Convallatoxin inhibits IL-1ß production by suppressing zinc finger protein 91 (ZFP91)-mediated pro-IL-1ß ubiquitination and caspase-8 inflammasome activity.

BACKGROUND AND PURPOSE: ZFP91 positively regulates IL-1ß production in macrophages and may be a potential therapeutic target to treat inflammatory-related diseases. We investigated whether this process is modulated by convallatoxin, which is a cardiac glycoside isolated from the traditional Chinese medicinal plant Adonis amurensis Regel et Radde. EXPERIMENTAL APPROACH: In vitro, the mechanisms by which convallatoxin inhibits ZFP91-regulated IL-1ß expression were investigated using molecular docking, western blotting, RT-PCR, ELISA, immunofluorescence and immunoprecipitation assays.In vivo, mice liver injury was induced by an intraperitoneal injection of D-GalN and LPS, colitis was induced by oral administration of dextran sulfate sodium (DSS) in drinking water and peritonitis was induced by an intraperitoneal injection of alum. KEY RESULTS: We confirmed that convallatoxin inhibited the release of IL-1ß by down-regulating ZFP91. Importantly, we found that convallatoxin significantly reduced K63-linked polyubiquitination of pro-IL-1ß regulated by ZFP91 and decreased the efficacy of pro-IL-1ß cleavage. Moreover, convallatoxin suppressed ZFP91-mediated activation of the non-canonical cysteine-requiring aspartate protease-8 (caspase-8) inflammasome and MAPK signalling pathways in macrophages. Furthermore, we showed that ZFP91 promoted the assembly of the caspase-8 inflammasome complex, whereas convallatoxin treatment reversed this result. Mice in vivo studies further demonstrated that convallatoxin ameliorated D-GalN/LPS-induced liver injury, DSS-induced colitis and alum-induced peritonitis by down-regulating ZFP91. CONCLUSION AND IMPLICATIONS: We show for the first time that convallatoxin-mediated inhibition of ZFP91 is an important regulatory event that prevents inappropriate inflammatory responses to maintain immune homeostasis. This mechanism provides new insight for the development of convallatoxin as a novel anti-inflammatory drug targeting ZFP91. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer.

BACKGROUND: Aberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties. PURPOSE: In this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells. METHODS: In vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells. RESULTS: Convallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model. CONCLUSION: The result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.

Convallatoxin induces HaCaT cell necroptosis and ameliorates skin lesions in psoriasis-like mouse models.

Psoriasis is considered an immune-mediated inflammatory skin disorder that affects the quality of life of nearly four percent of the world population. Considering the side effects of existing therapeutic drugs and the urgent need for new drug development, we screened more than 250 traditional Chinese medicine compounds to identify drugs that significantly reduced the viability of human HaCaT keratinocytes, a psoriasis-related model cell line. Convallatoxin (CNT) was found to be a highly effective inhibitor of HaCaT cell viability. Subsequent mechanistic studies revealed that CNT induced HaCaT cell death by necroptosis rather than by apoptosis. CNT destroyed the membrane integrity of HaCaT cells, as detected by nuclear propidium iodide (PI) staining and lactate dehydrogenase (LDH) release. Additionally, the intercellular levels of adenosine triphosphate (ATP) were lower in HaCaT cells treated with CNT than in control HaCaT cells, and typical necroptosis-associated characteristics were observed by electron microscopy in cells treated with CNT. Furthermore, compared with control HaCaT cells, CNT-treated HaCaT cells produced more reactive oxygen species (ROS), but this effect was inhibited by the antioxidants N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and apocynin and the necroptosis inhibitor Nec-1. In addition, antioxidant treatment attenuated necroptotic cell death, suggesting that CNT-induced HaCaT necroptosis is mediated by oxidative stress. More importantly, CNT ameliorated skin lesions and inflammation in imiquimod (IMQ)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced psoriasis-like mouse models. In conclusion, our results demonstrate that CNT is cytotoxic against HaCaT cells in vitro and exerts antipsoriatic activities in two mouse models of psoriasis in vivo, making CNT a potential promising candidate drug for future research.

