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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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Cell survival curves following KPU-300 treatment.(A) Dose dependency of surviving fractions after KPU-300 treatment for the indicated times. (B) Time course of surviving fractions after KPU-300 treatment at the indicated concentrations. After treatment with KPU-300, cells were prepared for colony-forming assays. Data represent means ± S.E. of values obtained from three independent experiments.
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pone.0145995.g002: Cell survival curves following KPU-300 treatment.(A) Dose dependency of surviving fractions after KPU-300 treatment for the indicated times. (B) Time course of surviving fractions after KPU-300 treatment at the indicated concentrations. After treatment with KPU-300, cells were prepared for colony-forming assays. Data represent means ± S.E. of values obtained from three independent experiments.

Mentions: Next, we examined cell survival in colony-forming assays. Up to 8 h after treatment, no significant decrease in surviving fraction was detected at concentrations from 5 to 100 nM, whereas more than 16 h after treatment, a significant decrease was observed at concentrations > 5 nM. Dose dependence was not detected in the range from 30 to 100 nM (Fig 2)(S2 Table). Similar results were obtained in other human tumor cell lines using the CCK-8 assay (S2 Fig)(S3 Table), confirming that the observed properties were not unique to HeLa-Fucci cells.


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)

Cell survival curves following KPU-300 treatment.(A) Dose dependency of surviving fractions after KPU-300 treatment for the indicated times. (B) Time course of surviving fractions after KPU-300 treatment at the indicated concentrations. After treatment with KPU-300, cells were prepared for colony-forming assays. Data represent means ± S.E. of values obtained from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145995.g002: Cell survival curves following KPU-300 treatment.(A) Dose dependency of surviving fractions after KPU-300 treatment for the indicated times. (B) Time course of surviving fractions after KPU-300 treatment at the indicated concentrations. After treatment with KPU-300, cells were prepared for colony-forming assays. Data represent means ± S.E. of values obtained from three independent experiments.
Mentions: Next, we examined cell survival in colony-forming assays. Up to 8 h after treatment, no significant decrease in surviving fraction was detected at concentrations from 5 to 100 nM, whereas more than 16 h after treatment, a significant decrease was observed at concentrations > 5 nM. Dose dependence was not detected in the range from 30 to 100 nM (Fig 2)(S2 Table). Similar results were obtained in other human tumor cell lines using the CCK-8 assay (S2 Fig)(S3 Table), confirming that the observed properties were not unique to HeLa-Fucci cells.

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