Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTRinh-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTRinh-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTRinh-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTRinh-172, but not GlyH-101, induced nuclear translocation of nuclear factor-κB (NF-κB). CFTRinh-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-κB signaling pathway, independently of CFTR inhibition.
The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cAMP-activated chloride channel expressed in epithelial cells of the lung, intestine, pancreas, and other tissues, where it facilitates transepithelial fluid transport. Mutations in CFTR that impair channel function cause the hereditary disease CF, characterized by abnormalities in fluid and electrolyte transport, dehydrated airway surface, chronic bacterial infection, and exaggerated inflammation (Ratjen and Doring, 2003). In the intestine, normal CFTR is involved in enterotoxin-mediated secretory diarrheas such as cholera and traveler's diarrhea. Cyst expansion in human autosomal dominant polycystic kidney disease (PKD) also involves progressive fluid accumulation, which is believed to require chloride transport by the CFTR protein (Sullivan et al., 1998). Therefore, identifying CFTR activators and inhibitors would be of interest, not only for potential therapeutic reasons but also for a better understanding of the physiological role of CFTR protein.
Recently, two potent and rather selective CFTR inhibitors with low micromolar affinities, the thiazolidinone CFTRinh-172 and the glycine hydrazide GlyH-101, have been discovered (Ma et al., 2002; Muanprasat et al., 2004). CFTRinh-172 blocks channel opening with a Ki of 0.3 to 5 μM from an intracellular site that involves interaction with Arg347, which is located near the CFTR cytosolic surface (Caci et al., 2008). Concerning GlyH-101, patch-clamp experiments have shown that it acts in a voltage-dependent way by occluding the pore from its extracellular side with a Ki of 5.6 μM at −60 mV (Muanprasat et al., 2004). The inhibitory effects of the two compounds are rapidly reversible. In vivo analysis of intestinal fluid secretion demonstrate the efficacy of CFTR inhibitors in reducing cholera toxin-induced intestinal fluid secretion (Thiagarajah et al., 2004). These molecules have also been proved to be able to reduce cyst expansion in PKD with in vitro and in vivo models (Yang et al., 2008; Li and Sheppard, 2009). Moreover, CFTR inhibitors have been suggested to be useful in the construction of CF animal models (Salinas et al., 2004).
CFTRinh-172 has also been used by several groups to evaluate in vitro the consequences of CFTR chloride transport disruption with particular attention on inflammatory parameters. Inflammation in CF begins at early infancy and, although clearly associated with infection, there is still some uncertainty about whether CF lungs are innately primed for a proinflammatory response (Khan et al., 1995). In this context, a growing number of experimental works describe greater baseline production of proinflammatory cytokines and exaggerated nuclear factor-κB (NF-κB) activation in CF bronchial epithelial cells compared with non-CF cells (Tabary et al., 1999; Weber et al., 2001; Perez et al., 2007; Verhaeghe et al., 2007; Bartling and Drumm, 2009). An elevated level in intracellular and mitochondrial reactive oxygen species (ROS) has also been described in CFTR-deficient cells IB3-1 versus their CFTR-sufficient counterpart C38 (Velsor et al., 2006) and in tracheal cells from CFTR knockout mice compared with their littermate wild-type controls (Trudel et al., 2009). In some of these studies, inhibition of CFTR activity by CFTRinh-172 was shown to increase ROS production (Chen et al., 2008; Maiuri et al., 2008), induce nuclear translocation of NF-κB (Perez et al., 2007; Vij et al., 2009), and enhance secretion of inflammatory markers like interleukin-8 (IL-8) (Perez et al., 2007; Chen et al., 2008; Bartling and Drumm, 2009; Vij et al., 2009) and eicosanoids (Borot et al., 2009) in CFTR-sufficient cells. These observations are in agreement with the hypothesis that dysfunctional CFTR is involved in ROS- and NF-κB-mediated cytokine up-regulation and that CFTR inhibition can mimic the CF inflammatory profile (Perez et al., 2007).
