Inhibition of M2-type macrophage-mediated migration and epithelial mesenchymal transition in colorectal cancer by bufalin / 安徽医科大学学报

Objective To investigate the role of bufalin(BU)in inhibiting M2-type macrophage-mediated colorec-tal cancer metastasis.Methods Human acute leukemia mononuclear cells(THP-1)were differentiated into M0 macrophages using phorbol ester induction(PMA)for 48 hours.The M0 macrophages were then treated with IL-4 and IL-13 medium.Surface markers and morphological changes were observed through ELISA,morphology,and RT-qPCR experiments.RT-PCR and ELISA experiments were conducted to detect the surface markers TGF-β and IL-10 of M2 macrophages.The secretion level of IL-6 in the supernatant of M2 macrophages and colorectal cancer cells HCT116 was compared using ELISA.Additionally,the effect of conditioned medium on colorectal cancer cell HCT116 was assessed through Transwell,Wound healing,RT-qPCR,and Western blot experiments.Subsequent-ly,bufalin was added to the conditioned medium and the changes in AKT/PI3K protein,migration,and epithelial-mesenchymal transition ability in HCT116 were observed using Western blot,Transwell,Wound healing and RT-qPCR experiments.Results THP-1 were successfully differentiated into M2 macrophages.The activation of AKT/PI3K protein in HCT116 cells was induced by the secretion of IL-6 from M2 macrophages,which in turn promoted the migration and epithelial-mesenchymal transition ability of the HCT116 cells.The migration and epithelial-mes-enchymal transition mediated by M2 macrophages in HCT116 cells were effectively inhibited by Bufalin.Conclu-sion The release of IL-6 from M2 macrophages activates the AKT/PI3K signaling pathway in colorectal cancer cells,thereby promoting their migration and epithelial-mesenchymal transition capacity.Moreover,bufalin exhibits inhibitory effects on this effect.

Bufalin induces apoptosis of HCT116 cells by activating endoplasmic reticulum stress pathway / 安徽医科大学学报

Objective @#To study the effect of bufalin on the proliferation and apoptosis of human colorectal cancer cell line HCT116，and to explore the role of endoplasmic reticulum stress ( ERS) in this process．@*Methods @#The effect of bufalin on the proliferation of HCT116 cells was determined by CCK-8 assay．After HCT116 cells were treated with different concentrations of bufalin for 48 hours，cell apoptosis was detected by Annexin V / PI assay， and the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot．At the same time，the expression of ERS-related proteins glucose regulated protein 78 ( GRP78) ，phosphorylated protein kinase R like endoplasmic reticulum kinase ( p-PERK) ，eukaryotic translation initiation factor 2 α ( eIF2 α) ，phosphorylated eukaryotic translation initiation factor 2 α (p-eIF2 α) and C / EBP homologous protein ( CHOP) was detected by Western blot．HCT116 cells were divided into control group，bufalin group and combination group (bufalin + 4-phenylbutyric acid) ，and the expression of apoptosis-related proteins Bax and Bcl-2 was observed by Western blot. @*Results@#CCK-8 assay showed that bufalin could inhibit the proliferation of HCT116 cells．Apoptosis assay showed that bufalin could induce apoptosis of HCT116 cells．The results of Western blot showed that bufalin could up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2．It could also induce ERS and activate PERK / eIF2 α/ CHOP pathway．When bufalin combined with 4-phenylbutyric acid，the apoptosis-promoting effect of bufalin was inhibited．@*Conclusion@#Bufalin can effectively inhibit the prolif- erative activity and induce apoptosis of HCT116，which is achieved to some extent by activating ERS.

