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1.
J. venom. anim. toxins incl. trop. dis ; 27: e20200073, 2021. tab, graf, ilus
Article in English | ID: biblio-1154769

ABSTRACT

he resistance against antimalarial drugs represents a global challenge in the fight and control of malaria. The Brazilian biodiversity can be an important tool for research and development of new medicinal products. In this context, toxinology is a multidisciplinary approach on the development of new drugs, including the isolation, purification, and evaluation of the pharmacological activities of natural toxins. The present study aimed to evaluate the cytotoxicity, as well as the antimalarial activity in silico and in vitro of four compounds isolated from Rhinella marina venom as potential oral drug prototypes. Methods: Four compounds were challenged against 35 target proteins from P. falciparum and screened to evaluate their physicochemical properties using docking assay in Brazilian Malaria Molecular Targets (BraMMT) software and in silico assay in OCTOPUS® software. The in vitro antimalarial activity of the compounds against the 3D7 Plasmodium falciparum clones were assessed using the SYBR Green I based assay (IC50). For the cytotoxic tests, the LD50 was determined in human pulmonary fibroblast cell line using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Results: All compounds presented a ligand-receptor interaction with ten Plasmodium falciparum-related protein targets, as well as antimalarial activity against chloroquine resistant strain (IC50 = 3.44 µM to 19.11 µM). Three of them (dehydrobufotenine, marinobufagin, and bufalin) showed adequate conditions for oral drug prototypes, with satisfactory prediction of absorption, permeability, and absence of toxicity. In the cell viability assay, only dehydrobufotenin was selective for the parasite. Conclusions: Dehydrobufotenin revealed to be a potential oral drug prototype presenting adequate antimalarial activity and absence of cytotoxicity, therefore should be subjected to further studies.(AU)


Subject(s)
Bufanolides/administration & dosage , Bufonidae , Biodiversity , Malaria/immunology , Antimalarials , In Vitro Techniques , Computer Simulation
2.
Article in Chinese | WPRIM | ID: wpr-828363

ABSTRACT

In order to observe the anti-tumor effect of cinobufotalin on H22 liver cancer mice and to explore its regulatory mechanism, 50 Kunming mice were subcutaneously inoculated with H22 intraperitoneal passage cells under the armpit to establish H22 hepatocellular carcinoma model. They were then randomly divided into model group, cinobufotalin low dose group, cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, which received 0.01% ethanol solution, 1 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cisplatin, 5 mg·kg~(-1)cisplatin + 5 mg·kg~(-1) cinobufotalin respectively for 10 days. The general condition of mice during the intervention was observed, and the inhibition rate, tumor mass, thymus index, histopathological changes of the tumors, apoptotic rate of the tumors, the expressions of phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), apoptosis related gene(Fas), Fas ligand(FasL) mRNA and protein phosphorylated Akt(pAkt) protein in the tumors of each group were compared. The results showed that during the modeling period, the mice showed a decline in food intake, dark fur, poor mental status, and gradually worsened over time. The mental status of mice in each intervention group was improved gradually, especially in the cisplatin+cinobufotalin group. As compared with the model group, the tumor mass of each intervention group was lower(P<0.05). As compared with the cinobufotalin low dose group, the tumor mass was lower and inhibition rate was higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, the tumor mass was lower and the inhibition rate was higher in cisplatin+cinobufotalin group(P<0.05). As compared with the model group, the thymus index was higher in cinobufotalin high dose group and cisplatin + cinobufotalin group, while was lower in cisplatin group(P<0.05). As compared with the cinobufotalin low dose group, the thymus index was higher in the cinobufotalin high dose group and lower in the cisplatin group(P<0.05). As compared with the cinobufotalin high dose group, the thymus index was lower in cisplatin group(P<0.05). As compared with cisplatin group, the thymus index was higher in cisplatin+cinobufotalin group(P<0.05). Pathological staining showed that a large number of heterogeneous cells and mitotic phenomena were observed in the model group. Cell fragments and neutrophils were observed in the tumor tissues of the intervention groups, showing diffuse necrosis, and the diffuse necrosis was more obvious in the cisplatin+cinobufotalin group. As compared with the model group, the apoptotic rate of the tumors and the relative expressions of Fas mRNA and protein were higher in the intervention groups, while the relative expressions of PI3 K, FasL mRNA and protein and the relative expression of pAkt protein were lower in the intervention groups(P<0.05). As compared with the cinobufotalin low dose group, the apoptotic rate of the tumors and relative expression of Fas and protein were higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, apoptotic rate of the tumors and the relative expression of Fas mRNA and protein were higher in the cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower in the cisplatin+cinobufotalin group(P<0.05). In summary, cinobufotalin has significant anti-tumor effect on H22 liver cancer mice, and can enhance the immune function of mice and synergistically enhance the effect of chemotherapy. Its mechanism may be associated with regulating PI3 K/Akt/Fas/FasL signaling pathway related genes and protein expression.


