Study On The Screening, Characterization And Anti-tumor Mechanism Of Anti-hepatocarcinoma Components From Cinobufacini

Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms worldwide and its incidence has been increasing over the past few decades. It is necessary to develop more effective and low toxic anti-hepatocarcinoma reagents for the prevention and treatment of HCC. Clinical observation has suggested that cinobufacini, used alone or in combination with other chemotherapeutic agents, had significant preventive and curative effect on hepatocarcinoma, and favorably prevented hepatocarcinoma from hepatitis B, influenced the course of advance HCC, and improved patient survival. Its anti-hepatocarcinoma activity may caused by inducing apoptosis in HCC cells. The chemical components of cinobufacini have been investigated since the 1980s, and several different classes of compounds have been isolated. However, the effective anti-tumor components of cinobufacini are still unclear, which limits the further application and development of cinobufacini. Therefore, it is necessary to study the effective components of cinobufacini.After reviewed all the relevant reports of this field, in the present study, we screened and identified two active compounds, cinobufagin and resibufogenin, from cinobufacini with anti-hepatocarcinoma effect, investigated anti-tumor activity and mechanism of cinobufagin in vitro and in vivo, determined the concentrations of cinobufagin and resibufogenin in cinobufacini, and studied anti-tumor activity and mechanism of SCE-TS in vitro. This study may provide an experimental basis for the clinical application and further development of cinobufacini and related anti-tumor agents.1 Screening and characterization of anti-hepatocarcinoma components from cinobufaciniAim:To screen and identify anti-hepatocarcinoma compounds from cinobufacini. Methods:Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by preliminary fractionation, silica column chromatography, preparative thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Nuclear magnetic resonance (NMR) and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. Results:Through bioassay-guided isolation, two effective anti-cancer components from cinobufacini were purified. TLC and HPLC results indicated that they were two highly homogeneous compounds.1H NMR,13C NMR, DEPT, and EI-MS data of these two compounds were obtained. After analyzing and comparing these data online (SciFinder Scholar) with reported compounds and references, the structure of these two compounds were identified as cinobufagin and resibufogenin. Conclusion:Cinobufagin and resibufogenin with anti-proliferation activity against HepG2 cells were screened and elucidated from cinobufacini.2 Study on anti-hepatocarcinoma and related mechanism of cinobufagin in vitroAim:To investigate the anti-proliferation and apoptosis-inducing effects of cinobufagin in HepG2 cells. Methods:MTT-based assay was used to assess the anti-proliferative effect of cinobufagin and resibufogenin. Hoechst 33258 staining and PI staining were used to detect cell apoptosis. Western blotting method was used to detect the expression of the apoptosis-related proteins Bax, Bcl-2, Caspase-3, and Caspase-9. Results:Both cinobufagin and resibufogenin treatment induced proliferation inhibition in HepG2 in a dose- and time-dependent manner. After treatment for 24h,48h, and 72h, the IC50 values of cinobufagin were 0.56μg/mL,2.29 x 10-3μg/mL, and 1.02×10-3μg/mL respectively, while the IC50 values of resibufogenin were 6.95μg/mL,3.59μg/mL, and 3.15μg/mL. Therefore, cinobufagin was selected for further investigation because of its significant cytotoxicity. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced significant changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. At 24 h of drug treatment, the percentage of apoptotic cells was 22.24%. Western blotting analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression, and the expression of pro-Caspase-3 and -9 decreased with the treatment of cinobufagin in vitro. Conclusion:The more effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells in vitro. This was potentially triggered by regulation of the Bax, Bcl-2, Caspase-3, and Caspase-9.3 Study on anti-hepatocarcinoma and related mechanism of cinobufagin in a HepG2 xenograft mouse modelAim:To investigate the anti-tumor and apoptosis-inducing effects of cinobufagin in HepG2 xenograft mouse model. Methods:40 HepG2 xenograft balb/c nude mice were modeling by injecting HepG2 cells into the left anterior oxter of nude mice. When the tumor volume had reached approximately 