Correlation between Potassium Channel Expression and ...

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Current Pharmaceutical Design, 2014, 20, 189-200 189

Correlation between Potassium Channel Expression and Sensitivity to Drug-induced Cell Death in Tumor Cell Lines

Luigi Leanza1, Paul O’Reilly2, Anne Doyle2, Elisa Venturini1, Mario Zoratti3, Eva Szegezdi2,# and Ildiko Szabo1,*,#

1Department of Biology, University of Padova, Italy; 2Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway; 3CNR Institute of Neurosciences and Department of Biomedical Sciences, University of Padova, Italy

Abstract: Plasma membrane (PM) and mitochondrial (mt) ion channels - particularly potassium channels - became oncological targets soon after the discovery that they are involved both in the regulation of proliferation and apoptosis. Some members of the Kv Shaker family, namely Kv1.1, Kv1.3, Kv1.5 and Kv11.1 (Herg), and the intermediate-conductance calcium-activated potassium KCa3.1 (IK) channels have been shown to contribute to apoptosis in various cell lines. Kv1.3, Kv1.5 and IK are located in the plasma membrane but also in the mitochondrial inner membrane, where they participate in apoptotic signalling. Interestingly, an altered protein expression of some of the channels mentioned above has been reported in neoplastic cell lines/tissues, but a systematic quantification addressing the protein expression of the above potassium channels in tumor cell lines of different origin has not been carried out yet. In the present study we investigated whether expression of specific potassium channels, at the mRNA and protein level, can be correlated with cell sensitivity to various apoptotic stimuli, including chemotherapeutic drugs, in a panel of cancer cell lines. The results show correlation between the protein expression of the Kv1.1 and Kv1.3 channels and susceptibility to death upon treatment with staurosporine, C2-ceramide and cis-platin. Furthermore, we investigated the correlation between Kv channel expression and sensitivity to three distinct membrane-permeant Kv1.3 inhibitors, since these drugs have recently been shown to be able to induce apoptosis and also reduce tumor volume in an in vivomodel. Higher protein expression of Kv1.3 significantly correlated with lower cell survival upon treatment with clofazimine, one of the Kv1.3 inhibitors. These results suggest that expression of Kv1.1 and Kv1.3 sensitizes tumour cells of various origins to cytotoxins. Data reported in this work regarding potassium channel protein expression in different cancer cell lines may be exploited for pharmacological manipulation aiming to affect proliferation/apoptosis of cancer cells.

Keywords: Potassium channels, mitochondria, apoptosis, chemotherapeutic drugs, Kv1.3 inhibitors.

INTRODUCTION Impaired apoptosis and/or abnormally enhanced proliferation leads to deregulated cell growth and to tumour formation. Existing evidence indicates a crucial role for K+ channels in regulating both cell growth and cell death [e.g. 1-3]. K+ channels are upregulated in actively proliferating cells and are thought to be important for the maintenance of the cell membrane potential, which is in turn re-quired for the proper entry of calcium into the cells, a prerequisite for proliferation upon stimulation with a wide variety of mitogenic factors. This is well known for lymphocytes [4, 5], but also for cancer cells [3, 6], and indeed there is differential expression of several K+ channel family members in various tumor cells with respect to normal cells [7]. The importance of K+ channels for tu-mour cell proliferation is highlighted by recent publications where either intravenous or intratumoral injection of a highly specific Kv1.3 channel blocker (Margatoxin) significantly reduced tumour volume in vivo [8,9]. While a correlation between K+ channel activ-ity and proliferation has been demonstrated in a variety of cells [for review see e.g. 5,7], no such correlation has been established for cell death [10]. K+ channels of the plasma membrane (PM) are shown to affect apoptosis by regulating volume changes that accompany pro-grammed cell death [2], and by mediating loss of intracellular K+

and subsequent reduction in ionic strength, suggested to release the inhibition of proapoptotic caspases (but see ref. 11 who recently challenged this hypothesis). The mechanistic features of apoptosis are broadly understood and it is widely accepted that mitochondria have a central role since the release of cytochrome c (cyt c) (and other proteins) contributes in a fundamental way to the activation of

*Address correspondence to this author at the Department of Biology, Uni-versity of Padova, Italy; Tel: +39 049 8276324; Fax: +39 049 8276300; E-mail: [email protected] #E.S. and I.S. contributed equally to this study.

caspases, which marks the point of no return. Accumulating evi-dence points to a crucial role of mitochondria-localised K+ channels in the regulation of apoptosis as well, through modulation of bio-energetics and cytochrome c release [10, 12]. The PM-located two-pore TWIK-related acid-sensitive TASK-1 and TASK-3, NMDA receptors (mediating K+ flux), inward rectifying Kir channels [e.g. 13, 14] and calcium-dependent K+ channels have all been shown to contribute to apoptosis regulation in various cell types. Further-more, various Kv channel � subunits including subtypes Kv1.1, Kv1.3, Kv1.5, Kv11.1 (hERG) and Kv2.1 (e.g. for review see e.g. 10, 15) are implicated. Voltage-gated potassium channels (Kv) comprise a large family of channels which are expressed in both excitable and non-excitable cells. They control the resting plasma membrane potential and the frequency of action potentials in excit-able cells while in non-excitable tissues, such as pancreatic islets, immune system, and epithelial cells, they are involved in feedback regulation of the plasma membrane potential, and thus in processes ranging from secretion to cell proliferation [5, 16]. Each Kv gene encodes a protein subunit, four of which may form either homo-tetramers or heterotetramers within the same family (Kv1-Kv12). Functional diversity of Kv activities can be ascribed to hetero-tetramerisation as well as to the association of accessory proteins, like the �-subunits, able to modulate gating properties and assist multimerization. Alternative splicing and posttranslational modifi-cations also contribute to variety of activity, and different mecha-nisms have been proposed to regulate protein expression itself. For example, KCNRG has been shown to cause the suppression of Kv currents by affecting tetramerisation [17]. In addition, N-glycosylation of Kv1.3 promotes PM-localisation of the channel [18]. KCNE4 ancillary subunit instead decreases current density due in part to the impairment of Kv1.3 channel targeting to lipid raft microdomains [19]. Similarly, the matrix metalloprotease MMP23-PD keeps the channel protein in the secretory pathway, decreasing thus its surface expression [20].

