Figure 4.

Expression of HH pathway members in immortalized pulmonary epithelial and lung cancer cell lines. Effects of Smo inhibition on lung cancer cell clonal growth, apoptosis and lung tumor formation were also examined. (A) Cyclopamine treatment of ED-1 cells significantly inhibited clonal growth of this lung cancer cell line (left panel). This treatment also increased apoptosis as scored by caspase 3/7 activity (right panel). In these panels, dose-responsive effects are displayed with comparisons made to tomatidine treatments. (B) ED-1 and ED-2 lung cancer cells expressed multiple HH pathway members. Findings were compared to C-10 immortalized murine lung epithelial cells. Analogous experiments were performed using BEAS-2B immortalized human bronchial epithelial cells and human lung cancer cell lines. The mRNA levels are shown. (C) Recombinant sonic hedgehog (sHH) significantly induced mRNA levels of the indicated HH regulated species in ED-1 cells. (D) Gli-BS-luciferase reporter activity in murine and human immortalized lung epithelial and lung cancer lines. (E) Repression of Smo by independent transfection of shRNAs (Smo1 and Smo2) versus controls. (F) Significant reduction of lung cancer formation in vivo after transplantation of ED-1 cells that express Smo1 or Smo2 shRNAs versus control shRNA. Each circle represents an individual mouse; horizontal lines indicate mean tumor numbers. (G) Ex vivo treatment of ED-1 cells with the Smo inhibitor MK-4101 before tail vein injection into syngeneic mice also reduced lung tumor formation. Each circle represents an individual mouse; horizontal lines indicate mean tumor numbers. Standard deviation bars are shown. ^*P<0.05; ^**P<0.01.