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Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

Yumita N, Iwase Y, Nishi K, Komatsu H, Takeda K, Onodera K, Fukai T, Ikeda T, Umemura S, Okudaira K, Momose Y - Theranostics (2012)

Bottom Line: Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine.The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis.These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Pharmacy, Yokohama College of Pharmacy, 601, Matano-cho, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan;

ABSTRACT
In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

No MeSH data available.


Related in: MedlinePlus

Diagram of the ultrasonic exposure apparatus.
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Figure 2: Diagram of the ultrasonic exposure apparatus.

Mentions: The apparatus for ultrasonic exposure is shown schematically in Figure 2. The ultrasound transducer uses a piezoelectric ceramic disk 24 mm in diameter and was driven at its resonance frequency (1.93 MHz). Low frequency ultrasound waves have greater depth of penetration but are less focused. On the other hands, ultrasound at a frequency of more than 1 MHz is absorbed primarily by tissues but it can be focused into small volume. In consideration of the absorption and the penetration, we employed the frequency of 1.93 MHz for exposure in this experiment 27. Before exposure, the cells were harvested and washed twice in phosphate-buffered saline (PBS, pH 7.4). HL-60 cells were washed, resuspended at a concentration of 1x106 cells/ml in 2.5 ml of RPMI 1640 (serum free), and transferred into a cylindrical 16×125 mm polystyrene tissue culture tube (Corning, Corning, NY, USA) for exposure to ultrasound. A polystyrene tissue culture tube was suspended 35 mm away from the surface of the plane-wave transducer in degassed water, and insonated at 37°C. Immediately before exposure, DCPH-P-Na(I) was added to the cell suspension. During exposure, the tube was rotated at 60 rpm by a synchronous motor to improve mixing and to provide a uniform exposure 28. HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of PF. The samples treated with PF alone were kept in the same position for the same period as the ultrasonic exposure. Ultrasonic output from the transducer was evaluated in degassed water by placing the axis of the transducer horizontally. The output acoustic power was calibrated by measuring the radiation force on a 2-mm-thick hollow aluminum plate with an area of 20 x 28 mm, suspended at an angle of 45° to the axis. Its horizontal projection was therefore 20 mm x 20 mm. The ultrasonic intensity was calculated by dividing the measured acoustic power by the projected area. The temperature rise in 2.5 ml RPMI 1640 in the container during a 5-min exposure at the highest ultrasonic intensity used in the experiments was less than 1°C. After the treatment procedure, the medium was replaced with fresh RPMI 1640 with 10% FBS and the cells was incubated in an atmosphere humidified with 5% CO2 at 37 °C for 6 h before apoptosis induction was evaluated


Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

Yumita N, Iwase Y, Nishi K, Komatsu H, Takeda K, Onodera K, Fukai T, Ikeda T, Umemura S, Okudaira K, Momose Y - Theranostics (2012)

Diagram of the ultrasonic exposure apparatus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475214&req=5

Figure 2: Diagram of the ultrasonic exposure apparatus.
Mentions: The apparatus for ultrasonic exposure is shown schematically in Figure 2. The ultrasound transducer uses a piezoelectric ceramic disk 24 mm in diameter and was driven at its resonance frequency (1.93 MHz). Low frequency ultrasound waves have greater depth of penetration but are less focused. On the other hands, ultrasound at a frequency of more than 1 MHz is absorbed primarily by tissues but it can be focused into small volume. In consideration of the absorption and the penetration, we employed the frequency of 1.93 MHz for exposure in this experiment 27. Before exposure, the cells were harvested and washed twice in phosphate-buffered saline (PBS, pH 7.4). HL-60 cells were washed, resuspended at a concentration of 1x106 cells/ml in 2.5 ml of RPMI 1640 (serum free), and transferred into a cylindrical 16×125 mm polystyrene tissue culture tube (Corning, Corning, NY, USA) for exposure to ultrasound. A polystyrene tissue culture tube was suspended 35 mm away from the surface of the plane-wave transducer in degassed water, and insonated at 37°C. Immediately before exposure, DCPH-P-Na(I) was added to the cell suspension. During exposure, the tube was rotated at 60 rpm by a synchronous motor to improve mixing and to provide a uniform exposure 28. HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of PF. The samples treated with PF alone were kept in the same position for the same period as the ultrasonic exposure. Ultrasonic output from the transducer was evaluated in degassed water by placing the axis of the transducer horizontally. The output acoustic power was calibrated by measuring the radiation force on a 2-mm-thick hollow aluminum plate with an area of 20 x 28 mm, suspended at an angle of 45° to the axis. Its horizontal projection was therefore 20 mm x 20 mm. The ultrasonic intensity was calculated by dividing the measured acoustic power by the projected area. The temperature rise in 2.5 ml RPMI 1640 in the container during a 5-min exposure at the highest ultrasonic intensity used in the experiments was less than 1°C. After the treatment procedure, the medium was replaced with fresh RPMI 1640 with 10% FBS and the cells was incubated in an atmosphere humidified with 5% CO2 at 37 °C for 6 h before apoptosis induction was evaluated

Bottom Line: Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine.The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis.These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Pharmacy, Yokohama College of Pharmacy, 601, Matano-cho, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan;

ABSTRACT
In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

No MeSH data available.


Related in: MedlinePlus