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An Evidence-based Perspective of Bufo Gargarizans (Asiatic Toad) for Cancer Patients

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Abstract

Bufalin and cinobufagin are cardiotonic steroids extracted from Chansu, a galenical preparation of the dried white venom of Chinese Bufo gargarizans (Asiatic toad). Chansu is also one of the major components of Kyushin, a traditional Chinese medicine. It has been shown that bufalin and cinobufagin increase vascular resistance, vasoconstriction, and blood pressure via an inhibition of Na+,K+-ATPase. This evidence indicates that bufalin and cinobufagin are endogenous digitalis-like factors. Since bufalin, cinobufagin, and digoxin share the similarity in the chemical structure, it is not surprising that bufalin and cinobufagin have digoxin-like effects. It is well-known that bufalin, cinobufagin, and digoxin as well as ouabain are specific blockers of the sodium pump. The digoxin-like immunoactivity has been observed in Chansu. It has been shown that bufalin and cinobufagin possess antitumor effects on human lung adenocarcinoma cells, leukemia cells, pancreatic cancer cells, gastric cancer cells, prostate cancer cells, endometrial cancer cells, ovarian cancer cells, and hepatocellular carcinoma via induction of growth inhibition, cell cycle arrest and/or apoptosis. Reactive oxygen species-dependent Bax translocation, PI3K/Akt and apoptotic modulators including Bax, cytochrome c, and caspases are involved in bufalin-induced apoptosis or anticancer pathways. In the present chapter, we shall review antitumor effects of bufalin on human cancer cells. Although in most studies, human carcinoma cells or cell lines were employed, we expect that more in vivo studies and clinical trials will be performed in the near future.

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... In recent years, traditional Chinese medicines have attracted increasing interests because of a broad spectrum of biological activities. Bufo bufo gargarizans Cantor, Bufo melanostictus Schneider, and Bufo raddei Sauch are species of toad found to be a source of bufalin, a soluble digoxin-like immunoreactive component also found in Chansu [7]. It has been demonstrated that bufalin exerts cardiotonic, anesthetic, blood pressure stimulation, respiration, and antineoplastic activities [8]. ...
... The mechanism of bufalin-induced apoptosis has been reported to be the activation of transcription factor AP-1, Rac1, cdc2 kinase, the induction of Tiam1, suppression PI3K/Akt, JNK1/2, ERK1/2 networks, and the elevation of intracellular calcium concentrations. These findings suggest potential clinical benefits of bufalin and drive an increase in ongoing clinical trials [7,19]. Although a few preliminary studies indicate cytotoxic effects of bufalin on HCC [12,20], the exact mechanism of action remains to be defined. ...
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The traditional Chinese medicine bufalin, extracted from toad's skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.
... The processed products Chan Pi (the toad skin) and Chansu (arenobufagin) which are the extracts from the dried toad skin and the white whey of parotoids are used to treat cardiovascular and cerebrovascular diseases and certain types of cancer, and proved to have antipyretic, detoxicant, diuretic, stasis-eliminative, and pus-discharging properties [8]. Chansu is also one of the major components of Kyushin, a traditional Chinese medicine [9]. Cinobufacini (Huachansu), a Chinese medicine prepared from Chan Pi, has been widely used in clinical therapy for various cancers in China [10]. ...
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Digoxin, a cardiac glycoside, is used to increase cardiac contractility via inhibition of Na(+)/K(+)-adenosinetriphosphatase (ATPase) and increase intracellular calcium in congestive heart failure. Inhibitory effects of digoxin have been demonstrated on the biosynthesis of gonadal hormones and adrenal glucocorticoids in rats. However, acute effects of digoxin on levels of adrenal corticosteroid hormones in the primates in vivo are uncertain. Therefore, we test the hypothesis that a single injection of digoxin decreases the secretion of aldosterone and cortisol in monkeys. An intravenous injection of digoxin (1 microg/kg) inhibited basal and adrenocorticotropin (ACTH)- or KCl-stimulated aldosterone release in monkeys. Furthermore, digoxin induced a decrease in ACTH- and KCl-stimulated cortisol release. Administration of digoxin did not alter plasma concentrations of Na(+) and K(+). Ouabain, a selective inhibitor of Na(+)/K(+)-ATPase, did not affect ACTH- or KCl-stimulated aldosterone and cortisol release. These results revealed that injection of digoxin induced an inhibitory effect on aldosterone and cortisol secretion in monkeys. Because ouabain did not affect levels of plasma aldosterone or cortisol, we suggest that (1) the Na(+)/K(+)-ATPase pathway may not be involved in the mechanism of action of digoxin on aldosterone or cortisol secretion in monkeys and/or (2) the Na(+)/K(+)-ATPase is more sensitive to digoxin than to ouabain in monkeys.
