GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells

Abstract

Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line was the most sensitive (IC50: 36 µM) to the tested compound. The compound’s effects on the expression of HH pathways components were analyzed by qPCR and Western blot; cell viability was analyzed by trypan blue assay and flow cytometry were used to investigate cell cycle phase, morphology, and death patterns in HSC3 cells. A significant reduction in mRNA levels of the GLI1 transcription factor was found after 12 h of treatment withGANT61. Protein expression levels of other HH pathway components (PTCH1, SHH, and Gli1) and HSC3 cell viability also decreased after 24 h of treatment. Cell cycle analysis and death pattern evaluations revealed significantly increased nuclear fragmentation in sub-G1 phase, as well as cell death due to apoptosis. In conclusion, the significantly reduced GLI1 gene expression seen in response to the GLI inhibitor indicates diminished downstream activation in HH pathway components. GANT61 significantly reduced cell viability in the metastatic cell line of OSCC and promoted a significant increase in nuclear fragmentation and cell death by apoptosis.

Hedgehog Signaling Pathway Orchestrates Human Lung Branching Morphogenesis

“…The second class of inhibitors targets the transcription factor GLI1, such as Curcumin or Gant61 [54,55]. The third class contains inhibitors that act on different phases of production, activation, and binding of the SHH ligand, such as RU-SKI 43 (SHH palmitoylation) or 5E1 (binding) [35,56]. Therefore, we used the SHH antibody 5E1, a strong candidate to safely interfere with HH signaling in clinical trials [57] with limited off-target effects, to abrogate HH activation in our human culture model [17,35].…”

The role of Hedgehog and Notch signaling pathway in cancer

“…Initially, identified from cellular screening assays in 2007, GANT58 and GANT61 were demonstrated to be selective inhibitors of Hh-driven tumor growth because of their capacity to downregulate the transcriptional activity of Gli1 and Gli2 [458][459][460][461]. Since then, a substantial number of studies have explored their antitumor effects and have shown that GANT61 can inhibit malignant behavior, induce autophagy and apoptosis, and enhance the therapeutic sensitivity of tumor cells both in vitro and in vivo [462][463][464][465][466]. However, no clinical trials The relevant clinical trial data were obtained from the registration on ClinicalTrials.gov using GANT61 for treating human cancer are currently ongoing.…”

A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells

“…Given these results and the regulation of GLI1 in human mast cells by the reduction in SUFU expression (Figure 1C and Supplementary Figure 2), we next focused on understanding the role of GLI proteins, particularly GLI1 and GLI2, in the regulation of human mast cell proliferation and survival. We employed the widely used GLI inhibitors GANT61 and ATO (31)(32)(33), and the new generation inhibitor HPI-1 (34), which as shown in Figure 1D, effectively downregulated both GLI1 and GLI2 mRNA expression, a readout of pathway inhibition.…”

“…All GLI inhibitors markedly reduced (by 40 to 95%) the growth of neoplastic human mast cells in culture (Figure 3A) in a concentration-dependent manner (Figure 3A). We used concentrations ranging from 5 to 40 µM of GANT61 and HPI-1, or up to 10 µM of ATO, as these were within the range of the reported IC 50 for the corresponding inhibitors in several cell line models (31,(34)(35)(36). Concentrations greater than 40 µM for HPI-1 and GANT61, or greater than10 µM for ATO were not tested due to concerns of potential off-target effects.…”