Experimental Research Of The Ubiquintin-proteasome Inhibitor Induced Apoptosis In Head And Neck Squamous Cell Carcinoma

ObjectiveTo explore the apoptotic effect of ubiquitin-proteasome inhibitor MG-132 on human tongue squamous cell carcinoma cell line Tca-8113 cells through endoplasmic reticulum stress.MethodsTca8113 cells were cultured in RPMI 1640 medium which supplemented with 10% fetal calf serum, the exponential phase cells were divided into 5 groups: negative control group is what the cell culture medium were added to 1640, positive control group were added to thapsigargin and the final concentration was 5μmol/L, and experimental groups were 10,20,30μmol/L MG-132. After 18 h, make the following experiments:MTT test measured the OD values of 490nm, detected the activity of the Tca-8113; the morphological changes of nuclear chromatin were observed by fluorescence microscopy after the cells stained with Hoechst 33258; agarose gel electrophoresis detected the formation of DNA ladder which formed in apoptotic Tca-8113 cells; apoptosis rate of Tca-8113 cells was determined by flow cytometry with annexinV-FITC/Pl double staining; the expression levels of GRP78 and caspase-12 mRNA were determined by RT-PCR; the expression levels of caspase-12 and GRP78 proteins were evaluated by Western blot; the human ubiquitin-protein ligase E3 concentrations were detected by ELISA.ResultsMTT test:the OD valus of negative control group, positive control group and MG-132 groups were 0.515±0.265,0.170±0.690,0.292±0.420,0.222±0.106, 0.148±0.205; Hoechst 33258 staining:positive control group and MG-132 groups showed typical apoptotic morphology of nuclear chromatin; DNA fragmentation detected:ladder with a gray value compared with the control group significantly increased of MG-132 groups and thapsigargin group, and had a concentration-dependent relationship, particularly in high concentration was significantly; the apoptotic rates of negative control group, positive control group and MG-132 groups were 0.43±0.25%,8.49±0.25%, 13.50±0.41%,19.55±0.32%å'Œ34.74±0.50%, respect-tively; the expression levels of GRP78 and caspase-12 mRNA were up-regulated; the similar results were demonstrated by Western blot detection for the treated cells; the human ubiquitin ligase E3 expressions of negative control group, positive control group and MG-132 groups were 40.88±4.52,38.96±0.33,28.75±2.28,18.16±0.65 and 8.85±0.72.Conclusions1. Ubiquitin-proteasome inhibitor MG-132 might induce the human tongue squamous cell carcinoma cell line Tca-8113 cells apoptosis;2. MG-132 might cause endoplasmic reticulum stress to raise GRP78 of Tca-8113 cells;3. MG-132 might induce the apoptosis of Tca-8113 cells through the endoplasmic reticulum stress pathway, activated procaspase-12 and elevated caspase-12 mRNA and protein;4. MG-132 might inhibit the human ubiquitin ligase E3 expression, this is different from a typical endoplasmic reticulum stress inducer thapsigargin.SignificanceThis study investigated the ubiquitin-proteasome inhibitor MG-132 induced human tongue squamous cell carcinoma Tca-8113 cells apoptosis and its relationship between the endoplasmic reticulum stress, revealed the effect of ubiquitin-proteasome pathway in tumor cell apoptosis, provide a theoretical basis of ubiquitin-proteasome inhibitor used in the treatment of head and neck squamous cell carcinoma.