Improvement of Quality Control Standard of Bufonis Venenum in Shexiang Baoxin Pills / 中国实验方剂学杂志

ObjectiveTo develop a quantitative analysis of multi-components by single marker （QAMS） for determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills， and to provide a method for improving the national standard of the pills. MethodHigh performance liquid chromatography （HPLC） was developed for simultaneous determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills and the methodology validation was carried out. The chromatographic separation was performed on a Nucleosil 100-5 C18 column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile -0.1% potassium dihydrogen phosphate aqueous solution （pH adjusted to 3.2 with phosphoric acid） （48∶52）， and the flow rate was 0.6 mL·min-1， the detection wavelength was set at 296 nm and the column temperature was 35 ℃. Taking cinobufagin as the internal standard， the relative correction factors （RCFs） of bufalin and resibufogenin were calculated， and the key influencing factors of RCFs were investigated. Relative retention time was used for the chromatographic peak location of the analyte， combining with the on-line ultraviolet spectroscopy and accurate relative molecular weight obtained by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF/MS）. The external standard method was used to verify the contents of three components obtained by QAMS. ResultQAMS was established for the determination of bufalin， cinobufagin and resibufogenin in the samples， and RCFs of cinobufagin to bufalin and resibufogenin were 0.922 and 1.01， respectively. The total content of the three marker compounds in 11 batches of Shexiang Baoxin pills was 33.7-36.0 µg per pill. There was no significant difference between the quantitative results of QAMS and external standard method. ConclusionThe established method can be used for the quality control of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills. It is suggested that bufalin should be considered as one of three marker compounds， and the sum of bufalin， cinobufagin and resibufogenin should be used for the content limit of this preparation.

Research on the mechanism of bufalin inhibiting migration and invasion of human prostate cancer PC3 cells / 药学学报

In this study, we investigated the inhibitory effects of bufalin on proliferation, migration and invasion of PC3 cells in vitro, and preliminarily explored the molecular mechanism of epithelial-mesenchymal transformation (EMT) inhibited by bufalin. The viability of PC3 cells was evaluated by MTT assay, and the migration and invasion abilities of PC3 cells were detected by wound healing and Transwell assay. Western blot was used to detect the expression of EMT and integrin family proteins. The results showed that the half maximal inhibitory concentration (IC50) value of bufalin against PC3 cells was 0.26 ± 0.03 μmol·L-1. After bufalin treatment, the migration rate of PC3 cells slowed down (P < 0.05), the number of PC3 cells passing through the microporous membrane decreased (P < 0.05), which indicated that bufalin could inhibit the proliferation, migration and invasion of PC3 cells in a concentration-dependent manner. We found that bufalin could affect the expression of EMT-related proteins,including up-regulation of E-cadherin and down-regulation of N-cadherin, β-catenin, matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 2 (MMP2), c-myc and Snail. Bufalin also inhibited the expression of integrin family proteins, including integrin α2 (ITGA2), integrin β1 (ITGB1), integrin β3 (ITGB3), integrin β5 (ITGB5), Yes-associated protein/transcriptional coactivator with a PDZ-binding motif (YAP/TAZ) and integrin-linked kinase (ILK). In addition, bufalin could also inhibit the protein expression level of phospho-focal adhesion kinase (p-FAK)/FAK, phospho-steroid receptor coactivator (p-Src)/Src and phospho-protein kinase B (p-Akt)/Akt. These results suggested that bufalin might inhibit the proliferation, metastasis and invasion of prostate cancer PC3 cells through the FAK/Src/phosphoinositide 3-kinase (PI3K)/Akt pathway. Therefore, bufalin provides reference value for the development of therapeutic drugs for prostate cancer.

