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
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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.
Twenty-five enzyme and transporter proteins and 6 metabolites were quantified in sensitive and resistant pancreatic cancer cell lines, PK9 and RPK9, respectively.
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.
Combining targeted proteomics and metabolomics suggests that acquisition of resistance in RPK9 cells is due to attenuation of dFdC phosphorylation via suppression of dCK.
KEY WORDSdrug resistance gemcitabine metabolomics proteomics pancreatic cancer
ATP binding cassette
breast cancer resistance protein
concentrative nucleoside transporter
cytidine 5′-triphosphate synthetase
2′,2′-difluorodeoxycytidine or gemcitabine
equilibrative nucleoside transporter
liquid chromatography-tandem mass spectrometer
multidrug resistance protein 1
multiple reaction monitoring
multidrug resistance-associated proteins
ribonucleotide reductase subunit 1
ribonucleotide reductase subunit 2
Acknowledgments & Disclosures
We thank K. Hamase for technical suggestions and Shiseido Co. Ltd for providing columns. This study was supported in part by a Grant-in-Aid for JSPS Fellows, a Global COE Program from the Japan Society for the Promotion of Science, and a Grant for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency. This study was also supported in part by the Industrial Technology Research Grant Program from the New Energy and the Industrial Technology Development Organization of Japan, and the Funding Program for Next Generation World-Leading Researchers by the Cabinet Office, Government of Japan.
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