The Research About ERK Inhibitor Increased Susceptibility Of Colon Cancer Cells For Raltitrexed And It’s Mechanism

Purpose This study aims to research that whether ERK inhibitor PD98059 increased the sensitivity of colon cancer cells HT-29 to raltitrexed through experiments in vitro,and the relevant function characteristics and mechanism are discussed,to provide reliable experimental data for clinical treatment of colon cancer.Methods 1.Cultivated human colon cancer cells HT-29,go down to posterity and plant the plates when the exponential phase,to group after cell adherent according to different processing factors :the control group,the pure raltitrexed group,the pure PD98059 group and the combination group.2.The state of cell growth and morphological changes of cells of each treatment group were observed under inverted microscope after treated 24 h.3.MTT method and cell counting method were used to detect the cell proliferation activity of the experimental group after treated 24 h.4.Cells apoptosis rate of each experimental group were detected to confirm whether any difference by flow cytometry instrument method after treated 24 h.5.Get total protein and detected the relative expression volume of Ras,ERK1/2,p-ERK1/2 protein of each group by using western blot method after treated 8 h.6.Extracted total RNA of each group under sterile state and without enzyme,get k-ras,erk1 and erk2 c DNA by reverse transcription,by real-time fluorescent quantitative PCR method to detect relative expression of related genes of each group.Result 1.Under inverted microscope,compared with control group,cell form and quantity change of the simple PD98059 group is not obvious,but the pure RTX group and the combination group were decreased significantly,and typical apoptosis morphological changes of the pure RTX group and the combination group are visible,for example:karyorrhexis and apoptotic body.The number of cells and morphology change of the combination group is more obvious than other groups.2.Cell proliferation rate of the simple PD98059 group was not a significant change,but the pure RTX group and the combination group were reduced,and cell proliferation rate of the combination group is the most reduction compared with control group by MTT method and cell counting method.3.Flow cytometry instrument was used to detect cell apoptosis rate change,visible that compared with control group,the apoptosis rate of each experimental group were significantly increased,and the trend of apoptosis of the combination group is the most significant.4.Cell related protein expression were detected by western blot method,when compared with the control group,the Ras,ERK1/2 protein expression has no obvious change,but p-ERK1/2 protein expression reduced in the simple PD98059 group;the Ras,ERK1/2 and p-ERK1/2 protein expression has no obvious change in simple RTX group;and the protein expression of the combination group as same as the simple PD98059 group.5.RT-PCR results are that k-ras,erk1 and erk2 mRNA expression of each experimental group have no significant difference compared to the control group(P>0.05).Conclusion 1.Raltitrexed has proliferate inhibition and apoptosis promotion to colon cancer cell HT-29.2.ERK inhibitors can increase proliferate inhibition and apoptosis promotion of HT-29 to raltitrexed to some extent,means that ERK inhibitor increase susceptibility of colon cancer cells for raltitrexed.3.ERK inhibitor can increase susceptibility of colon cancer cells for raltitrexed through the RAS-MEK-ERK pathway at the protein level rather than the gene level.