Bufalin inhibits the malignant development of non-small cell lung cancer by mediating the circ_0046264/miR-522-3p axis.

BACKGROUND: Bufalin is an active component of the traditional Chinese medicine "Chan Su" and is reported to play anti-tumor roles in cancer development, but its functional mechanism is largely unclear. This study intends to explore a potential action mode of bufalin in NSCLC. MATERIALS AND METHODS: The malignant properties of NSCLC, including cell viability, proliferation, adhesion capacity, migration and invasion, were monitored by cell counting kit-8 (CCK-8), adhesion assay and transwell assay, respectively. The expression of circ_0046264 and miR-522-3p was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of proliferation- and migration-related markers was examined by western blot. The putative relationship between circ_0046264 and miR-522-3p was verified by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Animal experiments in nude mice were performed to investigate the role of bufalin in vivo. RESULTS: Bufalin treatment inhibited cell viability, colony formation, cell adhesion capacity, migration and invasion in NSCLC cells. Bufalin facilitated the expression of circ_0046264, and circ_0046264 overexpression also inhibited NSCLC cell viability, colony formation, cell adhesion capacity, migration and invasion. Besides, circ_0046264 knockdown partially counteracted the effects of bufalin. Further, miR-522-3p was identified as a target of circ_0046264, and its deficiency reversed the effects of circ_0046264 knockdown to suppress malignant activities of NSCLC cells. In addition, bufalin restrained the tumor growth and development in vivo via enhancing the expression of circ_0046264. CONCLUSION: Bufalin played an anti-tumor role in NSCLC by modulating the circ_0046264/miR-522-3p pathway, which might be a potential functional mechanism of bufalin in NSCLC.

Efficacy and safety of cinobufacini injection combined with vinorelbine and cisplatin regimen chemotherapy for stage III/IV non-small cell lung cancer: A protocol for systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: The main component of cinobufacini injection is dry toad skin, which is used as adjuvant therapy for stage III/IV non-small cell lung cancer patients in long-term combination with vinorelbine and cisplatin. However, the efficacy and safety of this combination therapy remain unclear. METHODS: A systematic review and meta-analysis will be conducted following the preferred reported items for systematic review and meta-analysis guidelines. Two independent reviewers (LRL and ZLN) will carry out a comprehensive search of the PubMed, Web of Science, Cochrane Library, EMBASE, the Chinese Science and Technology Periodical Database, China National Knowledge Infrastructure, Wanfang Databases, China Biology Medicine. The last search date will be July 30, 2020. Reference list of all selected articles will independently screened to identify additional studies left out in the initial search. The Cochrane Risk of Bias Tool will be used to evaluate the risk of bias of the randomized controlled trials. Outcome index: The main efficacy indicators were based on the objective efficacy evaluation criteria of the World Health Organization antineoplastic drugs or the objective efficacy evaluation criteria of solid tumors established by RECIST. Secondary criteria Karnofsky performance scale (KPS) score, pain efficacy criteria, side effects of chemotherapy such as myelosuppression and gastrointestinal symptoms. Assessment of risk of bias and data synthesis will be conducted using Review Manager V5.3 software. RESULTS: This study will systematically evaluate the efficacy and safety of cinobufacini combined with vinorelbine and cisplatin in the treatment of stage III/IV non-small cell lung cancer. The results of this systematic review will be published in peer-reviewed scientific journals. ETHICS: The ethical approval is not required since systematic review is based on published studies. INPLASY REGISTRATION NUMBER: INPLASY202060091.

Cinobufotalin injection combined with chemotherapy for the treatment of advanced NSCLC in China: A PRISMA-compliant meta-analysis of 29 randomized controlled trials.

