and mitochondrial superoxideformation. It will be interesting to investigate whether theapplication of mtSK2 inhibitors alone or in combination withother chemotherapeutics could promote cell death in cancer cellsexpressing SK2.

To our knowledge, whether the mitochondrial large-conduc-tance Ca2þ -activated Kþ channel (BKCa/KCa1.1)150 has a role incancer has not been investigated. However, the channel openerCGS7184 has been reported to induce the glioma cell death thatwas accompanied by increased respiration and mitochondrialdepolarization,151 perhaps downstream of an increased Ca2þ

release from the ER.152

��m

apoptoticcascade

Kv1.3

Bax

MPTP Cyt c

K+

IMM

OMM

ClofaziminePAPPsora

Psora PAP Clofazimine

Bax

Bax Bax

ROS

ROS

Figure 3. Proposed mechanism of action of mitochondrial Kv1.3 during apoptosis. Normally mtKv1.3 acts as a conduit for the passive flow ofKþ ions across the IMM, that is, as a component of the machinery controlling mitochondrial volume, ion homeostasis and transmembranepotential. In cells challenged with membrane-permeable Kv1.3 inhibitors able to reach the IMM-localized Kv1.3, a transient hyperpolarizationand enhanced ROS production occurs. These ROS can stimulate Bax recruitment and/or induce mitochondrial permeability transition as wellas promote cytochrome c detachment from the outer IMM surface. Consequently, a loss of cytochrome c occurs (large, red) via Bax oligomersor OMM lacerations that are caused by swelling of the matrix compartment following MPTP induction. The latter event also results in thecollapse of the potential and cytoplasmic dispersion of small molecules, such as NADH and Krebs cycle intermediates, hampering respiration.Pharmacological inhibition of mtKv1.3 is sufficient to induce Bax–Bak-independent cell death in cancerous cells that are already subjected to ahigher than normal redox stress.


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TWIK-related acid-sensitive Kþ channel-3 (TASK-3)A protein recognized by an antibody against TASK-3 (TWIK-relatedacid-sensitive Kþ channel-3; KCNK9), a two-pore potassiumchannel, was identified in the mitochondria of melanoma,keratinocytes153 and healthy intestinal epithelial cells.154 Thereduction of TASK-3 expression in WM35 melanoma cellscompromised mitochondrial function and cell survival.155 TASK-3knockdown also decreased human keratinocyte HaCaT cellviability following ultraviolet B irradiation.156 In mitochondriaisolated from HaCaT cells, TASK-3-compatible activity has beenrecorded for the first time by electrophysiology (patch clamp).156

Two aspects of TASK-3 still require investigation: first, whether theobserved migration and invasion-reducing effects of TASK-3overexpression in breast cancer cells157 and increased apoptosisinduced by TASK-3 blockers (zinc and methanandamide) inovarian carcinoma158 are correlated with the mitochondrialchannel expression and, second, whether pharmacologicalaction on mtTASK-3 can affect cancer cell death. However,because no highly specific modulators are available, this taskremains difficult.

Uncoupling protein (UCP)Uncoupling proteins (UCP1–5) belong to the superfamily ofmitochondrial anion-carrier proteins, and UCP1 has recently beenshown to mediate fatty acid-regulated proton transport.159 Thewidely expressed UCP-2 has also been proposed to transportprotons,160 (for review, see Cannon and Nedergaard161), althoughits physiological importance and the occurrence of UCP2-mediated proton leak are not entirely clear and remaindebatable. UCP2 has been suggested to regulate cell survival bydecreasing mitochondrial ROS because it might limit the maximalvalue of the proton gradient across the IMM, and a depolarizingproton leak would be expected to diminish superoxideproduction162,163 (see, for example, Cannon et al.164 for adiscussion on this topic). Increased ROS production wasobserved in UCP2 knockout mice, whereas UCP2 overexpressionmay contribute to a higher apoptotic threshold promoting cancercell survival because it prevents oxidative injury. Indeed, UCP2overexpression, documented in numerous tumor types, includingleukemia, ovarian, bladder, esophageal, testicular, colorectal,kidney, pancreatic, lung and prostate tumors, has been shownto protect cells from oxidative stress165 and even to abolish theapoptosis-inducing effects of chemotherapeutic drugs.166

