低氧诱导因子-1α mRNA在克唑替尼诱导H2228细胞凋亡中的作用  

The role of HIF -1α in crizotinib- induced apoptosis in lung adenocarcinoma cell line H2228

作　　者：潘星辰[1] 周韶璋[2] 戴辉[1] 韦江[1] 彭海燕[1] 宋向群[2] 

机构地区：[1]广西医科大学研究生院,南宁530021 [2]广西医科大学附属肿瘤医院化疗二科,南宁530021

出　　处：《广东医学》2014年第13期1980-1983,共4页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81060188;81260357);广西自然科学基金资助项目(编号:桂科自0991233)

摘　　要：目的探讨低氧诱导因子-1α(HIF-1α)在克唑替尼诱导EML4-ALK阳性肺腺癌细胞株H2228凋亡中的作用。方法 (1)用10、30、90、270、810 nmol/L浓度梯度的克唑替尼处理H2228细胞48 h,MTT比色法测定细胞增殖能力。(2)用100、200、300 nmol/L克唑替尼处理H2228细胞48 h,Annexin V流式细胞仪检测凋亡细胞。(3)RT-PCR实验:1采用50、100、200、400、800μmol/L浓度的低氧模拟剂氯化钴(CoCl2)作用H2228细胞24 h,观察HIF-1α的mRNA表达水平变化。2常氧对照组(0.5%DMSO培养基48 h)、常氧+克唑替尼组(0.5%DMSO培养基24 h+500 nmol/L克唑替尼24 h)、低氧对照组(200μm/L CoCl224 h+0.5%DMSO培养基24 h)、低氧+克唑替尼组(200μm/L CoCl224 h+500 nmol/L克唑替尼24 h),采用RT-PCR方法检测各组细胞HIF-1α、Akt、VEGF mRNA的表达水平。结果 (1)MTT实验结果示:随着克唑替尼药物浓度升高,H2228细胞增殖抑制率逐渐升高,呈剂量依赖性,半增殖抑制浓度(IC50)为335 nmol/L。(2)凋亡实验结果示:H2228细胞凋亡率随克唑替尼浓度增加而升高,呈剂量依赖性。(3)RT-PCR实验结果示:1随着CoCl2浓度的增加,HIF-1α的mRNA表达水平逐渐下降,呈剂量及时间依赖性,于200μm/L浓度时下降最明显。2与对照组相比,无论是在常氧还是低氧,克唑替尼组H2228细胞HIF-1α、Akt的mRNA表达均上调,VEGF的表达下调。与常氧+克唑替尼组比较,低氧+克唑替尼组HIF-1αmRNA上调更明显(P<0.05)。结论克唑替尼对肺腺癌细胞株H2228的增殖抑制和诱导凋亡作用呈剂量依赖性,HIF-1αmRNA的上调在克唑替尼诱导肺癌细胞凋亡过程中发挥重要作用。Objective To investigate the role of HIF-1α in crizotinib-induced apoptosis in EML4-ALK positive lung adenocarcinoma cell line H2228. Methods H2228 cells were treated with crizotinib of 10,30,90,270 and810 nmol /L for 48 h,and using MTT method to measure the proliferation ability. H2228 cells were treated with crizotinib of 100,200 and 300 nmol /L for 48 h,and the levels of apoptosis were quantitated using Annexin V assay. In RT- PCR experimental groups,H2228 were treated with low oxygen simulation agent CoCl2 of 50,100,200,400 and 800 μmol /L for 24 h,and the change of HIF- 1α mRNA expression was detected. The expression of HIF- 1α,Akt and VEGF mRNA was assessed in oxygen control group（0. 5% DMSO medium for 48 h）,oxygen ＋ crizotinib group（0. 5% DMSO medium for 24 h ＋ 500 nmol /L crizotinib for 24 h）,hypoxia control group（200 μmol /L CoCl2 for 24 h ＋ 0. 5% DMSO medium for 24 h） and hypoxia ＋ crizotinib group（200 μmol /L CoCl2 for 24 h ＋ 500 nmol /L crizotinib for 24 h） by RT- PCR.Results Cellular proliferation inhibition rate of H2228 cells was increased along with the increasing concentration of crizotinib,presenting in a dose- dependent manner with the IC50 of 335 nmol /L. Apoptosis rate of H2228 cells was also increased along with the increasing concentration of crizotinib. HIF- 1α mRNA expression was reduced gradually with the increase of CoCl2 concentration in a dose- dependent manner,with greatest reduction concentration of 200 μmol /L. Crizotinib up- regulated HIF- 1α and Akt mRNA in H2228 cells,but down- regulated the expression of VEGF. Comparing to normoxia group,crizotinib up- regulated HIF- 1α mRNA expression significantly more in hypoxia group. Conclusion Crizotinib inhibits proliferation and promotes apoptosis in lung adenocarcinoma cell line H2228 in dose- dependent manner. The increase of HIF- 1α mRNA expression plays an important role in this process.

关 键 词：低氧诱导因子-1Α EML4-ALK阳性肺腺癌细胞株H2228细胞 凋亡 克唑替尼 

分 类 号：R36[医药卫生—病理学]