Helveticoside Exhibited p53-dependent Anticancer Activity Against Colorectal Cancer.

BACKGROUND: Investigation into the anti-cancer activities of natural products and their derivatives represents an efficient approach to develop safe and effective chemotherapeutic agents for the treatment of colorectal cancer. Helveticoside is a biologically active component of the seed extract of Descurainia sophia. This compound has been reported to regulate the genes related to cell proliferation and apoptosis in lung cancer cells, however its anticancer activity has not been fully explored yet. METHODS: Cell viability was evaluated by MTT and Trypan blue exclusion assay; cell apoptosis was measured by flow cytometry; mitochondrial membrane potential was determined by using JC1-mitochondrial membrane potential assay kit; protein levels were determined by western blot assay; in vivo tumor growth was assessed in a xenograft nude mice model. RESULTS: The current study demonstrated the in vitro anti-cancer activity of helveticoside against colorectal cancer using colorectal cancer cells SW480 and HCT116. Moreover, induction of apoptosis was found to mediate the cytotoxic action of helveticoside on SW480 and HCT116 cells. Based on the decrease in the mitochondrial membrane potential, upregulation of Bax, downregulation of Bcl-2 and cleavage of caspase-3 and 9, apoptosis was induced by helveticoside via mitochondria-mediated intrinsic apoptotic signaling pathways in colorectal cancer cells. Besides, using p53-knockout SW480 cells, the cytotoxic action of helveticoside was found to be p53-dependent. More importantly, administration of helveticoside inhibited the growth of HCT116 cells derived-colorectal cancer xenograft in mice via activation of apoptosis. CONCLUSIONS: Helveticoside might be a potential candidate for the development of novel chemotherapeutic agents for the treatment of colorectal cancer, while the potential toxic effects of helveticoside may be worthy of further investigations.

The sodium pump and digitalis drugs: Dogmas and fallacies.

The sodium pump (Na/K-ATPase) is a plasma membrane enzyme that transports Na+ and K+ against their physiological gradients in most eukaryotic cells. Besides pumping ions, the enzyme may also interact with neighboring proteins to activate cell signaling pathways that regulate cell growth. Digitalis drugs, useful for the treatment of heart failure and atrial arrhythmias, inhibit the pumping function of Na/K-ATPase and stimulate its signaling function. In the current field of research on the sodium pump and digitalis drugs, some issues that are commonly accepted to be well established are not so, and this may impede progress. Here, several such issues are identified, their histories are discussed, and their open discussions are urged. The covered unsettled questions consist of (a) the suggested hormonal role of endogenous digitalis compounds; (b) the specificity of Na/K-ATPase as the receptor for digitalis compounds; (c) the relevance of the positive inotropic action of digitalis to its use for the treatment of heart failure; (d) the conflicting findings on digitalis-induced signaling function of Na/K-ATPase; and (e) the uncertainties about the structure of Na/K-ATPase in the native cell membrane.

Aspectos novedosos de la intoxicación digitálica / New aspects on digitalis toxicity

Los digitálicos son fármacos con capacidad de aumentar la contractilidad miocárdica (inotrópico positivo), que han desempeñado un rol primordial en el tratamiento de pacientes con insuficiencia cardiaca; su uso inapropiado puede traer complicaciones serias a estos pacientes, incluso, hasta la muerte. La más importante de estas complicaciones es la intoxicación digitálica, originada por la sobredosis de dichos fármacos, a causa de la combinación del efecto inhibitorio en la conducción nodal y la estimulación sobre las fibras individuales auriculares y ventriculares. Debido al uso frecuente de estos medicamentos en todos los niveles de atención de salud y lo difícil que resulta diagnosticar dicha complicación por la complejidad de su cuadro clínico y de su expresión electrocardiográfica, se realizó una revisión bibliográfica exhaustiva sobre el tema para brindar amplia información, que permita una atención adecuada a los pacientes con este diagnóstico