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190 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Leanza et al.

Among Kv channels, Kv1.3 was one of the first voltage-gated potassium channels reported to be modulated during apoptosis [21] and was later shown to contribute to the increased K+ efflux detect-able during lymphocyte apoptosis [22]. Similarly, pharmacological targeting of calcium-dependent intermediate and big conductance (IK (KCa3.1) and BK (KCa1.1), respectively) potassium channels revealed that Staurosporine-induced apoptotic volume decrease (AVD) was dependent on K+ efflux through IK channel, while TRAIL-induced AVD was mediated by BK channels [23]. Most importantly, genetic evidence supports the crucial involvement of some Kv channels in the regulation of apoptosis. Downregulation of Kv1.3 expression by siRNA in human peripheral lymphocytes conferred resistance to apoptosis [24]. In addition, rat retinal gan-glion cells (RGC), which express Kv1.1, Kv1.2 and Kv1.3, under-went significantly diminished death upon transfection with siRNAs directed against Kv1.3 or Kv1.1 channels. In contrast, Kv1.2-targeted siRNAs had only a small effect on RGC death [25]. Kv1.3 depletion decreased expression of the pro-apoptotic molecules caspase-3, caspase-9, and Bad by a still unknown mechanism after axotomy, while Kv1.1 depletion increased mRNA expression of anti-apoptotic Bcl-XL. Kv1.1 is involved also in cerebellar granular neuronal apoptosis: its knock-down increases neuron viability [26]. In apparent contrast with the above results, Kv1.1 overexpression was recently shown to reduce glutamate-induced hippocampal neu-ronal apoptosis [27] and to attenuate neuron death resulting from glutamate toxicity [28]. As to Kv1.5, in pulmonary arterial hyper-tension down-regulation of Kv1.5 has been linked to impaired apoptosis [29]. Overexpression of Kv1.5 channel in human pulmo-nary artery smooth muscle cells and COS-7 cells caused enhanced cell apoptosis [30,31]. Interestingly, Kv1.5 has been shown to be activated by mitochondria-derived ROS [32] and a mitochondria-ROS-Kv1.5 axis has been proposed as an O2 sensing mechanism [33]. Kv11.1, whose expression in the nuclear membrane in addi-tion to the PM has been recently reported [34], mediates H2O2-induced apoptosis in various cancer cell lines [35]. Interestingly, the pro-apoptotic mediator cytochrome c can also activate Kv channels, while anti-apoptotic Bcl-2 inhibits them [36]. Finally, overexpres-sion of the background K+ channel TASK-3 throughout the hippo-campal structure resulted in protection of neurons from an oxygen-glucose deprivation (OGD) injury [37]. Instead, siRNA against TASK-2 of the same family attenuated apoptosis caused by BCR ligation in an immature B cell line (WEHI-231) [38]. Overall, these findings suggest that in many cell systems Kv channel down-regulation confers resistance to apoptosis, but exceptions exist. It is clear however, that in many cases pharmacological targeting of these channels affects cell survival making potassium channels promising oncological targets [16, 39, 40]. The present work focuses on the members of the Kv1 subfamily mentioned above and on IK, shown by various groups to contribute to death, resistance or sensitivity. Kv1.3, Kv1.5, BKCa and IKCa are present, at least in some cell types, not only in the plasma mem-brane but also in mitochondria (mt), where they regulate mitochon-drial function and participate in apoptotic signalling [see e.g. 12]. mtKv1.3 has been identified as a novel target of Bax, a pro-apoptotic Bcl-2 family protein [24, 41]. Evidence for the physio-logical relevance of the interaction of Bax with mtKv1.3 via Bax lysine 128 has been obtained in a cellular context, using Bax/Bak double knockout mouse embryonic fibroblasts [41]. Mutant BaxK128E was unable to mediate cell death in DKO MEFs chal-lenged with various apoptotic stimuli. It is of note that the action of Bax is not restricted to Kv1.3: Kv1.1, mtKv1.5 and mtKCa1.1 (BK) can also interact with Bax [42,43]. Indeed, in macrophages, which express both Kv1.3 and Kv1.5 in their mitochondria, down-expression of both channels is required to prevent staurosporine-induced apoptosis. Recently we reported for the first time that Psora-4, PAP-1 and clofazimine, three distinct membrane-permeant inhibitors of Kv1.3, induce death by directly targeting the mito-chondrial channel in multiple human and mouse cancer cell lines,

while membrane-impermeable, selective and high-affinity Kv1.3 inhibitors ShK or Margatoxin did not induce apoptosis, further proving the crucial role of mtKv1.3 for apoptosis [8]. Importantly, the membrane-permeant drugs killed MEF and Jurkat cells also in the absence of Bax and Bak, a result in agreement with the current mechanistic model for mtKv1.3 action. Genetic deficiency or siRNA-mediated downregulation of Kv1.3 abrogated the effects of the drugs, proving specificity of their action via Kv1.3. The impor-tance of our findings was validated in vivo: intraperitoneal injection of clofazimine reduced tumour size by 90% in an orthotopic mela-noma B16F10 mouse model in vivo, while no adverse effects were observed in several healthy tissues. Finally, mtIKCa3.1 inhibition by membrane-permeant inhibitor TRAM-34 was recently shown to enhance TRAIL-induced death [44]. Given the important role of Kv channels for the regulation of cell death and the accumulation of evidence pointing to an altered expression of Kv1.1, Kv1.3, Kv1.5, Kv11.1 and IK in cancerous tissues (see discussion), in the present work we have addressed whether a correlation exists between the expression of these channels and the susceptibility of tumour cell lines of different origin towards apoptotic stimuli and to widely used chemotherapeutics that activate the mitochondrial apoptotic pathway. Furthermore, in light of the results previously obtained via mtKv1.3 modulation by pharmacological means, we measured apoptosis induced by Psora-4, PAP-1 and clofazimine also.