BACKGROUND AND OBJECTIVE: Cinobufotalin injection (CFI), a kind of Chinese medicine, has been considered as a promising complementary therapy option for advanced non-small cell lung cancer (NSCLC), but their efficacy and safety remain controversial. This study aimed to systematically evaluate the efficacy and safety of CFI and chemotherapy-combined therapy for advanced NSCLC. METHODS: Clinical trials were searched from Web of Science, Cochrane Library, PubMed, Embase, China National Knowledge Infrastructure (CNKI), Chinese Biological Medicine Database (CBM), Chinese Medical Citation Index (CMCI), Wanfang database and Chinese Scientific Journal Database (VIP). Main measurements, including therapeutic efficacy, quality of life (QoL) and adverse events, were extracted from the retrieved publications and were systematically evaluated. RESULTS: The 29 trials including 2300 advanced NSCLC patients were involved in this study. Compared with chemotherapy alone, its combination with CFI significantly prolonged the patients' 1-, 2- and 3-year overall survival rate (OS) (1-year OS, OR = 1.94, 95% CI = 1.42-2.65, P < .0001; 2-year OS, OR = 2.31, 95% CI = 1.55-3.45, P < .0001; 3-year OS, OR = 4.69, 95% CI = 1.78-12.39, P = .002) and improved patients' overall response (ORR, OR = 1.84, CI = 1.54-2.18, P < .00001), disease control rate (DCR, OR = 2.09, 95% CI = 1.68-2.60, P < .00001) and QoL (quality of life improved rate, QIR, OR = 2.64, 95% CI = 1.98-3.52, P < .00001; karnofsky performance score, KPS, OR = 10.97, 95% CI = 5.48-16.47, P < .0001). Most adverse events caused by chemotherapy were obviously alleviated (P < .05) when CFI was also applied to patients. CONCLUSION: The combination of CFI and chemotherapy is safe, and is more effective in treating NSCLC than chemotherapy alone. Therefore, CFI mediated therapy could be recommended as an adjuvant treatment method for NSCLC.

Simultaneous determination of resibufogenin and its eight metabolites in rat plasma by LC-MS/MS for metabolic profiles and pharmacokinetic study.

BACKGROUND: Resibufogenin is one of the main active compounds of Venenum Bufonis and exhibits diverse pharmacological activities. It is brought into focus for its potency in heart failure and cancer therapy. PURPOSE: The purpose of this study was to establish a convenient and effective method which was used to simultaneously determine the resibufogenin and its metabolites in rat plasma for further understanding the metabolic profiles of resibufogenin in vivo and pharmacokinetic study by LC-MS/MS. METHODS: The analytes were separated on a BEH C18 column with a mobile phase of water containing 0.05% formic acid and acetonitrile under gradient elution at a flow rate of 0.4 ml/min. Resibufogenin and its eight metabolites were quantified in positive electrospray ionization and MRM mode with transitions of m/z 385.5→349.2 for resibufogenin; m/z 513.7→145.3 for IS (internal standard); m/z 401.23→365.21, m/z 417.23→285.21 and m/z 385.24→349.21 for three main metabolites (hydroxylated-resibufogenin; dihydroxylated-resibufogenin and 3-epi-resibufogenin, respectively). RESULTS: This method was successfully validated with a good linearity over the concentration ranges of 1-200 ng/ml for resibufogenin and the correlation coefficients was more than 0.990. The lower limit of quantification was 1 ng/ml and the precision and accuracy values were less than 15%. The method was applied to study the metabolic profiles of resibufogenin in rat plasma after oral administration of 20 mg/kg. The results indicated that the metabolic reactions of resibufogenin were mainly hydroxylation, dihydroxylation, dehydrogenation and isomerization. Totally eleven metabolites were identified, among which eight were successfully quantified. CONCLUSION: The results could provide further research foundation for the mechanisms study of activity and toxicity in vivo and facilitate the appropriate clinical application of resibufogenin.

Clinical effect of intravenous infusion of zoledronic acid combined with oral medication of cinobufagin in the treatment of metastatic bone tumors.

Aim of the study was to observe and analyze the clinical effect of intravenous infusion of zoledronic acid combined with oral medication of cinobufagin in treating metastatic bone tumors. The 120 patients who have been treated in the hospital for metastatic bone tumor from June 2014 to June 2017 were selected as research objects. They were randomly divided into research group and control group, each containing 60. The research group was treated with intravenous infusion of zoledronic acid combined with oral medication of cinobufagin. In the control group, only zoledronic acid intravenous infusion was administered. The overall treatment effect of the two groups was observed. The pain of two groups was evaluated using numerical rating scale (NRS). The results showed that compared with the control group, the research group achieved better clinical effect and had a higher quality of life, and the intergroup difference was of statistical significance, P<0.05. There was no difference in rate of adverse reactions between the two groups, P>0.05, without statistical significance. The combined therapy of intravenous infusion of zoledronic acid and oral medication of cinobufagin can obtain better therapeutic effect in treating metastatic bone tumors.