To raise more insight into the mechanisms involved in ROS production after CFTR inhibition, we compared the effects of these two CFTR inhibitors by using different cell lines: one CFTR-sufficient (C38), one CFTR-deficient (IB3-1), and two that do not express CFTR, HeLa and A549 (Bossard et al., 2007). Because mitochondria are a major source of cellular ROS (Turrens, 2003; Murphy, 2009), we focused on possible effects of inhibitors on mitochondrial functions. Mitochondria generate ROS as byproducts of oxidative phosphorylation. The incomplete coupling of electrons and H+ with oxygen in the electron transport chain generates superoxide anion, O2⨪, which is converted in mitochondria to hydrogen peroxide (H2O2) by superoxide dismutase. Subsequently, mitochondrial H2O2 either is eliminated by mitochondrial peroxidases or diffuses in the cytoplasm, where it can be degraded by cytosolic detoxifying enzymes (Murphy, 2009). We found substantial effects of both CFTR inhibitors on ROS levels, mitochondrial membrane potential (ΔΨm), and oxygen consumption in CFTR-null cells, independent of their inhibitory role on CFTR function.
Materials and Methods
Permeant probes 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methyl ester perchlorate (TMRM) were obtained from Invitrogen (Cergy Pontoise, France). CFTR inhibitors CFTRinh-172 and GlyH-101 were purchased from VWR International (Pessac, France). Stock solutions were prepared at 40 mM in DMSO. Culture media, serum, and antibiotics were from Invitrogen. All other chemicals were from Sigma-Aldrich (St-Quentin, France).
All cell lines were obtained from the American Type Culture Collection LGC Standards (Molsheim, France). The IB3-1 human CF bronchial epithelial cell line (mutations F508del/W1282X) and the stably rescued cell line C38 expressing CFTR were grown on fibronectin-coated culture flasks in Laboratory of Human Carcinogenesis basal medium 8 supplemented with 1% heat-inactivated fetal calf serum (FCS). HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FCS. The lung epithelium-derived cell line A549 was cultured in F-12K medium supplemented with 10% heat-inactivated FCS. For all cell lines, culture media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO2.
ROS Production Assays.
Cellular ROS production was measured by using the cell-permeant probe CM-H2DCFDA, which is oxidized to the highly fluorescent 2′-7′ dichlorodihydrofluorescein (DCF) by either H2O2, hydroxyl radical (•OH), or peroxynitrite (ONOO−). For ROS visualization cells were plated on Lab-Tek chambers mounted on glass slide (Nunc, Thermo Scientific, Cergy-Pontoise, France). Cells were grown to 80% confluence and incubated for 30 min at 37°C with 10 μM CM-H2DCFDA diluted in serum-free medium. At the end of the incubation period, cells were washed twice with phosphate-buffered saline (PBS), and drugs or vehicle were added to the cells in a salt-balanced solution (10 mM glucose, 5 mM KCl, 135 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES buffer adjusted to pH 7.4). Live-cell images were obtained at 37°C by laser confocal scanning microscopy (Zeiss LSM 510; Carl Zeiss GmbH, Jena, Germany) using a Plan-Apochromat 63/1.4 oil differential interference contrast objective, with a 488-nm excitation light from an argon laser and a 505- to 530-bandpass–barrier filter. For image recording, care was taken to keep laser intensity at low levels to avoid photooxidation of the probe. Z-series images were captured and fluorescence intensity was quantified on maximal projection images after thresholding, using Zeiss LSM 5 Pa and ImageJ (rsb.info.nih.gov/ij) software. Three fields of view were analyzed per experiment.
ROS quantification was also performed by flow cytometry. Cells were seeded to six-well plates and treated as above. Cells were then collected by trypsinization, washed, and resuspended in the salt-balanced solution containing the drugs or the vehicle. Fluorescence intensity was immediately analyzed by flow cytometry (FACSCalibur, BD Biosciences, Le Pont de Claix, France) with the FL1 (530 nm) channel set for detection of the oxidized product of CM-H2DCFDA.
Mitochondria Isolation and Aconitase Activity.