Interestingly, UCP2 overexpression has also been proposed todirectly contribute to the Warburg phenotype167 and to thedevelopment of tumors in an orthotopic in vivo model of breastcancer.168 In support of this hypothesis, UCP2 expression in MCF7breast cancer cells was shown to lead to a decreasedmitochondrial membrane potential and increased tumorigenicproperties. These studies suggest that UCP2 overexpression isinvolved in the development of a variety of cancers and that UCP2can be considered as a promising oncological target, although theactual ions transported by UCP2 under physiological conditionsare still uncertain. As recently suggested,162 UCP2 may act as anuniporter for pyruvate, presumably promoting pyruvate effluxfrom the matrix and thus restricting glucose availability formitochondrial respiration162 and promoting mitochondrial fattyacid oxidation. Efflux of pyruvate would aid highly glycolytic cells,in which large amounts of pyruvate would otherwise putenormous redox pressure on the mitochondria. Nevertheless, aplant-derived small molecule, genipin, has been identified as anagent capable of suppressing the tumor-promoting functions ofUCP2 presumably by abolishing UCP2-mediated proton leak.168 Insharp contrast with the above studies, when highly metastaticMDA-MB-231 breast cancer cells recombinantly overexpressingUCP1, UCP2 or UCP3 were injected into the flanks of athymic nudemice, in vivo tumor growth was reduced 2.2-fold compared with

empty-vector control cells.169 The authors interpreted their resultsas a consequence of UCP-induced increased autophagy anddecreased mitochondrial function. This unexpected observationwarrants further investigation.

Mitochondrial calcium uniporter MCUThe mitochondrial Ca2þ ‘uniporter’ (MCU), a calcium-selective ionchannel,29 is responsible for low-affinity calcium uptake into themitochondrial matrix. The recent identification of the proteinresponsible for this activity170,171 has produced new perspectivespertaining to the control of Ca2þ signaling in the cell. In fact, MCUmay be a very useful tool to influence a number of cellularcalcium-dependent processes, including cell death.51 For example,subthreshold apoptotic stimulation in synergy with cytosolic Ca2þ

waves induced MPTP opening172 and, moreover, MCUoverexpression resulted in increased apoptosis upon with H2O2

and C2-ceramide challenges.170 In addition, recent data suggestthat in the absence of MICU1, a proposed negative regulator ofMCU, mitochondria become constitutively loaded with Ca2þ ,resulting in excessive ROS generation and sensitivity to apoptoticstress.173 To avoid calcium overload, MCU activity is also regulatedby a dominant-negative subunit, MCUb.174 As for a possiblerelationship between MCU and cancer, the MCU-targetingmicroRNA miR-25 has been shown to affect Ca2þ homeostasisin colon cancer cells through the specific downregulation of MCU.The miR-25 caused a strong decrease in mitochondrial Ca2þ

uptake and, importantly, conferred resistance to Ca2þ -dependentapoptotic challenges.175 Thus, MCU could be an important proteinfor tumorigenesis, and its specific pharmacological activators, ifidentified, might become useful tools. However, the situation islikely more complex. MCU overexpression has been observed inbreast cancer samples and has been suggested to provide asurvival advantage against some cell death pathways.176

Pharmacological or small interfering RNA-mediated inhibition ofmitochondrial Ca2þ entry sensitized cancer cells to positivelycharged gold nanoparticles through the induction of endoplasmicreticulum stress.177 Further complications arose from recentwork reporting that epidermal growth-induced epithelial–mesenchymal transition in breast cancer cells was not associatedwith changes in MCU expression, as assessed by quantitativereverse transcriptase–PCR.178 Finally, in sharp contrast with theextensive literature suggesting that alterations in mitochondrialcalcium play a central role in acute metabolic regulation anddetermination of a threshold for cell death, the basal metabolismof knockout animals was not markedly altered in the absence ofMCU.179 Furthermore, although experiments with MCU� /�

mitochondria confirmed that MCU is required for calcium-induced MPTP opening, MCU� /� mice exhibited no evidence ofprotection from ischemia–reperfusion-mediated injury. In thisstudy, tumor incidence in knockout mice was not assessed. Theforthcoming elucidation of MCU regulation,180–182 as well as thecreation of a conditional knockout mouse, might provideexplanation for these apparently contrasting findings.