Subject(s)
Animals , Apoptosis , Bufanolides , Carcinoma, Hepatocellular , Cisplatin , Fas Ligand Protein , Liver Neoplasms , Mice
3.
Article in Chinese | WPRIM | ID: wpr-773481

ABSTRACT

OBJECTIVE@#To observe the effect of cinobufagin on transient outward potassium current () in rat dorsal root ganglion cells of cancer-induced bone pain (CIBP) and explore the possible analgesic mechanism of cinobufagin.@*METHODS@#Whole cell patch clamp technique was used to examine the effect of cionbufagin on in acutely isolated dorsal root ganglion (DRG) cells from normal SD rats and rats with bone cancer pain.@*RESULTS@#The DRG cells from rats with CIBP showed obviously decreased current density, an activation curve shift to the right, and an inactivation curve shift to the left. Cinobufagin treatment significantly increased the current density and reversed the changes in the activation and inactivation curves in the DRG cells.@*CONCLUSIONS@# current is decreased in DRG neurons from rats with CIBP. Cinobufagin can regulate the activation and inactivation of current in the DRG cells, which may be related to its analgesic mechanism.


Subject(s)
Analgesics , Pharmacology , Animals , Bufanolides , Pharmacology , Cancer Pain , Drug Therapy , Cells, Cultured , Ganglia, Spinal , Patch-Clamp Techniques , Potassium Channels , Metabolism , Rats , Rats, Sprague-Dawley
4.
Article in Chinese | WPRIM | ID: wpr-773157

ABSTRACT

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.


Subject(s)
Amphibian Venoms , Chemistry , Animals , Bufanolides , Bufo bufo , Chromatography, High Pressure Liquid , Medicine, Chinese Traditional , Quality Control
5.
Article in English | WPRIM | ID: wpr-327194

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>


Subject(s)
Apoptosis , Genetics , Bufanolides , Pharmacology , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cell Shape , DNA (Cytosine-5-)-Methyltransferases , Metabolism , DNA Methylation , Genetics , Gene Expression Regulation, Leukemic , Humans , Leukemia, Erythroblastic, Acute , Genetics , Pathology , RNA, Messenger , Genetics , Metabolism , Up-Regulation , Genetics , WT1 Proteins , Genetics , Metabolism
6.
Article in Chinese | WPRIM | ID: wpr-286873

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.</p><p><b>METHODS</b>MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.</p><p><b>RESULTS</b>Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.</p><p><b>CONCLUSION</b>Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.</p>


Subject(s)
Apoptosis , Bufanolides , Pharmacology , Caspase 9 , Metabolism , Cell Survival , Colorectal Neoplasms , Pathology , Humans , MAP Kinase Kinase Kinases , Metabolism , NF-KappaB Inhibitor alpha , Metabolism , Oxidative Stress , Poly (ADP-Ribose) Polymerase-1 , Metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53 , Metabolism , bcl-2-Associated X Protein , Metabolism , bcl-Associated Death Protein , Metabolism
7.
Chinese Journal of Oncology ; (12): 490-496, 2015.
Article in Chinese | WPRIM | ID: wpr-286793

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism.</p><p><b>METHODS</b>The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot.</p><p><b>RESULTS</b>The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly.</p><p><b>CONCLUSIONS</b>Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.</p>


Subject(s)
Antineoplastic Agents , Pharmacology , Antineoplastic Combined Chemotherapy Protocols , Pharmacology , Bufanolides , Pharmacology , Cadherins , Metabolism , Cell Line, Tumor , Cell Proliferation , Coloring Agents , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Hepatocyte Growth Factor , Pharmacology , Humans , Lung Neoplasms , Drug Therapy , Metabolism , Pathology , MAP Kinase Signaling System , Neoplasm Proteins , Metabolism , Phosphatidylinositol 3-Kinases , Quinazolines , Pharmacology , ErbB Receptors , Signal Transduction , Tetrazolium Salts , Thiazoles
8.
Acta Pharmaceutica Sinica ; (12): 244-248, 2014.
Article in Chinese | WPRIM | ID: wpr-297986

ABSTRACT

Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research.


