To study the pharmacokinetics of Chan Su, a sensitive and selective method was developed and validated for the determination of five main bufadienolides (cinobufagin, resibufogenin, bufalin, bufotalin and arenobufagin) in rat plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate after internal standard (IS, caudatin) spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 microm, 2.1 mmx100 mm) and a C18 guard column (5 microm, 4.0 mmx2.0 mm) with an isocratic mobile phase consisted of acetonitrile-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. The nominal retention times for cinobufagin, resibufogenin, bufalin, bufotalin, arenobufagin and caudatin were 3.07, 3.55, 2.30, 1.62, 1.22 and 3.43 min, respectively. All analytes showed good linearity in a wide concentration range (r>0.995) and their lower limits of quantification (LLOQ) were all 1.0 ng/mL. The method was linear for all analytes with correlation coefficients>0.995 for all analytes. The average extract recoveries of the five analytes from rat plasma were all over 85%, the precisions and accuracies determined were all within 15%. This method has been successfully applied to pharmacokinetic study of Chan Su in rats following oral administration.

Four bufotoxins were isolated by means of preparative reversed phase HPLC from the water soluble fraction of the skin of Bufo bufo gargarizans Cantor. On the basis of their spectral data (UV, IR, 1HNMR, 13CNMR, EIMS and FDMS), amino acid analyses and chemical properties, they were identified as bufotalin 3-succinoylarginine ester(I), bufalin 3-succinoylarginine ester(II), cinobufagin 3-succinoylarginine ester(III), and resibufogenin 3-succinoylarginine ester(IV). Compound I is a new bufotoxin.

The method of enzyme immunoassay (EIA) was developed for measurement of anti-resibufogenin IgG reactive substance (RRS) in plasma and urine of healthy volunteers receiving a Senso containing drug, Kyushin (KY). Unchanged bufalin (BF), cinobufagin (CB), and resibufogenin (RB) and their metabolites in plasma and urine were also measured by EIA after the separation of these compounds by high performance liquid chromatography. In a single dosing study, the concentration of BF, CB and RB in plasma reached maximum at 1-2 hr after drug administration, and the half-lives (T1/2 of these were 15.8, 8.94 and 11.5 hr, respectively. In a multiple dosing study, the concentration of BF, CB and RB measured fitted on the estimated curve calculated from data of the single dosing study. These results suggest that BF, CB and RB, important components of Senso, do not have a cumulative nature.

Microbial transformation of a cytotoxic bufadienolide, cinobufagin (1), was performed by Syncephalastrum racemosum. The six metabolites obtained were identified as 7beta-hydroxycinobufagin ( 2), 12beta-hydroxycinobufagin (3), cinobufotalin (4), 5,12beta-dihydroxycinobufagin (5), 4beta,11alpha-dihydroxycinobufagin (6), and 4beta,12alpha-dihydroxycinobufagin (7), respectively, on the basis of spectroscopic studies. Metabolites 2 and 5-7 were characterized as new compounds, and 2-7 proved to be cytotoxic against Bel-7402 human hepatoma cells.

Background. Cinobufagin has been widely used in the treatment of carcinoma and plays an important role in the relief of cancer pain. But the involved mechanism remains unknown. Aim. To investigate the changes in thermal and mechanical hyperalgesia in paw cancer pain in mice and the action mechanism of cinobufagin using a paw cancer pain model. Methods. 60 female mice were randomly divided into 5 groups: control group, model group, cinobufagin group, cinobufagin +NAL-M group, and morphine group; except ones in control group, mice were inoculated with H22 hepatoma cells in the right hind paw. From the 9th day after inoculation, mice were administrated drug once daily lasting for 8 days. The pain behavior was determined on the 2nd, 4th, 6th, and 8th days before and after administration. On the last day, they were sacrificed. The levels of β -END, CRF, and IL-1 β were analyzed by ELISA; immunohistochemistry was performed to detect the expressions of β -END, POMC, and μ -OR in the tumor and adjacent tissue. Results. The thresholds of thermal pain and mechanical pain were significantly increased by cinobufagin. Moreover, the expressions of β -END, CRF, POMC, and μ -OR were significantly upregulated by cinobufagin. The analgesic effect of cinobufagin was blocked by the peripheral opioid receptor antagonist NAL-M. Conclusions. Cinobufagin significantly relieved cancer pain in mice and raised their pain threshold, mainly upregulating the expression levels of β -END and μ -OR in the hind paw tumor and adjacent tissue.

Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1 μM concentration both RBG and CBG could alter some channel kinetics and gating properties of IK, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that IK is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides' pathological effects on central nervous system.

The objective of this study was to evaluate the immunomodulatory effects of cinobufagin (CBG) isolated from Chan Su (Venenum Bufonis) in vitro. In this paper, our results show that CBG significantly stimulated cell proliferation of splenocytes and peritoneal macrophages (PMΦ) and markedly enhanced the phagocytic activation of PMΦ. CBG also significantly increased CD4(+)CD8(+) double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes. The levels of several Th1 cytokines, including interferon-γ and tumor necrosis factor-α, are significantly increased after CBG treatment, whereas the levels of the Th2 cytokine interleukin-4 and interleukin-10 are significantly decreased. As a result, the ratio of Th1/Th2 also increased. Taken together, these results indicated that CBG had potential immune system regulatory effects and suggested that this compound could be developed as a novel immunotherapeutic agent to treat immune-mediated diseases such as cancer.

OBJECTIVE

To investigate the in vitro effects and mechanism of action of cinobufacini on apoptosis of lens epithelial cells (LEC).

METHODS

Rabbit LEC were cultured for 72 hours with cinobufacini at different concentrations(0.0 [control], 0.1, 0.2, 0.3mg/L). The inhibition ratio of cinobufacini acting on LEC was analyzed by ethyl thiazolyl tetrazolium(MTT); the changes in DNA structure, by electrophoresis, and the apoptosis rate, by flow cytometry. The mRNA expression of apoptosis-related genes bcl-2 and bax was examined using the reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

At concentrations of 0.1mg/L-0.3mg/L, cinobufacini inhibited LEC proliferation. The inhibition ratio increased as the concentration of the drug increased. The typical DNA-ladders on electrophoretic gels were observed for extracts of LEC in the treated groups. The higher the drug concentration (0.1, 0.2, and 0.3mg/L) was, the higher the apoptosis rate (20.47±0.65%, 27.14±0.95%, and 33.49±0.77%, respectively) would be. The apoptosis rates in these groups were significantly different from those of the control group (P<0.01). With the drug concentration increasing, the mRNA expression levels of the pro-apoptotic bax increased, whereas those of the anti-apoptotic bcl-2 decreased.

CONCLUSIONS

Cinobufacini can notably induce apoptosis of LEC by decreasing the ratio of bcl-2 to baxin vitro. With its low toxicity, this medication may be effective in the prevention and treatment of posterior capsule opacification.

UNASSIGNED

To discuss and assess the clinical value of treating lung cancer cachexia with thalidomide combined with cinobufagin.s.

UNASSIGNED

A cohort of 54 patients, who were diagnosed with lung carcinoma, was randomly divided into two groups, a trial group and a control group, respectively. The trial group was given 150 mg/day thalidomide and 2700 mg/day cinobufagin; the control group only received 2,700 mg/day cinobufagin. The therapy lasted for 12 weeks, and the nutritional status, quality of life, survival, and side effects in patients in the two groups were recorded.

UNASSIGNED

The nutritional status, quality of life, and survival of patients with lung cancer cachexia in the trial group were significantly improved compared to the control group. The trial group received 150 mg thalidomide, which by contrast reduced the incidence of side effect and increased tolerance.

UNASSIGNED

Using thalidomide combined with cinobufagin to treat patients with lung cancer cachexia will significantly improve their nutritional status and quality of life. This therapy is better than that using cinobufagin alone and is well tolerated.

Effects of bufadienolides such as bufalin (BF) and cinobufagin (CB), the main components of Senso (Ch'an Su), on myocardial Na+,K(+)-ATPase activity, the cardiotonic activity in vivo and the action potential of isolated guinea pig papillary muscle cells were compared with those of other cardiotonic drugs. 1) The rank order of potency for inhibition of myocardial Na+,K(+)-ATPase activity was BF greater than digoxin (DG) greater than digitoxin (DT) greater than telocinobufagin greater than gamabufotalin greater than cinobufotalin greater than CB greater than g-strophanthin (GS) greater than digitoxigenin (DTG) greater than resibufogenin (RB) when compared at the 50% inhibitory concentration. 2) In isolated papillary muscle cells, CB shortened the action potential duration (APD) dose-dependently. The order of potency for shortening of APD was GS greater than CB greater than DTG much greater than DT. 3) In open-chest guinea pigs, intraduodenal administration of BF or CB increased the myocardial contractile force (MCF), but did not affect the heart rate. The order of potency for increase in MCF was as follows: methyldigoxin, proscillaridin greater than BF greater than CB greater than DG greater than Senso much greater than DT, DTG, RB. These results indicate that CB has a shortening effect on APD and an inhibitory effect on Na+,K(+)-ATPase activity along with its cardiotonic effect, like GS.

