Decreased Endothelin-1 bioavailability impairs aggressiveness of gallbladder cancer cells.

Background: Gallbladder cancer (GBC) is a highly lethal malignancy, often diagnosed at advanced stages when curative options are limited. Its rapid progression and high metastatic potential result in a 5-year survival rate below 5%. Recent evidence highlights the role of Endothelin-1 (ET1), a bioactive peptide, in promoting tumor aggressiveness through activation of its receptors (ETRs). However, therapeutic strategies have mainly focused on receptor inhibition, neglecting the modulation of ET1 availability. Therefore, this study aimed to evaluate the therapeutic potential of modulating ET1 levels through the application of recombinant Neprilysin (rNEP) to degrade ET1 or inhibition of Endothelin Converting Enzyme-1 (ECE1) to reduce its production in GBC cells.

Methods: The effects of rNEP and the ECE1 inhibitor SM19712 were evaluated in GBC cell lines by assessing ET1 levels, nuclear β-catenin localization, transcript levels of target genes, and changes in proliferation, migration, invasion, and stemness-associated markers in vitro.

Results: Both rNEP and SM19712 significantly reduced extracellular ET1 levels, nuclear β-catenin localization, and expression of genes such as CCND1, VEGFA, and BIRC5. Treatment also decreased the expression of EMT and stemness markers CD44 and Vimentin. Functionally, rNEP reduced cell migration, invasion, and colony formation, while SM19712 affected migration and colony formation. Isoform analysis revealed predominant expression of ECE1c, suggesting potential ET1-independent roles in invasion.

Conclusion: Modulating ET1 bioavailability through enzymatic degradation or inhibition of its synthesis reduces aggressiveness in GBC cells. These findings support the use of rNEP and ECE1 inhibition as promising strategies for GBC treatment, although further in vivo validation is required.