Cistanoside F acts as a Monoacylglycerol Lipase inhibitor that synergizes the anti-tumor effect of 2-Arachidonoyl Glycerol on Bladder cancer

Background: Bladder Cancer (BCa) remains clinically challenging due to high recurrence rates. As an endocannabinoid, 2-arachidonoylglycerol (2-AG) plays a pivotal role in regulating numerous physiological and pathological processes, including tumorigenesis. The maintenance of its levels in the biological system is essential for 2-AG to exert its biological functions. Therefore, inhibitors targeting the 2-AG metabolic enzyme, such as monoacylglycerol Lipase (MGLL), are considered promising for clinical application.

Methods: The cell viability assay, colony formation, cell migration, and invasion assay were used to demonstrate 2-AG's anti-proliferative and anti-metastatic effects in BCa cells, inversely correlated with MGLL expression. MGLL overexpression or knockdown confirmed its regulatory role in 2-AG efficacy. Transcriptomics identified LKB1 as a potential 2-AG target. From a medicinal-food compound library, the MGLL activity assay was used to discover Cistanoside F (CF) as a potent MGLL inhibitor.

Results: The in vitro experiments confirmed 2-AG's selective anti-proliferative and anti-metastatic effects in BCa cells, while MGLL attenuates the anti-tumor effect of 2-AG in BCa cells via metabolizing 2-AG into AA. In mechanism, 2-AG activated the LKB1-AMPKα-mTOR axis to suppress BCa progression. At non-cytotoxic concentrations (4-8 nM), CF enhanced 2-AG's effects in BCa cells by sustaining endogenous 2-AG levels through MGLL suppression. In vivo, CF synergized with 2-AG to significantly inhibit tumor growth and lung metastasis compared to 2-AG monotherapy.

Conclusion: These findings establish CF as a novel MGLL-targeting Adjuvant that amplifies 2-AG's anti-BCa efficacy via LKB1 pathway activation, providing dual therapeutic strategies: MGLL inhibition for 2-AG potentiation and LKB1 modulation for pathway-directed therapy.