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.