Reserch On The Inhibitory Effect Of Cucurbitacin B On The Growth And Proliferation Of Colon Cancer Cells

Objective:To explore the effect and molecular mechanism of cucu- rbitacin B on human colon cancer LS-174-T cells, and provide the experimental basis for the treatment of colon cancer in human colon cancer LS-174-T cells as the research object.Methods: Human colon cancer cill lines LS-174-T cells in containing 10% fetal bovine serum,100 u/ml and 100 ug/ml penicillin streptomycin RPMI 1640 culture based on the temperature of 37℃ 5% CO2 incubator in training,when the cells adherencing,take the logarithmic phase cell to test.In the following experiments:1 CCK-8 method was used to detect the inhibitory effect of cucurbitacin B onthe growth of LS-174-T cellsTake the human colon cancer cells in the logarithmic growth phase to dissociate the cells into single cell suspension,adjust the final concentration of 5×103/ml cells/ml,vaccination in 96-well culture plate,respectively,to join the mass concentration of 0.01μmol/L,0.1μmol/L,1.0μmol/L,10.0μmol/L and 100.0μmol/L cucurbitacin B,meanwhile set up blank group(DMSO solvent control group)and the negative control group(group without drugs).Dosing role 24,48,72 h,with the CCK-8 method the Ascent microplate was used to detect the cell optical density value(OD value), calculate the inhibition rate of cell growth,draw the cell growth curve,Effects of 24,48 and 72 h.2 PI staining flow cytometry methodCollect the cells of the negative control group and drug group(0.1,1mol/L) cultivatied for 24 h,collecting cells,with heavy precooling PBS suspension cells,2000 RPM centrifuge for 10 min,washing cell,add in 300 ul Binging Buffer suspension,avoid light,incubation at room temperature for 15 min,5min before computer to join 5ul PI,upflow cell test.3 The expression level of STAT3,caspase-3 and tert m RNA determine- ated by realtime fluorescence quantitative PCRHuman colon cancer LS-174-T cells after suspension evently were distributed into four groups,every group of four samples,were inoculated in the culture flasks in 25 ml overnight,cucurbitacin B of 0.1,10 and 1mol/L was added in the flasks.Extracting RNA at 24 h and 48 h,then STAT3、caspase-3、and tert m RNA were cultured and tested with realtime fluorescence quantitative PCR.Results:1 Inhibitory effect of cucurbitacin B on human colon cancer cell line LS-174-TThe results of CCK-8 showed that cucurbitacin B has obvious inhibitory effect on the growth ofcolon cancer LS-174-T cells, and was time anddose dependent,with the cucurbitacin B( 0.01μmol/L,0.1μmol/L,1.0μmol/L, 10.0μmol/L and 100.0μmol/L) groups for 24 h,.the inhibition rate was(0.52±0.17)%,(11.34±0.13)%,(14.27±0.61)%,(19.73±2.54)%,(74.81±2.12)%,for 72 h,the inhibition rate was(0.89±0.21)%,(34.31±0.29)%,(43.01±0.59)%,(71.21±1.77)%,(98.91±0.49)%,the maximum inhibition rate was(98.91±0.49)%,the concentration of 100 μmol/L for 72 h.2 Analysis of cell cycle changes by flow cytometric PI stainingPI flow cytometry staining method to detect the drug group and negative control group cell cycle change,the results indicate that comparing with the negative control group,the drug groups(0.1,1.0μmol/L)for 24 h,The control group and 0.1 mol/L group, 1 mol/L concentration cell percentage of G0/G1 stage were 77.41±4.69%,58.31±3.15% and 41.13±5.31%; The proportion of G2/M cell stage were 5.32±0.73%, 14.17±0.82% and 31.28±4.02%; The proportion of S cell stage were 17.27±3.40%, 27.52±4.71% and 27.59±8.99%,With the concentration of cucurbitacin B increases, the percentage of cells in G2/M phase increased gradually, the percentage of cells in G0/G1 phase was decreased.3 The detection on the m RNA expression f stat3,tert and caspase3 with realtime fluorescence quantitative PCREffected for 24 and 48 h,the m RNA expression of stat3,tert and caspase3 of the drug group(10μmol/L,1μmol/L,0.1μmol/L) are detected by realtime fluorescence quantitative PCR.The results showed: the Stat3 and tert m RNA expression decreased, and the drug concentration and action time of presentation time-dose-dependent, and the expression of Caspase3 increased, and the drug concentration and action time of presentation time- dose- dependent.Conclusions:1 The proliferation of cucurbitacin B on human colon adenocarcinoma cell line LS174-T can inhibit the growth, in a time- and dose-dependent relationship.2 Inhibitory effect of cucurbitacin B on human colon adenocarcinoma cell line LS174-T proliferation may be related to its effect on cell cycle related.3 Inhibitory effect of cucurbitacin B on colon cancer cells may eventually lead to apoptosis.4 Cucurbitacin B act on Stat3, tert factor, the decreases of m RNA expression showed time- and dose dependence of,TERT, the increase of m RNA expression showed a time- dose- dependent.