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J. Biol. Chem., Vol. 279, Issue 39, 40629-40633, September 24, 2004
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F508-CFTR Cellular Processing by Curcumin in Cell Culture and Mouse Models*
¶
From the
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143 and the Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy
Received for publication, June 30, 2004 , and in revised form, July 19, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508-cystic fibrosis transmembrane conductance regulator (CFTR) cellular processing in homozygous mutant mice, restoring nasal potential differences and improving survival (Egan, M. E., Pearson, M., Weiner, S. A., Rajendran, V., Rubin, D., Glockner-Pagel, J., Canny, S., Du, K., Lukacs, G. L., and Caplan, M. J. (2004) Science 304, 600–602). Because of the implied potential use of curcumin or similar compounds in the therapy of cystic fibrosis caused by the F508 mutation, we tried to reproduce and extend the pre-clinical data of Egan et al. Fluorometric measurements of iodide influx in Fischer rat thyroid cells expressing F508-CFTR showed no effect of curcumin (1–40 µM) when added for up to 24 h prior to assay in cells grown at 37 °C. Controls, including 27 °C rescue and 4 mM phenylbutyrate at 37 °C, were strongly positive. Also, curcumin did not increase short circuit current in primary cultures of a human airway epithelium homozygous for F508-CFTR with a 27 °C rescue-positive control. Nasal potential differences in mice were measured in response to topical perfusion with serial solutions containing amiloride, low Cl–, and forskolin. Robust low Cl– and forskolin-induced hyperpolarization of 22 ± 3 mV was found in wild type mice, with 2.1 ± 0.4 mV hyperpolarization in F508 homozygous mutant mice. No significant increase in Cl–/forskolin hyperpolarization was seen in any of the 22 F508 mice studied using different curcumin preparations and administration regimens, including that used by Egan et al. Assay of serum curcumin by ethyl acetate extraction followed by liquid chromatography/mass spectrometry indicated a maximum serum concentration of 60 nM, well below that of 5–15 µM, where cellular effects by sarcoplasmic/endoplasmic reticulum calcium pump inhibition are proposed to occur. Our results do not support further evaluation of curcumin for cystic fibrosis therapy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508), is present in at least one allele in 
90% of CF subjects worldwide (3). Clinically, CF subjects manifest chronic lung infections, resulting in deterioration of lung function, and pancreatic insufficiency, resulting in intestinal malabsorption. The F508-CFTR protein appears to be mildly misfolded and retained at the endoplasmic reticulum in cells where it is expressed (4, 5). Interestingly, the F508-CFTR protein can be rescued by incubation of cell cultures for 18 h or more at reduced (<30 °C) temperature (6) or with high concentrations of 4-phenylbutyrate (7), resulting in partial restoration of F508-CFTR trafficking to the plasma membrane. However, the gating of the plasma membrane-rescued F508-CFTR protein remains defective, such that its open probability after cAMP stimulation is reduced by >3-fold compared with that of wild type CFTR (8, 9). Various small molecules have been identified that act as correctors of defective F508-CFTR chloride channel gating (9–11). Small molecule strategies to correct defective F508-CFTR folding and plasma membrane targeting are under development, although approval of potentially efficacious compounds for clinical use is many years away.
Recently, curcumin, the yellow colored component of the spice turmeric, has been reported to correct defective F508-CFTR processing and function in cell and mouse models (12). The stated rationale for the use of curcumin was its action as a weak inhibitor of the sarcoplasmic/endoplasmic reticulum calcium (SERCA) pump at 5–15 µM concentration, which was based on a prior study from the same group reporting that the potent SERCA pump inhibitor thapsigargin restored F508-CFTR function (13). In F508-CFTR homozygous mutant mice, Egan et al. (12) reported that oral curcumin (45 mg/kg/day in three doses for three days) corrected nasal potential differences (PDs) to the same level found in wild type mice and improved the survival of F508-CFTR mice. Additionally, inclusion of 5 µM curcumin for 18 h in the cell culture medium in F508-CFTR-transfected BHK cells resulted in a small increase in plasma membrane F508-CFTR protein expression and cell chloride permeability. Curcumin preparations are widely available as nutritional supplements and have been cited to have anti-tumor, anti-inflammatory, and anti-oxidant effects (14–17). However, studies in humans have indicated very low bioavailability and rapid hepatic metabolism of orally administered curcumin (18–20), with only low nanomolar levels detectable in the blood after high dose administration.