MATERIALS AND METHODS Reagents Membrane permeable Kv1.3 inhibitors, Psora-4, PAP-1 and clofazimine (CLZM) as well as etoposide (ETOP), cisplatin (CisPL), C2-ceramide (CRM) and staurosporine (STS) were from Sigma Aldrich. The concentrations used to induce cell death were the following: STS: 2 �M; CRM: 10 �M; CisPL: 10 �M; ETOP: 10 �M; Psora-4 and PAP-1: 20 �M; CLZM: 10 �M if not otherwise indicated. MDRi CSH and Probenecid were used at 4 and 100 �M concentration, respectively.

Cell Culture MCF-7 and MDA-MB-231 breast cancer, DLD-1 and Colo205 colon carcinoma and SHSY5Y neuroblastoma cells were main-tained in DMEM (Life Technologies) supplemented with 10 % foetal bovine serum (FBS), 10 mM Hepes (Sigma Aldrich), 1% non-essential amino acids (Life Technologies), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin. OCIAML-3, HL-60, K562, ML-1 and MOLM-13 acute myeloid leukemia cells were cultured in RPMI medium (Life Technologies) with 10% FBS, 10 mM Hepes, 1% non-essential amino acids, 100 U/ml penicillin and 0.1 mg /ml streptomycin. All cell lines were maintained in incubators at 37°C with a 5% of CO2. K562 cells were a gift from Prof. Arianna De-ana-Donella (University of Padova, Italy).

Enriched Membrane Fraction Isolation 6 x 107 cells were collected in a tube, washed with 5 ml of Phosphate Saline Buffer (PBS) and centrifuged at 400 g for 10 min at room temperature (RT). Cells were resuspended in 300 �L of TES buffer (100 mM TES + 1 M sucrose, 100 mM EGTA, 1X cocktail protease inhibitors and lysed by an electronic pestle (Kon-tes, Sigma Aldrich) for 5 min on ice. Unbroken cells were separated by centrifugation at 500 g for 10 min at 4°C. The supernatant was collected and the still intact cells were resuspended in 200 �l TES buffer and subjected for an additional 5 min to homogenization with the electronic pestle on ice. After a new centrifugation at 500 g for 10 min at 4°C, the supernatants were combined in an Eppendorf tube. The soluble cytosolic fraction was separated from the mem-brane-enriched fraction by centrifugation at 19,000 g for 10 min at 4°C. Finally, the pelleted membranes were resuspended in TES buffer and stored at -80°C. Protein concentration was determined using the BCA method in a 96 well plate (200 ul total volume for

Correlation between Potassium Channel Expression and Sensitivity Current Pharmaceutical Design, 2014, Vol. 20, No. 2 191

each well) incubating at 37°C in the dark for 30 min. Absorbance at 540 nm was measured by a Packard Spectra Count 96 well plate reader.

Western Blotting Membrane enriched fraction proteins from the different cell lines were separated by SDS-PAGE in a 10% polyacrylamide gel containing 6 M Urea. To enhance protein separation, samples were solubilized for 1 h at RT in Sample Buffer (30% Glycerol + 125 mM Tris/HCl pH 6.8 + 9% SDS + 0.1M DTT+ Bromophenol blue). After separation by electrophoresis, gels were blotted overnight at 4°C onto Polyvinylidene fluoride (PVDF) membranes. After block-ing with a 10% solution of defatted milk, the membranes were in-cubated with the following primary antibodies overnight at 4°C: anti-Kv1.1 (mouse monoclonal, Abnova); anti-Kv1.3 (rabbit poly-clonal, Alamone Labs APC-101); anti-Kv1.5 (rabbit polyclonal, Alamone Labs APC -004); anti-Kv11.1 (hHERG; rabbit polyclonal, Alamone Labs); anti-KCNN4 (IK, rabbit polyclonal, Abcam, ABC 65985); anti-SERCA (mouse monoclonal, Affinity Bioreagents MA3-910); anti-Na+/K+ ATPase (mouse monoclonal, Abcam ab7671); anti-Bcl-2 (mouse monoclonal, Santa Cruz SC-9746); Mcl-1 (rabbit monoclonal, Cell Signalling 4572); BclxL (mouse monoclonal, Santa Cruz SC-8392); Bax (rabbit monoclonal, Cell Signalling 2772); After washing off the excess primary antibody, the membranes were developed using corresponding anti-mouse or anti-rabbit secondary antibodies (Calbiochem). Antibody signal was detected with enhanced chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific). Densi-tometry was performed using the Quantity One 1-D software (Bio-Rad).

MTT Assay Cells were seeded at 40,000 (AML) or 5,000 (solid tumour) cells per well in a 96 well plate the day before treatment. Cells were treated with the different compounds, as indicated in the figure legends, for 24 h at 37°C. Cell survival was determined based on the proportionate transformation of MTT into formazan following the instructions by the supplier (CellTiter 96 Aqueous One Solu-tion, Promega). Formazan accumulation was quantified by measur-ing absorbance on a Packard Spectra Count 96 well plate reader at 490 nm. All measurements were performed in quadruplicate in each experiment.

Apoptosis by FACS Analysis Cells were seeded at 80,000 per well in a 96 well plate and treated for 24 h at 37°C with different compounds as indicated in figure legends. Cells were then incubated with 2 �L of FITC-conjugated Annexin-V (Roche) for 20 min at 37°C. After incuba-tion cells were collected in FACS tubes and analysed by cyto-fluorimetry (FACSAnto II, BD biosciences).

