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KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

Okuyama K, Kaida A, Hayashi Y, Hayashi Y, Harada K, Miura M - PLoS ONE (2015)

Bottom Line: Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction.Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided.Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

View Article: PubMed Central - PubMed

Affiliation: Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.

ABSTRACT
KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

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Radiosensitization of HeLa-Fucci cells by the sequence of irradiation after treatment with KPU-300 and their pedigree analysis.(A)Survival curves of HeLa-Fucci cells irradiated after treatment with variousconcentrations of KPU-300 for 24 h (left panel). Survival curves in HeLa-Fucci cells irradiated after treatment with 30 nM KPU-300 for various times (right panel). Surviving fractions (SFs) were determined by colony-forming assay. SFs were normalized such that SFs in the absence of irradiation had a value of 1. Data represent means ± S.E. of values obtained from three independent experiments. (B) Pedigree analysis in cells treated with KPU-300 alone (left panel) or in combination with irradiation (right panel). After treatment with 30 nM KPU-300 for 24 h, the medium was replaced by fresh medium without drug. Time-lapse imaging was performed in the presence or absence of 4 Gy irradiation, starting immediately after drug treatment and continuing for up to 72 h. Lines extending to the right end of the panel represent cells surviving for at least 72 h, and shorter lines represents cells that collapsed within 72 h; the position of the end of the line indicates the time of cell collapse. A unique cell death mode showing anaphase skipping was observed albeit with an infrequent event in irradiated cells following drug treatment (bidirectional arrow).
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pone.0145995.g005: Radiosensitization of HeLa-Fucci cells by the sequence of irradiation after treatment with KPU-300 and their pedigree analysis.(A)Survival curves of HeLa-Fucci cells irradiated after treatment with variousconcentrations of KPU-300 for 24 h (left panel). Survival curves in HeLa-Fucci cells irradiated after treatment with 30 nM KPU-300 for various times (right panel). Surviving fractions (SFs) were determined by colony-forming assay. SFs were normalized such that SFs in the absence of irradiation had a value of 1. Data represent means ± S.E. of values obtained from three independent experiments. (B) Pedigree analysis in cells treated with KPU-300 alone (left panel) or in combination with irradiation (right panel). After treatment with 30 nM KPU-300 for 24 h, the medium was replaced by fresh medium without drug. Time-lapse imaging was performed in the presence or absence of 4 Gy irradiation, starting immediately after drug treatment and continuing for up to 72 h. Lines extending to the right end of the panel represent cells surviving for at least 72 h, and shorter lines represents cells that collapsed within 72 h; the position of the end of the line indicates the time of cell collapse. A unique cell death mode showing anaphase skipping was observed albeit with an infrequent event in irradiated cells following drug treatment (bidirectional arrow).

Mentions: Because KPU-300 synchronized cells in M phase, the most radiosensitive stage of the cell cycle, we reasoned that cells would be radiosensitized following KPU-300 treatment. After 24 h treatment with various concentrations of KPU-300, cells were X-irradiated, and survival curves were obtained (Fig 5A)(S5 Table). As shown in Fig 2, the surviving fractions after 24 h treatment with KPU-300 alone at concentrations ≥ 30 nM were ~20%. Synergistic radiosensitization was observed for cells after treatment at ≥ 30 nM, whereas at 10 nM, only an additive effect was observed. When the concentration was fixed at 30 nM, synergistic effects were observed only when drug treatment duration was ≥ 16 h (Fig 5B)(S5 Table).


KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

Okuyama K, Kaida A, Hayashi Y, Hayashi Y, Harada K, Miura M - PLoS ONE (2015)

Radiosensitization of HeLa-Fucci cells by the sequence of irradiation after treatment with KPU-300 and their pedigree analysis.(A)Survival curves of HeLa-Fucci cells irradiated after treatment with variousconcentrations of KPU-300 for 24 h (left panel). Survival curves in HeLa-Fucci cells irradiated after treatment with 30 nM KPU-300 for various times (right panel). Surviving fractions (SFs) were determined by colony-forming assay. SFs were normalized such that SFs in the absence of irradiation had a value of 1. Data represent means ± S.E. of values obtained from three independent experiments. (B) Pedigree analysis in cells treated with KPU-300 alone (left panel) or in combination with irradiation (right panel). After treatment with 30 nM KPU-300 for 24 h, the medium was replaced by fresh medium without drug. Time-lapse imaging was performed in the presence or absence of 4 Gy irradiation, starting immediately after drug treatment and continuing for up to 72 h. Lines extending to the right end of the panel represent cells surviving for at least 72 h, and shorter lines represents cells that collapsed within 72 h; the position of the end of the line indicates the time of cell collapse. A unique cell death mode showing anaphase skipping was observed albeit with an infrequent event in irradiated cells following drug treatment (bidirectional arrow).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696839&req=5

pone.0145995.g005: Radiosensitization of HeLa-Fucci cells by the sequence of irradiation after treatment with KPU-300 and their pedigree analysis.(A)Survival curves of HeLa-Fucci cells irradiated after treatment with variousconcentrations of KPU-300 for 24 h (left panel). Survival curves in HeLa-Fucci cells irradiated after treatment with 30 nM KPU-300 for various times (right panel). Surviving fractions (SFs) were determined by colony-forming assay. SFs were normalized such that SFs in the absence of irradiation had a value of 1. Data represent means ± S.E. of values obtained from three independent experiments. (B) Pedigree analysis in cells treated with KPU-300 alone (left panel) or in combination with irradiation (right panel). After treatment with 30 nM KPU-300 for 24 h, the medium was replaced by fresh medium without drug. Time-lapse imaging was performed in the presence or absence of 4 Gy irradiation, starting immediately after drug treatment and continuing for up to 72 h. Lines extending to the right end of the panel represent cells surviving for at least 72 h, and shorter lines represents cells that collapsed within 72 h; the position of the end of the line indicates the time of cell collapse. A unique cell death mode showing anaphase skipping was observed albeit with an infrequent event in irradiated cells following drug treatment (bidirectional arrow).
Mentions: Because KPU-300 synchronized cells in M phase, the most radiosensitive stage of the cell cycle, we reasoned that cells would be radiosensitized following KPU-300 treatment. After 24 h treatment with various concentrations of KPU-300, cells were X-irradiated, and survival curves were obtained (Fig 5A)(S5 Table). As shown in Fig 2, the surviving fractions after 24 h treatment with KPU-300 alone at concentrations ≥ 30 nM were ~20%. Synergistic radiosensitization was observed for cells after treatment at ≥ 30 nM, whereas at 10 nM, only an additive effect was observed. When the concentration was fixed at 30 nM, synergistic effects were observed only when drug treatment duration was ≥ 16 h (Fig 5B)(S5 Table).

Bottom Line: Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction.Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided.Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

View Article: PubMed Central - PubMed

Affiliation: Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.

ABSTRACT
KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

Show MeSH
Related in: MedlinePlus