The purpose of this study was to confirm and extend the results of Egan et al. (12), motivated by our interest in identifying clinically useful small molecule correctors of F508-CFTR gating and cellular misprocessing (11, 21). Contrary to the report by Egan et al. (12), we were unable to demonstrate correction of nasal PDs in F508-CFTR homozygous mutant mice despite using different curcumin preparations and administration protocols, nor were we able to demonstrate functional correction in F508-CFTR-transfected epithelial cells or F508-CFTR homozygous human airway cells. Furthermore, we could detect very little curcumin in blood specimens from mice after oral bolus administration. The inability of curcumin to give a reproducible, robust response in mouse and cell models, together with the lack of a validated mechanism of action and pharmacological profile, do not support its development as a therapy for CF caused by the F508 mutation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508 gene replacement per Ref. 22) were used for nasal PD studies. The CF mice were back-crossed into a CD1 genetic background (>8 generations) and bred at the University of California, San Francisco animal facility. CF mice were genotyped in accordance with standard procedures using the primers 5'-GCCTAGAAAATGTCCCTGTATCATG-3' and 5'-CCCTTTTCAAGGTGAGTAGTCAAG-3' (Invitrogen) and SsPI restriction digestion to cut the targeted Cftr allele. Wild type mice were fed standard mouse chow, and the CF mice were maintained on a liquid diet (Peptamen). CF mice were studied at age 12–16 weeks, with weights ranging from 20 to 25 g. Animal protocols were approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Curcumin Preparations and Administration Protocols—Various preparations and administration methods were used. Most studies were done using curcumin from Sigma (catalog number C1386, lot number 053k3522), with a limited set of studies done with a purified curcumin preparation (Fluka, catalog number 28260) and a neutraceutical curcumin preparation (Curcumin 95TM, Jarrow Formulas, Los Angeles, CA). Three different vehicles were used for the oral administration of curcumin to mice as follows. 1) Curcumin was dissolved in Me2SO, and then ethanol was added and mixed with PBS (for 1 mg per 100 µl of curcumin solution containing 5.4 µlofMe2SO, 44.6 µl of ethanol, and 50 µl of PBS, pH 7.2). During brief anesthesia with halothane, a 25-gauge feeding needle (or PE-90 tubing) was used to infuse 45 mg/kg curcumin into the mouse stomach daily for 3 days, with the final dose delivered 2 h before the measurement of nasal PDs. 2) According to the method reported in Egan et al. (12), curcumin powder was mixed with Alimentum (50 mg/ml) and sonicated for 30 s just before oral delivery by presenting the liquid at the tip of an Eppendorf pipette to non-anesthetized mice. The presented liquid was rapidly ingested by most mice. Curcumin was given at 0, 45, or 100 mg/kg three times a day for 3 days, with the final dose delivered 2 h before the measurement of nasal PDs. 3) The third method was the same as the second one, except that Peptamen was used instead of Alimentum as the delivery vehicle.
Nasal Potential Difference Measurements—Following anesthesia with intraperitoneal ketamine (90–120 mg/kg) and xylazine (5–10 mg/kg), the airway was protected by orotracheal intubation with a 21-gauge angiocatheter as described (23). A PE-10 cannula pulled to a tip diameter of 0.3 mm was inserted into one nostril 5 mm distal to the anterior nares and connected through a 1 M KCl agar bridge to a silver/silver chloride electrode and high impedance digital voltmeter (IsoMillivolt Meter, World Precision Instruments). The nasal cannula was perfused at 50 µl/min using dual microperfusion pumps serially with PBS, PBS containing amiloride (100 µM), low chloride PBS (chloride reduced to 4.7 mM by substitution with gluconate), low chloride PBS containing forskolin (20 µM), and then PBS. The reference electrode was a PBS-filled 21-gauge needle inserted in the subcutaneous tissue in the abdomen and connected to a second silver/silver chloride electrode by a 1 M KCl agar bridge.
