Attenuation of phosphorylation by deoxycytidine kinase is key to acquired gemcitabine resistance in a pancreatic cancer cell line: Targeted proteomic and metabolomic analyses in PK9 cells

Purpose: Multiple proteins are involved in activation and inactivation of 2′,2′-difluorodeoxycytidine (gemcitabine, dFdC). We aimed to clarify the mechanism of dFdC resistance in a pancreatic cancer cell line by applying a combination of targeted proteomic and metabolomic analyses. Methods: Twenty-five enzyme and transporter proteins and 6 metabolites were quantified in sensitive and resistant pancreatic cancer cell lines, PK9 and RPK9, respectively. Results: The protein concentration of deoxycytidine kinase (dCK) in RPK9 cells was less than 0.02-fold (2%) compared with that in PK9 cells, whereas the differences (fold) were within a factor of 3 for other proteins. Targeted metabolomic analysis revealed that phosphorylated forms of dFdC were reduced to less than 0.2% in RPK9 cells. The extracellular concentration of 2′,2′-difluorodeoxyuridine (dFdU), an inactive metabolite of dFdC, reached the same level as the initial dFdC concentration in RPK9 cells. However, tetrahydrouridine treatment did not increase phosphorylated forms of dFdC and did not reverse dFdC resistance in RPK9 cells, though this treatment inhibits production of dFdU. Conclusions: Combining targeted proteomics and metabolomics suggests that acquisition of resistance in RPK9 cells is due to attenuation of dFdC phosphorylation via suppression of dCK.