Subject(s)
Amphibian Venoms , Chemistry , Animals , Bufanolides , Chemistry , Bufo bufo , Chromatography, High Pressure Liquid , Molecular Structure , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
9.
Article in Chinese | WPRIM | ID: wpr-299863

ABSTRACT

<p><b>OBJECTIVE</b>To study the apoptosis inducing effects of bufalin on various human osteosarcoma cells and the concerning molecular mechanisms.</p><p><b>METHOD</b>MTT assay was used to detect the growth inhibition rates of osteosarcoma cells U-20S, U-20S/MTX300, SaOS-2, IOR/OS9 treated with bufalin in different concentrations and times. The apoptosis of cells was observed flow cytometry 48 h following bufalin treatment. The proteomic techniques were used to separate and compare the treated and control groups 48 h after bufalin-incubation. Then, the proteomic results were validated by western blot.</p><p><b>RESULT</b>Bufalin inhibited the growth of human osteosarcoma cells U20S, U20S/MTX300 (methotrexate resistant cells), SAOS2, IOR/OS9 in a dose- and time-dependent manner. The 72 h IC50 were (37.43 +/- 4.1), (32.24 +/- 5.3) nmol x L(-1) in U20S,U20S/MTX300 cells,respectivly. Flow cytometry showed that the apoptosis cells were increased following bufalin treatment. The protein expression profile showed 24 differentiated expression proteins. Among these proteins, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27) decreased significantly and the result was then validated by western blot. Ectopic expression of Hsp27 could reduce the bufalin-induced apoptosis remarkably in U20S and U20S/MTX300 cells.</p><p><b>CONCLUSION</b>Bufalin could inhibit the cell growth and induce apoptosis on human osteosarcoma cells. The effect of bufalin may be related to the joint intervention with multiple protein targets. Among them, downregulation of Hsp27 plays a critical role in the bufalin-induced apoptosis in human osteosarcoma cells.</p>


Subject(s)
Antineoplastic Agents , Pharmacology , Apoptosis , Bufanolides , Pharmacology , Cell Line, Tumor , Cell Proliferation , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic , Humans , Osteosarcoma , Pathology , Proteomics
10.
Acta Pharmaceutica Sinica ; (12): 1446-1450, 2014.
Article in Chinese | WPRIM | ID: wpr-299113

ABSTRACT

To identify the active components in Bufo melanostictus Schneider and clarify the difference between fresh and dried Venenum Bufonis, a UPLC-Orbitrap MS method has been established. The separation was performed with gradient elution of acetonitrile and water (with 0.1% formic acid) as mobile phase. By comparing their retention time and high resolution mass data of Venenum Bufonis extracts, 39 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were bufadienolides. There were significant differences in the main composition between fresh and dried Venenum Bufonis. There are fewer bufadienolides in fresh toad venom.


Subject(s)
Amphibian Venoms , Chemistry , Animals , Bufanolides , Chemistry , Bufonidae , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry
11.
Acta Pharmaceutica Sinica ; (12): 1574-1577, 2014.
Article in Chinese | WPRIM | ID: wpr-299095

ABSTRACT

Cinobufacino injection is purified from water extraction of the skin of Bufo bufo gargarizans, which has been widely used for various cancers in clinic with significant anti-tumor effects. Bufadienolides were regarded as the main active constituents of cinobufacino injection in previous reports. In present study, 6 bufadienolides were isolated and purified from Cinobufacino injection. Their structures were identified as 3-epi-ψ-bufarenogin (1), ψ-bufarenogin (2), 3-epi-arenobufagin (3), arenobufagin (4), 3-epi-gamabufotalin (5), and 3-oxo-arenobufagin (6), separately. Among them, 1 and 3 were new compounds, 5 and 6 were new natural products. Compounds 1, 2 and compounds 3, 4 were two pairs configuration isomers at C-3, separately.


