Ectophosphatase Activity in Human Hepatocellular Carcinoma Huh7 Cells:Characterisation and Implications for the Tumour Microenvironment

Introduction/Objective: Hepatocellular carcinoma (HCC) is an aggressive liver malignancy characterised by extensive metabolic reprogramming and a complex tumour microenvironment (TME) in which inorganic phosphate (Pi) is crucial. Although ectophosphatases are known to regulate extracellular Pi through substrate dephosphorylation, their function in HCC has not been investigated. This study aimed to characterise ectophosphatase activity in HCC cells and to evaluate its potential contribution to tumour-associated processes. Methods: Ectophosphatase activity was assessed in the human HCC cell line Huh7. Enzymatic activity was determined by measuring p-nitrophenol (p-NP) release from p-nitrophenyl phosphate (p-NPP). Kinetic parameters, pH dependence, inhibitor sensitivity, and substrate specificity were analysed. Functional assays were performed to investigate the impact of enzyme inhibition on cell adhesion and migration. Results: Phosphatase activity was predominantly localised to the plasma membrane and followed Michaelis–Menten kinetics, with an apparent Km of 2.16 ± 0.49 mM and Vmax of 50.34 ± 4.45 nmol p-NP h⁻¹ per 10⁻⁵ cells. The activity increased under acidic conditions and was inhibited by sodium orthovanadate, sodium fluoride, and ammonium molybdate but remained unaffected by levamisole or tartrate. Hydrolysis of phosphotyrosine, phosphoserine, and phosphothreonine was detected, with orthovanadate selectively blocking p-NPP and phosphotyrosine hydrolysis, suggesting distinct catalytic sites. Inhibition of ectophosphatase activity significantly reduced Huh7 cell adhesion and migration. Discussion: These findings suggest that ectophosphatase activity contributes to key tumourrelated processes in HCC. By modulating phosphate metabolism in the extracellular environment, ectophosphatases may influence both the metabolic demands and the invasive properties of tumour cells. Conclusion: This study provides the first biochemical characterisation of ectophosphatase activity in HCC cells and highlights its potential role in shaping the TME, with implications for phosphate metabolism and tumour progression.