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Biochemical Modulation of Aracytidine (Ara-C) Effects by GTI-2040, a Ribonucleotide Reductase Inhibitor, in K562 Human Leukemia Cells

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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.

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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.

Conflict of interest statement

Lorus Therapeutics provided partial support for the development of the Ara-CTP and dNTP/NTP assays.

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Authors and Affiliations

  1. College of Pharmacy, The Ohio State University, Columbus, Ohio, 43210, USA

    Ping Chen, Josephine Aimiuwu, Zhiliang Xie, Xiaohui Wei, Guido Marcucci & Kenneth K. Chan

  2. Division of Hematology–Oncology, The Ohio State University, Columbus, Ohio, 43210, USA

    Shujun Liu, Rebecca Klisovic & Guido Marcucci

  3. College of Medicine, The Ohio State University, Columbus, Ohio, 43210, USA

    Shujun Liu, Rebecca Klisovic, Guido Marcucci & Kenneth K. Chan

  4. The Comprehensive Cancer Center, The Ohio State University, Room 302/308 OSU CCC, 410 W. 12th Avenue, Columbus, Ohio, 43210, USA

    Rebecca Klisovic, Guido Marcucci & Kenneth K. Chan

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  1. Ping Chen
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  2. Josephine Aimiuwu
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  3. Zhiliang Xie
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Correspondence to Kenneth K. Chan.

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Ping Chen and Josephine Aimiuwu contributed equally to this work.

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Chen, P., Aimiuwu, J., Xie, Z. et al. Biochemical Modulation of Aracytidine (Ara-C) Effects by GTI-2040, a Ribonucleotide Reductase Inhibitor, in K562 Human Leukemia Cells. AAPS J 13, 131–140 (2011). https://doi.org/10.1208/s12248-010-9246-5

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