Effects Of Echinacoside On 786-O Cells Apoptosis And Inductive Mechanisms In Vitro

Objective Echinacoside(ECH)is one of the main components of Cistanche deserticola.This experiment investigates effects of Echinacoside on cellular morphology of renal carcinoma cells(renal carcinoma cell,RCC)in vitro,analyzes the effects of Echinacoside on proliferation,appoptosis,viability and migration of 786-O cells and the mechanisms,and illustrate the anti-renal cancer effects of Echinacoside at the cellular level.Meanwhile,to explore the target of Echinacoside on renal carcinoma 786-O cells,the experiment investigates the effects of Echinacoside on Caspase cascade pathway and anti-apoptotic protein BCI-2 in renal cancer 786-O cells,elucidates the molecular mechanism of the effects of Echinacoside on renal cancer 786-O cells,and explore the functions and mechanisms of echinacoside for inhibiting renal cancer cells at the cellular and molecular level,and the experiment will provide basic data for the development of new renal cancer drugs.Method In this experiment,renal cancer 786-O cells were respectively cultured in vitro with Echinacoside concertration of 0,1.25,2.5,5,10,20,40 μmol/L for 24 h,and the proliferation of renal cancer 786-O cells was detected by MTT method.At the same time,the half-inhibition rate(IC50)of Echinacoside on renal cancer 786-O cells was measured,the concentration of Echinacoside in the experimental group was selected on the basis of this value before the following experiments were conducted.After different concentrations of Echinacoside(0,1,2,3,4,5,6 μmol/L)were applied to renal cancer 786-O cells for 24 h and 36 h,MTT method was used to detect the effects of different test drugs concentration on renal cancer 786-O cells.Transwell in vitro migration experiment was used to detect the impact of Echinacoside(0,1,2,3,4,5,6 μmol/L)on the migration ability of renal cancer 786-O cells after 24 h,and the relative expression levels of Echinacoside on BCI-2,Caspase-3 and Bax mRNA in renal cancer 786-O cells were furthher detected by RT-PCR experiment.Meanwhile,the effects of different drug concentrations on inhibition of the expression of apoptosis effector protein BCI-2 and apoptosis effector protein Caspase-3 in renal cancer 786-O cells were detached by Western blot experiment.Results(1)The inhibitory rate of proliferation of renal cancer 786-O cells with different concentrations of Echinacoside were increasing with the increasing of the concentrations of Echinacoside in drugs.Meanwhile,according to the experimental data,the IC50 semi-inhibitory concentration of Echinacoside against renal cancer 786-O cells was 5.443 μmol/L.(2)After observing the morphology of renal cancer 786-O treated with Echinacoside at different concentrations under a microscope,it was found that the morphology of the treated cells in the drug treatment group changed when compared with the cells without drug treatment.The cell morphology of the treated cells changed,the growth state became worse and the number of cells decreased significantly.(3)The drug treatment group can reduce the survival rate of renal cancer 786-O cells,and significantly promote the apoptosis of renal cancer 786-O cells(P<0.01).After the drug treatment group induced renal cancer 786-O cells for 24 h and 36 h,the cell proliferation rate in this group showed a downward trend with the increasing of drug concentration,which presented a significant inhibitory effect(P<0.01)that can cause cell apoptosis.Compared with the control group,the cell proliferation rate of the drug treatment group showed a concentration-dependent characteristic of the drug;After the treatment group induced the renal cancer 786-O cells for 24 h,the migration of the cells showed a significant downward trend(P<0.01)when compared with the control group.(4)According to the data obtained by RT-PCR and comparing with the control group,different concentrations of drug treatment can significantly promote the mRNA expression levels(P<0.05)of the intracellular apoptosis of Caspase-3,Bax and significantly inhibit the mRNA expression level of the BCI-2(P<0.05).(5)According to the data obtained by Western Blot and comparing with the control group,the expression level of BCI-2 in the cells in different concentrations of Echinacoside treatment group showed a significantly down trend,but but the expression level of Caspase-3 in the cell showed a clear upward trend,and it presented a drug concentration-dependent relationship.The results above suggested that Echinacoside caused programmed death of 786-O cells by activating Caspase-3 and BCl-2.Conclusion(1)Echinacoside can significantly inhibit the proliferation of renal cancer 786-O cells in a concentration-dependent manner and increase the apoptosis rate of the 786-O cells.(2)Echinacoside can significantly reduce the migration ability of renal cancer 786-O cells.(3)Echinacoside can further induce apoptosis of renal cancer 786-O cells.In this process,the expression level of Bax gene was up-regulated,the expression level of Caspase-3 was up-regulated,and the expression level of BCl-2 was down-regulated.The results indicates that Bax,Caspase-3 and BCl-2 are involved in the enchinacoside-induced apoptosis of renal cancer 786-O cells and that the apoptosis of renal cancer 786-O cells induced by Echinacoside is accomplished by affecting the mitochondrial pathway and the Caspase cascade.