Targeting UHRF1-dependent DNA repair selectively sensitizes KRAS mutant lung cancer to chemotherapy.

Kirsten rat sarcoma virus oncogene homolog (KRAS) mutant lung cancer remains a challenge to cure and chemotherapy is the current standard treatment in the clinic. Hence, understanding molecular mechanisms underlying the sensitivity of KRAS mutant lung cancer to chemotherapy could help uncover unique strategies to treat this disease. Here we report a compound library screen and identification of cardiac glycosides as agents that selectively enhance the in vitro and in vivo effects of chemotherapy on KRAS mutant lung cancer. Quantitative mass spectrometry reveals that cardiac glycosides inhibit DNA double strand break (DSB) repair through suppressing the expression of UHRF1, an important DSB repair factor. Inhibition of UHRF1 by cardiac glycosides was mediated by specific suppression of the oncogenic KRAS pathway. Overexpression of UHRF1 rescued DSB repair inhibited by cardiac glycosides and depletion of UHRF1 mitigated cardiac glycoside-enhanced chemotherapeutic drug sensitivity in KRAS mutant lung cancer cells. Our study reveals a targetable dependency on UHRF1-stimulated DSB repair in KRAS mutant lung cancer in response to chemotherapy.

Modulatory properties of cardiac and quercetin glycosides from Dacryodes edulis seeds during L-NAME-induced vascular perturbation.

Background Numerous food wastes have been identified to possess potent bioactive compounds used for the treatment of several diseases. Therefore this study evaluated the potentials of cardiac and quercetin glycosides extracted from Dacryodes edulis seeds to reverse vascular and endothelial damage (VAED). Methods The glycoside composition of the seeds was extracted using standard methods and characterized by gas chromatography. We then recruited rats with L-NAME-induced VAED based on confirmatory biomarkers cardiac troponin (CnT), cellular adhesion molecule (VCAM-1), lipoprotein associated phospholipase A2 (Lp-PLA2), RAAS, VWF, endothelin, eNOx, and homocysteine. Only rats that showed total alterations of all biomarkers were recruited into the respective experimental groups and treated with either metaprolol succinate (met.su) + losartan or glycoside extracts of D. edulis seeds (NPSG). Results Chromatographic isolation of glycosides in the seed showed predominance of artemetin (1.59 mg/100 g), amygdalin (3.68 mg/100 g), digitoxin (19.21 mg/100 g), digoxin (27.23 mg/100 g), avicularin (133.59 mg/100 g), and hyperoside (481.76 mg/100 g). We observed decreased water intake and higher heart beats under vascular damage as the experiment progressed up to the fourth week. The met.su + losartan and H.D NPSG proved effective in restoring troponin, but both doses of NPSG normalized the VCAM-1 and RAAS activities excluding aldosterone and Lp-PLA2. Among the endothelial dysfunction biomarkers, H.D NPSG produced equivalent effects to met.su + losartan towards restoring the eNOx and VWF activities, but showed higher potency in normalizing the endothelin and Hcy levels. Conclusions We thus propose that the synergistic effect of the isolated glycosides from D. edulis shown in our study proved potent enough at high doses in treatment of vascular and endothelial dysfunction.

GSH responsive nanomedicines self-assembled from small molecule prodrug alleviate the toxicity of cardiac glycosides as potent cancer drugs.

Cardiac glycosides (CGs) have been used to treat cancer for hundreds of years. However, the narrow therapeutic window and system toxicity have hindered their wide clinical applications. Herein, the small molecule prodrug strategy and nanotechnology were integrated into one drug delivery system with enhanced therapeutic effect. Using periplocymarin (PPM) as a target agent, we designed a novel redox-responsive prodrug conjugated with linoleic acid (PPM-ss-LA), which was capable of self-assembling independent of exogenous excipients. This prodrug could co-assemble with DSPE2k to form PEGylated prodrug nanoparticles (PPM-ss-LA/DSPE2k-NPs) with enhanced colloidal stability and blood circulation. Compared with free PPM, PPM-ss-LA/DSPE2k-NPs retained high anti-proliferative activity and showed increased cell uptake and therapeutic efficacy. Furthermore, the PPM-ss-LA/DSPE2k-NPs acquired a greatly enhancement of 50% lethal dose (LD50) in mice and reduced system toxicity compared with the free drug. Overall, the on-demand release of nanoprodrug delivery system could improve the therapeutic window and anticancer efficacy of CGs.

