Figure 7.

High expression of Shh and Nrf2 correlates with induced stem cell-like characteristics and contributes to chemoresistance of HNSCC cells. (a) FaDu cells were treated with three different concentrations of ShhsiRNA for 24 h and cell survival was analyzed. (b) FaDu cells were treated with Shh-siRNA and cyclopamine (10 µM) for 2 days, washed and treated with increasing concentrations of cisplatin and cell viability was assessed by alamarBlue assay. (c) FaDu cells were treated with Shh-siRNA and cyclopamine (10 µM) for 2 days, washed and treated with increasing concentrations of 5-fluorouracil (5-FU) and cell viability was assessed by alamarBlue assay. Dose–response data were analyzed by the R statistical software ‘DRC’ package. (d) Freshly isolated treatment-sensitive and treatment-resistant HNSCC cells and (e) FaDu cells were cultured in cancer stem cell (CSC) supplemented medium for 10 days. Resulting spheres were immunofluorescently stained for Shh and Nrf2 (green: Shh; red: Nrf2, blue: DAPI; scale bar, 100 µm). Patients’ tumor cells and FaDu cells were cultured in the supplemented sphere growth medium and analyzed for expression of (f) CD44, Oct4 and Nanog. (g, h) Shh, Gli1, Nrf2 and HO-1 by Real-time PCR and compared for relative expression. (i) FaDu cells were transfected with Shh-siRNA and analyzed by Western blots for expression of Shh, Gli1, Nrf2 and HO-1. (j) FaDu cells were cultured and transfected as in (i) and sphere growth and numbers were analyzed (p = 6.3×10⁻¹⁰, Student’s t test). (k) FaDu cells were transfected with Nrf2-siRNA and analyzed by Western blots for expression of Shh, Gli1, Nrf2 and HO-1. (l) FaDu cells were cultured and transfected as in (i) and sphere growth and numbers were analyzed (p < 0.0001, Student’s t test).

HNSCC, head and neck squamous cell carcinoma; PCR, polymerase chain reaction.