Mitochondria were isolated from cell pellets by differential centrifugation. After trypsinization, cells were washed in cold PBS and pelleted at 700g for 5 min at 4°C in isolation buffer [225 mM mannitol, 75 mM sucrose, 20 mM Tris (pH 7.4), 1 mM EDTA, and 0.1% bovine serum albumin (BSA)]. The pellet was resuspended in 750 μl of hypotonic lysis buffer [100 mM sucrose, 10 mM Tris (pH 7.4), 1 mM EDTA, and protease inhibitors] and homogenized on ice for 90 s at 1600 rpm with a Potter-Elvehjem Teflon homogenizer (Fisher Scientific, Illkirch France). The glass potter was rinsed once with 250 μl of lysis buffer, which was added to the previous fraction, and 160 μl of 1.25 M sucrose was added to restore isotonicity. The homogenate was centrifuged at 1000g for 10 min at 4°C to eliminate debris and nuclei. Mitochondria from the supernatant were isolated at 10,000g for 10 min at 4°C, and the pellet was frozen at −80°C.
Aconitase activity was determined in isolated mitochondria with an assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Activity was measured as the rate of NADP reduction by isocitrate dehydrogenase. NADPH formation was monitored at 340 nm for 20 min at 37°C, and results were expressed as nmol of NADPH produced per min and per mg of mitochondrial protein.
Measurement of ΔΨm.
ΔΨm was estimated by using the cationic fluorescent probe TMRM that preferentially accumulates in the mitochondria in response to the negative membrane potential across the inner mitochondrial membrane, emitting fluorescence at 560 nm when excited at 543 nm. The fluorescent signal in mitochondria decreases when ΔΨm is impaired. Cells were plated on Nalgen Nunc International (Rochester, NY) chambers and incubated for 20 min at 37°C in serum-free medium containing 20 nM TMRM. For fluorescence analysis, medium was replaced by salt-balanced solution containing 100 μM verapamil, and 10 min later it was replaced again by the same solution containing 100 μM verapamil plus the desired concentrations of drug or vehicle. Live cell images were obtained at 37°C by laser confocal scanning microscopy (Zeiss LSM 510) using a 543-nm excitation light and a 560-nm-long pass barrier filter. Z-series images were captured and TMRM intensity was quantified on maximal projection images after thresholding, using the Zeiss LSM and ImageJ software. A minimum of three fields of view was analyzed per experiment.
Measurement of Fluorescence of the TMRM Probe in Solution.
The mitochondrial probe TMRM was dissolved in PBS at a final concentration of 20 nM. The two CFTR inhibitors were added to the dye solution at the desired concentrations. Aliquots were transferred to a microplate and read on a spectrofluorimeter Victor3 (PerkinElmer France, Courtaboeuf, France), using excitation/emission wavelengths of 550/590 nm.
Oxygen Consumption Measurements.
HeLa cells were collected by trypsinization, centrifuged, and resuspended in either their culture medium or a salt-balanced solution as above. The JO2 of the suspension was recorded with an Oroboros Oxygraph-2k (Oroboros, Innsbruck, Austria). Data were acquired with DatLab4 software. Basal cell respiration was determined first in the absence of drug addition. The effects of CFTR inhibitors were determined either on the basal respiration rate or after addition of oligomycin (0.5 μg/ml) to induce state 4 respiration. Then, increasing concentrations of carbonyl cyanide m-chlorophenylhydrazone (CCCP; 0.05–1.5 μM) were used to estimate the maximum respiratory rate. Finally, antimycin A was added at 1 μM to evaluate the rate of oxygen consumption (JO2) caused by mitochondrial electron transfer. In all conditions, the antimycin-insensitive JO2 was subtracted from total JO2 and the results were expressed as percentage of basal antimycin-sensitive JO2.
Nuclear Translocation of NF-κB/p65.
HeLa cells grown in T75 culture flasks were incubated in serum-free medium with either 20 μM CFTRinh-172 or vehicle for 6 h, which we had found to correspond to the maximal activation. Nuclei were prepared as described (Brouillard et al., 2001). In brief, cells were harvested and suspended in a hypotonic ice-cold buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, and 0.5 mM DTT. The detergent Igepal CA-630 was added at 0.1% (v/v) and the mixture was incubated on ice for 10 min. After centrifugation (1100g for 10 min at 4°C), the supernatant containing the cytosolic fraction was discarded and the pellet was vigorously resuspended in a buffer containing 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 25% (v/v) glycerol and incubated on ice for 15 min. The nuclear extract was collected by centrifugation (15,000g for 20 min at 4°C).