The Associations of Atrial Fibrillation With the Risks of Incident Invasive Breast and Colorectal Cancer.

Atrial fibrillation (AF) is a common arrhythmia that poses a significant risk of stroke. Cross-sectional and case-control studies have shown evidence of associations between AF and breast or colorectal cancer, but there have been no longitudinal studies in which this has been assessed. We prospectively examined a cohort of 93,676 postmenopausal women enrolled in the Women's Health Initiative from 1994 to 1998 to determine whether there are relationships between baseline AF and the development of invasive breast or colorectal cancer. The prevalence of self-reported physician diagnosis of AF at baseline was 5.1%. Over approximately 15 years of follow-up, the incidence of invasive breast cancer was 5.7%, and the incidence of colorectal cancer was 1.6%. Adjusted hazard ratios and 95% confidence intervals were obtained using Cox proportional hazards models. We found no significant association between AF and incident colorectal cancer, but we did see a 19% excess risk of invasive breast cancer among those with AF (adjusted hazard ratio (HR) = 1.19, 95% confidence interval (CI): 1.03, 1.38). Additional adjustment for baseline use of cardiac glycosides attenuated the association between AF and invasive breast cancer (HR = 1.01, 95% CI: 0.85, 1.20). Cardiac glycoside use was strongly associated with incident invasive breast cancer (HR = 1.68, 95% CI: 1.33, 2.12) independent of AF and other confounders. Mechanisms of the associations among breast cancer, AF, and cardiac glycosides need further investigation.

Delivery of acetylthevetin B, an antitumor cardiac glycoside, using polymeric micelles for enhanced therapeutic efficacy against lung cancer cells.

Acetylthevetin B (ATB), a cardiac glycoside from the seed of Thevetia peruviana (Pers) K Schum (yellow oleander), exhibits not only antitumor activity but also potential cardiac toxicity. In the present study, we attempted to enhance its antitumor action and decrease its adverse effects via chitosan-Pluronic P123 (CP) micelle encapsulation. Two ATB-loaded CP micelles (ATB-CP1, ATB-CP2) were prepared using an emulsion/solvent evaporation technique. They were spherical in shape with a particle size of 40-50 nm, showed a neutral zeta potential, and had acceptable encapsulation efficiency (>90%). Compared to the free ATB (IC50=2.94 µmol/L), ATB-loaded CP micelles exerted much stronger cytotoxicity against human lung cancer A549 cells with lower IC50 values (0.76 and 1.44 µmol/L for ATB-CP1 and ATB-CP2, respectively). After administration of a single dose in mice, the accumulation of ATB-loaded CP1 micelles in the tumor and lungs, respectively, was 15.31-fold and 9.49-fold as high as that of free ATB. A549 xenograft tumor mice treated with ATB-loaded CP1 micelles for 21 d showed the smallest tumor volume (one-fourth of that in the control group) and the highest inhibition rate (85.6%) among all the treatment groups. After 21-d treatment, no significant pathological changes were observed in hearts and other main tissues. In summary, ATB may serve as a promising antitumor chemotherapeutic agent for lung cancer, and its antitumor efficacy was significantly improved by CP micelles, with lower adverse effects.

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.

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.

Cardiac glycosides use and the risk of lung cancer: a nested case-control study.

BACKGROUND: Two studies have reported statistically significant associations between the use of cardiac glycosides (CGs) and an increased risk of lung cancer. However, these studies had a number of methodological limitations. Thus, the objective of this study was to assess this association in a large population-based cohort of patients. METHODS: We used the United Kingdom Clinical Practice Research Datalink (CPRD) to identify a cohort of patients, at least 40 years of age, newly-diagnosed with heart failure, or supra-ventricular arrhythmia. A nested case-control analysis was conducted where each incident case of lung cancer identified during follow-up was randomly matched with up to 10 controls. Exposure to CGs was assessed in terms of ever use, cumulative duration of use and cumulative dose. Rate ratios (RRs) with 95% confidence intervals (CIs) were estimated using conditional logistic regression after adjusting for potential confounders. RESULTS: A total of 129,002 patients were included, and followed for a mean (SD) of 4.7 (3.8) years. During follow-up, 1237 patients were newly-diagnosed with lung cancer. Overall, ever use of CGs was not associated with an increased risk of lung cancer when compared to never use (RR = 1.09, 95% CI: 0.94-1.26). In addition, no dose-response relationship was observed in terms of cumulative duration of use and cumulative dose with all RRs around the null value across quartile categories. CONCLUSION: The results of this large population-based study indicate that the use of CGs is not associated with an increased risk of lung cancer.

