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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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Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, p < 0.05; **, p < 0.01 vs. lower values for the same time points.
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pone.0145995.g007: Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, p < 0.05; **, p < 0.01 vs. lower values for the same time points.

Mentions: We next examined the effect of reversing the combination sequence, i.e., by treating cells with KPU-300 for 24 h immediately after X-irradiation (Fig 7A)(S6 Table). At doses < 4 Gy, the SFs exhibited an additive effect, whereas at doses ≥ 4 Gy, they exhibited an antagonistic effect. Thus, this sequence of combination treatment did not result in synergistic radiosensitization. This observation is reasonable given that the observed radiosensitization could be attributed to synchronization in early M phase, as described above. To investigate the mechanism of the antagonistic effect at high doses of irradiation, we closely inspected the cell cycle kinetics (Fig 7B). DNA content analysis by flow cytometry revealed that cells were released later from G2 arrest after 6 Gy irradiation than after 2 Gy irradiation; however, when irradiation was combined with KPU-300 treatment, no useful information was obtained to explain the difference (Fig 7Ba). The elongation of the green phase represents G2 arrest [41]; therefore, we concluded that G2 arrest was more strongly induced during KPU-300 treatment following 6 Gy irradiation than following 2 Gy irradiation (Fig 7Ba). Consequently, the ratio of mitotic cells released from G2 arrest was significantly higher in cells exposed to 2 Gy than in those exposed to 6 Gy (Fig 7Bb, Fig 7Bc)(S6 Table). Thus, elongated G2 arrest results in a smaller proportion of mitotic cells during the limited period of KPU-300 treatment (24 h), leading to decreased cytotoxicity by KPU-300.


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)

Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, p < 0.05; **, p < 0.01 vs. lower values for the same time points.
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pone.0145995.g007: Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, p < 0.05; **, p < 0.01 vs. lower values for the same time points.
Mentions: We next examined the effect of reversing the combination sequence, i.e., by treating cells with KPU-300 for 24 h immediately after X-irradiation (Fig 7A)(S6 Table). At doses < 4 Gy, the SFs exhibited an additive effect, whereas at doses ≥ 4 Gy, they exhibited an antagonistic effect. Thus, this sequence of combination treatment did not result in synergistic radiosensitization. This observation is reasonable given that the observed radiosensitization could be attributed to synchronization in early M phase, as described above. To investigate the mechanism of the antagonistic effect at high doses of irradiation, we closely inspected the cell cycle kinetics (Fig 7B). DNA content analysis by flow cytometry revealed that cells were released later from G2 arrest after 6 Gy irradiation than after 2 Gy irradiation; however, when irradiation was combined with KPU-300 treatment, no useful information was obtained to explain the difference (Fig 7Ba). The elongation of the green phase represents G2 arrest [41]; therefore, we concluded that G2 arrest was more strongly induced during KPU-300 treatment following 6 Gy irradiation than following 2 Gy irradiation (Fig 7Ba). Consequently, the ratio of mitotic cells released from G2 arrest was significantly higher in cells exposed to 2 Gy than in those exposed to 6 Gy (Fig 7Bb, Fig 7Bc)(S6 Table). Thus, elongated G2 arrest results in a smaller proportion of mitotic cells during the limited period of KPU-300 treatment (24 h), leading to decreased cytotoxicity by KPU-300.

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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