Article
Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3-(4,5-dimethylthiazol-2-yle)-2,5-diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase-3 activity and protein expression of caspase-3, -8, and -9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide-treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen-dependent LNCaP cells and Fas in androgen-independent DU145 and PC3 cells.
Article
We have recently demonstrated that bufalin is a new potent inducer of the differentiation of human myeloid leukemia cells. The present work was carried out to examine further the effect of bufalin on the growth and characteristics of human leukemia-derived cell lines U937, ML1, and HL60. At concentrations of 5-10 nM, bufalin decreased the growth of ML1 cells preferentially at the G2 phase and U937 cells at the S and G2 phases of the cell cycle. Bufalin, under these conditions, induced the differentiation of U937, ML1, and HL60 cells to monocyte/macrophage-like cells by measuring the expression of various differentiation markers, as assessed by morphology and histochemistry, and ability to phagocytose latex particles, to reduce nitroblue tetrazolium, and to develop Fc receptors. U937 and ML1 cells started to differentiate at 4 and 6 h, respectively, after treatment with 10 nM bufalin and showed maximum differentiation 72 h later. At present, a mechanism for the bufalin-mediated induction of the differentiation of these human leukemia cells remains to be determined. The combination of bufalin with all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16), or human gamma-interferon synergistically induced the differentiation of HL60 and U937 cells. A similar effect on ML1 cells was observed with the combination of bufalin with VP16 or human rTNF-alpha. These results suggest that bufalin in combination with VP16, all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, rTNF-alpha, or gamma-interferon may be very useful in the differentiation of human leukemia.
Article
The bufadienolide compounds (bufalin, cinobufagin and resibufogenin), major constituents of Chansu in Liu-Shen-Wan (LSW), were determined by reverse phase high performance liquid chromatography. The procedure involves a preliminary extraction of the bufadienolides from LSW with chloroform using ultrasonication and subsequent evaporation to dryness of the chloroform extract. The residue of the chloroform extract was dissolved in methanol and separated on a Merck LiChrosorb RP-18 column. Methanol: water (74:26) was used as mobile phase. The compounds were satisfactorily separated with good chromatographic peaks. Good coefficients of correlation (r > 0.999) were obtained from the calibration of peak areas with concentrations for the 3 bufadienolides. Results of analysis showed that there were differences between the contents of bufadienolides in 11 LSW samples of different origin available to the public in Hong Kong where at present there is no legal control over the sale of traditional Chinese medicines. The variability of quantities of bufadienolides in Chansu may be a hazard to the public.
Article
The cardiotonic and arrhythmogenic effects, and the excitatory effect on respiration of "Kyushin," a drug containing toad venom, were studied in comparison with those of digoxin. In anesthetized rabbits, the maximum rate of rise of left ventricular systolic pressure (max dP/dt) was measured as an index of cardiotonic effect, and the respiratory flow was measured as an index of respiratory function. Intraduodenal (i.d.) administration of 80 mg/kg "Kyushin" produced a cardiotonic effect and an excitatory effect on respiration, but i.d. administration of 16 mg/kg digoxin produced only a cardiotonic effect, and conversely inhibited respiration. In anesthetized open-chest guinea pigs, myocardial contractile force was measured as an index of cardiotonic effect and the arrhythmogenic effect was evaluated from the appearance of arrhythmic myocardial contraction. By i.d. administration of a 20% ethanol suspension or solution, "Kyushin" and digoxin showed a cardiotonic activity with doses higher than 40 mg/kg and 0.25 mg/kg, respectively. The arrhythmogenic doses of "Kyushin" and digoxin by i.d. administration were 2560 mg/kg and 2 mg/kg, respectively, suggesting that the safety margin of "Kyushin" is broader than that of digoxin.
