CFTR and Genistein

Genistein and CFTR  (normal CFTR, G551D and delF508). 

Since some people with double delF508 mutations have tried the Curcumin – Genistein combination and have noticed positive effects on mucus thinning and digestion, I wondered how that could be explained. I’ve tried to simplify things as much as possible and I hope everyone understands how Genistein may work for delF508.  (Next I’ll be researching Curcumin and delF508, but that’ll be another page).

When reading research articles like the ones below, don’t take the information too literally. These are mostly laboratory findings. All kinds of different cell lines are used, different methods and different concentrations of compounds. Some research normal CFTR, some research delF508-CFTR. Some CFTR is from hamster kidneys, some is from human lung tissue, but mostly it’s pieces of tissue, or just a few cells, taken out of the body (mouse, hamster or human), taken out of its’ natural environment. It’s never the same as in the human body and than each human body is a little different also. These articles give an IDEA OF THE POSSIBILITIES.

We will not know for sure what Genistein and/or Curcumin can do (effects and side effects) until it is tried in a large group of patients. The problem here is: “who will pay for it?”. There is no money to be made from food-supplements, since they cannot be patented. Because there is no big profit to be made, who will invest in a large clinical trial?
For now, there is enough reason to believe it may be helpful for CF and the decision to try is completely up to you.
If you decide to try, I would really (really!) appreciate it when you would let me know how you are doing. If you’ve noticed effects and/or side effects etc. please let me know, so we can all learn from it. You can contact me through this blog (leave a reaction) or send me an e-mail at

CFTR, the simplified, layman’s version:

Normal CFTR:
1.   The nucleus of the cell makes a protein called CFTR (Cystic Fibrosis Transmembrane Regulator). (expression)
2.   It comes out of the nucleus and goes through a quality control mechanism (endoplasmic reticulum) and is processed in the Golgi apparatus (processing).
3.   It travels to the cell surface (trafficking).
4.   There it makes an opening (the chloride channel), which opens and closes continuously in a certain  rhythm (gating).
5.   Chloride moves out of the cell through this opening (conductance).
6.   In the meantime a ‘vacuum cleaner’ moves through the inside of the cell, cleaning up used CFTR , pulling it out of the cell surface, back into the cell to throw in the trash (endocytosis).

The defect of G551D and S1251N and other gating mutations is in step 4. There is a problem in the opening and closing of the channel.

The defect in delF508 is:
1.   The delF508-CFTR is not made properly in the nucleus.
2.   Only a very small part of the delF508-CFTR proteins make it through the quality control mechanism, the rest is ‘thrown in the trash’ (proteasome).
3.   Some delF508-CFTR that has made it through the quality control mechanism, makes it to the cell surface, some others just swim around in the inside of the cell.
4.   The ones that do make it to the cell surface, make an opening, (chloride channel) but it doesn’t open and close in the same rhythm as ‘normal’ CFTR. The open time is shorter, closed time longer.
5.   A little tiny bit of chloride moves out of the opening, much less than with normal CFTR.
6.   The vacuum cleaner comes by along the inside of the cell lining and cleans up, possibly sooner than in normal CFTR.

So in the end, a delF508-CFTR forms fewer chloride channels at the cell surface and the channels that are there, let out less chloride than normal and might stay there for a shorter period of time.
There are two (maybe three) problems: decreased quantity of delF508-CFTR at the cell surface and decreased function (and time?) of delF508-CFTR for chloride transport.

