Study On The Anti Pancreatic Cancer Activity And Mechanism Of The Bruguiera Gymnorhiza Leaves Of Marine Chinese Medicine

Objective: To estimate the antitumor activity of different solvent frations of alcohol extract of leaves of Bruguiera gymnorhiza and screen the antitumor fractions with MTS assay in vitro.Furthermore study on ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the proliferation,invasion,migration,and apoptosis of pancreatic cancer SW1990 cells through in vitro cell experiment,and expound the function and molecular mechanism of the ethyl acetate extracts from leaves in antipancreatic cancer.Methods: 1.MTS method was applied to detect the effects of different solvent frations of alcohol extract of leaves of Bruguiera gymnorhiza on the proliferation of tumor cell lines.The experimental groups are divided into as follows: five concentration gradient administration groups(31.25,62.50,125.00,250.00,500.00 ?g/ml);and control group and each group had two multiple holes.After the administration of 24,48,and 72 h,the cell viability was detected by MTS test according to the instructions.The most sensitive cell lines and the most effective extracts were screened out.2.The tablet cloning experiment was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the clone forming ability of pancreatic cancer SW1990 cells.400 pancreatic cancer cells were added into each hole of the six pore plates,and the administration group(50,30,40,20,10 ?g/ml)and the control group were set up.On the 8th day after the administration,the colony formation rate was calculated by scanning count with crystal violet staining.3.The scratch assay was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the migration ability of SW1990 cells in pancreatic cancer.The administration group(150,100,50,25 ?g/ml)and the control group were set up,photographed after the administration of 0,24 h respectively.The Image J was applied to conduct the analysis of the wound area to calculate the migration distance of cell to the middle.4.The Transwell invasive chamber was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the invasiveness of pancreatic cancer SW1990 cell line.The pancreatic cancer cells were treated with the administration group(60,30,15 ?g/ml)and the control group for 24 h and then were transferred to the Transwell chamber.The cells were stained with crystal violet and photographed after removing the upper chamber cells.The number of cells passing through the chambers was calculated by Image J and the ratio of cells passing through the chambers was calculated.5.The flow cytometry was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the apoptosis of pancreatic cancer SW1990 cell line.The administration group(150,100,50,25 ?g/ml)and the control group were set up.After 24,48,72 h treatment,the cells were treated according to the instructions of the apoptosis kit.Flow cytometry instrument was applied to test.6.The method of Western blot was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the relevant marker protein expression of the apoptosis protein of pancreatic cancer cell SW1990.The administration group(150,100,50 ?g/ml)and the control group were set up.After 24 h,the proteins were extracted.After the proteins were denatured,they were conducted the electrophoresis,transmembrane,and seal and incubated out the corresponding first antibodies and second antibodies.7.The method of the immunocytochemical(ICC)was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the relevant marker protein expression of the apoptosis protein of pancreatic cancer cell SW1990.The administration group(150,100,50,25 ?g/ml)and the control group were set up.After 24 h,the cells were fixed,closed,and incubated out the corresponding first antibodies and second antibodies.The high content imaging system was applied to take pictures so as to analyze the fluorescence intensity.Results: 1.The results of applying the proliferation test method of MTS showed that parts of the ethyl acetate of extracts from leaves of Bruguiera gymnorhiza could significantly inhibit the growth of SK-OV-3,SK-MES-1,MDA-MB-231,Bel-7402,T24,PC3,HT-29,and IC50 value were greater than 100 ?g/ml.The ethyl acetate extracts was minimum which the IC50 values of 4 extracts from leaves of Bruguiera gymnorhiza on pancreatic cancer cell SW1990,and IC50 values of the three time periods were 88.76?52.67?84.17 ?g/ml.2.The tablet cloning experiment showed that,with the increase of the concentration in parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza,the decrease tendency of the tablet cloning rate got more and more obvious.When the concentration was 20?g/ml and 10?g/ml,tablet cloning rate of SW1990 in pancreatic cancer cells was significantly decreased to 40% and 20% respectively.In comparison with the control group,there is statistically significant(P < 0.05).3.In the scratch assay,at 24 h,when the administration concentration of parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza were 100 ?g/ml and 150 ?g/ml,the migration distance(expressed in pixels)of pancreatic cancer SW1990 cells were 113.32 and 116.00 respectively.In comparison with the control group,there is statistically significant(P < 0.05),which indicated that pars of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza could inhibit the migration of pancreatic cancer cell line.4.In the Transwell chamber experiment,in comparison with the control group,at 24 h when the administration concentration of parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza was 60 ?g/ml,the number of SW1990 cells passing through the chamber of pancreatic cancer was 58.61,and concentration was 30 ?g/ml that cell passed through was75.67.In comparison with the control group,there is statistically significant(P < 0.05),which indicated that parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza could inhibit the invasion of SW1990 in pancreatic carcinoma.5.The results of the Flow Cytometry showed that the apoptosis rate of high concentration group was 2.85%,5.39%,10.34% at 24,48,72 h.In comparison with the control group,there is statistically significant(P < 0.01)6.The Western blot test method showed that the ethyl acetate extracts from leaves of Bruguiera gymnorhiza could up-regulate the protein expression of Caspase-3,Bax and Bcl-2,down-regulate the protein expression of Bcl-2/Bax in pancreatic cancer SW1990 cells.7.The method of the immunocytochemical(ICC)was applied to detect the effects of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on the apoptosis protein of pancreatic cancer cell SW1990.The results were as follows: the fluorescence intensity of Caspase-3,Bax,Bcl-2 increased,which was consistent with the results of western blot.Conclusion: 1.The ethanol extract,petroleum ether extract and ethyl acetate extract of leaves of Bruguiera gymnorhiza have obvious inhibitory effect on the proliferation of pancreatic cancer cell SW1990,and n-butanol extract is not obvious.Tablet cloning experiment also proved that the ethyl acetate fraction of the leaves of Bruguiera gymnorhiza had a significant inhibitory effect on the proliferation of pancreatic cancer SW1990.1.The parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza could significantly inhibit the proliferation,migration,invasion,and the clone formation of pancreatic cancer SW1990,and the inhibitory effect was positively correlated with time and concentration.It was suggested that parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza had the effects of anti-pancreatic cancer SW1990 proliferation.2.The inhibitory effects of the parts of the ethyl acetate extracts from leaves of Bruguiera gymnorhiza on SW1990 in pancreatic carcinoma may be related to its up-regulation of Caspase-3,Bcl and Bax protein expression,and the down-regulation of Bcl-2/Bax protein expression.