3B). However, Panc1 cells treated with another Smo antagonist (cyclopamine) did not show a reduction in cell growth (data not shown). Similar experiments were conducted in HT29 cells. About 2.5��105 6 Gy irradiated HT29 cells were seeded Pazopanib IC50 into 24 well plates in medium with or without cyclopamine at 2 ��M, 5 ��M or vehicle control respectively, onto which 1000 Fluc labeled live HT29 cells were seeded. Compared with vehicle control treated group, cyclopamine reduced HT29 cell growth in a dose-dependent manner (Fig. 3C). The HT29 cells grown in vehicle control showed a significantly greater luciferase activity than those cells grown in 2 ��M cyclopamine (P<0.05) and 5 ��M cyclopamine (P<0.01). We further tested the Gli1 antagonist Gant61 (Fig. 3D) and the Smo antagonist GDC-0449 (Fig. 3E).

In both cases, similar results were observed. The Gli1 antagonist Gant61 inhibited HT29 growth on dying feeder cells significantly. However, the difference between the vehicle control group and the GDC-0449 treated groups was not significant (P>0.05). Gli1 Knockdown by shRNA Reduces Dying Tumor Cell Stimulated Living Tumor Cell Growth We further confirmed the role of SHH signaling in dying tumor cell stimulated living tumor cell growth by using shRNA to knockdown Gli1 expression in feeder cells. HT29 and Panc1 cells infected with lentivirus carrying Gli1 shRNA were selected in media with 2 ��g/ml puromycin, and Western blot analysis for Gli1 expression was used to verify proper selection. The protein levels of Gli1 in both Panc1 and HT29 cells (Fig. 4A, 4B) were markedly reduced.

Panc1 or HT29 cells transduced by Gli1 shRNA or scramble shRNA were irradiated with 6 Gy and used as feeder cells, respectively. The growth of Fluc labeled living Panc1 or HT29 cells seeded onto Gli1 knockdown feeder cells was significantly attenuated as evidenced by the significantly lower luciferase activities in wells with Gli1 knockdown Panc1 or HT29 cells than wells with scramble shRNA transduced Panc1 or HT29 cells (P<0.05) (Fig. 4A, 4B). Figure 4 Effects of Gli1 knockdown on dying cell induced tumor cell growth. SHH Signaling Agonists Activate Tumor Cell Growth We next inquired if the SHH signaling pathway agonist, SAG, which acts by directly binding to downstream Smoothened, would promote cancer cell growth.

Panc1 and HT29 cells were irradiated at 6 Gy and seeded in 24 well plates as feeders in medium with or without 3 nM, 5 nM, 10 nM or 100 nM SAG respectively. SAG treatment resulted in increased reporter cell growth in a dose-dependent manner (Fig. 5A, 5B). To further verify the results obtained with SAG, we added an active form of Shh, i.e. recombinant N-terminal Cilengitide fragments of Shh at 600 ng/ml into the medium. The results suggested that the recombinant N-terminal fragment of Shh significantly enhanced tumor cell growth on dying feeder cells in comparison with vehicle control (P<0.01) (Fig. 5C, 5D).