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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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Confocal fluorescence imaging of spheroids after treatment with KPU-300.The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.
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pone.0145995.g004: Confocal fluorescence imaging of spheroids after treatment with KPU-300.The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.

Mentions: We next examined cell cycle kinetics of cells in spheroids following KPU-300 treatment. We previously reported that HeLa-Fucci cell spheroids with a diameter of ~500 μm have an outer growth fraction ~80 μm thick and an inner quiescent fraction. However, it was impossible to visualize the latter fraction under live conditions due to the optical limitations of the confocal fluorescence microscope [40]. Outer cells visualized at a depth of 65 μm from the spheroid surface responded to the KPU-300 treatment, as shown in Fig 4: the number of green cells increased gradually, and that of red cells increased 24 h after the treatment. Both signals reached a peak around 28 h after the treatment (data not shown), and fluorescence intensity gradually decreased thereafter. After drug treatment, the size of the spheroids significantly increased, indicating that cell-cell contact was loosened. When the spheroids were dispersed 24 h after the treatment, most of the cells were yellow (i.e., M phase) cells lacking a nuclear envelope, in contrast to the interphase cells from the untreated spheroid, in which fluorescence was localized in the nucleus (S3 Fig). These results suggest that the drug treatment loosened cell-cell contact, leading to recruitment of the quiescent fraction to growth fraction, which subsequently responded to the drug. As a control, fluorescence images of the untreated spheroid 24 h obtained under exactly the same observation conditions used for the KPU-300-treated spheroid are shown in S4 Fig.


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)

Confocal fluorescence imaging of spheroids after treatment with KPU-300.The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145995.g004: Confocal fluorescence imaging of spheroids after treatment with KPU-300.The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.
Mentions: We next examined cell cycle kinetics of cells in spheroids following KPU-300 treatment. We previously reported that HeLa-Fucci cell spheroids with a diameter of ~500 μm have an outer growth fraction ~80 μm thick and an inner quiescent fraction. However, it was impossible to visualize the latter fraction under live conditions due to the optical limitations of the confocal fluorescence microscope [40]. Outer cells visualized at a depth of 65 μm from the spheroid surface responded to the KPU-300 treatment, as shown in Fig 4: the number of green cells increased gradually, and that of red cells increased 24 h after the treatment. Both signals reached a peak around 28 h after the treatment (data not shown), and fluorescence intensity gradually decreased thereafter. After drug treatment, the size of the spheroids significantly increased, indicating that cell-cell contact was loosened. When the spheroids were dispersed 24 h after the treatment, most of the cells were yellow (i.e., M phase) cells lacking a nuclear envelope, in contrast to the interphase cells from the untreated spheroid, in which fluorescence was localized in the nucleus (S3 Fig). These results suggest that the drug treatment loosened cell-cell contact, leading to recruitment of the quiescent fraction to growth fraction, which subsequently responded to the drug. As a control, fluorescence images of the untreated spheroid 24 h obtained under exactly the same observation conditions used for the KPU-300-treated spheroid are shown in S4 Fig.

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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