Digitalis are drugs with the capacity of increasing myocardial contractility (inotropic positive agents) which have carried out an important role in the treatment of heart failure; their inappropriate use can bring severe complications to the patient, even, to death. The most important in these complications is the digitalis toxicity, originated by the overdose of these drugs, caused by the combination of the inhibitory effect in the nodal conduction and stimulation on the individual atrial and ventricular fibers. Due to the frequent use of these medications at all levels of medical care and to the difficulty in diagnosing this complication caused by the complexity of their clinical pattern and of their electrocardiographic expression, an exhaustive literature review was carried out on the topic to give a wide information that allows an appropriate care to the patients with this diagnosis

Clinical Use of Digitalis: A State of the Art Review.

The history of digitalis is rich and interesting, with the first use usually attributed to William Withering and his study on the foxglove published in 1785. However, some knowledge of plants with digitalis-like effects used for congestive heart failure (CHF) was in evidence as early as Roman times. The active components of the foxglove (Digitalis purpurea and Digitalis lanata) are classified as cardiac glycosides or cardiotonic steroids and include the well-known digitalis leaf, digitoxin, and digoxin; ouabain is a rapid-acting glycoside usually obtained from Strophanthus gratus. These drugs are potent inhibitors of cellular membrane sodium-potassium adenosine triphosphatase (Na+/K+-ATPase). For most of the twentieth century, digitalis and its derivatives, especially digoxin, were the available standard of care for CHF. However, as the century closed, many doubts, especially regarding safety, were raised about their use as other treatments for CHF, such as decreasing the preload of the left ventricle, were developed. Careful attention is needed to maintain the serum digoxin level at ≤ 1.0 ng/ml because of the very narrow therapeutic window of the medication. Evidence for benefit exists for CHF with reduced ejection fraction (EF), also referred to as heart failure with reduced EF (HFrEF), especially when considering the combination of mortality, morbidity, and decreased hospitalizations. However, the major support for using digoxin is in atrial fibrillation (AF) with a rapid ventricular response when a rate control approach is planned. The strongest support of all for digoxin is for its use in rate control in AF in the presence of a marginal blood pressure, since all other rate control medications contribute to additional hypotension. In summary, these days, digoxin appears to be of most use in HFrEF and in AF with rapid ventricular response for rate control, especially when associated with hypotension. The valuable history of the foxglove continues; it has been modified but not relegated to the garden or the medical history book, as some would advocate.

Why Whip the Starving Horse When There Are Oats for the Starving Myocardium?

Digoxin is the oldest drug for treatment of heart failure still in clinical use. Despite over 200 years of clinical experience with this drug, the optimal serum concentration required for both efficacy and safety remains unknown. It has been suggested that low doses have more favorable effects than higher ones. Cardiac glycosides act on the Na/K-ATPase (NKA). They show an inverted U-shaped dose-response curve with inhibition of pumping at high concentrations while increasing NKA activity at low concentrations. The classical sigmoidal dose-response curve describing an inhibition of the NKA by cardiac glycosides cannot explain this stimulatory effect. Cardiac glycosides are prototypical examples of hormetic substances. Biphasic dose-response curves of cardiac glycosides are also found in their neurohormonal effects. In low concentrations, vagomimetic effects are observed, whereas in high concentrations, sympathomimetic effects dominate. Lipophilic Digitalis glycosides have greater sympathomimetic effects; hydrophilic Strophanthus glycosides have greater vagomimetic effects. For digoxin, as a strong inotrope, there is evidence of only weak modulation of the autonomic nervous system. In ouabain, the modulation of the autonomic nervous system prevails over weak inotropic effects. Vagomimetic and sympatholytic effects characterize the therapeutic effects. In contrast to those of digoxin, the therapeutic effects of ouabain follow exactly the measurable serum concentration. Contrary to common prejudice ouabain is suitable for oral administration. Timely adjustments of dosage to patient therapeutic needs are easy to achieve with orally administered ouabain. Ouabain has the potential to crucially improve our arsenal of heart failure medications. Therefore, a clinical re-evaluation of ouabain is warranted. Randomized double-blind prospective clinical studies with ouabain, which meet today's standards, are worthwhile and necessary.

