ATF5蛋白对食管鳞癌细胞耐药性影响

Effect of ATF5 protein on the resistance of esophageal squamous cell carcinoma cells

目的食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)多药耐药严重影响化疗效果,其分子机制仍未阐明。本研究通过构建不同ATF5蛋白表达量的食管癌Eca-109细胞模型,探究过表达ATF5及低表达ATF5对食管癌Eca-109细胞耐药性及凋亡的影响。方法利用脂质体载体转染技术对食管癌细胞株进行转染,建立高、中(转染空质粒的对照组)、低3组不同ATF5蛋白表达量Eca-109细胞模型。蛋白质印迹法检测3组细胞ATF5蛋白表达水平。MTT实验和平板克隆实验观察3组细胞对紫杉醇、顺铂耐受性的变化;DAPI染料核染色后观察3组细胞在紫杉醇、顺铂作用下凋亡反应的差异;分析ATF5蛋白对食管鳞癌Eca-109细胞耐药的影响。结果利用转染技术成功构建的3组不同ATF5蛋白表达量的Eca-109细胞模型,表达模型组细胞中ATF5蛋白相对表达量为85.9±2.66,对照组为64.35±2.54,低表达模型组为45.3±3.11,3组差异有统计学意义,F=532.323,P<0.001。MTT实验显示,紫杉醇、顺铂作用72h后,3组细胞存活率差异有统计学意义,F_紫=163.382,P<0.001;F_顺=579.36,P<0.001;同一组细胞不同浓度对存活率的影响差异有统计学意义,F_紫=616.32,P<0.001;F_顺=2 558.05,P<0.001;不同浓度的紫杉醇、顺铂作用3组细胞后,过表达组细胞的半数抑制率浓度(IC50)分别为4.16±2.21和1.54±0.67;对照组分别为1.54±0.92和1.27±0.65;低表达组细胞的半数抑制率浓度分别为0.33±0.21和0.53±0.62,3组差异有统计学意义,F_紫=198 330.768,P<0.001;F_顺=64 298.048,P<0.001;表明过表达ATF5的细胞对紫杉醇、顺铂的药物耐受性均明显升高而低表达模型组细胞对紫杉醇、顺铂的药物耐受性均明显降低。DAPI染色实验显示,紫杉醇、顺铂作用3组细胞后,过表达组细胞的凋亡率分别为(14.04±1.66)%和(11.75±2.09)%;对照组分别为(26.44±2.99)%和(34.07±3.42)%;低表达组细胞的凋亡率分别为(54.85±5.88)%和(66.66±2.81)%,3组差异有统计学意义,F_紫=283.976,P<0.001;F_顺=954.464,P<0.001。提示过表达ATF5蛋白细胞在紫杉醇、顺铂作用下凋亡细胞均减少,低表达ATF5细胞在紫杉醇、顺铂作用下凋亡细胞均增加。平板克隆形成实验显示,紫杉醇、顺铂作用3组细胞后,过表达组细胞的克隆形成率分别为(56.09±1.37)%和(62.67±1.41)%;对照组分别为(38.7±1.37)%和(34.83±3.09)%;低表达组细胞的克隆形成率分别为(25.22±1.58)%和(18.17±3.64)%,3组差异有统计学意义,F_紫=1157.447,P<0.001;F_顺=612.595,P<0.001。表明过表达ATF5蛋白细胞在紫杉醇、顺铂作用下克隆形成数均更多,低表达ATF5蛋白细胞在紫杉醇、顺铂作用下克隆形成数减少。结论上调ATF5蛋白的表达能增加食管鳞癌Eca-109细胞对紫杉醇、顺铂的耐药性,而下调ATF5蛋白的表达则能降低Eca-109细胞对紫杉醇、顺铂的耐药性,提示食管鳞癌细胞的耐药性可能与ATF5蛋白的表达量相关,食管鳞癌细胞中ATF5蛋白的表达情况可能能间接干扰临床药物化疗的效果。

