| [Objective] To study the related molecular mechanism of the HSP90a protein inhibitors 17-AAG on hyperthermia-induced apoptosis in tongue squamous cell carcinoma Tca8113 cells.[Methods] (1) Tca8113 cells were cultured With RPMI 1640 containing 10% fetal bovine serum medium, with 37℃ and 5% CO2 incubator. (2)(a) Regular culturing Tca8113 cells. After hyperthermia-induced at 43℃ 80min, cultured in 3h,6h,12h,24h, 48h respectively. (b)① control group(Regular cultured Tca8113 cells);② hyperthermia-induced group(Tca8113 cells were treated with 43℃ 80min);③17-AAG group (Tca8113 cells were treated with lOμg/ml 17-AAG); ④ 17-AAG+ hyperthermia-induced group (10μg/ml 17-AAG+Tca8113 cells were treated with 43℃ 80min). (3) The expressions of HSP90a were analyzed through Western blot to study the optimal incubation time of Tca8113 cells which were hyperthermia-induced.(4) Observe the cell morphology by Inverted microscope. (5) CCK8 assay was used to evaluate the inhibition rate of cellular proliferation. (6) Apoptosis rates of Tca8113 cells were analyzed with Annexin V-FITC/PI labeling through flow cytometry. (7) Flow cytometry instrument detection of mitochondrial membrane potential. (8) Analysed the rate of the activated Caspase-3 in living cells of each group. (9) Western blot analyzed the Bel-2 and Bax protein expression relative to the amount of change and fracture activation of PKC-δ protein. (10) Statistical analysis was carried out with SPSS17.0.[Results] (1) The expressions of HSP90a were analyzed through Western blot. In normal control group, the relative expression of HSP90a was (0.14±0.01) in Tca8113 cells.3 hours after heating group (0.15±0.02) and 6 hours after heating group (0.20±0.06), They had no statistical difference with the normal control group.12 hours after heating group(0.72±0.06), compared with the normal control group, the expression is increased (P<0.05).24 hours after heating group(0.89±0.14), compared with the normal control group, the expression is obvious increased (P<0.01).48 hours after heating group(0.46±0.05), compared with the normal control group, the expression is increased (P< 0.05).(2) After treated with hyperthermia or 17-AAG alone, Tca8113 cells showed obvious morphological changes of apoptosis. The cells became round and shrinkage. When treated with 17-AAG combined hyperthermia, the number of round and shrinkage cells increased significantly.(3)The inhibition rate of proliferation of Tca8113 cells in hyperthermia-induced group was (12.93±3.07)%, the inhibition rate of proliferation of Tca8113 cells in the 17-AAG group was (16.85± 2.79)%,and 17-AAG+hyperthermia-induced group was (35.05±8.23)%.17-AAG+ hyperthermia-induced group had significantly statistical differences compared with the group that treated with hyperthermia or17-AAG alone (P<0.05), but there had no significantly statistical differences between 17-AAG and hyperthermia group.(4) With Annexin V-FITC/PI labeling and analysis by flow cytometry, the apoptosis rate in the control group was (3.19±0.89)%, in hyperthermia-induced group was (10.57±0.80)%, compared with the control group, it had statistical differences (P<0.05); The 17-AAG treated group was (20.99±1.65)% and the 17-AAG+hyperthermia-induced group was (40.67±2.04)% at 24h after treatment, it had significantly statistical differences compared with that of the group treated with hyperthermia or 17-AAG alone (P<0.05).(5) The content of mitochondrial membrane JC-1 monomer in control group was (5.67±1.20)%, in hyperthermia-induced group was (20.49±2.12)%,in 17-AAG group was (31.89±1.31)%,in 17-AAG+hyperthermia-induced group was (55.09 ±3.43)%. Compared with the normal control group, the hyperthermia-induced group or 17-AAG group both had statistical differences (P<0.05); The 17-AAG+ hyperthermia-induced group had significantly statistical differences compared with the group that treated with hyperthermia or 17-AAG alone (P<0.05).(6) The rate of the activated Caspase-3 in control group was (1.04±0.09)%, in hyperthermia-induced group was (3.21±0.17)%, in 17-AAG group was (5.16±0.23)%, in 17-AAG+ hyperthermia-induced group was (8.45 ± 0.58)%. Compared with the normal control group, the hyperthermia-induced group or 17-AAG group both had statistical differences (P<0.05); The 17-AAG+hyperthermia-induced group had significantly statistical differences compared with the group that treated with hyperthermia or 17-AAG alone (P<0.05).(7) The results of Western blot test, ①In normal control group, the Bcl-2 relative expression of was (1.06±0.07), in hyperthermia-induced group was (0.60±0.02), in 17-AAG group was (0.74±0.03);Compared with the normal control group, the Bcl-2 relative expression in both hyperthermia or 17-AAG alone group was decreased (P<0.05); The 17-AAG+hyperthermia-induced group (0.43±0.04) had significantly statistical differences compared with the group in hyperthermia or 17-AAG treated group (P<0.05).②In normal control group, the Bax relative expression of was (0.62±0.03),;In hyperthermia-induced group was (0.94±0.13), compared with the normal control group, the Bax relative expression was increased (P<0.05); In 17-AAG group was (1.21±0.11), compared with the normal control group, the expression was increased (P<0.05); In 17-AAG+hyperthermia-induced group was (1.21±0.11), compared with the rest of group, the expression was increased significantly (P<0.05). ③In 17-AAG group, the PKC-δ relative expression of was(0.42±0.20), compared with the normal control group(0.77±0.11), the expression was decreased (P<0.05); The 17-AAG+hyperthermia-induced group(0.42±0.18)compared with the normal control group, the expression was decreased (P<0.05). ④In 17-AAG group, the PKC δ-CF relative expression of was(0.50±0.04), compared with the normal control group(0.32±0.10) or the hyperthermia-induced group(0.32±0.07), the expression of PKC δ-CF was increased (P<0.05); The 17-AAG+hyperthermia-induced group(0.74 ±0.05) compared with the rest of group, the expression was increased significantly (P<0.05). [Conclusion] 17-AAG combined with hyperthermia can induced apoptosis through down-regulating the expression of Bcl-2, up-regulating the expression of Bax and promote the fracture activation of PKC-δ proteins in tongue squamous cell carcinoma Tca8113 cells. |
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