Emiko Sekine-Suzuki

Analysis of the antioxidative function of the radioprotective Japanese traditional (Kampo) medicine, hangeshashinto, in an aqueous phase

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH)–scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O2•−)–scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O2•−. Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.

High-throughput screening of radioprotectors using rat thymocytes.

We invented a high-throughput screening method for the examination of radioprotective activity of chemical compounds using rat thymocytes. X-irradiation of the rat thymocytes induced apoptosis, leading to a significant cell shrinkage, which could be easily detected and directly quantified by the flow cytometry analysis. The protective effect of some natural antioxidants against radiation induced apoptosis in the rat thymocytes, as well as their toxicities without X-irradiation, was successfully evaluated using this method. This method provides a powerful tool to develop novel radioprotectors without toxicity and can also be widely used to estimate other oxidative stress except for radiation.

Effectiveness of combined treatment using X-rays and a phosphoinositide 3-kinase inhibitor, ZSTK474, on proliferation of HeLa cells in vitro and in vivo.

ZSTK474 is a novel orally applicable phosphoinositide 3‐kinase‐specific inhibitor that strongly inhibits cancer cell proliferation. To further explore the antitumor effect of ZSTK474 for future clinical usage, we studied its combined effects with radiation. The proliferation of HeLa cells was inhibited by treatment with X‐rays alone or ZSTK474 alone. Combination treatment using X‐rays then ZSTK474 given orally for 8 days, starting 24 h post‐irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK‐3β by X‐irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X‐irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth. Combined treatment with X‐rays and ZSTK474 has greater therapeutic potential than radiation or drug therapy alone, both in vitro and in vivo. (Cancer Sci 2011; 102: 1176–1180)

A facile and rapid access to resveratrol derivatives and their radioprotective activity

A facile and rapid access to resveratrol derivatives has been achieved based on palladium-catalyzed oxidative Heck reaction of aryl boronic acids with styrenes followed by demethylation in moderate to good yields. A series of resveratrol derivatives with various functional groups has been synthesized easily. The radioprotective activity of synthesized compounds has also been evaluated using rat thymocytes. The results revealed that some resveratrol derivatives efficiently protected the thymocytes from radiation-induced apoptosis.

Non-invasive measurement of melanin-derived radicals in living mouse tail using X-band EPR

The aim of this experiment is to measure in vivo generation of melanin-derived radicals non-invasively, as a quantifiable index of radio-biological effect. Melanin-derived radicals in a living intact mouse tail tip were non-invasively measured in very simple way using an X-band electron paramagnetic resonance spectrometer. Colored mouse strains, C57BL/6NCr, BDF1, and C3H/He, have clear EPR signal corresponding to melanin-derived radicals in the tail tip; however, albino mouse strains, BALB/cCr, ddY, ICR, have no EPR signals. An X-ray fraction of 2 Gy/day (1 Gy/min) was repeatedly irradiated to a C3H/He mouse tail skin every Monday to Friday for 4 weeks. In comparison to before starting irradiation, the C3H/He mouse tail skin became darker, like a suntan. The melanin-derived radicals in C3H/He mouse tail skin were increased in association with X-ray fractions. Melanin-derived radicals in mouse tail skin can be readily and chronologically measurable by using X-band EPR spectrometer, and can be a marker for a radiobiological effect in the skin.

Efficient protective activity of a planar catechin analogue against radiation-induced apoptosis in rat thymocytes

About two thirds of biological damage due to low linear energy transfer (LET) radiation, such as X-rays and the plateau region of heavy-ion beams, is known to be caused by the hydroxyl radical (˙OH), the most powerful reactive oxygen species (ROS), generated via ionisation and excitation of water molecules. Thus, compounds having an efficient scavenging activity against ROS are expected to exhibit a radioprotective activity. A planar catechin analogue, where an isopropyl fragment was introduced into the catechol ring of (+)-catechin, showed an efficient protective effect against X-ray induced apoptosis in rat thymocytes compared to (+)-catechin. The planar catechin scavenged 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH˙) solubilised in water by β-cyclodextrin about 10-fold faster than (+)-catechin in phosphate buffer (0.1 M, pH 7.4) at 298 K. Furthermore, the experimental log P value of the planar catechin (1.22) is reported to be significantly larger than that of (+)-catechin (0.44). The higher radical-scavenging activity and lipophilicity of the planar catechin than those of (+)-catechin may contribute in part to the higher protective activity against X-ray-induced apoptosis in rat thymocytes.

Localized hydroxyl radical generation at mmol/L and mol/L levels in water by photon irradiation

The generation of localized hydroxyl radical (•OH) in aqueous samples by low linear energy transfer irradiation was investigated. Several concentrations of 5,5-dimethyl-1-pyrroline-N-oxid solution (from 0.5 to 1,680 mmol/L) were prepared and irradiated with an identical dose of X-ray or γ-ray. The density of •OH generation in aqueous solution was evaluated by the electron paramagnetic resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxid as an electron paramagnetic resonance spin-trapping agent. The relationship between the molecular density of 5,5-dimethyl-1-pyrroline-N-oxid in the samples and the concentration of 5,5-dimethyl-1-pyrroline-N-oxid-OH generated in the irradiated samples was analyzed. Two different characteristic linear trends were observed in the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots, which suggested •OH generation in two fashions, i.e., mmol/L- and mol/L-level local concentrations. The dose, dose rate, and/or the energy of photon irradiation did not affect the shapes of the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots. Moreover, the addition of 5 mmol/L caffeine could cancel the contribution of mmol/L-level •OH generation, leaving a trace of mol/L-level •OH generation. Thus, the localized mmol/L- and mol/L-level generations of •OH, which were independent of experimental parameters such as dose, dose rate, and/or the energy of photon of low linear energy transfer radiation, were established.