Analysis of Microarray Data Raw microarray data in .CEL file format produced by the Weinstein lab was acquired from the NIH cell-miner database (http: //discover.nci.nih.gov). Background correction and normalization of the dataset has been performed using the Affy package of Biocon-ductor (RMA algorithm) in the statistical environment, R (version 2.10.1) [45]. The microarray probeset best representing the genes of interest has been selected using the JetSet method designed for scoring and ranking probe sets for specificity, splice isoform coverage and ro-bustness against the degeneration of the transcript developed by Li et al [46]. The selected representative mRNA probeset (Probe Set IDs: KCNA1(230849_at), KCNA3(207237_at), KCNA5 (206762_at), KCNN4(204401_at) and KCNH2(205262_at)) for each gene of interest was extracted from the normalised microarray

data and graphed after sorting the cell lines by reducing expression level.

Correlation Studies: Acquisition of Cell Survival Data for Mi-croarray Data Cisplatin, etoposide and staurosporine-induced cytotoxicity in the NCI60 panel of cell lines has been obtained from the following public database. The drug concentration inducing 50% cell death (lethal concentration-50; LC50 value (Log10 (M)) has been deter-mined for multiple treatments of etoposide (n=4), staurosporine (n=3) and cisplatin (n=4) dosages. Cell death was measured as fol-lows. After 24 h, two plates of each cell line are fixed with TCA, this is a measurement of the cell population for each cell line at the time of drug application. Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the treatment is stopped by the addition of cold TCA. Cells are fixed by the gentle addition of 50 �l of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with water and air dried. Sulforhodamine B (SRB) solution (100 �l) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 �l of 80 % TCA (final concentration, 16 % TCA). These values were largely comparable; therefore the mean LC50 value across the treatments has been calculated and used for correlation analyses.

Correlation Studies: Statistical Analysis All analyses were done in R (version 2.15.1) [47], and correlation graphics were produced using the R corrgram package (version 1.4) [48]. The mRNA and/or protein expression values for potassium channels (Kv1.1, Kv1.3, Kv1.5, Kv11.1 and IK) and a selection of apoptosis regulators (Bcl-2, BclxL, Mcl-1, Bax and XIAP) were treated as continuous variables. The association between gene expression and response to the studied treatments has been assessed using the Pearson product moment correlation coefficient [49]. The correlation coefficient r, also called the linear correlation coefficient measures the strength and the direction of a linear relationship between two variables. The value of r is between -1 and +1. The closer the r value approaches -1 or +1, the stronger/closer the correlation is. A positive r value indicates that as one variable increases so does the other while a negative value describes an inverse relationship between the variables such that as one goes up the other goes down (e.g. as K+ channel expression increases, viability in response to staurosporine treatment decreases). An r value of 0 indicates no correlation. Significance was determined using two-tailed paired t-test. Due to the limited availability of a relatively small sample number due to the technical limitations imposed by the protein analysis, the significance level was set to p<0.1.

RESULTS Potassium Channel Expression in Different Tumour Cell Lines Limited information is available about the expression of potas-sium channels in general in tumours and tumour cell lines. To study this question, analysis of Affymetrix data obtained on 59 cancer cell lines (listed in Supplementary Table 1) was performed to evaluate the expression of mRNA for different potassium channel proteins, namely of Kv1.1 (KCNA1), Kv1.3 (KCNA3), Kv1.5 (KCNA5), IKCa (KCNN4), and Kv11.1 (KCNH2). With the exception of IK (KCNN4), all the examined K+ channels showed a narrow expres-


sion range, indicating that these genes are ubiquitously expressed at the mRNA level in most cell types (Fig. 1A). In order to study whether potassium channel mRNA expression is associated with a tumour cell’s response to chemotherapeutics, we have analysed whether there is a correlation between sensitivity to cisplatin or etoposide, genotoxic drugs activating the mitochon-drial apoptotic pathway and Kv1.1, Kv1.3, Kv1.5, IK, or Kv11.1 mRNA expression. Additionally, we have also analyzed correlation between the transcript expression of the above K+ channels and sensitivity to staurosporine, a classical inducer of the intrinsic apop-totic pathway. To this end, first the mean LC50 concentration (the concentration that caused 50% reduction in viability measured by a 50% drop in total protein content in the sample) of the cytotoxic drugs has been determined as detailed in the Materials and Methods

section. Using these values, the Pearson's product moment correla-tion coefficient has been determined as a measure of association. Interestingly, no association could be found between any of the channels and any cytotoxic drug, including staurosporine (Fig. 1B). mRNA levels frequently fail to reflect actual protein expression levels [50], which may explain the discrepancy between the lack of association for K+ channel mRNA expression against sensitivity to cytotoxic drugs and the well-established role for these channels in the regulation of apoptosis in the literature. Thus, we studied the expression of the K+ channel proteins in a set of tumour cell lines of different origins, including myeloid leukemia cell lines (OCIAML-3, HL-60, K562, ML-1, MOLM-13), breast adenocarcinomas (MCF-7, MDA-MB-231), colon carcinomas (DLD-1, Colo205) and a neuroblastoma (SHSY5Y) cell line. Due to the fact that ion chan-

Fig. (1). Lack of correlation between K+ channel mRNA expression and susceptibility to cytotoxic drugs. mRNA expression values for (A) KCNA1 (Kv1.1), KCNA3 (Kv1.3), KCNA5 (Kv1.5), KCNN4 (IK or KCa3.1) and KCNH2 (Kv11.1 or Herg) were extracted for 59 tumour cell lines from a publicly available microarray data set. All of the above-mentioned channels appear to be expressed across the 59 lines (bars). (B) Correlation between K+ channel mRNA expression and response (lethal concentration-50, LC50) to the cytotoxic agents cisplatin (cisPL), etoposide (ETOP) and staurosporine (STS). None of the correlation index values appeared to be significant using paired, two tailed t-test.