Helveticoside is a biologically active component of the seed extract of Descurainia sophia and induces reciprocal gene regulation in A549 human lung cancer cells.

BACKGROUND: Although the pharmacological activities of the seed extract of Descurainia sophia have been proven to be useful against cough, asthma, and edema, the biologically active components, particularly at the molecular level, remain elusive. Therefore, we aimed to identify the active component of an ethanol extract of D. sophia seeds (EEDS) by applying a systematic genomic approach. RESULTS: After treatment with EEDS, the dose-dependently expressed genes in A549 cells were used to query the Connectivity map to determine which small molecules could closely mimic EEDS in terms of whole gene expression. Gene ontology and pathway analyses were also performed to identify the functional involvement of the drug responsive genes. In addition, interaction network and enrichment map assays were implemented to measure the functional network structure of the drug-responsive genes. A Connectivity map analysis of differentially expressed genes resulted in the discovery of helveticoside as a candidate drug that induces a similar gene expression pattern to EEDS. We identified the presence of helveticoside in EEDS and determined that helveticoside was responsible for the dose-dependent gene expression induced by EEDS. Gene ontology and pathway analyses revealed that the metabolism and signaling processes in A549 cells were reciprocally regulated by helveticoside and inter-connected as functional modules. Additionally, in an ontological network analysis, diverse cancer type-related genes were found to be associated with the biological functions regulated by helveticoside. CONCLUSIONS: Using bioinformatic analyses, we confirmed that helveticoside is a biologically active component of EEDS that induces reciprocal regulation of metabolism and signaling processes. Our approach may provide novel insights to the herbal research field for identifying biologically active components from extracts.

On the differences between ouabain and digitalis glycosides.

Digoxin and digitoxin are widely used in the treatment of heart diseases. The exact mechanism of action of these drugs has remained an enigma. Ouabain has become the standard tool to investigate the mode of action of cardiotonic steroids, and results with ouabain are regarded as generally valid for all cardiac glycosides. However, there are marked differences between the effects of ouabain and digitalis glycosides. Ouabain has a different therapeutic profile from digitalis derivatives. Unlike digitalis glycosides, ouabain has a fast onset of action and stimulates myocardial metabolism. The inotropic effect of cardiotonic steroids is not related to inhibition of the Na-K-ATPase. Ouabain and digitalis derivatives develop their effects in different cellular spaces. Digitalis glycosides increase the intracellular calcium concentration by entering the cell interior and acting on the ryanodine receptors and by forming transmembrane calcium channels. Ouabain, by activation of the Na-K-ATPase from the extracellular side, triggers release of calcium from intracellular stores via signal transduction pathways and activates myocardial metabolism. These data no longer support the concept that all cardiotonic steroids exhibit their therapeutic effects by partial inhibition of the ion-pumping function of the Na-K-ATPase. Hence, it is suggested that this deeply rooted dogma be revised.

New cardenolide glycosides from the seeds of Digitalis purpurea and their cytotoxic activity.

A chemical investigation of Digitalis purpurea seeds led to the isolation of three new cardenolide glycosides (1, 8 and 11), together with 12 known cardenolide glycosides (2-7, 9, 10 and 12-15). The structures of 1, 8 and 11 were determined by 1D and 2D NMR spectroscopic analyses and the results of an acid or enzymatic hydrolysis. The cytotoxic activity of the isolated compounds (1-15) against HL-60 leukemia cells was examined. Compounds 2, 9, 11 and 12 showed potent cytotoxicity against HL-60 cells with respective 50% inhibition concentration (IC50) values of 0.060, 0.069, 0.038, and 0.034 µM. Compounds 2, 9 and 11 also exhibited potent cytotoxic activity against HepG2 human liver cancer cells with respective IC50 values of 0.38, 0.79, and 0.71 µM. An investigation of the structure-activity relationship showed that the cytotoxic activity was reduced by the introduction of a hydroxy group at C-16 of the digitoxigenin aglycone, methylation of the C-3' hydroxy group at the fucopyranosyl moiety, and acetylation of the C-3' hydroxy group at the digitoxopyranoyl moiety.

Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function.

Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with beta1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA-induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC(50) of 60 +/- 20 nmol/L. Digoxin also increased the fraction of some concanavalin A-binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients.

Electrocardiographic abnormalities associated with poisoning.

This article will review the cardiovascular toxicities of various medications, stressing the electrocardiographic presentation--both rhythm and morphological issues--and emphasizing recognition and management issues. Cardiovascular toxins are grouped into categories causing similar electrocardiographic effects, including the potassium efflux blockers, sodium channel blockers, sodium-potassium adenosine triphosphatase blockers (ie, digitalis compounds), calcium channel blockers, and beta-adrenergic blockers. This article reviews the various electrocardiographic abnormalities associated with these 5 classes of agents, ranging from morphological abnormalities and conduction blocks to brady- and tachyarrhythmias.

Screening of new chemopreventive compounds from Digitalis purpurea.

Chemopreventive agents induce a battery of genes whose protein products can protect cells from chemical-induced carcinogenesis. In this study, we isolated four different glycosides (1 acteoside; 2 purpureaside A; 3 calceolarioside B; and 4, plantainoside D) from the leaves of Digitalis purpurea and studied their abilities to induce glutathione S-transferase (GST) and their protective efficiencies against aflatoxin B1-induced cytotoxicity in H4IIE cells. Of these four glycosides, acteoside significantly inhibited the cytotoxicity induced by aflatoxin B1 (AFB1) and also selectively increased GSTalpha protein levels. Reporter gene analysis using an antioxidant response element (ARE) containing construct and subcellular fractionation assays, revealed that GSTalpha induction by acteoside might be associated with Nrf2/ARE activation. The results suggest that acteoside possesses a potent hepatoprotective effect against AFB1 and that it can be applied as a potential chemopreventive agent.

Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis.

In the HeLa tumor cell line, we studied the characteristics of the dual effect of digitalis compounds on cell growth (proliferation and death). In addition, we explored whether both effects occur by means of the same mechanism. HeLa cell cultures were exposed to increasing concentrations (0.01 nM-10 microM) of ouabain, strophantidin, digoxin, and digoxigenin at 24-96 h intervals. Cell growth in treated cultures was compared with cell growth under nontreated conditions. Additionally, we studied changes in nuclear morphology, as well as in genomic DNA degradation, cytochrome c release, and caspase-9 and -3 presence and processing induced by toxic concentrations of digitalis. Digitalis compounds increased HeLa cell number when exposed to concentrations <10 nM during a 48 h period. Ethacrynic acid (a nonsteroid inhibitor for Na+/K+-ATPase) did not induce cell growth at these concentrations. Digitalis concentrations >10 nM induced cell death in a concentration- and exposure period-dependent fashion. Changes in nuclear morphology, DNA fragmentation, mitochondrial cytochrome c release, and proteolytic processing of caspases-9 and -3, suggest apoptotic cell death. The IC50 for the inducing effect of apoptosis by ouabain at 96 h was 18 nM and corresponds with the IC50 for the Na+/K+-ATPase inhibition in HeLa cells. In conclusion, the dual effect of digitalis compounds on HeLa cells growth is concentration and time-dependent. The apoptosis-inducing effect correlates with inhibition of Na+/K+-ATPase. Proliferation does not appear to be mediated through this pathway. The apoptosis-induction pathway is possibly cytochrome c-dependent.

Sodium/potassium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: A new paradigm for development of anti- breast cancer drugs?

Prolonged exposure to 17beta-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERalpha and ERbeta. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.

On the importance and mechanism of amplification of digitalis signal through Na+/K+-ATPase.

