Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1alpha gene.
OBJECTIVE
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-1alpha RNA interference (RNAi) could reverse the hypoxia-induced resistance to chemotherapy.


METHODS
We transfected Human NSCLC cell lines, SPCA1 and A549 with HIF-1alpha specific RNAi lentiviral vectors as well as controls. HIF-1alpha silenced cells [SPCA1/HIF-1alpha(-) and A549/HIF-1alpha(-)] 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-1alpha 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-1alpha RNAi vector resulted in sequence specific silencing with 87.2 and 84.6% decreases of HIF-1alpha 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-1alpha(-) and A549/HIF-1alpha(-) 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-1alpha RNAi lentiviral vector with cisplatin-related chemotherapy regimens may enable us to develop more effective strategy for NSCLC therapy.
Song
Xianrang
X
Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, China. sxr@vip.163.com
Liu
Xianxi
X
Chi
Weiling
W
Liu
Yonglei
Y
Wei
Ling
L
Wang
Xingwu
X
Yu
Jinming
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010100:Oxygen / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D004317:Doxorubicin; D002945:Cisplatin; D010100:Oxygen