Suppression of NRF2/ARE by convallatoxin sensitises A549 cells to 5-FU-mediated apoptosis.

NF-E2-related factor 2 (NRF2) regulates transcription of phase II cytoprotective enzymes to protect normal cells against oxidative stress. However, a high level of NRF2 offers a growth advantage, chemoresistance, and radioresistance in cancer. In the present study, we have identified convallatoxin as a novel inhibitor of NRF2/ARE. Suppression of NRF2 by convallatoxin was not transcriptionally mediated, but regulated at the level of proteolysis. Convallatoxin activated GSK-3ß and suppression of NRF2 by convallatoxin required the Neh6 domain. Convallatoxin sensitised A549 cells to 5-fluorouracil-mediated cell death by promoting apoptosis. Together, our results provide evidence that convallatoxin might be useful as a chemotherapeutic adjuvant due to its ability to suppress NRF2/ARE.

Molecular mechanisms of anti-oxidant and anti-aging effects induced by convallatoxin in Caenorhabditis elegans.

Convallatoxin is widely used as a cardiac glycoside in acute and chronic congestive heart-failure and paroxysmal tachycardia, with many effects and underlying protective mechanisms on inflammation and cellular proliferation. However, convallatoxin has not been investigated in its antioxidant effects and lifespan extension in Caenorhabditis elegans. In this study, we found that convallatoxin (20 µM) could significantly prolong the lifespan of wild-type C. elegans up to 16.3% through daf-16, but not sir-2.1 signalling and increased thermotolerance and resistance to paraquat-induced oxidative stress. Convallatoxin also improved pharyngeal pumping, locomotion, reduced lipofuscin accumulation and reactive oxygen species levels in C. elegans, which were attributed to hormesis, free radical-scavenging effects in vivo, and up-regulation of stress resistance-related proteins, such as SOD-3 and HSP-16.1. Furthermore, aging-associated genes daf-16, sod-3, and ctl-2 also appeared to contribute to the stress-resistance effect of convallatoxin. In summary, this study demonstrates that convallatoxin can protect against heat and oxidative stress and extend the lifespan of C. elegans, pointing it as a potential novel drug for retarding the aging process in humans.

[Tolerance to cardiotoxic effect of strophanthane K in experimental total heart block and electrocardiostimulation]. / Izuchenie tolerantnosti k kardiotoksicheskomu deistviiu strofantina K pri éksperimental'noi polnoi poperechnoi blokade serdtsa i élektrokardiostimuliatsii.

Tolerance to the cardiotoxic effect of strophanthin K was studied in narcotized dogs with experimental complete heart block (CHB) and ventricular electrostimulation. A threshold to the toxic arrhythmogenic effect of strophanthin K was significantly reduced as compared to that in the control group. As the electrocardiostimulation (ECS) frequency increased, the minimum arrhythmogenic dose of strophanthin K showed a growth, while not reaching a level for the sinus rhythm. It is concluded that the basic mechanisms of the arrhythmogenic effect of strophanthin K under the conditions studied are the trigger activity in dogs with ECS and the increased peacemaker's activity in dogs with CHB.

Homeopathic effect on heart rate variability.

We record ECGs from healthy human subjects during 24-h long intervals, using ambulatory equipment. We calculate from the data various parameters, searching for those that change in a clear and systematic way under a homeopathic stimulus, (Strophantus hispidus 30c). The energy fraction at high frequencies in the power spectrum of heart rate variability fulfills this condition, and we are able to interpret our results in a way consistent with the information on this medicine in the homeopathic Materia Medica.

[The influence of kordaron on the pharmacodynamic effects of strophanthin in experiments on isolated myocardial preparations and in the modelling of heart failure]. / Vliianie kordarona na farmakodinamicheskie éffekty strofantina v éksperimentakh na izolirovannykh preparatakh miokarda i v usloviiakh modelirovaniia serdechnoi nedostatochnosti.