OBJECTIVE The multidrug resistance of esophageal squamous cell carcinoma seriously affects the efficacy of chemotherapy, and its molecular mechanism has not been elucidated. We observed the effect of up regulate of ATF5 or down regulate of ATF5 on drug resistance of Eca-109 cells in esophageal cancer, and investigated the effect of ATF5 protein on the drug resistance of Eca-109 cells in esophageal squamous cell carcinoma. METHODS We established the high ATF5 expressing groups, the middle ATF5 expressing groups （Control group transfected with empty plasmid） and the low ATF5 expressing groups by transfected the Eca-109 cells using the liposome transfection technique. Then we used the Western Blot to detect the expression the ATF5 protein of the 3 groups, and studied how the different expres- sions of ATF5 protein influenced the Eca-109 cells. We established the ATF5 high expressing vector and low expressing vector to analyze the effect of ATF5 protein on the drug resistance of Eca-109 cells in esophageal squamous cell carcinoma using plasmid construction technology and the transfection technology. We tried to find the evidences that the sensitivity and apoptosis of the three groups Eca-109 cells to paclitaxel and cisplatin had changed using the MTT experiments, plate clone formation experiments and DAPI dye nuclear staining experiments. RESULTS We used the liposome transfection technique to establish 3 different ATF5 expression groups of Eca-109 cells. The expression of ATF5 protein in the high ATF5 expression group was 85.9±2.66, the control group was 64.35±2.54, the low group was 45.3±3. 11（F= 532. 323,P〈0. 001）. With the effect of paclitaxel or cisplatin on the 3 groups of cells 72 h, the surviving fractions of the 3 group of cells were significantly different （Fpaclitaxel = 163. 382, P〈0. 001 ; Feisplatin = 579.36, P〈0. 001 ）, and the effects of different concentrations of the 3 groups of ceils on survival rate were significate different （Fpaclitaxel= 616.32, P〈0.001; Fcisplatin=2 558.05,P〈0. 001）. With the effect of different concentrations of paclitaxel and eisplatin on the 3 groups of cells, the IC50 of the high ATF5 expression group were 4.16±2.21 and 1.54±0.67 respectively, and the control group were 1.54±0.92 and 1.27±0.65 respectively, the low ATF5 expression group were 0.33±0.21 and 0.53±0.62 respectively（Fpaclitaxel=198 330. 768,P〈0. 001 ;Fcisplatin = 64 298. 048 ,P〈0. 001）. It was proved by the experiment that up regulated of ATF5 protein expression could increase the resistance of Eca-109 ceils to paelitaxel and cisplatin, and down regulated the expression of ATF5 protein could decrease the resistance of Eca-109 cells. The apoptosis rates of the high ATF5 protein expression group were （14. 04±1. 66）% and （11. 75 ± 2. 09）% respectively, the control group were （26.44±2.99）% and （34. 07 ±3. 42）% respectively, the low ATF5 expression group were （54. 85 ±5. 88）% and （66.66 ± 2.81）% respectively （Fpaclitaxel = 283. 976, P〈0. 001 ; Fcisplatin= 954. 464, P〈0. 001）. It was proved by the experiment that up regulated of ATF5 protein expression can decrease the apoptosis rate of Eca-109 cells to paclitaxel and cisplatin, and down regulated the expression of ATF5 protein can increase the apoptosis rate of Eca-109 cells. The colony forming efficiency of the high ATF5 protein expression group were （56.09 ± 1.37） % and （ 62.67 ± 1.41）% respectively, the control group were （38. 7 ± 1. 37）% and （34. 83 ± 3. 09） % respectively, the low ATF5 expression group were （25.22±1.58）% and （18.17±3.64）% respectively （Fpaclitaxel 1 157. 447, P〈0. 001;Fcisplatin=612. 595,P〈0. 001）. It was proved by the experiment that up regulated of ATF5 protein expression can increase the colony forming efficiency of Eca-109 cells to paclitaxel and eisplatin, and clown regulated the expression of ATF5 protein could decrease the colony forming efficiency of Eca-109 cells. CONCLUSIONS It suggests the expression of ATF5 protein is related to the drug resistance of esophageal squamous cell carcinoma that up regulated of ATF5 protein expression can increase the resistance of Eca-109 cells to paclitaxel and eispIatih in esophageal squamous cell carcinoma, while down regulated the expression of ATF5 protein can decrease the resistance of Eca-109 ceils to paclitaxel and cisplatin. It suggests that the expression of ATF5 protein in esophageal squamous cell carcinoma can indirectly interfere with the effect of clinical drug chemotherapy.