GLI1-selective inhibitor, Glabrescione B, impairs proliferation in intrahepatic cholangiocarcinoma

Cholangiocarcinoma (CCA) constitutes a heterogeneous group of epithelial malignancies that arise in the biliary tree. CCA is a very aggressive cancer and carries a poor prognosis [1]. Furthermore, in view of the constant increase in its incidence and mortality in recent decades throughout the world, it represents a growing global health problem [1,2]. CCAs are divided into three subtypes according to their anatomical site of origin: intrahepatic CCA (iCCA), perihilar (pCCA), and distal (dCCA). Specifically, iCCA is characterized by high intertumoral clinical-pathological and molecular heterogeneity, leading to an innovative histological classification approved by WHO (ICD-O-3.2), into small bile duct iCCA and large bile duct iCCA [3]. The molecular pathogenesis of CCA is very complicated and involves multiple molecular networks: among them, the Hedgehog (Hh) [4,6] signaling pathway. Indeed, the Hh pathway plays an important role in many aspects of iCCA, such as tumor survival, proliferation, migration and EMT reprogramming [5]. The impact of innovative therapies on the survival of patients with iCCA is an ongoing area of investigation. Evidence on the pathogenetic role of Hh in iCCA implies the possibility of targeting this signaling pathway for therapeutic purposes in iCCA subtypes. The aim of this study is to demonstrate the antitumor efficacy of a natural therapeutic compound, from an in-house library, that inhibits the Hh pathway in experimental models of human iCCA, in vitro, in established iCCA cell lines (CCLP1) and primary cell lines derived from small or large bile duct iCCA. The iCCA samples from resected specimens have been processed by mechanical and enzymatic digestion to obtain patient-derived primary cell lines. The effect of Glabrescione B (GlaB), a selective inhibitor of GLI1 (Hh downstream transcriptional factor), has been evaluated. The dose-dependent efficacy of GlaB [0, 1, 5, 10, 20, 50 μM] or hyaluronic acid (HA) nanohydrogel-encapsulated Glab [0, 0,5, 1, 5, 10 μM], have been evaluated by Trypan Blue Exclusion Test and MTS assay (cell viability), at different time points (24h, 48h, 72h, 96h). Its ability as inhibitor of Hh signaling pathway has been evaluated by Western Blot analysis. All experiments have been conducted in N.3 experimental replicates. Our research shows a dose-dependent and time-dependent reduction of cell proliferation by Trypan Blue Exclusion Test in all cell lines (p<0.05). The decrease already occurs at low concentrations and at 24-hour incubation (40-50%) and becomes more evident at higher concentrations and at 96-hour incubation (80-90%) (p<0.05). Likewise, at the protein level, Gli1 knockdown, in a dose and time-dependent manner, is demonstrated (p<0.05). MTS assays confirmed the cytotoxicity effect of GlaB at 20 μM and 50 μM (p<0.05). Therefore, we decided to pursue the study with lower concentrations (from 1 μM to 10 μM) capable of knocking down the cell viability by 50%. Thereafter, successful, and stable encapsulation of [0.5-10 μM] Glab in HA nanogels has been accomplished. Data showed a dose-dependent and time-dependent inhibitory activity of HA-encapsuled Glab [0.5-10 μM] in all cell lines (p<0.05). Aberrant Hh pathway activation is widely known to be closely related with the development and malignant progression of various human cancers. Preclinical experimental data indicate that dysregulation of this pathway is a determinant of cholangiocarcinoma progression and invasiveness. The achievements of this study could lay the groundwork for pre-clinical studies of HA-encapsuled Glab in iCCA. References [1] Banales JM. et al., Nat Rev Gastroenterol Hepatol. 2020 Sep;17(9):557-588. doi: 10.1038/s41575-020-0310-z. Epub 2020 Jun 30. PMID: 32606456. [2] Bertuccio P. et al., J Hepatol. 2019 Jul;71(1):104-114. doi: 10.1016/j.jhep.2019.03.013. Epub 2019 Mar 23. PMID: 30910538. [3] European Association for the Study of the Liver. Electronic address: easloffice@easloffice.eu; European Association for the Study of the Liver. EASL-ILCA Clinical Practice Guidelines on Intrahepatic Cholangiocarcinoma. J Hepatol. 2023 Mar 22:S0168-8278(23)00185-X. doi: 10.1016/j.jhep.2023.03.010. Epub ahead of print. PMID: 37084797. [4] Riedlinger D. et al., J Hepatobiliary Pancreat Sci. 2014 Aug;21(8):607-15. doi: 10.1002/jhbp.107. Epub 2014 Apr 15. PMID: 24733827. [5] Cigliano A. et al., Expert Opin Ther Targets. 2017 May;21(5):471-483. doi: 10.1080/14728222.2017.1310842. Epub 2017 Mar 30. PMID: 28326864. [6] Razumilava N. et al., J Hepatol. 2014 Mar;60(3):599-605. doi: 10.1016/j.jhep.2013.11.005. Epub 2013 Nov 14. Erratum in: J Hepatol. 2014 Jul;61(1):185. PMID: 24239776.