Subject(s)
Animals , Bufanolides , Chemistry , Bufo bufo , Injections , Skin , Chemistry
12.
Article in Chinese | WPRIM | ID: wpr-330350

ABSTRACT

Twelve compounds were isolated from the venom of Bufo bufo gargarizans. On the basis of their physical and chemical properties and spectral data, their structures were identified as resibufagenin (1), bufotalin (2), desacetylcinobufagin (3), 19-oxodesacetylcinobufotalin (4), cinobufotalin (5), 1beta-hydroxylbufalin (6), 12alpha-hydroxybufalin (7), bufotalinin (8), Hellebrigenin (9), telocinobufagin (10), hellebrigenol (11) and cinobufagin-3-hemisuberate methyl ester (12), respectively. Compounds 7 and 12 are new natural products.


Subject(s)
Animals , Bufanolides , Chemistry , Bufo bufo , Medicine, Chinese Traditional , Molecular Structure , Venoms , Chemistry
13.
Article in Chinese | WPRIM | ID: wpr-327883

ABSTRACT

Bufalin is an active compound of the traditional Chinese medicine Chansu, which exhibits significant anti-tumor activities in many solid tumors and leukemia cell lines. Bufalin could introduce apoptosis, reverse drug-resistance, and prevent migration and invasion of tumor cells. This paper reviewed the latest research progress of the in vitro and in vivo anti-tumor effect and mechanism of bufalin on a series of cancers, such as hepatocellular carcinoma, lung cancer, colon cancer, gastric cancer, leukemia, bladder cancer, and its formulation study is also summarized for the reference of its further study and application.


Subject(s)
Animals , Antineoplastic Agents , Chemistry , Pharmacology , Therapeutic Uses , Bufanolides , Chemistry , Pharmacology , Therapeutic Uses , Chemistry, Pharmaceutical , Methods , Neoplasms , Drug Therapy , Pathology
14.
Journal of Experimental Hematology ; (6): 1306-1310, 2014.
Article in Chinese | WPRIM | ID: wpr-340508

ABSTRACT

The aim of this study was to explore the reversing effect of cinobufacini on multidrug resistance of Raji/ADR cells and its mechanisms. The growth inhibitory rate, half inhibitory concentration (IC50), reversing multiples to adriamycin- resistance were detected by MTT, and the curve of growth inhibitory rate was drawn; the MDR-1 and MRP-1 gene transcription was determined by RT-PCR; the expressions of P-gp and MRP-1 proteins were assayed by Western blot and flow cytometry. The results showed that the inhibitory rates of cinobufacini on Raji and Raji/ADR cells at 72 h were 75.6% and 69.3% respectively, the IC50 were 3.9 mmol/L and 4.6 mmol/L without significant difference (P > 0.05). The reversing multiples to adriamycin-resistance were 255.7 multiples, the transcription of mdr-1 and mrp-1 genes and the expression of P-gp and MRP-1 proteins significantly decreased (P < 0.05) in Raji/ADR cells after the treatment with cinobufotalin. It is concluded that cinobufotalin can reverse the adriamycin-resistance in Raji/ADR cells and the expression of P-gp and MRP-1 proteins were down-regulated through the transcriptional pathway. The cinobufotalin is an effective reversal agent for the multidrug resistance of tumors.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Amphibian Venoms , Bufanolides , Pharmacology , Cell Line, Tumor , Doxorubicin , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Humans , Reverse Transcriptase Polymerase Chain Reaction
15.
Article in English | WPRIM | ID: wpr-812224

ABSTRACT

AIM@#To study the bufadienolides in the Chinese traditional drug "Ch'an Su" and their cytotoxic activity.@*METHOD@#Various chromatographic techniques were used to isolate the constituents, and their structures were elucidated through physical and spectroscopic data.@*RESULTS@#Twenty compounds were isolated, and eighteen were evaluated in vitro for their cytotoxic activity against A-549 and K-562 cells.@*CONCLUSION@#Compound 1 (bufalin 3β-acrylic ester) was a new bufadienolide and exhibited the most potent activity against the two tumor cell lines with IC50 values of 7.16 and 6.83 nmol · L(-1). The relationships between structure and activity are discussed.