 How to fix delF508-CFTR:
1.   Let the nucleus make more delF508-CFTR (increase expression), so in the end more delF508-CFTR will end up at the cell surface, which will result in increased amount of chloride transport.
2.   De-activate the quality control mechanism (not a good idea, because CFTR is not the only protein going through quality control, all kinds of misprocessed proteins will end up in the cell….)
3.   Increase movement/trafficking of delF508-CFTR to the cell surface. Get the ones that are swimming around, doing nothing, to move to the cell surface and get to work.
4.   Change the rhythm of opening and closing of delF508-CFTR. Make it stay open longer, or stay closed for a shorter period of time; or both, so more chloride can move out of the cell.
5.   Let chloride move out faster, with the speed of a firefighter’s water hose, in stead of our kitchen tab. So even when the door isn’t open long, chloride is pumped out of there, high speed.
6.   Lower the speed of the vacuum cleaner, so delF508-CFTR stays at the apical membrance longer, and has more time to move some chloride out of the cell, before being thrown in the trash.

There are also other proteins that can move chloride out of a cell, and many other mechanisms that  incluence this process, but let’s focus on the above first, it’s complicated enough, and I just barely understand this part. (Please remember that the above is an extremely simplified version of reality, and probably not 100% correct, but it doesn’t have to be to understand the idea of what I’m trying to explain with the articles below)

Below are pieces of articles referring to how Genistein can influence delF508-CFTR.


link to abstract:

Dietary intake of soy genistein is associated with lung function in patients with asthma.

Smith LJHolbrook JTWise RBlumenthal MDozor AJMastronarde JWilliams L;

The nutrition substudy included 1033 participants, aged 12-75. Intake of antioxidant vitamins, soy isoflavones, total fruits and vegetables, fats, and fiber was compared with asthma severity at baseline, peak expiratory flow rate (PEF), asthma symptoms and the rate of asthma exacerbations during the 2 weeks following influenza vaccination. The only nutrient that had a consistent association with asthma severity was genistein. Increasing consumption of genistein is associated with better lung function in patients with asthma.

Comment: this possibly has nothing to do with CFTR function. It could be related to anti-oxidant and anti-inflammatory action of Genistein, but it's positive and interesting.
Association of dietary soy genistein intake with lung function and asthma control: a post-hoc analysis of patients enrolled in a prospective multicentre clinical trial.
Bime CWei CYHolbrook JSmith LJWise RA.

Soy genistein has important anti-inflammatory and other biological effects that might be beneficial in asthma. A positive association was previously reported between soy genistein intake and lung function but not with asthma exacerbations.
Results: Participants with little or no genistein intake had a lower baseline FEV(1) than those with a moderate or high intake (2.26 L vs. 2.53 L and 2.47 L, respectively; p=0.01). EPACs were more common among those with no genistein intake than in those with a moderate or high intake (54% vs. 35% vs. 40%, respectively; p<0.001). These findings remained significant after adjustment for patient demographics and body mass index.
CONCLUSIONS: In patients with asthma, consumption of a diet with moderate to high amounts of soy genistein is associated with better lung function and better asthma control.

Comment: again, it’s about asthma, but still interesting.


Link to full article:

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein.
Illek BZhang LLewis NCMoss RBDong JYFischer H.

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P(o) = 0.047 +/- 0.019. Addition of genistein activated Cl currents approximately 10-fold, and the P(o) of G551D-CFTR increased to 0.49 +/- 0.12, which is a P(o) similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired DeltaF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane.
Comment: Adding Genistein to G551D increased the open time of the chloride channel from an average of 0.047 to 0.49 (more than 10 times). The open time of 0.49 after adding Genistein is similar to normal CFTR. Chloride transport increased 10-fold (concentration used?)


