Research Bafilomycin A1 Inhibits Gastric Cancer Cell Line SGC-7901 And Enhances Its Sensitivity To 5-FU

ObjectiveVerify 5-fluorouracil can induce the protective autophagy of SGC-7901 cells, andobserve the effect of bafilomycin A1(an autophagy inhibitor) on its autophagy.Investigate the change of autophagy to the cellular proliferation, apoptosis, migratoryand invasive ability, and to chemosensitivity to 5-fluorouracil.Methods1. Use different concentration of 5-furouacil(0, 0.625, 1.25, 2.5, 5.0, 10.0ug/m L)to culture the SGC-7901 cells for 24 h or 48 h, and then use MTT to detect the cellviability.2. Use 1.25ug/m L of 5-fluorouracil to culture SGC-7901 cells for 0, 3, 6, 12, 24 or48 h, and then use MTT to detect the cell viability and Western Blotting to detect thechange of autophagy-related and apoptosis-related protein.3. Divide the cells into four groups: the Control group in which cells were culturedin complete medium only; the BAF group in which cells were cultured in completemedium contained 100 n M of bafilomycin A1; the 5-FU group in which cells werecultured in complete medium contained 1.25ug/m L 5- fluorouracil; the 5+B group inwhich cells were cultured in complete medium contained 100 n M of bafilomycin A1 and1.25ug/m L 5- fluorouracil. Use scanning electron microscopic, IHC and WesternBlotting to observe autophagy level of the cells. Use MTT assay to observe cellviability.Use flow cytometry and Western Blotting to observe apoptosis. Use migrationassay and invasion assay to observe cellular migratory and invasive ability.Result1. 5- fluorouracil could depress the cell viability of SGC-7901 cells inconcentration-and time-dependent manners(0-48h). When the cells were cultured in thecomplete medium contained 1.25ug/m L 5- fluorouracil for 24 h and 48 h, the cellviability of the two groups were significantly lower than the control group(P=0.000).2.When the cells was cultured with 1.25 ug/m L of 5-fluorouracil for 48 h, with theextension of action time the expression of autophagy-related protein Beclin1 wasincreasing gradually until arriving at the peak at 24 h, and then turned into reducing;LC3-Ⅱ/Ⅰ was increasing gradually(0-48h); P62 was increasing gradually untilarriving at the peak at 12 h, and then turned into reducing; the expression ofapoptosis-related protein Bax was increasing gradually until arriving at the peak at 6h,and then turned into reducing, the Bcl-2 was increasing gradually(0-48h). The ratio ofBcl-2 and Bax(12-48h) was significantly higher than that in the Control group from12 h to 48 h.3. Compared with the 5-FU group, the 5+B group had more autophagosomes andless autolysosome. The IHC assay showed that the average optical density of Beclin1 inthe 5+B group was significantly lower than the 5-FU group(P=0.000). Western blotshowed that the expression of LC3Ⅱ/Ⅰin the 5+B group was significantly higher thanthat in the 5-FU group(P=0.000), the expression of P62 in the 5+B group wassignificantly higher than that in the 5-FU group(P=0.002). MTT assay showed that thecell viability of the 5+B group was significantly lower than that in the 5-FU group(P=0.004). The migration assay showed that the cells penetrated the filter in the 5+Bgroup was fewer than that in the 5-FU group(P=0.000). The invasion assay showed thatthe cells penetrated the filter in the 5+B group was fewer than that in the 5-FU group(P=0.001). The apoptosis rate of the 5+B group was significantly lower than it of the5-FU group(P=0.000). Western Blot showed that the expression of Bax in the 5+Bgroup was significantly higher than that in the 5-FU group(P=0.000), the expression ofBcl-2 in the 5+B group was significantly lower than that in the 5-FU group(P=0.000).The ratio of Bcl-2 and Bax in the 5+B group was significantly lower than that in the5-FU group(P=0.000).4. Compared with the Control group the addition of Bafilomycin A1 made moreautophagosomes and less autolysosomes gathered. The IHC assay showed that theaverage optical density of Beclin1 in the the BAF group was significantly lower thanthat in the Control group(P=0.008). Western blot showed that the expression of LC3Ⅱ/Ⅰin the BAF group was significantly higher than that in the Control group(P=0.000),the expression of P62 in the BAF group was significantly higher than that in the Controlgroup(P=0.000). MTT assay showed that the cell viability in the BAF group wassignificantly lower than that in the Control group(P=0.000). The migration assayshowed that the cells penetrated the filter in the BAF group was fewer than that in theControl group(P=0.000). The invasion assay showed that the cells penetrated the filterin the BAF group was fewer than that in the Control group(P=0.000). The apoptosisrate of the BAF group was significantly lower than that of the Control group(P=0.000).Western Blot showed that the expression of Bax in the BAF group was significantlyhigher than that in the Control group(P=0.005), the expression of Bcl-2 in the BAFgroup was lower than that in the Control group(P=0.046). The ratio of Bcl-2 and Bax inthe BAF group was significantly lower than that in the Control group(P=0.002).Conclusion1. 5-fluorouracil can induce the protective autophagy of the SGC-7901 cells, andthe autophagy may improve the resistance ability of the SGC-7901 cells to5-fluorouracil.2. Bafilomyicn A1 can inhibit the protective autophagy of the SGC-7901 cellsinduced by 5- fluorouracil, and also can improve its chemosensitivity to 5- fluorouracil.3. Bafilomycin A1 can inhibit the normal level autophagy of the SGC-7901 cells.Inhibiting autophagy can depress the proliferation, invasive and migratory ability of theSGC-7901 cells, and also can promote their apoptosis. Bafilomycin A1 can promoteapoptosis, which may involve in down-regulating the expression of Bcl-2 andup-regulating the expression of Bax.