| Objective:To investigate the role of endoplasmic reticulum stress (ERS) in adenosine-induced esophageal cancer (EC109) cells apoptosis.Methods:EC109 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (V/V) fetal bovine serum (FBS). Cells were trypsinized and plated into culture dishes, and then were used to the following experiments: (1) MTT assay: 2 mmol·L-1 adenosine treated EC109 cells in different time (24～72 h) or different concentrations of adenosine (0.5～4 mmol·L-1) treated EC109 cells for 36 h and the cell survival rates were analyzed with MTT method (2) TUNEL technique: Apoptotic effect of EC109 cells treated by adenosine in various concentrations (0.5, 1, 2 and 4 mmol·L-1) for 36 h respectively, assessed by TUNEL assay (3) Immunofluorescence: cells were treated with normal media (blank and control group) or 2.0 mmol·L-1 adenosine (adenosine treatment group) for 36 h after cells adherence, Immunofluorescence assay was used to detect the sub-cellular distributions of GRP78, Caspase-4, Caspase-3, CHOP. (5) Western blotting: cells were treated with different concentrations of adenosine (0.5～4 mmol·L-1) for 36 h, the total proteins were extracted from cells, western blotting was used to detect the expressions of ERS-related proteins (GRP78, Caspase-4, CHOP) and NF-κB, Caspase-3.Results:1, The survival rates of EC109 cells after adenosine treatment in different time (24～72 h) were gradually decreased (57.7±15.0%,56.5±11.1%,43.8±5.7%,28.8±4.1%; p<0.01, vs control ). The cell survival rates after treated with different concentrations of adenosine (0.5～4 mmol·L-1) decreased gradually ( 82.2±6.2%,67.1±4.1%,54.7±2.2%,45.6±3.9%, p<0.01, vs control )2, The apoptotic indexes after treated with different concentrations of adenosine (0.5～4 mmol·L-1) gradually increased (15.5±1.1%,28.2±0.8%,40.1±2.2%,50.6±1.3%, p<0.05, vs control ) in a dose-dependent manner after adenosine treatment.3, According to the results of immunofluorescence, GRP78 appeared significantly both in control and drug treated group cells'cytoplasm, and Caspase-4,CHOP and Caspase-3 appeared both in drug treated group cells'cytoplasm and nuclei.4, western blotting suggested that expressions of GRP78, Caspase-3, Caspase-4, CHOP and NF-κB increased in a dose-dependent manner with differernt adenosine concentrations (0.5, 1, 2 and 4 mmol·L-1) treatment (rGRP78=0.9471, rCaspase-4=0.8977,rCaspase-3=0.968, rCHOP=0.9762, rNF-κB=0.9471, P<0.05, vs control)Conclusion:1, Adenosine can induce EC109 cells apoptosis, which was consistent with our previous studies on HepG2 cells.2, In this process of apoptosis, the expressions of ERS related proteins such as GRP78,Caspase-4,CHOP,NF-κB were increased obviously, Caspase-4,Caspase-3,CHOP translocated from cytoplasm to cell nuclei, which means that the endoplasmic reticulum stress pathway is activated and ERS is related to adenosine-induced EC109 cells apoptosis. |
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