Functional Measurements in F508-CFTR-transfected Fisher Rat Thyroid (FRT) Cells—FRT cells stably expressing human F508-CFTR and YFP-H148Q-I152L were cultured on 96-well black wall plates as described previously (11). Cells were treated with different concentrations of curcumin (1–40 µM) diluted in the culture medium from a concentrated stock in Me2SO. Negative controls included cells treated with equivalent concentrations of Me2SO alone. In some studies curcumin was delivered in Alimentum as was done in nasal PD experiments. Two positive controls were used as follows: 1) incubation of cells for 24 h at 37 °C with culture medium containing 4 mM 4-phenylbutyrate (7), and 2) incubation of cells for 24 h at 27 °C ("low temperature rescue"). After treatment, cells were washed three times with a phosphate-buffered solution and then stimulated with forskolin (20 µM) and genistein (50 µM) to maximally activate halide conductance in the F508-CFTR protein that reached the plasma membrane (11). Iodide influx measurements were done on a fluorescence plate reader (Optima, BMG Lab Technologies) equipped with two syringe pumps and HQ500/20X (500 ± 10 nm) excitation and HQ535/30 M (535 ± 15 nm) emission filters (Chroma). YFP fluorescence was recorded for 2 s prior to and 12 s after creation of the iodide gradient. Initial rates of iodide influx were computed from the time course of decreasing fluorescence after the iodide gradient (11).
Short Circuit Current Measurements on Human Airway Epithelial Cells—Human airway epithelial cells from a CF subject homozygous for the F508 mutation were cultured on Snapwell filters with 1-cm2 surface area (Corning-Costar) to resistances >1,000 ohms·cm2 as described previously (24). Filters were mounted in an Easymount Chamber System (Physiologic Instruments, San Diego). Both hemichambers contained Krebs solution (120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose, pH 7.3). Solutions were bubbled with 95% O2, 5% CO2 and maintained at 37 °C. Short circuit current was recorded using a DVC-1000 voltage-clamp (World Precision Instruments) with silver/silver chloride electrodes and 1 M KCl agar bridges.
Oral Bioavailability of Curcumin in Mice—Blood samples were obtained at 2 h after the oral administration of curcumin (0, 45, or 100 mg/kg), and plasma was collected (after heparinization) by centrifugation at 4300 x g for 10 min. Plasma (0.5 ml) was acidified to pH 3 using 6 N HCl and extracted twice (1 ml each) using a mixture of ethyl acetate and isopropanol (9:1; v/v) by shaking for 6 min (25). In experiments to determine detection sensitivity, known quantities of curcumin (from Me2SO, final concentration10–1000 nM) were added to serum samples from mice that did not receive curcumin. The samples were centrifuged at 5000 x g for 20 min, and the upper ethyl acetate layer was used for the determination of curcumin by liquid chromatography/mass spectometry. The organic layer was dried under flowing argon, and the residue was dissolved in an eluent containing 1% acetic acid and filtered to remove insoluble material. To the resultant filtrate was added a known quantity of rhodamine 101 as an internal standard for ratiometric quantitative analysis.
