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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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Enhanced PRKDC expression in neuroblastoma patients correlates with a poor prognosis.(A) Comparison between the average Affymetrix PRKDC mRNA expression levels in two independent neuroblastoma datasets (dark grey) versus expression levels in a compiled library of normal tissues and in nonmalignant adrenal glands (light grey). (B) Affymetrix PRKDC mRNA expression in neuroblastoma of 88 individual patients, ordered by the 2log fold transformed level of PRKDC. Color coded tracks below the YY plot give information about neuroblastoma stage (INSS; green = stage 1, dark green = stage 2, light brown = stage 3, blue = stage 4s and red = stage 4), patient survival (green = yes and red = no) and the presence of MYCN amplification and ALK mutation (green = yes and red = no). (C) Kaplan-Meier curve of the survival of neuroblastoma patients with an Affymetrix PRKDC mRNA expression above 656 (n = 8) versus neuroblastoma patients with an Affymetrix PRKDC mRNA expression below 656 (n = 80). The cutoff of 656 was determined using the Kaplan scanner tool in the R2 web application (http://r2.amc.nl). Samples were sorted according to the expression of PRKDC and divided into two groups on the basis of a cutoff expression value. All cutoff expression levels and the resulting groups were analyzed for survival, with the provision that each group included at least eight samples. For each cutoff level and grouping, the log-rank significance of projected survival was calculated. The graph depicts the best p value corrected for multiple testing (Bonferroni correction).
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pone.0145744.g001: Enhanced PRKDC expression in neuroblastoma patients correlates with a poor prognosis.(A) Comparison between the average Affymetrix PRKDC mRNA expression levels in two independent neuroblastoma datasets (dark grey) versus expression levels in a compiled library of normal tissues and in nonmalignant adrenal glands (light grey). (B) Affymetrix PRKDC mRNA expression in neuroblastoma of 88 individual patients, ordered by the 2log fold transformed level of PRKDC. Color coded tracks below the YY plot give information about neuroblastoma stage (INSS; green = stage 1, dark green = stage 2, light brown = stage 3, blue = stage 4s and red = stage 4), patient survival (green = yes and red = no) and the presence of MYCN amplification and ALK mutation (green = yes and red = no). (C) Kaplan-Meier curve of the survival of neuroblastoma patients with an Affymetrix PRKDC mRNA expression above 656 (n = 8) versus neuroblastoma patients with an Affymetrix PRKDC mRNA expression below 656 (n = 80). The cutoff of 656 was determined using the Kaplan scanner tool in the R2 web application (http://r2.amc.nl). Samples were sorted according to the expression of PRKDC and divided into two groups on the basis of a cutoff expression value. All cutoff expression levels and the resulting groups were analyzed for survival, with the provision that each group included at least eight samples. For each cutoff level and grouping, the log-rank significance of projected survival was calculated. The graph depicts the best p value corrected for multiple testing (Bonferroni correction).

Mentions: For clinical purposes it would be desirable that anticancer agents exert their effects specifically on cancer cells, so unwanted side effects in healthy organs will be limited. We therefore evaluated PRKDC mRNA expression levels in human neuroblastoma patients and compared these expression levels with expression levels in non-diseased (normal) tissues. As shown in Fig 1A, PRKDC mRNA expression levels in two independent neuroblastoma tumor datasets were significantly higher than the expression levels in a compiled library of normal tissues and nonmalignant adrenal glands (the main primary tumor site) [39]. Elevated PRKDC mRNA levels in neuroblastoma were associated with poor clinical characteristics (Fig 1B) and a significant reduction in overall survival (Fig 1C).


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)

Enhanced PRKDC expression in neuroblastoma patients correlates with a poor prognosis.(A) Comparison between the average Affymetrix PRKDC mRNA expression levels in two independent neuroblastoma datasets (dark grey) versus expression levels in a compiled library of normal tissues and in nonmalignant adrenal glands (light grey). (B) Affymetrix PRKDC mRNA expression in neuroblastoma of 88 individual patients, ordered by the 2log fold transformed level of PRKDC. Color coded tracks below the YY plot give information about neuroblastoma stage (INSS; green = stage 1, dark green = stage 2, light brown = stage 3, blue = stage 4s and red = stage 4), patient survival (green = yes and red = no) and the presence of MYCN amplification and ALK mutation (green = yes and red = no). (C) Kaplan-Meier curve of the survival of neuroblastoma patients with an Affymetrix PRKDC mRNA expression above 656 (n = 8) versus neuroblastoma patients with an Affymetrix PRKDC mRNA expression below 656 (n = 80). The cutoff of 656 was determined using the Kaplan scanner tool in the R2 web application (http://r2.amc.nl). Samples were sorted according to the expression of PRKDC and divided into two groups on the basis of a cutoff expression value. All cutoff expression levels and the resulting groups were analyzed for survival, with the provision that each group included at least eight samples. For each cutoff level and grouping, the log-rank significance of projected survival was calculated. The graph depicts the best p value corrected for multiple testing (Bonferroni correction).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696738&req=5

pone.0145744.g001: Enhanced PRKDC expression in neuroblastoma patients correlates with a poor prognosis.(A) Comparison between the average Affymetrix PRKDC mRNA expression levels in two independent neuroblastoma datasets (dark grey) versus expression levels in a compiled library of normal tissues and in nonmalignant adrenal glands (light grey). (B) Affymetrix PRKDC mRNA expression in neuroblastoma of 88 individual patients, ordered by the 2log fold transformed level of PRKDC. Color coded tracks below the YY plot give information about neuroblastoma stage (INSS; green = stage 1, dark green = stage 2, light brown = stage 3, blue = stage 4s and red = stage 4), patient survival (green = yes and red = no) and the presence of MYCN amplification and ALK mutation (green = yes and red = no). (C) Kaplan-Meier curve of the survival of neuroblastoma patients with an Affymetrix PRKDC mRNA expression above 656 (n = 8) versus neuroblastoma patients with an Affymetrix PRKDC mRNA expression below 656 (n = 80). The cutoff of 656 was determined using the Kaplan scanner tool in the R2 web application (http://r2.amc.nl). Samples were sorted according to the expression of PRKDC and divided into two groups on the basis of a cutoff expression value. All cutoff expression levels and the resulting groups were analyzed for survival, with the provision that each group included at least eight samples. For each cutoff level and grouping, the log-rank significance of projected survival was calculated. The graph depicts the best p value corrected for multiple testing (Bonferroni correction).
Mentions: For clinical purposes it would be desirable that anticancer agents exert their effects specifically on cancer cells, so unwanted side effects in healthy organs will be limited. We therefore evaluated PRKDC mRNA expression levels in human neuroblastoma patients and compared these expression levels with expression levels in non-diseased (normal) tissues. As shown in Fig 1A, PRKDC mRNA expression levels in two independent neuroblastoma tumor datasets were significantly higher than the expression levels in a compiled library of normal tissues and nonmalignant adrenal glands (the main primary tumor site) [39]. Elevated PRKDC mRNA levels in neuroblastoma were associated with poor clinical characteristics (Fig 1B) and a significant reduction in overall survival (Fig 1C).

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