Application of QAMS for quality evaluation and control of Chinese patent medicines:taking Bufonis Venenum-contained preparations as examples / 中国中药杂志

As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

Design of dual targeting immunomicelles loaded with bufalin and study of their anti-tumor effect on liver cancer / 中西医结合学报

OBJECTIVE@#Bufalin is an effective drug for the treatment of liver cancer. But its high toxicity, poor water-solubility, fast metabolism and short elimination half-life limit its use in tumor treatment. How to make the drug accumulate in the tumor and reduce side effects while maintaining its efficacy are urgent problems to be solved. The goal of this study is to solve these problems.@*METHODS@#A copolymer with tunable poly-N-isopropylacrylamide and polylactic acid was designed and synthesized. The corresponding dual targeting immunomicelles (DTIs) loaded with bufalin (DTIs-BF) were synthesized by copolymer self-assembly in an aqueous solution. The size and structure of DTIs-BF were determined by ZetaSizer Nano-ZS and transmission electron microscopy. Then, its temperature sensitivity, serum stability, critical micelle concentration (CMC), entrapment efficiency (EE), drug release and non-cytotoxicity of blank block copolymer micelles (BCMs) were evaluated. Next, the effects of DTIs-BF on cellular uptake, cytotoxicity, and tumor cell inhibition were evaluated. Finally, the accumulation of DTIs-fluorescein isothiocyanate (FITC) and the in vivo anti-tumor effect were observed using an interactive video information system.@*RESULTS@#DTIs-BF had a small size, spherical shape, good temperature sensitivity, high serum stability, low CMC, high EE, and slow drug release. The blank BCMs had very low cytotoxicity. Compared with free bufalin, the in vitro cellular internalization and cytotoxicity of DTIs-BF against SMMC-7721 cells were significantly enhanced, and the effects were obviously better at 40 °C than 37 °C. In addition, the therapeutic effect on SMMC-7721 cells was further enhanced by the programmed cell death specifically caused by bufalin. When DTIs-FITC were injected intravenously in BALB/c nude mice bearing liver cancer, the accumulation of FITC was significantly increased in tumors.@*CONCLUSION@#DTIs-BF is a potentially effective nano-formulation and has broad prospects in the clinical treatment of liver cancer.

Effects of Bufalin on Human Esophageal Cancer Cells Proliferation and Mechanism Study / 中国药学杂志

OBJECTIVE: To investigate the effect of bufalin on the proliferation of esophageal cancer and its possible molecular mechanism. METHODS: The effects of bufalin on the activity of esophageal cancer cell line KYSE-70 were determined by MTT assay and LDH assay kit. Colony-forming and EdU (5-ethynyl-2-deoxyuridine) assay were performed to detect the inhibitory effect of bufalin on the proliferation of KYSE-70 cells. DAPI staining, TUNEL and flow cytometry were used to assess the effects of bufalin on the apoptosis of KYSE-70 cells. Quantitative real-time PCR (qPCR) and Western blotting were used to determine the relevant expression changes in mRNA and protein of apoptosis-related factors Bcl-2, Bax, NF-κBp65, NF-κBpp65. The mitochondrial function changes of KYSE-70 cells were studied by using JC-1 fluorescent probe kit and ATP detection kit after bufalin treatment. Finally, the effect of bufalin on esophageal cancer proliferation in vivo was studied by xenograft model in nude mice. RESULTS: The results of MTT and LDH assay shown that bufalin inhibited the activity of KYSE-70 cells. Colony-forming and EdU assay showed that bufalin significantly suppressed KYSE-70 cells proliferation. DAPI staining showed that chromatin heterogeneity, nuclear concentration and fragmentation were observed after bufalin treatment. It was found that bufalin treatment significantly promoted KYSE-70 cells apoptosis by TUNEL staining and flow cytometry assay of qPCR and Western blot showed that Bcl-2 expression was down-regulated, Bax expression was up-regulated and Bax/ Bcl-2 expression ratio was increased after bufalin treatment. The mitochondrial membrane potential and the ATP production of KYSE-70 cells were significant reduced after bufalin treatment. In vivo, the growth of xenograft tumors was significantly inhibited in bufalin group. CONCLUSION: Bufalin markedly inhibits KYSE-70 cells proliferation by promoting apoptosis, and the possible mechanism of apoptosis may be related to mitochondrial pathway. Our results indicate that bufalin may be a potential therapeutic agent for esophageal cancer.