Resibufogenin suppresses transforming growth factor-ß-activated kinase 1-mediated nuclear factor-κB activity through protein kinase C-dependent inhibition of glycogen synthase kinase 3.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has received considerable attention for its potency in cancer therapy. However, the anticancer effects and the underlying mechanisms of RB on pancreatic cancer remain elusive. Here, we found that RB inhibited the viability and induces caspase-dependent apoptosis in human pancreatic cancer cells Panc-1 and Aspc. Resibufogenin-induced apoptosis was through inhibition of constitutive nuclear factor-κB (NF-κB) activity and its target genes' expression, which was caused by downregulation of transforming growth factor-ß-activated kinase 1 (TAK1) levels and suppression of IκB kinase activity in Panc-1 and Aspc cells. This induction of TAK1-mediated NF-κB inactivation by RB was associated with increased glycogen synthase kinase-3 (GSK-3) phosphorylation and subsequent suppression of its activity. Moreover, RB-induced GSK-3 phosphorylation/inactivation acted through activation of protein kinase C but not Akt. Finally, RB suppressed human pancreatic tumor xenograft growth in athymic nude mice. Thus, our findings reveal a novel mechanism by which RB suppresses TAK1-mediated NF-κB activity through protein kinase C-dependent inhibition of GSK-3. Our findings provide a rationale for the potential application of RB in pancreatic cancer therapy.

Systemic delivery of the anticancer agent arenobufagin using polymeric nanomicelles.

Arenobufagin (ABG) is a major active component of toad venom, a traditional Chinese medicine used for cancer therapy. However, poor aqueous solubility limits its pharmacological studies in vivo due to administration difficulties. In this study, we aimed to develop a polymeric nanomicelle (PN) system to enhance the solubility of ABG for effective intravenous delivery. ABG-loaded PNs (ABG-PNs) were prepared with methoxy poly (ethylene glycol)-block-poly (d,l-lactic-co-glycolic acid) (mPEG-PLGA) using the solvent-diffusion technique. The obtained ABG-PNs were 105 nm in size with a small polydispersity index of 0.08. The entrapment efficiency and drug loading were 71.9% and 4.58%, respectively. Cellular uptake of ABG-PNs was controlled by specific clathrin-mediated endocytosis. In addition, ABG-PNs showed improved drug pharmacokinetics with an increased area under the curve value (a 1.73-fold increase) and a decreased elimination clearance (37.8% decrease). The nanomicelles showed increased drug concentrations in the liver and lung. In contrast, drug concentrations in both heart and brain were decreased. Moreover, the nanomicelles enhanced the anticancer effect of the pure drug probably via increased cellular uptake of drug molecules. In conclusion, the mPEG-PLGA-based nanomicelle system is a satisfactory carrier for the systemic delivery of ABG.

Bufalin reverses acquired drug resistance by inhibiting stemness in colorectal cancer cells.

Drug resistance is an obstacle to chemotherapy in tumor patients. Recent studies have shown that the high stemness of cancer cells may be induced by chemotherapeutic drugs, which is correlated with drug resistance. In the present study, we investigated the effects of bufalin on the stemness of colorectal cancer. We found that cisplatin could induce high stemness through the tumorsphere formation assay in vitro and in vivo in the colorectal cancer cell lines HCT116 and LoVo. In addition, cisplatin-treated tumorsphere cells showed drug­resistant properties. These results suggested that acquired drug resistance induced by cisplatin in colorectal cancer cells occurred via high stemness. On assessing the effects of bufalin, a traditional Chinese medicine monomer, we found that it could reverse the high stemness and drug resistance induced by cisplatin in colorectal cancer. These findings suggest that bufalin plays an adjuvant role in colorectal cancer chemotherapy and may help reverse acquired drug resistance. These findings may aid in the development of new therapeutic strategies.