nels are generally low-abundance proteins, instead of whole cell lysates, we used membrane-enriched subcellular fractions to deter-mine K+ channel protein expression using Western blotting (Fig. 2A). As a loading control we used antibodies against the endoplas-mic reticulum calcium ATPase (SERCA) and plasma membrane sodium-potassium ATPase (Na+-K+ ATPase), given that the expres-sion of the Na+/K+ ATP-ase mRNA expression was found to be fairly constant across the NCI60 cell line panel (not shown). K+ channel protein expression was quantified by densitometric analysis and the obtained values were normalized taking into account the Na+-K+ ATPase signal in a given cell line with respect to that ob-served in Colo205 cells. Normalized values were then used for subsequent correlation analyses (Fig. 2B). Please note that the val-ues reported on the y scale do not reflect the relative quantity of the different channels in a given cell line (given that the used antibodies have different affinities), instead, permit a comparison for expres-sion of a given channel among the different lines. The results re-vealed that K+ channel expression at the protein level varied to a much bigger extent than at the mRNA level in case of all the stud-ied channels (compare e.g. (Fig. 2B) and (Fig. S1)), indicating sig-nificant post-transcriptional regulation of K+ channel expression.

Tumour Cell Sensitivity to Pro-apoptotic and Chemotherapeu-tic Agents which Activate the Mitochondrial Apoptotic Pathway Experimental evidence in various systems implicate that the low potassium channel expression may confer resistance against apoptotic stimuli while their overexpression may increase sensitiv-ity. Considering the differences observed in K+ channel protein expression in the nine cell lines tested, we determined the sensitiv-ity of these cell types to cytotoxic and chemotherapeutic drugs which activate the mitochondrial apoptotic pathway, namely staurosporine (STS), ceramide (CRM), and to two chemotherapeu-tic compounds, Etoposide (ETOP) and Cisplatin (cisPL) (Fig. 3). Staurosporine, isolated from the bacterium Streptomyces staurosporeus, is a classical inducer of the intrinsic apoptotic path-way: it inhibits serine threonine kinases in a non-selective way by preventing ATP binding to the kinases. Ceramide is a lipid second messenger that has been found to be released from the plasma membrane and induces apoptosis [e.g. 51]. Although the naturally occurring long-chain ceramides are often used for induction of cell death and have been shown to be very efficient to reduce tumor growth also in vivo [52-54], we used C2-ceramide since induction of apoptosis by C2-ceramide has been shown to affect the mito-chondrial network and the ability of mitochondria to accumulate less Ca2+ released by the ER than control cells [55]. Interestingly, ceramides also directly modulate potassium channel function [e.g. 56-59]. Etoposide is a chemotherapeutic agent, which forms a ter-nary complex with DNA and topoisomerase II enzyme and causes DNA strands to break, thus promoting apoptosis of the cancer cell which relies heavily on topoisomerase II for proliferation. Cisplatin is a platinum-containing anti-cancer drug, that acts by crosslinking DNA in several different ways and thus inhibits both replication and transcription of DNA. The cells were treated with the indicated doses of the different agents for 24 hours, and cell survival was determined by MTT as-say (Fig. 3). Induction of cell death by the same treatments has also been determined for several of the cell lines in the panel by measur-ing Annexin V exposure to confirm that the reduced viability meas-ured in the MTT assays was due to induction of cell death rather than reduced proliferation. Data from a representative experiment are shown in (Fig. 3C). The response of the cells to the cytotoxic drugs varied on a broad scale, but within each cell line, the four drugs showed closely correlated effects (Fig. 3B). Staurosporine and ceramide showed a similar effect in six out of the nine cell lines (OCIAML3, ML-1, MOLM-13, SHSY5Y, DLD-1, MDA-MB-231). Similarly, the two genotoxic drugs, etoposide and cisplatin had comparable effects in

two thirds of the cell lines (OCIAML3, ML-1, SHSY-5Y, Colo205, DLD-1, MCF-7), in line with the similar mechanism of action of these agents (Fig. 3A, 3B). Of note, a significant positive correla-tion was also found between the effect of ceramide and both etoposide or cisplatin, whereby the cell lines which showed high sensitivity to ceramide appeared to also be sensitive to the other two compounds (although to a lesser extent) (Fig. 3B). Next it was analysed whether expression of the K+ channels at the protein level correlate with sensitivity to the cytotoxic drugs using the protein expression values obtained from the densitometric analysis (Fig. 2B) against the percentage of viability values (Fig. 3A). The analysis revealed a good correlation between Kv1.1 and sensitivity to staurosporine (p=0.03) as well as between Kv1.3 channel expression and sensitivity to cisplatin (p= 0.09) and cera-mide (p=0.10) (Fig. 3D). This correlation was negative, which indi-cates that with decreasing expression of Kv1.1 and Kv1.3 the resis-tance of the tumour cells against the cytotoxic drugs increased. Kv1.1 expression significantly correlated with STS sensitivity in accordance with our previous data showing that otherwise resistant CTLL-2 cells, when expressing Kv1.1, underwent apoptosis upon STS treatment [8, 24]. In the case of Kv1.3, we have previously shown that sensitivity of CTLL-2 cells to ceramide and STS-induced apoptosis was restored upon transfection with Kv1.3 [24]. The correlation analysis shows a slightly lower value for Kv1.3/STS than for Kv1.1/STS although further cell lines will have to be tested to render the former correlation statistically significant. For the rest of the K+ channels (Kv1.5, IKCa and Kv11.1) no sig-nificant correlations were detectable with any of the apoptotic stim-uli (p values ranging from 0.46 to 0.93), underlying the predomi-nant effect of Kv1.1 and 1.3 in regulating intrinsic apoptosis signal-ling.

