Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells.

BACKGROUND: Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. METHODS: We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. RESULTS: Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. CONCLUSIONS: Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SIGNIFICANCE: Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

Anti-tumorigenic effects of a novel digitoxin derivative on both estrogen receptor-positive and triple-negative breast cancer cells.

While there are targeted treatments for triple positive breast cancers, lack of specific biomarkers for triple-negative breast cancers (TNBC) has hindered the development of therapies for this subset of cancers. In this study, we evaluated the anticancer properties of cardiac glycoside Digitoxin (Dtx) and its synthetic analog MonoD on breast cancer cell lines MCF-7 (estrogen receptor-positive breast cancer) and MDA-MB-468 (triple-negative breast cancer). Both cardiac glycosides, at concentrations within the therapeutic range, increased the fraction of cells in the G0/G1 phase of the cell cycle, decreased viability, and inhibited the migration of MCF-7 and MDA-MB-468 cells. Both cardiac glycosides increased production of superoxide and induced apoptosis in both cell types. Reduced protein levels of nuclear factor kappa B and IkappaB kinase-beta were found in cardiac glycoside-treated cells, indicating that the cellular effects of these compounds are mediated via nuclear factor kappa B pathway. This study demonstrates the cytotoxic potential of digitoxin, and more importantly its synthetic analog MonoD, in the treatment of triple-positive breast cancer and more importantly the aggressive triple-negative breast cancer. Collectively, this study provides a basis for the reevaluation of cardiac glycosides in the treatment of breast cancer and more importantly reveals their potential in the treatment of triple-negative breast cancers.

Autophagy-Induced Apoptosis in Lung Cancer Cells by a Novel Digitoxin Analog.

We have synthesized a novel derivative of Digitoxin, termed "MonoD", which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within 1 h of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles, and increased expression of pro-autophagic markers Beclin-1 and LC3-II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3-II and Beclin-1. Prolonged treatment with MonoD-induced apoptosis in lung cancer cells. We investigated MonoD-dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3-II and Beclin-1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD-dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. J. Cell. Physiol. 231: 817-828, 2016. © 2015 Wiley Periodicals, Inc.

The New Semisynthetic Cardenolide Analog 3ß-[2-(1-Amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) Efficiently Suppresses Cell Growth in Human Leukemia and Urological Tumor Cell Lines.

BACKGROUND/AIM: The use of cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3ß-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines. MATERIALS AND METHODS: The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay. RESULTS: AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both cardenolides. CONCLUSION: The new cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.

Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation.

Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50=11.9 nM) than digitoxin (IC50=90.7 nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3ß signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.

Catalytic stereoselective synthesis of ß-digitoxosides: direct synthesis of digitoxin and C1'-epi-digitoxin.

A mild and atom-economic rhenium(V)-catalyzed stereoselective synthesis of ß-D-digitoxosides from 6-deoxy-D-allals has been described. This ß-selective glycosylation was achieved probably because of the formation of corresponding α-digitoxosides disfavored by 1,3-diaxial interaction. In addition, this method has been successfully applied to the synthesis of digitoxin trisaccharide glycal for the direct synthesis of digitoxin and C1'-epi-digitoxin.

Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin.

The cardiac glycoside natural product digitoxin was selectively glycosylated at one of its five hydroxyl groups using a borinic acid derived catalyst. This method provided access to the glycosylation pattern characteristic of a subclass of natural products from Digitalis purpurea. Variation of the glycosyl donor was tolerated, enabling the synthesis of novel cardiac glycoside analogs from readily available materials.

C3'/C4'-Stereochemical Effects of Digitoxigenin α-L-/α-D-Glycoside in Cancer Cytotoxicity.

Sweet'n low in stereo: A Wharton reaction was employed along with a diastereoselective palladium-catalyzed glycosylation and other post-glycosylation transformations to synthesize digitoxin analogues. Cytotoxic evaluation against a panel of cancer cell lines uncovered the stereochemical and substitutional limits of the C3'/C4'-hydroxy functionality in digitoxin monosaccharide.

Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells.

Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potency than digitoxin. In comparison, non-tumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.

Modifying the glycosidic linkage in digitoxin analogs provides selective cytotoxins.

A chemoselective reaction between oxyamines and unprotected, unactivated reducing sugars was used to construct for the first time a panel of linkage-diversified neoglycosides. This panel of digitoxin analogs included potent and selective tumor cytotoxins; cytotoxicity was dependent on the structure of the glycosidic linkage. These results validate linkage diversification through neoglycosylation as a unique and simple strategy to powerfully complement existing methods for the optimization of glycoconjugates.

The de novo synthesis of oligosaccharides: application to the medicinal chemistry SAR-study of digitoxin.

To address the medicinal chemist's need for new synthetic methods for the preparation of unnatural carbohydrates, a new de novo method for carbohydrate synthesis has been developed. These routes use a palladium catalyzed glycosylation reaction to stereoselectively control the anomeric center and subsequent diastereoselective post glycosylation to install the remaining sugar stereocenters. The utility of this method was demonstrated by the syntheses and biological evaluation of several digitoxin oligosaccharide analogues.

A stereoselective synthesis of digitoxin and digitoxigen mono- and bisdigitoxoside from digitoxigenin via a palladium-catalyzed glycosylation.

