Dual luciferase assay

HEK293 cells were transfected with GLI1 expression plasmid, together with the reporter plasmids 12×GliBSLuc and R-Luc on 10-cm plates (day 0). Twenty-four hrs later, cells were seeded in white 96-well plates with clear bottom at a density of 15,000 cells per well. Cells were allowed to attach, and compounds were added at a final concentration of 10 μM in DMSO (0.5% final DMSO concentration) (day 1.5). Cells were grown for another 24 hrs, subsequently lysed, and then analyzed by using the Dual Luciferase kit.

Cell lines

22Rv1 and PANC1 cells

Preparation method

The solubility of this compound in DMSO is limited. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

5 μM; 48 hrs

Applications

The expressions of GLI1 and PTCH were reduced in 22Rv1 or PANC1 cells incubated with 5 μM GANT61 for 48 hrs. GANT61 efficiently inhibited tumor cell proliferation in vitro.

Animal models

Nude mice injected s.c. with GLI1-positive 22Rv1 prostate cancer cells

Dosage form

50 mg/kg; s.c.; for 18 days

Applications

In nude mice injected s.c. with GLI1-positive 22Rv1 prostate cancer cells, GANT61 significantly inhibited tumor growth. During the 18-day treatment period, GANT61 showed no adverse side effects, such as weight loss, ulcerations, or general non-wellbeing of the animals.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

GANT61 is a selective inhibitor of GLI with IC50 value of 13.56 μM (NBL-W-S cells, 48 hours) [1].
GLI genes, GLI1 and GLI2, are transcription activator and regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH->PTCH->SMO-
>GLI). It is well known that HH signaling plays a pivotal role in normal cellular processes, like embryonic development, tissue patterning and differentiation. As oncogenes, GLI1 and GLI2 are found to be constitutively activated in a variety types of human cancers [2].
GANT61 is a potent GLIs inhibitor. In SK-N-EB(2) cells expressing MYCN, GANT61 treatment reduced the cell viability with the IC50 value of 10.9μM and 7.96μM at 48 hours and 72 hours, respectively [1]. When tested with human RMS cell lines—RD and RH30, GANT-61 treatment exhibited anti-proliferative effects and induced cell death in a dose-dependent manner (5-25 μM) via decreasing GLI1/2 expression which resulted in the arrest of cell cycle at Go/G1 phase [3].
In nude mice model with RD or RH30 cells xenograft, intraperitoneal injected GANT-61 (50mg/kg, body weight in 200μl PBS; three times a week) markedly reduced tumor growth [3].
References:
[1].Wang, J., et al., Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells. BMC Cancer, 2014. 14: p. 768.
[2].Agyeman, A., et al., Mode and specificity of binding of the small molecule GANT61 to GLI determines inhibition of GLI-DNA binding. Oncotarget, 2014. 5(12): p. 4492-503.
[3].Srivastava, R.K., et al., GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis. Oncotarget, 2014. 5(23): p. 12151-65.