Isolation And Identification Of The Side Population In Human Gastric Carcinoma Cell Lines And The Related Regulation Pathway

ObjectiveRecent studies suggest that tumor may be a stem cell disease and cancerstem cells may be responsible for tumorigenesis and contribute to someindividuals’ resistance to cancer therapy. Although their ratio are very low incancer cells, cancer stem cells are more important than other tumor cellsbecause they are capable of self-renewing, differentiating, maintaining tumorgrowth and heterogeneity, playing an important role in both tumorigenesis andtherapeutics. At the same time side population(SP)cells are also used to enrichcancer stem-like cells in many cell lines and even some fresh tumor samples.Gastric carcinoma has become a disease which is serious harmful tohuman health. It occurs at very high frequency in certain areas of China. Itsincidence and mortality are very high. To our knowledge there was no detailedresearch of these cells in gastric carcinoma(GC). It is worth studying SP cells inGC to understand the character of GC cancer stem cells, in order to developeffective strategies for targeted treatment.MethodsHuman gastric carcinoma cell lines BGC-823and MGC-803cells werestained by Hoechst33342and analyzed by FACS when cells in a logarithmicgrowth phase, at the same time added reserpine as control sample. They weresorted into two subpopulations, Hoechst33342negative (SP), andHoechst33342positive population (non-SP, NSP). We collected the fluorescene weakest SP cells and did series of experiments to detect the stem cellcharacteristics with SP and NSP cells. Freshly sorted SP and NSP cells wereplanted in6-well plate to determine their clone formation ability. We analyzedthe fluorescence of Hoechst33342to detect whether they could differentiationinto subpopulation cells with different phenotype after cultured for several days.We used NOD/SCID mice to determine the tumor formation ability of SP andNSP cells, which was one of the standard criteria for proving the cancer stemcell characterization.To get better understanding of the mechanism of the maintenance of SPcells in gastric cancer, we used the special inhibitor of PI3K/Akt pathway todetect the variation of the SP cells through FCM and the ability of the gastric cellline cells in cloning.ResultsSP cells in human gastric carcinoma cell lines were isolated and identifiedsuccessfully. BGC-823and MGC-803cells labeled with Hoechst33342. Both ofthe two cell lines contained SP cells, and the cells that had the strongest dyeefflux activity (SP cell) in MGC-803had higher clone formation efficiency thannon-SP cells. When transplanted into non-obese diabetic/severe combinedimmunodeficiency (NOD/SCID) mice, SP cells showed at least50times highertumorigenicity than non-SP cells. To get better understanding of the mechanismof the maintenance of SP cells in GC, we used the special inhibitor of PI3K/Aktpathway to detect the variation of the SP cells through FCM and the ability of thegastric cell line cells in cloning, and we found that PI3K/Akt pathway wasessential to SP cells.ConclusionsTaken together, our results supported that the minority populationdescribed as SP cells owned cancer stem cell character in gastric carcinomacell lines. These findings indicated that SP cells enriched cancer stem-like cells in GC cell lines and PI3K/Akt pathway regulated this stem-like population.Further understanding of tumor-stem cell specific traits will offer insights on theearly stages of tumorigenesis for prevention and enhanced selectivity ofanti-tumor therapeutics.