The experiments with isolated rat atria isometrically contracting and those with simulated rat heart failure were performed to study the effects of the alpha-, beta-, and X-blocker cordarone on the pharmacodynamic effects of strophanthin. Cordarone was demonstrated to greatly decrease the toxicity of the cardiotonic in circulatory decompensation, without causing negative effects of cardiac inotropic function. Cordarone in combination with strophanthin slightly diminished the magnitude of the negative chronotropic effect of the cardiac glycoside and slowed down the rate of its cardiotonic effect.

[Antiviral properties of various pharmacologic groups of drugs]. / Antivirusnye svoistva razlichnykh farmakologicheskikh grupp preparatov.

Currently used cardiovascular drugs such as nicotinamide, strophanthin, corglycon, curantyl, cavinton, papaverin hydrochloride, nicotinic acid, xantinole nicotinate, isoptin, parmidin and halidor were studied by the program of antiviral drug screening. The majority of them (9 out of 11) were shown to have antiviral activity which was rather individual by its specificity and level. Laboratory strains of 9 viruses inducing the most common infections in man and animals, i.e. Herpes simplex, poxvaccine, influenza, vesicular stomatitis, respiratory syncytial infection, VEE, ECHO. Lassa fever and rotavirus infection were tested. The characteristic feature of the drugs was their high specific activity against the DNA-containing viruses and rotavirus. The three drugs papaverin hydrochloride, strophanthin and corglycon proved to be the most promising. Their antiviral activity was confirmed on a model of herpes infection in mice. The paper discusses whether the phenomenon discovered in the official drugs is important in the therapy of somatic patients.

[Inotropic action of Astragalus membranaceus Bge. saponins and its possible mechanism].

Astragalus membranaceus saponins (AMS) 50-200 micrograms/ml had a positive inotropic action on the isolated working heart of rats, but in the case of 30 micrograms/ml the inotropic action turned negative. The contractility recovered after washing out the AMS. Strophanthin K performed in a similar manner as AMS. AMS 500 micrograms/ml decreased the resting potential of cultured rat heart cells by 10 mV. The results suggest that AMS plays its inotropic role through influencing the Na-K-ATPase.

[Effect of the anti-arrhythmia compounds etmozin and ethacizine on the contractile activity of myocardial preparations from heart defect patients]. / Vliianie antiaritmicheskikh soedinenii étmozina i étatsizina na sokratitel'nuiu aktivnost' preparatov miokarda bol'nykh porokami serdtsa.

Changes in contraction amplitudes, contraction peak time and relaxation time were evaluated at 50% and 80% of contraction peak, with adrenaline or strophanthin acting on the myocardium pretreated with antiarrhythmic substances, using atrial specimens from patients with acquired heart diseases and septal defects. Etmozin and ethacizine were shown to have a negative inotropic effect, reducing contraction intensity by about 50%. Adrenaline and strophanthin are capable of restoring myocardial contractility in the presence of antiarrhythmic effects without actually inducing spontaneous auto-arrhythmic activity. Causes of the recovery of myocardial mechanical activity, effectuated by adrenaline or strophanthin in the presence of antiarrhythmic agents are discussed. It is suggested that a combination of effective antiarrhythmic properties and negative inotropic effect of phenothiazines is significant for cases of arrhythmias with and without signs of heart failure.

[Comparative characteristics of the activity of lithium preparations and anti-arrhythmia agents in strophanthin, calcium chloride and barium chloride arrhythmias in an experiment]. / Sravnitel'naia kharakteristika aktivnosti preparatov litiia i antiaritmicheskikh sredstv pri strofantinovoi, khloridkal'tsievoi i khloridbarievoi aritmiiakh v éksperimente.

During disorders of cardiac rhythm induced by strophanthine K in anesthesized cats, by calcium chloride in mice and anesthesized rats, and by barium chloride in rabbits, lithium hydroxybutyrate (0.55-3.2 mg/kg according to cation) compares very favourably with lithium chloride, novocainamide, lidocaine, trimecaine, etmozine, quinidine and verapamil as regards antiarrhythmic activity and the range of therapeutic action.