Acid-sensing ion channel 1aIn a recent study, the sodium-permeable acid-sensing ion channel1a (ASIC1a) was documented in mitochondria, although thetransport properties of this mitochondrial protein were notdefined.183 Interestingly, neurons from ASIC1a� /� mice wereresistant to cytochrome c release and inner mitochondrialmembrane depolarization, and mitochondria from these cellsdisplayed an enhanced Ca2þ retention capacity and oxidativestress response. The authors proposed that mitochondrial ASIC1amay serve as a regulator of MPTP through a still ill-definedmechanism, thus contributing to oxidative neuronal cell death.Interestingly, lack of ASIC1 prevented axonal degeneration inexperimental autoimmune encephalomyelitis; however, MPTP


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function was not assessed.184 Unfortunately, no information iscurrently available on mtASIC1a expression in cancer tissues.

THERAPEUTIC PERSPECTIVESThe concept that mitochondria can serve as promising pharma-cological targets in oncology is gaining increasing support. Oneintervention strategy envisions direct targeting of mitochondrialchannels to induce the death of unwanted, cancerous cells.Although much work remains in this relatively new field, studiesinvolving VDAC, inner membrane Kþ and Ca2þ channels and thepermeability transition pore justify high expectations. To produceadvances in this field, further progress should focus on under-standing the functions of these and other channels in normal cells,mechanisms involved in cell death induction upon their manip-ulation, characterization of other mitochondrial channels (forexample, the 107-pS anion channel) in an oncological context,screening of potential drugs and improvements specificallytargeting drugs to mitochondria and to cancerous cells. Table 1summarizes the currently available pharmacological data target-ing mitochondrial ion channels obtained in various cancer cells.

To expand this field, studies should address the expression levelof a set of ion channels in cancer tissues. For example, among thechannels that are also present in the mitochondrial membranes,IKCa (KCa3.1) expression has been shown to be significantlyincreased in breast tumors bearing a p53 mutation, and acorrelation was detected between high expression of this channeland high tumor grades.185 The same situation occurs for VDAC1,VDAC2 and VDAC3.185 Conversely, BKCa (KCa1.1) expression wasreduced in patients with higher grade tumors compared withpatients with lower grade tumors.185 Emerging data onmitochondrial ion channels might help to identify specific drugsthat can be used to obtain maximal cancer-killing efficacy.

The major hurdle from a pharmacological perspective concernsthe specificity and membrane permeability of drugs acting onmitochondrial channels. In many cases, the used inhibitors havemultiple off-target effects as well (for example, methyl jasmonate,honokiol). Because several channels are present in both theplasma membrane and the mitochondrial membrane, drugstargeting the mitochondrial channels might exert undesiredeffects on processes linked to plasma membrane channels ofhealthy cells (for example, proliferation). Furthermore, only cellmembrane-permeable drugs will reach mitochondrial channels,and a portion of these drugs is likely to be retained in cellularmembranes. Therefore, higher drug concentrations than thoseconcentrations sufficient to block the channels are needed toexert an effect on cell viability, and these concentrations may leadto a loss of specificity. Different strategies can be envisioned tocreate more efficient targeting of mitochondrial channels. Cell-penetrating peptides (such as the VDAC-related peptides) are onepossibility, but clinical problems could arise, as these peptides areable to cross the blood–brain barrier. Another strategy might bederivatization of the drugs by adding a permeating positivelycharged moiety186–188 that is able to drive accumulation of thedrugs in mitochondria.

In summary, to exploit mitochondrial channels as oncologicaltargets, we should explore exactly how these ion channelscontribute to cell fate, and state-of-the-art pharmacological toolsshould be developed and used for specific targeting.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe are grateful to all co-authors for their important contributions to the work carriedout in their laboratories and reported in this review. We apologize for not citing all

publications that have been published on this topic. The work carried out in theauthors’ laboratories was supported in part by grants from the Italian Association forCancer Research (AIRC; Grant 11814 to IS), the EMBO Young Investigator Programgrant (to IS), the Progetti di Rilevante Interesse Nazionale (PRIN) program(2010CSJX4F to IS and 20107Z8XBW_004 to MZ), the Fondazione Cassa di Risparmiodi Padova e Rovigo (to MZ), the CNR Project of Special Interest on Aging (to MZ), theDFG Grant Gu 335/13–3 (to EG), the International Association for Cancer Research (toEG) and the Progetto Giovani Studiosi 2012 (to LL).

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