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Experimental Research of the Ubiquintin-Proteasome Inhibitor Induced Apoptosis in Head and Neck Squamous Cell Carcinoma

Author: LiuXianBin
Tutor: ZhangBin
School: Taishan Medical College
Course: Surgery
Keywords: Ubiquitin - proteasome inhibitor MG-132 Endoplasmic reticulum stress Tca-8113 cells Apoptosis Ubiquitin- protein ligase
CLC: R739.91
Type: Master's thesis
Year: 2010
Downloads: 24
Quote: 0
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Abstract


Objective To investigate the endoplasmic reticulum stress in the role of the ubiquitin - proteasome inhibitor MG-132-induced human tongue squamous carcinoma cell line Tca-8113 cell apoptosis and its related mechanism. Method using RPMI-1 640 with 10% fetal bovine serum-containing medium was routinely cultured Tca-8113 cells, the exponential growth phase cells were randomly divided into 5 groups: the culture solution were added 1640 medium (negative control group), thapsigargin 5μmol / L (positive control group), and 10,20,30 μmol / L of MG-32 group. Cultured for 18h, the following experiment: MTT experiments measure the absorbance at 490nm wavelength detection MG-132 Tca-8113 cell activity; using fluorescent dye Hoechst 33258 staining, fluorescence microscopy the morphological changes of nuclear chromatin; agar the sugar gel electrophoresis detection of apoptotic cells in the formation of the DNA ladder; Annexin Ⅴ -FITC/PI double staining to detect apoptosis rate; RT-PCR detection of glucose-regulated protein 78 (glucose regulated protein 78, GRP78), caspase-12 mRNA expression; Western blotting analysis of glucose-regulated protein 78, caspase-12 protein expression; ELISA detection of human ubiquitin-protein ligase E3 concentration. Results of MTT assay: negative control group, thapsigargin group and low, medium and high concentrations of MG-132 group absorbance were 0.515 ± 0.265,0.170 ± 0.690,0.292 ± 0.420,0.222 ± 0.106,0.148 ± 0.205, with the negative control group, were statistically significant, in which low concentrations of MG-132 P lt; 0.05, each group P lt; 0.01; Hoechst 33258 staining showed that: thapsigargin group, and 10,20,30 μmol / L MG detection of DNA fragmentation typical apoptotic morphology of nuclear chromatin -132 cells, Tca-8113 cell apoptosis morphological changes in the MG-132 concentration-dependent manner;: MG-132 group and thapsigargin group trapezoidal striped gray increased significantly compared with the control group, and the concentration dependence, especially evident in the high concentration group; flow cytometry: negative control group, thapsigargin group and MG-132 in each experimental group apoptosis rate were 0.43 ± 0.25%, 8.49 ± 0.25%, 3.50 ± 0.41% 1 9.55 ± 0.32% and 34.74 ± 0.50%; RT-PCR test results: MG-1 32 can cause GRP78, caspase-12 mRNA expression; Western- blot results: MG-132 can cause of GRP78, caspase-12 protein expression enhancement: ELISA test results: negative control group, thapsigargin group and MG-32 experimental group of ubiquitin ligase enzymes E3 expression levels were 40.88 ± 4.52,38.96 ± 0.33,28.75 ± 2.28,1 8.16 ± 0.65 and 8.85 ± 0.72. Conclusion 1, ubiquitin - proteasome inhibitor MG-1 32 can induce apoptosis in human tongue squamous carcinoma cell line Tca-8113 cells; 2, MG-132 Tca-8113 cells can cause endoplasmic reticulum stress, manifested in GPR78 mRNA and protein expression; 3, MG-132 can cause the Tca-8 113 cell endoplasmic reticulum stress-induced apoptosis, manifested in the shear activation upregulated both procaspase-12 to enhance the expression of caspase-12 mRNA and protein ; 4, MG-132 inhibited ubiquitin ligase enzyme E3 expression, which is different from the typical endoplasmic reticulum stress inducer thapsigargin. Significance of this study is to explore the ubiquitin - proteasome inhibitor MG-132 induces apoptosis tongue squamous cell carcinoma Tca-8113 role and its relationship with the endoplasmic reticulum stress-induced apoptosis, reveals ubiquitin - proteasome pathway role in tumor cell apoptosis and its possible mechanism for the ubiquitin - proteasome inhibitor used in the treatment of head and neck squamous cell carcinoma at the same time to provide a theoretical basis, and expanded new ideas for cancer treatment.

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CLC: > Medicine, health > Oncology > Other parts of the tumor > Head, neck, shoulder tumor
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