Cancer Cell Sensitivity to Membrane Permeant Kv1.3 Inhibi-tors Acting on Mitochondrial Kv1.3 Potassium Channel As mentioned above, we have recently demonstrated that mem-brane-permeant inhibitors of mitochondrial Kv1.3 can selectively kill cancer cells by inducing apoptosis both in vitro and in vivo [8]. Considering that the cancer cell lines analysed in (Fig. 2) all ex-pressed Kv1.3 at variable levels (except K562 cells, which were included as a negative control since they do not express Kv1.3 [60] and have been shown to be resistant to Kv1.3 inhibitors), we de-cided to verify the effect of Kv1.3 inhibitors, at the doses previ-ously shown to induce apoptosis. Psora-4 (20 �M), PAP-1 (20 �M) or Clofazimine (10 �M) were applied in the presence of MDR in-hibitors (MDRi) in order to avoid export of these drugs from the cells. Clofazimine itself is acting also as an MDR inhibitor [8]. Previous publications have shown that a correlation exists for the distribution of Kv1.3 between the plasma- and inner mitochondrial membrane in a number of cell types (i.e. if the channel is present in the plasma membrane, it is also present in the mitochondrial mem-brane) [e.g. 8, 43, 61]. If we incubate intact cells with membrane-impermeant Kv1.3 inhibitors, e.g. MgTx or Shk, no apoptosis oc-curs. Instead, the membrane-permeant inhibitors induce a series of mitochondrial changes via mtKv1.3, leading to apoptosis induction clearly indicating that the mitochondrial population of the channel is crucial for cell death. Thus, if the Kv1.3 channel was functional in the tumour cell, it was expected that the cell permeable inhibitors could trigger apoptosis. To test this hypothesis, the tumour cell line panel was treated with the indicated doses of the Kv1.3 inhibitors for 24 hours after which cell survival was evaluated by MTT assay (Fig. 4). Again, for the myeloid cell lines the sensitivity towards the drugs was studied by FACS analysis confirming that the observed decrease in cell survival is due to increased apoptosis (an example is shown in (Fig. 4C)). Staurosporine was used at 2 �M concentra-tion. The study revealed an inverse and significant correlation be-tween Kv1.3 expression and susceptibility to cell death upon clo-fazimine treatment (p=0.06904), as expected and confirming the specific action of the Kv1.3 inhibitor clofazimine. Possible explana-


tions for the lack of strong correlation between Kv1.3 expression and the effects of PAP-1 and Psora-4 are discussed below. No other significant correlations could be observed, except for Herg expres-sion and Psora-4 treatment: interestingly, the higher Herg expres-sion was, the better the cells survived Psora-4 treatment (p=0.07793) (Fig. 4D). Finally, to examine whether Kv1.3 inhibitors act upstream or downstream of the Bcl-2 family the expression of Bcl-2 family proteins, including Bcl-2, BclxL, Mcl-1 and Bax has been deter-mined in whole cell lysates of the cell line panel using Western blotting quantifying protein expression by densitometry (Supple-mentary Fig. 2). The levels of the four proteins were normalized using the levels of the same proteins in an HCT116 human colon tumour cell lysate. There was no relationship between the expres-sion of any of the Bcl-2 family members examined and reduction of cell survival induced by K+ channel inhibitors, confirming that in-hibition of K+ channels induces cell death that bypasses regulation

by the Bcl-2 family (Fig. 5). However, a negative significant corre-lation exist between Bcl-2 expression and sensitivity to clofazimine, indicating that cells expressing high levels of Bcl-2 (myeloid cell lines) tend to be more sensitive to clofazimine. On the other hand, expression of XIAP correlated with reduced susceptibility to the Kv1.3 inhibitors, in line with the current understanding that inhibi-tion of Kv channels induces cell death by triggering mitochondrial depolarisation and cytochrome c release, which is inhibitable by downstream blockage of caspase activation/activity, for example by XIAP (Fig. 5).

DISCUSSION In the present work we examined the expression of five differ-ent potassium channels, previously shown to be linked to the regu-lation of apoptosis, in nine cancer cell lines of different origin. In order to understand whether potassium channels impact on the sen-sitivity of cancer cells to apoptosis inducers as well as to che-motherapeutics, a correlation analysis was performed revealing a

Fig. (2). Expression of Kv1.1, Kv1.3, Kv1.5, IK and Kv11.1 proteins in nine different cancer cell lines. (A) Membrane-enriched fractions obtained from the indicated cell lines were loaded on SDS-PAGE (40 �g proteins/lane). Western blot was performed with specific antibodies against the indicated channel proteins. Na/K ATP-ase was used as loading control. Apparent molecular weights of the bands are the following: : Kv1.3: 65 kDa; Kv1.1: 50 kDa; Kv1.5: 66 kDa; IKCa: 50 kDa; Herg: 135 kDa; PMCA: 140 kDa; SERCA: 104 kDa; Na+/K+ ATP-ase: 100 kDa. The blots shown derive from 2 gels processed together. Similar results were obtained in another experiment. (B) Quantification of K+ channel expression based on densitometry. Values refer to those obtained fol-lowing normalization as described in the text, from two WBs including the one shown in (A).