Genistein potentiates wild-type and delta F508-CFTR channel activity.
T. C. Hwang , F. Wang , I. C. Yang , W. W. Reenstra
In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time. Our results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein.
Comment: they made sure that step 1 trough 3 and step 5 were steady and than tested what happened when adding Genistein to delF508-CFTR. They conlude that Genistein works by increasing open time of the delF508-CFTR channel, increasing the amount of chloride transport by 2-fold.
Tzyh-Chang Hwang, PhD
Research Interests
The most common CF- associated mutation, F508, causes an abnormal retention of the mutant protein in the endoplasmic reticulum. A small portion of F508 CFTR proteins can reach the plasma membrane and function as chloride channels. However, kinetic studies of these mutant CFTR channels in cell-attached patches indicate a lower open probability in response to cAMP stimulation compared to the wild-type channels. Genistein, a plant isoflavone that is abundant in legumes, can dramatically enhance F508 CFTR channel currents activated via the cAMP pathway. This effect of genistein appears to be caused by a direct binding of genistein to the CFTR. Current studies are focused on understanding the molecular nature of genistein binding site(s) and on the kinetic mechanism of genistein's action. Our work on CFTR modulation could potentially provide information useful for drug design and therapeutic intervention for cystic fibrosis.
Comment: Genistein increases chloride current by influencing CFTR at the cell surface.


Activation of delF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

C. Andersson, G.M. Roomans

Genistein and 8-cyclopentyl-1,3-dipro- pylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open.

Comment: confirms that Genistein can potentiate delF508-CFTR by increasing open time of chloride channel.

Modulation of delF508 Cystic Fibrosis Transmembrane Regulator Trafficking and Function with 4-Phenylbutyrate and Flavonoids
Meerana Lim, Karen McKenzie, Alexandra D. Floyd, Edwin Kwon, and Pamela L. Zeitlin

Over 70% of patients with cystic fibrosis have the delF508 mutation. This protein is a partially functional chloride (Cl-) channel that is prematurely degraded in the endoplasmic reticulum. Specific members of the flavonoid class of compounds have been shown to increase Cl- conductance of wild-type and delF508 cystic fibrosis transmembrane regulator (CFTR). Although flavonoid effects on CFTR processing are unknown, evidence of effects on heat shock proteins, specifically those that have been shown to interact with CFTR, led us to believe that there would be an effect on CFTR processing through modulation of CFTR–chaperone interactions. We sought to determine the effect of apigenin, genistein, kaempferol, and quercetin on CFTR processing in IB3–1 cells (F508/ W1282X) and whether sequential treatment with 4-phenylbutyrate (4-PBA) to increase CFTR processing and flavonoid to directly stimulate CFTR would increase Cl- conductance. Our results show no significant effect on CFTR processing as measured by immunoblotting with 1 mM or 5 mM of apigenin, genistein, kaempferol, or quercetin. However, despite no effect on CFTR processing as determined by immunoblot, immunofluorescence demonstrated a favorable change in the intracellular distribution of CFTR with 24 h treatments of apigenin, kaempferol, and genistein.

Furthermore, we observed an increase in Cl- conductance as measured by Cl- efflux in cells that were treated for 24 h with 4-PBA and then assayed with forskolin and 1 mM or 5 mM genistein, and also with cells treated for 24 h with either 4-PBA, 5 mM apigenin, or 1 mM quercetin. Thus, a combination of chronic treatment with 4-PBA or select flavonoids, followed by acute flavonoid exposure, may be beneficial in cystic fibrosis.

Flavonoids exhibit features that make them ideal candidates for therapeutic agents. Their antimicrobial, antiinflammatory, and antioxidant properties, combined with the fact that they are naturally found in foods and appear to be well tolerated thus far in human clinical trials (27), make them an ideal candidate as a therapeutic agent.

Comment: with different flavonoids they tried to get more CFTR to the cell surface, among which, genistein. They didn’t really notice more CFTR AT the cell surface, but did see more CFTR movement TOWARDS the cell surface. Adding Genistein also increased chloride transport through the channel.

Vasoactive Intestinal Peptide, Forskolin, and Genistein Increase Apical CFTR Trafficking in the Rectal Gland of the Spiny Dogfish, Squalus acanthias
Acute Regulation of CFTR Trafficking in an Intact Epithelium
Rüdiger W. Lehrich, Stephen G. Aller, Paul Webster, Christopher R. Marino, and John N. Forrest, Jr.