Article
Effects of a Senso (toad venom)-containing drug KY on systemic hemodynamics were examined, and participation of beta-adrenoceptor in its action was evaluated by using propranolol in anesthetized dogs. KY produced a positive inotropic action, and decreased total peripheral (TPR) and coronary vascular resistances (CR), while renal vascular resistance (RR) was increased. After propranolol, KY significantly increased TPR, CR, vertebral vascular resistance and RR. KY-induced positive inotropic action was partly diminished but not abolished by beta-blockade. These results indicate that the beta-adrenergic action may be involved in the vasodilating effect of KY and partly in the positive inotropic action.
Article
Reports from several laboratories suggest the presence of an ouabainlike compound in plasma and various animal tissues, particularly during acute volume expansion and in low-renin hypertension. It has been hypothesized that this compound, through inhibition of the Na(+)-K+ pump, can constrict blood vessels, enhance vasoconstriction in response to agonists, increase cardiac contractility, raise blood pressure, and cause natriuresis/diuresis and therefore is implicated in the pathophysiology of the low-renin, volume-expanded type of hypertension. However, so far, only two steroid Na(+)-K+ pump inhibitors (namely, a bufodienolide derivative [resibufogenin], obtained from toad skin and plasma and a factor with the same carbon, oxygen, and hydrogen content as ouabain obtained from the plasma of volume-expanded humans) have been purified and structurally characterized. To determine whether such endogenous Na(+)-K+ pump inhibitors can in fact produce the above effects on the cardiovascular and renal systems, we infused commercially available bufalin (aglycone, identical to resibufogenin except for one H+), ouabain, and ouabagenin (aglycone) at equimolar doses in normotensive rats. Relative to ouabain, bufalin produced significantly greater dose-dependent increases in blood pressure, left ventricular rate of pressure change, heart rate, and excretion of urinary volume and sodium. Ouabagenin was without effect on any of these parameters. These data indicate that a Na(+)-K+ pump inhibitor can cause an increase in blood pressure despite potent diuretic and natriuretic effects and that, in rats, bufalin is much more potent in this respect than ouabain or ouabagenin.
Article
Bufalin was found to be a potent inducer of differentiation in human erythroleukemia K562 cells by examination of various differentiation markers (as assessed by the morphology, histochemistry, and the abilities to phagocytose latex particles, to reduce nitro-blue tetrazolium and to develop Fc receptors). Bufalin, at a concentration as low as 10 nM, also produced a strong differentiation-inducing activity in three other human leukemia-derived cell lines (human promyelocytic HL60, monoblastic U937 and myeloblastic ML1). Treatment of K562 cells with other cardiotonic steroids, such as cinobufagin, ouabain and digitoxigenin, at the concentration of 10 nM for four days resulted in weak or no effect on the cells. These findings suggest that bufalin might have potentiality as a new agent in the differentiation therapy for human myelogenous leukemia.
Article
Studies in Lichstein's laboratory suggest that the endogenous digitalislike substance implicated in low renin hypertension might be a steroidal dienolide derivative. If this is true, the bufadienolides should block potassium vasodilation and enhance norepinephrine vasoconstriction, constrict blood vessels, raise blood pressure, and produce natriuresis and diuresis. We have therefore examined these parameters while infusing bufalin (aglycone), a bufadienolide, intrabrachially and intravenously in the anesthetized dog. Intrabrachial infusion of 5-25 micrograms/min with brachial arterial blood flow held constant at 100 ml/min produced a dose-dependent increase in perfusion pressure with rapid onset and offset, a progressive decrease in the vasodilator response to intrabrachial injection of 1 ml iso-osmotic potassium chloride solution (but not to acetylcholine), and an increase in the vasoconstrictor response to intrabrachial injection of 0.1 microgram norepinephrine. Intravenous infusion at 5-50 micrograms/min produced a dose-dependent increase in systemic arterial blood pressure, rate of change of ventricular pressure (dP/dt), and after the highest dose, cardiac irregularities. Natriuresis and diuresis were not observed. Thus, bufalin does in fact have some of the physiological properties required to be considered a candidate for the digitalislike substance found in low renin hypertension.
