Mechanism Of Madecassic Acid Inhibiting Human Tongue Cancer Tca8113 Cells Proliferation

ObjectiveIn this experiment,human tongue cancer Tca8113 cells were cultured in vitro,and related experiments were used to explore the effect and mechanism of Madecassic acid on the growth and apoptosis of human tongue cancer Tca8113 cells.It is hoped to provide preliminary theoretical support for the selection of Madecassic acid as a clinical drug.MethodsTca8113 cells were cultured in vitro,and the treatment group with a concentration gradient of Madecassic acid was（40,80,120,160）μmol·L^-1 and the control group without adding medicine.MTT method was used to evaluate the effects of different concentrations of Madecassic acid on the viability and proliferation of Tca8113 cells.The absorbance of each well at a wavelength of490nm was measured,calculate IC₅₀ with Graph Pad Prism software;The colony formation test was used to detect the effect of different concentrations of Madecassic acid on cell colony forming ability;Hoechst 333422 staining method was used to observe the cell apoptosis;Annexin V-FITC/PI double staining method was used to calculate the cell apoptosis rate;Western blot was used to detect the expression of apoptosis-related proteins Bcl-2,P53,Bax,and Cleaved caspase-3.Results1.After different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)were applied to tongue cancer Tca8113 cells for 24 hours,the MTT results showed that the viability and proliferative capacity of the cells in the treatment group were reduced.Compared with the control group,the cell viability rate of each treatment group were（89.13±4.54）%,（81.71±5.93）%,（57.86±6.09）%,and（32.58±7.87）%,respectively,and the differences were statistically significant（P<0.05）).The IC ₅₀ at 24 h was 123.70μmol·L^-1.2.After one week of different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)in tongue cancer Tca8113 cells,the formation of Tca8113 cell colonies was significantly blocked.The colony forming ability of the control group was 100%.Compared with the control group,the cell colony forming rate of each treatment group were（89.21±4.70）%,（68.57±3.02）%,（27.76±9.31）%,（2.73±1.37）%,and the differences were statistically significant（P<0.05）.3.Different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)acted on tongue cancer Tca8113 cells for 24 hours.After staining,it was observed by an inverted fluorescence microscope.The cells in the control group had a regular shape with weak blue light.The cytoplasmic condensed nuclei of the group were deeply stained,showing high-brightness fluorescence,and the change became more significant with increasing drug concentration.Different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)acted on tongue cancer Tca8113 cells for 24 hours.After staining,it was observed by inverted fluorescence microscopy that the cells in the control group had no obvious apoptosis,while the drug group apoptosis is obvious,and this change becomes more significant with increasing drug concentration.Flow cytometry results showed that after different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)were applied to tongue cancer Tca8113 cells for 12 hours,the apoptotic rate increased with the increase in the concentration of Madecassic acid,and mainly the late apoptosis.Flow cytometry results showed that after different concentrations of Madecassic acid(40,80,120,160μmol·L^-1)were applied to tongue cancer Tca8113 cells for 12 hours,the apoptosis rate increased with the increase of Madecassic acid concentration.The apoptosis rates of the control group and each treatment group were（1.94±0.87）%,（5.31±0.12）%,（13.80±2.75）%,（20.86±0.45）%,（38.10±2.50）%,respectively.Compared with the control group,the difference in the apoptosis rate of each treatment group was statistically significant（P<0.05）.4.After treatment with Madecassic acid for 24 hours,compared with the control group,the expression levels of pro-apoptotic protein Bax,transcription activation factor P53,and Cleaved caspase-3 protein were increased in each treatment group,and the difference was statistically significant（P<0.05）;In contrast,the expression level of anti-apoptotic protein Bcl-2 in each treatment group decreased,except for the 40μmol·L^-1 group,the difference was statistically significant（P<0.05）.ConclusionsMadecassic acid can inhibit the growth and proliferation of human tongue cancer Tca8113 cells in vitro and promote its apoptosis.The mechanism may be through up-regulating the expression of Bax,P53,Cleaved Caspase-3 and down-regulating the expression of Bcl-2 and activate the mitochondrial apoptotic pathway to induce Tca8113 cell apoptosis.