| Objective: To study the relationship between the high performance liquid chromatography(HPLC)fingerprints and the anti-tumor effect of toad skin from different origins,revealing the anti-tumor effect constituents of toad skin,and to explore the effect of the pharmacological components of toad skin on the apoptosis of breast cancer cells.Methods:1.To extract the active ingredients of toad skin from different areas by methanol reflux.2.Establishment and validation of HPLC analysis method.2.1 Chromatographic conditionsThe chromatographic column was Thermo ODS C18 column(150 mm × 2.1 mm,3 ?m).The mobile phase was water(0.1% acetic acid)-acetonitrile,gradient elution program: 0 ~ 2 min,10% acetonitrile,2 ~ 15 min,25% to 25% acetonitrile,25 to 30% acetonitrile,25 to 60 minutes,30 to 40% acetonitrile,60 to 65 minutes,40 to 10 percent acetonitrile 65 to 80 minutes,10% acetonitrile,Flow rate 0.2 m L · min-1.Temperature 25 ?,detection wavelength of 296 nm,injection volume of 3 ?L.2.2 The preparation of the reference substanceThe reference substance powders were precision weighed and placed in 10 m L volumetric flask,then diluted to the scale by adding methanol.Reference solution were then dissolved through ultrasound and shaking.Mixture of reference solution were prepared as follows: each reference solution 1 m L respectively were accurately transferred to 10 m L volumetric flask,then diluted to the scale by adding methanol and mixed by ultrasonic oscillation.3.Analysis of chemical profile-bioactivity relationships3.1 The peak area of each of the ten producing areas was averaged and processed into quantitative characteristic peak data.X = i peak area value / i peak average area value3.2 Principal Component Analysis(PCA)of fingerprints of toad skin.3.3 Correlative coefficients of common peaks and antitumor effects of fingerprints of toad skin in different areas.4.MTT assay was used to determine the proliferation ability of the tumor cells.Colony cloning was used to detect the colony formation after five days of drug treatment.5.PI staining was employed to detect the apoptotic rate of human breast cancer cells after breast cancer cells were treated with bioactive components of toad skin for 24 h using flow cytometry.DAPI staining was used to detect the late cell apoptosis.The mitochondrial membrane potential assay kit(JC-1)was used to detect the change of cell membrane potential after treated for 24 h.6.Western blot assay was used to explore the expression of Mcl-1?Bax?Bcl-2?p-Akt?Akt?c IAP-1.7.The tumor inhibition effect of bioactive components of toad skin was observed by using tumor-bearing mouse model.8.Data were expressed as mean ± standard deviation((?)±SD),and SPSS 16.0 software was used to statistical analysis.Dunnette-t test was used to test the significance of each group.* P <0.05 refers to a significant difference.XResults:1.Similarity evaluation was carried out by using vectorial angle cosine method,the correlation coefficients between the fingerprints of reference standard and ten batch toad skin samples were 0.90.2.The proliferation inhibitory rates of MCF-7 and MDA-MB-231 cells induced by ten batches different sources were determined.Proliferation inhibition rate of Bufo bufo gargarizans skin extracts on MCF-7 and MDA-MB-231 cells were higher.3.The PCA was analyzed by SPSS 16.0 statistical analysis software.The principal component variance and the initial factor load matrix were obtained.In the initial factor load matrix,each load represents the correlation coefficient between principal component and the corresponding variable.The data of main component load matrix divided by the eigenvalues that correspond to variable,then took square root,we could obtained coefficient each index that correspond of two main components.The main component model were as follows:F1 = 0.384X1 + 0.41X2 + 0.364X3 + 0.368X4 + 0.228X5 + 0.4X6 + 0.28X7 – 0.097X8 + 0.342X9 F2 = 0.066X1 – 0.121X2 – 0.276X3 – 0.181X4 + 0.259X5 – 0.161X6 + 0.492X7 + 0.641X8 + 0.354X9 Where F1,F2 refer to two main components while X1,X2,X3,...,X9 represent the peaks 1 to 9 respectively.F1 is the index