| Background Glycyrrhetinic acid(glycyrrhetinic acid,GA)is also called the licorice pavilion acid,belonging to the type pier fruit alkanes rings triterpenoid compounds.The GA is the main one of the active substances of licorice.GA has good antitumor activity on liver cancer,cervical cancer,breast cancer,early young granulocyte leukemia and other tumors,and the mechanism is related to cell apoptosis,with the reduce of antiapoptosis protein Bcl-2 gene.The study found that the GA can also inhibit the proliferation of tumor cells by inducting the autophagy mechanism.The research of GA inhibiting gastric cancer cell’s proliferation is less,there are reports that GA and its derivatives can inhibit proliferation of gastric cancer cells BGC823 and SGC7901 with concentration and time dependence,but the research of inhibition mechanism is less.Objective The article studies the effect of cell proliferation,apoptosis and autophagy,which the 6.25~200 μmol/L concentration scope of GA effect gastric SGC7901 cells,and the time increased to 48 h,provided the theoretical basis for GA inhibiting the proliferation effect of gastric cancer cell and its molecular mechanism.Method 1.Cell culture: SGC7901 cells were seeded in culture medium containing 10% fetal bovine serum,the double resistance of 100u/ml penicillin and 100u/ml streptomycin of RPMI1640,placed in incubator with 5% CO2,37 ℃,95% humidity conditions.2.Experimental groups: experimental group 1~7,negative control group and cultivate tone zero group,experimental group 1~7 is the cultured cells of dimeth-ylsulfoxide(DMSO)solution of GA,make the final concentration of GA were 6.25,12.5,25,50,100,150,200 μmol/L,and the negative control group added the end concentration of GA the same to volume of DMSO.3.To determined the effect of proliferation on GA in SGC7901 gastric cancer cells by MTT method: In addition to the experimental group 1 ~ 7 and the negative control group,adding cultivate tone zero group is the no cell’s culture medium.The logarithmic phase of SGC7901 cells,the drug effect on the cells after 48 hours,added on the final 0.5 mg/ml concentration of MTT solution and continue to develop 4 h;Gently pour out the broth,add on DMSO solution,avoided light and vibration waved 15~30 min;the absorbance value(OD)of wavelength of 492 nm measured by Enzyme standard instrument.the experimental group1~7,negative control group and the cultivate tone zero group all has three holes,repeated the experiment 3 times.Calculate the inhibition rate of tumor cells on GA and IC50.4.The influence of DNA and apoptosis on GA in gastric cancer SGC7901 cells: experimental group 1~7 and negative control group is cultured 48 h and to detect the DNA contents of SGC7901 cells by propyliodide organism(PI)single dye and flow cytometry instrument;to detect the apoptosis rate of SGC7901 cells by Annexin V-FITC and PI staining and flow cytometry instrument,the end oncentration of GA using IC50,effecting SGC7901 cells 48 h,separately dyeing by Annexin V-FITC and PI single dye as the control.5.To detect the influence of autophagy involved in protein Beclin1,Lc-3 and the antiapoptotic proteins Bcl-2 in SGC7901 gastric cancer cells by Western blot: the total protein of the experimental group and the negative control group cells,protein quantitative,taking 30 μg proteins in SDS polyacrylamide gel electrophoresis,voltage 200 V,60 min.Power transferred to the NC membrane,current 300 m A,90 min.Enclosed 2h with 5% skim milk.Contrast dye protein Marker,cropping the related bands range NC membrane of Beclin1,Lc-3,the Bcl-2,β-actin;Respectively,and the 1:1000 anti rabbit Beclin1,anti rabbit Lc-3,anti rat β-actin and the 1:500 anti rabbit Bcl-2 incubated 2 h at room temperature or 4 for the night;Then 1:3000℃ sheep anti rat Ig G or sheep anti rabbit Ig G shake 1 h at room temperature;Luminescent imaging method record the results in Amersham Imager 600 instruments,detecting the average gray level of western blot strip by Image J software.6.Statistical analysis: Using SPSS19.0 statistical software for the data processing,data presentation with the mean standard deviation,Comparison between different groups by analysis of variance,P < 0.05 represents the difference was statistically significant.Results 1.The effects of proliferation on GA in gastric cancer cell SGC7901 gastric cancer cells: MTT method results show that concentrations of GA treated gastric cancer SGC7901 cells after 48 h,the proliferation inhibition role showed statistically significant in 12.5 ~ 100 μmol/L(P < 0.05),with the concentration dependence,the difference showed no statistical significance in 100 ~ 200 μmol/L(P > 0.05),and the IC50 was 74.25 μmol/L.2.The effects of autophagy on GA in gastric cancer SGC7901 cells: Western Blot results show: compared with control group,the GA effected SGC7901 cells after 48 h,the expression of autophagy related Beclin1 protein significantly increased in 6.25~ 50 μmol/L,the expression weakened or disappeared more than 50 μmol/L;the expression of Lc-3 was similar to those of control group in in 6.25 ~ 200 μmol/L,the expression weakened or disappear more than 100 μmol/L.3.The effects of apoptosis on GA in gastric cancer cell SGC7901 gastric cancer cells: The results by PI single dye flow cytometry instrument showed that the influence of different concentrations of GA on DNA had difference in SGC7901 gastric cancer cells,the number of the diploid cell gradually increased in 12.5~150 μmol/L with concentration dependence and showed statistically significant(P < 0.05);the results by Annexin V-FITC and PI staining flow cytometry instrument showed that compared with control group,different concentrations of GA effect on SGC7901 cells after 48 h,of the late early apoptosis,the cell number of late poptosis and necrosis is different,the less than 150 μmol/L concentration of GA effect on cell,early apoptosis rate increased with concentration dependence,the late apoptosis and necrosis rate also increased.4.The expression of Bcl-2 protein on GA in gastric cancer SGC7901cells: Western blot detection results showed that: Compared with control group,the GA effected SGC7901 cells after 48 h,the expression of Bcl-2 protein was similar to those of control group in 6.25~100 μmol/L,the expression gradually weakened more than 100 μmol/L.The quantitative analysis shows that the expression of Bcl-2 protein showed increasing trend less than 12.5 μmol/L;the trend decreased with concentration dependence after above 12.5 μmol/L.Conclusion 1.GA can inhibit the proliferation of gastric cancer SGC7901 cells with concentration dependence,when the concentration of GA more than 100 μmol/L,the efficiency of inhibiting cell proliferation declined.2.The autophagy of GA has two-way character in SGC7901 cells,below the concentration of IC50,GA mainly accelerated the formation of autophagy body of SGC7901 cells,above the concentration of IC50,GA reduced the form and quantity of autophagy body in SGC7901 cells.3.GA can promote the early apoptosis,late apoptosis and necrosis with concertration dependence in gastric cancer SGC7901 cells,and inhibition rate reached 90%,cell apoptosis is still increasing;the apoptosis effect of GA on Bcl-2 has two-way character in SGC7901 cells,below the 17% oncentration of IC50 was enhanced,above the 17% oncentration of IC50 was reduced with concentration dependent. |
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