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Pharmaceutical Research

, Volume 29, Issue 7, pp 2006–2016 | Cite as

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

  • Ken Ohmine
  • Kei Kawaguchi
  • Sumio Ohtsuki
  • Fuyuhiko Motoi
  • Shinichi Egawa
  • Michiaki Unno
  • Tetsuya Terasaki
Research Paper

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.

KEY WORDS

drug resistance gemcitabine metabolomics proteomics pancreatic cancer 

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

Notes

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.

Supplementary material

11095_2012_728_MOESM1_ESM.doc (234 kb)
ESM 1 (DOC 234kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ken Ohmine
    • 1
  • Kei Kawaguchi
    • 2
  • Sumio Ohtsuki
    • 1
  • Fuyuhiko Motoi
    • 2
  • Shinichi Egawa
    • 2
  • Michiaki Unno
    • 2
  • Tetsuya Terasaki
    • 1
  1. 1.Division of Membrane Transport and Drug Targeting Department of Biochemical Pharmacology and Therapeutics Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.Division of Hepato-Biliary Pancreatic Surgery Department of Surgery Graduate School of MedicineTohoku UniversitySendaiJapan

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