Establishment of HPLC fingerprint of Bufonis Venenum pulp and determination of eight components / 中草药

Objective: To establish the HPLC fingerprint of Bufonis Venenum pulp and the determination methods of eight components in Bufonis Venenum pulp, and compare the differences of components of Bufonis Venenum pulp in different origins for quality evaluation. Methods: The mobile phase was acetonitrile and water with a gradient elution program. The detection wavelength was set at 296 nm. The flow rate was 1.2 mL/min. The column temperature was set at 30 ℃, and 10 μL of the test solution was injected. HPLC fingerprint of Bufonis Venenum pulp was established and eight components were determined. The results were analyzed by cluster analysis and principal component analysis. Results: There were 12 common peaks in the HPLC fingerprints of 18 batches of samples, and the similarity of each sample was different. The linear relationship of eight components was good (r2 > 0.999 5), RSD of precision and repeatability was less than 0.5%, and the stability was also good within 30 h (RSD < 0.7%). The average recoveries of gamabufotalin, arenobufagin, telocinobufagin, bufotaline, cinobufotalin, bufalin, cinobufagin and resibufogenin were 103.7%, 103.0%, 102.9%, 103.0%, 103.9%, 100.3%, 103.4%, and 103.2% respectively, and RSD was all less than 1.2%. The results of the content determination, cluster analysis and principal component analysis of eight components showed that Bufonis Venenum pulp in different habitats were different from each other. Conclusion: The method is simple and reliable, which can provide some basis and reference for quality control of Bufonis Venenum pulp.

Bufalin-induced cardiotoxicity: new findings into mechanisms / 中国天然药物

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), human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.03-0.3 μmol·L) and eight human cardiac ion channel currents (0.01-100 μmol·L) 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, while were weakened at 0.3 μmol·L after application. Bufalin blocks I in a concentration-dependent manner with half maximal inhibitory concentration of 74.5 μmol·L. Bufalin respectively increased the late sodium current and Na-Ca exchange current with a concentration for 50% of maximal effect of 2.48 and 66.06 μmol·L 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.

Correlative study of bufogenin constituents in venom of Bufo bufo gargarizans / 中国中药杂志

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.

Study on chemical constituents and pharmacodynamic changes of Bufonis Venenum with different drying methods / 中草药

Objective To study the changes of chemical constituents and pharmacodynamics with different drying methods (sun- drying, 50 ℃ vacuum-drying, 50 ℃ and 80 ℃ heat-drying, freeze drying method) in Bufonis Venenum. Methods HPLC method and TLC method was established for studying the changes of chemical constituents from B. Venenum before and after being dried, and determine the inhibitory effect of dried samples on five different tumor cell lines proliferation by MTT assay. Results The B. Venenum processed by five different methods were different in character, while no significant differences in the types and content of chemical constituents; The total content of resibufogenin and cinobufagin was more than 6%, which was consistent with the 2015 edition of Chinese Pharmacopeia; 50 ℃ and 80 ℃ heat-drying of B. Venenum showed more effective inhibitiory effect than other dry methods. Conclusion The appearance of B. Venenum met the criterion of pharmacoperia by sun-drying and 50 ℃ heat-drying method. Although the color of B. Venenum under the vacuum-drying and freeze drying method did not meet the requirements of Chinese Pharmacopeia, the main effective components have a few changes.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<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>

Mechanism of active ingredients of Venenum Bufonis in down-regulating expression of aurora kinase and promoting cell cycle arrest in human liver cancer HepG2 cells / 中草药

