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DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Dolman ME, van der Ploeg I, Koster J, Bate-Eya LT, Versteeg R, Caron HN, Molenaar JJ - PLoS ONE (2015)

Bottom Line: Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on.Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone.Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

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NU7026 more potently radiosensitizes neuroblastoma cells than gemcitabine.(A) Cell viability of NGP cells after co-treatment with gemcitabine and IR. Cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to study effects on cell viability. Data represent the mean (n = 4) +/- SD. (B) Radiosensitizing effects of 1, 5 and 10 nM gemcitabine for neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2). Cells were pre-treated with gemcitabine for 3 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to assess cell viability (n = 4 for each cell line). Radiosensitizing effects were estimated by calculating combination indices (CI) according to Chou and Talalay [40], given on the Y-axis. CI > 1.1 is antagonistic (dark grey), 1.1 ≥ CI ≥ 0.9 is additive (white) and CI < 0.9 is synergistic (light grey). Horizontal lines between the symbols represent the mean combined effects.
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pone.0145744.g005: NU7026 more potently radiosensitizes neuroblastoma cells than gemcitabine.(A) Cell viability of NGP cells after co-treatment with gemcitabine and IR. Cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to study effects on cell viability. Data represent the mean (n = 4) +/- SD. (B) Radiosensitizing effects of 1, 5 and 10 nM gemcitabine for neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2). Cells were pre-treated with gemcitabine for 3 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to assess cell viability (n = 4 for each cell line). Radiosensitizing effects were estimated by calculating combination indices (CI) according to Chou and Talalay [40], given on the Y-axis. CI > 1.1 is antagonistic (dark grey), 1.1 ≥ CI ≥ 0.9 is additive (white) and CI < 0.9 is synergistic (light grey). Horizontal lines between the symbols represent the mean combined effects.

Mentions: Gemcitabine is a chemotherapeutic agent which is currently evaluated extensively in clinical trials for its additional radiosensitizing potential [41]. It is suggested that gemcitabine exerts its radiosensitizing effect via inhibition of ribonucleotide reductase by its active metabolite gemcitabine diphosphate, thereby reducing the synthesis of deoxynucleoside triphosphates [41]. Current study compared the radiosensitizing effects of NU7026 in neuroblastoma cell lines with the radiosensitizing effects of gemcitabine. As for NU7026, we first investigated the optimal gemcitabine dose for combination treatment with 0.63 Gy IR in NGP cells. Based on literature [41–43], NGP cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before irradiation. Effects on cell viability were studied at 96 h after IR-exposure of the cells. Twenty-four hours pre-treatment with gemcitabine did not improve the inhibitory effect of low-dose IR treatment on the cell viability of NGP cells, while 3 h pre-treatment did (Fig 5A). Monotherapy of NGP cells with ≥ 100 nM gemcitabine furthermore resulted into almost complete cell death (i.e. 97–99%). For the comparison with NU7026, gemcitabine was therefore administered to the cells in doses below 100 nM (i.e. 1, 5 and 10 nM) at 3 h before IR-exposure.


DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Dolman ME, van der Ploeg I, Koster J, Bate-Eya LT, Versteeg R, Caron HN, Molenaar JJ - PLoS ONE (2015)

NU7026 more potently radiosensitizes neuroblastoma cells than gemcitabine.(A) Cell viability of NGP cells after co-treatment with gemcitabine and IR. Cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to study effects on cell viability. Data represent the mean (n = 4) +/- SD. (B) Radiosensitizing effects of 1, 5 and 10 nM gemcitabine for neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2). Cells were pre-treated with gemcitabine for 3 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to assess cell viability (n = 4 for each cell line). Radiosensitizing effects were estimated by calculating combination indices (CI) according to Chou and Talalay [40], given on the Y-axis. CI > 1.1 is antagonistic (dark grey), 1.1 ≥ CI ≥ 0.9 is additive (white) and CI < 0.9 is synergistic (light grey). Horizontal lines between the symbols represent the mean combined effects.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696738&req=5

pone.0145744.g005: NU7026 more potently radiosensitizes neuroblastoma cells than gemcitabine.(A) Cell viability of NGP cells after co-treatment with gemcitabine and IR. Cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to study effects on cell viability. Data represent the mean (n = 4) +/- SD. (B) Radiosensitizing effects of 1, 5 and 10 nM gemcitabine for neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2). Cells were pre-treated with gemcitabine for 3 h before exposure to 0.63 Gy IR. At 96 h after IR-exposure, MTT cell proliferation assays were performed to assess cell viability (n = 4 for each cell line). Radiosensitizing effects were estimated by calculating combination indices (CI) according to Chou and Talalay [40], given on the Y-axis. CI > 1.1 is antagonistic (dark grey), 1.1 ≥ CI ≥ 0.9 is additive (white) and CI < 0.9 is synergistic (light grey). Horizontal lines between the symbols represent the mean combined effects.
Mentions: Gemcitabine is a chemotherapeutic agent which is currently evaluated extensively in clinical trials for its additional radiosensitizing potential [41]. It is suggested that gemcitabine exerts its radiosensitizing effect via inhibition of ribonucleotide reductase by its active metabolite gemcitabine diphosphate, thereby reducing the synthesis of deoxynucleoside triphosphates [41]. Current study compared the radiosensitizing effects of NU7026 in neuroblastoma cell lines with the radiosensitizing effects of gemcitabine. As for NU7026, we first investigated the optimal gemcitabine dose for combination treatment with 0.63 Gy IR in NGP cells. Based on literature [41–43], NGP cells were pre-treated with 0, 1, 5, 10 or 100 nM or 1 or 10 μM gemcitabine for 3 or 24 h before irradiation. Effects on cell viability were studied at 96 h after IR-exposure of the cells. Twenty-four hours pre-treatment with gemcitabine did not improve the inhibitory effect of low-dose IR treatment on the cell viability of NGP cells, while 3 h pre-treatment did (Fig 5A). Monotherapy of NGP cells with ≥ 100 nM gemcitabine furthermore resulted into almost complete cell death (i.e. 97–99%). For the comparison with NU7026, gemcitabine was therefore administered to the cells in doses below 100 nM (i.e. 1, 5 and 10 nM) at 3 h before IR-exposure.

Bottom Line: Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on.Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone.Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Show MeSH
Related in: MedlinePlus