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


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.
    PLoS ONE 08/2014; 9(8):e104125. DOI:10.1371/journal.pone.0104125 · 3.23 Impact Factor
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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.
    OncoTargets and Therapy 11/2013; 6:1707-1717. DOI:10.2147/OTT.S54217 · 2.31 Impact Factor
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    • "3-AP (NSC #663249) is an investigational RNR inhibitor provided to Case Western Reserve University (Cleveland, OH) under an agreement with the National Cancer Institute Cancer Therapy Evaluation Program (Bethesda, MD) and Nanotherapeutics, Inc. (Alachua, FL). Cells were exposed to therapeutic doses of 3-AP (1, 5, or 10 μM) and cisplatin (5 μM) as indicated, and then, subjected to clonogenic cell survival, RNR activity, and γH2Ax DNA damage assays [10,11]. Because in vivo human minimum inhibitory concentrations of RNR blockade by 3-AP lasts 6 hours [15-18] and cisplatin adduct formation reaches peak six hours after initial cisplatin exposure [19], four conditions were studied: (a) six-hour 3-AP; (b) six-hour cisplatin; (c) six-hour 3-AP followed by six-hour cisplatin sequential exposure (i.e., modeling GOG #126O); or (d) six-hour 3-AP plus cisplatin co-exposure. "
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    ABSTRACT: The potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer. Preclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP (96 mg/m(2), day one to four) and cisplatin (25 mg/m(2), day two and three) infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response. 3-AP preceding cisplatin exposure in platinum-resistant ovarian cancer cells was not as effective as sequencing cisplatin plus 3-AP together in cell survival assays. Platinum-mediated DNA damage (i.e., γH2AX foci) resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual. When sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/m(2)) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron. NCT00081276.
    Journal of Translational Medicine 04/2012; 10(1):79. DOI:10.1186/1479-5876-10-79 · 3.93 Impact Factor
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