Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1α gene
Objectives: Hypoxia is associated with human non-small cell lung cancers (NSCLC), which are highly resistant to chemotherapy. The hypoxia inducible factor (HIF) as a transcription factor in response to hypoxia indicates that it could be a novel, tumor-specific target for anticancer therapy. We hypothesized that disruption of HIF pathway through lentiviral vector-mediated HIF-1α RNA interference (RNAi) could reverse the hypoxia-induced resistance to chemotherapy. Methods: We transfected Human NSCLC cell lines, SPCA1 and A549 with HIF-1α specific RNAi lentiviral vectors as well as controls. HIF-1α silenced cells [SPCA1/HIF-1α(-) and A549/HIF-1α(-)] were screened by blasticidin. They were incubated in 19 or 0.5% O2 for 16 h followed by the assessment of chemosensitivity to cisplatin and doxorubicin with MTT and clonogenic assays. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of HIF-1α mRNA and protein, respectively. Moreover, flow cytometry was used to monitor the expression of P-glycoprotein. Results: Exposure of SPCA1 and A549 cells to 0.5% O2 significantly increased resistance to cisplatin and doxorubicin, in contrast to cells incubated in normoxia. Transduction of SPCA1 with HIF-1α RNAi vector resulted in sequence specific silencing with 87.2 and 84.6% decreases of HIF-1α mRNA transcription and 97.3 and 94.8% of protein expressions in normoxia and hypoxia, respectively. Correspondingly, they are 89.2, 89.9% and 97.2, 88.4% decreases in A549 cells. Hypoxia-induced resistance to cisplatin and doxorubicin were reversed in SPCA1/HIF-1α(-) and A549/HIF-1α(-) cells. There was no significant P-glycoprotein increase induced by hypoxia in NSCLC cells. Conclusions: Our studies demonstrated that hypoxia-induced chemoresistance to cisplatin and doxorubicin in NSCLC cells is through the HIF pathway. MDR1 regulation may not be involved in hypoxia-induced chemoresistance. Combining delivery of HIF-1α RNAi lentiviral vector with cisplatin-related chemotherapy regimens may enable us to develop more effective strategy for NSCLC therapy.
KeywordsNon-small cell lung cancer RNA interference Hypoxia inducible factor-1 alpha Drug resistance
We thank Dr. Fabin Han in Ottawa Health Research Institute, University of Ottawa for revising the manuscript. This work was supported by grants from the National Science Foundation of China (30570547).
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