Effects Of Pachymaran On Proliferation,Migration And Pro-apoptosis Of Human Cervical Carcinoma HeLa Cells And Its Mechanism

ObjectiveTo research the effects of pachymaran on proliferation inhibition,migration and pro-apoptosis of human cervical carcinoma HeLa cells and their related mechanisms.MethodsTreated with Pachymaran at the dosage of 10,20,30,40,50,60,80,100 and 120 mg/ml for 24 hours.MTT assay was used to detect the proliferation of HeLa cells,and appropriate low,medium and high concentrations of pachymaran were screened for subsequent experiments.After having dealt the HeLa cells with Pachymaran at the level of low,medium and high for 24 hours,the author observed the morphological effects of the cells by inverted phase contrast microscope.Observe the change of the cell nucleus after Hoechst 33342 stain.Test the ability of cell cloning by the plate cloning assay.Test the ability of cell migration ability by the cell scratch experiment.Detect the apoptotic rate of the HeLa cells by flow cytometry in the method of AV/PI double stain.Test the cell cycle by flow cytometry.The expression of apoptosis-related proteins(Cleaved Caspase 3,Cleaved Caspase 8,Cleaved Caspase 9,Bcl-2,Bax),migration-related proteins(MMP-9,VEGFA)and ERK pathway-related proteins(P-ERK1/2,ERK1/2)was detected by Western-blotting.The control group without adding drugs was set up at the same time.ResultsCompared with the control group,when the concentration of pachymaran was more than 30 mg/ml,the survival rate of HeLa cells significantly decreased(P<0.05).When the concentration of pachymaran was more than 60 mg/ml,the cell viability decreased by nearly 50%.In order to avoid the possibility that the high concentration of drugs may directly kill the cells and to get the larger errors in the experiment due to the deviation of the number of cells obtained in the condition of cell culture,therefore,the experiment used pachymaran of 30,40 and 50 mg/ml as the low,medium and high dosage for subsequent experiments.Compared with the control group,the medium and high concentration of pachymaran could cause significant apoptosis morphological changes in cells and nucleus,but no significant morphological changes at low concentration.In the contrast of control group,the low,medium and high concentrations of pachymaran can reduce the ability of cell cloning and cell migration(P<0.05),increase cell apoptotic rate(P<0.05),reduce S-phase cells and block them in G2/M phase(P<0.05)in a dose-dependent manner.The expressions of Cleaved Caspase 3,Cleaved Caspase 8,Cleaved Caspase 9 and Bax increased significantly.While the expressions of Bcl-2,MMP-9,VEGFA and P-ERK1/2 obviously declined(P<0.05).The ERK1/2 expression did not change notably.ConclusionsPachymaran can inhibit proliferation,migration and promoting apoptosis of HeLa cells.The mechanism of inducing apoptosis may be related to the inhibition of phosphorylation of ERK signaling pathway by downregulating the expression of P-ERK 1/2.In addition,pachymaran can reduce the migration of HeLa cells to a certain extent.