title: Effects of lentivirus-mediated HIF-1alpha knockdown on hypoxia-related cisplatin resistance and their dependence on p53 status in fibrosarcoma cells.
abstract: 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-1alpha-specific RNAi lentiviral vectors and selected with blasticidin. Real-time PCR and western blot analysis of HIF-1alpha 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-1alpha 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-1alpha 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-1alpha silencing in cells with different p53 status.
authors.affiliation: Department of Oncology, Cancer Center, Qilu Hospital of Shandong University, Jinan, China.
authors.forename: J
authors.initials: J
authors.lastname: Hao
authors.forename: X
authors.initials: X
authors.lastname: Song
authors.forename: B
authors.initials: B
authors.lastname: Song
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: L
authors.initials: L
authors.lastname: Wei
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Yu
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D017931:DNA Primers / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics*; D005434:Flow Cytometry; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D016086:Lentivirus / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D017304:Annexin A5; D017931:DNA Primers; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016159:Tumor Suppressor Protein p53; D002945:Cisplatin