Article
There is a narrow margin between therapeutic and toxic doses and serum levels of digitalis glycosides. Clinicians should therefore be knowledgeable and attentive to early signs and symptoms of toxicity in order to optimize the risk:benefit ratio when using this class of drugs. A number of pharmacokinetic interactions with specific implications for digitalis toxicity have recently been delineated. Serious cardiac rhythm disturbances should initially be treated with lidocaine or phenytoin. Digitalis toxicity resistant to conventional measures may now be treated with digoxin-specific Fab fragments.
Article
General pharmacological properties of two kinds of Senso-containing drugs were studied. There were no prominent differences in the pharmacological profile between the two prescriptions. Inhibition of writhing (60 mg/kg, p.o.), prolongation of hexobarbital-induced hypnosis, hypothermia, antipyretic effect, and inhibition of acetic acid-induced capillary permeability (600 mg/kg, p.o.) in mice were observed after administration of these drugs. These effects were suggested to originate from cinobufagin, a constituent of Senso. Augmentation of blood sugar level and inhibition of gastric juice secretion (600 mg/kg, p.o.) in rats were also observed after administration of these drugs. These effects were suggested to originate from constituents of Senso other than cinobufagin. Isolated ileum and aorta of guinea pigs and vas deferens and fundus strip preparation of rats contracted, and isolated trachea of guinea pigs relaxed after the application of these drugs. The majority of these effects were suggested to originate from epinephrine-like or serotonin-like compounds, constituents of Senso.
Article
Efforts to study the endogenous sodium pump inhibitor (ESPI) have been complicated by the limited specificity of available assays. We recently developed an assay of [Na,K]ATPase inhibition more sensitive than conventional assays. This enhancement reflects a prereaction step that increases binding affinity of digitalislike molecules to the digitalis receptor. We tested the possibility that this enhanced inhibition is limited to inhibitors acting specifically through the digitalis-binding site. Using both the standard and sensitive [Na,K]ATPase methods, known specific inhibitors of the sodium pump (ouabain, digoxin, bufalin) showed significant increases in the inhibition in the sensitive as compared with the standard [Na,K]ATPase assay (ouabain 34.4 +/- 7.3 vs. 8.3 +/- 0.5%, digoxin 43.2 +/- 9.1 vs. 7.2 +/- 3.1%, bufalin 46.9 +/- 5.0 vs. 22.6 +/- 1.6%). Some proposed candidates for the ESPI, acknowledged to be nonspecific inhibitors, showed no enhancement (oleic acid 36.0 +/- 4.5 vs. 34.8 +/- 5.6%, lysophosphatidyl choline 10.8 +/- 3.5 vs. 12.8 +/- 5.2%, and vanadate 34.3 +/- 8.8 vs. 33.8 +/- 1.4%). Other candidates, whose inhibitory specificity is unknown, including an ESPI from the peritoneal dialysate of patients with renal failure were also studied. The ESPI showed enhanced inhibition (24.1 +/- 4.9 vs. 5.3 +/- 2.0%). These studies suggest that the sensitive assay in conjunction with a standard [Na,K]ATPase assay may allow the early determination of candidates interacting specifically with the digitalis receptor to inhibit the sodium pump.
Article
A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chan'su, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.
Article
In order to evaluate the developmental toxicity and teratogenicity of a Chinese drug, the toad venom Chan Su, the drug was administered in a single intraperitoneal injection to pregnant ICR mice during the period of embryonic organogenesis. At the end of the gestation period, the mice were sacrificed, and the fetuses were collected, fixed and sectioned. A dose of Chan Su lower than 50 mg (dry weight)/kg body weight did not produce detectable changes in the recipient mice, but at a dose of 50 mg/kg the drug induced a decrease in body weight and structural abnormalities in livers and kidneys of the pregnant mice as well as increases in the number of resorbed and dead fetuses. There was a reduction in fetal weight although there were no changes in fetal crown-rump length nor tail length. No abnormalities were observed in the fetuses.
