Cell experiment:

HEK293 cells are transfected with GLI1 expression plasmid, together with the reporter plasmids 12× GliBS-Luc and R-Luc on 10 cm plates (day 0). Twenty-four hours later, cells are seeded in white 96 well plates with clear bottom at a density of 15,000 cells per well. Cells are allowed to attach, and compounds are added at a final concentration of 10 μM in DMSO (0.5% final DMSO concentration) (day 1.5). Cells are grown for another 24 h, subsequently lysed, and then analyzed by using the Dual Luciferase kit. Plates are read on a Berthold Technologies microplate luminometer. Subconfluent cells are grown in reduced FBS (2.5%) for 48 h in the presence of 5 μM test compound (or DMSO) on white 96 well plates with clear bottom. Subsequently, cells are labeled for 2 h with BrdU, fixed, and analyzed. Samples are read on a Molecular Devices SpectraMax Gemini EM[1].

Animal experiment:

Mice[1] 5×106 22Rv1 cells are suspended in a total volume of 100 μL of a 1:1 mixture of RPMI medium 1640:Matrigel (E1270). The cell suspension is injected s.c. at the posterior flank of female BALB/c nude mice (nu/nu). Tumors are grown until they reached a median size of ≈250 mm3 (5-6 days). Animals are randomly divided into four groups (n=4-5) and treated with solvent only (corn oil:ethanol, 4:1) or compounds in solvent (50 mg/kg) for 16 days s.c. injections of compounds are performed several centimeters away from the tumor. Tumor volumes are calculated by the formula length×width×0.5×(length+width). At the end of the treatment period, animals are given a BrdU pulse (50 mg/kg) for 30 min, and tumors are removed. All animal experiments are approved by local ethics authorities.

GANT 58 is an antagonist of GLI that inhibits GLI1-induced transcription with an IC50 value of 5 μM [1].

GLI1 is a human glioblastoma isolated protein which belongs to GLI protein family. GLI proteins act as effectors in Hedgehog signaling pathway and get involved in cell proliferation, determination and patterning during embryo development.

GANT 58 is considered to inhibit GLI1-mediated gene trans-activation with a high selectivity for Hedgehog signaling pathway. When TNF signaling/NFkB activation, glucocorticoid, receptor gene transactivation, and the Ras-Raf-Mek-Mapk cascade were treated with GANT 58, no inhibition was observed. When SAG-treated shh-L2 cells were treated with 10 μM GANT 58 for 48 hr, the reduction of Hedgehog pathway signaling was observed, whereas Gli1 mRNA level reduction was induced by the treatment of 10 μM GANT 58 for 2-3 days. Indeed, GANT 58 was confirmed as inhibitor of Hedgehog signaling pathway downstream Sufu and Smo [1]. Furthermore, GANT 58 treatments for 48 hr resulted in the significant reduction of cell viability in a dose-dependent manner for CCRFeCEM, CEM7e14, CEM1e15 and Jurkat cells. When CCRFeCEM cells were treated with 10 μM GANT 58 for 48 hr, the percentage of cells in G1/S phase increased whereas the percentage in G2/M phase decreased [2].

In mouse model with xenograft, daily s.c. injection of GANT 58 resulted in significant suppression of tumor cell growth. 50 mg/kg/d GANT 58 injections for 18 days induced the suppression of additional xenograft growth and restrict the increase of tumor size. No side effects were observed during the treatment [1].

References:
[1] Lauth M, Bergstr?m ?sa, Shimokawa T, Toftg?rd R.  Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists.?Proceedings of the National Academy of Sciences of the United States of America. 2007,104(20):8455-8460.
[2] Hou X, Chen X, Zhang P, et al.  Inhibition of hedgehog signaling by GANT58 induces apoptosis and shows synergistic antitumor activity with AKT inhibitor in acute T cell leukemia cells. Biochimie. 2014, 101:50-9