Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest.

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.

Vasoconstrictor and hemodynamic effects of a methanolic extract from Rhinella marina toad poison.

Rhinella marina toad is abundant in Brazil. Its poison contains cardiac glycosides called bufadienolides, which are extensively investigated for their bioactivity. Our aim was to characterize the vasoactivity of Rhinella marina poison (RmP) on the aorta of male Wistar rats. For this, the RmP was first collected and processed to obtain an alcoholic extract. To determine cardiovascular effects of RmP, we performed in vivo tests by administering RmP intravenously in doses of 0.1-0.8 mg/kg. Vascular reactivity was also performed through concentration-response curves to RmP (10 ng/mL to 200 µg/mL) in aortic segments with and without endothelium. RmP induced a concentration-dependent contraction in rat aorta which was partly endothelium-mediated. Nitric oxide contributes with this response in view that incubation with L-NAME increased the contractile response. Additionally, treatment with indomethacin [cyclooxygenase, (COX) inhibitor], nifedipine (L-type voltage-gated calcium channels blocker), and BQ-123 (ETA receptors antagonist) decreased maximum response, and ketanserin (5-HT2 receptors antagonist) decreased pEC50, suggesting active participation of these pathways in the contractile response. On the other hand, apocynin (NADPH oxidase inhibitor) did not alter contractility. Incubation with prazosin (α1-adrenergic receptor antagonist) abolished the contractile response, suggesting that the RmP-induced contraction is dependent on the adrenergic pathway. In the Na+/K+ ATPase protocol, a higher Emax was observed in the RmP experimental group, suggesting that RmP potentiated Na+/K+ATPase hyperpolarizing response. When this extract was injected (i.v.) in vivo, increase in blood pressure and decrease in heart rate were observed. The results were immediate and transitory, and occurred in a dose-dependent manner. Overall, these data suggest that the poison extract of R. marina toad has an important vasoconstrictor action and subsequent vasopressor effects, and its use can be investigated to some cardiovascular disorders.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14ß-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(ß→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Life-threatening pediatric poisoning due to ingestion of Bufo bufo toad eggs: A case report.

Bufo parotid glands and eggs contain cardiac glycosides also known as bufadienolides. This class of molecules can cause digoxin-like cardiac toxicity, as they can block the sodium potassium-adenosine triphosphatase (Na/K-ATPase) pump. Poisoning with these toxins is rare but carries a high mortality risk. There are only a few cases of toad poisoning that have been reported worldwide, mainly in the southern hemisphere. We will describe the case of a child on the autistic spectrum disorder who developed an acute and severe cardiac bradyarrhythmia soon after being in a mountain creek. The child ingested a large quantity of Bufo bufo toad eggs and developed bradycardia (35/min) associated with junctional rhythm with narrow QRS complexes. The poison control center (PCC) indicated the use of atropine on the way to the nearest hospital and the administration of antidotal therapy, i.e., anti-digoxine fragment antibodies (DigiFab), as soon as possible. The patient was transferred by air ambulance to the Regional Referral Pediatric Hospital (RRPH), tested for digoxin blood level by immuno-essay (0.68 ng/mL) and successfully treated with five vials of DigiFab, since atropine administration produced only a fleeting effect on the cardiac rhythm. Patient was discharged 48 hours after poisoning. The presence of bufadienolides in the toad eggs was also confirmed. To our knowledge, this is the first report of toad egg poisoning in Europe. The administration of Digifab helped to reverse the bufadienolide cardiac toxicity.

Surfactant Assisted Rapid-Release Liposomal Strategies Enhance the Antitumor Efficiency of Bufalin Derivative and Reduce Cardiotoxicity.