Objective To investigate the function of aurora kinase (AURK) in liver cancer and the mechanism of cinobufagin and bufalin-induced liver cancer HepG2 cells growth inhibition by down-regulating AURK family. Methods Kaplan-Meier survival method analyzed the relationship between mRNA expression levels of AURKA and AURKB and survival periods. The viability and cell cycle of HepG2 cells were detected by MTT method and flow cytometry. Western blotting analyzed the expression levels of spindle-associated protein AURKA, AURKB, TPX2, SMC2, TOP2A, and cyclin-dependent kinase CDK1. Results Kaplan-Meier survival analysis presented a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin inhibited the growth of HepG2 cells in a time- and dose-dependent manner, and induced the cell cycle G2/M phase arrest. They all down-regulated the expression of AURKA, AURKB, TPX2, SMC2, TOP2A, and CDK1 (P < 0.05). Conclusion There is a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin could induce HepG2 cells growth inhibition and cell cycle arrest by down-regulating the expression of AURKA and AURKB and other mitosis-regulating proteins.

Simultaneous determination of nine bufadienolides in Liushen Pill by HPLC-MS / 中草药

Objective: To establish an HPLC-MS method for determining nine bufadienolides (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin, and resibufogenin) in Bufonis Venenum and Liushen Pill. Methods: HPLC analysis was performed on ODS-2 column (250 mm×4.6 mm, 5 μm) with the mobile phase made up of acetonitrie and 0.15% phosphoric in water. Flow rate was set at 1 mL/min and column temperature was 40℃. The detection wavelength was 296 nm. Nine bufadienolides were determined by HPLC-ESI-TOF-MS with the mobile phase made up of acetonitrie and 0.2% formic acid in water. Eletrospray ionization source was applied and operated in the positive ion mode. Results: Nine bufadienolides were separated at baseline with good linearity (r≥0.9996). The recovery rates were 92%-105% (RSD<5%). Conclusion: The method established is rapaid, simple, and accurate, which is helpful to control the quality of Liushen Pill.

Improved anti-tumor efficacy and pharmacokinetics of bufalin via PEGylated liposomes / 中国药理学与毒理学杂志

OBJECTIVE To determine the characterization, anti-tumor efficacy and pharmacokinetics of bufalin- loaded PEGylated liposomes compared with bufalin entity. METHODS Bufalin- loaded PEGylated liposomes and bufalin- loaded liposomes were prepared reproducibly with homogeneous particle size by the combination of thin film evaporation method and high pressure homogenization method. The particle size and zeta potential of the liposomes were determined by dynamic light scattering technique. The direct imaging of morphology of liposomes was charactered by transmission electron microscope. The content of bufalin in liposomes was analysed by HPLC method. The entrapment efficiency and the particle size was applied to assess the stability profile, after storage at 4℃ on day 0, 7, 15, 30 and 90. The in-vitro release behaviours of bufalin from liposomes were conducted using dialysis bag technique at 37℃. In-vitro cytotoxicity studies were carried out using MTT〔3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide〕assay on several kinds of tumor cell lines including SW620, PC-3, MDA-MB-231, A549, U251, U87 and HepG2. In-vivo pharmacokinetic study of bufalin liposomes was evaluated by HPLC method. RESULTS Their mean particle sizes were 127.6 nm and 155.0 nm, mean zeta potentials were 2.24 mV and - 18.5 mV, entrapment efficiencies were 76.31% and 78.40% , respectively. In- vitro release profile revealed that the release of bufalin in bufalin- loaded PEGylated liposomes was slower than that of bufalin-loaded liposomes. The cytotoxicity of blank liposomes has been found within acceptable range, whereas bufalin-loaded PEGylated liposomes showed enhanced cytotoxicity to U251 cells compared with bufalin entity. In-vivo pharmacokinetics indicated that bufalin-loaded PEGylated liposomes could extend eliminate half-life time of bufalin in plasma in rats. CONCLUSION The results suggested that bufalin-loaded PEGylated liposomes improved the solubility and increased the drug concentration in plasma.

