The Effect And Mechanism Of Group Ⅰ Metabotropic Glutamate Receptor On Proliferation And Migration Of Esophageal Carcinoma Cells

Background Esophageal cancer is a common digestive systemic neoplasm,which accounts for the second death of tumors in various parts of the digestive system.Due to the high degree of malignancy of esophageal cancer and the prone to early metastasis,most patients have been in advanced stage at the time of treatment and lost the chance of surgery.Due to the side effects of chemotherapitic drugs,it is necessary to find new therapeutic targets.Studies have shown that activation/blocking of metabotropic glutamate receptors can affect the growth and migration of multiple tumor cells.However,the relationship between metabotropic glutamate receptors and esophageal cancer remains unclear.This study aims to elucidate the effects of metabotropic glutamate receptor on proliferation and migration of esophageal cancer cells and the possible mechanisms.Objective In this study,a metabotropic glutamate receptor agonist DHPG was applied to esophageal cancer cell lines,Eca109 and KYSE-450,to elucidate the effect of DHPG on proliferation and migration of esophageal cancer cells and its downstream signaling pathways.Method Western blot was performed to examine the expression of metabotropic glutamate receptors in esophageal cancer tissues and adjacent normal tissues,as well as normal esophageal epithelia cell line Het-1A and esophageal squamous carcinoma cell lines Eca109 and KYSE-450.Eca109 and KYSE-450 cells were treated with group I metabotropic glutamate receptor agonist DHPG,(1)The effect of DHPG on the proliferation of esophageal cancer cells was detected by MTT assay,the effect of migration on the ability of migration by Transwell assay,and the effect of flow cytometry on cell cycle and apoptosis.(2)Western blot was used to detect the changes of p21,Bcl2、Bax、E-cadherin,β-catenin,vimentin,AKT,p-AKT,m TOR,p-m TOR,p-p70S6 K and p-4EBP1.Result 1.Western blot was performed to examine the expression of two subtypes of group I metabotropic glutamate receptors m Glu R1 and m Glu R5 in esophageal cancer tissues and their adjacent tissues,as well as normal esophageal cell Het-1A and esophageal squamous carcinoma cells Eca109 and KYSE-450.We demonstrated that both m Glu R1 and m Glu R5 were expressed in all tested tissues and cell lines.This result provides a theoretical basis for this project.2.MTT assay was performed to investigate the effect of DHPG on the proliferation of Eca109 and KYSE-450 cells.The results showed that DHPG inhibited cell proliferation of Eca109 and KYSE-450 cells in a time and dose dependent manner.According to the MTT assay results,Eca109 cell line was choosen for following experiments.3.Transwell assay was utilized to assess the effect of DHPG on the migration of Eca109 cells.The results showed that the migrtated cell number of the control group was(457.82 ± 47.25)/field,and that of the DHPG-treated group was(258.76 ± 38.53)/fields.DHPG significantly inhibited the cell migration(p<0.01)compared with the control group.4.The cell cycle distribution analyzed by flow cytometry showed that the cell number at S phase was increased in DHPG-treated cells compared with control cells(26.52 ± 4.35% vs 45.44 ± 6.73%,p<0.01),indicating that DHPG blocks cells in cell clycle S phase.The apoptosis of Eca109 cells determined by flow cytometry was promoted by DHPG.5.Western blot results showed that after treatment with DHPG for 48 h,p-AKT,p-m TOR,p-p70S6 K,p-4EBP1,and the ratio of apoptosis-related protein Bcl2/Bax was downregulaed,the cell cycle-related protein p21 was up-regulated.The migration-associated protein E-cadherin was up-regulated while β-catenin and vimentin were significantly down-regulated after DHPG application.Conclusion 1.Group I metabotropic glutamate receptors were expressed in esophageal cancer tissues and esophageal cancer cell lines.2.DHPG inhibits the proliferation of esophageal cancer cells,which may be mediated by cell cycle arrest in S phase and activation of cell apoptotic process.3.DHPG previented the migration of esophageal cancer cells,which may be mediated by blocking epithelial-to-mesenchymal transition.4.Activation of metabotropic glutamate receptors in group Ⅰ may be related to the AKT/ m TOR/p70S6K-4EBP1 signaling pathway.