[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

Inhibitory Effects of Digoxin and Digitoxin on Cell Growth in Human Ovarian Cancer Cell Line SKOV-3.

BACKGROUND: Cardiac glycosides (CGs) possess a chemical structure similar to steroids, and are inhibitors of the sodium potassium pump. An anti-tumor effect of CGs in breast and prostate cancers has been reported, but the effect of CGs on ovarian cancer is still unclear. AIMS: In this study, the effects of CGs on proliferation, cytotoxicity and cell cycle of ovarian cancer cell line (SKOV-3) have been investigated. PROCEDURE: The cell proliferation and cytotoxicity were detected by MTT assay and LDH activity assay, respectively. CGs, at concentrations higher than IC50, decreased cell proliferation and showed increased cytotoxicity toward SKOV-3 cells. The colony-formation ability was reduced after treatment with digoxin and digitoxin for 10 days. Furthermore, we explored the effect of digoxin and digitoxin on the distribution of cell cycle by flow cytometry. RESULTS: Results revealed that both digoxin and digitoxin led to cell cycle arrest in G0/G1 phase with 24 or 48 hours, but the arrest of G0/G1 phase was not observed at 72 hours. We evaluated the percentage of hypodiploid cell population as an index of the cellular fragments through flow cytometry. The data indicated that cellular fragments were significantly increased by treating with digitoxin at the concentrations of IC50 and 10-6 M for 72 hours. CONCLUSION: Taken together, these data suggest that CGs decreased cell proliferation and increased cytotoxicity through cell cycle arrest at the G0/G1 phase. CGs have anti-tumor effect in SKOV-3 cells and might be a potential therapeutic drug for ovarian cancer. Since this study is a preliminary investigation of CGs on SKOV-3 cells, more experiments might be performed in the future. Furthermore, more ovarian cancer cell lines might also be employed in the future studies to confirm the effect of CGs in ovarian cancer.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Compound Library Screening Identified Cardiac Glycoside Digitoxin as an Effective Growth Inhibitor of Gefitinib-Resistant Non-Small Cell Lung Cancer via Downregulation of α-Tubulin and Inhibition of Microtubule Formation.

Non-small-cell lung cancer (NSCLC) dominates over 85% of all lung cancer cases. Epidermal growth factor receptor (EGFR) activating mutation is a common situation in NSCLC. In the clinic, molecular-targeting with Gefitinib as a tyrosine kinase inhibitor (TKI) for EGFR downstream signaling is initially effective. However, drug resistance frequently happens due to additional mutation on EGFR, such as substitution from threonine to methionine at amino acid position 790 (T790M). In this study, we screened a traditional Chinese medicine (TCM) compound library consisting of 800 single compounds in TKI-resistance NSCLC H1975 cells, which contains substitutions from leucine to arginine at amino acid 858 (L858R) and T790M mutation on EGFR. Attractively, among these compounds there are 24 compounds CC50 of which was less than 2.5 µM were identified. We have further investigated the mechanism of the most effective one, Digitoxin. It showed a significantly cytotoxic effect in H1975 cells by causing G2 phase arrest, also remarkably activated 5' adenosine monophosphate-activated protein kinase (AMPK). Moreover, we first proved that Digitoxin suppressed microtubule formation through decreasing α-tubulin. Therefore, it confirmed that Digitoxin effectively depressed the growth of TKI-resistance NSCLC H1975 cells by inhibiting microtubule polymerization and inducing cell cycle arrest.

Digitoxin enhances the growth inhibitory effects of thapsigargin and simvastatin on ER negative human breast cancer cells.