Our studies provide the first morphological evidence in an intact tissue that acute hormonal stimulation of chloride transport is accompanied by trafficking of CFTR from intracellular compartments to the apical membrane.
We interpret these findings as compelling evidence that CFTR traffics to the apical membrane during hormonal stimulation of chloride secretion in the rectal gland. Our data strongly support the concept that under physiological conditions, intracellular CFTR shifts to the apical membrane, and that secretagogue-stimulated insertion of CFTR-containing vesicles leads to an increase in chloride transport

Comment: This basically says that Genistein increases trafficking of normal CFTR (not in particular delF508-CFTR) to the apical membrance (cell surface). Tests were done in intact tissue of shark rectal gland, not just the CFTR containing cells.


Cholesterol Depletion and Genistein as Tools to Promote F508delCFTR Retention at the Plasma Membrane
Christina H. Lim, Marcel J. Bijvelds, Alex Nigg, Kees Schoonderwoerd, Adriaan B. Houtsmuller, Hugo R. de Jonge and Ben C. Tilly

The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis.
In the Western population more than 70% of the mutations in CFTR appear to be a deletion of a phenylalanine residue at amino- acid position 508 (F508delCFTR) [1, 4], resulting in an incompletely processed protein, defective in its ability to traffic to the plasma membrane [5, 6]. Although a small fraction of F508delCFTR protein may reach the plasma membrane and retain its function as cAMP-activated chloride channel [7-9], the reduced number of channels at the cell surface leads to dramatically impaired transepithelial transport of chloride [8]. In addition, although glycosylation of CFTR is not required for CFTR function [10], F508delCFTR exhibits a significant decrease in the open probability [11, 12] and a strongly reduced cell surface half-life as compared to wild type [8, 13, 14], which also contribute to the diminished Cl- conductance and, consequently, water flux.
In addition, the results suggest inhibition of endocytosis as a novel mechanism by which genistein is able to enhance CFTR activity in intact cells.
This novel finding suggest that the beneficial effect of genistein on CFTR-mediated Cl- secretion may result from a dual action: potentiation of the CFTR Cl- channel by binding to CFTR and stabilizing the channel in its open state [41-43] and promotion of its retention in the plasma membrane through inhibition of endocytosis.

Comment: Suprisingly Genistein seems to have a second (or third) mechanism of action on delF508-CFTR! Not only does it stabilize the open channel state (the open time), but it also keeps delF508-CFTR at the cell surface longer, by slowing down the vacuum cleaner (step 6 = endocytosis).


Genistein: a natural isoflavone with a potential for treatment of genetic diseases
GrzegorzWegrzyn et al.

Although several hundred mutations in the CFTR gene have been described to date [15], between 70 and 90% of CF patients (depending on population) bear the most frequent defective allele of this gene, called delF508. This mutation leads to a deletion of a phenylalanine residue at position 508 in the 1480-amino-acid polypeptide, which has two dramatic effects on the protein properties. First, the product of the mutant allele has severe problems with proper folding. This causes its recognition by the ERQC (endoplasmic reticulum quality control) system, and exposition to proteolytic degradation.
As a consequence, little, if any, delF508 CFTR protein reaches the cellular membrane, the normal compartment of wild-type CFTR function. Secondly, the activity of delF508 CFTR as a Cl− ion channel is severely impaired. Therefore the mutated protein is not able to both reach its proper localization in the cells and perform its biochemical function [15].
Although several other potentiators of delF508 CFTR were found [23,24], genistein is still one of the most efficient activators of the mutant protein, and, because of its low toxicity [4], it can be placed among the best candidates for a CF therapeutic. In fact, genistein has been demonstrated to be more effective in partial restoration of the delF508 CFTR activity than other potentiators, including scopoletin, isopsoralen, osthole, imperatorin, praeruptorin A and UCCF - 029 (7,8-benzoflavone) [23,24].
Interestingly, recent studies on the mechanism of genistein- mediated potentiation of delF508 CFTR revealed that this isoflavone acts not only by Cl− channel gating. Moderate concentrations of genistein augmented CFTR maturation and increased its localization to the cell surface [25]. These results indicate that genistein may be an even more potent drug in the treatment of CF than suggested previously, as it may enhance expression, proper localization and activity of the mutant protein.