BACKGROUND: BF211, a derivative of bufalin (BF), shows significantly improved solubility and potent antitumor efficiency compared to BF. Unfortunately, the unwanted toxicity such as cardiotoxicity caused by unspecific distribution has hindered its clinical use. METHODS: PEGylated BF211 liposomes (BF211@Lipo) were designed and optimizely prepared based on the pre-prescription research. In vitro and in vivo cardiotoxicity was evaluated. In vivo pharmacokinetics and biodistribution of BF211@Lipo were investigated. In vivo antitumor activity and toxicity were evaluated in HepG2 cell xenograft models. The rapid-release triggered by Poloxamer 188 (P188) was assessed in vitro and in vivo. RESULTS: The optimized BF211@Lipo displayed a spherical morphology with a size of (164.6 ± 10.3) nm and a high encapsulation efficiency of (93.24 ± 2.15) %. The in vivo concentration-time curves of BF211 loaded in liposomes showed a prolonged half-life in plasma and increased tumor accumulation. No obvious abnormality in electrocardiograms was observed in guinea pigs even at 9 mg/kg. Moreover, to improve the efficient release of BF211@Lipo, a surfactant-assisted rapid-release strategy was developed, and the release-promoting mechanism was revealed by the fluorescence resonance energy transfer (FRET) and fluorescence nanoparticle tracking analysis (fl-NTA) technology. Sequential injection of BF211@Lipo and P188 could ignite the "cold" liposomes locally in tumor regions, facilitating the burst release of BF211 and enhancing the therapeutic index. CONCLUSION: Our progressive efforts that begin with preparation technology and dosage regimen enable BF211 to like a drug, providing a promising nano platform to deliver the cardiac glycosides and alleviate the side effects by decreasing unspecific biodistribution.

Variation in size and shape of toxin glands among cane toads from native-range and invasive populations.

If optimal investment in anti-predator defences depends on predation risk, invading new regions (and thus, encountering different predators) may favour shifts in that investment. Cane toads offer an ideal system to test this prediction: expensive anti-predator toxins are stored mainly in parotoid glands whose dimensions are easy to measure, and toad invasions have changed the suites of predators they encounter. Although plasticity may influence parotoid morphology, comparisons between parents and progeny revealed that gland dimensions were highly heritable. That heritability supports the plausibility of an evolved basis to variation in gland dimensions. Measurements of 3779 adult toads show that females have larger glands than males, invasive populations have larger glands than in the native-range, and that parotoid sexual size dimorphism varies strongly among invaded areas. Geographic variation in parotoid morphology may be driven by predation risk to both adult toads and offspring (provisioned with toxins by their mother), with toxins allocated to eggs exacerbating the risk of cannibalism but reducing the risk of interspecific predation. Investment into chemical defences has evolved rapidly during the cane toad's international diaspora, consistent with the hypothesis that organisms flexibly adjust resource allocation to anti-predator tactics in response to novel challenges.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Endogenous Bufadienolide, Blood Pressure and Alcohol Withdrawal.

BACKGROUND AND OBJECTIVE: Previously, it was demonstrated that marinobufagenin (MBG) is implicated in the development of ethanol withdrawal in rats. It has been shown that ethanol withdrawal is associated with a pressor response in the alcoholics. We hypothesized that elevated levels of sodium pump ligand, MBG, would underline the increase in systolic blood pressure during alcohol withdrawal in humans. METHODS: The cohort included 9 patients with the diagnosis "alcohol dependence syndrome" (F10.(1-3) according to ICD-10). The blood samples for measurement of MBG concentration were collected from the subjects on the first day of withdrawal and after 7 days treatment of the abstinence. Arterial blood pressure was measured via plethysmography at the same time points. RESULTS: The beginning of the alcoholic abstinence was associated with the rise of arterial blood pressure with enhanced levels of plasma MBG. At day 7 following withdrawal, the systolic blood pressure and MBG levels were decreased to normal values. CONCLUSION: The development of alcohol withdrawal is accompanied by an increase in arterial blood pressure, which is associated with increased plasma MBG concentration.

Bufalin-induced cardiotoxicity: new findings into mechanisms.