Thirty micrograms of nuclear extracts was diluted with Laemmli sample buffer and heated at 95°C for 3 min. Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. After transfer, analysis was performed following the manufacturer's recommendations for the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Dilution of rabbit polyclonal antibodies to NF-κB/p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was 1:2000 and 1:10,000 for the fluorescent IRDye TM700 secondary antibody (ScienceTec, Paris, France). Images were acquired with the Odyssey infrared imaging system. Quantification of protein bands was performed with the Odyssey software.
HeLa cells were preincubated for 24 h in FCS-free medium. The production of IL-8 in supernatants was quantified after 6 h of treatment with either 20 μM CFTRinh-172 or the vehicle in culture medium supplemented with 0.1% BSA, using the Quantikine enzyme-linked immunosorbent assay (R&D Systems, Lille, France). The assay was carried out according to the manufacturer's instructions, and quantification was determined with a microplate reader MR5000 (Dynatech, Paris, France) set to 450 nm with a wavelength correction set at 550 nm.
Data shown are given as mean ± S.E.M. Student's t test was applied to determine the statistical significance. p values of 0.05 or lower were considered significant.
CFTRinh-172 and GlyH-101 Increase Cellular ROS Levels Independently of CFTR Function.
We have compared the effects of two potent CFTR inhibitors, CFTRinh-172 and GlyH-101, on cellular ROS levels in CF (IB3-1) versus non-CF (C38) epithelial cells, as well as in HeLa cells and the lung carcinoma cell line A549, which do not express CFTR. Exposure of C38 cells to 20 μM CFTRinh-172 induced a marked increase in DCFDA fluorescence, indicating an increase in ROS levels. The same effect was observed with the CFTR inhibitor GlyH-101 (Fig. 1). However, similar results were obtained in IB3-1, HeLa, and A549 cells (Fig. 1), suggesting that the impact on ROS levels is independent of the inhibition of CFTR activity. This increase in ROS levels was time-dependent, detectable after 5 min of cell treatment and peaking at 30 min (data not shown).
Quantitative analysis of DCFDA fluorescence by flow cytometry in HeLa cells showed a dose-dependent stimulatory effect of CFTRinh-172 and GlyH-101 (Fig. 2A). Although both inhibitors induced similar increases in DCFDA fluorescence at 20 μM, GlyH-101 was more effective than CFTRinh-172 at 5 μM. Because CFTRinh-172 is known to bind to plastic surfaces and serum proteins (Perez et al., 2007), the effect of drugs was also evaluated in the presence of either 0.1% BSA or 10% FCS. Figure 2B shows that both inhibitors still enhanced ROS production at 20 μM in the presence of 10% FCS, although at a lesser extent than in FCS-free medium. By contrast, the effects of both compounds were unchanged in the presence of 0.1% BSA.
CFTR Inhibitors Decrease Mitochondrial Aconitase Activity.
Although DCFDA represents a widely used marker of oxidative stress, self-induced production of O2⨪/H2O2 can occur in complex systems possessing peroxidase activity (Bonini et al., 2006). For this reason we performed an independent assay, consisting of the determination of aconitase activity, which is often applied to assess the presence of oxidative stress in mitochondria. Aconitase contains an iron-sulfur cluster in its active site that is sensitive to oxidation and leads to enzyme inactivation (Gardner et al., 1994). As shown in Fig. 3, incubation of HeLa cells with 20 μM CFTR inhibitors for 2 h induced a slight, but significant, decrease in aconitase activity, in agreement with the results described above, supporting the idea that CFTR inhibitors induce oxidative stress independently of CFTR inhibition.
CFTR Inhibitors Induce ΔΨm Depolarization.
We next examined the effects of both inhibitors on ΔΨm. ΔΨm was measured by incorporation of the potentiometric dye TMRM and analyzed by confocal microscopy and fluorescence quantification. It has been shown in previous work that TMRM staining is sensitive to multidrug resistance (MDR) phenotype and MDR inhibitors like verapamil or progesterone prevent spontaneous loss of dye in several cell types, including HeLa cells (Diaz et al., 2001). Because CFTR belongs to the same family (ABC transporters) as MDR proteins and we wanted to discard a nonspecific increase in dye efflux, TMRM-loaded cells were first incubated with 100 μM verapamil for 10 min, and then CFTR inhibitors were added at 20 μM for 30 min in the presence of verapamil. As shown in Fig. 4, addition of both inhibitors resulted in a decrease of the fluorescent signal in the four cell types (C38, IB3-1, A549, and HeLa), reflecting mitochondrial membrane depolarization induced by the drugs. The depolarizing effect was also observed when cells were pretreated by the drugs for 30 min and then incubated with the TMRM probe. In these conditions, no accumulation of the probe in mitochondria could be observed (data not shown).