Liver-targeting Resibufogenin-loaded poly(lactic-co-glycolic acid)-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles for liver cancer therapy.

Liver cancer remains a major problem around the world. Resibufogenin (RBG) is a major bioactive compound that was isolated from Chansu (also called toad venom or toad poison), which is a popular traditional Chinese medicine that is obtained from the skin secretions of giant toads. RBG has strong antitumor effects, but its poor aqueous solubility and its cardiotoxicity have limited its clinical use. The aim of this study was to formulate RBG-loaded poly(lactic-co-glycolic acid) (PLGA)-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticle (RPTN) to enhance the treatment of liver cancer. RPTN, RBG-loaded PLGA nanoparticle (RPN), and RBG/coumarin-6-loaded PLGA-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticle (RCPTN) were prepared. The cellular uptake of RCPTN by HepG2 and HCa-F cells was analyzed using confocal laser scanning microscopy. Apoptosis was induced in HepG2 cells by RPTN, RBG solution (RS), and 5-fluorouracil solution (used as the negative controls), as assayed using flow cytometry. LD50 (median lethal dose) values were determined for RS and RPTN, and the liver-targeting properties were determined for RCPTN in intravenously injected mice. A pharmacokinetic study was conducted in rats, and the in vivo therapeutic effects of RPTN, RPN, and RS were examined in a mouse tumor model. The results showed that RCPTN simultaneously delivered both coumarin-6 and RBG into HepG2 and HCa-F cells. The ratio of apoptotic cells was increased in the RPTN group. The LD50 for RPTN was 2.02-fold higher than the value for RS. Compared to RS, RPTN and RPN both showed a significant difference in vivo not only in the pharmacodynamic study but also in anticancer efficacy, and RPTN performed much better than RPN. The detection indexes for drug concentration and fluorescence inversion microscopy images both demonstrated that RCPTN was much better at targeting the liver than RS. The liver-targeting RPTN, which displayed enhanced pharmacological effects and decreased toxicity for the loaded drug RBG, is therefore a promising intravenous dosage form that may be useful in the treatment of liver cancer.

Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin Pill following oral administration to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), widely used to treat cardiovascular diseases such as angina pectoris and myocardial infarction. Bufadienolides are major active compounds of Venenum Bufonis, which is one of the seven materiamedicas that comprise the Shexiang Baoxin Pill. Previous pharmacokinetics studies of bufadienolides have typically used a single medicinal material delivered to rats. In this study, we have chosen the mouse, a more proper animal model than the rat, to investigate the in vivo pharmacokinetics and tissue distribution of bufadienolides from the Shexiang Baoxin Pill. MATERIALS AND METHODS: The concentrations of bufadienolides in plasma and tissues were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS). The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3 column by gradient elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. RESULTS: The results showed that the five bufadienolides were rapidly absorbed and distributed into the body. The pharmacokinetic curve showed double peaks after oral administration. The major tissue depots for resibufogenin, bufalin, and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. CONCLUSION: The information gained from this research provides a meaningful insight for the clinical applications of the Shexiang Baoxin Pill.

[Research on therapeutic effects of bufonis venenum on L7212 leukemia and its mechanism].

OBJECTIVE: To study the therapeutic effects of Bufonis Venenum on L7212 leukemia and the potential mechanism. METHODS: L7212 leukemia model mice were randomly divided into four groups: model group, low and high dose Bufonis Venenum groups and chemotherapy group. Normal mice were treated as control group. Mice were injected intraperitoneally for 10 days continually. The body weight, survival time, peripheral blood leukocyte, hepatic and splenic indexes, bone marrow leukocyte and T lymphocyte were observed and determined. RESULTS: Body weight of L7212 leukemia model group mice were decreased significantly. Compared with other groups, high dose Bufonis Venenum group's weight loss was the least. Bufonis Venenum groups survived longer than L7212 model group. Compared with model group, high dose Bufonis Venenum group's liver index was higher (P < 0.05). After inoculation for 1 day, leukocyte count as well as percentage of leukemic cells within five groups had no significant difference (P > 0.05). Compared with the model group, after inoculation for 10 days, leukocyte count in Bufonis Venenum groups and the chemotherapy group were significantly reduced (P < 0.05). Percentage of leukemia cells in blood and bone marrow in high dose Bufonis Venenum group was significantly decreased (P < 0.05). Compared with model group, CD3+ and CD4+ in Bufonis Venenum groups and the chemotherapy group were increased, CD8+ was decreased, but had no significant difference (P > 0.05). CONCLUSION: Bufonis Venenum has therapeutic effects on the L7212 leukemia by inducing apoptosis and improving immune system.

