Identification Of Side Population Cells In Human Gastric Carcinoma

Part I Isolation and stem cell related gene characterization of the side population cells from human gastric cancer cell linesObjective:To isolate and characterize the side population cells from gastric cancer cell lines with a series of stem cell related gene.Methods:Side populations of four human gastric cancer cell lines with different differentiation grades, named MKN-28, SGC-7901, BGC-823, and AGS were analyzed and isolated by fluorescence-activated cell sorting after being stained by fluorochrome Hoechst33342 and PI; expression of drug transporters ABCG2, stem cell related cellular membrane phenotype(CD 133, CD24, CD44) and signaling molecules(OCT-4, NOTCH, NANOG, BMI-1) were detected by reverse transcriptase PCR.Results:Side population were detected in three of the four gastric cancer cell lines, with a proportion of 0.46%-3.55%, without SP cells detected in AGS. The SP cells showed a higher expression of ABCG2, CD24, CD44, OCT-4, NANOG and NOTCH, compared to nonSP cells.Conclusion:Side population could be detected in the cell lines of gastric cancer, with a higher expression of a series of stem cell related gene compared to nonSP cells.Part II Identification of the side population cells from human gastric cancer cell linesObjective:To identify the side population cells from human gastric cell lines.Methods:Sorted SP and nonSP cells from gastric cancer cell lines were analyzed by cell growth assay(CCK-8), clonogenic assay, and matrigel invasion assay in vitro, furthermore by tumorigenicity assay, differentiation study in vivo.Results:Only SP cells from cell line MKN-28 showed a higher proliferation activity and clonogenicity compared to the nonSP cells in vitro(P<0.05). Sorted SP and nonSP cells from cell line SGC-7901 and BGC-823 showed similar proliferation activity, clonogenicity in vitro, tumorigenicity in vivo(P>0.05). Matrigel invasion assay showed that the number of nonSP cells which penetrated artificial basement membrane was more than SP cells (P<0.05). Additionally, culturing sorted SP and non-SP cells showed that the populations are interconvertible in vitro, and both conversed to different degree of differentiation cells in vivo.Conclusion:Sorted SP and nonSP cells from gastric cancer cell lines showed similar proliferation activity, clonogenicity, tumorigenicity and multi-directional differentiation potential, which standed in contrast to the observations that tumor SP cells were enriched in stem cells.Partâ…¢Chemoresistance study of the side population cells from human gastric cancer cell linesObjective:To examine chemotherapeutic drug resistance of the side population cells from human gastric cancer cell lines.Methods:Cell growth assay was performed for sorted SP and nonSP cells from gastric cancer cell line by drug intervention. Gene profile for tumor chemotherapeutic drug resistance was analyzed with a Quantitative Real-Time PCR Arrays.Results:The half maximal inhibitory concentration (IC50) of 5-FU for SP cells from gastric cancer cell line was significant higher than nonSP cells(0.3346 mg/ml vs 0.0142mg/ml, P<0.05). Similarly, the IC50 value of DDP for SP cells from gastric cancer cell line was significant higher than nonSP cells(0.0055 mg/ml vs 0.0032mg/ml, P<0.05). Quantitative Real-Time PCR Arrays displayed a series of 15 higher expression gene and 1 lower expression gene in SP cells rom gastric cancer cell line compared to nonSP cells, substantially including ABCB1, ABCG2, BAX, MYC, GSTP1 and AR (P<0.05)Conclusion:Sorted SP cells from gastric cancer cell line showed higher resistance to chemotherapeutic drugs, with a stronger expression of a series of tumor drug resisitance related genes.