Biochemical modulation of aracytidine (Ara-C) effects by GTI-2040, a ribonucleotide reductase inhibitor, in K562 human leukemia cells

Ping Chen, Josephine Aimiuwu, Zhiliang Xie, Xiaohui Wei, Shujun Liu, Rebecca Klisovic, Guido Marcucci, Kenneth K. Chan

Research output: Contribution to journalReview articlepeer-review

4 Scopus citations

Abstract

GTI-2040 is a potent antisense to the M2 subunit of the ribonucleotide reductase (RNR), an enzyme involved in the de novo synthesis of nucleoside triphosphates. We hypothesized that combination of GTI-2040 with the cytarabine (Ara-C) could result in an enhanced cytotoxic effect with perturbed intracellular deoxynucleotide/nucleotide (dNTP/NTP) pools including Ara-C triphosphate (Ara-CTP). This study aims to provide a direct experimental support of this hypothesis by monitoring the biochemical modulation effects, intracellular levels of Ara-CTP, dNTPs/NTPs following the combination treatment of Ara-C, and GTI-2040 in K562 human leukemia cells. GTI-2040 was introduced into cells via electroporation. A hybridization-ligation ELISA was used to quantify intracellular GTI-2040 concentrations. Real-time PCR and Western blot methods were used to measure the RNR M2 mRNA and protein levels, respectively. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium, inner salt assay was used to measure the cytotoxicity following various drug treatments. A non-radioactive HPLC-UV method was used for measuring the intracellular Ara-CTP, while a LC-MS/MS method was used to quantify intracellular dNTP/NTP pools. GTI-2040 was found to downregulate M2 mRNA and protein levels in a dose-dependent manner and showed significant decrease in dNTP but not NTP pool. When combining GTI-2040 with Ara-C, a synergistic cytotoxicity was observed with no further change in dNTP/NTP pools. Importantly, pretreatment of K562 cells with GTI-2040 was found to increase Ara-CTP level for the first time, and this effect may be due to inhibition of RNR by GTI-2040. This finding provides a laboratory justification for the current phase I/II evaluation of GTI-2040 in combination with Ara-C in patients with acute myeloid leukemia.

Original languageEnglish (US)
Pages (from-to)131-140
Number of pages10
JournalAAPS Journal
Volume13
Issue number1
DOIs
StatePublished - Mar 2011
Externally publishedYes

Bibliographical note

Funding Information:
Acknowledgment This work was partially supported by National Cancer Institute/National Institute of Health grant R21 CA133879 (GM/RBK) and grant UO1-CA76576 and Lorus Therapeutics.

Keywords

  • Ara-CTP
  • GTI-2040
  • HPLC-UV

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