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. 2004 May 18;101(20):7693-8.
doi: 10.1073/pnas.0402030101. Epub 2004 May 10.

Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells

Meera Srivastava  1 , Ofer Eidelman, Jian Zhang, Cloud Paweletz, Hung Caohuy, QingFeng Yang, Kenneth A Jacobson, Eliahu Heldman, Wei Huang, Catherine Jozwik, Bette S Pollard, Harvey B Pollard
Affiliations

Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells

Meera Srivastava et al. Proc Natl Acad Sci U S A. .

Abstract

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-kappa B (I kappa B alpha). I kappa B alpha phosphorylation is a required step in the activation of the NF-kappa B signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF.

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Figures

Fig. 2.
Fig. 2.
Concentration–response curves for different cardiac glycosides as a function of IL-8 secretion from IB-3 CF lung epithelial cells. (Upper) Roman numerals correspond to fitted curves and common names for the compounds in the table. Curves are prepared from at least three independent assays. IL-8 data are normalized to DNA content of each well, as defined by propidium iodide fluorescence. (Lower) Cardiac glycoside compounds are numbered as in Fig. 1. The lower and upper ranges are calculated from the same data used to compute the K50.
Fig. 1.
Fig. 1.
Structures of cardiac glycosides used to set the structure activity relation for suppression of constitutive IL-8 secretion from IB-3 CF lung epithelial cells. Roman numerals correspond to titrations and column names in Fig. 2.
Fig. 3.
Fig. 3.
Pharmacogenomic analysis of cardiac glycoside drugs. (a) Pharmacogenomic analysis of cardiac glycosides with representation by a hierarchical clustering algorithm. Cardiac glycoside concentrations were chosen at ID90 points, as calculated from Fig. 1. The exact concentrations are given in Methods. Genes for the analysis were selected from those differing significantly (P < 0.05) between CF lung epithelial IB-3 cells and the wild-type CFTR-repaired daughter cells, IB-3/S9. Drugs 1, 2, and 3, corresponding to cells treated with drugs I, II, and III, respectively, cluster on the immediate right. The inactive cardiac glycoside (VIII) clusters with control cells, with (E) or without (C) 0.01% ethanol vehicle. A separate cluster contains a subcluster for drugs 4 and 6 (drugs IV and VI) and drugs 5 and 7 (drugs V and VII). IB-3/S9 cells corrected with wild-type CFTR are shown as controls with (e) or without (c) 0.01% ethanol vehicle. The diadic hierarchical cluster marked in red corresponds to most of the genes affected in common by both cardiac glycoside therapy and CFTR gene therapy. (b) Pharmacogenomic comparison between IB-3 cells treated with active cardiac glycosides or with wild-type CFTR. The effects of drug treatment or wild-type CFTR gene therapy are shown as the log of the ratio of the respective treatments to IB3-1 cells treated with vehicle only. Genes with relative expression in the range of 0.1–1.0 are relatively highly correlated (symbols with red borders) compared with those in the relative expression range of >1.0 (symbols with black borders). The symbols with red borders correspond to 27 genes marked by an asterisk in a and further denoted in a by a red-bordered gene cluster and text box. Eight genes marked by asterisks are also located in the lower diad. Red-bordered symbols are further differentiated by circles with white fill (drugs I, II, and III; y = 0.88x + 0.19, R2 = 0.75) or triangles with yellow fill (drugs IV, V, VI, and VII; y = 1.16x + 0.16, R2 = 0.76). Thus, regression analysis indicates that the slopes are close to 1 and the intercepts are close to 0. Three black-bordered “outlier” genes, somewhat more heavily expressed by drugs than by gene therapy, are marked by red fill (see text for gene identification). Symbols in black borders correspond principally to genes in the lower cluster in a. The symbols and fill colors code for the same drug categories as for the red-bordered symbols. However, for the latter genes, there is virtually no correlation between cardiac glycosides and gene therapy: drugs I, II, and III (y = 0.32x - 0.15, R2 = 0.05) and drugs IV, V, VI, and VII (y = 0.29x - 0.13, R2 = 0.03).
Fig. 4.
Fig. 4.
Cardiac glycosides block activation of IκBα and NFκB,p65 in CF lung epithelial IB-3 cells. (a) Cell extracts from IB3-1 and IB3-1/S9, repaired with wild-type CFTR, were robotically arrayed in serial 2-fold titremetric series on nitrocellulose-coated slides. Arrays were probed with antibodies against proteins known to be associated with activation of the NF-κB signaling pathway. Cells were incubated with ID90 concentrations of compounds I–VIII. Quantitative analyses are shown for total ERK and (T202,Y204)phospho-ERK; IKKβ and (Ser-176,Ser-180)phospho-IKKβ; total IκBα and (Ser-32,Ser-36)phospho-IκBα; and total p65 and (Ser-536)phospho-p65. Total caspase-3 and cleaved caspase-3 are included as a control, as is total protein concentration. The negative control (neg ctrl) is isotype-matched primary mouse IgG with common second antibody. An equivalent negative control is prepared for rabbit antibodies (data not shown). The data are representative of three independent experiments. (b) Fractional phosphorylation values of ERK, IKKβ, IκBα, and p65 are calculated as a function of cardiac glycoside treatment. The control (ctrl) value is calculated as the average of untreated cells, cells treated with ethanol vehicle (0.01%), and cells treated with the inactive compound VIII. The cardiac glycoside (CG) value is calculated as the average of effects for compounds I–VII. The asterisk represents differences between control and cardiac glycoside, with a significance of P < 0.05. RFU, relative fluorescence unit.
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