Bufalin is one of the main pharmacological and toxicological components of Venenum Bufonis and many traditional Chinese medicine preparations. The cardiotoxicity clearly limits its application to patients living in countries. Hence, an investigation of its toxicological mechanism is helpful for new drug development and treatment of the related clinical adverse reactions. We investigate the cardiotoxicity of bufalin using human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.003-0.1 µmol·L-1), human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.03-0.3 µmol·L-1) and eight human cardiac ion channel currents (0.01-100 µmol·L-1) combined with an impedance-based bioanalytical and patch clamp method. Biphasic effect of bufalin on the contractility in hiPSC-CMs, which has been shown to strengthen myocardial contractility, accelerate conduction, and increase beating rate at the earlier stage of administration, whereas weakened myocardial contractility, abolished conduction, and ceased beating rate at the later stage of administration. Bufalin decreased the action potential duration (Action potential duration at 30%, 50% and 90% repolarization), cardiac action potential amplitude, and maximal depolarization rate and depolarized the resting membrane potential of hiPSC-CMs. Spontaneous beating rates of hiPSC-CMs were markedly increased at 0.03 µmol·L-1, while were weakened at 0.3 µmol·L-1 after application. Bufalin blocks INav1.5 in a concentration-dependent manner with half maximal inhibitory concentration of 74.5 µmol·L-1. Bufalin respectively increased the late sodium current and Na+-Ca2+ exchange current with a concentration for 50% of maximal effect of 2.48 and 66.06 µmol·L-1 in hiPSC-CMs. Whereas, bufalin showed no significant effects on other cardiac ion channel currents. The enhancement of the late sodium current is one of the main mechanism for cardiotoxicity of bufalin.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

Metabolites from Bufo gargarizans (Cantor, 1842): A review of traditional uses, pharmacological activity, toxicity and quality control.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufo gargarizans (Cantor, 1842) (BGC), a traditional medicinal animal distributed in many provinces of China, is well known for the pharmaceutical value of Chansu and Chanpi. As traditional Chinese medicines (TCMs), Chansu and Chanpi, with their broad-spectrum of therapeutic applications, have long been applied to detoxification, anti-inflammation, analgesia, etc. OVERARCHING OBJECTIVE: We critically analyzed the current evidence for the traditional uses, chemical profiles, pharmacological activity, toxicity and quality control of BGC (Bufonidae family) to provide a scientific basis for future in-depth studies and perspectives for the discovery of potential drug candidates. METHODOLOGY: All of the available information on active constituents and TCMs derived from BGC was obtained using the keywords "Bufo gargarizans", "Chansu", "Chanpi", "Huachansu", or "Cinobufacini" through different electronic databases, including PubMed, Web of Science, Chinese National Knowledge Infrastructure (CNKI), the Wanfang Database, and Pharmacopoeia of China. In addition, Chinese medicine books from different times were used to elucidate the traditional uses of BGC. Electronic databases, including the "IUCN Red List of Threatened Species", "American Museum of Natural History" and "AmphibiaWeb Species Lists", were used to validate the scientific name of BGC. RESULTS: To date, about 118 bufadienolide monomers and 11 indole alkaloids have been identified from BGC in total. The extracts and isolated compounds exhibit a wide range of in vitro and in vivo pharmacological effects. The literature search demonstrated that the ethnomedicinal uses of BGC, such as detoxification, anti-inflammation and the ability to reduce swelling and pain associated with infections, are correlated with its modern pharmacological activities, including antitumor, immunomodulation and attenuation of cancer-derived pain. Bufadienolides and indole alkaloids have been regarded as the main active substances in BGC, among which bufadienolides have significant antitumor activity. Furthermore, the cardiotoxicity of bufadienolides was discussed, and the main molecular mechanism involves in the inhibition of Na+/K+-ATPase. Besides, with the development of modern analytical techniques, the quality control methods of BGC-derived TCMs are being improved constantly. CONCLUSIONS: An increasing number of reports suggest that BGC can be regarded as an excellent source for exploring the potential antitumor constituents. However, the future antitumor research of BGC needs to follow the standard pharmacology guidelines, so as to provide comprehensive pharmacological information and aid the reproducibility of the data. Besides, to ensure the efficacy and safety of BGC-derived TCMs, it is vital to construct a comprehensive quality evaluation model on the basis of clarifying pharmacodynamic-related and toxicity-related compositions.

Analgesic and toxic effects of venenum bufonis and its constituent compound cinobufagin: A comparative study.