Cardiac glycosides and the risk of breast cancer in women with chronic heart failure and supraventricular arrhythmia.

The aim of this study is to determine whether the use of cardiac glycosides (CGs), drugs used in the treatment of congestive heart failure (CHF) and supra-ventricular arrhythmia, is associated with an increased risk of breast cancer. A cohort of 53,454 women newly diagnosed with CHF or supra-ventricular arrhythmia between January 1, 1988 and December 31, 2010, followed until December 31, 2012, was identified using the United Kingdom Clinical Practice Research Datalink. A nested case-control analysis was performed, where all incident cases of breast cancer occurring during follow-up were identified and matched with up to 10 controls on age, cohort entry date, and duration of follow-up. Conditional logistic regression models were used to estimate adjusted odds ratios (ORs) with 95 % confidence intervals (CIs) of incident breast cancer associated with the use of CGs, along with measures of cumulative duration of use and dose. All analyses considered a one year lag period prior to the event, necessary for latency considerations and to minimize detection bias. The 898 breast cancer cases diagnosed beyond one year of follow-up were matched to 8,940 controls. Overall, use of CGs was not associated with an increased risk of breast cancer when compared to non-use (OR 1.07, 95 % CI 0.90-1.26). Furthermore, the risk did not vary with cumulative duration of use or cumulative dose. The findings of this large population-based study indicate that the use of CGs is not associated with an increased risk of breast cancer. This should provide reassurance to physicians and patients using these drugs.

The H1-H2 domain of the α1 isoform of Na+-K+-ATPase is involved in ouabain toxicity in rat ventricular myocytes.

The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H1-H2 domain of α1 and α2 isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α1 or α2 isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²âº](i)), attenuated mitochondrial Ca²âº overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 µM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²âº](i) and the contractility induced by 1 µM but not that induced by 1 mM OUA. These results indicate that the H1-H2 domain of the NKA α1 isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease.

[Pharmacotherapy of patients with chronic heart failure. Analysis of multicenter international studies].

Leterature data and own investigations are summarized in the article. New experimental facts about mechanism of action of cardiac glycosides (especially digoxin) were obtained last year. It was shown that in human organism there are endogenous cardiosteroids (digitalis-like substances, endogenous oubain and other). They are in different organs (adrenal glands, hypothalamus, pituitary gland) and have influence on different organs and systems. Indications, contraindications, side effects, interactions with other drugs have been studied by cardiologists. Results of international multicentere studies demonstrate high efficacy of digoxin in treatment of chronic heart failure caused by compromised contractile activity of myocardium, atrium fibrillation and is used for controlling number of heart contractions and supraventricular paroxysmal tachycardia. More than 200 years history of scientific researches and clinical use of cardiac glycosides show that this group of medicine may be applied in a combine pharmacotherapy of chronic heart failure together with antihypertensive drugs.

Evaluation of the pharmacokinetics of digoxin in healthy subjects receiving etoricoxib.

AIMS: Digoxin is a commonly prescribed cardiac glycoside with a narrow therapeutic index. The aim was to investigate whether the cyclooxygenase-2 selective nonsteroidal anti-inflammatory drug etoricoxib affects the steady-state pharmacokinetics of digoxin. METHODS: This was a double-blind, randomized, placebo-controlled, two-period cross-over study. In each period, 14 healthy volunteers ranging in age from 21 to 35 years received oral digoxin 0.25 mg daily and were randomized to either etoricoxib 120 mg or matching placebo tablets once daily for 10 days. Trough digoxin plasma concentrations were analysed by linear regression to examine digoxin accumulation over time. RESULTS: The geometric mean ratios (etoricoxib/placebo) for AUC(0-24h), C(max) and urinary excretion were 1.06 (90% confidence interval 0.97, 1.17), 1.33 (1.21, 1.46) and 1.10 (1.00, 1.20), respectively. The median (range) for digoxin T(max) (h) values with etoricoxib and placebo were 0.5 (0.5, 1.5) and 1.0 (0.5, 1.5), respectively. Steady-state digoxin plasma concentrations were achieved by day 7 in each treatment period. No serious adverse experiences were reported. CONCLUSIONS: Although etoricoxib 120 mg did produce an approximately 33% increase in digoxin C(max), this increase does not appear to be clinically meaningful, as cardiotoxicity with digoxin has been associated with elevations in steady-state rather than peak concentrations. From these results, it appears that etoricoxib does not cause any changes in digoxin steady-state pharmacokinetics that would necessitate a dose adjustment.