Subject(s)
Amphibian Venoms , Chemistry , Pharmacology , Therapeutic Uses , Antineoplastic Agents , Chemistry , Pharmacology , Therapeutic Uses , Biological Products , Chemistry , Pharmacology , Therapeutic Uses , Bufanolides , Chemistry , Pharmacology , Therapeutic Uses , Humans , Inhibitory Concentration 50 , K562 Cells , Medicine, Chinese Traditional , Molecular Structure , Neoplasms , Drug Therapy , Structure-Activity Relationship
16.
Article in Chinese | WPRIM | ID: wpr-359251

ABSTRACT

<p><b>OBJECTIVE</b>To observe the effect of bufalin combined Gefitinib on lung cancer H1975 cells, and to explore its potential mechanisms for anti-tumor.</p><p><b>METHODS</b>The cytostatic effects of bufalin (1 -100 nmol/L), gefitinib (0.1-20 micromol/L), and bufalin plus gefitinib on H1975 cells were evaluated by MTT assay. Their effects on apoptosis of H1975 cells were determined by flow cytometry (FCM). Their effects on expressions of epidermal growth factor receptor (EGFR) and Met signal pathway related proteins in H1975 cells were detected by Western blot.</p><p><b>RESULTS</b>Results of MTT assay showed that gefitinib over 5 micromol/L could inhibit H1975 cells. But combined therapy of bufalin and gefitinib could potently inhibit the growth of H1975 cells. Results of FCM showed the apoptotic rate was 61.64% +/- 5.61% in the bufalin plus gefitinib group, obviously higher than that of the bufalin group (18.34% +/- 3.42%) and the gefitinib group (7.32% +/- 1.08%), showing statistical difference (P < 0.01). Results of Western blot showed the protein expressions of p-EGFR, p-Met, p-Akt, and p-mTOR in H1975 cells could be markedly down-regulated by bufalin plus gefitinib.</p><p><b>CONCLUSIONS</b>Combination of bufalin and gefitinib potently inhibited the growth of H1975 cells, and induced cell apoptosis. The potential mechanism for anti-tumor might be involved in blocking EGFR-PI3k/Akt pathway.</p>


Subject(s)
Bufanolides , Pharmacology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Drug Synergism , Humans , Lung Neoplasms , Metabolism , Pathology , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Quinazolines , Pharmacology , ErbB Receptors , Metabolism , Signal Transduction
17.
Article in English | WPRIM | ID: wpr-17408

ABSTRACT

Preeclampsia is one of the leading causes of maternal mortality/morbidity and preterm delivery in the world, affecting 3% to 5% of pregnant women. The pathophysiology of preeclampsia likely involves both maternal and fetal/placental factors. Abnormalities in the development of placental vessels early in pregnancy may result in placental hypoperfusion, hypoxia, or ischemia. Hypoperfusion, hypoxia, and ischemia are critical components in the pathogenesis of preeclampsia because the hypoperfused placenta transfers many factors into maternal vessels that alter maternal endothelial cell function and lead to the systemic symptoms of preeclampsia. There are several hypotheses to explain the pathogenesis of preeclampsia, including altered angiogenic balance, circulating angiogenic factors (such as marinobufagenin, a bufadienolide trigger), and activation of the renin-angiotensin system. Epigenetically-modified cell-free nucleic acids that circulate in plasma and serum might be novel markers with promising non-invasive clinical applications in the diagnosis of preeclampsia.


Subject(s)
Angiogenesis Inducing Agents , Hypoxia , Bufanolides , Endothelial Cells , Female , Humans , Ischemia , Nucleic Acids , Placenta , Plasma , Pre-Eclampsia , Pregnancy , Pregnant Women , Renin-Angiotensin System
18.
Article in Chinese | WPRIM | ID: wpr-236893

ABSTRACT

<p><b>OBJECTIVE</b>To study the role and possible mechanisms of gap junctional intercellular communication (GJIC) involved in mesangial cell (MC) proliferation which could be inhibited by bufalin.</p><p><b>METHODS</b>Rat mesangial cells were cultured in vitro. The effect of bufalin on platelet-derived growth factor-BB (PDGF-BB)-induced MC proliferation was evaluated by MTT assay. The function of GJIC was detected by Lucifer Yellow scrape loading and dye transfer (SLDT). mRNA levels of Cx43, Cx45 and Cx40 were measured by RT-PCR. Intracellular calcium concentrations ([Ca(2+)]i) were examined in laser scanning confocal microscopy after loading by Fura-3/AM.</p><p><b>RESULTS</b>MTT indicated that bufalin could inhibited PDGF-BB-induced MC proliferation (P<0.01). Compared with the hormal control group, PDGF-BB inhibited GJIC function, increased the expression of Cx45 and Cx40 (P<0.01) without altering the Cx43 (P>0.05) in gene level and also increased [Ca(2+)]i. However, bufalin treatment enhanced GJIC function, decreased Cx45 mRNA and Cx40 mRNA expression (P<0.01), and reduced [Ca(2+)]i (P<0.01).</p><p><b>CONCLUSIONS</b>Bufalin inhibits PDGF-BB-induced MC proliferation, and its possible mechanisms may be related to regulation of Cx45 and Cx40 expression in the gene level, reduction of [Ca(2+)]i and enhancement of GJIC function.</p>


