Researches On The Effects Of Advanced Glycation End Products On The Proliferation And Apoptosis Of Colon Cancer Stem Cells And Its Mechanism

Objectives To investigate the effects of advanced glycation end products(AGEs)on the proliferation and apoptosis of colon cancer stem cells,and the effects of AGEs on the expression of RAGE and the ERK1 / 2 signaling pathway,and to explore the possible mechanism of AGEs acts on colon cancer stem cells.Methods 1.The colon cancer stem cells obtained by indirect immunomagnetic bead sorting method in our group were chosen as the research object,and then the colon cancer stem cells were cultured in serum-free medium and identified by immunocytochemistry.2.The colon cancer stem cells were divided into five groups,which contains BSA group(300 ?g/mL BSA),control group(without any intervention),AGEs 1 group(100 ?g/mL AGEs),AGEs 2 group(200 ?g/mL AGEs)and AGEs 3 group(300 ?g/mL AGEs).The mRNA and protein expressions of RAGE in each group were detected by qPCR and Western blot respectively after 24 hours of intervention.3.CCK-8 assay:(1)Colon cancer stem cells were divided into AGEs group(100 ?g/mL,200 ?g/mL,300 ?g/mL,500 ?g/mL)and BSA group(100 ?g/mL,200 ?g/mL,300 ?g/mL,500 ?g/mL).The OD values of each group were detected by CCK-8 assay after 24 hours of intervention,and comparison of cell proliferation rate in different concentrations.(2)Colon cancer stem cells were divided into BSA group(200 ?g/mL BSA),AGEs group(200 ?g/mL AGEs),AGEs+PD group(200 ?g/mL AGEs+50 ?M PD98059)and PD group(50 ?M PD98059),and the OD values were detected by CCK-8 assay.4.Apoptosis test: colon cancer stem cells were induced by metformin.The test was divided into BSA group(200 ?g/mL BSA),control group(without any intervention),MET group(40 mmol/L MET),MET+AGEs group(40 mmol/L MET+200 ?g/mL AGEs),MET+AGEs+PD group(40 mmol/L MET+200 ?g/mL AGEs+50 ?M PD98059)and MET+PD group(40 mmol/L MET+50 ?M PD98059),after 24 hours of intervention,flow cytometry was used to detect the apoptosis in different groups.5.Colon cancer stem cells were divided into BSA group(200 ?g/mL BSA),control group(without any intervention),AGEs group(200 ?g/mL),AGEs+PD group(200 ?g/mL AGEs+50 ?M PD98059)and PD group(50 ?M PD98059).Western blot was chosen to detect the protein expression of p-ERK/ERK and RAGE.Results 1.Colon cancer stem cells were suspended growth in serum-free medium,then stem cell spheres gradually formed and the spheres further increased and became round.CD133 antigen was observed on the cell membrane surface of colon cancer stem cells by immunocytochemical.2.(1)The mRNA expression of RAGE: when different concentration AGEs acted on colon cancer stem cells,compared with control group,the mRNA expression of RAGE did not change significantly when treated with 100 ?g/mL AGEs,while increased when treated with 200 ?g/mL AGEs and 300 ?g/mL AGEs(P <0.05),and the highest mRNA expression of RAGE was found at 200?g/mL AGEs concentration.(2)The protein expression of RAGE: The protein expression of RAGE did not change when colon cancer stem cells were treated with 100 ?g/mL AGEs and 300 ?g/mL AGEs,while increased with 200 ?g/ mL AGEs,and the difference was statistically significant(P<0.05).3.When 100,200,300,500 ?g/mL AGEs were acted on colon cancer stem cells respectively,the cell proliferation rates were all increased compared with the corresponding BSA concentration group(P<0.05),and the cell proliferation rate was the highest at the concentration of 200 ?g/mL.The OD value of AGEs group was higher than BSA group,and the difference was statistically significant(P<0.05),and the OD value of AGEs+PD group decreased when compared with AGEs group(P<0.05).4.Flow cytometry showed that the apoptosis rate in MET group was significantly higher than that in control group(P<0.05).Compared with MET group,the apoptosis rate of MET+AGEs group decreased(P<0.05),and the apoptosis rate of MET + AGEs + PD group increased compared with MET + AGEs group(P <0.05).5.Western blot showed that the protein expression of RAGE and p-ERK / ERK in AGEs group were significantly higher than those in control group(P <0.05),and the protein expression of RAGE and p-ERK / ERK in AGEs+PD group were significantly decreased compared with AGEs group(P <0.05).Conclusion 1.AGEs can promote the proliferation of colon cancer stem cells and inhibit its apoptosis.2.The effect of AGEs on colon cancer stem cells may be related to increasing the expression of RAGE and activating the ERK1 / 2 signaling pathway.