A rapid cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC) method was proposed for the determination of resibufogenin and cinobufagin in the Chinese herbal extracts from toad venom and its medicinal preparation (Liushen tablet). The two components have the close structural similarity and similar hydrophobicity, which result in poor resolution in normal MEKC. The addition of neutral beta-CD to the MEKC system was found to improve the separation of the studied compounds. The effects of several CD-MEKC parameters on the resolutions were evaluated systematically. Based on the investigation, a background electrolyte solution consisting of 10 mM borate buffer adjusted to pH 8.5, 40 mM sodium dodecyl sulfate (SDS), 12 mM beta-CD and 10% (v/v) of methanol was found to be optimal conditions for the fast separation. The contents of resibufogenin and cinobufagin were successfully determined within 5 min, with satisfactory repeatability and recovery.

Chansu, a galenical preparation of the dried white venom of Chinese Bufo bufo gargarizans, is one of the major components of Kyushin, a traditional Chinese medicine. Kyushin is reported to have a cardiotonic effect that has been suggested to be due to the action of bufadienolides such as bufalin and cinobufagin. Recently, we found that administration of bufalin in male rats diminished the luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) and the secretion of testosterone both in vivo and in vitro. These observations suggest that Chansu may possess hypogonadal effects in male rats. In the present study, the effects of the methanol extract of Chansu on hypothalamic-pituitary-testicular function in male rats were examined. Crude Chansu was extracted by methanol and purified by a Sep-Pak C18 column. No activity of bufalin, cinobufagin, estradiol, or digoxin in purified methanol extract was detected; all Chansu used in this study was the purified methanol extract. A single intravenous injection of Chansu resulted in a decrease of the basal (20% to 55%) and human chorionic gonadotropin (hCG)-induced (35% to 40%) levels of plasma testosterone and the GnRH-induced level of plasma LH (25% to 30%). Administration of Chansu in vitro decreased basal and hCG-stimulated testosterone production by 60% to 70% and 40% to 60%, respectively, as well as spontaneous and forskolin- or 3-isobutyl-1-methylxanthine (IBMX)-induced accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) by 30% to 45% in rat testicular interstitial cells. Although LH release by rat anterior pituitary glands was diminished, GnRH release by the rat mediobasal hypothalamus was enhanced by administration of Chansu in vitro. These results suggest that the bufalin-free extracts of Chansu inhibit testosterone secretion in rats, in part, due to (1) a decreased production of testicular cAMP, (2) a decreased response of testosterone to gonadotropin, and (3) a reduction of the LH response to GnRH.

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

Keywords: Anti-AChE activity; avian; chick neurobiological preparations; cytotoxicity; neuromuscular blockade; toad poison.

BACKGROUND

Chansu is a transitional Chinese medicine that has been used for centuries as therapy for inflammation, anaesthesia and arrhythmia in China and other Asian countries. Recently, it has also been used for anti-cancer purposes. We have previously shown that Chansu has a huge pro-apoptotic potential on colon cancer cells, but to date the detailed mechanism of this action is not well understood.

METHODS

One of the major components of Chansu, Cinobufagin (CBF) was used to treat cancer cells. The expressions of levels of cortactin, an important factor in tumour progression and cancer invasion, were assessed in in vitro and in vivo experiments. Additional analyses were performed in subcellular protein fractions and immune-fluorescent staining was used to define cortactin protein expression and the changes of location in CBF-treated cells.

RESULTS

CBF strongly inhibited the expression of cortactin in HCT116 cells. There were reductions of both mRNA transcription and protein synthesis, which were more significant in the absence of oxygen in vitro. In addition, nuclear translocation of cortactin was observed in HCT116 cells post CBF exposure but not in the negative control, indicating that CBF is likely to interrupt co-localisation of cortactin to cytoskeletal proteins. Most importantly, CBF could diminish the expression of cortactin in human HCT116 xenograft tumours in nude mouse in vivo.

