热疗通过下调Notch1/Jagged1/Hes1诱导舌鳞癌细胞凋亡的初步研究

A preliminary study of apoptosis in tongue squamous cell carcinoma cells induced by hyperthermia via downregulation of Notch1/Jagged1/Hes1

目的观察舌鳞癌细胞Cal-27短时多次热疗的最佳间隔时间并探究Notch信号通路在热疗诱导舌鳞癌细胞凋亡中的作用。方法将Cal-27细胞置入42℃培养箱热疗45 min,分别在6、12、24、48、72 h后进行第2次热疗,并在热疗结束后0、12、24 h通过CCK-8实验检测细胞增殖能力变化。确定最佳热疗间隔时间后进行多次热疗,分别通过CCK-8实验及流式细胞术检测热疗对Cal-27细胞增殖及凋亡能力的影响。通过实时反转录PCR实验及蛋白质印迹法实验分析Notch1、Jagged1、发状分裂相关增强子1(Hes1)的mRNA及蛋白表达变化。实验数据以x^(-)±s表示,两组间比较采用单因素方差分析。结果每隔12 h给予42℃45 min热疗1次可持续抑制舌鳞癌Cal-27细胞的增殖(P<0.05)。在热疗持续进行7次后与对照组相比,热疗可明显抑制舌鳞癌Cal-27细胞的增殖并诱导其凋亡(P<0.05),且热疗组细胞的Notch1、Jagged1、Hes1的mRNA及蛋白表达均显著降低(P值均<0.05)。结论12 h热疗1次为抑制舌鳞癌Cal-27细胞增殖的最佳间隔时间,其机制可能与热疗通过抑制Notch信号通路的表达诱导舌鳞癌Cal-27细胞凋亡有关。

Objective To observe the optimal time interval of multiple short-term hyperthermia in tongue squamous carcinoma Cal-27 cells,and investigate the role of Notch signaling pathway in hyperthermia-induced apoptosis of Cal-27 cells.Methods Cal-27 cells were placed into a 42℃incubator for 45 min,and the second hyperthermia was performed at 6 h,12 h,24 h,48 h and 72 h later,respectively.Cell proliferation capacity was detected by CCK-8 assay at 0 h,12 h and 24 h after the end of hyperthermia.After determining the optimal time interval of hyperthermia,multiple cycles of hyperthermia were performed,and the effects of hyperthermia on the proliferation and apoptosis of Cal-27 cells were detected by CCK-8 assay and flow cytometry,respectively.The changes in the expression of Notch1,Jagged1 and hairy and enhancer of split homolog-1(Hes1)mRNA and proteins were analyzed by real-time reverse transcription PCR(qRT-PCR)and Western blot.All data were expressed as mean±SD.Two-group comparison was performed by one-way ANOVA.Results Hyperthermia at 42℃for 45 min every 12 h consistently inhibited the proliferation of Cal-27 cells(P<0.05).Compared with the control group,hyperthermia in the experiment group significantly inhibited the proliferation and induced the apoptosis of Cal-27 cells after 7 cycles of hyperthermia(P<0.05),and the expression levels of Notch1,Jagged1 and Hes1 mRNA and proteins in the hyperthermia group were significantly down-regulated(all P<0.05).Conclusions The optimal time interval of hyperthermia to inhibit the proliferation of tongue squamous cells carcinoma Cal-27 cells is 12 h.Hyperthermia might induce the apoptosis of Cal-27 cells by inhibiting the Notch signaling pathway.