including largest eigenvalue,the largest contribution rate of variance,the most comprehensive expression of information,which were mainly associated with X2 and X6,which correspond to peak 2(arenobufagin)and peak 6(cinobufotalin),respectively.These findings suggest that these two components(peak 2(arenobufagin)and peak 6(cinobufotalin))has a relatively important role in the quality control of toad skin.4.Pearson correlation analysis was employed to associate common.Chromatography peaks with observed bioactivity in MCF-7 and MDA-MB-231 cells from 10 batches different source.The results showed that peak 8(cinobufagin)of the HPLC fingerprints were the most strongly associated with an inhibitory effect on MCF-7 cells when compared to the other peaks,while peak 7(bufalin)and peak 8(cinobufagin)of the HPLC fingerprints were the most strongly associated with an inhibitory effect on MDA-MB-231 cells when compared to the other peaks.5.Peak 8(cinobufagin)was adopted to study the cell proliferation inhibition on MCF-7 and MDA-MB-231 cells.According to the effect of cinobufagin on the proliferation of human breast cancer cells,cells were stimulated with cinobufagin,which was significantly lower than the half inhibitory concentration,and the colony formation of the cells was observed.The results showed that proliferation of breast cancer cells could be inhibited by low concentration cinobufagin.6.Cinobufagin induces cell apoptosis in MCF-7 cells.Breast cancer cells were treated with different concentrations of cinobufagin for 24 h,PI staining results showed that followed cinobufagin concentration increased,the rate of apoptosis increased.Late apoptosis of the tumor cells were detected by DAPI staining,the results demonstrated that the nucleus showed a dense condensate,nuclear fissure phenomenon gradually increased followed cinobufagin concentration increased.JC-1 kit was used to detect the change of mitochondrial membrane potential.Compared with the blank control group,the fluorescence of red fluorescence decreased and the green fluorescence increased gradually.The results showed that the mitochondrial membrane potential decreased and the early apoptosis occurred.7.Cinobufagin probably induced apoptosis of breast cancer cells by inhibiting PI3 K / Akt signaling pathway.The proliferation inhibition rate and the apoptotic rate of cinobufagin combined with PI3K/Akt signaling pathway inhibitors(LY294002)were significantly higher.Also,The expression of Mcl-1,Bcl-2,Bax,c IAP-1,Akt and p-Akt proteins were detected by Western blotting,The results showed that the expression of anti-apoptotic protein Mcl-1,Bcl-2,c IAP-1 down-regulate while the expression of pro-apoptotic protein Bax increased.Thus,we concluded that cinobufagin induced apoptosis of breast cancer cells through inhibiting PI3K/Akt signaling pathway.8.Nude mouse model.In order to determine the tumor growth inhibition of cinobufagin in vivo,breast cancer cells were implanted in nude mice.Nude mice tumor formation curve showed the growth curve of cinobufagin group is less steep than that of blank group,but the anti-tumor effect of cinobufagin group is slightly worse than that of positive group.On the 28 th day,the rats were sacrificed by cervical dislocation and the wet weight of the tumor was weighed.The average tumor weight of the blank group,cinobufagin group,cisplatin group was 1.08 g,0.68 g,0.29 g,respectively,while the inhibition rates were 36%,74%,respectively.HE staining showed that cisplatin group had serious tumor necrosis and bleeding.Conclusions: 1.Proliferation inhibition rate of Bufo bufo gargarizans skin extracts on MCF-7 and MDA-MB-231 cells were higher.2.Chemical fingerprints of Bufo bufo gargarizans skin extracts were established by high performance liquid chromatography(HPLC),while the similarity was 0.9 evaluated by vectorial angle cosine method.3.Cinobufagin had the strongest correlation to the inhibitory effect of MCF-7 breast cancer cell.4.Cinobufagin probably induced apoptosis of breast cancer cells by inhibiting PI3 K / Akt signaling pathway. |
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