The detection of plasma membrane-localized ΔF508 CFTR is a vital step in the development of therapies designed to treat the underlying cause of CF in the majority of patients with CF. In this study, we evaluated several flavonoid compounds for the activation of surface ΔF508 CFTR, and identified a number of agents that robustly activate ΔF508 CFTR-dependent Cl− transport in airway monolayers after temperature correction of aberrant processing. One agent in particular, quercetin, was selected for further investigation because it demonstrated efficient activation of ΔF508 CFTR-dependent Isc, and has a well-established safety profile in human subjects. Two other flavonoids, equol and apigenin, also demonstrated potent ΔF508 CFTR activation, but were not studied further because of their inferior safety profile in the literature. Furthermore, additional studies indicated that these agents affect ΔF508 CFTR misprocessing, a characteristic that could confound studies intended to detected ΔF508 CFTR correction. Using two polarized epithelial models, we showed that quercetin produces a dose-dependent activation of ΔF508 CFTR after cell surface localization (low temperature rescue) (). In both ΔF508 CFTR expressing FRT and CFBE41o− cells, the effect was specific for CFTR at concentrations below 100 μg/mL, as indicated by the lack of effect on anion transport in ΔF508 CFTR-transduced cells without temperature correction (or in cells lacking the ΔF508 CFTR transgene). When repeated in a sequence analogous to a standard NPD protocol, quercetin stimulated CFTR-dependent Isc without evidence of tachyphylaxis, indicating that the compound may be suitable for clinical protocols requiring repeated exposure (). Together with results demonstrating the stimulation of CFTR-dependent anion transport across the nasal epithelium in vivo ( and ), the findings provide support for further studies of this agent in human NPD protocols as a means to enhance the detection of ΔF508 CFTR at the plasma membrane. Based on these findings, a multicenter study is being conducted to examine the response of ΔF508 homozygotes to quercetin perfusion compared with isoproterenol, and is intended to determine whether the response to quercetin is associated with a more mild pulmonary phenotype.
In the Calu-3 serous glandular cell line, Illek and Fischer showed that quercetin (at concentrations >40 μM, equivalent to 12.1 μg/ml) stimulated Cl−
secretion (half-maximal doses were 22.1 ± 4.5 μM) in vitro
, whereas higher doses were inhibitory (26
). Our findings indicate that quercetin produced maximal Isc
at approximately 10 μg/ml (33 μM) in vitro
, and hyperpolarized the NPD response between 5 to 20 μg/ml. These results concerning the effects of quercetin in the murine airway extend earlier studies, and firmly establish the CFTR dependence of observations at the concentrations tested. Interestingly, Illek and Fischer (26
) suggested that pretreatment of Calu-3 cells with forskolin may sensitize to subsequent quercetin stimulation. Our results in ΔF508 CFTR-expressing FRT cells did not show this effect, insofar as quercetin stimulated Isc
to a similar degree, regardless of forskolin pretreatment. This could indicate differences in the response of wild-type CFTR versus the ΔF508 protein, or other variables attributable to the cell types being tested (26
). The inhibition of anion conductance at higher concentrations observed in our studies was reported with other flavonoids (26
) and in studies of colonic epithelia (41
). These results emphasize the importance of using the appropriate concentration for studies intended to activate CFTR. Moreover, tissue-specific differences may mediate anion conductance in response to quercetin. These concerns may be abrogated through the use of the NPD assay, which requires topical administration directly to the nasal epithelia, as opposed to systemic treatment in which pharmacokinetics are more difficult to control, and unintended consequences of CFTR inhibition may be more likely in off-target tissues.
In other studies, Lim and colleagues reported no activation of chloride efflux with the addition of quercetin (1 or 5 μM) to forskolin (13 μM) in IB3–1 bronchial epithelial cells after the correction of endogenous ΔF508 CFTR with 4-phenylbuturate (42
). This disparity may be attributable to the low concentrations of quercetin tested, because our experiments also showed that no ΔF508 CFTR activation occurred with 3.3 μM quercetin. Plasma membrane ΔF508 CFTR levels can also be markedly influenced by cell polarity and by growth at an air–liquid interface (13
). Because the studies with IB3–1 cells were conducted under nonpolarizing conditions, this fundamental difference may have contributed to the previously reported negative results. These effects may also represent additional cell model-specific responses to flavonoid exposure, as reported by Hansen and colleagues regarding the quercetin modulation of heat shock protein-70 synthesis (45
Although previous work with quercetin in Calu-3 cells demonstrated a cAMP/PKA-dependent activation of CFTR (26
), our results provide evidence that the quercetin signaling that promotes corrected ΔF508 CFTR activation may differ from that produced by classic PKA-dependent stimuli, and this effect may be unique to flavonoids. Quercetin produced a small increase in cAMP relative to forskolin, despite potent stimulation of ion transport compared with cAMP agonist (). The cAMP levels resulting from quercetin treatment, however, were greater than those produced by genistein. Previous work from our laboratory has shown that ΔF508 CFTR channel activation (by genistein) can be partially blocked by inhibiting PKA in CFBE41o−
). This implies that ΔF508 CFTR requires some level of PKA activity (and thus PKA dependent RD phosphorylation) for genistein to confer Cl−
transport. While no RD phosphorylation was detected following quercetin treatment (further distinguishing the mechanistic basis of flavonoid versus PKA activation pathways; see
), we speculate that quercetin may provide two stimuli that together optimize ΔF508-CFTR activation (including both cAMP-dependent and -independent effects), particularly in cells where endogenous cAMP levels could be limiting.