To check that the decrease in TMRM fluorescence did not result from a direct quenching effect of CFTR inhibitors on the mitochondrial probe, either the drugs or the vehicle were added directly to aqueous solutions of TMRM, and fluorescence was measured. At 20 μM, neither CFTRinh-172 nor GlyH-101 changed fluorescence significantly (not shown). This excluded the idea that a direct quenching effect was responsible for the decrease in TMRM fluorescence.
Figure 5A shows the time course of the decrease in TMRM fluorescence in HeLa cells treated by either 20 μM CFTRinh-172, 20 μM GlyH-101, or the vehicle alone. The effect of both inhibitors was evident at less than 5 min of treatment, and no further decrease was observed after 10 min of incubation. The dose-dependent effects of both inhibitors are summarized in Fig. 5B. TMRM fluorescence was significantly decreased by concentrations ≥2 μM. Fluorescence intensity decreased to approximately 40% of the control value when cells were treated with each compound at 20 μM. Mitochondrial membrane depolarization was reversible, because after 30 min of incubation of cells with each inhibitor at 20 μM and a 1-h washout, TMRM incorporation resulted in the same fluorescence intensities as before treatment (data not shown). Figure 5C shows the effects of both inhibitors when cells were maintained in the saline solution complemented with 0.1% BSA or 10% FCS. The presence of FCS, but not BSA, attenuated the drug-induced decrease in fluorescence intensity.
Mitochondrial depolarization can result from several mechanisms, such as a decoupling effect, inhibition of the activities of the respiratory chain complexes, and opening of the mitochondrial permeability transition pore (mPTP) or the mitochondrial inner membrane anion channel (IMAC). We analyzed whether the mPTP inhibitor cyclosporine A and the IMAC inhibitor disodium 4,4′-diisothiocynatostilbene-2,2′-disulfonate were able to prevent CFTR inhibitor-induced mitochondrial depolarization. Preincubation of cells for 15 min with either 1 μM cyclosporine A or 10 μM disodium 4,4′-diisothiocynatostilbene-2,2′-disulfonate neither affected basal TMRM fluorescence nor prevented the fluorescence decrease induced by CFTR inhibitors (not shown), suggesting that their effect does not involve mPTP or IMAC opening. Therefore, inhibition/uncoupling of the respiratory chain is more likely to be responsible for mitochondrial membrane depolarization.
Inhibition of Mitochondrial Respiration.
ΔΨm is generated by the components of the electron chain, which consume O2 and pump protons across the inner mitochondrial membrane. Mitochondrial polarization results from the balance between proton pumping by the mitochondrial respiratory chain and proton return through different pathways. Mitochondrial depolarization would result either from increased proton return or decreased proton pumping. Increasing proton return (uncoupling) is expected to increase cellular respiratory rate. If inhibition of mitochondrial respiration takes place this would reduce proton pumping. To test whether the depolarizing effects of CFTR inhibitors were caused by inhibition of electron transport or a protonophoric/uncoupling effect, the JO2 was evaluated in intact HeLa cells under different conditions. In all experiments, antimycin A was added at the end of recordings to evaluate the JO2 caused by electron transport in the mitochondrial respiratory chain. It should be recalled that with the cell line used the antimycin-resistant oxygen consumption (nonmitochondrial) accounted for up to 50% of the cellular oxygen consumption rate in basal state (no addition). Addition of CFTR inhibitors decreased basal respiration rate in a dose-dependent way. As shown in Fig. 6A, significant inhibitory effects were observed for ≥2 μM GlyH-101 and CFTRinh-172. The inhibitory effect of 20 μM GlyH-101 was 2-fold that of 20 μM CFTRinh-172. In another series of experiments, oligomycin was added first to establish a state 4 respiration rate. Afterward, gradual concentrations of the uncoupler CCCP were added to evaluate the maximum respiration rate. The percentage of maximum rate reached 2-fold the basal rate in control conditions (Fig. 6B, left). When CFTR inhibitors were added to cells after oligomycin treatment, the maximum rate was greatly reduced compared with control conditions, in agreement with an inhibitory effect of both compounds on basal and maximal electron transport (Fig. 6B, middle and right). When oligomycin was added to initiate a state 4 respiratory rate inside cells the addition of 20 μM CFTRinh-172 afterward increased the respiration rate, whereas 20 μM GlyH-101 had no effect. This increase, not observed in absence of oligomycin (Fig. 6A), indicates that in addition to its toxic effect on the electron transfer CFTRinh-172 increases the proton leak of the mitochondrial inner membrane (uncoupling).