A convergent and stereocontrolled route to trisaccharide natural product digitoxin has been developed. The route is amenable to the preparation of both the digitoxigen mono- and bisdigitoxoside. This route featured the iterative application of the palladium-catalyzed glycosylation reaction, reductive 1,3-transposition, diastereoselective dihydroxylation, and regioselective protection. The natural product digitoxin was fashioned in 15 steps starting from digitoxigenin 2 and pyranone 8a or 18 steps from achiral acylfuran.

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization.

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycoside-induced cytotoxicity and Na+/K+-ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.

Application of fluorescence correlation spectroscopy to hapten-antibody binding.

Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations must be monitored carefully. Clearly, concentration assays based on IgG rely on accurate knowledge of the hapten-IgG binding strengths. The protocols for measuring and determining the dissociation constants for both IgG-hapten pairs are outlined and discussed.

Desymmetrization of enone-diones via rhodium-catalyzed diastereo- and enantioselective tandem conjugate addition-aldol cyclization.

Catalytic tandem conjugate addition-enolate trapping represents an effective strategy for the design of catalytic transformations that enable formation of multiple C-C bonds. Recently, using enantioselective rhodium-catalyzed conjugate addition methodology, we developed a catalytic tandem conjugate addition-aldol cyclization of keto-enones. Here, we report related desymmetrizations and parallel kinetic resolutions involving the use of diones as terminal electrophiles. The Rh-enolate generated on enone carbometallation effectively discriminates among four diastereotopic pi-faces of the appendant dione, ultimately providing products that embody four contiguous stereocenters, including two adjacent quaternary centers, with quantitative diastereoselection and high levels of enantiomeric excess. This methodology allows concise entry to optically enriched seco-B ring steroids possessing a 14-hydroxy cis-fused C-D ring junction, as found in naturally occurring cardiotonic steroids derived from digitalis.

Pharmacodynamics, pharmacokinetics and metabolism of digitoxin and derivatives in cats.

Derivatives of dihydro-digitoxin (DHD) were studied in the search for a glycoside with a primarily extrarenal clearance and a faster elimination rate than digitoxin. The positive inotropic doses of the derivatives of DHD were higher than those of digitoxin and digoxin. There was no significant difference in the therapeutic margin. After injection of 3H-digoxin in unaesthetized cats, no metabolites were found in the serum which did not bind with the antibody used for the RIA. After injection of 3H-digitoxin and its derivatives, the radioactivity was cleared from the serum at a much lower rate than the concentrations assayed by RIA. The metabolites which did not bind to the digitoxin antibody were hydrophilic and had a low protein binding. Digitoxin-bisdigitoxoside (Dt-2) determined by RIA rapidly disappeared from the serum. The radioactivity remaining after 24 h was eliminated with a half-life of 219 h. Ten min after injection of DHD the serum contained no unchanged DHD, but 36% digitoxin suggesting that the reduction of digitoxin to DHD is reversible and that the conversion of DHD to Dt-2 is the rate limiting step in the metabolism of digitoxin. The total body clearance of digitoxin, its metabolites and derivatives determined by RIA increased in the order DHD-oxime less than or equal to digitoxin less than DHD less than or equal to DHD-acetyloxime less than DHD-methyloxime. The clearance and the elimination rate of DHD-methyloxime were significantly higher than those of digitoxin (P = 0.05).

[Pharmacologic study of a semisynthetic digitalis derivative lacking the hydroxyl group on position 14 of the steroid nucleus]. / Estudio farmacológico de un digitálico semisintético desprovisto del grupo oxhidrilo en la posición 14 del núcleo esteroideo.

All natural cardiac glycosides (CG) have a hydroxyl (OH) group attached to carbon 14 (C 14) of the steroid nucleus which has been considered important for their pharmacological action. To investigate the relation between chemical structure and biological activity of CG, we studied the cardiac effects of a semisynthetic derivative if gluco-digitoxigenin that lacks the C 14 hydroxyl group; the compound was named Dig-3 and corresponds to the number of a series of semisynthetic glycosides being studied. On the failing heart of the Starling's heart-lung preparation Dig-3 reverts experimental cardiac failure. Electrophysiological experiments in anesthetized dogs (morphine chloralose) have shown that Dig-3 shortens the functional refractory period of the ordinary atrial myocardium (OAM), and lengthens that of the specialized atrial tissue (SAT). The basal excitability is reduced in both tissues, however OAM is more susceptible to Dig-3 action. The conduction velocity of impulses in SAT diminishes 50% with one tenth of the lethal dose (LD) of Dig-3 whereas in the OAM, an equivalent decrease is achieved with 60% of LD. A-V dissociation induced by the infusion of toxic doses of Dig-3 reverted to sinus rhythm in about 6 min after the administration was stopped. We conclude that the presence of the OH group attached to C 14 of digitalis molecule does not constitute a structural requirement for the preservation of its inotropic and electrophysiological actions on the heart, although its potency is greatly diminished.

Effects of digoxin and acetyl-digitoxin on basal and CO2-stimulated ventilation.

Eight healthy volunteers were studied to ascertain the effect of digoxin and the relatively more lipophylic cardiac glycoside, acetyl-digitoxin on ventilation. Baseline ventilation as well as the response to the inspiration of 2.2% and 4.8% carbon dioxide were assessed. Digoxin produced a depression of minute volume and oxygen consumption whereas acetyl-digitoxin produced the opposite effect. This could be the result of a relatively greater vagomimetic effect with digoxin and a greater symphatomimetic effect with acetyl-digitoxin. These findings might have clinical implications in cardiac patients who have pulmonary disease.