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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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Abstract

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.

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Abbreviations

ABC:

ATP binding cassette

BCRP:

breast cancer resistance protein

CDA:

cytidine deaminase

CNT:

concentrative nucleoside transporter

CTPS:

cytidine 5′-triphosphate synthetase

dCK:

deoxycytidine kinase

DCTD:

deoxycytidylate deaminase

dFdC:

2′,2′-difluorodeoxycytidine or gemcitabine

dFdCDP:

gemcitabine diphosphate

dFdCMP:

gemcitabine monophosphate

dFdCTP:

gemcitabine triphosphate

dFdU:

2′,2′-difluorodeoxyuridine

dFdUMP:

2′,2′-difluorodeoxyuridine monophosphate

ENT:

equilibrative nucleoside transporter

Fara-AMP:

9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate

LC-MS/MS:

liquid chromatography-tandem mass spectrometer

MDR1:

multidrug resistance protein 1

MRM:

multiple reaction monitoring

MRP:

multidrug resistance-associated proteins

5′-NT:

cytosolic 5′-nucleotidase

RRM1:

ribonucleotide reductase subunit 1

RRM2:

ribonucleotide reductase subunit 2

THU:

tetrahydrouridine

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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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Authors and Affiliations

  1. Division of Membrane Transport and Drug Targeting Department of Biochemical Pharmacology and Therapeutics Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan

    Ken Ohmine, Sumio Ohtsuki & Tetsuya Terasaki

  2. Division of Hepato-Biliary Pancreatic Surgery Department of Surgery Graduate School of Medicine, Tohoku University, 2-1 Seiryou-machi, Aoba-ku, Sendai, 980-8574, Japan

    Kei Kawaguchi, Fuyuhiko Motoi, Shinichi Egawa & Michiaki Unno

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  1. Ken Ohmine
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  2. Kei Kawaguchi
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  4. Fuyuhiko Motoi
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Correspondence to Tetsuya Terasaki.

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Ohmine, K., Kawaguchi, K., Ohtsuki, S. et al. 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. Pharm Res 29, 2006–2016 (2012). https://doi.org/10.1007/s11095-012-0728-2

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