Article
The existence of a mammalian natriuretic substance similar to plant digitalis, which inhibits Na,K-ATPase, has been speculated about, but as yet no definite substance has been found. Digoxin-like immunoreactive substance (DLIS) has been reported in various clinical states including new born infants. Using bufalin (a cardioactive substance of animal origin) as antigen, four polyclonal antisera have been produced from 2 separate rabbits and characterised for cross-reactivity with 32 compounds. One antiserum showed a marked change in its cross-reactivity after resting the animal for a year. Of the endogenous substances tested, progesterone was found to be the most cross-reactive. Radioimmunoassay of foetal cord sera with different antisera, gave different levels of bufalin-like immunoactivity. However, after a novel “affinity-immunoassay” procedure, this apparent bufalin-like immunoactivity disappeared. It is concluded that bufalin-like immunoactivity in the cord blood is caused by the cross-reaction of endogenous steroids with bufalin antiserum, and the same may be true for DLIS.
Article
Bufalin, an active principle of the traditional Chinese medicine chan'su, has been proved to be a potent differentiation inducer in human leukemia cells. To study the mechanism of the differentiation of human leukemia ML1 cells induced by bufalin, we measured the effect of 10 nM bufalin on cell growth, activities of various protein kinases, and cell cycle. The ML1 cell growth was inhibited significantly at 24 hr and the inhibiting effect persisted for 6 days. Activities of PKC, PKA, cdc2 kinase and CK II in ML1 cells were changed early by bufalin; PKA and PKC activities were inhibited, and cdc2 kinase and CK II activities were increased. These results suggest that bufalin induces differentiation of ML1 cells by modulating several protein kinase activities in a distinct way from RA and 1 alpha, 25(OH) 2D3. Cell cycle changes, measured by flow cytometry, became evident at 12 hr after treatment of ML1 cells with bufalin and the cells were preferentially arrested in the G2/M phase. This effect of bufalin on the cell cycle of leukemia cells is similar to that of topoisomerase inhibitors. Indeed, the activity of topoisomerase II but not topoisomerase I of ML1 cells was inhibited remarkably by the treatment of the cells with 10 nM bufalin.
Article
Endogenous Na+,K(+)-ATPase inhibitors may have a role in the mechanism of low-renin hypertension. Two such compounds have been characterized: ouabain from human plasma and resibufogenin from toad plasma. Previously, we examined the acute effects of ouabain and bufalin (which has the same structure as resibufogenin except for one H+) in normal rats. Bufalin raised blood pressure, but ouabain had little effect. In contrast, given chronically, ouabain substantially increased blood pressure in normal rats and 70% reduced renal mass rats on a salt-free diet. We have now examined the chronic effects of bufalin in rats. Normal rats received 14.8 micrograms/kg per day bufalin or an equimolar dose of ouabain intraperitoneally for 6 weeks; 70% reduced renal mass rats also received 14.8 micrograms/kg per day bufalin. Another group of normal rats received 29.6 micrograms/kg per day bufalin intraperitoneally for 6 weeks. Respective control animals received vehicle. In contrast to ouabain, blood pressure did not increase in normal rats receiving the 14.8 micrograms dose of bufalin. However, normal rats receiving 29.6 micrograms bufalin and 70% reduced renal mass rats receiving 14.8 micrograms bufalin developed significant increases in blood pressure. Increases in blood pressure were associated with decreases in myocardial Na+,K(+)-ATPase activity and correlated with increased plasma Na+,K(+)-ATPase inhibitory activity. Thus, although bufalin is a more potent pressor agent than ouabain when both agents are given acutely, ouabain is at least as potent a vasopressor agent as bufalin when given chronically. Thus, both are pressor agents, more so in the presence of reduced renal mass, when given chronically in the rat.
