绿茶提取物对口腔鳞癌抗肿瘤效应及作用机制的研究

Experimental Study on Anti-tum or Effect and Mechanism of Green Tea Extrac

目的探讨绿茶提取物(green tea extract,GTE)对不同的人口腔鳞癌细胞株的抗肿瘤效应及其相关机制。方法体外培养人舌鳞癌CAL-27细胞株、人舌鳞癌SCC-25细胞株、人口腔上皮癌KB细胞株,应用MTT法检测GTE对细胞增殖的影响,筛选出敏感细胞株,用流式细胞术检测GTE对CAL-27细胞周期的影响,采用蛋白芯片技术检测GTE对CAL-27细胞株蛋白表达的影响,并应用Western blot检测周期蛋白依赖性激酶4(cyclin-dependent kinases,CDK4)、周期蛋白依赖性激酶6(cyclin-dependent kinases,CDK6)、磷酸化3-磷酸肌醇依赖性蛋白激酶-1(phospho-3-phosphoinositide-dependent protein kinase-1,p-PDK1)蛋白表达情况。结果与对照组比较,50、100、200、400μg/m L GTE作用于CAL-27细胞抑制率升高,100、200、400μg/m L GTE作用于KB细胞抑制率升高,25、50、100、200、400μg/m L GTE作用于SCC-25细胞抑制率亦升高,差异均有统计学意义(P<0.01),并呈剂量依赖性。流式细胞术显示:与对照组比较,50μg/m L GTE介导CAL-27细胞G2/M期阻滞(P<0.05);100μg/m L GTE能够介导CAL-27细胞S期及G2/M期阻滞(P<0.01),G0/G1期细胞减少(P<0.01)。应用蛋白芯片技术共分析107种蛋白,应用GTE处理后,CAL-27细胞共有13种蛋白发生明显变化。应用Western blot技术确认了25、50、100μg/m L GTE作用于CAL-27细胞后p-PDK1、CDK4、CDK6蛋白表达,药物浓度越高,抑制率越高,差异有统计学意义(P<0.05)。结论 GTE能够抑制多种类型人口腔鳞癌细胞株的增殖,敏感细胞株为CAL-27。GTE主要影响表皮生长因子受体(EGFR)和Notch信号传导通路,并通过以上信号传导通路影响细胞周期相关蛋白表达水平的变化,导致细胞周期S期及G2/M期阻滞。

Objective To explore anti-cancer effect and mechanism of green tea extract（ GTE） in three human oral squamous carcinoma cell lines（ CAL-27,SCC-25 and KB）. Methods The cell lines were in vitro cultured and its growth inhibition was detected by MTT. After screening most sensitive cell line,effect of GTE on CAL-27 cell cycle was analyzed by flow cytometry. The protein expression of GTE on CAL-27 cell strain was determined by protein chip technique. The protein expression of CDK4,CDK6,and p-PDK1 was verified by using Western blot. Results Compared with the control group,the inhibition rate on CAL-27 increased significantly after treated by 50,100,200,and 400μg /m L GTE; the inhibition rate on KB increased after treated by 100,200,and 400 μg /m L GTE; the inhibition rate on SCC-25 increased after treated by 25,50,100,200,and 400 μg / m L GTE,all with statistical difference and in dose dependant manner（ P 〈0. 01）. Flow cytometric analysis showed that,when compared with the control group,50μg /m L GTE arrested CAL-27 cells in the G2/ M phase（ P 〈0. 05）,and 100 μg / m L GTE arrested CAL-27 cells in the G2/ M phase with concurrent decreased cells in the G0/ G1phase（ P 〈0. 01）. Totally 107 proteins were analyzed by protein chip technique. After treated by GTE,a total of 13 proteins significantly changed in CAL-27 cell line. West-ern blot showed that 25,50,and 100 μg / m L GTE inhibited the expression of phopho-phosphoinositide-dependent protein kinase 1（ p-PDK1）,cyclin-dependent kinase 4（ CDK4）,and CDK6 of CAL-27 cell line with statistical difference（ P 〈0. 05）. The higher the drug concentration,the higher the inhibition rate（ P 〈0. 05）. Conclusions GTE could inhibit the proliferation of different human oral squamous carcinoma cell lines. CAL-27 is a sensitive cell line.GTE significantly affected EGFR and Notch signal network,and influenced changes of cell cycle related protein expression levels through the aforesaid channels,resulting in cell cycle arrest in S and G2/ M phases.