Preliminary Analysis Of SP Phenomenon, Chemotherapy Resistance And Stem Cell Related Genes Expression In Gastric Cancer Side Population Cells

Objective:To analyze the relationship among the side population phenomenon,chemotherapy resistance and ABCG2 in gastric cancer cell lines.To compare stem cell related gene expressions between SP cells and NSP cells.Methods:1.We choose gastric cell line MKN28(well differentiated) and BGC823(poor differentiated) as candidates,labeled them with Hoechst33242 and PI,then SP detection and fluorescence-activated cell sorting(FACS) were performed using flow cytometry.2.Observe morphologic differences between SP cells and NSP cells after FACS by inverted microscope,especially diameters.3.Detect SP cells percentages in BGC823 and MKN28 under different Hoechst33342 concentrations,and compare dye refluxing ability.Meanwhile,detect ABCG2 expression in both cell lines by semi-quantitative polymerase chain reaction(PCR) and Western-blot.4.Carry out chemotherapy resistant test in both cell lines by using 5-FU and cisplatin, then compare ABCG2 expression in SP cells and its counterpart by semi-quantitative PCR and fluorescent-quantitative PCR.5.Compare expression of OCT-4,NOTCH,NANOG,BMI-1,CD133,CD24 and CD44 in BGC823 SP cells and NSP cells by semi-quantitative PCR.Results:1.After FACS and plated 4 hours,BGC823 SP cells' diameter is longer than NSP cells'(SP=30.03,NSP=28.23,P<0.05),however the difference disappeared after cells were plated 24 hours(SP=53.48,NSP=52.01,P=0.335).2.The percentage of SP cells in BGC823 is about 1%under 5μg/ml dye,but there is no SP cells found until dye concentration is reduced to 1μg/ml in MKN28(0.4%).Results of Semi-quantitative PCR and Western-blot show ABCG2 is expressed more in BGC823 than in MKN28.3.Chemotherapy resistant test in BGC823 show SP cells grew more quickly than NSP cells in 72h-cisplatin(1μg/ml) Group and 120h-5FU(10μg/ml) Group.PCR results indicate ABCG2 is expressed more in SP cells than in NSP cells.4.Semi-quantitative PCR results show OCT-4,NOTCH,NANOG,CD24 and CD44 were expressed more in BGC823 SP cells than NSP cells.Conclusion:Side population cells in gastric cell line BGC823 possess stronger chemotherapy resistant ability,which may have some relationship with ABCG3,than its counterpart.Compare with NSP cells,SP cells are expressed more stem cell related genes,such as OCT-4,NOTCH, NANOG,CD24 and CD44.