| Objective:The gastric cancer is one of the deterioration malignant tumors of the alimentary canal, which morbidity and mortality are high. The gastric cancer is one of the most health-threatening diseases in our country. Energy metabolism were changed in cancer cells. Glutamine is an significant metabolic substrate that provide sufficient materials,like carbon source, nitrogen source for the rapid proliferation of cells. In addition, glutamine metabolism can also participate in signal transduction, even in such aspects as maintaining redox homeostasis is of great significance. In recent years,changes of glutamine metabolism in cancer cells were gradually valued, the effect of abnormal glutamine metabolism on proliferation of cancer cell has become a hot area of research. GLS is the foremost and imiting velocity enzyme of glutamine metabolic,so inhibition of GLS will influence energy supply of cancer cells which living dependence on glutamine metabolism. The disorder of glutamine metabolism were determined at organ and cell levels.And then this study choosed CB-839 as inhibitor,because its specific inhibition effect on GLS. In a word, the objective of this issue was explored the glutamine metabolism and the effect of GLS inhibition in gastric cancer cells MGC–803 and SGC–7901, to provide a more effective way to kill gastric cancer cells.Methods:1) Collect the normal gastric mucosa and gastric cancer tissue samples,immunohistochemical was used to detected GLS protein expression in those samples.2) Cultivate gastric cancer cells MGC-803 and SGC-7901 as experimental subject,and normal gastric epithelial cell GES-1 for reference.3) Set up blank control group culture with normal medium, experimental group culture with excluding glutamine medium to study the implication of gastric cancer cells dependence on the effects of glutamine metabolism.4) Proteins were extracted from exponential cells of GES-1, MGC-803,SGC–7901.GLS protein expression was detected by western blotting.5) Proteins were extracted from exponential cells of GES-1, MGC-803, SGC-7901.According to the kit manual processing, GLS activity was detection by visible spectrophotometry.6) Set up blank control group, experimental group culture with CB-839 1μmol/l.Effects of CB-839 on proliferation of experimental groups was measured by MTT assay. The test was to identify the effect of GLS inhibition on survival of gastric cancer cells SGC-7901, MGC-803 and normal GES-1.7) Set up blank control group, experimental group culture with CB-839 1μmol/l.Effects of CB-839 on cell cycle of experimental groups was measured by flow cytometry. The test was to identify the effect of GLS inhibition on cell cycle of gastric cancer cells SGC-7901, MGC-803 and normal GES-1.Results:1) Positive expression rate of GLS in normal gastric mucosa tissues was very low,but in gastric cancer tissues, it was rather high(P<0.05), in 30 cases of gastric cancer tissues, there were 19 cases with high expression of GLS, and found that the lower the degree of differentiation, the higher positive expression rate.2) The dependence of gastric cancer cells on glutamine was measured by cell counting: The results showed that MGC-803 cells have absolute requirement for glutamine compared with the control group. The lethal effec has been observed in the third day. In the 5th day, MGC-803 cells showed typical apoptotic patterns.In the 7th day, almost all the adherent cell falls off, and leading MGC-803 cells to fatal influence because glutamine had been deprived(P<0.01). Effect of glutamine deprivation on SGC-7901 was smaller relative to MGC-803, the differences between blank and experimental groups showed in the 7th day( P<0.05). The contrast was not statistically significant in both groups of GES-1.3) GLS protein expression was detected by western blotting: GLS expression were no differences in SGC-7901 and GES-1(P>0.05), but in MGC-803, the expression of GLS had much higher expression rate(P<0.01).4) GLS activity was determined by visible spectrophotometry: GLS activity in SGC-7901 and MGC-803 are much higher than in GES-1, and the statistical analysis showed significant difference(P<0.05).5) The cell survival inhibitory effect of CB-839: the results showed that, no apparent changes of cell survival rate of GES-1 were observed, the difference was not statistically significant(P>0.05). But in gastric cancer cells, the statistical analysis showed significant difference. Cell survival rate of SGC-7901 and MGC-803 were(88.32±5.44)% and(31.63±8.62)% respectively, contrast with GES-1, survival rate of MGC-803 showed significant difference(P<0.01).6) The cell cycle inhibitory effect of CB-839: the results showed that, no apparent changes of cell cycle of GES-1 were observed, but in MGC-803, the cell cycle can obviously influenced. The ratio of G0/G1 phase, S phase and G2/M phase were changed from 51.52%, 39.38%, 9.10% to 38.78%, 54.62%, 6.60%,respectively. The percentage of S phase of MGC-803 cells was significantly been influenced(P<0.01). The ratio of G0/G1 phase, S phase and G2/M phase of SGC-7901 were changed from 41.06%, 43.58%, 15.36% to 47.10%, 42.95%,9.95%, respectively. The percentage of G0/G1 phase of SGC-7901 cells was increased(P<0.01).Conclusion:The result suggest that there are abnormal metabolism in gastric cancer cells,expression of GLS are significantly increased; the influential of glutamine metabolic on proliferation of gastric cancer cells is connect with it’s differentiated degree;suggest that GLS may be potential therapeutic targets for gastric carcinoma. |
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