Fig. (3). Sensitivity of cancer cell lines to cytotoxic drugs staurosporine, ceramide, etoposide and cisplatin as a function of K+ channel expression. (A)The indicated cell lines were treated with the drugs for 24 hours and cell survival was determined by MTT assay (mean ±SD are shown). Measurements were performed in quadruplicate in 3 sets of separate experiments. (B) Correlation between all the cytotoxic drugs tested against each other. As can be observed from the graph each compound correlated 100% with itself (r=+/-1). This acted as a control for all correlation data analysis carried out. Significant correlations were observed for cisplatin/ceramide, cisplatin/Staurosporine, and cisplatin/etoposide. (C) Representative FACS analysis determining apoptosis (as assessed by Annexin binding) and percentage of apoptotic death following the indicated treatments (n=3)(mean ±SD are shown). (D) All treatments with cytotoxic compounds were shown to have a positive correlation with all K channels examined in this study. Potassium channels that were shown to have a significantly strong positive correlation with cytotoxic treatments included Kv1.1 with STS (p=0.0302) and Kv1.3 with cisPL (p=0.09144).


Fig. (4). Sensitivity of cancer cell lines to membrane-permeant Kv1.3 inhibitors as a function of K+channel expression. (A) MTT assay and analysis as in (Fig. 3A). (B). The correlation between the Kv channels inhibitors were all in a positive direction. PAP-1 (PAP) correlated against Psora-4 (Pasora) showed the most significant correlation indicating the similarity of functionality in these compounds. As can be observed in the graph above each treatment correlates fully (r = +/-1) with itself. They therefore were included to act as experimental controls for data analysis in R (bioconductor). (C) A representative experiment in MOLM-13 cells, performed as in (Fig. 3C). (D) Association between K+ channel protein expression and Kv1.3 channel inhibitor-induced cell death. The * denotes significant values determined by paired, two-tailed t-test.


Fig. (5). Relationship between the expression of Bcl-2 family members and reduction of cell survival induced by K+ channel inhibitors. Correlation analysis was performed on the basis of Western blot and densitometry regarding expression of the indicated proteins (Supplementary Fig. 2) and results of the MTT assay. Asterisks denote significant values determined by paired, two-tailed t-test.

significant relationship for Kv1.1/Kv1.3 levels on one side and staurosporine, ceramide and cisplatin toxicity on the other. Higher expression of these channels renders cells more sensitive towards cytotoxins. Considering that for Kv1.3 in particular an altered ex-pression has been observed in several cancer tissues, and highly specific channel blockers exist, this information may well be of practical value. The Kv1.3 gene is up-regulated in cancers such as large B cell lymphoma [62] and gliomas (along with Kv1.5)[63]. In prostate cancers Kv1.3 is mainly expressed in early stages of pro-gression [64, 65]. Human breast cancer specimens show a positive immunoreaction for Kv1.3 in all the examined samples [66] and Jang and colleagues reported that the expression level of Kv1.3 is positively correlated to the stage of breast cancer [67]. Kv1.3, alongwith other Kv � subunits, was detected in primary human samples of colon carcinoma [68, 69]. Its expression was instead decreased in pancreatic adenocarcinomas and down-regulation of the channel was associated with metastatic tumours [70]. Both Kv1.3 and Kv1.5 showed a high expression in leiomyosarcoma (LMS) tumors with respect to healthy muscle and a correlation of Kv1.3 and Kv1.5 expression with tumor aggressiveness was observed [71]. Two re-cent studies, one using a xenograft model of human lung adenocar-cinoma in nude mice [9] and another using an orthotopic melanoma model [8] showed that Margatoxin (MgTx), a specific non-permeant toxin inhibiting Kv1.3 significantly reduces tumor size by blocking proliferation. Importantly, in the melanoma model inhibi-tion of Kv1.3 by membrane-permeant drug clofazimine reduces tumor volume to a much higher extent (90%), in accordance with the apoptosis-inducing ability of these drugs. As to other Kv chan-nels, a Kv1.1-specific blocker, dendrotoxin-�, reduced tumor for-mation induced by the human lung adenocarcinoma cell line A549 in a xenograft model [72]. DTX-� exerted its anti-tumor effects through the pathway governing the G1-S transition. Kv1.5 has been shown to be overexpressed in several cancer tissues [69]. However, since macrophages express both Kv1.3 and Kv1.5 and these cells are known to infiltrate tumour tissues, the possibility cannot be excluded at present that high tissue staining is due in part to the presence of macrophages. Multiplex reverse transcription-PCR

showed increased mRNA expression for Kv1.3, Kv1.5 and mem-bers of the Kv10 (Eag) channel family with documented oncogenic potential [73], in drug-induced colon cancer [74]. Furthermore, Kv10.1 was detected in malignant mouse colon and human colonic cancers (74). Kv11.1 is also known to be overexpressed in different types of tumour cells and is a promising oncological target [for recent review see 75]. Our present work provides information on the expression of potassium channels in cancer cell lines which had not been charac-terized from this point of view. For example no such information is available on OCIAML-3 cells from the literature. In general, acute myeloid leukemia cells were shown to express high level of Kv11.1 and to correlate with a more aggressive phenotype [39]. Pharma-cological block of this channel in primary AML cells by a specific inhibitor induced cell cycle block in the G1 state [76]. In our study, OCIAML-3 cells express Kv11.1, as well as all other Kv channels studied. HL-60 expressed more Kv1.1 but less Kv1.3 with respect to OCIAML-3. Previous studies documented the presence of IK [77] and of Kv11.1 [78] in HL-60. ML-1 expressed a considerable amount of Kv1.1 with respect to the other two AML cell lines, but no Kv1.5. The presence of Kv1.1 and Kv1.3 is compatible with that of 4-AP sensitive current [79]. MOLM-13 does not express signifi-cant levels of Kv11.1 and IK while the other Kv channels are pre-sent. Finally, K562 did not express Kv1.3 but showed positivity for Kv11.1, in accordance with previously published results [60]. In the MCF-7 breast cancer cell line all the channels studied are ex-pressed, with the exception of Kv1.5. Kv1.3 has been detected by various groups in MCF-7 [66, 80], and was identified also in mito-chondria where it plays the crucial role we have already discussed. Kv1.1 [81], Kv11.1 [82] and IK [81] are also present in accordance with our data. Inhibition of IK blocks cell cycle at the level of G1 in these MCF-7 cells. MDA-MB-231, deriving from MCF-7 but char-acterized by a higher metastatic ability, had a higher expression of Kv1.1 and Kv11.1 and a lower level of Kv1.5 with respect to MCF-7 in our experiments. MDA-MB-231 was shown to express Kv11.1 [83]. Colo205 and DLD-1 are colon cancer cell lines. On the former no information about channel expression is present in the literature,