Synergy with the active ingredients of toad venom and sorafenib suppresses hepatocellular carcinoma HepG2 cells proliferation through down-regulating Akt/NF-κB signaling pathways / 中国药理学通报

Aim To investigate the effect of the active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib on the growth of hepatocellular carcinoma HepG2 cells,and to explore the possible mechanism.Methods The rates of inhibition after treated with drugs 12,24,48 h were detected by MTT assay.The changes of cell morphology were detected by Hoechst 33342 fluorescent staining.The changes of cell cycle were detected by flow cytometry.The expressions of proteins such as Akt,p-Akt (Ser473),IκB,NF-κB,p-NF-κB p65,Bcl-2,Bax,cyclin A,PCNA were detected by Western blot.Results Bufalin,cinobufagin and sorafenib could inhibit the proliferation of HepG2 cells,presenting a dose-and time-dependent manner.Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of single treatment,and they performed a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment (P ＜ 0.01).The results of fluorescence staining showed the observation of the morphological features of nuclear condensation.Sorafenib induced the cell cycle G0/G1 phase arrest (P ＜0.01),and bufalin,cinobufagin and the combination treatment generated the cell cycle S phase arrest (P ＜0.01).The results of Western blot showed that the expressions of Akt,NF-κB were not obviously changed between control and all other treatment.The expression levels of p-Akt (Ser473),p-NF-κB p65,Bcl-2,PC-NA and cyclin A in combination treatment significantly decreased,and the expression levels of IκB and Bax significantly increased compared to those in single treatment (P ＜ 0.01).Conclusion The active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib performs a synergetic effect on the anti-cancer of HepG2 cells by down-regulating Akt/ NF-κB signaling pathway.

Inhibition Effect of Bufalin on Invasion and Metastasis of Gastric Cancer Cells / 医药导报

Objective To study effect of bufalin on invasion and metastasis of gastric cancer cells and related mechanism.Methods AGS human gastric cancer cell line was used for in vitro experiments.Cultured cells were treated by negative control group,bufalin group and oxaliplatin group.Cell proliferation was determined by MTT.Invasion and metastasis were observed by Wound-Healing Assay and Transwell Assay.Expression levels of E-cadherin,matrix metalloproteinases (MMP)-2,MMP-9 were detected by Western blotting.Results Bufalin was found to significantly inhibit the proliferation of AGS cells in a dose and time dependent manner.Wound-Healing Assay and Transwell Assay showed that as compared with the blank control group,bufalin inhibited invasion and metastasis of AGS cells (P＜ 0.05),but there was no statistically significant difference between bufalin group and oxaliplatin group (P＞0.05).Western blotting showed the expression of E-cadherin was increased in bufalin group as compared with the blank control group while the expression levels of MMP-9 and MMP-2 were down-regulated (P＜0.05),but there was no statistically significant difference between bufalin group and oxaliplatin group (P＞ 0.05).Conclusion Bufalin has anti-cancer activity on gastric cancer cells,and it has the ability of inhibiting cancer cell invasion and metastasis,and regulating the expression of some related gene.

Effects of bufalin combined with doxorubicin on the proliferation and apoptosis of human lung cancer cell line A549 in vitro / 中南大学学报(医学版)

Objective:To explore the effects ofbufalin (BUF) combined with doxorubicin (DOX) on the proliferation and apoptosis in human lung cancer cell line A549 in vitro.Methods:Methyl thiazolyl tetrazolium (MTT) assay was used to measure the inhibitory effects of BUF,DOX and their combination on the growth ofA549 cells.Hoechst 33342 staining was used to observe the changes of nucleus.Flow cytometry was used to investigate the apoptosis and cell cycle distribution of A549 cells.Western blot was used to examine the expression of apoptotic protein.Results:BUF and DOX showed inhibitory effect on the A549 cells in a dose and time-dependent manner.Compared with BUF or DOX alone,combination of BUF (1,20,100 nmol/L) with DOX (1.0 μg/mL) could significantly increase the growth inhibition rate ofA549 cells at 24,36,72 h,respectively (all P＜0.05).BUF and DOX alone could induce apoptosis,and their combination could significantly increase the apoptosis ratio.In addition,BUF combined with DOX could block the cell stage of A549 cells,keep the cell stage stay in S stage and up-regulate the expression of caspase-3.Conclusion:BUF combined with DOX can significantly inhibit the proliferation ofA549 cells,which might be related to the induction of apoptosis,cell cycle S phase arrest and caspase-3 up-regulation.

