腺病毒载体介导的p53基因可增强乳腺癌细胞系MDA—MB-468对表皮生长因子受体抑制剂的敏感性  

Effect of recombinant human p53 adenovirus （Ad-p53） combined with EGFR inhibitor gefitinib on the sensitivity of breast cancer MDA-MB-468 cells

作　　者：王新昭[1] 管西运 王蕾蕾[1] 谢丽[1] 刘琪[1] 于志勇[1] 

机构地区：[1]山东省肿瘤医院山东省医学科学院济南大学山东省医学科学院医学与生命科学学院,250117 [2]山东省莒县中医医院普外科

出　　处：《中华肿瘤杂志》2014年第12期886-891,共6页Chinese Journal of Oncology

基　　金：山东省自然科学基金（ZR2012HL34）

摘　　要：目的观察腺病毒载体介导的p53基因（Ad—p53）能否增加三阴性乳腺癌细胞系MDA—MB-468对表皮生长因子受体（EGFR）抑制剂吉非替尼的敏感性。方法以Ad-p53和（或）吉非替尼处理MDA—MB-468细胞系，采用四甲基偶氮唑蓝（MTT）法检测细胞增殖，流式细胞术检测细胞凋亡，Western blot法检测p53和EGFR蛋白的表达以及P13K／Akt通路和凋亡通路相关蛋白的表达。建立裸鼠移植瘤模型，观察Ad—p53联合吉非替尼对裸鼠移植瘤的治疗作用，免疫组织化学法检测裸鼠移植瘤组织中p53的表达。结果MTY法检测结果显示，Ad—p53感染MDA—MB-468细胞后，MDA—MB-468细胞对吉非替尼的敏感性增加，半数抑制浓度为4．5μmol／L。流式细胞术检测结果显示，对照组、Ad-p53组、吉非替尼组和联合组MDA—MB-468细胞的凋亡率分别为8．5％、17．4％、20．5％和32．6％，Ad—p53联合吉非替尼能够明显增加细胞凋亡（P〈0．05）。Westernblot法检测结果显示，Ad-053联合吉非替尼后，MDA—MB-468细胞中P—Akt蛋白的表达明显下调（P〈0．01），caspase-9和活化caspase-3蛋白的活性增加（均P〈0．01）。裸鼠体内实验的结果显示，Ad—p53组、吉非替尼组和联合组的肿瘤生长抑制率分别为35．7％、28．7％和74．4％，Ad-p53和吉非替尼对MDA—MB-468细胞裸鼠移植瘤的生长均具有抑制作用，且二者联合可明显抑制肿瘤生长（均P〈0．05）。免疫组化检测结果显示，对照组、Ad-p53组、吉非替尼组和联合组裸鼠移植瘤肿瘤组织中p53蛋白的阳性率分别为45．2％、80．1％、50．7％和90．6％，Ad-p53组和联合组裸鼠移植瘤中p53蛋白的表达明显增加（P〈0．01）。结论Ad-p53可能通过下调P13K／Akt通路相关蛋白的表达增加MDA-MB-468细胞对吉非替尼的敏感性；Ad—p53联合吉非替尼可能通过启动easpase级联反应促进MDA—MB-468细胞凋亡。Objective To observe the impact of concurrent administration of recombinant human p53 adenovirus （Ad-p53） with EGFR inhibitor gefitinib on breast cancer MDA-MB-468 cells. Methods MDA-MB-468 cells were treated with Ad-p53 and/or gefitinib. The effect of Ad-p53 and gefitinib on the growth of MDA-MB-468 cells was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to detect the alteration of p53, EGFR, phosphatidylinositol 3-kinase （PI3K）/ Akt signaling pathway and apoptosis-related proteins. Ad-p53 combined with gefitinib was used in vivo to explore their effect on tumor xenograft in nude mice. Immunohistochemistry was used to detect the p53 expression in vivo. Results The MTT assay showed a stronger inhibitory effect of gefitinib on MDA-MB-468 cells infected with Ad-p53 than on the control cells. Cell apoptosis assay revealed that the apoptosis rates of MDA-MB-468 cells in vehicle-treated group, Ad-p53 group, gefitinib group, and combination group were 8.5%, 17.4%, 20.5% and 32.6%, respectively. The apoptosis rate of MDA-MB-468 cells in the combination group was higher than that in other groups（ P 〈 0.05, for all）. Western blot analysis revealed that the expression of p53 was significantly up-regulated in the presence of Ad-p53. The combination of Ad- p53 and gefitinib significantly down-regulated p-Akt （ S473 ） （ P 〈 0.01 ） and up-regulated caspase-9 and cleaved caspase-3 （ P 〈 0.01 for both）. Tumor inhibition rates （TIR） in the Ad-p53, gefitinib, and combination groups were 35.7%, 28.7% and 74.4%, respectively. Ad-p53 and gefitinib combination therapy significantly reduced the tumor burden in nude mice （ P 〈 0.05 for all）. Immunohistochemistry showed that after intratumoral administration of Ad-p53, wild-type p53 was increased （ P 〈 0.01 ）. p53 expressions in the vehicle-treated, Ad-p53, gefitinib and combination groups were 45.2%, 80.1%, 50.7% and 90.6% , respectively. Conclusions Wild-type p53 may reverse the sensit

关 键 词：乳腺肿瘤 三阴性 p53 表皮生长因子受体 吉非替尼 腺病毒感染 MDA—MB-468细胞 

分 类 号：R737.9[医药卫生—肿瘤]