Therapeutic concentrations of digitalis drugs inhibit the proliferation of breast cancer cells by inducing the interaction of Na+/K+-ATPase with Src/EGFR, activation of ERK1/2, and the resulting upregulation of cell cycle inhibitor p21Cip1. Quantitative comparison of ouabain dose-response curves for growth arrest and pump inhibition shows that ratio of Ki (pump)/Ki (proliferation) = 7.2. Such large gains in sensitivity are characteristic of several signal transducing pathways of other receptors. Making the reasonable assumption that Na+/K+-ATPase is the only receptor for ouabain, the large amplification factor clearly shows that occupation of a small fraction of pumping Na+/K+-ATPase by digitalis drugs, or endogenous digitalis-like factors, is sufficient to cause near complete inhibition of cell growth. The likely causes of large amplification factor in the signaling function of Na+/K+-ATPase include (a) interactions among the protomers of Na+/K+-ATPase in the membrane; and (b) induced clustering of Na+/K+-ATPase oligomers with neighboring proteins. The upstream location of both mechanisms suggests that similar amplifications also occur in other cell types with different digitalis downstream effects; e.g., stimulation of proliferation or hypertrophy.

Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells.

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.

Simplified digitalis-like compounds acting on Na(+), K(+)-ATPase.

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na(+),K(+)-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5beta,14beta-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na(+), K(+)-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.

Hallmarks of ion channel gene expression in end-stage heart failure.

Electrical conductance is greatly altered in end-stage heart failure, but little is known about the underlying events. We therefore investigated the expression of genes coding for major inward and outward ion channels, calcium binding proteins, ion receptors, ion exchangers, calcium ATPases, and calcium/calmodulin-dependent protein kinases in explanted hearts (n=13) of patients diagnosed with end-stage heart failure. With the exception of Kv11.1 and Kir3.1 and when compared with healthy controls, major sodium, potassium, and calcium ion channels, ion transporters, and exchangers were significantly repressed, but expression of Kv7.1, HCN4, troponin C and I, SERCA1, and phospholamban was elevated. Hierarchical gene cluster analysis provided novel insight into regulated gene networks. Significant induction of the transcriptional repressor m-Bop and the translational repressor NAT1 coincided with repressed cardiac gene expression. The statistically significant negative correlation between repressors and ion channels points to a mechanism of disease. We observed coregulation of ion channels and the androgen receptor and propose a role for this receptor in ion channel regulation. Overall, the reversal of repressed ion channel gene expression in patients with implanted assist devices exemplifies the complex interactions between pressure load/stretch force and heart-specific gene expression.

The Na, K-ATPase in the failing human heart.

The Na, K-ATPase consists of alpha- and beta-subunits and actively transports Na out and K into the myocyte. It is the receptor for cardiac glycosides exerting its positive inotropic effect by inhibiting enzyme activity, decreasing the driving force for the Na/Ca-exchange and increasing cellular content and release of Ca during depolarization. The specific binding capacity for cardiac glycosides is utilized as a tool for Na, K-ATPase quantification with high accuracy and precision. In treatment of patients with heart failure cardiac glycosides improve symptoms and reduce the need for hospitalization without affecting mortality. In endomyocardial biopsies from patients with compromised cardiac function total Na, K-ATPase concentration is decreased by approximately 40% and a correlation between decrease in heart function and decrease in Na, K-ATPase concentration exists. At the subunit level, the alpha1-, alpha3- and beta1-proteins are reduced in human heart failure. During digitalization approximately 30% of remaining Na, K-pumps are occupied by digoxin. Thus, a total of not less than half the Na, K-pumps may be out of function in the myocardium of digitalised heart failure patients. It is still a matter of debate whether a digitalis-like factor exists. There is a pressing need for the identification of its precise chemical structure, properties and quantitative relation to the Na, K-ATPase. It is recommended that cardiac glycosides are prescribed to heart failure patients who are still having heart failure symptoms after institution of mortality reducing therapy. Cardiac glycoside treatment is still the only safe inotropic drug for oral use that improves hemodynamics in patients with compromised cardiac function.