First proof-of-concept study on targeting Hedgehog pathway in intrahepatic cholangiocarcinoma histological subtypes

Intrahepatic Cholangiocarcinoma (iCCA) represents a highly heterogeneous cancer that emerge in the bile ducts, displaying dismal prognosis (5-year survival rate <20%). iCCA is still considered a rare type of cancer, however, its global incidence and mortality is alarmingly increasing. The silent growth of iCCA strongly impact its early diagnosis, resulting in its late detection, compromising patients’ access to curative options. According to the WHO classification (ICD-O-3.2), the two histological subtypes of iCCA are the large-duct type and the small-duct type, exhibiting different clinical-pathological characteristics, prognosis and molecular profile. The Hedgehog (Hh) pathway is one of the molecular network entangled in the pathogenesis of iCCA. It is actually cardinal in various iCCA properties, including tumor growth, survival, invasiveness, cancer stem cells activation and epithelial-mesenchymal transition. Considering these observations, targeting this pathway for therapeutic reasons is feasible in account of the evidence of Hh's pathogenetic function in iCCA. This study intends to evaluate, in vitro, a novel natural chemical compound called Glabrescione B (GlaB) that specifically inhibits Gli1, the final effector of Hh pathway. Trypan Blue Exclusion test and MTS assay have been used to evaluate the dose- and time-dependent response and the IC50 of free GlaB and/or hyaluronic acid (HA)-encapsulated GlaB (HA-GlaB). Western blot has been performed to analyze the target protein levels. Wound-healing assay has been achieved to assess cells migratory activity. Colony formation assay has been performed to rate the colony-forming ability of cancer cells. Flow cytometry analysis has been accomplished to investigate cell death. Our data display that both free GlaB and HA-GlaB treatments significantly narrow iCCA cell rate proliferation, migration, colony-forming ability, and Gli1 levels in a dose- and time-dependent manner (0,05<0,001), leading to a decisive reduction in cancer cells invasiveness. Moreover, evidence from flow cytometry exhibit an induction of cell death as a result of drug administration. Hedgehog pathway aberration is associated with the development and progression of various cancers, including iCCA. Glabrescione B is able to inhibit cancer cells growth, survival, invasiveness and to induce cancer cell death. Taken together, these data provide insight into a new potentially beneficial natural compound for the management of iCCA in vivo.