Bufalin inhibits proliferation and downregulates expression of WT1 in K562 cells in vivo and vitro / 中国药理学通报

Aim To investigate the effect of bufalin on proliferation and expression of WT1 in K562 cells. Methods The colony number of K562 cell was detec-ted with semi-solid culture assay. The cell cycle was measured by flowcytometry, and the expression of WT1 was observed with immunocytochemistry. Subcutaneous tumor models established by K562 cells in BALB/C nu/nu mice were divided into three groups, including model group, bufalin group and positive control group. After 21 days, the subcutaneous tumors were removed for calculating the inhibitory rate of tumor growth. HE staining and immunohistochemistry were used to ob-serve the morphological changes and the expression of WT1 . Results ① Bufalin could significantly decrease the colony number of K562 cell, arrest it at G0/G1 phase and down-regulate its expression of WT1 in a dose-dependent manner. ② Compared with the model group, the tumor inhibitory rate was much higher, while the volume and the weight were obviously lower in the other two groups. ③Bufalin could induce apop-tosis, necrosis, hemorrhage and fibrosis with HE stai-ning, and down-regulate the expression of WT1. Con-clusion Bufalin could inhibit the proliferation, arrest the cell cycle at G0/G1 phase and down-regulate the expression of WT1 in vitro. Bufalin could inhibit the tumor inhibitory rate, the volume and the weight of the subcutaneous tumors, induce apoptosis, necrosis, hemorrhage and fibrosis with HE staining and down-regulate the expression of WT1 .

Bufalin alleviates adriamycin-induced podocyte injury by up-regulating the expression of vitamin D receptor / 中华肾脏病杂志

Objective To investigate the role of vitamin D receptor (VDR) in the protection of bufalin on podocyte injury induced by adriamycin (ADR).Methods (1) In vitro:the toxic effect of different concentrations of bufalin (10-9,10-8,10-7,104 mol/L) on podocyte was evaluated by lactate dehydrogenase (LDH) test;Annexin V-FITC and RT-PCR were utilized for podocyte apoptosis and VDR mRNA level respectively.Western blotting was used to analyze the protein expression of VDR and nephrin.SiRNA intervene was also applied to evaluate the role of VDR in bufalin's protective effect on podocyte injury induced by ADR.(2) In vitro:24 SD rats were randomly divided into three groups:control group,ADR group and ADR+bufalin group.TUNEL assay was applied to detect the apoptosis of podocytes in the kidney.Immunofluorescence and transmission electron microscope (TEM) were applied to analyze the expression of VDR and the ultrastructure of the glomerulus.Results Bufalin concentration lower than 10-7 mol/L had no toxicity on normal podocyte.Bufalin reduced the urinary protein excretion (P ＜ 0.05),alleviated the removal of podocyte foot processes and attenuated the changes in nephrin expression in the glomerulus of the adriamycin (ADR) rats (P ＜ 0.05).Bufalin notably inhibited the down-regulation of VDR in protein levels on the glomerulus of the ADR rats.Additionally,bufalin inhibited the down-regulation of VDR in both mRNA levels and protein levels (P ＜ 0.05),nephrin protein expression (P＜ 0.05),and apoptosis induced by ADR in cultured podocytes.Additionally,VDR specific siRNA intervene abolished the protective effect of bufalin in ADR-induced podocyte injury.Conclusion Bufalin can alleviate ADR-induced podocyte injury via enhancing VDR expression.