Hypocrellin A against intrahepatic Cholangiocarcinoma via multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive and highly lethal cancer with an increasing incidence worldwide that lacks effective treatment regimens. Hypocrellin A (HA), a natural small compound isolated from S. bambusicola, has multiple biomedical activities, including antitumor activity. PURPOSE: We intended to investigate the therapeutic effects of HA on ICC and its potential mechanisms. METHODS: RBE and HuccT1 cell lines were utilized for in vitro experiments. CCK8 assay, colony formation analysis, RTCA, and immunofluorescence staining of ki67 were employed to evaluate the suppression effects of HA on proliferation. The inhibitory effects of HA on cell migration and invasion were evaluate through transwell and wound healing assays, and Hoechst 33,258 staining was performed to evaluate apoptosis. Additionally, we performed transcriptome sequencing and molecular docking for targeting identification, and immunoblotting and immunofluorescence of key molecules for validation. Two in vivo models, HuccT1 xenografts, and the primary ICC model (KRAS/P19/SB) established via hydrodynamic tail-vein injection were implemented. Multiplex immunohistochemistry (mIHC) was used to illustrate the multi-target inhibitory effects of HA. RESULTS: The IC50 values of HA against RBE and HuccT1 cells were 4.612 μM and 10.01 μM for 24 h, as determined through the CCK8 assay. Our results confirmed that HA significantly repressed the proliferation, migration, invasion, and promoted the apoptosis of ICC cells at low concentrations. Moreover, HA exerted its anti-cancer effects through multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. This inhibitory effect was rescued by Recilisib, an activator of the PI3K-AKT-mTOR pathway. Bioinformatics analysis of a multi-center RNA-Seq cohort (n = 90) demonstrated significant associations between these target pathways and the occurrence and poor prognosis of ICC. Animal studies suggested that HA strongly inhibited tumor growth in xenograft ICC models, and repressed the tumor number and size in the liver of primary ICC models by suppressing these three crucial pathways. CONCLUSION: HA, a novel natural small molecule, demonstrated promising therapeutic efficacy against ICC through its multi-target inhibitory effects on the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Moreover, it exhibited notable therapeutic benefits in a primary ICC model (KRAS/P19/SB), positioning it as a novel therapeutic agent for ICC.