BACKGROUND: The cardiac glycoside digitoxin preferentially inhibits the growth of breast cancer cells and targets the Erk pathway. Digitoxin alters the expression of genes that mediate calcium metabolism and IAP genes. PURPOSE: Since the optimal treatment for cancer involves the use of agents in combination, we assessed the growth inhibitory effects of digitoxin combined with agents that alter calcium metabolism, thapsigargin, a sarcoplasmic/ER Ca(2+)-ATPase inhibitor, and the statin simvastatin, as well as digitoxin's effect on the IAP pathway of apoptosis. METHODS: To reveal signaling pathways, we treated human cancer cells with digitoxin, alone or combined with thapsigargin or simvastatin, and measured cell growth using the MTT and colony formation assays. We used histology and Western blot analysis of HEK293 cells to assay effects on IAPs. RESULTS: Digitoxin inhibited the growth of breast, colon and ovarian cancer cells. Consistent with an effect on calcium metabolism, digitoxin exhibited synergy with thapsigargin and simvastatin on ER-negative breast cancer cells. Digitoxin activates expression of Erk pathway genes and suppresses expression of IAP genes. The growth inhibitory effects on HEK293 cells are not blocked by the pancaspase inhibitor zVAD-FMK, indicating that digitoxin may act by a caspase independent pathway of apoptosis. Furthermore, digitoxin does not have an effect on XIAP protein, a major anti-apoptotic protein. CONCLUSION: Digitoxin appears to act through the Erk and stress response pathways and is worthwhile to study to prevent and treat cancer. Our findings warn of possible safety issues for cardiac patients who take a combination of digitoxin and statins.

Use of digitoxin and digoxin as internal standards in HPLC analysis of triterpene saponin-containing extracts.

INTRODUCTION: Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives. OBJECTIVE: To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis. METHODOLOGY: The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water-methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using electrospray ionisation in negative ion mode (ESI(-)). RESULTS: Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol-water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol-water partitioning. CONCLUSION: Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves.

Actein inhibits the Na+-K+-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells.

The Na+K+-ATPase is a known target of cardiac glycosides such as digitoxin and ouabain. We determined that the enzyme also is a target of the structurally-related triterpene glycoside actein, present in the herb black cohosh. Actein's inhibition of Na+-K+-ATPase activity was less potent than that of digitoxin, but actein potentiated digitoxin's inhibitory effect on Na+-K+-ATPase activity and MDA-MB-453 breast cancer cell growth. We observed different degrees of signal amplification for the two compounds. Actein's inhibitory effect on ATPase activity was amplified 2-fold for cell growth inhibition, whereas digitoxin's signal was amplified 20-fold. Actein induced a biphasic response in proteins downstream of ATPase: low dose and short duration of treatment upregulated NF-kappaB promoter activity, p-ERK, p-Akt and cyclin D1 protein levels, whereas higher doses and longer exposure inhibited these activities. Actein and digitoxin may be a useful synergistic combination for cancer chemoprevention and/or therapy.

Anti-HSV activity of digitoxin and its possible mechanisms.

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC(50)) of 0.05 microM. The 50% cytotoxicity concentration (CC(50)) of digitoxin is 10.66 microM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.

Interference of Uzara glycosides in assays of digitalis glycosides.

OBJECTIVE: Presentation of a case report and pharmacokinetic investigation in healthy volunteers on the potential interference between cardiac glycosides and glycosides of Uzara, a herbal antidiarrheal preparation. METHODS: Pharmacokinetic pilot investigation of apparent digitoxin and digoxin serum concentrations in 4 healthy volunteers after single-dose administration of 30 drops Uzara (approximately 1.5 ml approximately = 22 mg glycosides). RESULTS: Maximal apparent serum concentrations of digitoxin between 198.0 microg/l and 919.8 microg/l (therapeutic range: 10-25 microg/l) occurred at 4-8 hours after administration. The terminal half-life of the glycosides was 8.87 +/- 2.20 hours. For digoxin, maximal apparent serum concentrations ranged between 1.4 microg/l and 6.34 microg/l (therapeutic range: 0.9-2.0 microg/l) at 6 hours post dosing. CONCLUSIONS: Administration of a single dose of an Uzara preparation, an over-the-counter product, results in false high serum concentrations of digitoxin and digoxin. As described in the manufacturers Summary of Product Characteristics, this preparation should not be given to patients with cardiac failure or arrhythmia who require treatment with cardiac glycosides because of the demonstrated pharmacological actions of uzara glycosides.

A screen for drugs that protect against the cytotoxicity of polyglutamine-expanded androgen receptor.

