| Objective:To investigate the inhibitory effect of PI3 K / AKT / mTOR dual channel inhibitor NVP-BEZ235 combined with 5-fluorouracil(5-FU)on human gastric cancer cell line MKN-45,and to explore its possible mechanism.Methods:1 The gastric cancer cell line MKN-45 was cultured in RMPI-1640 complete medium containing NVP-BEZ235(8 n M,16 nM,32 nM,64 nM,250 nM,250 nM,500 nM,1 μM NVP-BEZ235 respectively).The MTT assay was used to detect the proliferation of gastric cancer MKN-45 cell which culture with 5-FU for 48 hours.2 The gastric cancer cell line MKN-45 was cultured in RMPI-1640 complete medium containing 5-FU(4μM,8μM,16μM,32μM,125μM,250μM,500 M,1mM 5-FU respectively).The MTT assay was used to detect the proliferation of gastric cancer MKN-45 cell which culture with 5-FU for 48 hours.3 The cells of MKN-45 cells were cultured with RMPI-1640 complete medium containing NVP-BEZ235(200 nM,400 nM,800 nM),RMPI-1640 complete medium containing5-FU(40 μM,80 μM,160 μM)and RMPI-1640 complete medium containing NVP-BEZ235 combined with 5-FU(200 nM NVP-BEZ235 + 40 μM 5-FU,400 nM NVP-BEZ235 + 80μM 5-FU,800nMNVP-BEZ235 + 160μM 5-FU)for 48 hours,and then MTT assay was used to detect the proliferation of gastric cancer MKN-45 cell.4 The MKN-45 cells were divided into four groups: The control group was cultured with RMPI-1640 complete medium;The BEZ group was cultured in RMPI-1640 complete medium containing 400 nM NVP-BEZ235;The FU group was cultured in RMPI-1640 complete medium containing 80μM 5-FU;The Comb group was cultured in RMPI-1640 complete medium containing 400 nM NVP-BEZ235 and 80 μM 5-FU.Western blot is used to detect the autophagy and apoptosis of MKN-45 cells.The apoptotic rate of cells was detected by flow cytometry.The MKN-45 cells’ s ability of migration and invasion were detected by transwell chamber.Result:1 NVP-BEZ235 and 5-FU were treated with different concentrations on gastric cancer MKN-45 cells.After 48 h culture,the results showed that the cell proliferation ability decreased with the increase of drug concentration.2 NVP-BEZ235,5-FU and the combination of both drugs inhibited the proliferation of human gastric cancer MKN-45 cells compared with the control group after 48 hours(P <0.01).Compared with the respective of two drugs,the inhibitory effect of combination group was obvious(P <0.01).3 Western Blot to detect the drugs after treatment MKN-45 cell PI3K/Akt/mTOR related proteins: Akt results shows,BEZ was significantly lower than the Control Group,Comb set significantly lower than FU group,statistically significant differences(P=0.00);p-Akt(308)relative expression levels,BEZ was significantly lower than the Control Group,Comb set significantly lower than FU group,statistically significant differences(P=0.00);P-Akt(473)relative expression levels showed that BEZ group was significantly lower than the Control Group,Comb set significantly lower than FU group,statistically significant differences(P=0.00);displayed relative expression of mTOR,BEZ was significantly lower than the Control Group,Comb set significantly lower than FU group,statistically significant difference(P=0.00).Expression of apoptosis-related protein Caspase3 display,BEZ group was significantly higher than the Control Group,Comb set significantly lower than FU group,statistically significant differences(P=0.00);Bax/bcl-2 the relative expression level,the difference between treatment groups is not exactly the same,BEZ group was significantly higher than the Control Group,Comb group was significantly higher than that of FU group was statistically significant(P=0.00).4 The apoptotic rate was measured by flow cytometry.The results showed that the apoptosis rate of MKN-45 cells in BEZ group was significantly higher than that in Control group.The apoptosis rate of MKN-45 cells in the Comb group was significantly higher than that in FU group(P = 0.00).The number of MKN-45 migratory cells in the BE group was(150.00 ± 9.17 / field)less than that in the control group(275.67 ± 11.59 / field);the percentage of MKN-45 migratory cells in the Comb group was(47.33 ± 8.14 / field)In the FU group(200.00 ± 25.16 / field),the difference was statistically significant(P = 0.00).The number of MKN-45 invasive cells in the BEZ group was(150.00 ± 9.17 / field)and less than that in the control group(275.67 ± 11.59 / field).The MKN-45 invasion cells in the Comb group were(47.33 ± 8.14 field),In the FU group(200.00 ± 25.16 / field),the difference was statistically significant(P=0.00).Conclusion:1 NVP-BEZ235 can increase the level of autophagy in tumor cells,increase the apoptosis of tumor cells,reduce the migration and invasion of tumor cells.2 NVP-BEZ235 and 5-FU can improve the sensitivity of gastric cancer MKN-45 cells to 5-FU3 NVP-BEZ235 Increases the sensitivity of gastric cancer MKN-45 cells to 5-FU may be associated with a decrease in activity of PI3 K / AKT / mTOR pathway levels. |
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