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Chemokine Mig Mediate Thermotolerance Through Rescuing the Activity of Akt in the Tongue Squamous Cell Carcinoma Tca8113Cells

Author: ZhangChangBin
Tutor: HeYongWen
School:
Course: Clinical Stomatology
Keywords: tongue squamous cell carcinoma chemokineMig CXCR3 hyperthermia apoptosis
CLC: R739.86
Type: Master's thesis
Year: 2013
Downloads: 1
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Abstract


[Objective]Explore the role of chemokine Mig which mediate thermotolerance of the tongue squamous cell carcinoma Tca8113cells in vitro, and also the possible related molecular mechanisms.[Methods](1) Cultured Tca8113cells conventionally, grouping:Mig treated group(Tca8113cells were treated with100nM Mig for6hours), Mig+AKT inhibitor group (Tca8113cells were treated with100nM Mig+12nM AKT inhibitor MK-2206simultaneously for6hours), heat-induced group (Tca8113cells were treated with43℃80min in the water bath)、Mig+heat-induced group (Tca8113cells were treated with100nM Mig for6hours, and then43℃C for80min water bath). Mig+AKT inhibitor+heat-induced group (Tca8113cells were treated with100nM Mig+12nM AKT inhibitor MK-2206for6hours, and then43°C for80min water bath).(2) Harvested Tca8113cells and the Mig treated, heat-induced treatment group cells, then to detected the phosphorylation level of relevant protein with Full Moon PCS248phosphorylated protein microarray technology, analysed the differences and related signal pathway.(3) Observed the morphology changes of cells with inverted microscope.(4) Harvested cells to analyse cell apoptosis rate by FCM with TUNEL.(5) Analysed the rate of the activated Caspase-3in living cells of each grop, and also the mitochondrial transmembrance potential by FCM.(6) Further validated and analysed the phosphorylation level of relevant target protein through Western blot.(7) SPSS17.0statistical software for statistical analysis of the datas.[Results](1) Full moon PCS248phosphorylated protein chip hybridization detection, the results showed that, treated Tca8113cells with heat induced,5kinds of proteins’ phosphorylation levels increased significantly (more than two-fold);6proteins’ phosphorylation levels decreased significantly (more than0.6times). Significantly, the phosphorylation levels of the cell survival key factor Akt2(phospho-Ser474) and its downstream signaling pathway transcription factor eIF4E (phospho-Ser209) phosphorylation levels decrease significantly, while pretreated with Mig and heat-induced, both whose phosphorylation levels decreased no obviously.(2) Treated Tca8113cells with heat-induced for24h, cells showed obvious morphological changes of apoptosis, after pretreated with Mig, the number of apoptotic cells were significantly decreased, however, pretreated Tca8113cells with Mig+AKT inhibitor, the apoptosis was restored.(3) Detected apoptosis rate with TUNEL labeling by flow cytometry, the results showed that after treated Tca8113cells with heat-induced, the apoptosis rate increased from (2.77±0.47)%to (33.97±1.08)%(p=0.000), pretreated Tca8113cells with Mig, the apoptosis rate decreased to (17.78±2.1)%, pretreated Tca8113cells with Mig+AKT inhibitor, the apoptosis rate restored to (25.43±0.77)%.(4) After treated Tca8113cells with heat-induced, the rate of the activated Caspase-3in living cells increased from (10.93±1.17)%to (49.73±2.04)%(p=0.000), but pretreated Tca8113cells with Mig, the rate decreased to (30.90±2.42)%, pretreated Tca8113cells with Mig+AKT inhibitor, the rate restored to (44.53±1.59)%.(5) After treated Tca8113cells with heat-induced, the content of mitochondrial membrane JC-1monomer increased from (10.3±0.92)%to (30.5±2.86)%(p=0.000), but pretreated Tca8113cells with Mig, the content of JC-1monomer decreased to (16.97±1.53)%, pretreated Tca8113cells with Mig+AKT inhibitor, the content of JC-1monomer restored to (25.34±1.79)%.(6)The results of Western blot showed that, after treated Tca8113cells with heat-induced, the relative expression rate of phosphorylation of Akt2(p-Akt2)(Phospho-Ser474) decreased from (0.50±0.09) to (0.10±0.04)(p=0.000), but pretreated Tca8113cells with Mig, the relative expression rate restored to (0.43±0.08), pretreated Tca8113cells with Mig+AKT inhibitor, the relative expression rate decreased to (0.27±0.04). After treated Tca8113cells with heat-induced, the relative expression rate of phosphorylation of eIF4E(p-eIF4E)(Phospho-Ser209) decreased from (0.45±0.05) to (0.16±0.02)(p=0.000), but pretreated Tca8113cells with Mig, the relative expression rate restored to (0.39±0.04), pretreated Tca8113cells with Mig+AKT inhibitor, the relative expression rate decreased to (0.21±0.04)%. After treated Tca8113cells with heat-induced, the relative expression rate of Bcl-2decreased from (1.20±0.06) to (0.51±0.07), but pretreated Tca8113cells with Mig, the relative expression rate restored to (1.30±0.12), pretreated Tca8113cells with Mig+AKT inhibitor, the relative expression rate decreased to (1.03±0.10).[Conclusion] Chemokine Mig inhibit the apoptosis of the tongue squamous cell carcinoma Tca8113cells and mediate thermotolerance through rescuing the activity of Akt signaling pathway, promoting the phosphorylation of eIF4E, increasing the expression of apoptosis inhibition gene Bcl-2.

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CLC: > Medicine, health > Oncology > Oral cavity, maxillofacial tumors > Tongue cancer, sublingual tumor
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