Comment: they describe results of other studies and conclude that Genistein has a dual action, as it brings more delF508-CFTR to the cell surface and once there Genistein increases the open time of the chloride channel.

Towards Pharmacological Treatment of Cystic Fibrosis  (dissertation)

Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ∆F508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.

Comment: that’s exactly what we are doing here! Thanks for the tip! What took everyone so long???

(continued) Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. Our study on 19 CF patients with severe mutations showed that three of these patients had a still functional cAMP dependent chloride transport in their nasal epithelial cells.

Comment: This caught my attention! The fact that some CF patients with severe genotype have functioning CFTR in the nose….this could explain the runny noses some patients experience that are trying the Curcumin-Genistein supplement combination. That would mean CFTR in the nose is now overstimulated and moving more chloride out than normal CFTR…..

(continued) CFTR is a ~180kDa glycosylated protein (114) present in epithelial cells where it forms a cAMP regulated chloride channel (3,8). The protein consists of two putative membrane spanning domains each consisting of six α-helices, two nucleotide binding domains (NBDs) and one regulatory domain (103) (Fig. 1). CFTR belongs to the ABC (ATP-binding cassette) superfamily of transporters. The majority of ABC proteins are active transporters using the energy of ATP hydrolysis to pump solutes across the membrane. In the case of CFTR ATP hydrolysis is needed for the opening (NBD1) and closing (NBD2) of the chloride channel (18). The regulatory domain has many sites that are phosphorylated by primarily cAMP-dependent PKA but also PKC (103). Phosphorylation is needed for opening of the channel. The regulatory domain and the NBDs seem to be connected so that phosphorylation of the regulatory domain alters the ATP- sensitivity at NBD1 (78). Mutations that affect the ATP sensitivity (i.e., ∆F508) can on the other hand change the stability of the phosphorylation state (52,116) suggesting bi-directional interaction between the R-domain and the NBDs. NBD1 has been shown to interact with the plasma membrane and may be part of the channel pore (6,66). Depletion of the R-domain leaves a constitutively active chloride channel, which suggests that it has an inhibitory function in its unphosphorylated state CFTR is a chloride ion channel with a conductance of ~ 9 pS that has a linear I-V relation (13,23), but it also functions as a regulator of sodium ion channels (81,126), potassium ion channels (82,84), as well as other chloride ion channels (31,38,117,135). It may also conduct other molecules such as bicarbonate ions (85,123) and ATP (28,117) as well as regulate water transport through aquaporins (115). Involvement of CFTR in water transport may contribute to the dehydration in the CF airways. CFTR has also been suggested to be critical for the cAMP-dependent regulation of membrane recycling .
The possible conductance of ATP is interesting since purinergic receptors in the plasma membrane may activate calcium activated chloride channels through the PLC and IP3 pathway (28,117).
CFTR is synthesized on ER-associated ribosomes and incorporated into the ER membrane (121). Calnexin, an ER chaperone, binds to core oligosaccharide chains attached to the CFTR. Also cytosolic chaperones, Hsp70, Hdj-2, Hsc70 and Hsp90 bind to the CFTR to assist in folding and prevent aggregation of folding intermediates (77,90,96,140,141). An ATP dependent conformational maturation is required for calnexin and the cytosolic chaperones to dissociate. Not fully folded CFTR is degraded via the ubiquitinin/proteasome pathway. Interestingly, as much as 75% of the wildtype CFTR, and all or almost all of the ∆F508-CFTR is degraded in experiments performed on cell lines (20,58,134). The correctly folded CFTR travels to the Golgi apparatus where it becomes further glycosylated. The CFTR is then transported from the trans-Golgi network to the plasma membrane. The CFTR that reaches the plasma membrane is recycled or degraded by lysosomal proteases by endocytosis.