We also evaluated the effects of CFTR inhibitors in the presence of either 10% FCS or 0.1% BSA. When cells were resuspended in the presence of 10% FCS, CFTR inhibitors did not modify basal JO2, even at the highest concentration (data not shown). When 0.1% BSA was added to the cell suspension, both inhibitors still decreased basal respiration rate; yet at 20 μM their inhibitory effects were slightly reduced compared with the results observed without BSA. In the presence of either CFTRinh-172 or GlyH-101, respiration rates decreased to 73.6 ± 1.5% (n = 3) and 50.1 ± 1.3% (n = 3), respectively, of the control levels. These values are similar to the effects produced by drugs at 10 μM in the absence of BSA (Fig. 6A).
NF-κB Is Activated by CFTRinh-172 but Not GlyH-101 in HeLa Cells.
By elevating cellular ROS levels, it could be expected that CFTR inhibitors modulate ROS-dependent pathways, such as that of NF-κB (Gloire et al., 2006). We tested the effect of incubation with each inhibitor for 30 min, 3 h, and 6 h on NF-κB nuclear translocation. Immunoblot analysis of nuclear p65 indicated no effect at either 30 min or 3 h (not shown). However, 6 h of treatment with 5 or 20 μM CFTRinh-172 increased the level of nuclear p65 by 5-fold (Fig. 7). This effect is comparable with that of stimulation by 20 ng/ml tumor necrosis factor α (TNFα) (a well-known NF-κB activator) for 30 min (Fig. 7). By contrast, GlyH-101 had no effect at any time of treatment (only shown at 6 h in Fig. 7). This discrepancy may result from the more pronounced decrease by GlyH-101 of the mitochondrial electron chain transport (Fig. 6A). Indeed, previous results have shown that impairment of the mitochondrial electron chain transport prevents NF-κB activation by hydrogen peroxide (Josse et al., 1998).
IL-8 Production Is Modulated by CFTR Inhibitors in HeLa Cells.
Because NF-κB controls the transcription of a number of inflammatory genes, it is considered a key regulator of tissue inflammation, including IL-8 secretion (Barnes and Karin, 1997). Figure 8 shows that, contrary to what could be expected, treatment of HeLa cells for 6 h with 20 μM CFTRinh-172 resulted in a small decrease in IL-8 secretion, whereas 20 μM GlyH-101 induced a slight increase. This suggests that other undefined signaling pathways contribute to the modulation of IL-8 production in these cells independently of NF-κB.
The main finding of the present study is the demonstration that the two CFTR inhibitors, CFTRinh-172 and GlyH-101, impair mitochondrial function in cells that do not express CFTR, leading to mitochondrial depolarization and elevation of ROS, independently of blockade of CFTR chloride channel function. This may have important implications in both the potential therapeutic use of these compounds and their applications in the development of in vitro models of CF.
