Ribonucleic acid interference knockdown of IL-6 enhances the efficacy of cisplatin in laryngeal cancer stem cells by down-regulating the IL-6/STAT3/HIF1 pathway

Qiang Fu, Pengruofeng Liu, Xiumei Sun, Shanshan Huang, Fengchan Han, Lili Zhang, Yannan Xu, Tingyan Liu

Cisplatin has been used in the treatment of many cancers, including laryngeal cancer; however, its efficacy can be reduced due to the development of drug resistance. This study aimed to investigate whether interleukin-6 (IL-6) knockdown may enhance the efficacy of cisplatin in laryngeal cancer stem cells (CSC) and the potential involvement of the signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1 (HIF1) in this effect. The ALDH+ and CD44+ CSC in Hep2 human laryngeal squamous cancer cells were identified by the fluorescence-activated cell sorting technique. IL-6, STAT3 and HIF1 mRNA and protein expressions were examined with quantitative real-time polymerase chain reaction and Western blot, respectively. Cell proliferation was measured by MTT assay. Tumorigenicity was measured by a colony formation assay and invasion was determined by a cell invasion assay. Apoptotic cells were counted by flow cytometry. Immunohistochemistry was performed to detect immunoreactive IL-6, STAT3 and HIF1 cells in xenografts. The mRNA and protein levels of IL-6, STAT3 and HIF1 were significantly increased in Hep2-CSC as compared with those from Hep2 cells. Application of siRNA-IL-6 to knockdown IL-6 resulted in significantly decreased IL-6, STAT3 and HIF1 mRNA and protein levels. IL-6 knockdown reduced cell proliferation, tumorigenicity and invasion and increased apoptosis within CSC. Enhanced degrees of suppression in these parameters were observed when IL-6 knockdown was combined with cisplatin in these CSC. Results from the xenograft study showed that the combination of IL-6 knockdown and cisplatin further inhibited the growth of xenografts as compared with that obtained in the cisplatin-injected group alone. Immunoreactive IL-6, STAT3 and HIF1 cell numbers were markedly reduced in IL-6 knockdown tumor tissues. IL-6, STAT3 and HIF1 immunoreactive cell counts were further reduced in tissue where IL-6 knockdown was combined with cisplatin treatment as compared with tissue receiving cisplatin alone. IL-6 knockdown can increase chemo-drug efficacy of cisplatin, inhibit tumor growth and reduce the potential for tumor recurrence and metastasis in laryngeal cancer. The IL-6/STAT3/HIF1 pathway may represent an important target for investigating therapeutic strategies for the treatment of laryngeal cancer.