| Objective: Existing theory has shown that tumors is closely related with stem cells. Cancer stem cells play an important role in the development of the malignant disease and are the source of defective therapy. Cancer stem cells was involved in human gastric cancer cell lines. Diallyl trisulfide can inhibit growth of human gastric cancer cells, while it is not clear if its anti-cancer role is related with CSCs. In this study, after sorting subpopulation of CD44(+) CSCs-like cells in the the human gastric cancer SGC-7901, the suppression effect of DATS in CSCs-like cells was observed, and the mechanism was analyzed, aiming at offering credible academic and experimental bases for clinical use.Methods:The proliferation inhibition effect was detected in SGC-7901 cells treated with DATS by MTT assay. The expression of c-FLIP was investigated by immunohistochemistric assay and Western blot. Subpopulation of CD44(+) was sorted in human gastric cancer SGC-7901 by MACS and immuno-fluorescence flow cytometry analysis. Human gastric CSCs-like cells were identified by Spheroid Colony Formation Assay and colony formation in soft agar. Tumor stem cells were morphologically observed under the light mocroscope. The proliferation effect and cell cycle ratio were observed by colony formation in soft aga experiment and flow cytometry analysis in gastric cancer stem cells respectively. After exposed to DATS, c-FLIP expression in CD44(+) cells was detected by immunohistochemistric assay.Results:1. After SGC-7901 cells were treated with 6,8,10,12,14,16mg/L-1 DATS for 24h, the proliferation inhibition ratio ranged from 20.4% , 29.4% , 32.2%, 49.1%, 71.3%, 79.2%; the inhibition ratio was 36%, 37.3%, 65.7%, 78.2%, 84%, 89.1% respectively after exposed to DATS for 48h, which exhibited a dose-dependent model by MTT test.2. Immunohistochemistry and western-blot indicated that after exposed to DATS for 24h and 48h, c-FLIP expression of SGC-7901 cells down-regulated significantly compared to untreated group(P<0.05).3. Immuno-fluorescence flow cytometry analysis showed that only 2.4% cells was CD44(+) in the total population of SGC-7901 cell line, but the ratio of CD44(+) cells in the CD44(+) population reached 97.4% after cell–sorting. CD44-positive gastric cancer cells produced spheroid colonies in serum-free medium after two weeks, while CD44(-) cell lines did not (P<0.05). Colony formation in soft agar experiment showed that CD44(+) cells exhibited more powerful proliferation capacity than CD44(-) cells after cultured in soft agar medium for two weeks (P<0.05). Flow cytometry results showed that S phase ratio of CD44(+) cells was higher than that of CD44(-) cells(P<0.05). Under the optical microscope, the typical morphological changes were no difference in cells of three groups.4. After treated with DATS, colony formation assay in soft agar experiment showed that sphere cells of CD44(+) group were smaller than untreated CD44(+) group(P<0.05). 5. Both 10 mg·L-1 and 20 mg·L-1 DATS for 48h could induced G2/M phase ratio of CD44(+) cells significantly higher than untreated CD44(+) cells (P<0.05). 6. After exposed to DATS , c-FLIP protein expression down-regulated significantly in CD44(+) cells compared to the untreated group(P<0.05).Conclusion:1. CD44(+) CSCs-like cells were involved in gastric cancer cell line SGC-7901.2. Inhibition effect and G2/M phase arrest was induced by DATS in gastric cancer SGC-7901 CSCs-like cells .3. Down-regulation of c-FLIP expression may be one of the molecular mechanism of DATS-induced growth inhibition in SGC-7901 cells and its CSCs-like cells.
|
PDF Full Text Request