As shown in the experiments (Fig. 6), both inhibitors decreased basal and maximal respiration rates. This indicates that mitochondrial depolarization can be explained primarily by an inhibitory effect of these compounds on mitochondrial electron transfer. The possible intervention of the mitochondrial transition pore and IMAC has been ruled out. However, from our experiments one should conclude that CFTRinh-172 has a dual activity: on one side, it inhibits respiration (like GlyH-101); on the other side, (unlike GlyH-101) it uncouples mitochondria (increase of respiration in presence of oligomycin). It is well known that the chemical protonophores commonly used to uncouple mitochondria (CCCP and carbonylcyanide p-trifluoromethoxyphenylhydrazone) show a relatively narrow concentration range in which uncoupling results in a significant increase of respiratory rate. Above this range inhibition of respiration takes place. In this respect CFTRinh-172 acts similarly with the difference that the inhibiting action is relatively stronger and takes advantage of the protonophoric action for the final outcome on respiratory rate when CFTRinh-172 is administrated to cells where mitochondria drive ATP production (mitochondrial state 3). When mitochondria within cells were set to state 4 (oligomycin present) the respiratory rate was decreased and the membrane potential was increased. In these conditions the protonophoric action of CFTRinh-172 is unmasked before the inhibitory effect becomes dominant. It should be recalled that both effects of CFTRinh-172 are synergistically deleterious for cellular bioenergetics.
Impaired mitochondrial respiration is expected to enhance superoxide anion production at the mitochondrial level. This has been verified by the concomitant decrease in aconitase activity coupled to mitochondrial depolarization and the decrease in oxygen consumption. Accordingly, a significant part of cellular ROS produced after administration of these inhibitors is likely to be of mitochondrial origin.
The molecular mechanisms involved in these effects have not been investigated precisely in the present work. However, our results suggest that CFTR inhibitors target the mitochondrial membrane, which may result in an ineffective transport of electrons through the various subunits of the electron transport chain. Mitochondria have been shown to accumulate lipophilic compounds and a number of therapeutic substances like thiazolidinediones, not primarily directed to mitochondria, have been shown to interact with the mitochondrial membrane (Feinstein et al., 2005). Thiazolidinediones have repeatedly been reported to depolarize mitochondria (Bova et al., 2005; Konrad et al., 2005; Perez-Ortiz et al., 2007), induce ROS elevation (Perez-Ortiz et al., 2007), and reduce oxygen consumption (Brunmair et al., 2004; Perez-Ortiz et al., 2007) through a mechanism that does not involve their agonistic action on peroxisome proliferator-activated receptor γ (PPARγ).
The reported effect of CFTR inhibitors on mitochondrial function raises the point of cell toxicity. A previous study in cultured cells demonstrated that the inhibitory effect of CFTRinh-172 on chloride transport was diminished in the presence of 10% FCS caused by binding of the drug to serum proteins and in the presence of 0.1% BSA in FCS-depleted cells. Consequently, a concentration of 20 μM seemed to be necessary to achieve complete block of channel activity (Perez et al., 2007). We have also found that the presence of 10% FCS (unlike BSA) attenuated, but did not abolish the effect of both inhibitors at 20 μM on ROS production and ΔΨm. This probably occurs by decrease of the available concentrations of both drugs. In these conditions the effect of 20 μM was equivalent to that of 5 to 10 μM in the absence of FCS (Figs. 2B and 5C). In addition, we observed that in the presence of 10% FCS the decrease in JO2 was no longer detectable. These results can explain the absence of toxic effects of high concentrations of inhibitors in cultured cells (Ma et al., 2002; Muanprasat et al., 2004; Perez et al., 2007), which otherwise would be expected from the results presented here. Nevertheless, it has to be noted that we have observed a decrease in cell viability when the inhibitors were applied for 24 h at 20 μM on C38 and IB3-1 cells in the presence of 1% FCS.
A fundamental issue concerning the study of CF pathogenesis is the deregulation of the inflammatory response characteristic of CF phenotype. A number of studies have shown an exaggerated NF-κB activation and increased levels of proinflammatory cytokines in CF cells compared with controls (Tabary et al., 1999; Weber et al., 2001; Perez et al., 2007; Bartling and Drumm, 2009; Vij et al., 2009), but the link between loss of chloride channel function and innate inflammatory response remains unclear.
Oxidative stress, but not activation of NF-κB, in CF lung cells has been proposed to be responsible for the deregulation of inflammatory mediators by increased acetylation of the cytokine gene promoters (Bartling and Drumm, 2009). The relationship between ROS elevation and cytokine production may also involve either PPARγ down-regulation (Maiuri et al., 2008) or a decreased expression and activity of the transcription factor NF-E2-related factor 2 (Chen et al., 2008).
