The Inhibitory Effect Of Beta-aescin On Colon Cancer Cells And Its Molecular Mechanism

Nowadays,cancer is one of the most difficult diseases to conquer.As a malignant tumor,colon cancer is a fatal disease with rapid progression and poor prognosis.How to prevent and cure colon cancer is one of the important subjects in medical.Chinese traditional medicine is the great treasure of the Chinese nation.As a natural triterpenoid glycoside compound,beta-aescin is extracted from Aesculus Wilsonii Rebd or Chinese Semen Aesculi.It is mainly used for the treatment of cerebral edema,anti-edema,enhancement of venous tension,improvement of microcirculation and chronic venous insufficiency and other diseases.It has the effects of vascular regulation,inhibition of gastric emptying,protection of experimental liver injury in mice,nerve protection and anti-tumor.The anti-cancer effect of beta-aescin to HT-29 cells,which are common in colorectal cancer,have been covered.However,the inhibitory effect on Lo Vo cells in colorectal and its molecular mechanism have been rarely reported.We used MTT assay,immunofluorescence analysis,flow cytometry(FCW),western blot(WB),laser scanning confocal microscopys imaging(LSCM)and other techniques to study the anti-colon cancer effect of beta-aescin and the influence of Wnt/ beta-catenin signal transduction pathway.To investigate the effect of beta-aescin on colon cancer cells,three aspects were elaborated : the growth inhibitory effect of beta-aescin on Lo Vo cells,the effect of beta-aescin on the cycle and apoptosis of colon cancer cells,the expression level of the core molecule beta-catenin and the intracellular distribution.Firstly,is to study the growth inhibitory effect of beta-aescin on Lo Vo cells.Quantitatively,cells were treated with 0,10,30,50 and 100 ?g/ml beta-aescin for 24,48 and 72 h by MTT assay.MTT assay revealed that the cells showed significant concentration(dose)and time correlation.The higher the concentration,the longer the action time and the more obvious the inhibitory effect.Meanwhile,Its IC50 was 44.1 ?g/ml.Qualitatively,cells were treated with 0,10,30,50 and 100 ?g/ml beta-aescin for 72 h by indirect immunofluorescence.The results revealed that with the increase of concentration,the growth inhibitory effect of beta-aescin on Lo Vo cells was also enhanced,and the proliferation activity of Lo Vo cells was significantly inhibited,which also showed a dose-action correlation.Secondly,is to study the effect of beta-aescin on the cycle and apoptosis of Lo Vo cells.Macroscopically,the cells were treated with beta-aescin at a certain concentration(50 ?g/ml)for 0,24,48 and 72 h,and the morphological changes were observed under an optical microscope after PI staining.Apoptosis such as nuclear condensation,membrane puffing into vesicles,and plasmid wall separation were observed gradually.Microscopically,the cycle and apoptosis of Lo Vo cells were analyzed using DNA content at the level of molecular biology.The cells were treated with different concentrations(0,10,30,50 and 100 ?g/ml)for 72 h in beta-aescin,and the cell DNA content was detected by flow cytometry after PI staining.The results showed that the cycle of Lo Vo cells became shorter and the S phase was blocked,resulting in the decrease of cells entering the G2/M phase.A small peak of apoptosis appeared,and the apoptosis period was significantly prolonged,and the cells were induced by beta-aescin.Thirdly,is to study the effect of beta-aescin on the expression level and intracellular distribution of the core molecule of Wnt/ beta-catenin signal pathway.With western blot(WB),at low concentrations(10 and 30 ?g/ml),the expression inhibitory effect of beta-catenin protein was not obvious.However,at higher concentrations(50 and 100 ?g/ml),the expression/inhibition ratios of beta-catenin protein were 0.53 and 0.27,respectively,with statistically significant differences(P<0.05).With laser scanning confocal microscopy(LSCM),it can be seen that the fluorescence of beta-catenin is mainly distributed in the cytoplasm(cytoplasm,organelle)and the cell membrane,especially in the connection part of the cell membrane.The fluorescence in the nucleus is distributed in multiple clumps,weakens,darkens and gradually decreases.The results showed that the nucleus was the main pathway affecting the expression of DNA protein in tumor cells.In conclusion,beta-aescin had a significant inhibitory effect on the growth of colon cancer Lo Vo cells.It can block the cell cycle and induce the apoptosis.The Wnt/beta-catenin pathway plays an important role in the anti-colon cancer mechanism in beta-aescin.