Spinobulbar muscular atrophy is a neurodegenerative disorder caused by expansion of a CAG triplet repeat sequence encoding a polyglutamine tract in the androgen receptor. It has been shown that the mutant protein is toxic in cell culture and triggers an apoptotic cascade resulting in activation of caspase-3. We developed an assay of caspase-3 activation in cells expressing the mutant androgen receptor. This assay was used to screen 1040 drugs, most of which are approved for clinical use. Drugs that inhibit polyglutamine-dependent activation of caspase-3 were subjected to follow-up screens to identify compounds that reproducibly prevent polyglutamine-induced cytotoxicity. Four drugs satisfied these criteria. Three of these (digitoxin, nerifolin and peruvoside) are structurally and functionally related compounds of the cardiac glycoside class and known inhibitors of Na(+)K(+)-ATPase. The fourth compound, suloctidil, is a calcium channel blocker.

Acaricidal effects of cardiac glycosides, azadirachtin and neem oil against the camel tick, Hyalomma dromedarii (Acari: Ixodidae).

The cardiac glycoside, digitoxin, from Digitalis purpurea L (Scrophulariaceae), a cardiac glycosidal (cardenolide) extract from Calotropis procera (Ait) R Br (Asclepiadaceae), azadirachtin and neem oil from Azadirachta indica A Juss (Meliaceae) were tested for their effects against larvae and adult stages of the camel tick, Hyalomma dromedarii Koch (Acari: Ixodidae). The contact LC50 values of the first three materials against adults were 4.08, 9.63 and >40.7 microg cm(-2), respectively, whereas the dipping LC50 values of the four materials were 409.9, 1096, >5000 and >5000 mg litre(-1), respectively. Contact and dipping LC50 values of the extract and azadirachtin against larvae were 6.16, >20.3 microg cm(-2) and 587.7 and >2500 mg litre(-1), respectively. Azadirachtin had no effects on egg production or feeding of adults up to 5000 mg litre(-1); however at 2500 mg litre(-1), it caused significant reduction in feeding activity of larve, prolonged the period for moulting to nymphal stage, and caused 60% reduction in moultability. Results of the two cardiac glycoside materials are comparable with those of several commercial acaricides. The risks and benefits associated with the use of cardiac glycosides are considered.

A neutrophil multitarget functional bioassay to detect anti-inflammatory natural products.

A multitarget functional bioassay was optimized as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes, mainly to release elastase detected by p-nitroanilide (pNA) formation. Using this bioassay, 100 fractionated extracts of 96 plants were screened, with results presented in a manner that links recorded biological activity to phylogenetic information. The plants were selected to represent a major part of the angiosperms, with emphasis on medicinal plants, Swedish anti-inflammatory plants, and plants known to contain peptides. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. The extract of Digitalis purpurea enhanced pNA formation, and digitoxin, the active compound, was isolated and identified. Plant extracts that exhibited potent nonselective inhibition (>80% inhibition) were evaluated further for direct inhibition of isolated elastase and trypsin enzyme. The inhibitory effect of most tested extracts on the isolated enzyme elastase was similar to that of PAF- and fMLP-induced pNA formation. Compared to trypsin, inhibition of elastase by extracts of Rubus idaeus and Tabernaemontana dichotoma was significantly higher (80% and 99%, respectively). Inhibition of trypsin by the extract of Reseda luteola was high (97%). Orders such as Lamiales and Brassicales were shown to include a comparably high proportion of plants with inhibitory extracts.

Uremic sera contain inhibitors that block digitoxin-valproic acid interaction.

BACKGROUND: Digitoxin and valproic acid show strong binding to serum albumin. Thus, when present simultaneously in serum, digitoxin and valproic acid compete for binding sites. We studied digitoxin-valproic acid interaction in normal and uremic sera. METHODS: Fluorescence polarization immunoassays were used for measuring total digitoxin and total valproic acid concentrations. We used a modified protocol of improved sensitivity to measure free digitoxin concentrations. We supplemented 2 normal and 2 uremic pools with digitoxin and then aliquots of these pools were further supplemented with various concentrations of valproic acid. After incubation at 37 degrees C for 2 hours in a water bath, specimens were allowed to re-equilibrate at room temperature for 20 minutes. Free digitoxin concentrations were measured. We also investigated digoxin-valproic acid interaction using 1 normal and 1 uremic serum pool. RESULTS: We observed significant increases in free digitoxin concentrations in normal sera in the presence of valproic acid. In contrast, we observed a slight decline in free digitoxin concentration in the presence of valproic acid in uremic sera. We speculated that uremic sera contained inhibitors that block digitoxin-valproic acid interaction and identified indoxyl sulfate as an inhibitor. However, another uremic compound, hippuric acid showed no inhibitory effect. Interestingly, we observed no significant interaction between digoxin and valproic acid in either normal or uremic serum pool. This is probably because of poor protein binding of digoxin. CONCLUSION: We conclude that valproic acid significantly displaces digitoxin from protein binding sites in normal serum. However, uremic sera contain inhibitors that block digitoxin-valproic acid interaction.

