| Objective: Cancer stem cells (CSCs) is a unique tumor cells with the ability ofself-renewal and can produce a heterogeneous tumor cells, and is the source of tumorgrowth. Studies had showed that there are gastric cancer stem cells (GCSCs) in gastriccancer. This paper following two aspects, one was that used the magnetic cell sorting(MACS) to separate CD44(+) AGS cells from AGS cells, to elucidate the biologicalcharacteristic of GCSCs-like in CD44(+) AGS cells from proliferation,differentiation, migration, invasion resistance and tumorigenic capacity in vivo. Theother one was, explored the affection of cell growth inhibition and cell cycle of diallyldisulfide (DADS) to CD44(+) AGS, provided important experimental basis for thetreatment of gastric cancer by DADS.Methods: The first, CD44(+) and CD44(-) subpopulation cells in the human gastriccancer AGS cell line were sorted by MACS. Single isolated CD44(+) and CD44(-)AGS cells was cultured in serum-freeDMEN/F12medium.Cell cycle of the twosubpopulation was analyzed by flow cytometry (FCM). Monoclonal spherical colonyforming experiment was performed to investigate the capacity of self-rewal andclonogenic potential of the subpopulation cells.Immunofluorescence staining wasperformed to investigate the multidiferentiation of CD44(+)AGS cells and tumorsphere,formed by CD44(+)AGS cells cultured in serum-free medium. MTT wasperformed to analyze the drug sensitivity of the two subpopulation to anti-tumor drug,5-FU and VP-16. Invasion and migration experiment was performed to investigate thecapacity of migration and invasion of the subpopulation cells. And in order toinvestigate the tumorigenic potential of the two subpopulation in vivo，xenograft inSubcutaneous of BALB/c nude mice was performed. MTT, flow cytometry and softagar colony formation assay was performed to study the cell proliferation and Cycleof the subpopulation cells by DADS. Results:1. Flow cytometry results showed that, only2.8%cells was CD44(+) in the totalpopulation of AGS cell line，and the purity of CD44(+)AGS cells in the CD44(+)population can reach99.9%after cell-sorting.2. The CD44(+) AGS cells exhibited powerful proliferation capacity. The singleCD44(+) AGS cells could proliferate and form typical tumor spheroid, whileCD44(-) AGS cells couldn’t.3. The cell proliferation index of CD44(+) and CD44(-)AGS cells was59.6%and28.3%respectively, showed by FCM.And the monoclonal spherical colonyformation rate of CD44(+)AGS cells was up to74.3%.Itnmunofluoresceneestaining showed that, CD44(+)AGS cells and the tumor sphere, the greenfluorescence was clearly observed in the cell membrane of CD44(+)AGS cells,and only the individual CD44(-)AGS cells had weak green fluorescence, positiveexpression rate was extremely low.4. Differentiation experiment showed that the cells of tumor sphere were adherentgrowth, cytoplasmic outwardly extending protrusions, the expression of CD44fluorescence decreased obviously, the cell envelope of migrated cell was withoutgreen fluorescence.5. The drug resistance experiment showed that cell number decreased muchobviously in CD44(+)AGS cells than those in CD44(-)AGS cells after treated withchemotherapy drug. MTT assay showed that, CD44(-)AGS cells were moresensitive to5-fu and vp-16than CD44(+)AGS cells.6. Invasion and migration experiment showed that, CD44(+)AGS cells entered thelayer of the microporous membrane more than CD44(-)AGS cells.7. Xenograft experiment showed that, CD44(+)AGS cells had tumorigenic potentialin vivo, while CD44(-)AGS cells couldn’t.8. DADS could inhibit the proliferation of CD44(+) AGS cells, and arrested thecycle of CD44(+) AGS cells in G2/M phase with the dependence of time andconcentration. Conclusion:1. CD44positive gastric cancer cell line AGS Gastric cancer stem cells were separatedfrom human gastric cancer AGS cells by MACS technology.2. DADS can inhibit the proliferation of gastric cancer stem cells and make its cyclearrest in G2/M phase. |
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