100 mm3, they were divided into five groups:1.5 mg/kg cinobufagin group,1.0 mg/kg cinobufagin group, 0.5 mg/kg cinobufagin group, ADM group, and NS group randomly. To estimate the toxicity of cinobufagin, appetite, mental condition, and body weight were observed. To investigate the anti-tumor effect of cinobufagin, tumor volumes were determined. HE staining was used to investigate the tumor necrosis. TUNEL assay was used to detect cell apoptosis. Western blotting method was used to detect the expression of the apoptosis-related proteins Bax, Bcl-2, Caspase-3, and Caspase-9. Results:The nude mice in ADM group were present some toxic response. In contrast, the three doses of cinobufagin,1.5 mg/kg,1.0 mg/kg and 0.5 mg/kg, were in safe. Cinobufagin treatment induced tumor inhibition in HepG2 xenograft mouse model in a does-dependent manner. HE staining indicated that cinobufagin treatment induced significant changes in tumor tissues and tumor necrosis in HepG2 xenograft mouse model in a does-dependent manner. TUNEL assays suggested that cinobufagin significantly increased the proportion of apoptotic cells in HepG2 xenograft mouse model in a does-dependent manner. After treatment with 1.5 mg/kg,1.0 mg/kg, and 0.5 mg/kg cinobufagin, the apoptotic index were 13.4±1.68,8.8±1.04, and 6.2±1.04. Western blotting analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression, and the expression of pro-Caspase-3 and-9 decreased with the treatment of cinobufagin in HepG2 xenograft mouse model in a does-dependent manner. Conclusion:Cinobufagin inhibited tumor growth by inducing the apoptosis of HepG2 cells in vivo. This was potentially triggered by regulation of the Bax, Bcl-2, Caspase-3, and Caspase-9.5 Determination of cinobufagin and resibufogenin in cinobufaciniAim:To determine the concentrations of cinobufagin and resibufogenin in cinobufacini. Methods and Results:Established a HPLC method for the determination of cinobufagin and resibufogenin in cinobufacini. HPLC conditions were as follows:reversed-phase COSMOSIL Cholester C-18 column (Waters,4.6 mm x 150 mm,5μm), mobile phase was 45% acetonitrile, flow rate was 1 mL/min, detection wavelength was 296 nm, and samples were dissolved in methanol. The method has good linearity in the ranges of 8.5μg/mL-85μg/mL for cinobufagin (r2= 0.9998), and 15μg/mL-75μg/mL for resibufogenin (r2=0.9998). The relative standard deviations (RSD) of cinobufagin and resibufogenin were 1.22% and 0.73% respectively. The average recoveries were 98.2%(RSD=1.76%) for cinobufagin and 100.8%(RSD=2.61%) for resibufogenin. The contents of cinobufagin and resibufogenin were greatly different between different batches of cinobufacini, which may relate to the storage stability and quality stability. Conclusion:The results indicated that the method is simple, accurate, and reproducible. The contents of cinobufagin and resibufogenin were greatly different between different batches of cinobufacini. 6 Study on anti-hepatocarcinoma and related mechanism of SCE-TS in vitroAim:To investigate the anti-proliferation and apoptosis-inducing effects of SCE-TS on HepG2 cells. Methods:The HPLC method was used to determine the contents of cinobufagin and resibufogenin in SCE-TS. MTT-based assay was used to assess the anti-proliferative effect of cinobufacini and resibufogenin. Hoechst 33258 staining and PI staining were used to detect cell apoptosis. Western blotting and caspase colorimetric assay were used to detect the expression of the apoptosis-related proteins Bax, Bcl-2, Caspase-3, and Caspase-9. Results:The average contents of cinobufagin and resibufogenin in SCE-TS were 334.3μg/mL and 1211.7μg/mL. Both cinobufacini and SCE-TS treatment induced proliferation inhibition for HepG2 in a dose-and time-dependent manner. After treatment for 24h,48h, and 72h, the IC50 values of cinobufacini were 9.310 mg/mL,0.122 mg/mL, and 0.033 mg/mL, while the IC50 values of SCE-TS were 0.026 mg/mL,0.012 mg/mL, and 0.011 mg/mL. Hoechst 33258 staining and flow cytometric analysis indicated that SCE-TS induced significant changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. At 48 h of 0.001 mg/mL and 0.01 mg/mL SCE-TS treatment, the percentages of apoptotic cells were 18.45% and 26.95%. Western blotting analysis and caspase colorimetric assay showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression, and the expression of pro-Caspase-3 and -9 decreased with the treatment of cinobufagin in vitro. Conclusion:SCE-TS inhibited cell proliferation by inducing the apoptosis of HepG2 cells in vitro. This was potentially triggered by regulation of the Bax, Bcl-2, Caspase-3, and Caspase-9.