Subject(s)
Animals , Bufanolides , Pharmacology , Calcium , Metabolism , Cell Communication , Cell Proliferation , Cells, Cultured , Gap Junctions , Mesangial Cells , Physiology , Proto-Oncogene Proteins c-sis , Pharmacology , Rats
19.
Article in Chinese | WPRIM | ID: wpr-284333

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of bufalin on nucleus-mitochondria localization of human telomerase reverse transcriptase(hTERT) by exploring its effect on proliferation and apoptosis in human esophageal squamous carcinoma EC9706 cells.</p><p><b>METHODS</b>EC9706 cells were treated with bufalin at various concentrations, and then the cell growth inhibition of EC9706 cells was examined by CCK-8 assay and the 50% inhibitory concentration (IC(50)) was calculated.Cell cycle analysis was performed by flow cytometry with PI staining, and nucleus morphology of apoptosis were observed by fluorescence microscopy with Hoechst 33342 staining. The apoptotic index was measured by flow cytometry with Annexin V-FITC/PI double staining. hTERT subcellular localization and protein expression were determined by Western blotting and multiple immunofluorescence labling combined with laser confocal scanning microscopy.</p><p><b>RESULTS</b>The proliferation of EC 9706 cells was significantly inhibited by bufalin along with the increase of processing time and concentrations (p<0.01). After the EC9706 cells were exposed to 100 nmol/L bufalin,the number of cells gradually decreased in G(1) phase and increased in S and G(2)/M phases(p<0.05). The typical nucleus morphological changes of apoptosis were observed and the apoptotic index was increased(p<0.01). The expression of hTERT decreased in nucleus but increased in mitochondria(p<0.05).</p><p><b>CONCLUSIONS</b>Bufalin can inhibit the proliferation of human esophageal squamous carcinoma EC9706 cells in a time- and dose-dependent manner. It can arrest cell cycle in S and G(2)/M phases and induce the apoptosis of EC 9706 cells. hTERT is localized in both nucleus and mitochondria,and can be partially translocated from nucleus to mitochondria during the bufalin-induced apoptosis.</p>


Subject(s)
Apoptosis , Bufanolides , Pharmacology , Carcinoma, Squamous Cell , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Esophageal Neoplasms , Metabolism , Pathology , Humans , Telomerase , Metabolism
20.
Acta Pharmaceutica Sinica ; (12): 1200-1204, 2012.
Article in Chinese | WPRIM | ID: wpr-274677

ABSTRACT

The aim of this paper is to report the development of a method for the determination of the binding rates of cinobufagin (CBG) and resibufogenin (RBG) with different plasma proteins. Equilibrium dialysis method was used to imitate the binding process between cinobufagin, resibufogenin and plasma proteins. HPLC was employed to determine the concentration of cinobufagin and resibufogenin inside and outside of the dialysis membrane, and then on the basis of which protein binding rates were calculated. The calibration curves of both cinobufagin and resibufogenin with human, rats' and Beagle dogs' plasma were linear in the range of 0.50-40.00 microg x mL(-1), while the buffer solution was 0.25-4.00 microg x mL(-1). The extract recovery of cinobufagin was in the range of (68.73 +/- 2.16)%--(79.27 +/- 1.62)%, while that of resibufogenin was (71.59 +/- 4.31)%--(83.47 +/- 2.63)%. The average plasma protein binding rates with cinobufagin were 85.63%, 80.21% and 70.10% in human, rats' and Beagle dogs' plasma, whereas those binding rates with resibufogenin were 84.51%, 75.11% and 70.60%, respectively. The results suggested that the protein binding rates of cinobufagin and resibufogenin were of middle strength, which in rats', Beagle dogs' and human plasma were decreased in the following order: human plasma > rats' plasma > Beagle' dogs plasma.


Subject(s)
Animals , Blood Proteins , Metabolism , Bufanolides , Chemistry , Metabolism , Chromatography, High Pressure Liquid , Methods , Dialysis , Dogs , Humans , Molecular Structure , Protein Binding , Rats , Species Specificity
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