| Objective: To observe the effects of Fentanyl,PI3 K inhibitor(LY294002)and MMP-9 inhibitor(SB-3CT)on the growth of human gastric carcinoma cell line MGC-803,and explore the possible mechanism of PI3K/Akt/MMP-9 of fentanyl in inhibiting the progression of gastric cancer.Methods: Human gastric carcinoma cell line MGC-803 were cultured to logarithmic growth phase and randomly divided into 6 groups: 0.1 ?mol/L fentanyl group(F group),20 ?mol/L LY294002 group(LY group),10 ?mol/L SB-3CT group(SB group),20 ?mol/L LY294002+0.1 ?mol/L fentanyl(FLY group),10 ?mol/L SB-3CT+0.1 ?mol/L fentanyl group(FSB group)and set up a blank control group(C group).Fentanyl,LY294002,and SB-3CT were added to the cell culture medium in the experimental group.The final concentration of fentanyl in the F group was 0.1 ?mol/L,LY294002 in the LY group was 20 ?mol/L and SB-3CT in the SB group was 10 ?mol/L.In the FLY group,the culture medium containing 20 ?mol/L LY294002 was added to incubate for 30 min and then added fentanyl to a final concentration of 0.1 ?mol/L for incubation.Likewise,the culture medium containing 10 ?mol/L SB-3CT was added first to incubate for 30 min in the FSB group and then added fentanyl to a final concentration of 0.1 ?mol/L for incubation.While the C group did not add any drugs.CCK-8 was used to detect the change of cell viability.Transwell invasion and wound healing test were used to detect the invasion and migration of cells in each group.Flow cytometry was used to detect apoptosis and cell cycle distribution.The ultrastructure of cells was observed by transmission electron microscope.The expression of Akt,pAkt,MMP-9,caspase-9 and DAPK-1 were detected by quantitative real-time PCR(qPCR)and Western Blot.Results: Compared with the C group,the cell viability of experimental group was decreased(P <0.05).The activity of cells in combination group was lower than that of single drug group.Transwell invasion experiments showed that the invasive ability of the experiment groups was significantly lower than C group(P<0.05).The combination group was lower than the single drug group,and the difference was statistically significant.The results of wound healing test showed that the healing rate was significantly reduced compared with the C group(P<0.05),and the healing ability of the combination group was even lower than the single drug group.The results of cell apoptosis detected by flow cytometry showed that the apoptosis rates of experimental groups increased compared with the C group(P <0.05).The apoptosis rate of the combination group was higher than the single drug group.Compared with the C group,the proportion of cells in G0/G1 increased in each experiment group,while S phase decreased,and the cell proliferation rate was significantly reduced(P<0.05).The combined drug group was lower than the single drug group.Moreover,transmission electron microscopy showed morphological changes of apoptosis in cells of each experimental group.The results of qPCR and Western Blot showed that the expression of Akt and pAkt was increased in F group while the others was decreased(P<0.05).The expression of MMP-9 was decreased in experiment groups while the expression of caspase-9 and DAPK-1 was higher than the C group.Conclusions: Fentanyl can reduce the activity of human gastric cancer MGC-803 cells,promote its apoptosis,arrest its cell cycle in G0/G1 phase,and reduce its invasion and migration ability.The mechanism of fentanyl inhibiting the growth,invasion and migration of MGC-803 cells may be inhibiting the expression of MMP-9 and enhancing the expression of apoptosis promoting factors such as caspase-9 and DAPK-1 through PI3K/Akt signaling pathway. |
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