Several laboratories showed that ΔF508 CFTR, in addition to a well-described cellular processing defect, also exhibits significant abnormalities in channel gating at the cell surface. For example, ΔF508 CFTR exhibits reduced sensitivity to phosphorylation dependent activation in membrane patch and whole-cell studies (46
). More recent findings by Wang and colleagues using a series of molecules (including genistein, NPPB-AM, and curcumin), indicate that ΔF508 CFTR activation required stimuli independent of cAMP and PKA to rescue mutant channel function fully (47
). Studies from our laboratory and from others using intact airway monolayers established that low temperature or pharmacologically corrected ΔF508 CFTR is not fully activated by cAMP stimuli, including the β2
agonists albuterol and isoproterenol or the A2B adenosine receptor agonists adenosine or 5′-N-ethylcarboxamide adenosine. This defect appears to be more pronounced in pulmonary epithelia, including human alveolar and airway cells (13
). These findings provide a compelling rationale to investigate more sensitive biomarkers of ΔF508 CFTR activity after processing correction, including agents such as quercetin. For example, current methods may not detect effective biochemical corrections of ΔF508 CFTR, insofar as chemically corrected ΔF508 CFTR may not be activated by the cAMP pathway because of persistent gating defects. As such, improved surface activation of CFTR by an agent such as quercetin would be expected to decrease the number of subjects required to observe a relevant improvement in CFTR-dependent NPD response, simplifying the designs of clinical trials.
In addition, agents such as quercetin could help detect and discriminate the ΔF508 CFTR that resides in the plasma membrane, but that is unresponsive to endogenous activation because of a persistent gating defect, thus helping define the mechanistic basis underlying a ΔF508 CFTR processing corrector under study. Although the mechanism by which quercetin and other flavonoids activate CFTR is unknown, our findings support a direct effect on the CFTR Cl−
channel that can overcome the defective gating of cell surface-localized ΔF508 CFTR. These results are further supported by observations from whole-cell studies with quercetin (26
) and single-channel studies with other flavonoids (47
Because the endoplasmic reticulum-associated degradation of ΔF508 CFTR was postulated to be less than 100% efficient in vitro
), and because small amounts of ΔF508 CFTR at the cell membrane were speculated to account for phenotypic differences among ΔF508 CFTR homozygous individuals (48
), the evaluation of CFTR-dependent Cl−
conductance by NPD using quercetin (or other potentiating stimuli) could be used as an approach to detect individuals with small but relevant amounts of ΔF508 CFTR at the plasma membrane. Such an approach could identify ΔF508 CFTR homozygotes who are potential candidates for treatment with other CFTR potentiators, including the agent VX-770, currently under investigation in patients with CF harboring the G551D CFTR mutation (49
). The relative safety of the approach, as confirmed during the human NPD studies described here, makes this assessment feasible.
Flavonoids are ubiquitous in the human diet, are highly bioavailable, and have been extensively studied with regard to their pharmacology and toxicology (50
). Multiple laboratory clinical trials point to an excellent safety profile of quercetin, including studies using systemic, oral, and intravenous administration at levels far exceeding those found to activate ΔF508 CFTR (51
). For example, Shoskes and colleagues reported that oral quercetin (500 mg, twice daily) was well tolerated and significantly improved symptoms in men with chronic prostatitis (54
). The intravenous administration of high doses of quercetin was well tolerated over a 6- to 7-month period in a study examining quercetin as a tyrosine kinase inhibitor (51
). More recently, oral quercetin (730 mg daily) for 28 days produced no significant adverse events in a hypertension trial (55
). Carcinogenic testing in hamsters by Morino and colleagues (56
), and additional studies involving systemic exposure in rats (57
), further indicate the safety of the agent in animal models. This extensive preclinical and clinical experience, coupled with our results in vitro
and in vivo
, provide strong support for further studies to examine quercetin in subjects with CF as a means to activate ΔF508 CFTR.