Comment: step 1 to 6 explained the correct way.

(continued) CF cells have an impaired ability to transport chloride ions in response to cAMP.

There are five mechanisms by which mutations disrupt CFTR function (129):

1) Defective protein production
2) Defective protein processing
3) Defective regulation
4) Defective conductance
5) Decreased abundance

In the first two classes of mutations no or little CFTR is present at the plasma membrane and these mutations are often coupled to a severe form of the disease. The most common mutation, ∆F508, belonging to class two of the CF mutations, leads to omission of phenylalanine residue 508, which is part of nucleotide-binding domain 1. Although the deletion of the phenylalanine residue occurs in the NBD1, the primary fault is a folding defect (97) that traps the CFTR in the ER where it is destroyed by the ubiquitinin/proteasome pathway (134). At the plasma membrane the ∆F508-CFTR functions as a chloride channel although with changed channel activity. It has reduced ATP sensitivity, reduced open probability and is more likely to inactivate than the wildtype CFTR

However, only classifying mutations after chloride transport characteristics might not be sufficient to understand the clinical course of the disease in different organs. For example, deficient chloride transport might not be the main cause of pancreas insufficiency. Choi et al. (21) recently showed that CF mutations with normal or almost normal chloride transport, but with deficient bicarbonate transport were correlated with pancreas insufficiency, whereas in mutations with defective chloride transport but functional HCO3- transport pancreatic function was sufficient.

More general info on CFTR:

CFTR is a massive and complex protein, that must be properly folded, embedded in a membrane, and shuttled to the cell surface. The fully functional, or mature, form weighs in at almost 190,000 Daltons (g/mol for the chemists out there ;)), which is more than 3 times the size of a typical protein. It’s initially expressed as a smaller precursor protein (~135,000 Daltons), but as CFTR passes quality control checkpoints, it’s decorated with sugar chains that tell cellular machinery that this CFTR protein is OK and ready for the next step of the process. With normal CFTR, only about 30% of CFTR that’s expressed actually transits the checkpoints successfully. The remaining protein is trashed without ever having done the job it was made to do. Just as sugars flag proteins that have passed quality control, ubiquitin commonly flags proteins that need to be degraded. Ubiquitin is a small regulatory protein that can be attached to a specific group (lysine) on other proteins. Ubiquitin can be conjugated to itself to create poly-ubiquitin chains, which target the proteins to the proteasome, a large multi-protein cylinder that cuts proteins into smaller pieces, or to the lysosome, an acidic intracellular sack filled with degradative proteins; in both cases, the end result is destruction of ubiquitinated proteins.


In three mutation classes, insufficient CFTR is made — Classes I, II and V. 
Class I mutations are nonsense mutations. Because of missing genetic information, these mutations stop cells from making the complete CFTR protein.
In Class II mutations, CFTR folds incorrectly. The malformed protein cannot move from the inside of the cell to the surface, where it is needed. The most common CF mutation, Delta F508, is a Class II mutation.
Class V mutations decrease the amount of functioning CFTR that makes it to the cell surface.

In the other mutation classes — III, IV and VI — CFTR is produced and delivered to the cell surface, but the mutations keep it from working properly once it is there. Patients with these mutations sometimes have less severe CF symptoms because there is some functioning CFTR on the cell surface.
The “engine” of CFTR is dysfunctional in Class III mutations. The protein does not respond to chemical signals that tell it to work, explains Steven Rowe, M.D., assistant professor of medicine at the University of Alabama.

In Class IV mutations, not enough chloride moves through the opening of the chloride channel. Class VI mutations cause the protein to pull away from the cell surface too quickly, creating a shortage of CFTR.

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