Severe thrombocytopenia caused by digitoxin intoxication in a patient with heart failure associated with Sjögren's syndrome.

Congestive heart failure (CHF) related to Sjögren's syndrome is extremely rare. This report concerns a patient who presented with CHF and severe thrombocytopenia (5,000/microl). Serum concentrations of K, Mg and digitoxin were 3.2mmol/L, 1.4mg/L and 57.2ng/ml, respectively. Digitoxin intoxication was evident, seemingly evoked by hypokalemia, hypomagnesemia, hepatorenal dysfunction and hypothyroidism. The severe thrombocytopenia was considered to have been caused by this intoxication, as it disappeared soon after the digitoxin was discontinued and potassium was supplemented.

Identification of an amino acid substitution in human alpha 1 Na,K-ATPase which confers differentially reduced affinity for two related cardiac glycosides.

The ouabain-resistant cell line H1C1 displays a 30-fold differential of reduced sensitivity to the structurally related cardiac glycosides digoxin and digitoxin (Baker, R. M. (1976) in Biogenesis and Turnover of Membrane Macromolecules (Cook, J.S., ed) pp. 93-103, Raven Press, New York). Since these ligand congeners differ only by the presence of a hydroxyl group at C-12 of digoxin we predicted that the H1C1 phenotype must reflect a mutation which alters the binding site of the cardiac glycoside receptor (Na,K-ATPase). Complementary DNA encoding the alpha 1 Na,K-ATPase was prepared from H1C1 cell total RNA by reverse transcription-coupled polymerase chain reaction and these cDNAs were cloned. Sequence analysis of the reverse transcriptase-polymerase chain reaction clones revealed several independent isolates containing a G > A transition at nucleotide 332 of the propeptide coding sequence, generating the amino acid substitution C108Y. The ability of this substitution to confer differential sensitivity for digoxin and digitoxin was tested and confirmed by expressing a human alpha 1 C108Y-Na,K-ATPase in wild type HeLa cells and assaying for inhibition of cell growth and inhibition of Na,K-ATPase activity. Phenylalanine or alanine substitutions of this cysteine also confer this pattern of ligand discrimination. Ouabain-resistant Na,K-ATPase substitutions, at positions other than Cys-108 failed to exhibit differential sensitivity indicating that this ligand discrimination is unique to Cys-108 substitutions rather than a general property of cardiac glycoside-resistant mutants. It is proposed that differential resistance of the C108Y receptor for these ligands is a consequence of altering two features of the ligand-receptor interaction; one, a disruption of a common hydrogen bond resulting in general loss of affinity for cardiac glycosides and the other, formation of a new H-bond between the C-12 hydroxyl of digoxin and the receptor, specifically augmenting the stability of this ligand-receptor complex.

Treatment of foxglove extract poisoning with digoxin-specific Fab fragments.

A 22-year-old man presented to our emergency department after an intentional overdose of a homemade foxglove extract. Clinical symptoms with symptomatic bradyarrhythmia and ECG changes were consistent with cardiac glycoside poisoning. Treatment with digoxin-specific Fab fragments resulted in transient clinical and ECG improvement. Serum immunoassay demonstrated a digitoxin-like glycoside. The serum levels showed no evidence of altered elimination or distribution with Fab therapy despite temporary improvements in the clinical course. The use of Fab did not result in a shortened clinical course in this episode of foxglove poisoning, as one would expect in the setting of commercial glycoside product poisoning.

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.