[Chronic systolic heart failure - dosing recommendations at beginning and end of treatment]. / Chronische systolische Herzinsuffizienz - Arzneimittel-Dosierung am Anfang und Ende einer Therapie.

As a multi-system disease, chronic heart failure requires a complex, multimodal therapy. Achieving maximum guideline adherence in (advanced) chronic heart failure can therefore become a challenge. The important decrease in morbidity and mortality seen lately largely depends on consequent and sometime tedious implementation of guideline targets. The present paper provides a comprehensive overview on medical therapeutic strategies with a particular focus of onset and discontinuation of drugs in patients with chronic systolic dysfunction.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Signalling pathways involving sodium pump stimulate endothelin-1 secretion and nitric oxide production in endothelial cells.

The cardiac steroid ouabain, a known inhibitor of the sodium pump, or Na+,K+-ATPase, has been shown to induce a variety of signaling cascades in various cells. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied to human umbilical artery endothelial cells (HUAECs) in culture at low concentrations that do not cause global sodium pump inhibition, induces a reaction cascade that leads to the release of the vasoactive peptide endothelin-1 (ET-1). While ouabain-induced ET-1 release seems to be accomplished within 10 min, ouabain also stimulates a second signaling cascade that involves activation of Akt (also known as protein kinase B, or PKB), activation of endothelial nitric oxide synthase (eNOS) and increased NO production in HUAECs. This reaction cascade reaches its maximum approximately 30 min after exposure to the steroid. The results indicate that ouabain or similar compounds might actively participate in the regulation of vascular tone.

Vascular endothelium as a target for endogenous ouabain: studies on the effect of ouabain on human endothelial cells.

It has been suggested that an endogenous inhibitor of the sodium pump, identified as ouabain, contributes to the regulation of blood pressure and the pathogenesis of certain forms of hypertension. Vascular endothelial cells, whose functional integrity is crucial for the maintenance of blood flow and the antithrombotic activity, could be a target for endogenous ouabain. We studied the effect of ouabain on human umbilical vein endothelial cells (HUVEC) and found that nanomolar concentrations of the glycoside have an antiapoptotic activity that is dependent on the activation of phosphatidylinositol 3 kinase (PI-3K) and extracellular signal-regulated kinases (ERKs). At the same concentrations we found that ouabain affects the endocytosis of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) through the activation of signaling proteins such as Src kinase. This review sumarizes our findings on the effect of ouabain on HUVEC, the signal transduction pathways involved and the significance of these observations on the pathophysiology of endothelial function.

The rapid decline of MTT reduction is not a marker of death signaling in ouabain-treated cells.

Decreased staining with dimethylthiazol diphenyltetrazolium (MTT) is widely used for cell death detection. This study examined MTT assay as a marker of the Na+i,K+i-independent mode of cell death revealed in ouabain-treated C7-MDCK cells derived from distal tubule of the Madin-Darby canine kidney. The action of 3-M ouabain on MTT reduction in C7-MDCK cells exhibited bipartite kinetics with a rapid ~2-fold decline occurring in 30-120 min and a delayed ~8-10-fold decrease after 10 hr of ouabain addition. Treatment with ouabain for 18 hr led to 6-fold activation of caspase-3, 4-fold elevation of chromatin fragmentation, and massive cell detachment. Caspase-3 activation, chromatin fragmentation and cell detachment were completely abolished by acidification of the incubation medium from pH 7.2 to 6.7. In contrast, the 2-fold inhibition of MTT reduction seen in 5 hr of ouabain addition was not affected by medium acidification. Within the 5-hr time window, we did not observe any significant impact of ouabain on the cellular redox state estimated by the autofluorescence ratio of reduced pyridine nucleotides and oxidized flavoproteins. In rat aortic endothelial cells and primary astrocytes, exposure to 5-mM ouabain attenuated MTT reduction but did not affect cell survival. Thus, our results show that diminished staining with MTT in ouabain-treated cells is not sufficient proof of triggering of the cell death machinery. We speculate that altered endo- and exocytoses evoked by cardiotonic steroids contribute to decreased MTT reduction.