Stanford BIOC 230 - Curcumin, a Major Constituent of Turmeric, Corrects Cystic Fibrosis Defects

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30. A. V. Budanov, A. A. Sablina, E. Feinstein, E. V. Koonin,P. M. Chumakov, unpublished data.31. Single-letter abbreviations for the amino acid resi-dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn;P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.32. G. D. Schuler, S. F. Altschul, D. J. Lipman, Proteins 9,180 (1991).33.We thank I. Verma for the lentivirus vector system used forexpression experiments, M. Chernov for sharing expertise in2D gel separations, and G. Stark and A. Gudkov for criticalreading of the manuscript. The work was supported byfunds provided by the Lerner Research Institute to P.M.C.Supporting Online Materialwww.sciencemag.org/cgi/content/full/304/5670/596/DC1Materials and MethodsFigs. S1 to S8References and Notes12 January 2004; accepted 23 March 2004Curcumin, a Major Constituentof Turmeric, Corrects CysticFibrosis DefectsMarie E. Egan,1,2Marilyn Pearson,1Scott A. Weiner,1Vanathy Rajendran,2Daniel Rubin,1Judith Glo¨ckner-Pagel,2Susan Canny,2Kai Du,3Gergely L. Lukacs,3Michael J. Caplan2*Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosistransmembrane conductance regulator (CFTR). The most common mutation,⌬F508, results in the production of a misfolded CFTR protein that is retainedin the endoplasmic reticulum and targeted for degradation. Curcumin is anontoxic Ca–adenosine triphosphatase pump inhibitor that can be administeredto humans safely. Oral administration of curcumin to homozygous ⌬F508 CFTRmice in doses comparable, on a weight-per-weight basis, to those well toleratedby humans corrected these animals’ characteristic nasal potential differencedefect. These effects were not observed in mice homozygous for a completeknockout of the CFTR gene. Curcumin also induced the functional appearanceof ⌬F508 CFTR protein in the plasma membranes of transfected baby hamsterkidney cells. Thus, curcumin treatment may be able to correct defects asso-ciated with the homozygous expression of ⌬F508 CFTR.The ⌬F508 CFTR mutation accounts for⬃69% of all cystic fibrosis (CF) alleles,and ⬃90% of CF patients carry at least onecopy of ⌬F508 CFTR (1). Although it isfunctional as a chloride channel (2), themisfolded ⌬F508 CFTR protein is retainedin the endoplasmic reticulum (ER) throughinteractions with elements of the ER’squality control chaperone machinery andtargeted for subsequent degradation in theproteasome (3–5). Many ER lumen chaper-ones are calcium-binding proteins (6, 7 ),suggesting the possibility that treatmentscapable of reducing the concentration ofcalcium in the ER lumen might interferewith chaperone function and thus permit⌬F508 CFTR to escape. Incubation of hu-man cell lines expressing ⌬F508 CFTRwith sarcoplasmic/endoplasmic reticulumcalcium pump (SERCA) inhibitors resultsin the release of the ER-retained ⌬F508CFTR protein, permitting it to achieve itscharacteristic functional residence at thecell surface (8). To identify nontoxic com-pounds that may work through similarmechanisms, we tested the capacity of cur-cumin, a relatively low-affinity SERCApump inhibitor (apparent Ki⬃ 5to15␮M)(9–11), to correct aspects of the CF defect incell lines and mice expressing ⌬F508 CFTR.To determine whether oral curcumin treat-ment could safely effect the functional redis-tribution of the ⌬F508 protein in the contextof a living CF-affected organism, gene-targeted mice homozygous for the ⌬F508mutation were given 45 mg of curcumin perkilogram of body weight by mouth daily for 3days. The dose administered was chosen toapproximate, on a weight per weight basis,curcumin doses that have been well toleratedin humans in previous studies (12). The cur-cumin was given either as a once per daybolus or as a divided dose three times daily.Following treatment, the membrane potentialdifference across the nasal epithelia [nasalpotential difference (NPD)] was measured(13). In untreated CF-affected animals thenasal epithelium exhibited a large, lumen-negative potential that was sensitive to amilo-ride, reflecting electrogenic Na⫹absorption(Fig. 1A) (8, 14 ). Removal of luminal Cl–and exposure to isoproterenol did not sub-stantially alter the potential in untreated⌬F508 animals (15, 16). After curcumintreatment, the average baseline NPD de-creased from –27.9 ⫾ 0.77 mV to –10.8 ⫾0.62 mV, approaching the values in wild-typemice (– 8.36 ⫾ 0.55 mV). In addition, perfu-sion with the low Cl–solution and subsequentaddition of isoproterenol each resulted inhyperpolarizations of the NPD similar tothose seen with wild-type animals (Fig.1A). Thus, there was a correction of boththe baseline and isoproterenol-stimulatedcomponents of the NPD trace after treat-ment with orally administered curcumin. Incontrast, phenylbutyrate treatment of theCF mice produced an effect only on theisoproterenol-stimulated component, con-sistent with what has been observed inhuman clinical trials (17 ) (fig. S1).To test the specificity of these effects for⌬F508 CFTR, curcumin was administered tohomozygous CFTR knockout mice (18, 19).These animals do not express any CFTRprotein but retain the remaining complementof transport systems that is associated withepithelial fluid and electrolyte secretion. Wefound that curcumin treatment did not correctthe abnormal NPD measured in the CFTRknockout mice (Fig. 1B).To assess whether curcumin altered intes-tinal ion transport, we measured the rectalpotential difference (RPD) in ⌬F508 CF micebefore and after treatment with curcumin.RPD measurements obtained from ⌬F508 CFmice differ in two major characteristics fromthose derived from wild-type littermates (20).First, the baseline RPD of the ⌬F508 CFmice is less negative than the baseline RPDof the wild-type mice. Furthermore, the RPDof wild-type mice, but not ⌬F508 CF mice,hyperpolarizes in response to forskolin. Thelack of response in the CF animals is believedto result from the absence of functionalCFTR in the rectal mucosa. We found thatafter treatment with curcumin, the CF micehad a 4.46 ⫾ 1.0 mV hyperpolarization inresponse to forskolin (Fig. 2A). This responseis approximately 91% of the response ob-served in the wild-type and heterozygotemice examined in this study.We examined the processing of ⌬F508CFTR protein expressed in the baby hamsterkidney (BHK) cell line. The ER-retained ⌬F508CFTR protein is core-glycosylated (4), whereaswild-type CFTR acquires


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Stanford BIOC 230 - Curcumin, a Major Constituent of Turmeric, Corrects Cystic Fibrosis Defects

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