Antiproliferative activity and new argininyl bufadienolide esters from the "cururú" toad Rhinella (Bufo) schneideri.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads known as "cururú" (Rhinella schneideri) have been used in the Brazilian Pantanal and Paraguayan Chaco wetlands to treat erysipelas and cancer. The aim of the study was to assess the antiproliferative effect of the venom obtained from Rhinella schneideri and to identify its constituents by spectroscopic and spectrometric methods. MATERIALS AND METHODS: The venom was obtained by gentle pressing the parotid glands of the toads. The dry crude drug was analyzed by HPLC-MS-MS and chromatographed on Sephadex LH-20 to obtain purified compounds and fractions for spectroscopic analysis. The venom and fractions were evaluated for antiproliferative activity towards normal human lung fibroblasts (MRC-5) and four human cancer cell lines: gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1), bladder carcinoma (J82) and promyelocytic leukemia (HL-60). RESULTS: From the Rhinella schneideri venom, 29 compounds were isolated and/or identified by spectroscopic and spectrometric means. Three known alkaloids and five argininyl diacids were identified in the complex mixture by HPLC-MS-MS. Nine out of fifteen argininyl diacid derivatives of the bufadienolides bufalin, marinobufagin and telocinobufagin are reported for the first time and four argininyl diacids are described for the first time as natural products. The venom and the fractions 9-13 showed a remarkable antiproliferative effect, with IC50 values in the range 0.019-0.022, 0.035-0.040, 0.028-0.064, 0.042-0.056 and 0.044-0.052 µg/mL for MRC-5, AGS, SK-MES-1, J82 and HL-60 cell lines, respectively. Under the same experimental conditions, IC50 values of the reference compound etoposide were 2.296, 0.277, 1.295, 1.884 and 1.059 µg/mL towards MRC-5, AGS, SK-MES-1, J82 and HL-60 cells, respectively. CONCLUSIONS: The venom showed a strong antiproliferative effect towards human cancer cells and presented a high chemical diversity in its constituents, supporting its use as anticancer agent. These findings encourage further work on the chemistry and bioactivity of South American toad venoms.

Bufalin loaded biotinylated chitosan nanoparticles: an efficient drug delivery system for targeted chemotherapy against breast carcinoma.

Bufalin is a traditional oriental medicine which is known to induce apoptosis in many tumor cells, and it is thus considered as a new anticancer therapeutic. By now, most of the studies of bufalin are in vitro, however in vivo evaluations of its therapeutic efficacy are less and are in great demand for its development toward anticancer drug. One of the problems probably hampering the development of bufalin is the lack of tumor selectivity, which may reduce the therapeutic effect as well as showing side effects. To overcome this drawback, in this study, we designed a tumor-targeted drug delivery system of bufalin based on enhanced permeability and retention (EPR) effect, by using biotinylated chitosan, resulting in bufalin encapsulating nanoparticles (Bu-BCS-NPs) with mean hydrodynamic size of 171.6 nm, as evidenced by dynamic light scattering and transmission electron microscope. Bu-BCS-NPs showed a relative slow and almost linear release of bufalin, and about 36.8% of bufalin was released in 24 h when dissolved in sodium phosphate buffer. Compared to native bufalin, Bu-BCS-NPs exhibited a stronger cytotoxicity against breast cancer MCF-7 cells (IC50 of 0.582 µg/ml vs 1.896 µg/ml of native bufalin). Similar results were also obtained in intracellular reactive oxygen species production, apoptosis induction, and decrease in mitochondria membrane potential. These results may contribute to the rapid intracellular uptake of nanoparticles, partly benefiting from the highly expressed biotin receptors in tumor cells. In vivo studies using MCF-7 tumor models in nude mice confirmed the remarkable therapeutic effect of Bu-BCS-NPs. These findings suggest the potential of Bu-BCS-NPs as an anticancer drug with tumor targeting property.