Because treatment of CFTR-sufficient cells with CFTRinh-172 has been shown to reproduce ROS elevation (Perez et al., 2007; Chen et al., 2008; Maiuri et al., 2008; Bartling and Drumm, 2009) and increase IL-8 (Perez et al., 2007; Vij et al., 2009), it has been suggested that lack of chloride transport was the primary event that leads to a raise in ROS levels and cytokine production. Conversely, our results showing that CFTRinh-172 and GlyH-101 induce ROS elevation in non-CFTR-expressing cells, even at low concentrations, suggest that this assumption remains questionable, even though the inhibitors did not exhibit a marked proinflammatory action on these CFTR null cells.
Because CFTR inhibitors have potential implications in the treatment of secretory diarrheas (Ma et al., 2002, Muanprasat et al., 2004; Thiagarajah et al., 2004; Sonawane et al., 2005) and PKD (Yang et al., 2008; Li and Sheppard, 2009), their potential toxic effects have been explored in vivo in rodents after intraperitoneal or oral administration (Ma et al., 2002; Thiagarajah et al., 2004; Sonawane et al., 2005). There is evidence that CFTRinh-172 is concentrated in the liver of animals after intravenous administration and undergoes enterohepatic circulation in mice (Sonawane et al., 2005). In general the inhibitor is well tolerated in the range of 1 to 3 mg/kg daily for 1 to 6 weeks. Only in adult mice, a higher concentration of CFTRinh-172 (25–50 mg/kg per day) was shown to induce a mild weight loss and a 2- to 5-fold increase in serum transaminases after 1 week. This reflects a mild hepatotoxicity that might be explained by our results (Sonawane et al., 2005). Potential drug-induced mitochondrial dysfunction is particularly important in chronic diseases necessitating long-term therapy. This is not the case for secretory diarrheas, but represents a relevant issue to the treatment of PKD, because of its slow evolution in humans. Note that to improve either water solubility of CFTRinh-172 or to better retain GlyH-101 in the case of rapid flushing from the intestine during diarrhea, the development of analogs of the two drugs is in progress (Sonawane et al., 2006; Sonawane and Verkman, 2008; Yang et al., 2008). Nevertheless, extensive analysis of in vivo pharmacology and toxicity has not been performed yet for these compounds. Therefore, it would be important to verify whether these new compounds affect mitochondrial function.
In summary, CFTR inhibitors induce ROS production and mitochondrial failure in non-CFTR-expressing cells. This unexpected result indicates that, even though several studies have demonstrated a significant elevation in cellular ROS and inflammatory markers in CF versus non-CF cells, the link between these observations and the loss of chloride transport needs further research to be clearly elucidated. In addition, although CFTR inhibitors represent an attractive alternative to the design of large animal models of CF (Salinas et al., 2004), their effects must be taken cautiously, because a potential risk of mitochondrial failure may generate pathological symptoms not directly related to chloride channel inhibition.
We thank Corinne Cordier (Plateau Tri Cellulaire) and Meriem Garfa-Traoré (Plateau d'Imagerie Cellulaire) for technical assistance with flow cytometry experiments and confocal imaging, respectively.
- Received September 25, 2009.
- Accepted January 4, 2010.
M.K. and S.T. contributed equally to this work.
This work was supported by Institut National de la Santé et de la Recherche Médicale; Centre National de la Recherche Scientifique; Chancellerie des Universités de Paris (Legs POIX); the European Community [Grant NEUPROCF LSHG-CT-2005-512044]; French Association Vaincre La Mucoviscidose; and French Association ABCF 2.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- cystic fibrosis
- CF transmembrane regulator
- bovine serum albumin
- carbonyl cyanide m-chlorophenylhydrazone
- 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester
- mitochondrial membrane potential
- dimethyl sulfoxide
- fetal calf serum
- N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide
- mitochondrial inner membrane anion channel
- rate of oxygen consumption
- multidrug resistance
- nuclear factor-κB
- phosphate-buffered saline
- polycystic kidney disease
- peroxisome proliferator-activated receptor γ
- mitochondrial permeability transition pore
- reactive oxygen species
- tetramethylrhodamine methyl ester perchlorate
- tumor necrosis factor α.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics