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.

Kohei Okuyama,Yoshiki Hayashi,Masahiko Miura,Yoshio Hayashi,Atsushi Kaida,Kiyoshi Harada,Robert M Hoffman

doi:10.1371/journal.pone.0145995

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.

Highlights

Microtubules, which consist of polymers of α- and β-tubulins, are major cytoskeletal components that play an important role in the regulation of chromosome separation in mitosis [1,2,3]
We examined the kinetics of fluorescent ubiquitination-based cell cycle indicator (Fucci) fluorescence in HeLa-Fucci cells following treatment with various concentrations of KPU-300 (Fig 1C, upper panel)
Because colchicine-type anti-microtubule drugs are mostly vascular disrupting agents (VDA) [31,32,33, 42,43,44], we closely examined their effect on cell cycle kinetics and investigated the possibility of using such agents as a radiosensitizer in vitro

Summary

Introduction

Microtubules, which consist of polymers of α- and β-tubulins, are major cytoskeletal components that play an important role in the regulation of chromosome separation in mitosis [1,2,3]. Many anti-microtubule agents have been developed; in general, they can be classified into three types depending on the binding site in β-tubulin: the taxane site, the vinca domain, and the colchicine site. Drugs belonging to the former two types, such as paclitaxel and vincristine, have been already extensively used as chemotherapeutic agents in the clinic [4, 5]. Several drugs with colchicine-like activities have been recently developed; these compounds exert vascular disruption [10] in addition to their cytotoxic effects on tumor cells themselves Agents of this type include combretastatin A-4 (CA-4) [11] and plinabulin (NPI-2358) [12, 13]. The simpler structure of KPU-300 makes chemical synthesis much easier

Methods

Results

Conclusion