while in DLD-1 a proliferation-blocking effect of a general potas-sium channel inhibitor, 4-aminopyridine, has been shown [84]. Transcripts for Kv1.3, Kv1.5 and Kv11.1 have been revealed in primary human colon cancer tissues [68, 74]. The expression level of Kv11.1 and the tumour stage were correlated [85] and a negative correlation between channel expression and sensibility to doxorubi-cin was found in colon cancer cells [86]. Finally, the neuroblastoma cell line SH-SY5Y has been shown to harbour Kv11.1 [73]. In summary, the expression analysis performed here at the level of proteins provides additional information exploitable for further pharmacological studies in the context of cancer cell prolifera-tion/apoptosis. Further work will be required to establish whether protein expression corresponds to the presence of functional chan-nels in the plasma membrane and/or mitochondria (this latter local-ization is expected for Kv1.3, Kv1.5 and IK). Information on Kv channel expression might be useful, given that Kv channels have been shown to be involved in apoptotic volume decrease, which is largely attributed to K+ efflux and precedes apoptotic events like cytochrome c release from mitochondria and apoptosome formation [87, 88]. Elevation of extracellular [K+], or treatment with K+ chan-nel blockers such as TEA and 4-AP have been shown to block apoptosis in various cell types [89-91]. As previously mentioned above, in the case of Kv1.3, the pres-ence of this potassium channel in the PM correlates with its location also in the mitochondria, at least in the case of the cell lines exam-ined up to now (Jurkat, CTLL-2, SAOS-2, B16F10, MCF-7, PC-3). In mitochondria, K+ influx and efflux pathways (via channels and transporters) can potentially modulate the tightness of coupling between respiration and ATP synthesis, thereby maintaining a bal-ance between energy supply and demand in the cell. Furthermore, mtK+ channels regulate ROS production and mitochondrial volume, maintaining the structural integrity of the organelle [12]. In light of our previous results on mtKv1.3 and apoptosis induction, we ex-pected to observe a significant correlation between Kv1.3 expres-sion and death upon treatment with Psora-4, PAP-1 and clo-fazimine. The fact that this latter substance indeed correlated with Kv1.3 expression in the different cell lines examined (i.e. more Kv1.3 expression was associated with higher extent of death in-duced by clofazimine) is in line with our expectations and previous data showing that this substance induces apoptosis via mtKv1.3. Clofazimine acted also in the case of Bcl-2 overexpression (Fig. 5)known to occur in myeloid cancer cells and confirmed also in B cells from chronic lymphocytic leukemia patients [92]. Further-more, since clofazimine is already in clinical use with an excellent safety profile, this correlation suggests that the drug might be used against various tumours expressing/overexpressing Kv1.3. On the other hand, no such significant correlation was observed for Psora-4 and PAP-1. One possible explanation for this difference is that clo-fazimine itself has been shown to act also as an MDR inhibitor and therefore its concentration in the cells and reaching the mitochon-dria might be higher than that of Psora-4 and PAP-1. Furthermore, we show here that expression of Kv11.1 decreased the susceptibil-ity of the cells to Psora-4. The molecular mechanism for this corre-lation is not clear; both Psora-4 and PAP-1 show and EC50 of 5 �Mfor Kv11.1 block [93,94]. As to clofamizimine, its effect on Kv11.1 current has not been established to our knowledge [95]. The possi-bility that clofazimine acts in an Kv1.3-independent manner is not likely, given that siRNA against Kv1.3 prevents its apoptosis-inducing effects [8]. The question also arises why myeloid and neuroblastoma cell lines are more sensitive to these drugs than breast and colon adenocarcinoma lines, even though Kv1.3 is ex-pressed also in these latter ones, at least to some extent. Clarifica-tion of this result requires further experiments, but possible expla-nations include e.g. the hypothesis of a different expression of MDR pumps, the lack of functional and/or mitochondria-located Kv1.3, or the lack of pro-apoptotic proteins downstream of the mitochondrial events. For example, increased expression of XIAP

reduced the effect of Kv1.3 inhibitors. Similarly, we expect a re-duced effect if executor caspases are downregulated. In summary, the present work shows for the first time that in contrast to what is expected on the basis of a correlation analysis between mRNA of potassium channels and susceptibility to apop-totic stimuli and cytotoxins, this correlation instead can be revealed when the protein levels are taken into account, at least for Kv1.1 and Kv1.3. Experiments on numerous other cell lines will be re-quired to strengthen this correlation. The information described in this work might be exploited for pharmacological manipulation of the channels in the various cell lines in attempt to affect prolifera-tion/apoptosis of cancer cells.

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS The authors are grateful to AIRC (to I.S) and Cariparo (to M.Z) for funding. The work presented here was supported by Science Foundation Ireland (09/SIRG/B1575) and an Irish Cancer Society Research Fellowship to ES (CRF09SZE) L.L. is recipient of a sen-ior post-doc fellowship and of "Progetto Giovani Studiosi 2012" of the University of Padova. Current address of E.V. is: Department of Molecular Biology, University of Duisburg-Essen. The authors are grateful to Profs. E. Gulbins. K. Chandy, G. Panyi and M. Djamgoz for useful discussions.

SUPPLEMENTARY MATERIAL Supplementary material is available on the publishers Web site along with the published article.

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Received: March 11, 2013 Accepted: May 15, 2013

Correlation between Potassium Channel Expression and ...

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