The Thermosensitivity Effect And The Mechanism Of Artemether On Hyperthermia-induced Apoptosis In Tongue Squamous Cell Carcinoma Cal27Cells

[Objective] To study the thermosensitivity effect and the mechanism of artemether on hyperthermia-induced apoptosis in tongue squamous cell carcinoma Cal27cells.[Methods](1Experimental groups:control group(Cal27cells), hyperthermia-induced group (Cal27cells were treated with43℃60min,43℃80min,43℃120min in the water bath or Cal27cells were treated with41℃,43℃,45℃80min in the water bath), Artemether treated group(Cal27cells were treated with150mmol/L,250mmol/L,350mmol/L,450mmol/L,550mmol/LArtemether), Artemether+hyperthermia-induced group(After treated with150mmol/L,250mmol/L,350mmol/L,450mmol/L or550mmol/L Artemether for30min, Cal27cells were treated with43℃for80min in water bath).(2) The changes of cell morphology were observed by inverted microscope.(3) MTT (Methy thiazolyl tetrazolium) assay was used to evaluate the inhibition rate of cellular proliferation.(4) Apoptosis rates of Cal27cells were analyzed with TUNEL labeling through flow cytometry.(5) Mitochondrial membrane potentials of Cal27cells were analyzed with JC-1staining through flow cytometry.(6) Caspase-3activities were measured with colorimetric assay.(7) The expressions of apoptosis-related proteins were detected with protein chip hybridization.(8) The The expressions of relevant target protein were further analyzed through Western blot.(9) Statistical analysis was carried out with SPSS17.0.[Results](1) After treated with hyperthermia or Artemether alone, Cal27cells showed obvious morphological changes of apoptosis. The cells became round and shrinkage. When treated with Artemether combined hyperthermia, the number of round and shrinkage cells increased significantly.(2) The inhibition rate of proliferation of Cal27cells raised with temperature improvement and time prolonging of hyperthermia-induction; Compared with hyperthremia-induced treatment alone, proliferation inhibition rate of was Cal27cells significantly higher, and increased with improvement of Artemether concentration (P<0.05).(3) With TUNEL labeling and analysis by flow cytometry, the apoptosis rate of Cal27cells in the control group was (1.37±0.50)%. The apoptosis rate of Cal27cells in hyperthermia-induced group (43℃80min) and Artemether treated group was (26.60±4.09)%and (20.17±.67)%respectively at24h after treatment. The apoptosis rate of Cal27were increased to (46.80±1.04)%after treated with Artemether combined hyperthermia, there was statistically significant differences compared with that of the group treated with hyperthermia or Artemether alone (P<0.05).(4) The population rate of Cal27cells with low mitochondrial membrane potential was0in control group,(47.1±4.3)%in hyperthermia-induced group,(27.1±3.4)%in Artemether treated group and (68.0±2.7)%in Artemether+hyperthermia-induced group (p<0.05).(5) The OD value of Caspase-3activity was (0.059±0.017) in control group.(0.466±0.138) in hyperthermia-induced group,(0.258±0.122) in Artemether treated group and (0.589±0.145) in Artemether+hyperthermia-induced group (p<0.05).(6) Through apoptosis array (35proteins) analysis, expressions of Bad、Bax、Cleaved Caspase-3、Catalase、Cytochrome c、FADD、 HSP60. HSP70and Phospho-p53(S392) in hyperthermia-induced group (43℃80min) were found increased significantly compared with that in control group (P<0.05). No protein was found with decreased expression level. Expressions of Cleaved Caspase-3、Catalase、P21/C1P1/CDKN1A、P27/Kip1、Phospho-p53(S392) and Phospho-Radl7(S635) were increased significantly, and expressions of Pro-Caspase-3. Claspin、Cytochrome c、FADD、HSP60、HSP70、HTRA2/Omi、 SMAC/Diablo and XIAP were decreased significantly in Artemether treated group(P<0.05). Expressions of Bad、Bax、Cleaved Caspase-3、Catalase Fas/TNFRSF6/CD、P21/CIP1/CDKN1A、P27/Kip1、Phospho-p53(S15). Phospho-p53(S46)、Phospho-p53(S392) and Phospho-Rad17(S635) in Artemether+hyperthermia-induced group were increased compared with that in the control group and hyperthermia-induced group(P<0.05). Expressions of Pro-Caspase-3、 Cytochrome c、FADD、HSP60、HSP70、HTRA2/Omi、SMAC/Diablo and XIAP in Artemether+hyperthermia-induced group were decreased compared with that in the control group and hyperthermia-induced group(P<0.05).[Conclusion] Artemether can increase the thermosensitivity of tongue squamous cell carcinoma Cal27cells through promoting expressions of pro-apoptosis proteins and inhibiting expressions of anti-apoptosis proteins such as heat shock proteins induced by hyperthermia.