The Effects Of Endoplasmic Reticulum Stress In Adenosine-induced HepG2 Cell Apoptosis

Objective:To investigate the effects and molecular mechanisms of endoplasmic reticulum stress in adenosine-induced apoptosis of human hepatoma HepG2 cells.Materials And Methods:The human hepatoma HepG2 cells were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10%(V/V) fetal bovine serum(FBS). The cells were trypsinized and plated in culture dishes, and then they were used to the following experiments:①MTT assay: Cells were plated in 96-wells culture dishes, exposed to different concentrations of ADO(0—6.0 mmol/L) for 36 h after cell adherence, MTT assay were used to measure the effects of ADO on cell proliferation.②Cell nuclei DAPI staining: Cells were plated in cover glass, treated with different concentrations of ADO(0—4.0 mmol/L) for 36 h or 2.0 mmol/L ADO for different time(0—48 h)after cell adherence, the nuclei were stained by DPAI to view the morphological changes.③Immunofluorescence: Cells were plated in cover glass, treated with normal media(blank and control group) or 2.0 mmol/L ADO(ADO treatment group) for 36 h after cell adherence, Immunofluorescence assay technique was used to detect the sub-cellular distributions of CHOP and Caspase-3.④Western blot: Cells were plated in culture-flasks, treated with different concentrations of ADO (0—4.0 mmol/L)for 36 h, the total proteins were extracted from the cells, western blot was used to detect the expressions of ERS-related proteins(GRP78,Caspase-4,CHOP,JNK) and Caspase-3. Results:1,The results of MTT assay showed that the optical density (OD) value of ADO-treated group was gradually decreased as the ADO concertrations increased, there was a statistical difference compared with the control group(p<0.05).2,DAPI stainning showed that the cell nuclei displayed condensation after treated with 1.0 mmol/L ADO for 36 h or 2.0 mmol/L ADO for 12 h; the nuclei displayed fragmentation after treated with 2.0 mmol/L ADO for 36 h; and the nuclei displayed break down after treated with 4.0 mmol/L ADO for 36 h or 2.0 mmol/L for 48 h.3,The results of immunofluorescence showed that the protein expressions of CHOP and Caspase-3 increased after treated with 2.0 mmol/L ADO, and they translocated from cytoplasm to cell nuclei.4,Western blot tests demonstrated that the expressions of ERS related proteins (GRP78,Caspase-4,CHOP) increased after treated with different concentrations of ADO(0—4.0 mmol/L), and there was a statistical difference compared with the control group(p<0.05); while the expression difference of JNK was not observed. The protein expression of Casspase-3 increased as the ADO concentrations increased, and reached a peak value at 2.0 mmol/L ADO; with a statistical difference compared with the control group(p<0.05).Conclusion:1,Adenosine can induce apoptosis of human hepatoma HepG2 cells, which was consistent with our previous studies.2,In the process of cell apoptosis, the expressions of ERS related proteins such as GRP78,CHOP and Caspase-4 were increased obviously, CHOP and Caspase-3 translocated from cytoplasm to the cell nuclei ,which means that the endoplasmic reticulum stress pathway is activated and ERS is related to ADO-induced HepG2 cell apoptosis .