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Ribonucleotide Reductase Inhibition Enhances Chemoradiosensitivity of Human Cervical Cancers

Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA.
Radiation Research (Impact Factor: 2.91). 11/2010; 174(5):574-81. DOI: 10.1667/RR2273.1
Source: PubMed

ABSTRACT

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

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    • "Blockade of dNTP supply needed for replication and DNA repair is one such strategy. Inhibition of thymidylate synthase [61] and ribonucleotide reductase [62], [63], two critical enzymes of the de novo dNTP synthesis, has been utilized for tumor radiosensitization. The importance of dN salvage in IR-induced DNA repair makes this pathway a possible target for novel sensitizers to genotoxic therapy. "
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    ABSTRACT: Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Other RR inhibitors, such as triapine, are also reported to increase both radiation and chemotherapy sensitivity through a mechanism involving blocking RR activity which results in sustained DNA damage in cervical cancer cells.36 Further, the synergistic cytotoxicity between cisplatin and gemcitabine has been observed in human ovarian cancer cell lines and head and neck cancer xenografts,13,37 and one clinical study has shown that carboplatin plus gemcitabine is an active combination for patients with metastatic breast cancer.38 "
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    ABSTRACT: The present study aims to investigate the subunit expression and enzyme activity of ribonucleotide reductase in cervical cancer patients, and detect the combined effect of the ribonucleotide reductase inhibitor gemcitabine and the chemotherapeutic agent carboplatin on cervical cancer cell lines. Using quantitative reverse transcription polymerase chain reaction, Western blotting, and cytidine 5'-diphosphate reduction assays, we tested the expression and activity of ribonucleotide reductase in cervical cancer patients. The antitumor activity of gemcitabine and/or carboplatin treatments to SiHa and CaSki human cervical cancer cell lines were assessed by Cell Counting Kit-8 viability assay, EdU incorporation assay, immunofluorescence assay, flow cytometry assay, and Western blotting methods. Additionally, synergistic efficacy was quantitatively analyzed using a combination index based on the Chou-Talalay method. The mRNA levels of three ribonucleotide reductase subunits were all upregulated in the cervical cancer tissues compared with normal tissues (P<0.0001). Consistently, the protein expression and enzyme activity of ribonucleotide reductase were also increased in the cervical cancer tissues. Interestingly, gemcitabine inhibited DNA synthesis and carboplatin induced DNA damage. Further, the combined drug regime had a significant synergistic effect on inhibiting cervical cancer cell viability (log10[combination index] <0) via enhanced DNA damage and cell apoptosis. The expression and activity of ribonucleotide reductase was increased in cervical cancer. Our study demonstrated the synergistic cytotoxicity of gemcitabine and carboplatin, through inhibiting DNA synthesis and increasing cell apoptosis in cervical cancer cell lines. This evidence might provide a rational clue of their combined application to improve cervical cancer treatment.
    Full-text · Article · Nov 2013 · OncoTargets and Therapy
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    • "Envision a biologic anti cancer agent that nestles around an iron-stabilized radical in the ribonucleotide reductase enzyme and disrupts the vital proton-coupled electron transfer of its radical to its active site [5,6]. When radiation and chemotherapy damage DNA, and thus increase the demand for DNA building blocks, a blocked ribonucleotide reductase cannot provide the demanded supply of DNA precursors and cells die [1]. Clinically, the targeting of ribonucleotide reductase by such a potent inhibitor during radio chemotherapy has found success [2]. "

    Preview · Article · Jan 2013
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