Reversed phase high performance liquid chromatography (HPLC) was carried out using a Supelco C18 column (2.1 x 100 mm; 5-µm particle size) connected to a Waters solvent delivery system, UV/visible detector, and a mass spectrometer. The mobile phase consisted of 40% tetrahydrofuran and 60% water (v/v) containing 1% citric acid adjusted to pH 3 (KOH) (25, 26), and system was run isocratically. The flow rate was 0.2 ml/min, and the injection volume was 20 µl. In most experiments the solvent system consisted of a 20-min linear gradient of acetonitrile/water (0.05% trifluoroacetic acid) with acetonitrile increasing from 10 to 95%. Calibration curves over the range of 50 nM to 20 µM were established using rhodamine 101 as an internal standard. Curcumin was detected at 424 nm, and its peak was confirmed by a molecular ion peak in mass chromatogram (m/z) at 369 daltons (M+1)+ using the positive electrospray mode. Mass spectra were acquired using positive ion detection, scanning from 200 to 600 Da. The electrospray ion source parameters were as follows: capillary voltage, 3.5 volts (positive ion mode); cone voltage, 37 volts; source temperature, 120 °C; desolvation temperature, 250 °C; cone gas flow, 62 liters/h, and desolvation gas flow, 362 liters/h. The limit of detection for serum curcumin in this assay was <20 nM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508-CFTR Rescue—A series of experiments was done using FRT epithelial cells that stably coexpressed F508-CFTR with a green fluorescent protein-based fluorescence halide indicator. These cells were selected from many clones to give a robust low temperature rescue in which incubation at 27 °C for 18–24 h results in F508-CFTR targeting the plasma membrane and restoration of halide transport function (11). Fig. 1A shows representative fluorescence data. Basal iodide influx in cells grown at 37 °C in the absence of a corrector of defective F508-CFTR cellular processing was 0.094 ± 0.006 mM/s in this assay. In positive control studies, the influx rate increased to 0.28 ± 0.01 mM/s for cells grown at 27 °C for 24 h and 0.18 ± 0.01 mM/s for cells grown at 37 °C for 24 h in the presence of 4 mM phenylbutyrate (Fig. 1B). The increased iodide influx in both positive controls was inhibited by the thiazolidinone CFTR blocker CFTRinh-172 (not shown). In dose-response measurements involving the 24-h incubation of cells with different concentrations of curcumin (1–40 µM, from Me2SO stock), there was no significant increase in iodide influx over the Me2SO (vehicle-alone) control (Fig. 1B). Also, no significant increase in iodide influx was found for the pure and neutraceutical curcumin preparations (1–40 µM; 40 µM data is shown in Fig. 1B). No increase in iodide influx was seen for different times of incubation (0–24 h) with 40 µM curcumin (Fig. 1C). Also, the possibility that curcumin could function as a potentiator of defective F508-CFTR gating was investigated. In low temperature-rescued cells, curcumin (40 µM) did not increase the rate of iodide influx above that induced by forskolin (not shown). Also, a 24-h growth of cells at 27 °Cin the presence of curcumin did not increase iodide influx above that seen in the absence of curcumin (not shown).
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F508 mutation, which we showed previously to have a low temperature rescue response (11). Short circuit current data shown in Fig. 2A indicate a substantial increase in current in response to forskolin/genistein after a 24-h growth of cells at 27 °C. Also, a reduction in the amiloride response was often seen. Averaged data are summarized in Fig. 2B, including data showing no significant effect of incubation with 40 µM curcumin at 37 °C for 24 h prior to short circuit current measurements.
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PD, PBS/amiloride versus low chloride/forskolin) for all wild type and F508 mutant mice. Although robust hyperpolarizations were seen in all curcumin-treated and control wild type mice, no effect of curcumin treatment was seen in any CF mouse, including mice treated with a dose equivalent to the maximum tolerated by humans (8 g/day, 100 mg/kg) (27), given in three divided doses in Alimentum for 3 days. Data are also shown for mice given the pure and neutraceutical curcumin preparations (45 mg/kg/day in three doses in Alimentum; Fig. 4C, right-most lanes). Averaged PD was 22 ± 3 mV in wild type mice (all study conditions averaged) and 2.1 ± 0.4 mV in F508 mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508-CFTR processing in epithelial cells in culture models and CF mice. Although a negative study cannot be definitive, the data raise significant doubt about the potential utility of curcumin and related compounds in the therapy of CF. Although we cannot rule out the possibility that under some conditions curcumin may correct F508-CFTR processing, the data here show that such correction, if it can occur, is not robust or explicable on the basis of measured curcumin pharmacology or the proposed curcumin inhibition of the SERCA pump.