[Effects of cinobufagin on apoptosis in U-2OS osteosarcomas cells].

OBJECTIVE: To investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism. METHODS: The cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. RESULTS: The results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P < 0.05). The apoptotic rate of experimental group (46.87% +/- 11.23%) was significantly higher than that of the control group (2.34% +/- 0.98%) and blank control group (1.04% +/- 0.25%) (P < 0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14 +/- 0.32, 1.31 +/- 0.41, and 1.92 +/- 0.54, respectively, which were significantly higher than that in control group (P < 0.05). Compared with 20and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P < 0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P < 0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P < 0.05). CONCLUSION: Cinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.

Cyto-/genotoxic effects of the ethanol extract of Chan Su, a traditional Chinese medicine, in human cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Chan Su, an ethanolic extract from skin and parotid venom glands of the Bufo bufo gargarizans Cantor, is widely used as a traditional Chinese medicine for cancer therapy. Although the anti-cancer properties of Chan Su have been investigated, no information exists regarding whether Chan Su has genotoxic effects in cancer cells. The aim of the present study was to examine the cyto-/genotoxic effect of Chan Su in human breast carcinoma (MCF-7 cells), human lung carcinoma (A-549 cells), human T cell leukemia (Jurkat T cells), and normal human lymphocytes. MATERIALS AND METHODS: Effects on the viability of MCF-7, A-549, Jurkat T cells, and normal lymphocytes were evaluated by Trypan blue exclusion assays. The DNA content in the sub-G1 region was detected by propidium iodide (PI) staining and flow cytometry. The genotoxicity of Chan Su was assessed by single-cell gel electrophoresis (comet assay) and the cytokinesis-block micronucleus assay (CBMN assay). RESULTS: Chan Su significantly inhibited the viability of MCF-7, A-549, and Jurkat T cells dose dependently, but had no effect on normal human lymphocytes. Apoptotic death of the cancer cells was evident after treatment. Chan Su also induced genotoxicity in a dose-dependent manner, as indicated by the comet and cytokinesis-block micronucleus assays. CONCLUSIONS: These findings suggest that Chan Su can induce apoptotic death of, and exert genotoxic effects on, MCF-7, A-549, and Jurkat T cells.

Bufalin induces G2/M phase arrest and triggers autophagy via the TNF, JNK, BECN-1 and ATG8 pathway in human hepatoma cells.

Liver cancer is the fifth most common cause of cancer death worldwide. The study of more effective anti-hepatoma drugs is urgently required. Bufalin has been isolated from a traditional Chinese medicine and possesses less toxicity to normal cells. However, it has been found to inhibit growth of cancer cells. In this study, we aimed to investigate the efficacy and mechanism of bufalin in Huh7, Hep3B and HA22T human hepatoma cells. The three cell lines were treated with bufalin, the proliferation was detected by WST-1 assay and cell cycle was detected by flow cytometry analysis. The results showed that bufalin inhibited the proliferation of hepatoma cells and regulated the hepatoma cell death program in a dose- and time-dependent manner without typical features of apoptosis. RT-PCR arrays were used to investigate the autophagy transcriptional response triggered by bufalin and 13 genes were altered and further confirmed by real-time PCR. The translation levels of selected genes were examined by western blot analysis to reveal the bufalin-induced autophagy cascade. Bufalin synergized with the JNK pathway to induce autophagy of hepatoma cells and is closely associated with the upregulation of TNF, BECN-1, MAPK and ATG8, together with the downregulation of Bcl-2 and Bid. Our study provided a multi-angle evaluation system for anti-hepatoma pharmacology for pre-clinical drug investigation. In this case, bufalin was capable of inducing hepatoma cell autophagy, suggesting a potential regimen for single or combined chemotherapy to overcome hepatoma in clinical practice.

Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells.

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho­mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.

[A comparison study of pharmacokinetics between bufalin-loaded bovine serum albumin nanoparticles and bufalin in rats].

