Effects of lentivirus-mediated HIF-1α knockdown on hypoxia-related cisplatin resistance and their dependence on p53 status in fibrosarcoma cells
Effects of lentivirus-mediated HIF-1α knockdown on hypoxia-related cisplatin resistance and their dependence on p53 status in fibrosarcoma cells
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DOI：
10.1038/cgt.2008.4
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发表时间：
2008-07-01
影响因子：
6.4
通讯作者：
Yu, J.
中科院分区：
文献类型：
--
作者：
Hao, J.;Song, X.;Yu, J.
以缺氧诱导因子-1（hypoxia-inducible factor-1，HIF-1）为靶点逆转缺氧相关耐药的研究受到广泛关注。尽管HIF-1和p53之间存在密切的相互作用，并且在> 50%的肿瘤中观察到p53突变，但靶向治疗是否使HIF-1沉默取决于肿瘤p53状态仍然未知。用HIF-1 α特异性RNAi慢病毒载体转导两种等基因纤维肉瘤细胞HT 1080（野生型p53）和HT 1080 - 6 TG（突变型p53），并用杀稻瘟菌素选择。Real-time PCR和Western blot检测HIF-1 α mRNA和蛋白表达，验证了HIF-1 α基因沉默的效果。细胞首先在缺氧（0.5%O-2）下预处理4 h，然后与顺铂共处理另外24 h。MTT法检测顺铂对化疗药物的敏感性。流式细胞仪检测膜联蛋白V和碘化丙啶染色，分析顺铂诱导的细胞凋亡。Western blotting检测Bcl-2家族部分成员的变化。暴露于缺氧显着增加耐顺铂比暴露常氧。HIF-1 α基因敲低可逆转HT 1080细胞对顺铂的耐药性和耐凋亡性，但对HT 1080 - 6 TG细胞的耐药性影响不大。HIF-1 α基因敲减后，缺氧时HT 1080中Bid的表达高于HT 1080 - 6 TG。总之，逆转缺氧相关顺铂耐药的HIF-1靶向治疗依赖于正常的p53状态。低氧条件下Bid表达水平的变化可能部分导致了不同p53状态细胞对HIF-1 α沉默的不同反应。
Therapy targeting hypoxia-inducible factor-1 (HIF-1) to reverse the hypoxia-related drug resistance has received much interest. Despite a close interaction between HIF-1 and p53 and that p53 mutation is seen in > 50% of tumors, whether HIF-1 silencing by targeted therapy depends on tumor p53 status remains unknown. Two isogenic fibrosarcoma cells HT1080 (wild-type p53) and HT1080-6TG (mutant p53) were transduced with HIF-1 alpha-specific RNAi lentiviral vectors and selected with blasticidin. Real-time PCR and western blot analysis of HIF-1 alpha mRNA and protein respectively validated the silencing effects. Cells were first preconditioned under hypoxia (0.5% O-2) for 4 h and then co-treated with cisplatin for another 24 h. MTT was used for assessment of chemosensitivity to cisplatin. Moreover, annexin V and propidium iodide staining was detected on flow cytometry for analysis of cisplatin-induced apoptosis. Furthermore, changes of some Bcl-2 family members were detected on western blotting. Exposure to hypoxia significantly increased resistance to cisplatin than exposure to normoxia. HIF-1 alpha knockdown could reverse hypoxia-related resistance to cisplatin and apoptotic resistance only in HT1080 cells, but had little effect on HT1080-6TG cells. With HIF-1 alpha knockdown, Bid expression was higher in HT1080 than in HT1080-6TG under hypoxia. In summary, HIF-1 targeted therapy to reverse hypoxia-related cisplatin resistance depends on normal p53 status. Changes of Bid expression levels under hypoxia might contribute in part to the differential response to HIF-1 alpha silencing in cells with different p53 status.