CONCLUSIONS

CBF inhibits cortactin expression and nuclear translocation in colon cancer cells in vitro and in mouse models bearing human colon tumour in vivo, suggesting it might disrupt actin-regulated cell movement. Thus, CBF or Chansu could be developed as an effective anti-cancer therapy to stop local invasion and metastasis.

Cinobufagin (1) is a major bufadienolide in ChanSu (a traditional Chinese medicine) with a wide range of pharmacological activities. In this paper, the in vivo metabolites of 1 in rats were studied. Nine metabolites were isolated from the bile of rats, and their structures were identified as: desacetylcinobufagin (2), 3-ketodesacetylcinobufagin (3), 3-epi-desacetylcinobufagin (4); 5beta-hydroxy-3-epi-desacetylcinobufagin (5), 1alpha-hydroxy-3-epi-desacetylcinobufagin (6), 12beta-hydroxy-3-epi-desacetylcinobufagin (7), 1beta-hydroxy-3-epi-desacetylcinobufagin (8), 1alpha,5alpha-dihydroxy-3-epi-desacetylcino-bufagin (9), and 2alpha, 5beta-dihydroxy-3-epi-desacetylcinobufagin (10), respectively, on the basis of widely spectroscopic studies including two-dimensional-nuclear magnetic resonance (NMR). Among them, metabolites 6-10 are new compounds. The results show that hydroxylation is the main reaction involved in metabolism of 1, and the preferred hydroxylation sites were C-1 and C-5.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was in the range of 0.5-10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74-13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications.

Emerging evidence has shown that cinobufagin, as an active ingredient of Venenum Bufonis, inhibits tumor development. The aim of this study was to investigate the inhibitory effects of cinobufagin on A375 human malignant melanoma cells. MTT and colony formation assays showed that cinobufagin significantly inhibited A375 cell proliferation and cell colony formation. Additional studies demonstrated that cinobufagin markedly increased the levels of ATM serine/threonine kinase (ATM) and checkpoint kinase 2 (Chk2) and decreased the levels of cell division cycle 25C (CDC25C), cyclin-dependent kinase 1 (CDK1), and cyclin B, subsequently inducing G2/M cell cycle arrest in A375 cells. Moreover, cinobufagin clearly inhibited the levels of phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), AKT, p-AKT, and B-cell lymphoma 2 (Bcl-2). By contrast, it increased the levels of Bcl-2-associated death promoter, Bcl-2-associated X, cytoplasmic cytochrome C, and apoptotic protease activating factor 1, leading to increased levels of cleaved caspase-9 and cleaved caspase-3, resulting in the apoptosis of A375 cells. Together, these results indicate that cinobufagin can induce cell cycle arrest at the G2/M phase and apoptosis, leading to inhibition of A375/B16 cell proliferation. Thus, cinobufagin may be useful for melanoma treatment.

Chemical defences are widespread in nature, yet we know little about whether and how climatic and geographic factors affect their evolution. In this study, we investigated the natural variation in the concentration and composition of the main bufogenin toxin in adult Asian toads (Bufo gargarizans Cantor) captured in twenty-two regions. Moreover, we explored the relative importance of eight climatic factors (average temperature, maximum temperature, minimum temperature, average relative humidity, 20-20 time precipitation, maximum continuous precipitation, maximum ground temperature, and minimum ground temperature) in regulating toxin production. We found that compared to toads captured from central and southwestern China, toads from eastern China secreted higher concentrations of cinobufagin (CBG) and resibufogenin (RBG) but lower concentrations of telocinobufagin (TBG) and cinobufotalin (CFL). All 8 climatic variables had significant effects on bufogenin production (r_i>0.5), while the plastic response of bufogenin toxin to various climate factors was highly variable. The most important climatic driver of total bufogenin production was precipitation: the bufogenin concentration increased with increasing precipitation. This study indicated that the evolution of phenotypic plasticity in chemical defences may depend at least partly on the geographic variation of defensive toxins and their climatic context.