OBJECTIVE: To determine the bufalin concentration in rats' plasma by establishing a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, and to evaluate and compare the pharmacokinetic characteristics of bufalin-loaded bovine serum albumin nanoparticles (bufalin-BSA-NP) and bufalin. METHODS: Thirty Wistar rats were randomly divided into six groups with five rats in each group, and administered with a single dose of 0.6, 0.3 and 0.15 mg/kg of bufalin-BSA-NP or bufalin, respectively. After the administration, blood samples were collected from the orbital venous plexus at designed time points (1, 5, 8, 10, 15, 20, 30, 45, 60, 120, 180, 300 and 480 min). The concentration of bufalin in plasma at different sampling time points was determined by HPLC-MS/MS. The pharmacokinetic parameters were calculated and compared. RESULTS: The established HPLC-MS/MS method had high linearity, precision and accuracy. The blood plasma area under curve, the mean retention time and the terminal half life of bufalin-BSA-NP were 1.19 to 1.81, 2.12 to 3.61 and 2.17 to 2.94 times of bufalin, respectively. CONCLUSION: Bufalin-BSA-NP has the function of sustained release thus to prolong the bufalin remaining in blood.

[Clinical re-evaluation of effects of two different "cocktail therapy" to prevent from phlebitis induced by Chansu injection].

OBJECTIVE: To re-evaluate the effects of different "cocktail therapy" to prevent from phlebitis induced by Chansu injection. METHOD: Patients treated with Chansu injection were divided randomLy into 4 groups with 90 per group, control group, phentolaminum group, the magnesium sulfate group-phentolaminum group, and anisodamine-phentolaminum group. Patients in the control group only received the routine nursing treatment, and patients in the various experiment group received different interventions. The comparison was made in the morbidity and the starting time of occurrence of phlebitis, the severity of pain, duration of pain. RESULT: The morbidity of phlebitis was 8%, 8%, 6%, respectively. The starting time of phlebitis occurrence was (22 +/- 4), (27 +/- 5), (28 +/- 7) h, respectively. The NRS of pain was (4.75 +/- 1.51), (3.27 +/- 1.02), (2.71 +/- 1.63), respectively. The duration time of pain was (4.25 +/- 1.36), (2.51 +/- 1.05), (2.19 +/- 1.13) d respectively. In control group, the morbidity of phlebitis, the starting time of occurrence of phlebitis, the severity of pain, duration of pain was 30%, (16 +/- 4) h, (6.34 +/- 1.21), (5.47 +/- 1.07) d, respectively. As compared with the control group, a significance difference was found between every group in three test groups and control group respectively (P<0.05). CONCLUSION: The morbidity and the starting time of occurrence of phlebitis, the severity of pain, duration of pain was significantly reduced respectively by two different "cocktail therapy".

Improved antitumor efficacy and reduced toxicity of liposomes containing bufadienolides.

Long-circulating liposomes are used extensively nowadays for enhancing the therapeutic effect and reducing the toxicity of anticancer drugs. In this paper, a traditional Chinese medicine, toad venom, which has long been used in the clinic for tumor therapy with unpleasant side effects, was incorporated into poloxamer modified liposomes to increase its antitumor effect and reduce its toxicity. Our preparation of bufadienolides liposomes had a particle size of around 70 nm and an entrapment efficiency of about 87.6%. Lyophilized liposomes well retained their appearance, particle size and encapsulation efficiency for 3 months. The in vitro release results verified the sustained release properties of the bufadienolides liposomes. The concentration of bufadienolides in modified liposomes that caused 50% cell killing was much lower than that of free drug for both Lovo cells and NCI-H157 cells. Compared to the bufadienolides solution and the unmodified liposomes, the bufadienolides liposomes significantly prolonged the retention time and increased the area under the curve in vivo. The antitumor efficiency of the bufadienolides liposomes against mice bearing H22 liver cancer cells and Lewis pulmonary cancer cells were 2.15 and 2.96, respectively, times that of a bufadienolides solution at the same toxicity. The safety test results demonstrated that the bufadienolides liposomes had an LD(50) that was 3.5 times the LD(50) of bufadienolides solution and caused no allergen-related or blood vessel irritation effects. All these results proved that poloxamer modified bufadienolides liposomes have improved antitumor efficacy and safety.