OBJECTIVE: In this study, we aimed to compare the analgesic and toxic effects of Venenum Bufonis (VB) with those of cinobufagin (CBG), a monomer isolated from VB, to provide the experimental basis for further research and development of VB. METHODS: After intragastric administration, the analgesic activities of VB and CBG were compared using the hot plate test and acetic acid-induced writhing test. Their side effects were also compared using hepatotoxicity, acute toxicity, cytotoxicity, and hemolytic toxicity tests, as well as by evaluating their effects on rat heart rate. RESULTS: In the hot plate test, both drugs prolonged paw withdrawal latency; however, CBG-treated mice exhibited significantly longer latency than VB-treated mice. In the acetic acid-induced writhing test, both drugs inhibited mouse writhing; however, the inhibitory effects of CBG were stronger. In addition, VB significantly increased serum aspartate aminotransferase (AST) levels, whereas CBG did not these levels. The LD50 of VB and CBG was 36.25 and 4.78 mg/kg, respectively. Both drugs increased the heart rate with CBG exhibiting stronger effects. Moreover, results showed that the cytotoxicity of CBG was more dose-dependent than that of VB. Both VB and CBG showed low hemolytic toxicity. CONCLUSIONS: Both VB and CBG exhibited analgesic effects and low hemolytic toxicity; however, the latter showed stronger analgesic activity and less hepatotoxicity. Additionally, both VB and CBG increased the heart rate; however, CBG had stronger effects and higher acute toxicity.

In Vitro Cytotoxicity Induced by the Bufadienolides 1α,2α-Epoxyscillirosidine and Lanceotoxin B on Rat Myocardial and Mouse Neuroblastoma Cell Lines.

Consumption of bufadienolide-containing plants are responsible for many livestock mortalities annually. Bufadienolides are divided into two groups; non-cumulative bufadienolides and cumulative bufadienolides. Cumulative bufadienolides are referred to as neurotoxic, as the chronic intoxication with this type of bufadienolide results in a paretic/paralytic syndrome known as 'krimpsiekte'. The in vitro cytotoxicity of a non-cumulative bufadienolide, 1α,2α-epoxyscillirosidine, and a cumulative bufadienolide, lanceotoxin B, were compared using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction) assay after exposing rat myocardial (H9c2) and mouse neuroblastoma (Neuro-2a) cell lines. The effect of these two bufadienolides on cell ultrastructure was also investigated using transmission electron microscopy (TEM). H9c2 cells exhibited greater cytotoxicity when exposed to 1α,2α-epoxyscillirosidine, compared to lanceotoxin B. In contrast, Neuro-2a cells were more susceptible to lanceotoxin B. The EC50 (half maximal effective concentration) of lanceotoxin B exposure of Neuro-2a cells for 24⁻72 h ranged from 4.4⁻5.5 µM compared to EC50s of 35.7⁻37.6 µM for 1α,2α-epoxyscillirosidine exposure of Neuro-2a cells over the same period. 1α,2α-Epoxyscillirosidine induced extensive vacuolization in both cell types, with swollen RER (rough endoplasmic reticulum) and perinuclear spaces. Lanceotoxin B caused swelling of the mitochondria and sequestration of cytoplasmic material within autophagic vesicles. These results corroborate the notion that cumulative bufadienolides are neurotoxic.

Hybrids of arenobufagin and benzoisoselenazol reducing the cardiotoxicity of arenobufagin.

Arenobufagin is a naturally occurring bufadienolide showing promising antitumor activity accompanied however with apparent cardiac toxicity. Following the recent discovery that oxidative damage possibly be an important cause of the cardiac toxicity of cardenolides, a strategy fusing the antitumor agent arenobufagin with a benzoisoselenazol fragment, a reactive oxygen species (ROS) scavenger, has been developed. Six novel hybrids were synthesized and their ROS scavenging activities as well as their in vitro cytotoxicity against the human hepatocellular carcinoma cell line HepG2, an adriamycin-resistant subline HepG2/ADM, and the human myocardial cell line AC16 were evaluated. The results indicate that the hybrids exhibit various degrees of in vitro ROS scavenging activities, and weaker cytotoxicity than that of arenobufagin against the myocardial cell line AC16. These findings suggest the feasibility of a strategy in which the cardiotoxicity of the potential antitumor agent arenobufagin is reduced.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Cinobufagin Induces Apoptosis in Osteosarcoma Cells Via the Mitochondria-Mediated Apoptotic Pathway.