We were unable to detect correction of nasal PDs in any F508-CFTR homozygous mutant mouse receiving curcumin using a variety of different vehicles, curcumin concentrations, and administration protocols. Robust hyperpolarization in response to low chloride and forskolin-containing solutions was seen in all wild type mice. We reported previously that the forskolin-dependent hyperpolarization in wild type mice was blocked by the CFTR inhibitors CFTRinh-172 and GlyH-101 (24, 29, 30) and thus involved chloride transport through the CFTR pore. The low chloride-induced hyperpolarization in the absence of cAMP agonists was not blocked by CFTR inhibitors, was partially inhibited by DIDS, and was absent in CF mice, suggesting CFTR-regulation of chloride transport by an as yet unidentified non-CFTR pathway. The nasal PD measurement method used here has several improvements over previous methods, including the use of orotracheal intubation and micropositional cannula placement (23), yielding reproducible PDs with excellent sensitivity for detection of activator/inhibitor effects.
Our negative nasal PD data are at variance with the large correction in nasal PDs by curcumin reported by Egan et al. (12). We think it is unlikely that genetic factors could account for the difference in results, because no correction was found here in any mouse in an outbred CD1 strain having mouse-to-mouse genetic variations. The hybrid inbred strain of mice and the identical lot of curcumin used by Egan et al. were not available for our study. It was puzzling that Egan et al. found full correction in nasal PDs in CF mice to the same level seen in wild type mice, particularly because F508 mutant mice express considerably less CFTR than wild type mice, and the apparent extent of correction was very small in their cell culture studies. Also, it is difficult to understand how full correction of nasal PDs could occur because curcumin did not correct defective F508-CFTR gating in our studies, and thus only a minor response is predicted even if curcumin corrected the reduced F508-CFTR expression level and the defective cellular processing.
Egan et al. also reported improved survival in CF mice given oral curcumin. However, the improved survival cannot be ascribed to a curcumin effect because appropriate controls were not done, including survival studies on CFTR-null mice and a vehicle (Alimentum)-alone control. The improved survival of F508 CF mice may be related to the pro-diarrheal effects of mouse handling or the administration of Alimentum and/or curcumin (31), as other pro-diarrheal agents such as Colyte are known to improve the survival of CF mice (32). It is quite possible that the high concentrations of curcumin in the mouse gut may reduce mucus accumulation and obstruction by effects unrelated to the correction of F508-CFTR processing.
We were unable to detect functional correction in F508-CFTR-transfected FRT epithelial cells or primary cultures of human airway epithelium homozygous for the F508 mutation. The transfected FRT cell model has been used for the identification of small molecule correctors of defective F508-CFTR gating and cellular processing by high throughput screening (11). These cells manifest a robust low temperature rescue response as demonstrated by fluorescence, electrophysiological, and biochemical assays, as well as a large response to the small molecule correctors of F508-CFTR processing identified by high throughput screening. Our YFP-based assay for F508-CFTR activity is sensitive and accurate with a Z'-factor of >0.7. By statistical analysis we estimated the ability to detect with confidence a level of correction for curcumin equivalent to 10% that of low temperature. Curcumin was also without effect in human F508-CFTR homozygous airway cells as assayed electrophysiologically with a positive low temperature rescue control. In contrast to our negative data, Egan et al. (12) reported a small amount of F508-CFTR correction (40% of the low temperature response) in F508-CFTR-transfected BHK cells as assayed biochemically and by 36Cl– uptake.
In summary, we were unable to document a significant effect of curcumin in the functional correction of defective F508-CFTR processing in transfected cells, native airway cells, and mutant mice. The measured low oral bioavailability of curcumin suggests maximal serum concentrations many orders of magnitude lower than that proposed to produce SERCA pump inhibition. Our results do not support further evaluation of curcumin as a therapy of CF caused by the F508 mutation.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: 1246 Health Sciences East Tower, Cardiovascular Research Inst., University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman{at}itsa.ucsf.edu.
1 The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; FRT, Fischer rat thyroid; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PD, potential difference; SERCA, sarcoplasmic/endoplasmic reticulum calcium; YFP, yellow fluorescent protein.
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REFERENCES |
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