[Effect of plant glycosides on resistance and capacitance vessels]. / Zur Wirkung pflanzlicher Glykoside auf Widerstandsgefässe und Kapazitätsgefässe.

In the anaesthetized cat, SCOA ( Miroton ), a product which contains extracts from Scilla , Convallaria , Oleander and Adonis , displays not only its well-known positive inotropic effect but has also constrictor effects on veins when applied in intravenous doses of 21.5-100 GPU /kg ( GPU = guinea-pig units, i.e. cardiotoxic equivalents related to 1 g body weight of guinea-pigs). The latter effect differs in that it is somewhat more prolonged. With intraduodenal administration the doses required to achieve equal peak effects as with intravenous injection are about 4 times larger and this suggests a relatively good enteral availability in the cat. SCOA constricts not only veins but also arteries. However, this latter effect is comparatively small and occurs only after intraarterial infusion of high doses (9.1 and 91 GPU /min, respectively).--The cardiac glycosides contained in the drug product primarily account for its vasoactive qualities. The venous constrictor effect correlates with the guinea-pig units. In qualitative respects, the pure glycosides cymarin , convallatoxin , proscillaridin , and scillaren exert equal effects. There is, however, evidence that the correlation between the effect on veins and on the heart differs for the glycosides tested. Based on equal guinea-pig units, the adonis extract, for instance, acts on capacitance vessels about twice as much as scilla , oleander and convallaria extracts. Cymarin , too, has a stronger effect on veins than would be expected from its cardiotoxic effect. The action on arteries and veins are based on different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

[Anti-arrhythmic action of preparations of the dihydropyridine series]. / Antiaritmicheskoe deistvie nekotorykh preparatov digidropiridinovogo riada.

The derivatives of the dihydropyridine series, sodium salt of 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydronicotinic acid (drug I) and disodium salt of 2,6-dimethyl-1,4-dihydropyridine-3,6-bis-carbonyloxyacetic acid (drug II), reduce or completely prevent (depending on the dose) arrhythmias of different types (disturbances of atrioventricular conduction, extrasystoles, heart fibrillation) induced by vasopressin, calcium chloride and strophantine. Study of the lipid composition and activity of lipid-dependent enzymes of sarcoplasmic reticulum membranes has demonstrated that the antiarrhythmic effect of the drugs under study is linked with their antioxidant action favouring the preservation of enzymes involved in Ca2+ transport.

[Effects of lithium preparations in certain forms of experimental arrhythmias]. / Effekty preparatov litiia pri nekotorykh formakh éksperimental'nykh aritmii.

In tests on cats with cut vagus and aortal nerves, clipped common carotid arteries caused a sharp increase in sympathic activity (SA) in the cardiac nerves, a rise of arterial pressure (AP) and ventricular arrhythmias. Lithium chloride and hydroxibutirate injected intravenously reduced SA, AP and restored sinus rhythm. While administering arrhythmogenic doses of strophantin R, similar rhythm disturbances were observed in addition to increase in SA in animals with denervated cardio-aortal and sinocarotid areas. In these cases lithium drugs also had an antiarrhythmic action and reduced SA. In cats with the preserved innervation of the above reflexogenic sites, reflexogenic sites, strophantine arrythmias developed against the background of SA reduction and lithium drugs were inefficacious. In experiments on rats, while injecting large doses of intravenous adrenalin secondary ventricular rhythm disturbances have been shown to occur 1-1.5 min after the drug injection and to result in death, Lithium chloride and hydroxibutirate prevent the development of these arrhythmias and the animals' death.

Nifedipine for digitalis-induced arrhythmias.

The calcium antagonist, nifedipine (Adalat; Bayer), has previously been thought by most workers to lack cardiac-anti-arrhythmic properties, thus differing from verapamil. We have found that nifedipine was successful in converting cardiac glycoside (K-strophanthoside)-induced ventricular tachycardia to sinus rhythm in 4 of 5 anaesthetized dogs when given intravenously. In this respect, therefore, nifedipine is similar to verapamil. Further work is advocated before applying these results to the clinical situation in man.