BACKGROUND/AIMS: Osteosarcoma is a common primary malignant bone tumor that mainly occurs in childhood and adolescence. Despite developments in the diagnosis and treatment of osteosarcoma, the prognosis is still very poor. Cinobufagin is an active component in the anti-tumor Chinese medicine called "Chan Su", and we previously revealed that cinobufagin induced apoptosis and reduced the viability of osteosarcoma cells; however, the underlying mechanism remains to be elucidated. Herein, the present study was undertaken to illuminate the molecular mechanism of cinobufagin-induced apoptosis of osteosarcoma cell. METHODS: U2OS and 143B cells were treated with different concentrations of cinobufagin. Cell viability, colony formation ability and morphological changes were assessed by a CCK-8 assay, a clonogenic assay and light microscopy, respectively. Cell apoptosis was detected by Hoechst 33258 and Annexin V-FITC/PI staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. Glutathione (GSH) levels were detected by a GSH and GSSG assay kit. The levels of apoptosis-related proteins were determined by western blotting, and 143B cells were introduced to establish a xenograft tumor model. The effect of cinobufagin on osteosarcoma was further investigated in vivo. RESULTS: Our results showed that cinobufagin significantly reduced the viability of U2OS and 143B cells in vitro in a dose-and time-dependent manner. In addition, cinobufagin-induced apoptosis in U2OS and 143B cells was concentration-dependent. Moreover, we found that cinobufagin treatment increased the level of intracellular ROS, decreased ΔΨm, reduced GSH and inhibited GSH reductase (GR). The effects of cinobufagin on cell proliferation, apoptosis, ROS generation and ΔΨm loss were dramatically reversed when the cells were pretreated with the thiol-antioxidants NAC or GSH. Moreover, cinobufagin treatment increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptitic protein Bcl-2, thus altering the ratio of Bax to Bcl-2. Furthermore, Cinobufagin treatment caused cytochrome c release from the mitochondria to cytoplasm, thus increasing the protein levels of cleaved-caspase family members to induce apoptosis. Ac-DEVD-CHO or Z-LEHD-FMK significantly reduced cinobufagin-induced apoptosis. Finally, a subcutaneous xenograft animal study verified that cinobufagin also significantly suppressed osteosarcoma growth in vivo. CONCLUSIONS: Our present data demonstrated that cinobufagin triggered cell apoptosis in osteosarcoma cells via the intrinsic mitochondria-dependent apoptosis pathway by the accumulation of ROS and the loss of ΔΨm. In an in vivo subcutaneous xenograft model, cinobufagin exhibited excellent tumor inhibitory effects. These results suggest that cinobufagin might potentially be further developed as an anti-tumor candidate for treating osteosarcoma patients in the clinic.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Bufalin Inhibits Cellular Proliferation and Cancer Stem Cell-Like Phenotypes via Upregulation of MiR-203 in Glioma.

BACKGROUND/AIMS: Prior studies have shown that bufalin inhibits cellular proliferation and induces apoptosis in various human cancers. MicroRNA-203 (miR-203) has been shown to function as an important regulator of tumor progression at various stages. In this study, we investigated the effect of miR-203 expression and bufalin treatment on glioma cell proliferation and stem cell-like phenotypes. METHODS: We used cell viability assay, colony formation assay, cell apoptosis assay and neurosphere formation assay to dectect the treatment effect of bufalin on U251 and U87 cells. Cells were transfected with the miR-203 mimic without bufalin treatment or cells were transfected with anti-miR-203 under bufalin treatment, the above expreiments were repeated. RT-PCR was employed to quantify miR-203 expression. Western blot was performed to detect the stem cell-like (CSC) markers, OCT4 and SOX2. Luciferase activity assay was used to determine whether the SPARC is the target of miR-203. RESULTS: Bufalin treatment inhibited cell proliferation, colony formation, and CSC phenotypes and increased cell apoptosis and expression of miR-203. Furthermore, overexpression of miR-203 led to similar outcomes as bufalin treatment with respect to the cell viability, colony formation, cell apoptosis and the phenotypes of glioma cells. While anti-miR-203 attenuated the inhibitory effects of bufalin as promoting cell proliferation, colony formation and CSC phenotyes and inhibiting cell apoptosis. In addition, we identified SPARC as a novel target gene of miR-203. CONCLUSIONS: These findings suggest that miR-203 plays an important role in bufalin's ability to inhibit the growth of glioma cells and the development of stem cell-like phenotypes.