In this study, the application of ultrasound-assisted extraction (UAE) method was shown to be more efficient in extracting anti-tumor bufadienolides (bufalin, cinobufagin and resibufogenin) from important animal medicine of ChanSu than the maceration extraction (ME) and soxhlet extraction (SE) method. The effects of ultrasonic variables including extraction solvent, solvent concentration, solvent to solid ratio, ultrasound power, temperature, extraction time and particle size on the yields of three bufadienolides were investigated. The optimum extraction conditions found were: 70% (v/v) methanol solution, solvent to solid ratio of 10ml/g, ultrasound power of 125W, temperature of 20°C, extraction time of 20min and particle size of 60-80 mesh. The extraction yields of bufalin, cinobufagin and resibufogenin were 43.17±0.85, 52.58±1.12, 137.70±2.65mg/g, respectively. In order to achieve a similar yield as UAE, soxhlet extraction required 6h and maceration extraction required much longer time of 18h. The results indicated that UAE is an alternative method for extracting bufadienolides from ChanSu.

Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca(2+) current (I Ca-L) of ventricular myocytes remains undefined. The aim of the present study was to characterize the effect of CBG on intracellular Ca(2+) ([Ca(2+)]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence on I Ca-L, Ca(2+) transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10(-8) M) decreased the maximal inhibition of CBG by 47.93%. CBG reduced I Ca-L in a concentration-dependent manner with an IC50 of 4 × 10(-10) M, upshifted the current-voltage curve of I Ca-L, and shifted the activation and inactivation curves of I Ca-L leftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca(2+) transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca(2+) channels, and reduced [Ca(2+)]i and contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.

The metabolism of cinobufagin (CB) in rat liver microsomes was studied. By comparison of retention times and fragmentation patterns obtained by thermospray LC/MS with those of authentic standards, four metabolites of CB were also identified, in addition to deacetylCB (M1) and 3-epideacetylCB (M2), which have been reported previously. The additional four metabolites were identified to be 3-epiCB (M4), 3-ketoCB (M5), 3-keto-16 beta-OH-deacetylCB (M3), and 3-keto-16 alpha-OH-deacetylCB (M6). The primary metabolic products of CB were M1 and M2, with a small amount of M4 and trace amounts of M3, M5, and M6. These results indicate that major metabolic routes of CB were deacetylation at the 16-position and epimerization at the 3-position via the 3-keto intermediate.

An efficient separation method of using high-speed counter-current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:6:3:4, v/v) was used in high-speed counter-current chromatography. A total of 9 mg of 4beta,12alpha-dihydroxyl-cinobufagin (1), 15 mg of 12beta-hydroxyl-cinobufagin (2), 8 mg of 5beta-hydroxyl-cinobufagin (3), 12 mg of deacetylcinobufagin (4) and 6 mg of 3-keto-cinobufagin (5) were obtained in a one-step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of (1)H-NMR and (13)C-NMR technology. All products (1-5) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.

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).

CONCLUSIONS

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.

To assess the clinical effect and safety of Chinese traditional medicine injection combined with radiotherapy for esophageal cancer. The relative randomized controlled trials (RCTs) of Chinese medical injections (CMI) combined with radiotherapy as well as simple radiotherapy for esophageal cancer were searched from PubMed, Cochrane Library, EMBASE, Chinese Biomedical Literature Database(CBM), China National Knowledge Infrastructure (CNKI), Wanfang Database and VIP Database as at September 2014. Two researchers completed the data extraction and quality evaluation independently. The data were analyzed by GeMTC 0.14.3 and Stata 12. 0 software. Finally, 43 RCTs involving 3 289 patients were finally included. The star network was constructed by different comparison groups. The results of network meta-analysis showed that the seven CMIs combined with radiotherapy was superior to simple radiotherapy in the treatment of esophageal cancer in efficacy, quality of life, and reduction in the incidence of nausea and leucopenia, but with no significant difference among the seven CMIs. Probability ranking result showed a great possibility for Shenqi Fuzheng and astragalus polysaccharide injections in improving the overall response rate and quality of life, which were followed by cinobufagin and kangai injections. However, only one study was included for Shenqi Fuzheng and astragalus polysaccharide injections. Therefore, cinobufagin or kangai injections were preferred in improving the overall response rate and quality of life. Aidi or compound sophora injections were better than other CMIs in reducing? the incidences of nausea (III-IV) and leukopenia. More RCTs of Shenqi Fuzheng and astragalus polysaccharide injections combined with radiotherapy for patients with esophageal cancer were expected in the future to confirm our results. Moreover, study findings will be reported, particularly for the adverse events in radiotherapy for esophageal cancer.