Article
Speculations about development of tolerance to the inotropic effect of digitalis have been engendered since studies in various in vitro systems and tissues not representative of the heart have shown up-regulation of sodium potassium adenosine triphosphatase (Na,K-ATPase) when exposed to digitalis. Moreover the digitalis receptor (i.e., Na,K-ATPase) concentration in the normal, vital human left ventricle has not been previously determined. On this basis, digitalis receptor concentration was quantified in the left ventricle of explanted hearts from subjects without heart disease and from patients with end-stage heart failure who had received digitalis therapy. This was performed using vanadate-facilitated 3H-ouabain binding to intact tissue samples giving values of 728 +/- 58 (n = 5) and 467 +/- 55 pmol/g wet weight (n = 6) (mean +/- SEM) (p < 0.005), respectively. However, some of the digitalis receptors may have retained digoxin before 3H-ouabain binding and thus may have escaped detection. To eliminate this effect of retained digoxin, samples were exposed to prolonged washing in buffer containing excess digoxin antibody, a method recently shown to clear digoxin from receptors and allow subsequent complete digitalis receptor quantification by 3H-ouabain binding. After washing in digoxin specific antibody, specific digitalis receptor concentration was 760 +/- 58 pmol/g (n = 5) and 614 +/- 47 pmol/g (n = 6) wet weight in samples of the normal and failing hearts, respectively (p < 0.08). Thus, digitalization was associated with occupancy of digitalis receptors in the failing human heart of 24% (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)
Article
1. The presence of an endogenous digitalis-like factor (EDLF) in the plasma of both normal and volume expanded animals is well documented. In this study we have used ouabain and bufalin as pharmacological analogues to mimic the renal effects of EDLF and to investigate whether any interaction occurs between atrial natriuretic factor (ANF) and EDLF. 2. Conscious Na replete sheep with chronically indwelling catheters in the renal artery received renal arterial infusion of ouabain (1000 micrograms h-1) or bufalin (500 micrograms h-1) for 60 min. Renal arterial infusion of bufalin increased sodium excretion (UNaV) from 120 +/- 13 to 596 +/- 161 mumol min-1 after 45 min. Bufalin infusion did not alter glomerular filtration rate (GFR), effective renal plasma flow (ERPF), or lithium clearance. Ouabain infusion increased UNaV from 124 +/- 57 to 764 +/- 123 mumol min-1 in the first hour after infusion. 3. ANF infusion increased UNaV from 159 +/- 34 to 583 +/- 134 mumol min-1. When renal arterial bufalin infusion was followed by renal arterial ANF infusion (50 micrograms h-1) UNaV was increased from 155 +/- 31 to 795 +/- 96 mumol min-1. This increase in UNaV is approximately equal to the sum of the separate effects of bufalin and ANF. 4. The natriuretic effects of ouabain at pharmacological doses in sheep are confirmed by this study. The data presented here do not support the hypothesis that EDLF sensitizes the kidney to the natriuretic effects of ANF.
Article
Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.
Article
A Na+,K(+)-ATPase inhibitor, bufalin, has been shown previously to induce leukemia cell differentiation. The presence of a circulating Na+,K(+)-ATPase inhibitor has been proposed in mammals. The aim of this study was to explore an endogenous bufalin-like factor that induces leukemia cell differentiation. We found a fraction, designated as fraction A, obtained from human plasma extract that inhibits the growth of several human-derived leukemia cell lines. The effect of the fraction was retained after protease digestion or heat treatment. Murine leukemia cells and ouabain-resistant cells, which are insensitive to bufalin, appeared to be refractory to fraction A in terms of growth inhibition. Fraction A also induced functional and morphological maturation in THP-1 cells. Fraction A was recognized by anti-bufalin anti-serum and inhibited 3H-bufalin binding to K562 cells. These findings suggest that fraction A shows a similar behavior to that of bufalin on leukemia cells by inhibiting Na+,K(+)-ATPase. We propose that an endogenous Na+,K(+)-ATPase inhibitor in human plasma may play a role in cell differentiation.
Article
Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.
Article
Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1 beta (IL-1 beta). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto- oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K(+)-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.