Study On Expression And Significance Of PERK/eIF2? Protein In Human Tongue Squamous Cell Carcinoma Apoptosis Induced By Anti-tumor Component-? From Agkistrodon Acutus Venom

Objective:To investigate the detection of protein kinase R like endoplasmic reticulum kinase?PERK?/eukaryotic translation initiation factor 2??eIF2??molecular protein in apoptosis of human tongue squamous cell carcinoma Tca8113 induced by anti-tumor component-I from agkistrodon acutus venom?AAVC-I?and analyze the mechanism of signal transduction pathway of PERK/eIF2?molecular protein under endoplasmic reticulum stress?ERS?.Methods:In this study,human tongue squamous cell carcinoma Tca8113 cultured in vitro was selected as the research object,logarithmic growth cells were selected for the experiment,ERS inducer DL-dithiothreitol?DTT?as positive control drug.1.MTT assay was used to detect the proliferation inhibition rate of Tca8113 cells treated with DTT?1.0?2.0?4.0?8.0mM?and AAVC-I?2.0?4.0?8.0?16.0?24.0?g/ml?at different concentrations for 24h,and according to the half maximal inhibitory concentration(IC₅₀)value of cell proliferation inhibition rate,the experimental concentration of positive control drug DTT was screened,and AAVC-I may induce ERS of Tca8113 cells optimal experimental concentration range.2.Tca8113 cells were treated with according to the selected experimental concentration groups of DTT and AAVC-I,24h later,the cell haematoxylin&eosin?HE?staining was used to observe the morphological changes of the cells under inverted microscope.3.Annexin V-FITC/PI double fluorescence staining was used to observe the apoptosis of Tca8113 cells treated with the selected experimental concentration groups of DTT and AAVC-I for 24h,and flow cytometry was used to detect the apoptosis rate.4.When the Tca8113 cells were treated with the selected experimental concentration groups of DTT and AAVC-I for 24h,the cells undergo apoptosis,western blot?WB?was used to detect the expression levels of ERS landmark molecular protein glucose-regulated protein 78?GRP78?,phosphorylated-PERK?p-PERK?and phosphorylated-eIF2??p-eIF2??in PERK/eIF2?molecular proteins signal transduction pathway,downstream activating transcription factor 4?ATF4?and enhance-binding protein-homologous protein?CHOP?molecular proteins,apoptosis-related molecular proteins B cell lymphoma-2?Bcl-2?and Bcl-2 associated X protein?Bax?.Results:1.Human tongue squamous cell carcinoma Tca8113 were treated with different concentrations of DTT?1.0,2.0?4.0,8.0mM?and AAVC-I?2.0,4.0,8.0,16.0,24.0?g/ml?for 24h,MTT test results show that with the increase of DTT and AAVC-I concentration the inhibition rate of cells proliferation increased gradually?P<0.01?,meanwhile,the DTT positive control experimental concentration group?4.0mM?and and the optimal experimental concentration range of AAVC-I?4.0,8.0,16.0?g/ml?that caused ERS in Tca8113 cells were successfully screened.2.The Tca8113 cells were treated with 4.0mM DTT positive control experimental concentration group and 4.0?g/ml,8.0?g/ml,16.0?g/ml AAVC-I experimental concentration groups for 24h,the observation under an inverted microscope by cell HE staining showed that cells in the normal control group grew adherent,cells were spindle or polygon,and the cytoplasmic density was evenly distributed,the observation of cells in the concentration groups of AAVC-I experiment showed that with the increase of the concentration groups of AAVC-I experiment,cells gradually shrunk and became smaller,adherent cells gradually decreased,intercellular space increased,cytoplasmic cytonuclear concentration,and cell rupture,in particular,the 4.0mM DTT positive control experimental concentration group and the 16.0?g/ml AAVC-I experimental concentration group showed more significant changes in cell morphology.3.The Tca8113 cells were treated with 4.0mM DTT positive control experimental concentration group and 4.0?g/ml,8.0?g/ml,16.0?g/ml AAVC-I experimental concentration groups for 24h,Annexin V-FITC/PI double fluorescence reagent was used to observe the apoptotic cells by fluorescence staining and the apoptotic rate was analyzed by flow cytometry,the results showed that,compared with the normal control group,fluorescently labeled apoptotic cells were observed under fluorescence microscope in the DTT positive control experimental concentration group and the AAVC-I experimental concentration groups,the AAVC-I experimental concentration group cells with the increase of AAVC-I experimental concentration,apoptotic cells increased and the apoptosis rate increased gradually?P<0.01?.4.The Tca8113 cells were treated with4.0mM DTT positive control experimental concentration group and 4.0?g/ml,8.0?g/ml,16.0?g/ml AAVC-I experimental concentration groups for 24h,the expression levels of related proteins by WB method to detect the ERS landmark molecular protein GRP78,the p-PERK and p-eIF2amolecular proteins of PERK/eIF2amolecular protein signal transduction pathway,downstream ATF4 and CHOP molecular proteins,apoptosis-related molecular proteins Bcl-2 and Bax.The results showed that the expression levels of molecular proteins in the concentration group of DTT positive control experiment and the concentration groups of AAVC-I experiment were statistically different compared with the normal control group?P<0.05?,and with the increase of AAVC-I experimental concentration,the expression levels of GRP78,p-PERK,p-eIF2a,ATF4,CHOP and Bax molecular proteins gradually increased,while the expression levels of Bcl-2 molecular proteins gradually decreased?P<0.05?.Conclusion:1.DTT and AAVC-I can inhibit the proliferation of human tongue squamous cell carcinoma Tca8113,4.0mM DTT and 4.0?g/ml,8.0?g/ml,16.0?g/ml AAVC-I were successfully screened as the optimal experimental concentration groups for ERS in human tongue squamous cell carcinoma Tca8113.2.DTT concentration of 4.0mM was used as the experimental positive control group,4.0?g/ml,8.0?g/ml,16.0?g/ml AAVC-I experimental concentration group can cause human tongue squamous cell carcinoma Tca8113 to develop ERS.3.AAVC-I can induce apoptosis of human tongue squamous cell carcinoma Tca8113.4.The ERS PERK/eIF2amolecular protein signaling pathway promotes apoptosis in human tongue squamous cell carcinoma Tca8113 induced by AAVC-I.